ORCID Profile
0000-0002-2135-1294
Current Organisations
National University of Singapore
,
Singapore Center for Environmental Life Sciences Engineering
,
Danderyds Sjukhus AB
,
Karolinska Institutet
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Publications
2022 World Hypertension League, Resolve To Save Lives and International Society of Hypertension dietary sodium (salt) global call to action
Publisher: Springer Science and Business Media LLC
Date: 17-05-2022
Author response: Structure of dual BON-domain protein DolP identifies phospholipid binding as a new mechanism for protein localisation
Publisher: eLife Sciences Publications, Ltd
Date: 07-12-2020
Effects of deep and superficial experimentally induced acute pain on skin sympathetic nerve activity in human subjects
Publisher: Springer Science and Business Media LLC
Date: 14-04-2009
DOI: 10.1007/S00221-009-1790-9
Abstract: There is evidence in experimental animals that deep and superficial pain exert differential effects on cutaneous sympathetic activity. Skin sympathetic nerve activity (SSNA) was recorded from the common peroneal nerve of awake human subjects and injections of 0.5 ml hypertonic saline was made into the tibialis anterior muscle (causing a deep, dull ache) or 0.2 ml into the overlying skin (causing a sharp burning pain) at unexpected times. Both deep and superficial pain caused increases in SSNA immediately on injection and preceding the onset of pain for both muscle and skin pain (10.1 +/- 2.4 vs. 15.3 +/- 5.3 s muscle versus skin, respectively). SSNA increases were short lasting (104.2 +/- 13.4 vs. 81.8 +/- 11.7 s muscle versus skin pain) and did not follow muscle and skin pain profiles. Sweat release occurred following both intramuscular and subcutaneous injections of hypertonic saline. While muscle or skin pain invariably caused changes in skin blood flow as well as increases in sweat release, skin blood flow increased in females and decreased in males. We conclude that both acute muscle and skin pain cause an increase in SSNA, sweat release and gender-dependent changes in skin blood flow.
Structure of dual-BON domain protein DolP identifies phospholipid binding as a new mechanism for protein localization
Publisher: eLife Sciences Publications, Ltd
Date: 14-12-2020
DOI: 10.7554/ELIFE.62614
Abstract: The Gram-negative outer-membrane envelops the bacterium and functions as a permeability barrier against antibiotics, detergents, and environmental stresses. Some virulence factors serve to maintain the integrity of the outer membrane, including DolP (formerly YraP) a protein of unresolved structure and function. Here, we reveal DolP is a lipoprotein functionally conserved amongst Gram-negative bacteria and that loss of DolP increases membrane fluidity. We present the NMR solution structure for Escherichia coli DolP, which is composed of two BON domains that form an interconnected opposing pair. The C-terminal BON domain binds anionic phospholipids through an extensive membrane:protein interface. This interaction is essential for DolP function and is required for sub-cellular localisation of the protein to the cell ision site, providing evidence of subcellular localisation of these phospholipids within the outer membrane. The structure of DolP provides a new target for developing therapies that disrupt the integrity of the bacterial cell envelope.
MCE domain proteins: conserved inner membrane lipid-binding proteins required for outer membrane homeostasis
Publisher: Springer Science and Business Media LLC
Date: 17-08-2017
DOI: 10.1038/S41598-017-09111-6
Abstract: Bacterial proteins with MCE domains were first described as being important for M ammalian C ell E ntry. More recent evidence suggests they are components of lipid ABC transporters. In Escherichia coli , the single-domain protein MlaD is known to be part of an inner membrane transporter that is important for maintenance of outer membrane lipid asymmetry. Here we describe two multi MCE domain-containing proteins in Escherichia coli , PqiB and YebT, the latter of which is an orthologue of MAM-7 that was previously reported to be an outer membrane protein. We show that all three MCE domain-containing proteins localise to the inner membrane. Bioinformatic analyses revealed that MCE domains are widely distributed across bacterial phyla but multi MCE domain-containing proteins evolved in Proteobacteria from single-domain proteins. Mutants defective in mlaD, pqiAB and yebST were shown to have distinct but partially overlapping phenotypes, but the primary functions of PqiB and YebT differ from MlaD. Complementing our previous findings that all three proteins bind phospholipids, results presented here indicate that multi-domain proteins evolved in Proteobacteria for specific functions in maintaining cell envelope homeostasis.
