Danny Hatters

Human apolipoprotein C-II forms twisted amyloid ribbons and closed loops

Publisher: American Chemical Society (ACS)

Date: 20-06-2000

DOI: 10.1021/BI000002W

Abstract: Human apolipoprotein C-II (apoC-II) self-associates in solution to form aggregates with the characteristics of amyloid including red-green birefringence in the presence of Congo Red under cross-polarized light, increased fluorescence in the presence of thioflavin T, and a fibrous structure when examined by electron microscopy. ApoC-II was expressed and purified from Escherichia coli and rapidly exchanged from 5 M guanidine hydrochloride into 100 mM sodium phosphate, pH 7.4, to a final concentration of 0.3 mg/mL. This apoC-II was initially soluble, eluting as low molecular weight species in gel filtration experiments using Sephadex G-50. Circular dichroism (CD) spectroscopy indicated predominantly unordered structure. Upon incubation for 24 h, apoC-II self-associated into high molecular weight aggregates as indicated by elution in the void volume of a Sephadex G-50 column, by rapid sedimentation in an analytical ultracentrifuge, and by increased light scattering. CD spectroscopy indicated an increase in beta-sheet content, while fluorescence emission spectroscopy of the single tryptophan revealed a blue shift and an increase in maximum intensity, suggesting repositioning of the tryptophan into a less polar environment. Electron microscopy of apoC-II aggregates revealed a novel looped-ribbon morphology (width 12 nm) and several isolated closed loops. Like all of the conserved plasma apolipoproteins, apoC-II contains hipathic helical regions that account for the increase in alpha-helix content on lipid binding. The increase in beta-structure accompanying apoC-II fibril formation points to an alternative folding pathway and an in vitro system to explore the general tendency of apolipoproteins to form amyloid in vivo.

A biosensor of protein foldedness identifies increased “holdase” activity of chaperones in the nucleus following increased cytosolic protein aggregation

Publisher: Elsevier BV

Date: 08-2022

DOI: 10.1016/J.JBC.2022.102158

A biosensor-based framework to measure latent proteostasis capacity

Publisher: Springer Science and Business Media LLC

Date: 18-01-2018

DOI: 10.1038/S41467-017-02562-5

Abstract: The pool of quality control proteins (QC) that maintains protein-folding homeostasis (proteostasis) is dynamic but can become depleted in human disease. A challenge has been in quantitatively defining the depth of the QC pool. With a new biosensor, flow cytometry-based methods and mathematical modeling we measure the QC capacity to act as holdases and suppress biosensor aggregation. The biosensor system comprises a series of barnase kernels with differing folding stability that engage primarily with HSP70 and HSP90 family proteins. Conditions of proteostasis stimulation and stress alter QC holdase activity and aggregation rates. The method reveals the HSP70 chaperone cycle to be rate limited by HSP70 holdase activity under normal conditions, but this is overcome by increasing levels of the BAG1 nucleotide exchange factor to HSPA1A or activation of the heat shock gene cluster by HSF1 overexpression. This scheme opens new paths for biosensors of disease and proteostasis systems.

Application of flow cytometry to analyze intracellular location and trafficking of cargo in cell populations

Publisher: Springer New York

Date: 30-12-2015

DOI: 10.1007/978-1-4939-2309-0_17

Abstract: Pulse shape analysis (PulSA) is a flow cytometry-based method that involves the measurement of the pulse width and height of a fluorescently labeled molecule simultaneously, enabling a multidimensional analysis of protein localization in a cell at high speed and throughput. We have used the method to detect morphological changes in organelles such as Golgi fragmentation, track protein trafficking from the cell surface, and also discriminate cells with different target protein localizations such as the Golgi, lyso-endosomal network, and the plasma membrane. Here, we describe the basic experimental setup and analytical methods for performing PulSA to examine membrane trafficking processes. We illustrate in particular the application of PulSA for monitoring the trafficking of the membrane-bound enzyme furin and morphological changes to the Golgi caused by Brefeldin A.

High-throughput quantitation of intracellular trafficking and organelle disruption by flow cytometry

Publisher: Wiley

Date: 11-03-2014

DOI: 10.1111/TRA.12161

Abstract: Current methods for the quantitation of membrane protein trafficking rely heavily on microscopy, which has limited quantitative capacity for analyses of cell populations and is cumbersome to perform. Here we describe a simple flow cytometry-based method that circumvents these limitations. The method utilizes fluorescent pulse-width measurements as a highly sensitive indicator to monitor the changes in intracellular distributions of a fluorescently labelled molecule in a cell. Pulse-width analysis enabled us to discriminate cells with target proteins in different intracellular locations including Golgi, lyso-endosomal network and the plasma membrane, as well as detecting morphological changes in organelles such as Golgi perturbation. The movement of endogenous and exogenous retrograde cargo was tracked from the plasma membrane-to-endosomes-to-Golgi, by decreasing pulse-width values. A block in transport upon RNAi-mediated ablation of transport machinery was readily quantified, demonstrating the versatility of this technique to identify pathway inhibitors. We also showed that pulse-width can be exploited to sort and recover cells based on different intracellular staining patterns, e.g. early endosomes and Golgi, opening up novel downstream applications. Overall, the method provides new capabilities for viewing membrane transport in thousands of cells per minute, unbiased analysis of the trafficking of cargo, and the potential for rapid screening of inhibitors of trafficking pathways.

