ORCID Profile
0000-0001-8052-5584
Current Organisation
La Trobe University
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Research Topics
In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Top 5 Research Topics
ANZSRC Field of Research (FoR)
Biochemistry and Cell Biology | Characterisation Of Macromolecules | Bacteriology | Enzymes | Biological And Medical Chemistry | Macromolecular and Materials Chemistry | Analytical Biochemistry | Biochemistry And Cell Biology Not Elsewhere Classified | Nanochemistry and Supramolecular Chemistry | Nanoscale Characterisation | Structural Biology (incl. Macromolecular Modelling) | Enzymes | Proteomics and Intermolecular Interactions (excl. Medical Proteomics) | Medical Biochemistry and Metabolomics | Medical Biochemistry: Proteins And Peptides | Nanotechnology | Crop and Pasture Biochemistry and Physiology | Genetic Engineering And Enzyme Technology | Medical Biochemistry: Proteins and Peptides (incl. Medical Proteomics) | Nanobiotechnology | Cell Neurochemistry
ANZSRC Socio-Economic Objective (SEO)
Prevention—biologicals (e.g. vaccines) | Diagnostics | Treatments (e.g. chemicals, antibiotics) | Living resources (flora and fauna) | Wheat | Preventive Medicine | Infectious diseases | Health related to ageing | Nervous system and disorders | Human Diagnostics | Cardiovascular system and diseases | Biological sciences | Cancer and related disorders | Chemical sciences | Teaching and Instruction Technologies | Computer software and services not elsewhere classified | Expanding Knowledge in the Chemical Sciences | Expanding Knowledge in the Agricultural and Veterinary Sciences | Expanding Knowledge in the Biological Sciences | Treatments (e.g. chemicals, antibiotics) |
Related Links
Publications
X-ray crystal structure and specificity of the plasmodium falciparum malaria aminopeptidase PfM18AAP
Publisher: Elsevier BV
Date: 09-2012
DOI: 10.1016/J.JMB.2012.06.006
Abstract: The malarial aminopeptidases have emerged as promising new drug targets for the development of novel antimalarial drugs. The M18AAP of Plasmodium falciparum malaria is a metallo-aminopeptidase that we show demonstrates a highly restricted specificity for peptides with an N-terminal Glu or Asp residue. Thus, the enzyme may function alongside other aminopeptidases in effecting the complete degradation or turnover of proteins, such as host hemoglobin, which provides a free amino acid pool for the growing parasite. Inhibition of PfM18AAP's function using antisense RNA is detrimental to the intra-erythrocytic malaria parasite and, hence, it has been proposed as a potential novel drug target. We report the X-ray crystal structure of the PfM18AAP aminopeptidase and reveal its complex dodecameric assembly arranged via dimer and trimer units that interact to form a large tetrahedron shape that completely encloses the 12 active sites within a central cavity. The four entry points to the catalytic lumen are each guarded by 12 large flexible loops that could control substrate entry into the catalytic sites. PfM18AAP thus resembles a proteasomal-like machine with multiple active sites able to degrade peptide substrates that enter the central lumen. The Plasmodium enzyme shows significant structural differences around the active site when compared to recently determined structures of its mammalian and human homologs, which provides a platform from which a rational approach to inhibitor design of new malaria-specific drugs can begin.
Crystallization of dihydrodipicolinate synthase from a clinical isolate ofStreptococcus pneumoniae
Publisher: International Union of Crystallography (IUCr)
Date: 25-12-2009
Size-Distribution Analysis of Proteins by Analytical Ultracentrifugation: Strategies and Application to Model Systems
Publisher: Elsevier BV
Date: 02-2002
Cloning, expression and crystallization of dihydrodipicolinate reductase from methicillin-resistantStaphylococcus aureus
Publisher: International Union of Crystallography (IUCr)
Date: 25-12-2009
A new robust kinetic assay for DAP epimerase activity
Publisher: Elsevier BV
Date: 10-2013
DOI: 10.1016/J.BIOCHI.2013.06.026
Abstract: DAP epimerase is the penultimate enzyme in the lysine biosynthesis pathway. The most versatile assay for DAP epimerase catalytic activity employs a coupled DAP epimerase-DAP dehydrogenase enzyme system with a commercial mixture of DAP isomers as substrate. DAP dehydrogenase converts meso-DAP to THDP with concomitant reduction of NADP(+) to NADPH. We show that at high concentrations, accumulation of NADPH results in inhibition of DAPDH, resulting in spurious kinetic data. A new assay has been developed employing DAP decarboxylase that allows the reliable characterisation of DAP epimerase enzyme kinetics.
Imperfect centered miRNA binding sites are common and can mediate repression of target mRNAs
Publisher: Springer Science and Business Media LLC
Date: 2014
Antigen Ligation Triggers a Conformational Change within the Constant Domain of the αβ T Cell Receptor
Publisher: Elsevier BV
Date: 06-2009
DOI: 10.1016/J.IMMUNI.2009.03.018
Abstract: Ligation of the alphabeta T cell receptor (TCR) by a specific peptide-loaded major histocompatibility complex (pMHC) molecule initiates T cell signaling via the CD3 complex. However, the initial events that link antigen recognition to T cell signal transduction remain unclear. Here we show, via fluorescence-based experiments and structural analyses, that MHC-restricted antigen recognition by the alphabeta TCR results in a specific conformational change confined to the A-B loop within the alpha chain of the constant domain (Calpha). The apparent affinity constant of this A-B loop movement mirrored that of alphabeta TCR-pMHC ligation and was observed in two alphabeta TCRs with distinct pMHC specificities. The Ag-induced A-B loop conformational change could be inhibited by fixing the juxtapositioning of the constant domains and was shown to be reversible upon pMHC disassociation. Notably, the loop movement within the Calpha domain, although specific for an agonist pMHC ligand, was not observed with a pMHC antagonist. Moreover, mutagenesis of residues within the A-B loop impaired T cell signaling in an in vitro system of antigen-specific TCR stimulation. Collectively, our findings provide a basis for the earliest molecular events that underlie Ag-induced T cell triggering.
Homodimerization attenuates the anti-inflammatory activity of interleukin-37
Publisher: American Association for the Advancement of Science (AAAS)
Date: 24-02-2017
DOI: 10.1126/SCIIMMUNOL.AAJ1548
Abstract: Interleukin-37 homodimerization regulates its anti-inflammatory activity.
Role of salt bridges in the dimer interface of 14-3-3ζ in dimer dynamics, N-terminal α-helical order, and molecular chaperone activity
Publisher: Elsevier BV
Date: 2018
Structural and Dynamic Requirements for Optimal Activity of the Essential Bacterial Enzyme Dihydrodipicolinate Synthase
Publisher: Public Library of Science (PLoS)
Date: 07-06-2012
High density lipoproteins bind A beta and apolipoprotein C-II amyloid fibrils
Publisher: Elsevier BV
Date: 04-2006
The 2.1Å Crystal Structure of the Far-red Fluorescent Protein HcRed: Inherent Conformational Flexibility of the Chromophore
Publisher: Elsevier BV
Date: 05-2005
DOI: 10.1016/J.JMB.2005.03.020
Abstract: We have determined the crystal structure of HcRed, a far-red fluorescent protein isolated from Heteractis crispa, to 2.1A resolution. HcRed was observed to form a dimer, in contrast to the monomeric form of green fluorescent protein (GFP) or the tetrameric forms of the GFP-like proteins (eqFP611, Rtms5 and DsRed). Unlike the well-defined chromophore conformation observed in GFP and the GFP-like proteins, the HcRed chromophore was observed to be considerably mobile. Within the HcRed structure, the cyclic tripeptide chromophore, Glu(64)-Tyr(65)-Gly(66), was observed to adopt both a cis coplanar and a trans non-coplanar conformation. As a result of these two conformations, the hydroxyphenyl moiety of the chromophore makes distinct interactions within the interior of the beta-can. These data together with a quantum chemical model of the chromophore, suggest the cis coplanar conformation to be consistent with the fluorescent properties of HcRed, and the trans non-coplanar conformation to be consistent with non-fluorescent properties of hcCP, the chromoprotein parent of HcRed. Moreover, within the GFP-like family, it appears that where conformational freedom is permissible then flexibility in the chromophore conformation is possible.
Disruption of quaternary structure in Escherichia coli dihydrodipicolinate synthase (DHDPS) generates a functional monomer that is no longer inhibited by lysine
Publisher: Elsevier BV
Date: 11-2010
DOI: 10.1016/J.ABB.2010.08.009
Abstract: Escherichia coli dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52), a natively homotetrameric enzyme was converted to a monomeric species through the introduction of destabilising interactions at two different subunit interfaces allowing exploration of the roles of the quaternary structure in affecting catalytic competency. The double mutant DHDPS-L197D/Y107W displays gel filtration characteristics consistent with a single non-interacting monomeric species, which was confirmed by sedimentary velocity experiments. This monomer was shown to be catalytically active, but with reduced catalytic efficiency (k(cat)=9.8±0.5s(-1)), displaying 8% of the specific activity of the wild-type enzyme. The Michaelis constants for the substrates pyruvate and for (S)-aspartate semialdehyde increased by an order of magnitude, indicating that quaternary structure plays a significant role in substrate specificity. This monomeric species exhibited an enhanced propensity for aggregation and inactivation, indicating that whilst the oligomerization is not an intrinsic criterion for catalysis, higher oligomeric forms may benefit from both increased catalytic efficiency and diminished aggregation propensity. Furthermore, allosteric inhibition by (S)-lysine was abolished for DHDPS-L197D/Y107W, confirming the importance of the dimeric unit as the minimal functional assembly for efficient (S)-lysine binding.
Exploring the dihydrodipicolinate synthase tetramer: How resilient is the dimer–dimer interface?
Publisher: Elsevier BV
Date: 02-2010
DOI: 10.1016/J.ABB.2009.11.014
Abstract: Dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) is a tetrameric enzyme that catalyses the first committed step of the lysine biosynthetic pathway. Dimeric variants of DHDPS have impaired catalytic activity due to aberrant protein motions within the dimer unit. Thus, it is thought that the tetrameric structure functions to restrict these motions and optimise enzyme dynamics for catalysis. Despite the importance of dimer-dimer association, the interface between subunits of each dimer is small, accounting for only 4.3% of the total monomer surface area, and the structure of the interface is not conserved across species. We have probed the tolerance of dimer-dimer association to mutation by introducing amino acid substitutions within the interface. All point mutations resulted in destabilisation of the 'dimer of dimers' tetrameric structure. Both the position of the mutation in the interface and the physico-chemical nature of the substitution appeared to effect tetramerisation. Despite only weak destabilisation of the tetramer by some mutations, catalytic activity was reduced to approximately 10-15% of the wild-type in all cases, suggesting that the dimer-dimer interface is finely tuned to optimise function.
Identification of thebona fideDHDPS from a common plant pathogen
Publisher: Wiley
Date: 11-06-2014
DOI: 10.1002/PROT.24539
Abstract: Agrobacterium tumefaciens is a Gram-negative soil-borne bacterium that causes Crown Gall disease in many economically important crops. The absence of a suitable chemical treatment means there is a need to discover new anti-Crown Gall agents and also characterize bona fide drug targets. One such target is dihydrodipicolinate synthase (DHDPS), a homo-tetrameric enzyme that catalyzes the committed step in the metabolic pathway yielding meso-diaminopimelate and lysine. Interestingly, there are 10 putative DHDPS genes annotated in the A. tumefaciens genome, including three whose structures have recently been determined (PDB IDs: 3B4U, 2HMC, and 2R8W). However, we show using quantitative enzyme kinetic assays that nine of the 10 dapA gene products, including 3B4U, 2HMC, and 2R8W, lack DHDPS function in vitro. A sequence alignment showed that the product of the dapA7 gene contains all of the conserved residues known to be important for DHDPS catalysis and allostery. This gene was cloned and the recombinant product expressed and purified. Our studies show that the purified enzyme (i) possesses DHDPS enzyme activity, (ii) is allosterically inhibited by lysine, and (iii) adopts the canonical homo-tetrameric structure in both solution and the crystal state. This study describes for the first time the structure, function and allostery of the bona fide DHDPS from A. tumefaciens, which offers insight into the rational design of pesticide agents for combating Crown Gall disease.
From Knock-Out Phenotype to Three-Dimensional Structure of a Promising Antibiotic Target from Streptococcus pneumoniae
Publisher: Public Library of Science (PLoS)
Date: 13-12-2013
Molecular evolution of an oligomeric biocatalyst functioning in lysine biosynthesis.