Characterization of Interactions and Phospholipid Transfer between Substrate Binding Proteins of the OmpC-Mla System
Publisher: American Chemical Society (ACS)
Date: 04-10-2018
DOI: 10.1021/ACS.BIOCHEM.8B00897
Abstract: The outer membrane (OM) of Gram-negative bacteria is a permeability barrier that impedes the entry of external insults, such as antibiotics and bile salts. This barrier function depends critically on the asymmetric lipid distribution across the bilayer, with lipopolysaccharides (LPS) facing outside and phospholipids (PLs) facing inside. In Escherichia coli, the OmpC-Mla system is believed to maintain OM lipid asymmetry by removing surface exposed PLs and shuttling them back to the inner membrane (IM). How proteins in the pathway interact to mediate PL transport across the periplasm is not known. Evidence for direct transfer of PLs between these proteins is also lacking. In this study, we mapped the interaction surfaces between the two PL-binding proteins, MlaC and MlaD, using site-specific in vivo photo-cross-linking, and obtained a physical picture for how these proteins may transfer PLs. Furthermore, we demonstrated using purified proteins that MlaD spontaneously transfers PLs to MlaC, suggesting that the latter has a higher affinity for PLs. Our work provides insights into the mechanism of bacterial intermembrane lipid transport important for the maintenance of OM lipid asymmetry.
Potency Increase of Spiroketal Analogs of Membrane Inserting Indolyl Mannich Base Antimycobacterials Is Due to Acquisition of MmpL3 Inhibition
Publisher: American Chemical Society (ACS)
Date: 15-05-2020
Structure-Function Characterization of the Conserved Regulatory Mechanism of the Escherichia coli M48 Metalloprotease BepA
Publisher: American Society for Microbiology
Date: 18-12-2020
DOI: 10.1128/JB.00434-20
Abstract: M48 metalloproteases are widely distributed in all domains of life. E. coli possesses four members of this family located in multiple cellular compartments. The functions of these proteases are not well understood. Recent investigations revealed that one family member, BepA, has an important role in the maturation of a central component of the lipopolysaccharide (LPS) biogenesis machinery. Here, we present the structure of BepA and the results of a structure-guided mutagenesis strategy, which reveal the key residues required for activity that inform how all M48 metalloproteases function.
Defining key roles for auxiliary proteins in an ABC transporter that maintains bacterial outer membrane lipid asymmetry
Publisher: eLife Sciences Publications, Ltd
Date: 16-08-2016
DOI: 10.7554/ELIFE.19042
Abstract: In Gram-negative bacteria, lipid asymmetry is critical for the function of the outer membrane (OM) as a selective permeability barrier, but how it is established and maintained is poorly understood. Here, we characterize a non-canonical ATP-binding cassette (ABC) transporter in Escherichia coli that provides energy for maintaining OM lipid asymmetry via the transport of aberrantly localized phospholipids (PLs) from the OM to the inner membrane (IM). We establish that the transporter comprises canonical components, MlaF and MlaE, and auxiliary proteins, MlaD and MlaB, of previously unknown functions. We further demonstrate that MlaD forms extremely stable hexamers within the complex, functions in substrate binding with strong affinity for PLs, and modulates ATP hydrolytic activity. In addition, MlaB plays critical roles in both the assembly and activity of the transporter. Our work provides mechanistic insights into how the MlaFEDB complex participates in ensuring active retrograde PL transport to maintain OM lipid asymmetry.
Structure-function analysis of the Escherichia coli β–barrel assembly enhancing protease BepA suggests a role for a self-inhibitory state
Publisher: Cold Spring Harbor Laboratory
Date: 02-07-2019
DOI: 10.1101/689117
Abstract: The asymmetric Gram-negative outer membrane (OM) is the first line of defence for the bacteria against environmental insults and attack by antimicrobials. The key component of the OM barrier is the surface exposed lipopolysaccharide, which is transported to the surface by the essential lipopolysaccharide transport (Lpt) system. Correct folding of the Lpt system OM component, LptD, is essential and is regulated by a periplasmic metalloprotease, BepA. Here we present the crystal structure of BepA, solved to a resolution of 1.9 Å. Our structure comprises the zinc-bound m48 protease domain and a tetratricopeptide repeat (TPR) domain, consisting of four 2-helix TPR motifs and four non-TPR helices, leading to a nautilus-like shape in which the TPR repeats cup the protease domain. Using targeted mutagenesis approaches, we demonstrate that the protein is auto-regulated by the active-site plug. Further to this we reveal that mutation of a negative pocket, formed at the interface between the m48 and TPR domains, impairs BepA activity suggesting the pocket as a possible substrate binding site. We also identify a potential protein interaction site within the TPR cavity as being important for BepA function. Lastly, we provide evidence to show that increased antibiotic susceptibility in the absence of correctly functioning BepA occurs through disruption of OM lipid asymmetry, leading to reduced barrier function and increased cell permeability.
Loss of YhcB results in dysregulation of coordinated peptidoglycan, LPS and phospholipid synthesis during Escherichia coli cell growth
Publisher: Public Library of Science (PLoS)
Date: 23-12-2021
DOI: 10.1371/JOURNAL.PGEN.1009586
Abstract: The cell envelope is essential for viability in all domains of life. It retains enzymes and substrates within a confined space while providing a protective barrier to the external environment. Destabilising the envelope of bacterial pathogens is a common strategy employed by antimicrobial treatment. However, even in one of the best studied organisms, Escherichia coli , there remain gaps in our understanding of how the synthesis of the successive layers of the cell envelope are coordinated during growth and cell ision. Here, we used a whole-genome phenotypic screen to identify mutants with a defective cell envelope. We report that loss of yhcB , a conserved gene of unknown function, results in loss of envelope stability, increased cell permeability and dysregulated control of cell size. Using whole genome transposon mutagenesis strategies, we report the comprehensive genetic interaction network of yhcB , revealing all genes with a synthetic negative and a synthetic positive relationship. These genes include those previously reported to have a role in cell envelope biogenesis. Surprisingly, we identified genes previously annotated as essential that became non-essential in a Δ yhcB background. Subsequent analyses suggest that YhcB functions at the junction of several envelope biosynthetic pathways coordinating the spatiotemporal growth of the cell, highlighting YhcB as an as yet unexplored antimicrobial target.
Effect of Finerenone on Chronic Kidney Disease Outcomes in Type 2 Diabetes
Publisher: Massachusetts Medical Society
Date: 03-12-2020
YhcB coordinates peptidoglycan and LPS biogenesis with phospholipid synthesis during Escherichia coli cell growth
Publisher: Cold Spring Harbor Laboratory
Date: 18-04-2021
DOI: 10.1101/2021.04.16.440158
Abstract: The cell envelope is essential for viability in all kingdoms of life. It retains enzymes and substrates within a confined space while providing a protective barrier to the external environment. Destabilising the envelope of bacterial pathogens is a common strategy employed by antimicrobial treatment. However, even in one of the most well studied organisms, Escherichia coli , there remain gaps in our understanding of how the synthesis of the successive layers of the cell envelope are coordinated during growth and cell ision. Here, we used a whole genome phenotypic screen to identify mutants with a defective cell envelope. We report that loss of yhcB , a conserved gene of unknown function, results in loss of envelope stability, increased cell permeability and dysregulated control of cell size. Using whole genome transposon mutagenesis strategies we report the complete genetic interaction network of yhcB , revealing all genes with a synthetic negative and a synthetic positive relationship. These genes include those previously reported to have a role in cell envelope biogenesis. Surprisingly, we identified genes previously annotated as essential that became non-essential in a Δ yhcB background. Subsequent analyses suggest that YhcB sits at the junction of several envelope biosynthetic pathways coordinating the spatiotemporal growth of the cell, highlighting YhcB as an as yet unexplored antimicrobial target.
Structure-function characterization of the conserved regulatory mechanism of theEscherichia coliM48 metalloprotease BepA
Publisher: Cold Spring Harbor Laboratory
Date: 31-07-2020
DOI: 10.1101/2020.07.30.230011
Abstract: The asymmetric Gram-negative outer membrane (OM) is the first line of defence for bacteria against environmental insults and attack by antimicrobials. The key component of the OM is lipopolysaccharide, which is transported to the surface by the essential lipopolysaccharide transport (Lpt) system. Correct folding of the Lpt system component LptD is regulated by a periplasmic metalloprotease, BepA. Here we present the crystal structure of BepA from Escherichia coli, solved to a resolution of 2.18 Å, in which the M48 protease active site is occluded by an active site plug. Informed by our structure, we demonstrate that free movement of the active site plug is essential for BepA function, suggesting that the protein is auto-regulated by the active site plug, which is conserved throughout the M48 metalloprotease family. Targeted mutagenesis of conserved residues reveals that the negative pocket and the TPR cavity are required for function and degradation of the BAM complex component BamA under conditions of stress. Lastly, we show that loss of BepA causes disruption of OM lipid asymmetry, leading to surface exposed phospholipid.
The value of a new cardiac magnetic resonance imaging protocol in Myocardial Infarction with Non-obstructive Coronary Arteries (MINOCA) – a case-control study using historical controls from a previous
Publisher: Springer Science and Business Media LLC
Date: 24-07-2017
Related Organisations
Singapore Center For Environmental Life Sciences Engineering
Location: Singapore
Related Funding Activities
No related grants have been discovered for Jonas Spaak.