Publisher: Springer Science and Business Media LLC
Date: 05-12-2017
Dimerization of Bacterial Diaminopimelate Decarboxylase Is Essential for Catalysis
Publisher: Elsevier BV
Date: 04-2016
CED-4 forms a 2 : 2 heterotetrameric complex with CED-9 until specifically displaced by EGL-1 or CED-13
Publisher: Springer Science and Business Media LLC
Date: 16-09-2005
Abstract: The pathway to cell death in Caenorhabditis elegans is well established. In cells undergoing apoptosis, the Bcl-2 homology domain 3 (BH3)-only protein EGL-1 binds to CED-9 at the mitochondrial membrane to cause the release of CED-4, which oligomerises and facilitates the activation of the caspase CED-3. However, despite many studies, the biophysical features of the CED-4/CED-9 complex have not been fully characterised. Here, we report the purification of a soluble and stable 2 : 2 heterotetrameric complex formed by recombinant CED-4 and CED-9 coexpressed in bacteria. Consistent with previous studies, synthetic peptides corresponding to the BH3 domains of worm BH3-only proteins (EGL-1, CED-13) dissociate CED-4 from CED-9, but not from the gain-of-function CED-9 (G169E) mutant. Surprisingly, the ability of worm BH3 domains to dissociate CED-4 was specific since mammalian BH3-only proteins could not do so.
qmotif: determination of telomere content from whole-genome sequence data
Publisher: Oxford University Press (OUP)
Date: 2022
Abstract: Changes in telomere length have been observed in cancer and can be indicative of mechanisms involved in carcinogenesis. Most methods used to estimate telomere length require laboratory analysis of DNA s les. Here, we present qmotif, a fast and easy tool that determines telomeric repeat sequences content as an estimate of telomere length directly from whole-genome sequencing. qmotif shows similar results to quantitative PCR, the standard method for high-throughput clinical telomere length quantification. qmotif output correlates strongly with the output of other tools for determining telomere sequence content, TelSeq and TelomereHunter, but can run in a fraction of the time—usually under a minute. qmotif is implemented in Java and source code is available at github.com/AdamaJava/adamajava, with instructions on how to build and use the application available from adamajava.readthedocs.io/en/latest/. Supplementary data are available at Bioinformatics Advances online.
The structural basis for autonomous dimerization of the pre-T-cell antigen receptor.
Publisher: Springer Science and Business Media LLC
Date: 10-2010
DOI: 10.1038/NATURE09448
Abstract: The pre-T-cell antigen receptor (pre-TCR), expressed by immature thymocytes, has a pivotal role in early T-cell development, including TCR β-selection, survival and proliferation of CD4(-)CD8(-) double-negative thymocytes, and subsequent αβ T-cell lineage differentiation. Whereas αβTCR ligation by the peptide-loaded major histocompatibility complex initiates T-cell signalling, pre-TCR-induced signalling occurs by means of a ligand-independent dimerization event. The pre-TCR comprises an invariant α-chain (pre-Tα) that pairs with any TCR β-chain (TCRβ) following successful TCR β-gene rearrangement. Here we provide the basis of pre-Tα-TCRβ assembly and pre-TCR dimerization. The pre-Tα chain comprised a single immunoglobulin-like domain that is structurally distinct from the constant (C) domain of the TCR α-chain nevertheless, the mode of association between pre-Tα and TCRβ mirrored that mediated by the Cα-Cβ domains of the αβTCR. The pre-TCR had a propensity to dimerize in solution, and the molecular envelope of the pre-TCR dimer correlated well with the observed head-to-tail pre-TCR dimer. This mode of pre-TCR dimerization enabled the pre-Tα domain to interact with the variable (V) β domain through residues that are highly conserved across the Vβ and joining (J) β gene families, thus mimicking the interactions at the core of the αβTCR's Vα-Vβ interface. Disruption of this pre-Tα-Vβ dimer interface abrogated pre-TCR dimerization in solution and impaired pre-TCR expression on the cell surface. Accordingly, we provide a mechanism of pre-TCR self-association that allows the pre-Tα chain to simultaneously 's le' the correct folding of both the V and C domains of any TCR β-chain, regardless of its ultimate specificity, which represents a critical checkpoint in T-cell development. This unusual dual-chaperone-like sensing function of pre-Tα represents a unique mechanism in nature whereby developmental quality control regulates the expression and signalling of an integral membrane receptor complex.
Insight into the self-association of key enzymes from pathogenic species
Publisher: Springer Science and Business Media LLC
Date: 25-06-2005
DOI: 10.1007/S00249-005-0491-Y
Abstract: Self-association of protein monomers to higher-order oligomers plays an important role in a plethora of biological phenomena. The classical biophysical technique of analytical ultracentrifugation is a key method used to measure protein oligomerisation. Recent advances in sedimentation data analysis have enabled the effects of diffusion to be deconvoluted from s le heterogeneity, permitting the direct identification of oligomeric species in self-associating systems. Two such systems are described and reviewed in this study. First, we examine the enzyme dihydrodipicolinate synthase (DHDPS), which crystallises as a tetramer. Wild-type DHDPS plays a critical role in lysine biosynthesis in microbes and is therefore an important antibiotic target. To confirm the state of association of DHDPS in solution, we employed sedimentation velocity and sedimentation equilibrium studies in an analytical ultracentrifuge to show that DHDPS exists in a slow dimer-tetramer equilibrium with a dissociation constant of 76 nM. Second, we review works describing the hexamerisation of GDP-mannose pyrophosphorylase (GDP-MP), an enzyme that plays a critical role in mannose metabolism in Leishmania species. Although the structure of the GDP-MP hexamer has not yet been determined, we describe a three-dimensional model of the hexamer based largely on homology with the uridyltransferase enzyme, Glmu. GDP-MP is a novel drug target for the treatment of leishmaniasis, a devastating parasitic disease that infects more than 12 million people worldwide. Given that both GDP-MP and DHDPS are only active in their oligomeric states, we propose that inhibition of the self-association of critical enzymes in disease is an emerging paradigm for therapeutic intervention.
Letter: Collision-Induced Dissociation of [Metal(L)2]2+Complexes (Metal = Cu, Ca and Mg) of 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine Allows Distinction of the Acyl Groups
Publisher: SAGE Publications
Date: 12-2007
DOI: 10.1255/EJMS.897
Abstract: The collision-induced dissociation (CID) spectra of the alent metal complexes of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, [Metal II (L) 2 ] 2+ (where Metal = Cu 2+ , Mg 2+ and Ca 2+ , L = [16:0/18:1GPCho]), formed by electrospray ionization, reveal interesting metal dependent fragmentation chemistry. Six main classes of reaction are observed corresponding to: two competing carboxylate abstraction pathways (from the sn1 and sn2 positions) phosphate abstraction competing losses of the two different carboxylic acids from the sn1 and sn2 positions loss of a protonated ligand, [L + H] + . The relative ratios of the competing carboxylate abstraction reactions are dependent on the metal, with the Cu and Ca complexes favoring the abstraction of the larger carboxylate (18:1) and the Mg complex favoring the abstraction of the smaller carboxylate (16:0).
A family of helminth molecules that modulate innate cell responses via molecular mimicry of host antimicrobial peptides
Publisher: Public Library of Science (PLoS)
Date: 12-05-2011
Phospholipid interaction induces molecular level polymorphism in apolipoprotein C-II amyloid fibrils via alternative assembly pathways
Publisher: Elsevier BV
Date: 2008
DOI: 10.1016/J.JMB.2007.10.038
Abstract: A common feature of many of the most important and prominent amyloid-forming proteins is their ability to bind lipids and lipid complexes. Lipids are ubiquitous components of disease-associated amyloid plaques and deposits in humans, yet the specific roles of lipid in the process of amyloid fibril formation are poorly understood. This study investigated the effect of phospholipids on amyloid fibril formation by human apolipoprotein (apo) C-II using phosphatidylcholine derivatives comprising acyl chains of up to 14 carbon atoms. Submicellar concentrations of short-chain phospholipids increase the rate of apoC-II fibril formation in an acyl-chain-length- and concentration-dependent fashion, while high micellar concentrations of phospholipids completely inhibited amyloid formation. At lower concentrations of soluble phospholipid complexes, fibril formation by apoC-II was only partially inhibited, and under these conditions, aggregation followed a two-phase process. Electron microscopy showed that the fibrils resulting from the second phase of aggregation were straight, cablelike, and about 13 nm wide, in contrast to the homogeneous twisted-ribbon morphology of apoC-II fibrils formed under lipid-free conditions. Seeding experiments showed that this alternative fibril structure could be templated both in the presence and in the absence of lipid complex, suggesting that the two morphologies result from distinct assembly pathways. Circular dichroism spectroscopy studies indicated that the secondary structural conformation within the straight-type and ribbon-type fibrils were distinct, further suggesting ergent assembly pathways. These studies show that phospholipid complexes can change the structural architecture of mature fibrils and generate new fibril morphologies with the potential to alter the in vivo behaviour of amyloid. Such lipid interactions may play a role in defining the structural features of fibrils formed by erse amyloidogenic proteins.
A tetrameric structure is not essential for activity in dihydrodipicolinate synthase (DHDPS) from Mycobacterium tuberculosis
Publisher: Elsevier BV
Date: 08-2011
DOI: 10.1016/J.ABB.2011.05.014
Abstract: Dihydrodipicolinate synthase (DHDPS) is a validated antibiotic target for which a new approach to inhibitor design has been proposed: disrupting native tetramer formation by targeting the dimer-dimer interface. In this study, rational design afforded a variant of Mycobacterium tuberculosis, Mtb-DHDPS-A204R, with disrupted quaternary structure. X-ray crystallography (at a resolution of 2.1Å) revealed a dimeric protein with an identical fold and active-site structure to the tetrameric wild-type enzyme. Analytical ultracentrifugation confirmed the dimeric structure in solution, yet the dimeric mutant has similar activity to the wild-type enzyme. Although the affinity for both substrates was somewhat decreased, the high catalytic competency of the enzyme was surprising in the light of previous results showing that dimeric variants of the Escherichia coli and Bacillus anthracis DHDPS enzymes have dramatically reduced activity compared to their wild-type tetrameric counterparts. These results suggest that Mtb-DHDPS-A204R is similar to the natively dimeric enzyme from Staphylococcus aureus, and highlight our incomplete understanding of the role played by oligomerisation in relating protein structure and function.
Clinical and molecular characterization of HER2 amplified-pancreatic cancer
Publisher: Springer Science and Business Media LLC
Date: 2013
DOI: 10.1186/GM482
Age-Related Differences in Plasma Proteins: How Plasma Proteins Change from Neonates to Adults
Publisher: Public Library of Science (PLoS)
Date: 18-02-2011
Catalytic mechanism and cofactor preference of dihydrodipicolinate reductase from methicillin-resistant Staphylococcus aureus
Publisher: Elsevier BV
Date: 08-2011
DOI: 10.1016/J.ABB.2011.06.006
Abstract: Given the rapid rise in antibiotic resistance, including methicillin resistance in Staphylococcus aureus (MRSA), there is an urgent need to characterize novel drug targets. Enzymes of the lysine biosynthesis pathway in bacteria are ex les of such targets, including dihydrodipicolinate reductase (DHDPR, E.C. 1.3.1.26), which is the product of an essential bacterial gene. DHDPR catalyzes the NAD(P)H-dependent reduction of dihydrodipicolinate (DHDP) to tetrahydrodipicolinate (THDP) in the lysine biosynthesis pathway. We show that MRSA-DHDPR exhibits a unique nucleotide specificity utilizing NADPH (K(m)=12μM) as a cofactor more effectively than NADH (K(m)=26μM). However, the enzyme is inhibited by high concentrations of DHDP when using NADPH as a cofactor, but not with NADH. Isothermal titration calorimetry (ITC) studies reveal that MRSA-DHDPR has ∼20-fold greater binding affinity for NADPH (K(d)=1.5μM) relative to NADH (K(d)=29μM). Kinetic investigations in tandem with ITC studies show that the enzyme follows a compulsory-order ternary complex mechanism with inhibition by DHDP through the formation of a nonproductive ternary complex with NADP(+). This work describes, for the first time, the catalytic mechanism and cofactor preference of MRSA-DHDPR, and provides insight into rational approaches to inhibiting this valid antimicrobial target.
Crystal structure of the Leishmania major MIX protein: A scaffold protein that mediates protein–protein interactions
Publisher: Wiley
Date: 27-04-2011
DOI: 10.1002/PRO.631
Abstract: Infection by Leishmania and Trypanosoma causes severe disease and can be fatal. The reduced effectiveness of current treatments is largely due to drug resistance, hence the urgent need to develop new drugs, preferably against novel targets. We have recently identified a mitochondrial membrane‐anchored protein, designated MIX, which occurs exclusively in these parasites and is essential for virulence. We have determined the crystal structure of Leishmania major MIX to a resolution of 2.4 Å. MIX forms an all α‐helical fold comprising seven α‐helices that fold into a single domain. The distribution of helices is similar to a number of scaffold proteins, namely HEAT repeats, 14‐3‐3, and tetratricopeptide repeat proteins, suggesting that MIX mediates protein–protein interactions. Accordingly, using copurification and mass spectroscopy we were able to identify several proteins that may interact with MIX in vivo . Being parasite specific, MIX is a promising new drug target and, thus, the structure and potential interacting partners provide a basis for structure‐guided drug discovery.
Whole-genome landscapes of major melanoma subtypes
Publisher: Springer Science and Business Media LLC
Date: 05-2017
DOI: 10.1038/NATURE22071
Abstract: Melanoma of the skin is a common cancer only in Europeans, whereas it arises in internal body surfaces (mucosal sites) and on the hands and feet (acral sites) in people throughout the world. Here we report analysis of whole-genome sequences from cutaneous, acral and mucosal subtypes of melanoma. The heavily mutated landscape of coding and non-coding mutations in cutaneous melanoma resolved novel signatures of mutagenesis attributable to ultraviolet radiation. However, acral and mucosal melanomas were dominated by structural changes and mutation signatures of unknown aetiology, not previously identified in melanoma. The number of genes affected by recurrent mutations disrupting non-coding sequences was similar to that affected by recurrent mutations to coding sequences. Significantly mutated genes included BRAF, CDKN2A, NRAS and TP53 in cutaneous melanoma, BRAF, NRAS and NF1 in acral melanoma and SF3B1 in mucosal melanoma. Mutations affecting the TERT promoter were the most frequent of all however, neither they nor ATRX mutations, which correlate with alternative telomere lengthening, were associated with greater telomere length. Most melanomas had potentially actionable mutations, most in components of the mitogen-activated protein kinase and phosphoinositol kinase pathways. The whole-genome mutation landscape of melanoma reveals erse carcinogenic processes across its subtypes, some unrelated to sun exposure, and extends potential involvement of the non-coding genome in its pathogenesis.
Crystallization and preliminary X-ray diffraction analysis of diaminopimelate epimerase fromEscherichia coli
Publisher: International Union of Crystallography (IUCr)
Date: 25-12-2009
Dual roles for a non-conserved amino acid in protein homodimerization and inhibitor binding to biotin protein ligase from Staphylococcus aureus
Publisher: Wiley
Date: 21-11-2014
DOI: 10.1111/MMI.12446
Abstract: Protein biotinylation is catalysed by biotin protein ligase (BPL). The most characterized BPL is from Escherichia coli where it functions as both a biotin ligase and a homodimeric transcriptional repressor. Here we investigated another bifunctional BPL from the clinically important Staphylococcus aureus (SaBPL). Unliganded SaBPL (apo) exists in a dimer-monomer equilibrium at low micromolar concentrations - a stark contrast to E. coli BPL (EcBPL) that is monomeric under the same conditions. EMSA and SAXS analysis demonstrated that dimeric apo SaBPL adopted a conformation that was competent to bind DNA and necessary for it to function as a transcription factor. The SaBPL dimer-monomer dissociation constant was 5.8-fold tighter when binding the inhibitor biotin acetylene, but unchanged with biotin. F123, located in the dimer interface, was critical for homodimerization. Inhibition studies together with surface plasmon resonance analyses revealed a strong correlation between inhibitor potency and slow dissociation kinetics. A 24-fold difference in Ki values for these two enzymes was explained by differences in enzyme:inhibitor dissociation rates. Substitution of F123 in SaBPL and its equivalent in EcBPL altered both inhibitor potency and dissociation. Hence, F123 in SaBPL has novel roles in both protein dimerization and ligand-binding that have not been reported in EcBPL.
Dihydrodipicolinate synthase is absent in fungi
Publisher: Elsevier BV
Date: 09-2018
DOI: 10.1016/J.BIOCHI.2018.06.017
Abstract: The class I aldolase dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of the diaminopimelate (DAP) lysine biosynthesis pathway in bacteria, archaea and plants. Despite the existence, in databases, of numerous fungal sequences annotated as DHDPS, its presence in fungi has been the subject of contradictory claims. We report the characterization of DHDPS candidates from fungi. Firstly, the putative DHDPS from Coccidioides immitis (PDB ID: 3QFE) was shown to have negligible enzyme activity. Sequence analysis of 3QFE showed that three out of the seven amino acid residues critical for DHDPS activity are absent however, exact matches to catalytic residues from two other class I aldolases, 2-keto-3-deoxygluconate aldolase (KDGA), and 4-hydroxy-2-oxoglutarate aldolase (HOGA), were identified. The presence of both KDGA and HOGA activity in 3QFE was confirmed in vitro using enzyme assays, the first report of such dual activity. Subsequent analyses of all publically available fungal sequences revealed that no entry contains all seven residues important for DHDPS function. The candidate with the highest number of identities (6 of 7), KIW77228 from Fonsecaea pedrosoi, was shown to have trace DHDPS activity in vitro, partially restored by substitution of the seventh critical residue, and to be incapable of complementing DHDPS-deficient E. coli cells. Combined with the presence of all seven sequences for the alternative α-aminoadipate (AAA) lysine biosynthesis pathway in C. immitis and F. pedrosoi, we believe that DHDPS and the DAP pathway are absent in fungi, and further, that robust informed methods for annotating genes need to be implemented.
Structure and Function of Cyanobacterial DHDPS and DHDPR
Publisher: Springer Science and Business Media LLC
Date: 15-11-2016
DOI: 10.1038/SREP37111
Abstract: Lysine biosynthesis in bacteria and plants commences with a condensation reaction catalysed by dihydrodipicolinate synthase (DHDPS) followed by a reduction reaction catalysed by dihydrodipicolinate reductase (DHDPR). Interestingly, both DHDPS and DHDPR exist as different oligomeric forms in bacteria and plants. DHDPS is primarily a homotetramer in all species, but the architecture of the tetramer differs across kingdoms. DHDPR also exists as a tetramer in bacteria, but has recently been reported to be dimeric in plants. This study aimed to characterise for the first time the structure and function of DHDPS and DHDPR from cyanobacteria, which is an evolutionary important phylum that evolved at the ergence point between bacteria and plants. We cloned, expressed and purified DHDPS and DHDPR from the cyanobacterium Anabaena variabilis . The recombinant enzymes were shown to be folded by circular dichroism spectroscopy, enzymatically active employing the quantitative DHDPS-DHDPR coupled assay, and form tetramers in solution using analytical ultracentrifugation. Crystal structures of DHDPS and DHDPR from A. variabilis were determined at 1.92 Å and 2.83 Å, respectively, and show that both enzymes adopt the canonical bacterial tetrameric architecture. These studies indicate that the quaternary structure of bacterial and plant DHDPS and DHDPR erged after cyanobacteria evolved.
Dimerization of Plant Defensin NaD1 Enhances Its Antifungal Activity
Publisher: Elsevier BV
Date: 06-2012
An optimized coupled assay for quantifying diaminopimelate decarboxylase activity
Publisher: Elsevier BV
Date: 08-2015
DOI: 10.1016/J.BIOCHI.2015.05.004
Abstract: Diaminopimelate decarboxylase (DAPDC) catalyzes the conversion of meso-DAP to lysine and carbon dioxide in the final step of the diaminopimelate (DAP) pathway in plants and bacteria. Given its absence in humans, DAPDC is a promising antibacterial target, particularly considering the rise in drug-resistant strains from pathogens such as Escherichia coli and Mycobacterium tuberculosis. Here, we report the optimization of a simple quantitative assay for measuring DAPDC catalytic activity using saccharopine dehydrogenase (SDH) as the coupling enzyme. Our results show that SDH has optimal activity at 37 °C, pH 8.0, and in Tris buffer. These conditions were subsequently employed to quantitate the enzyme kinetic properties of DAPDC from three bacterial species. We show that DAPDC from E. coli and M. tuberculosis have [Formula: see text] of 0.97 mM and 1.62 mM and a kcat of 55 s(-1) and 28 s(-1), respectively, which agree well with previous studies using more labor-intensive assays. We subsequently employed the optimized coupled assay to show for the first time that DAPDC from Bacillus anthracis possesses a [Formula: see text] of 0.68 mM and a kcat of 58 s(-1). This optimized coupled assay offers excellent scope to be employed in high throughput drug discovery screens targeting DAPDC from bacterial pathogens.
Crystal structure and kinetic study of dihydrodipicolinate synthase from Mycobacterium tuberculosis
Publisher: Portland Press Ltd.
Date: 27-03-2008
DOI: 10.1042/BJ20071360
Abstract: The three-dimensional structure of the enzyme dihydrodipicolinate synthase (KEGG entry Rv2753c, EC 4.2.1.52) from Mycobacterium tuberculosis (Mtb-DHDPS) was determined and refined at 2.28 Å (1 Å=0.1 nm) resolution. The asymmetric unit of the crystal contains two tetramers, each of which we propose to be the functional enzyme unit. This is supported by analytical ultracentrifugation studies, which show the enzyme to be tetrameric in solution. The structure of each subunit consists of an N-terminal (β/α)8-barrel followed by a C-terminal α-helical domain. The active site comprises residues from two adjacent subunits, across an interface, and is located at the C-terminal side of the (β/α)8-barrel domain. A comparison with the other known DHDPS structures shows that the overall architecture of the active site is largely conserved, albeit the proton relay motif comprising Tyr143, Thr54 and Tyr117 appears to be disrupted. The kinetic parameters of the enzyme are reported: KMASA=0.43±0.02 mM, KMpyruvate=0.17±0.01 mM and Vmax=4.42±0.08 μmol·s−1·mg−1. Interestingly, the Vmax of Mtb-DHDPS is 6-fold higher than the corresponding value for Escherichia coli DHDPS, and the enzyme is insensitive to feedback inhibition by (S)-lysine. This can be explained by the three-dimensional structure, which shows that the (S)-lysine-binding site is not conserved in Mtb-DHDPS, when compared with DHDPS enzymes that are known to be inhibited by (S)-lysine. A selection of metabolites from the aspartate family of amino acids do not inhibit this enzyme. A comprehensive understanding of the structure and function of this important enzyme from the (S)-lysine biosynthesis pathway may provide the key for the design of new antibiotics to combat tuberculosis.
Electron capture dissociation of complexes of diacylglycerophosphocholine and divalent metal ions: Competition between charge reduction and radical induced phospholipid fragmentation
Publisher: American Chemical Society (ACS)
Date: 07-2008
DOI: 10.1016/J.JASMS.2008.03.006
Abstract: Divalent metal complexes of phosphocholines, [Metal(II)(L)(n)](2+) (where Metal=Cu(2+), Co(2+), Mg(2+), and Ca(2+), L=1,2-dihexanoyl-sn-glycero-3-phosphocholine [6:0/6:0GPCho] and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine [16:0/18:1GPCho] and n=2-5), were formed upon electrospray ionization mass spectrometry (ESI/MS) of 8 mM solution of phosphocholine (L) with 4 mM metal salt (Metal). The electron capture dissociation (ECD) reactions of these [Metal(II)(L)(n)](2+) complexes were examined via Fourier-transform ion-cyclotron resonance mass spectrometry. A rich and complex chemistry was observed, including charge reduction and fragmentation involving losses of a methyl radical, trimethylamine, and the acyl chains. The predominant reaction channel was dependent on the size (n) of the complex, the metal and ligand used, and the size of the acyl chain. Thus charge reduction dominates the ECD spectra of the larger phosphocholine, 16:0/18:1GPCho, but is largely absent in the smaller 6:0/6:0GPCho. For complexes of 16:0/18:1GPCho, n=4-5, fragmentation from the head group mainly occurs via loss of the methyl radical and trimethylamine. At n=3, the relative abundance of fragments due to loss of acyl chain radicals increases. The abundances of ions arising from these radical losses increase further for the n=2 complexes, thereby providing information on the composition and position of the 16:0 and 18:1 acyl groups. Thus ECD of metal complexes provides structurally useful information on the phosphocholine, including the nature of the head group, the acyl chains, and the positions of the acyl chains.
Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples
Publisher: Springer Science and Business Media LLC
Date: 21-09-2020
DOI: 10.1038/S41467-020-18151-Y
Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA s les, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological ergences between two reproducible somatic variant detection efforts.
The Sodium Sialic Acid Symporter From Staphylococcus aureus Has Altered Substrate Specificity
Publisher: Frontiers Media SA
Date: 04-07-2018
Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer
Publisher: Elsevier BV
Date: 2021
Aromatic residues in the C-terminal helix of human apoC-I mediate phospholipid interactions and particle morphology
Publisher: Elsevier BV
Date: 07-2009
Dynamic Modelling Reveals ‘Hotspots’ on the Pathway to Enzyme-Substrate Complex Formation
Publisher: Public Library of Science (PLoS)
Date: 11-03-2016
Whole genomes redefine the mutational landscape of pancreatic cancer
Publisher: Springer Science and Business Media LLC
Date: 25-02-2015
DOI: 10.1038/NATURE14169
A partially structured region of a largely unstructured protein, Plasmodium falciparum merozoite surface protein 2 (MSP2), forms amyloid‐like fibrils
Publisher: Wiley
Date: 21-09-2007
DOI: 10.1002/PSC.910
Abstract: Merozoite surface protein 2 (MSP2) from the human malaria parasite Plasmodium falciparum is expressed as a GPI-anchored protein on the merozoite surface. It has been implicated in the process of erythrocyte invasion and is a leading vaccine candidate. MSP2 is an intrinsically unstructured protein (IUP), and recombinant MSP2 forms amyloid-like fibrils upon storage. We have examined synthetic peptides corresponding to sequences in the conserved N-terminal region of MSP2 for the presence of local structure and the ability to form fibrils related to those formed by full-length MSP2. In a 25-residue peptide corresponding to the entire N-terminal region of mature MSP2, structures calculated from NMR data show the presence of nascent helical and turn-like structures. An 8-residue peptide from the central region of the N-terminal domain (residues 8-15) also formed a turn-like structure. Both peptides formed fibrils that were similar but not identical to the amyloid-like fibrils formed by full-length MSP2. Notably, the fibrils formed by the peptides bound both Congo Red and Thioflavin T, whereas the fibrils formed by full-length MSP2 bound only Congo Red. The propensity of peptides from the N-terminal conserved region of MSP2 to form amyloid-like fibrils makes it likely that this region contributes to fibril formation by the full-length protein. Thus, in contrast to the more common pathway of amyloid formation by structured proteins, which proceeds via partially unfolded intermediates that then undergo beta-aggregation, MSP2 is an ex le of a largely unstructured protein with at least one small structured region that has an important role in fibril formation.
Identification of a dimeric KDG aldolase from Agrobacterium tumefaciens
Publisher: Wiley
Date: 10-08-2017
DOI: 10.1002/PROT.25359
Abstract: Agrobacterium tumefaciens is a Gram-negative bacterium and causative agent of Crown Gall disease that infects a variety of economically important plants. The annotated A. tumefaciens genome contains 10 putative dapA genes, which code for dihydrodipicolinate synthase (DHDPS). However, we have recently demonstrated that only one of these genes (dapA7) encodes a functional DHDPS. The function of the other nine putative dapA genes is yet to be determined. Here, we demonstrate using bioinformatics that the product of the dapA5 gene (DapA5) possesses all the catalytic residues canonical to 2-keto-3-deoxygluconate (KDG) aldolase, which is a class I aldolase involved in glucose metabolism. We therefore expressed, purified, and characterized recombinant DapA5 using mass spectrometry, circular dichroism spectroscopy, analytical ultracentrifugation, and enzyme kinetics. The results show that DapA5 (1) adopts an α/β structure consistent with the TIM-barrel fold of KDG aldolases, (2) possesses KDG aldolase enzyme activity, and (3) exists as a tight dimer in solution. This study shows for the first time that dapA5 from A. tumefaciens encodes a functional dimeric KDG aldolase.
Dihydrodipicolinate synthase from Thermotoga maritima
Publisher: Portland Press Ltd.
Date: 14-11-2006
DOI: 10.1042/BJ20060771
Abstract: DHDPS (dihydrodipicolinate synthase) catalyses the branch point in lysine biosynthesis in bacteria and plants and is feedback inhibited by lysine. DHDPS from the thermophilic bacterium Thermotoga maritima shows a high level of heat and chemical stability. When incubated at 90 °C or in 8 M urea, the enzyme showed little or no loss of activity, unlike the Escherichia coli enzyme. The active site is very similar to that of the E. coli enzyme, and at mesophilic temperatures the two enzymes have similar kinetic constants. Like other forms of the enzyme, T. maritima DHDPS is a tetramer in solution, with a sedimentation coefficient of 7.2 S and molar mass of 133 kDa. However, the residues involved in the interface between different subunits in the tetramer differ from those of E. coli and include two cysteine residues poised to form a disulfide bond. Thus the increased heat and chemical stability of the T. maritima DHDPS enzyme is, at least in part, explained by an increased number of inter-subunit contacts. Unlike the plant or E. coli enzyme, the thermophilic DHDPS enzyme is not inhibited by (S)-lysine, suggesting that feedback control of the lysine biosynthetic pathway evolved later in the bacterial lineage.
New method for purifying histidine‐rich glycoprotein from human plasma redefines its functional properties
Publisher: Wiley
Date: 10-04-2013
DOI: 10.1002/IUB.1168
Abstract: Histidine-rich glycoprotein (HRG) is a relatively abundant plasma protein that has been implicated in multiple biological processes including immunity, tumor progression, and vascular biology. However, current protocols for purifying HRG from plasma result in the copurification of contaminating proteins and raise questions over the validity of biological activities ascribed to HRG. In this study, we describe a two-step protocol for the large-scale purification of HRG from human plasma using a combination of metal affinity and ion exchange chromatography. The protocol employs a rapid and simple strategy to isolate highly purified HRG that minimizes proteolytic cleavage of the protein. The purification of HRG was assessed at each stage by measuring the amount of HRG immunoreactive protein using a specific monoclonal antibody against total protein, and demonstrated ~1,000-fold purification with an overall yield of ~32%. Mass spectrometry analysis demonstrated that plasma-derived HRG was free of contaminating proteins and gel electrophoresis showed it to have minimal proteolytic degradation. Characterization of protein by physical method showed that the protein exists as a single, monodisperse species. In contrast to the previous studies of HRG purified by different methods, HRG purified using the new procedure demonstrated a reduced profile of functions. Although the HRG retained binding to heparin and phosphatidic acid, it did not interact with necrotic cells or other cellular lipids. These data demonstrate that HRG does not exhibit the broad interactive properties that have been reported previously, suggesting that copurification of HRG-binding partners or other impurities are responsible for some of the reported functional properties. The findings in this study demonstrate that the new purification procedure can provide a ready source of pure HRG to assess ligand specificity and biological function of this important plasma protein.
Disarming Bacterial Virulence through Chemical Inhibition of the DNA Binding Domain of an AraC-like Transcriptional Activator Protein
Publisher: Elsevier BV
Date: 10-2013
Cloning, expression, purification and crystallization of dihydrodipicolinate synthase fromAgrobacterium tumefaciens
Publisher: International Union of Crystallography (IUCr)
Date: 30-08-2012
Self-association of Human Apolipoprotein E3 and E4 in the Presence and Absence of Phospholipid
Publisher: Elsevier BV
Date: 11-2000
Inhibiting dihydrodipicolinate synthase across species: Towards specificity for pathogens?
Publisher: Elsevier BV
Date: 2008
DOI: 10.1016/J.BMCL.2007.11.026
Abstract: Dihydrodipicolinate synthase (DHDPS) is a key enzyme in lysine biosynthesis and an important antibiotic target. The specificity of a range of heterocyclic product analogues against DHDPS from three pathogenic species, Bacillus anthracis, Mycobacterium tuberculosis and methicillin-resistant Staphylococcus aureus, and the evolutionarily related N-acetylneuraminate lyase, has been determined. The results suggest that the development of species-specific inhibitors of DHDPS as potential antibacterials is achievable.
Comparison of untagged and his-tagged dihydrodipicolinate synthase from the enteric pathogen Vibrio cholerae
Publisher: Elsevier BV
Date: 05-2018
DOI: 10.1016/J.PEP.2018.01.003
Abstract: Given the emergence of multi drug resistant Vibrio cholerae strains, there is an urgent need to characterize new anti-cholera targets. One such target is the enzyme dihydrodipicolinate synthase (DHDPS EC 4.3.3.7), which catalyzes the first committed step in the diaminopimelate pathway. This pathway is responsible for the production of two key metabolites in bacteria and plants, namely meso-2,6-diaminopimelate and L-lysine. Here, we report the cloning, expression and purification of untagged and His-tagged recombinant DHDPS from V. cholerae (Vc-DHDPS) and provide comparative structural and kinetic analyses. Structural studies employing circular dichroism spectroscopy and analytical ultracentrifugation demonstrate that the recombinant enzymes are folded and exist as dimers in solution. Kinetic analyses of untagged and His-tagged Vc-DHDPS show that the enzymes are functional with specific activities of 75.6 U/mg and 112 U/mg, K
Crystal Structure of a BCL-W Domain-Swapped Dimer: Implications for the Function of BCL-2 Family Proteins
Publisher: Elsevier BV
Date: 10-2011
DOI: 10.1016/J.STR.2011.07.015
Abstract: The prosurvival and proapoptotic proteins of the BCL-2 family share a similar three-dimensional fold despite their opposing functions. However, many biochemical studies highlight the requirement for conformational changes for the functioning of both types of proteins, although structural data to support such changes remain elusive. Here, we describe the X-ray structure of dimeric BCL-W that reveals a major conformational change involving helices α3 and α4 hinging away from the core of the protein. Biochemical and functional studies reveal that the α4-α5 hinge region is required for dimerization of BCL-W, and functioning of both pro- and antiapoptotic BCL-2 proteins. Hence, this structure reveals a conformational flexibility not seen in previous BCL-2 protein structures and provides insights into how these regulators of apoptosis can change conformation to exert their function.
Caspase Inhibitors of the P35 Family Are More Active When Purified from Yeast than Bacteria
Publisher: Public Library of Science (PLoS)
Date: 14-06-2012
HNF4A and GATA6 Loss Reveals Therapeutically Actionable Subtypes in Pancreatic Cancer
Publisher: Elsevier BV
Date: 05-2020
Sources of artefacts in the electrospray ionization mass spectra of saturated diacylglycerophosphocholines: From condensed phase hydrolysis reactions through to gas phase intercluster reactions
Publisher: American Chemical Society (ACS)
Date: 03-2006
DOI: 10.1016/J.JASMS.2005.11.009
Abstract: The mass spectra of diacylglycerophosphocholine phospholipids comprised of saturated fatty acids (1,2-dipentanoyl-sn-glycero-3-phosphocholine (D5PC) 1,2-dihexanoyl-sn-glycero-3-phosphocholine (D6PC), and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (D14PC)) are sensitive to the electrospray ionization (ESI) conditions. When fresh solutions of phospholipid in 10 mM ammonium acetate are subjected to ESI, protonated oligomeric clusters, [DxPCn + H]+ (x = 5, 6, and 14) are observed in the following different types of mass spectrometers: 3D-quadrupole ion trap linear ion trap, and triple quadrupole. The formation of the protonated cluster ions is not unique to the ion trap instruments, although they tend to be more abundant in these instruments. As the ESI solutions age, new ions are observed, which correspond to acid-catalyzed solution phase deacylation reactions. The collision induced dissociation fragmentation reactions of the oligomer cluster ions exhibit a distinct dependence on the cluster size, with the larger clusters (n > 2) simply fragmenting via the loss of lipid monomers. In contrast, the fragmentation of the dimeric cluster ion is unique, resulting in a number of additional reactions including covalent bond formation via intermolecular cluster SN2 reactions and SN2 transfer of a methyl group. The nature of the charge has a significant role in the formation of products via these intermolecular cluster reactions. Changing the head group to phosphoethanolamine "switches off" the SN2 reactions, while changing the cation from a proton to either a sodium or a potassium ion, diminishes the intermolecular reactions relative to monomer loss. Semi empirical PM3 calculations on [D6PC2 + H]+ suggest that the SN2 reactions are thermodynamically favored over simple monomer loss. These results have important implications in the field of lipidomics.
Crystal, Solution and In silico Structural Studies of Dihydrodipicolinate Synthase from the Common Grapevine
Publisher: Public Library of Science (PLoS)
Date: 25-06-2012
Lost in translation: Returning germline genetic results in genome-scale cancer research
Publisher: Springer Science and Business Media LLC
Date: 28-04-2017
Irreversible inhibition of dihydrodipicolinate synthase by 4-oxo-heptenedioic acid analogues
Publisher: Elsevier BV
Date: 12-2008
DOI: 10.1016/J.BMC.2008.10.026
Abstract: We report the synthesis of (2E,5E)-4-oxoheptadienedioic acid and (2E)-4-oxoheptenedioic acid and evaluation of both diester and diacid analogues as inhibitors of bacterial dihydrodipicolinate synthase. Enzyme kinetic studies allowed the determination of second-order rate constants of inactivation and substrate co-incubation studies have shown the inhibitors act at the active-site. Mass spectrometric analyses have further explored the enzyme-inhibitor interaction and determined the sites of enzyme alkylation.
Membrane Core-Specific Antimicrobial Action of Cathelicidin LL-37 Peptide Switches Between Pore and Nanofibre Formation
Publisher: Springer Science and Business Media LLC
Date: 30-11-2016
DOI: 10.1038/SREP38184
Abstract: Membrane-disrupting antimicrobial peptides provide broad-spectrum defence against localized bacterial invasion in a range of hosts including humans. The most generally held consensus is that targeting to pathogens is based on interactions with the head groups of membrane lipids. Here we show that the action of LL-37, a human antimicrobial peptide switches the mode of action based on the structure of the alkyl chains, and not the head groups of the membrane forming lipids. We demonstrate that LL-37 exhibits two distinct interaction pathways: pore formation in bilayers of unsaturated phospholipids and membrane modulation with saturated phospholipids. Uniquely, the membrane modulation yields helical-rich fibrous peptide-lipid superstructures. Our results point at alternative design strategies for peptide antimicrobials.
Crystallization and preliminary X-ray analysis of dihydrodipicolinate synthase fromClostridium botulinumin the presence of its substrate pyruvate
Publisher: International Union of Crystallography (IUCr)
Date: 14-02-2009
Estimating the costs of genomic sequencing in cancer control
Publisher: Springer Science and Business Media LLC
Date: 03-06-2020
DOI: 10.1186/S12913-020-05318-Y
Abstract: Despite the rapid uptake of genomic technologies within cancer care, few studies provide detailed information on the costs of sequencing across different applications. The objective of the study was to examine and categorise the complete costs involved in genomic sequencing for a range of applications within cancer settings. We performed a cost-analysis using gross and micro-costing approaches for genomic sequencing performed during 2017/2018 across different settings in Brisbane, Australia. Sequencing was undertaken for patients with lung, breast, oesophageal cancers, melanoma or mesothelioma. Aggregated resource data were captured for a total of 1433 patients and point estimates of per patient costs were generated. Deterministic sensitivity analyses addressed the uncertainty in the estimates. Estimated costs to the public health system for resources were categorised into seven distinct activities in the sequencing process: s ling, extraction, library preparation, sequencing, analysis, data storage and clinical reporting. Costs were also aggregated according to labour, consumables, testing, equipment and ‘other’ categories. The per person costs were AU$347–429 (2018 US$240–297) for targeted panels, AU$871–$2788 (2018 US$604–1932) for exome sequencing, and AU$2895–4830 (2018 US$2006-3347) for whole genome sequencing. Cost proportions were highest for library preparation/sequencing materials (average 76.8% of total costs), s le extraction (8.1%), data analysis (9.2%) and data storage (2.6%). Capital costs for the sequencers were an additional AU$34–197 (2018 US$24–67) per person. Total costs were most sensitive to consumables and sequencing activities driven by commercial prices. Per person sequencing costs for cancer are high when tumour/blood pairs require testing. Using the natural steps involved in sequencing and categorising resources accordingly, future evaluations of costs or cost-effectiveness of clinical genomics across cancer projects could be more standardised and facilitate easier comparison of cost drivers.
Pursuing DHDPS: an enzyme of unrealised potential as a novel antibacterial target
Publisher: Royal Society of Chemistry (RSC)
Date: 2019
DOI: 10.1039/C9MD00107G
Abstract: DHDPS represents a novel enzyme target for the development of new antibiotics to combat multidrug resistance.
Inhibition of lysine biosynthesis: an evolving antibiotic strategy
Publisher: Royal Society of Chemistry (RSC)
Date: 2007
DOI: 10.1039/B705624A
Abstract: Bacterial biosynthesis of lysine has come under increased scrutiny as a target for novel antibacterial agents as it provides lysine for protein synthesis and both lysine and meso-diaminopimelate for construction of the bacterial peptidoglycan cell wall. In this Highlight article we review recent advances in the validation of antibiotic targets, studies of the enzymes of the lysine biosynthetic pathway and development of inhibitors of these enzymes.
Genomic analyses identify molecular subtypes of pancreatic cancer
Publisher: Springer Science and Business Media LLC
Date: 24-02-2016
DOI: 10.1038/NATURE16965
Abstract: Integrated genomic analysis of 456 pancreatic ductal adenocarcinomas identified 32 recurrently mutated genes that aggregate into 10 pathways: KRAS, TGF-β, WNT, NOTCH, ROBO/SLIT signalling, G1/S transition, SWI-SNF, chromatin modification, DNA repair and RNA processing. Expression analysis defined 4 subtypes: (1) squamous (2) pancreatic progenitor (3) immunogenic and (4) aberrantly differentiated endocrine exocrine (ADEX) that correlate with histopathological characteristics. Squamous tumours are enriched for TP53 and KDM6A mutations, upregulation of the TP63∆N transcriptional network, hypermethylation of pancreatic endodermal cell-fate determining genes and have a poor prognosis. Pancreatic progenitor tumours preferentially express genes involved in early pancreatic development (FOXA2/3, PDX1 and MNX1). ADEX tumours displayed upregulation of genes that regulate networks involved in KRAS activation, exocrine (NR5A2 and RBPJL), and endocrine differentiation (NEUROD1 and NKX2-2). Immunogenic tumours contained upregulated immune networks including pathways involved in acquired immune suppression. These data infer differences in the molecular evolution of pancreatic cancer subtypes and identify opportunities for therapeutic development.
Defining the interaction of perforin with calcium and the phospholipid membrane
Publisher: Portland Press Ltd.
Date: 22-11-2013
DOI: 10.1042/BJ20130999
Abstract: Following its secretion from cytotoxic lymphocytes into the immune synapse, perforin binds to target cell membranes through its Ca2+-dependent C2 domain. Membrane-bound perforin then forms pores that allow passage of pro-apoptopic granzymes into the target cell. In the present study, structural and biochemical studies reveal that Ca2+ binding triggers a conformational change in the C2 domain that permits four key hydrophobic residues to interact with the plasma membrane. However, in contrast with previous suggestions, these movements and membrane binding do not trigger irreversible conformational changes in the pore-forming MACPF (membrane attack complex erforin-like) domain, indicating that subsequent monomer–monomer interactions at the membrane surface are required for perforin pore formation.
A Helical Conotoxin from Conus imperialis Has a Novel Cysteine Framework and Defines a New Superfamily
Publisher: Elsevier BV
Date: 04-2012
Targeting of a natural killer cell receptor family by a viral immunoevasin.
Publisher: Springer Science and Business Media LLC
Date: 12-05-2013
DOI: 10.1038/NI.2605
Abstract: Activating and inhibitory receptors on natural killer (NK) cells have a crucial role in innate immunity, although the basis of the engagement of activating NK cell receptors is unclear. The activating receptor Ly49H confers resistance to infection with murine cytomegalovirus by binding to the 'immunoevasin' m157. We found that m157 bound to the helical stalk of Ly49H, whereby two m157 monomers engaged the Ly49H dimer. The helical stalks of Ly49H lay centrally across the m157 platform, whereas its lectin domain was not required for recognition. Instead, m157 targeted an 'aromatic peg motif' present in stalks of both activating and inhibitory receptors of the Ly49 family, and substitution of this motif abrogated binding. Furthermore, ligation of m157 to Ly49H or Ly49C resulted in intracellular signaling. Accordingly, m157 has evolved to 'tackle the legs' of a family of NK cell receptors.
Characterisation of the First Enzymes Committed to Lysine Biosynthesis in Arabidopsis thaliana
Publisher: Public Library of Science (PLoS)
Date: 05-07-2012
Crystal structure of a SFPQ/PSPC1 heterodimer provides insights into preferential heterodimerization of human DBHS family proteins
Publisher: Elsevier BV
Date: 04-2018
Specificity versus catalytic potency: The role of threonine 44 in Escherichia coli dihydrodipicolinate synthase mediated catalysis
Publisher: Elsevier BV
Date: 08-2009
DOI: 10.1016/J.BIOCHI.2009.05.013
Abstract: In plants and bacteria, the branch point of (S)-lysine biosynthesis is the condensation of (S)-aspartate-beta-semialdehyde and pyruvate, a reaction catalysed by dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52). In this study, we probe the function of threonine 44 in Escherichia coli DHDPS, with respect to its role in the proton relay. Removal of the hydroxyl moiety of threonine 44, by mutation to valine, significantly attenuates activity (0.1% of wild-type) because the proton relay is broken. It was thus predicted that mutation of threonine 44 to serine would re-establish the proton relay and thus enzymatic activity. Following site-directed mutagenesis and purification to yield the DHDPS-Thr44Ser mutant enzyme, kinetic and structural studies were undertaken. The crystal structure of DHDPS-Thr44Ser showed that the active site was intact and that Ser44 and Tyr107 have some conformational flexibility, which is consistent with the observed loss of activity compared to the wild-type enzyme. Electron density was observed at the active site of DHDPS-Thr44Ser, which was identified as a trapped pyruvate analogue, alpha-ketoglutarate. The activity was indeed found to be increased relative to DHDPS-Thr44Val, but was still reduced to only approximately 8% of that of the wild-type enzyme. Interestingly, there was a shift in the kinetic mechanism, from the substituted-enzyme mechanism, observed in the wild-type, to the ternary-complex mechanism, consistent with the trapped substrate analogue. Increased flexibility in the active site appears to facilitate the binding/reaction of substrate analogues, suggesting that wild-type DHDPS has evolved a relatively rigid active site in order to maintain substrate specificity for pyruvate.
The antigen 43 structure reveals a molecular Velcro-like mechanism of autotransporter-mediated bacterial clumping
Publisher: Proceedings of the National Academy of Sciences
Date: 13-12-2013
Abstract: Many persistent and chronic bacterial infections are associated with the formation of large cell aggregates and biofilms that are difficult to treat. This includes respiratory and urinary tract infections, infections on medical devices, and infections of the ear, gums, and heart. One mechanism used by bacteria to aggregate and form biofilms involves the expression of self-associating surface-located autotransporter proteins such as Antigen 43 (Ag43). Here we present the crystal structure of the functional passenger domain of Ag43 and demonstrate that its unique L-shaped structure drives the formation of cell aggregates via a molecular Velcro-like handshake mechanism. This work provides insight into the structure–function mechanisms that facilitate bacterial interactions during infection.
The crystal structure of myelin oligodendrocyte glycoprotein, a key autoantigen in multiple sclerosis
Publisher: Proceedings of the National Academy of Sciences
Date: 05-09-2003
Abstract: Myelin oligodendrocyte glycoprotein (MOG) is a key CNS-specific autoantigen for primary demyelination in multiple sclerosis. Although the disease-inducing role of MOG has been established, its precise function in the CNS remains obscure. To gain new insights into the physiological and immunopathological role of MOG, we determined the 1.8-Å crystal structure of the MOG extracellular domain (MOG ED ). MOG ED adopts a classical Ig (Ig variable domain) fold that was observed to form an antiparallel head-to-tail dimer. A dimeric form of native MOG was observed, and MOG ED was also shown to dimerize in solution, consistent with the view of MOG acting as a homophilic adhesion receptor. The MOG 35-55 peptide, a major encephalitogenic determinant recognized by both T cells and demyelinating autoantibodies, is partly occluded within the dimer interface. The structure of this key autoantigen suggests a relationship between the dimeric form of MOG within the myelin sheath and a breakdown of immunological tolerance to MOG that is observed in multiple sclerosis.
Dimerization of Bacterial Diaminopimelate Epimerase Is Essential for Catalysis
Publisher: Elsevier BV
Date: 03-2013
A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation
Publisher: Public Library of Science (PLoS)
Date: 21-05-2015
The structure of the atypical killer cell immunoglobulin-like receptor, KIR2DL4
Publisher: Elsevier BV
Date: 04-2015
i-bodies, Human Single Domain Antibodies That Antagonize Chemokine Receptor CXCR4
Publisher: Elsevier BV
Date: 06-2016
Characterization of monomeric dihydrodipicolinate synthase variant reveals the importance of substrate binding in optimizing oligomerization
Publisher: Elsevier BV
Date: 12-2011
DOI: 10.1016/J.BBAPAP.2011.07.016
Abstract: To gain insights into the role of quaternary structure in the TIM-barrel family of enzymes, we introduced mutations to the DHDPS enzyme of Thermotoga maritima, which we have previously shown to be a stable tetramer in solution. These mutations were aimed at reducing the number of salt bridges at one of the two tetramerization interface of the enzyme, which contains many more interactions than the well characterized equivalent interface of the mesophilic Escherichia coli DHDPS enzyme. The resulting variants had altered quaternary structure, as shown by analytical ultracentrifugation, gel filtration liquid chromatography, and small angle X-ray scattering, and X-ray crystallographic studies confirmed that one variant existed as an independent monomer, but with few changes to the secondary and tertiary structure. Reduction of higher order assembly resulted in a loss of thermal stability, as measured by a variety of methods, and impaired catalytic function. Binding of pyruvate increased the oligomeric status of the variants, with a concomitant increase in thermal stability, suggesting a role for substrate binding in optimizing stable, higher order structures. The results of this work show that the salt bridges located at the tetramerization interface of DHDPS play a significant role in maintaining higher order structures, and demonstrate the importance of quaternary structure in determining protein stability and in the optimization of enzyme catalysis.
Structure and Evolution of a Novel Dimeric Enzyme from a Clinically Important Bacterial Pathogen
Publisher: Elsevier BV
Date: 10-2008
PGTools: A Software Suite for Proteogenomic Data Analysis and Visualization
Publisher: American Chemical Society (ACS)
Date: 17-04-2015
The trans-Golgi network GRIP-domain proteins form α-helical homodimers
Publisher: Portland Press Ltd.
Date: 07-06-2005
DOI: 10.1042/BJ20041810
Abstract: A recently described family of TGN (trans-Golgi network) proteins, all of which contain a GRIP domain targeting sequence, has been proposed to play a role in membrane transport. On the basis of the high content of heptad repeats, GRIP domain proteins are predicted to contain extensive coiled-coil regions that have the potential to mediate protein–protein interactions. Four mammalian GRIP domain proteins have been identified which are targeted to the TGN through their GRIP domains, namely p230, golgin-97, GCC88 and GCC185. In the present study, we have investigated the ability of the four mammalian GRIP domain proteins to interact. Using a combination of immunoprecipitation experiments of epitope-tagged GRIP domain proteins, cross-linking experiments and yeast two-hybrid interactions, we have established that the GRIP proteins can self-associate to form homodimers exclusively. Two-hybrid analysis indicated that the N- and C-terminal fragments of GCC88 can interact with themselves but not with each other, suggesting that the GRIP domain proteins form parallel coiled-coil dimers. Analysis of purified recombinant golgin-97 by CD spectroscopy indicated a 67% α-helical structure, consistent with a high content of coiled-coil sequences. These results support a model for GRIP domain proteins as extended rod-like homodimeric molecules. The formation of homodimers, but not heterodimers, indicates that each of the four mammalian TGN golgins has the potential to function independently.
The purification, crystallization and preliminary X-ray diffraction analysis of dihydrodipicolinate synthase fromClostridium botulinum
Publisher: International Union of Crystallography (IUCr)
Date: 29-02-2008
Structural features of apolipoprotein B synthetic peptides that inhibit lipoprotein(a) assembly
Publisher: Elsevier BV
Date: 12-2004
Identification of the CIMP-like subtype and aberrant methylation of members of the chromosomal segregation and spindle assembly pathways in esophageal adenocarcinoma
Publisher: Oxford University Press (OUP)
Date: 10-02-2015
A Biophysical Analysis of the Tetratricopeptide Repeat-rich Mitochondrial Import Receptor, Tom70, Reveals an Elongated Monomer That Is Inherently Flexible, Unstable, and Unfolds via a Multistate Pathw
Publisher: Elsevier BV
Date: 11-2004
Expression, purification, crystallization and preliminary X-ray diffraction analysis of dihydrodipicolinate synthase fromBacillus anthracisin the presence of pyruvate
Publisher: International Union of Crystallography (IUCr)
Date: 31-01-2009
Identification of unique neoantigen qualities in long-term survivors of pancreatic cancer
Publisher: Springer Science and Business Media LLC
Date: 11-2017
DOI: 10.1038/NATURE24462
Crystal Structure of a UDP-glucose-specific Glycosyltransferase from a Mycobacterium Species
Publisher: Elsevier BV
Date: 10-2008
Ultramarine, a Chromoprotein Acceptor for Förster Resonance Energy Transfer
Publisher: Public Library of Science (PLoS)
Date: 16-07-2012
The BECN1 N-terminal domain is intrinsically disordered
Publisher: Informa UK Limited
Date: 03-03-2016
Characterization of a novel JNK (c-Jun N-terminal kinase) inhibitory peptide
Publisher: Portland Press Ltd.
Date: 24-02-2011
DOI: 10.1042/BJ20101244
Abstract: An improved understanding of the roles of protein kinases in intracellular signalling and disease progression has driven significant advances in protein kinase inhibitor discovery. Peptide inhibitors that target the kinase protein substrate-binding site have continued to attract attention. In the present paper, we describe a novel JNK (c-Jun N-terminal kinase) inhibitory peptide PYC71N, which inhibits JNK activity in vitro towards a range of recombinant protein substrates including the transcription factors c-Jun, ATF2 (activating trancription factor 2) and Elk1, and the microtubule regulatory protein DCX (doublecortin). Analysis of cell culture studies confirmed the actions of a cell-permeable version of PYC71 to inhibit c-Jun phosphorylation during acute hyperosmotic stress. The analysis of the in vitro data for the kinetics of this inhibition indicated a substrate–inhibitor complex-mediated inhibition of JNK by PYC71N. Alanine-scanning replacement studies revealed the importance of two residues (PYC71N Phe9 or Phe11 within an FXF motif) for JNK inhibition. The importance of these residues was confirmed through interaction studies showing that each change decreased interaction of the peptide with c-Jun. Furthermore, PYC71N interacted with both non-phosphorylated (inactive) JNK1 and the substrate c-Jun, but did not recognize active JNK1. In contrast, a previously characterized JNK-inhibitory peptide TIJIP [truncated inhibitory region of JIP (JNK-interacting protein)], showed stronger interaction with active JNK1. Competition binding analysis confirmed that PYC71N inhibited the interaction of c-Jun with JNK1. Taken together, the results of the present study define novel properties of the PYC71N peptide as well as differences from the characterized TIJIP, and highlight the value of these peptides to probe the biochemistry of JNK-mediated substrate interactions and phosphorylation.
Ribosome-inactivating proteins: current status and biomedical applications
Publisher: Elsevier BV
Date: 07-2012
DOI: 10.1016/J.DRUDIS.2012.03.007
Abstract: Ribosome-inactivating proteins (RIPs) are mainly present in plants and function to inhibit protein synthesis through the removal of adenine residues from eukaryotic ribosomal RNA (rRNA). They are broadly classified into two groups: type I and type II. Type I RIPs are a erse family of proteins comprising a single polypeptide chain, whereas type II RIPs are heterodimeric glycoproteins comprising an A-chain (functionally equivalent to a type I RIP) linked via a disulphide bond to a B chain, mediating cell entry. In this review, we describe common type I and type II RIPs, their erse biological functions, mechanism of cell entry, stability in plasma and antigenicity. We end with a discussion of promising applications for RIPs in biomedicine.
An intact membrane is essential for small extracellular vesicle‐induced modulation of α‐synuclein fibrillization
Publisher: Wiley
Date: 12-2020
DOI: 10.1002/JEV2.12034
Methods for sample labeling and meniscus determination in the fluorescence-detected analytical ultracentrifuge
Publisher: Elsevier BV
Date: 07-2009
Abstract: The fluorescence detection system for the analytical ultracentrifuge (AU-FDS) enables the measurement of hydrodynamic properties and interactions of biomolecules at subnanomolar concentrations. In this study, we describe methods for (i) preparing and purifying fluorescently labeled biomolecules and (ii) determining the meniscus position in the AU-FDS using BODIPY 493/503 fluorescent dye suspended in light oil. We subsequently use these methods to measure the interaction of DNA with Escherichia coli Klenow fragment (KF) and show that KF binds matched DNA to form 1:1 and 2:1 (protein/DNA) complexes with dissociation constants of 4.2 and 22 nM, respectively.
Solution Conformation, Backbone Dynamics and Lipid Interactions of the Intrinsically Unstructured Malaria Surface Protein MSP2
Publisher: Elsevier BV
Date: 05-2008
Structural and functional characterization of TesB fromYersinia pestisreveals a unique octameric arrangement of hotdog domains
Publisher: International Union of Crystallography (IUCr)
Date: 27-03-2015
DOI: 10.1107/S1399004715002527
Abstract: Acyl-CoA thioesterases catalyse the hydrolysis of the thioester bonds present within a wide range of acyl-CoA substrates, releasing free CoASH and the corresponding fatty-acyl conjugate. The TesB-type thioesterases are members of the TE4 thioesterase family, one of 25 thioesterase enzyme families characterized to date, and contain two fused hotdog domains in both prokaryote and eukaryote homologues. Only two structures have been elucidated within this enzyme family, and much of the current understanding of the TesB thioesterases has been based on the Escherichia coli structure. Yersinia pestis , a highly virulent bacterium, encodes only one TesB-type thioesterase in its genome here, the structural and functional characterization of this enzyme are reported, revealing unique elements both within the protomer and quaternary arrangements of the hotdog domains which have not been reported previously in any thioesterase family. The quaternary structure, confirmed using a range of structural and biophysical techniques including crystallography, small-angle X-ray scattering, analytical ultracentrifugation and size-exclusion chromatography, exhibits a unique octameric arrangement of hotdog domains. Interestingly, the same biological unit appears to be present in both TesB structures solved to date, and is likely to be a conserved and distinguishing feature of TesB-type thioesterases. Analysis of the Y. pestis TesB thioesterase activity revealed a strong preference for octanoyl-CoA and this is supported by structural analysis of the active site. Overall, the results provide novel insights into the structure of TesB thioesterases which are likely to be conserved and distinguishing features of the TE4 thioesterase family.
The Merozoite Surface Protein 1 Complex Is a Platform for Binding to Human Erythrocytes by Plasmodium falciparum
Publisher: Elsevier BV
Date: 09-2014
Integrated activities of two alternative sigma factors coordinate iron acquisition and uptake by Pseudomonas aeruginosa
Publisher: Wiley
Date: 26-10-2017
DOI: 10.1111/MMI.13855
Abstract: Alternative sigma (σ) factors govern expression of bacterial genes in response to erse environmental signals. In Pseudomonas aeruginosa σ
Novel Bcl-2 Homology-3 Domain-like Sequences Identified from Screening Randomized Peptide Libraries for Inhibitors of the Pro-survival Bcl-2 Proteins
Publisher: Elsevier BV
Date: 11-2009
Pan-cancer analysis of whole genomes
Publisher: Springer Science and Business Media LLC
Date: 05-02-2020
DOI: 10.1038/S41586-020-1969-6
Abstract: Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale 1–3 . Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4–5 driver mutations when combining coding and non-coding genomic elements however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution in acral melanoma, for ex le, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter 4 identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation 5,6 analyses timings and patterns of tumour evolution 7 describes the erse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity 8,9 and evaluates a range of more-specialized features of cancer genomes 8,10–18 .
Whole-genome landscape of pancreatic neuroendocrine tumours
Publisher: Springer Science and Business Media LLC
Date: 15-02-2017
DOI: 10.1038/NATURE21063
Abstract: The diagnosis of pancreatic neuroendocrine tumours (PanNETs) is increasing owing to more sensitive detection methods, and this increase is creating challenges for clinical management. We performed whole-genome sequencing of 102 primary PanNETs and defined the genomic events that characterize their pathogenesis. Here we describe the mutational signatures they harbour, including a deficiency in G:C > T:A base excision repair due to inactivation of MUTYH, which encodes a DNA glycosylase. Clinically sporadic PanNETs contain a larger-than-expected proportion of germline mutations, including previously unreported mutations in the DNA repair genes MUTYH, CHEK2 and BRCA2. Together with mutations in MEN1 and VHL, these mutations occur in 17% of patients. Somatic mutations, including point mutations and gene fusions, were commonly found in genes involved in four main pathways: chromatin remodelling, DNA damage repair, activation of mTOR signalling (including previously undescribed EWSR1 gene fusions), and telomere maintenance. In addition, our gene expression analyses identified a subgroup of tumours associated with hypoxia and HIF signalling.
Conserved main‐chain peptide distortions: A proposed role for Ile203 in catalysis by dihydrodipicolinate synthase
Publisher: Wiley
Date: 12-2008
Bicarbonate-Mediated Stimulation of RegA, the Global Virulence Regulator from Citrobacter rodentium
Publisher: Elsevier BV
Date: 12-2009
DOI: 10.1016/J.JMB.2009.10.033
Abstract: The global virulence regulatory protein RegA, an AraC-like regulator, controls the expression of more than 60 genes in the mouse enteric pathogen Citrobacter rodentium. In the presence of bicarbonate, RegA activates the transcription of a number of virulence determinants and inhibits the expression of a series of housekeeping genes. To elucidate the molecular mechanism by which bicarbonate stimulates RegA activity, we carried out biophysical and mutational analyses. Our data indicate that RegA exists as a dimer in solution regardless of bicarbonate concentration. A leucine zipper, located in the region downstream of the N-terminal domain, is responsible for dimerisation. The N-terminal arm itself is involved in modulating the response to bicarbonate, which appears to bind to a region comprising a series of beta-sheets within the N-terminal domain. The presence of bicarbonate relieves the autoinhibition of RegA activity by its N-terminal arm. RegA is the first ex le of a bacterial virulence regulator that utilises the light switch mechanism, previously described for the Escherichia coli AraC protein, to respond to a gut-associated effector that controls its activity.
DNA methylation patterns identify subgroups of pancreatic neuroendocrine tumors with clinical association
Publisher: Springer Science and Business Media LLC
Date: 03-02-2021
DOI: 10.1038/S42003-020-01469-0
Abstract: Here we report the DNA methylation profile of 84 sporadic pancreatic neuroendocrine tumors (PanNETs) with associated clinical and genomic information. We identified three subgroups of PanNETs, termed T1, T2 and T3, with distinct patterns of methylation. The T1 subgroup was enriched for functional tumors and ATRX , DAXX and MEN1 wild-type genotypes. The T2 subgroup contained tumors with mutations in ATRX , DAXX and MEN1 and recurrent patterns of chromosomal losses in half of the genome with no association between regions with recurrent loss and methylation levels. T2 tumors were larger and had lower methylation in the MGMT gene body, which showed positive correlation with gene expression. The T3 subgroup harboured mutations in MEN1 with recurrent loss of chromosome 11, was enriched for grade G1 tumors and showed histological parameters associated with better prognosis. Our results suggest a role for methylation in both driving tumorigenesis and potentially stratifying prognosis in PanNETs.
Structural Basis for the Recognition and Cross-linking of Amyloid Fibrils by Human Apolipoprotein E
Publisher: Elsevier BV
Date: 12-2007
MicroRNA-182-5p targets a network of genes involved in DNA repair
Publisher: Cold Spring Harbor Laboratory
Date: 18-12-2012
Abstract: MicroRNAs are noncoding regulators of gene expression, which act by repressing protein translation and/or degrading mRNA. Many have been shown to drive tumorigenesis in cancer, but functional studies to understand their mode of action are typically limited to single-target genes. In this study, we use synthetic biotinylated miRNA to pull down endogenous targets of miR-182-5p. We identified more than 1000 genes as potential targets of miR-182-5p, most of which have a known function in pathways underlying tumor biology. Specifically, functional enrichment analysis identified components of both the DNA damage response pathway and cell cycle to be highly represented in this target cohort. Experimental validation confirmed that miR-182-5p-mediated disruption of the homologous recombination (HR) pathway is a consequence of its ability to target multiple components in that pathway. Although there is a strong enrichment for the cell cycle ontology, we do not see primary proliferative defects as a consequence of miR-182-5p overexpression. We highlight targets that could be responsible for miR-182-5p-mediated disruption of other biological processes attributed in the literature so far. Finally, we show that miR-182-5p is highly expressed in a panel of human breast cancer s les, highlighting its role as a potential oncomir in breast cancer.
Cloning to crystallization of dihydrodipicolinate synthase from the intracellular pathogen Legionella pneumophila
Publisher: International Union of Crystallography (IUCr)
Date: 30-09-2013
Fluorescence and analytical ultracentrifugation analyses of the interaction of the tyrosine kinase inhibitor, tyrphostin AG1478-mesylate, with albumin
Publisher: Elsevier BV
Date: 07-2005
Abstract: Quantifying the interaction of drugs with carrier proteins in plasma is of importance for understanding effective drug delivery to disease-affected tissues. In this study, we employed analytical ultracentrifugation and steady-state fluorescence spectroscopy to characterize the interaction of a potential new anticancer drug, AG 1478-mesylate, with plasma proteins in a suspension of normal serum albumin (NSA). We found that mesylate salt of AG 1478, an epidermal growth factor receptor kinase inhibitor, sediments in 0.1%(w/v) NSA as a complex with a sedimentation coefficient of 3.8 S. This is consistent with the size of human serum albumin. This interaction was quantitated by meniscus depletion sedimentation and fluorescence titration analyses. AG 1478-mesylate binds to albumin with an apparent single-site affinity (K(d)) of 120 microM. In this article, we show that the cyclodextrin carrier molecule, Captisol, increases the apparent affinity of the hydrophobic AG 1478-mesylate for albumin (K(d)=4-6 microM), and we propose that the AG 1478-mesylate-Captisol (1:1) complex binds to albumin with at least 10-fold higher affinity than does AG 1478-mesylate ligand alone. A fluorenylmethoxycarbonyl-sulfonic acid (FMS) derivative of the 6-aminoquinazoline analog of AG 1478, which was designed to have improved serum-binding properties, was shown by fluorescence analysis to bind with approximately 100-fold greater affinity than the parent compound. This has significant implications in the effective delivery of therapeutic agents in vivo.
Differential lysine‐mediated allosteric regulation of plant dihydrodipicolinate synthase isoforms
Publisher: Wiley
Date: 02-03-2021
DOI: 10.1111/FEBS.15766
Abstract: Lysine biosynthesis in plants occurs via the diaminopimelate pathway. The first committed and rate‐limiting step of this pathway is catalysed by dihydrodipicolinate synthase (DHDPS), which is allosterically regulated by the end product, l ‐lysine (lysine). Given that lysine is a common nutritionally limiting amino acid in cereal crops, there has been much interest in probing the regulation of DHDPS. Interestingly, knockouts in Arabidopsis thaliana of each isoform (AtDHDPS1 and AtDHDPS2) result in different phenotypes, despite the enzymes sharing 85% protein sequence identity. Accordingly, in this study, we compared the catalytic activity, lysine‐mediated inhibition and structures of both A. thaliana DHDPS isoforms. We found that although the recombinantly produced enzymes have similar kinetic properties, AtDHDPS1 is 10‐fold more sensitive to lysine. We subsequently used X‐ray crystallography to probe for structural differences between the apo‐ and lysine‐bound isoforms that could account for the differential allosteric inhibition. Despite no significant changes in the overall structures of the active or allosteric sites, we noted differences in the rotamer conformation of a key allosteric site residue (Trp116) and proposed that this could result in differences in lysine dissociation. Microscale thermophoresis studies supported our hypothesis, with AtDHDPS1 having a ~ 6‐fold tighter lysine dissociation constant compared to AtDHDPS2, which agrees with the lower half minimal inhibitory concentration for lysine observed. Thus, we highlight that subtle differences in protein structures, which could not have been predicted from the primary sequences, can have profound effects on the allostery of a key enzyme involved in lysine biosynthesis in plants. Structures described are available in the Protein Data Bank under the accession numbers 6VVH and 6VVI .
Size matters! Fragmentation chemistry of [Cu(L)n]2+ complexes of diacylglycerophosphocholines as a function of coordination number (n = 2–7)
Publisher: Wiley
Date: 05-02-2007
DOI: 10.1002/RCM.2891
Abstract: [Cu(L)(n)](2+) complexes of 1,2-dihexanoyl-sn-glycero-3-phosphocholine (L = D6PC) are formed upon electrospray ionization mass spectrometry (ESI-MS) of an 8 mM solution of D6PC with 4 mM CuCl(2) in 10 mM ammonium acetate buffer, pH 6.1. The collision-induced dissociation (CID) reactions of the [Cu(L)(n)](2+) complexes were examined in a linear ion trap mass spectrometer. A rich fragmentation chemistry was observed, including: loss of a neutral ligand intermolecular ligand-ligand S(N)2 methylation metal ion induced ligand fragmentation via carboxylate abstraction and phosphate abstraction. The dominant reaction channel depends on the size (n) of the complex. Thus loss of neutral ligand(s) is the sole reaction channel for n = 5-7. At n = 4, S(N)2 methylation and carboxylate abstraction start to compete with neutral ligand loss. At n = 2 the carboxylate abstraction and phosphate abstraction reactions dominate the CID spectrum. The carboxylate abstraction and phosphate abstraction reactions are likely to be driven via neighboring group pathways. PM3 calculations, carried out to compare competing neighboring pathways based on the relative stabilities of the product ions, suggest a preference for five-membered ring formation for ligand fragmentation involving both carboxylate and phosphate abstraction.
Characterisation of dihydrodipicolinate synthase (DHDPS) from Bacillus anthracis
Publisher: Elsevier BV
Date: 10-2009
DOI: 10.1016/J.BBAPAP.2009.06.020
Abstract: Bacillus anthracis is a Gram-positive spore-forming bacterium that is the causative agent of anthrax disease. The use of anthrax as a bioweapon has increased pressure for the development of an effective treatment. Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step in the biosynthetic pathway yielding two essential bacterial metabolites, meso-diaminopimelate (DAP) and (S)-lysine. DHDPS is therefore a potential antibiotic target, as microbes require either lysine or DAP as a component of the cell wall. This paper is the first biochemical description of DHDPS from B. anthracis. Enzyme kinetic analyses, isothermal titration calorimetry (ITC), mass spectrometry and differential scanning fluorimetry (DSF) were used to characterise B. anthracis DHDPS and compare it with the well characterised Escherichia coli enzyme. B. anthracis DHDPS exhibited different kinetic behaviour compared with E. coli DHDPS, in particular, substrate inhibition by (S)-aspartate semi-aldehyde was observed for the B. anthracis enzyme (K(si(ASA))=5.4+/-0.5 mM), but not for the E. coli enzyme. As predicted from a comparison of the X-ray crystal structures, the B. anthracis enzyme was not inhibited by lysine. The B. anthracis enzyme was thermally stabilised by the first substrate, pyruvate, to a greater extent than its E. coli counterpart, but has a weaker affinity for pyruvate based on enzyme kinetics and ITC studies. This characterisation will provide useful information for the design of inhibitors as new antibiotics targeting B. anthracis.
Properties of GDP-mannose Pyrophosphorylase, a Critical Enzyme and Drug Target in Leishmania mexicana
Publisher: Elsevier BV
Date: 03-2004
Development of the procedures for high-yield expression and rapid purification of active recombinant Csk-homologous kinase (CHK): Comparison of the catalytic activities of CHK and CSK
Publisher: Elsevier BV
Date: 12-2010
DOI: 10.1016/J.PEP.2010.07.007
Abstract: Csk-homologous kinase (CHK) is an important endogenous inhibitor constraining the oncogenic actions of Src-family kinases (SFKs) in cells. It suppresses SFK activity by specifically phosphorylating the conserved regulatory tyrosine near the C-terminus of SFKs. In addition to phosphorylation, CHK employs a novel non-catalytic inhibitory mechanism to suppress SFK activity. This mechanism involves direct binding of CHK to the active forms of SFKs to form stable protein complexes. Since aberrant activation of SFKs contributes to cancer formation and progression, small-molecule inhibitors mimicking the non-catalytic inhibitory mechanism of CHK are potential anti-cancer therapeutics. Elucidation of the catalytic and regulatory properties and the structural basis of the CHK non-catalytic inhibitory mechanism would facilitate the development of these small-molecule inhibitors. To this end, we developed procedures for higher level expression in insect cells of active recombinant CHK with a hexa-histidine tag attached to its C-terminus (referred to as CHK-His(6)) and its rapid purification by a two-step method. Analyses by size-exclusion column chromatography and analytical ultracentrifugation revealed that the purified CHK-His(6) exists as a monomeric species in solution. Biochemical analyses demonstrated that CHK-His(6) exhibits efficiencies comparable to those of CSK in phosphorylating artificial protein and peptide substrates as well as an intact SFK protein. Our results indicate that the recombinant CHK-His(6) can be used for future studies to decipher the three-dimensional structure, and regulatory and catalytic properties of CHK.
Cloning, expression, purification and crystallization of dihydrodipicolinate synthase from the psychrophileShewanella benthica
Publisher: International Union of Crystallography (IUCr)
Date: 29-10-2010
Purification, crystallization and preliminary X-ray diffraction studies to near-atomic resolution of dihydrodipicolinate synthase from methicillin-resistantStaphylococcus aureus
Publisher: International Union of Crystallography (IUCr)
Date: 28-06-2008
Substrate-mediated Stabilization of a Tetrameric Drug Target Reveals Achilles Heel in Anthrax
Publisher: Elsevier BV
Date: 02-2010
Evolution of Quaternary Structure in a Homotetrameric Enzyme
Publisher: Elsevier BV
Date: 07-2008
DOI: 10.1016/J.JMB.2008.05.038
Abstract: Dihydrodipicolinate synthase (DHDPS) is an essential enzyme in (S)-lysine biosynthesis and an important antibiotic target. All X-ray crystal structures solved to date reveal a homotetrameric enzyme. In order to explore the role of this quaternary structure, dimeric variants of Escherichia coli DHDPS were engineered and their properties were compared to those of the wild-type tetrameric form. X-ray crystallography reveals that the active site is not disturbed when the quaternary structure is disrupted. However, the activity of the dimeric enzymes in solution is substantially reduced, and a tetrahedral adduct of a substrate analogue is observed to be trapped at the active site in the crystal form. Remarkably, heating the dimeric enzymes increases activity. We propose that the homotetrameric structure of DHDPS reduces dynamic fluctuations present in the dimeric forms and increases specificity for the first substrate, pyruvate. By restricting motion in a key catalytic motif, a competing, non-productive reaction with a substrate analogue is avoided. Small-angle X-ray scattering and mutagenesis data, together with a B-factor analysis of the crystal structures, support this hypothesis and lead to the suggestion that in at least some cases, the evolution of quaternary enzyme structures might serve to optimise the dynamic properties of the protein subunits.
Telomere sequence content can be used to determine ALT activity in tumours
Publisher: Oxford University Press (OUP)
Date: 30-04-2018
DOI: 10.1093/NAR/GKY297
Comparative structure and function analyses of native and his-tagged forms of dihydrodipicolinate reductase from methicillin-resistant Staphylococcus aureus
Publisher: Elsevier BV
Date: 09-2012
DOI: 10.1016/J.PEP.2012.06.017
Abstract: Given the rise of multi drug resistant bacterial strains, such as methicillin-resistant Staphylococcus aureus (MRSA), there is an urgent need to discover new antimicrobial agents. A validated but as yet unexplored target for new antibiotics is dihydrodipicolinate reductase (DHDPR), an enzyme that catalyzes the second step of the lysine biosynthesis pathway in bacteria. We report here the cloning, expression and purification of N-terminally his-tagged recombinant DHDPR from MRSA (6H-MRSA-DHDPR) and compare its secondary and quaternary structure with the wild type (MRSA-DHDPR) enzyme. Comparative analyses demonstrate that recombinant 6H-MRSA-DHDPR is folded and adopts the native tetrameric quaternary structure in solution. Furthermore, kinetic studies show 6H-MRSA-DHDPR is functional, displaying parameters for K(m)(NADH) of 6.0 μM, K(m)(DHDP) of 22 μM, and k(cat) of 21s(-1), which are similar to those reported for the native enzyme. The solution properties and stability of the 6H-MRSA-DHDPR enzyme are also reported in varying physicochemical conditions.
A mitochondrial protein affects cell morphology, mitochondrial segregation and virulence in Leishmania
Publisher: Elsevier BV
Date: 12-2006
DOI: 10.1016/J.IJPARA.2006.08.006
Abstract: The single mitochondrion of kinetoplastids ides in synchrony with the nucleus and plays a crucial role in cell ision. However, despite its importance and potential as a drug target, the mechanism of mitochondrial ision and segregation and the molecules involved are only partly understood. In our quest to identify novel mitochondrial proteins in Leishmania, we constructed a hidden Markov model from the targeting motifs of known mitochondrial proteins as a tool to search the Leishmania major genome. We show here that one of the 17 proteins of unknown function that we identified, designated mitochondrial protein X (MIX), is an oligomeric protein probably located in the inner membrane and expressed throughout the Leishmania life cycle. The MIX gene appears to be essential. Moreover, even deletion of one allele from L. major led to abnormalities in cell morphology, mitochondrial segregation and, importantly, to loss of virulence. MIX is unique to kinetoplastids but its heterologous expression in Saccharomyces cerevisiae produced defects in mitochondrial morphology. Our data show that a number of mitochondrial proteins are unique to kinetoplastids and some, like MIX, play a central role in mitochondrial segregation and cell ision, as well as virulence.
A Synthetic Peptide That Inhibits Lipoprotein(a) Assembly
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 03-2003
DOI: 10.1161/01.ATV.0000055741.13940.15
Abstract: Objective— We previously reported that human apolipoprotein B100 (apoB) amino acids 4330–4397 were important for the initial noncovalent binding to apolipoprotein(a) [apo(a)] that facilitates lipoprotein(a) [Lp(a)] assembly. In this study, we aimed to further define the apoB sequences within the 4330–4397 region that were important for the noncovalent binding to apo(a). Methods and Results— Alignment of the human apoB4330–4397 sequence with mouse apoB, which also noncovalently binds apo(a), revealed stretches of similar sequence, including a lysine-rich sequence spanning apoB amino acids 4372–4392. Structural analysis of the apoB4372–4392 sequence using the WHEEL program predicted an hipathic α-helix. Circular dichroism studies of a synthetic peptide spanning human apoB amino acids 4372–4392, both in the absence and presence of dimyristoylphosphatidylcholine, confirmed the α-helical nature of the sequence. We tested the ability of the apoB 4372–4392 peptide to bind to apo(a) and found that the peptide bound to apo(a) with high affinity but not to Lp(a). The apoB 4372–4392 peptide inhibited Lp(a) assembly in Lp(a) formation assays far more effectively than the lysine analogue, ε-amino-n-caproic acid (IC 50 =40 μmol/L versus 10 mmol/L, respectively). Incorporation of the apoB 4372–4392 peptide onto dimyristoylphosphatidylcholine vesicles yielded an even more effective inhibitor (IC 50 =4 μmol/L). Conclusions— Our study shows that the apoB4372–4392 sequence mediates the initial noncovalent binding to apo(a) and has demonstrated that the apoB 4372–4392 peptide is a novel and effective inhibitor of Lp(a) assembly.
Characterization of recombinant dihydrodipicolinate synthase from the bread wheat Triticum aestivum
Publisher: Springer Science and Business Media LLC
Date: 09-05-2018
DOI: 10.1007/S00425-018-2894-X
Abstract: Recombinant wheat DHDPS was produced for the first time in milligram quantities and shown to be an enzymatically active tetramer in solution using analytical ultracentrifugation and small angle X-ray scattering. Wheat is an important cereal crop with an extensive role in global food supply. Given our rapidly growing population, strategies to increase the nutritional value and production of bread wheat are of major significance in agricultural science to satisfy our dietary requirements. Lysine is one of the most limiting essential amino acids in wheat, thus, a thorough understanding of lysine biosynthesis is of upmost importance to improve its nutritional value. Dihydrodipicolinate synthase (DHDPS EC 4.3.3.7) catalyzes the first committed step in the lysine biosynthesis pathway of plants. Here, we report for the first time the expression and purification of recombinant DHDPS from the bread wheat Triticum aestivum (Ta-DHDPS). The optimized protocol yielded 36 mg of > 98% pure recombinant Ta-DHDPS per liter of culture. Enzyme kinetic studies demonstrate that the recombinant Ta-DHDPS has a K
Structure of Leishmania mexicana Phosphomannomutase Highlights Similarities with Human Isoforms
Publisher: Elsevier BV
Date: 10-2006
DOI: 10.1016/J.JMB.2006.08.023
Abstract: Phosphomannomutase (PMM) catalyses the conversion of mannose-6-phosphate to mannose-1-phosphate, an essential step in mannose activation and the biosynthesis of glycoconjugates in all eukaryotes. Deletion of PMM from Leishmania mexicana results in loss of virulence, suggesting that PMM is a promising drug target for the development of anti-leishmanial inhibitors. We report the crystallization and structure determination to 2.1 A of L. mexicana PMM alone and in complex with glucose-1,6-bisphosphate to 2.9 A. PMM is a member of the haloacid dehalogenase (HAD) family, but has a novel dimeric structure and a distinct cap domain of unique topology. Although the structure is novel within the HAD family, the leishmanial enzyme shows a high degree of similarity with its human isoforms. We have generated L. major PMM knockouts, which are avirulent. We expressed the human pmm2 gene in the Leishmania PMM knockout, but despite the similarity between Leishmania and human PMM, expression of the human gene did not restore virulence. Similarities in the structure of the parasite enzyme and its human isoforms suggest that the development of parasite-selective inhibitors will not be an easy task.
Identification of two dihydrodipicolinate synthase isoforms from Pseudomonas aeruginosa that differ in allosteric regulation
Publisher: Wiley
Date: 05-08-2020
DOI: 10.1111/FEBS.15014
Abstract: Pseudomonas aeruginosa is one of the leading causes of nosocomial infections, accounting for 10% of all hospital-acquired infections. Current antibiotics against P. aeruginosa are becoming increasingly ineffective due to the exponential rise in drug resistance. Thus, there is an urgent need to validate and characterize novel drug targets to guide the development of new classes of antibiotics against this pathogen. One such target is the diaminopimelate (DAP) pathway, which is responsible for the biosynthesis of bacterial cell wall and protein building blocks, namely meso-DAP and lysine. The rate-limiting step of this pathway is catalysed by the enzyme dihydrodipicolinate synthase (DHDPS), typically encoded for in bacteria by a single dapA gene. Here, we show that P. aeruginosa encodes two functional DHDPS enzymes, PaDHDPS1 and PaDHDPS2. Although these isoforms have similar catalytic activities (k
Non-fibrillar Components of Amyloid Deposits Mediate the Self-association and Tangling of Amyloid Fibrils
Publisher: Elsevier BV
Date: 05-2004
Shark Variable New Antigen Receptor (VNAR) Single Domain Antibody Fragments: Stability and Diagnostic Applications
Publisher: MDPI AG
Date: 25-01-2013
DOI: 10.3390/ANTIB2010066
Substrate Locking Promotes Dimer-Dimer Docking of an Enzyme Antibiotic Target
Publisher: Elsevier BV
Date: 07-2018
DOI: 10.1016/J.STR.2018.04.014
Abstract: Protein dynamics manifested through structural flexibility play a central role in the function of biological molecules. Here we explore the substrate-mediated change in protein flexibility of an antibiotic target enzyme, Clostridium botulinum dihydrodipicolinate synthase. We demonstrate that the substrate, pyruvate, stabilizes the more active dimer-of-dimers or tetrameric form. Surprisingly, there is little difference between the crystal structures of apo and substrate-bound enzyme, suggesting protein dynamics may be important. Neutron and small-angle X-ray scattering experiments were used to probe substrate-induced dynamics on the sub-second timescale, but no significant changes were observed. We therefore developed a simple technique, coined protein dynamics-mass spectrometry (ProD-MS), which enables measurement of time-dependent alkylation of cysteine residues. ProD-MS together with X-ray crystallography and analytical ultracentrifugation analyses indicates that pyruvate locks the conformation of the dimer that promotes docking to the more active tetrameric form, offering insight into ligand-mediated stabilization of multimeric enzymes.
Whole-genome characterization of chemoresistant ovarian cancer
Publisher: Springer Science and Business Media LLC
Date: 27-05-2015
DOI: 10.1038/NATURE14410
Abstract: Patients with high-grade serous ovarian cancer (HGSC) have experienced little improvement in overall survival, and standard treatment has not advanced beyond platinum-based combination chemotherapy, during the past 30 years. To understand the drivers of clinical phenotypes better, here we use whole-genome sequencing of tumour and germline DNA s les from 92 patients with primary refractory, resistant, sensitive and matched acquired resistant disease. We show that gene breakage commonly inactivates the tumour suppressors RB1, NF1, RAD51B and PTEN in HGSC, and contributes to acquired chemotherapy resistance. CCNE1 lification was common in primary resistant and refractory disease. We observed several molecular events associated with acquired resistance, including multiple independent reversions of germline BRCA1 or BRCA2 mutations in in idual patients, loss of BRCA1 promoter methylation, an alteration in molecular subtype, and recurrent promoter fusion associated with overexpression of the drug efflux pump MDR1.
Structural, kinetic and computational investigation of Vitis vinifera DHDPS reveals new insight into the mechanism of lysine-mediated allosteric inhibition
Publisher: Springer Science and Business Media LLC
Date: 26-01-2013
DOI: 10.1007/S11103-013-0014-7
Abstract: Lysine is one of the most limiting amino acids in plants and its biosynthesis is carefully regulated through inhibition of the first committed step in the pathway catalyzed by dihydrodipicolinate synthase (DHDPS). This is mediated via a feedback mechanism involving the binding of lysine to the allosteric cleft of DHDPS. However, the precise allosteric mechanism is yet to be defined. We present a thorough enzyme kinetic and thermodynamic analysis of lysine inhibition of DHDPS from the common grapevine, Vitis vinifera (Vv). Our studies demonstrate that lysine binding is both tight (relative to bacterial DHDPS orthologs) and cooperative. The crystal structure of the enzyme bound to lysine (2.4 Å) identifies the allosteric binding site and clearly shows a conformational change of several residues within the allosteric and active sites. Molecular dynamics simulations comparing the lysine-bound (PDB ID 4HNN) and lysine free (PDB ID 3TUU) structures show that Tyr132, a key catalytic site residue, undergoes significant rotational motion upon lysine binding. This suggests proton relay through the catalytic triad is attenuated in the presence of lysine. Our study reveals for the first time the structural mechanism for allosteric inhibition of DHDPS from the common grapevine.
Vaccinia virus anti-apoptotic F1L is a novel Bcl-2-like domain-swapped dimer that binds a highly selective subset of BH3-containing death ligands
Publisher: Springer Science and Business Media LLC
Date: 13-06-2008
DOI: 10.1038/CDD.2008.83
Abstract: Apoptosis is an important part of the host's defense mechanism for eliminating invading pathogens. Some viruses express proteins homologous in sequence and function to mammalian pro-survival Bcl-2 proteins. Anti-apoptotic F1L expressed by vaccinia virus is essential for survival of infected cells, but it bears no discernable sequence homology to proteins other than its immediate orthologues in related pox viruses. Here we report that the crystal structure of F1L reveals a Bcl-2-like fold with an unusual N-terminal extension. The protein forms a novel domain-swapped dimer in which the alpha1 helix is the exchanged domain. Binding studies reveal an atypical BH3-binding profile, with sub-micromolar affinity only for the BH3 peptide of pro-apoptotic Bim and low micromolar affinity for the BH3 peptides of Bak and Bax. This binding interaction is sensitive to F1L mutations within the predicted canonical BH3-binding groove, suggesting parallels between how vaccinia virus F1L and myxoma virus M11L bind BH3 domains. Structural comparison of F1L with other Bcl-2 family members reveals a novel sequence signature that redefines the BH4 domain as a structural motif present in both pro- and anti-apoptotic Bcl-2 members, including viral Bcl-2-like proteins.
Cloning, expression, purification and crystallization of dihydrodipicolinate synthase from the grapevine Vitis vinifera
Publisher: International Union of Crystallography (IUCr)
Date: 25-11-2011
DOI: 10.1107/S1744309111038395
Abstract: Dihydrodipicolinate synthase (DHDPS) catalyses the first committed step of the lysine-biosynthesis pathway in bacteria, plants and some fungi. This study describes the cloning, expression, purification and crystallization of DHDPS from the grapevine Vitis vinifera (Vv-DHDPS). Following in-drop cleavage of the hexahistidine tag, cocrystals of Vv-DHDPS with the substrate pyruvate were grown in 0.1 M Bis-Tris propane pH 8.2, 0.2 M sodium bromide, 20%( w / v ) PEG 3350. X-ray diffraction data in space group P 1 at a resolution of 2.2 Å are presented. Preliminary diffraction data analysis indicated the presence of eight molecules per asymmetric unit ( V M = 2.55 Å 3 Da −1 , 52% solvent content). The pending crystal structure of Vv-DHDPS will provide insight into the molecular evolution in quaternary structure of DHDPS enzymes.
Genome‐wide DNA methylation patterns in pancreatic ductal adenocarcinoma reveal epigenetic deregulation of SLIT‐ROBO, ITGA2 and MET signaling
Publisher: Wiley
Date: 09-05-2014
DOI: 10.1002/IJC.28765
Abstract: The importance of epigenetic modifications such as DNA methylation in tumorigenesis is increasingly being appreciated. To define the genome-wide pattern of DNA methylation in pancreatic ductal adenocarcinomas (PDAC), we captured the methylation profiles of 167 untreated resected PDACs and compared them to a panel of 29 adjacent nontransformed pancreata using high-density arrays. A total of 11,634 CpG sites associated with 3,522 genes were significantly differentially methylated (DM) in PDAC and were capable of segregating PDAC from non-malignant pancreas, regardless of tumor cellularity. As expected, PDAC hypermethylation was most prevalent in the 5' region of genes (including the proximal promoter, 5'UTR and CpG islands). Approximately 33% DM genes showed significant inverse correlation with mRNA expression levels. Pathway analysis revealed an enrichment of aberrantly methylated genes involved in key molecular mechanisms important to PDAC: TGF-β, WNT, integrin signaling, cell adhesion, stellate cell activation and axon guidance. Given the recent discovery that SLIT-ROBO mutations play a clinically important role in PDAC, the role of epigenetic perturbation of axon guidance was pursued in more detail. Bisulfite licon deep sequencing and qRT-PCR expression analyses confirmed recurrent perturbation of axon guidance pathway genes SLIT2, SLIT3, ROBO1, ROBO3, ITGA2 and MET and suggests epigenetic suppression of SLIT-ROBO signaling and up-regulation of MET and ITGA2 expression. Hypomethylation of MET and ITGA2 correlated with high gene expression, which was associated with poor survival. These data suggest that aberrant methylation plays an important role in pancreatic carcinogenesis affecting core signaling pathways with potential implications for the disease pathophysiology and therapy.
Structural insights into BCL2 pro-survival protein interactions with the key autophagy regulator BECN1 following phosphorylation by STK4/MST1
Publisher: Informa UK Limited
Date: 09-01-2019
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