The Allosteric Mechanism Induced by Protein Kinase A (PKA) Phosphorylation of Dematin (Band 4.9)

Publisher: Elsevier BV

Date: 03-2013

DOI: 10.1074/JBC.M112.438861

Polyalanine expansions drive a shift into α-helical clusters without amyloid-fibril formation

Publisher: Springer Science and Business Media LLC

Date: 16-11-2015

DOI: 10.1038/NSMB.3127

Abstract: Polyglutamine (polyGln) expansions in nine human proteins result in neurological diseases and induce the proteins' tendency to form β-rich amyloid fibrils and intracellular deposits. Less well known are at least nine other human diseases caused by polyalanine (polyAla)-expansion mutations in different proteins. The mechanisms of how polyAla aggregates under physiological conditions remain unclear and controversial. We show here that aggregation of polyAla is mechanistically dissimilar to that of polyGln and hence does not exhibit amyloid kinetics. PolyAla assembled spontaneously into α-helical clusters with erse oligomeric states. Such clustering was pervasive in cells irrespective of visible aggregate formation, and it disrupted the normal physiological oligomeric state of two human proteins natively containing polyAla: ARX and SOX3. This self-assembly pattern indicates that polyAla expansions chronically disrupt protein behavior by imposing a deranged oligomeric status.

A Platform to View Huntingtin Exon 1 Aggregation Flux in the Cell Reveals Divergent Influences from Chaperones hsp40 and hsp70

Publisher: Elsevier BV

Date: 12-2013

DOI: 10.1074/JBC.M113.486944

Transcriptional profiles for distinct aggregation states of mutant Huntingtin exon 1 protein unmask new Huntington's disease pathways

Publisher: Elsevier BV

Date: 09-2017

DOI: 10.1016/J.MCN.2017.07.004

Abstract: Huntington's disease is caused by polyglutamine (polyQ)-expansion mutations in the CAG tandem repeat of the Huntingtin gene. The central feature of Huntington's disease pathology is the aggregation of mutant Huntingtin (Htt) protein into micrometer-sized inclusion bodies. Soluble mutant Htt states are most proteotoxic and trigger an enhanced risk of death whereas inclusions confer different changes to cellular health, and may even provide adaptive responses to stress. Yet the molecular mechanisms underpinning these changes remain unclear. Using the flow cytometry method of pulse-shape analysis (PulSA) to sort neuroblastoma (Neuro2a) cells enriched with mutant or wild-type Htt into different aggregation states, we clarified which transcriptional signatures were specifically attributable to cells before versus after inclusion assembly. D ened CREB signalling was the most striking change overall and invoked specifically by soluble mutant Httex1 states. Toxicity could be rescued by stimulation of CREB signalling. Other biological processes mapped to different changes before and after aggregation included NF-kB signalling, autophagy, SUMOylation, transcription regulation by histone deacetylases and BRD4, NAD+ biosynthesis, ribosome biogenesis and altered HIF-1 signalling. These findings open the path for therapeutic strategies targeting key molecular changes invoked prior to, and subsequently to, Httex1 aggregation.

Widespread remodeling of proteome solubility in response to different protein homeostasis stresses

Publisher: Proceedings of the National Academy of Sciences

Date: 21-01-2020

DOI: 10.1073/PNAS.1912897117

Abstract: The accumulation of protein deposits in neurodegenerative diseases has been hypothesized to depend on a metastable subproteome vulnerable to aggregation. To investigate this phenomenon and the mechanisms that regulate it, we measured the solubility of the proteome in the mouse Neuro2a cell line under six different protein homeostasis stresses: 1) Huntington’s disease proteotoxicity, 2) Hsp70, 3) Hsp90, 4) proteasome, 5) endoplasmic reticulum (ER)-mediated folding inhibition, and 6) oxidative stress. Overall, we found that about one-fifth of the proteome changed solubility with almost all of the increases in insolubility were counteracted by increases in solubility of other proteins. Each stress directed a highly specific pattern of change, which reflected the remodeling of protein complexes involved in adaptation to perturbation, most notably, stress granule (SG) proteins, which responded differently to different stresses. These results indicate that the protein homeostasis system is organized in a modular manner and aggregation patterns were not correlated with protein folding stability (Δ G ). Instead, distinct cellular mechanisms regulate assembly patterns of multiple classes of protein complexes under different stress conditions.

Apolipoprotein E structure: insights into function

Publisher: Elsevier BV

Date: 08-2006

DOI: 10.1016/J.TIBS.2006.06.008

Abstract: Human apolipoprotein E (apoE) is a member of the family of soluble apolipoproteins. Through its interaction with members of the low-density lipoprotein receptor family, apoE has a key role in lipid transport both in the plasma and in the central nervous system. Its three common structural isoforms differentially affect the risk of developing atherosclerosis and neurodegenerative disorders, including Alzheimer's disease. Because the function of apoE is dictated by its structure, understanding the structural properties of apoE and its isoforms is required both to determine its role in disease and for the development of therapeutic strategies.

Cross-Linking and Amyloid Formation by N- and C-Terminal Cysteine Derivatives of Human Apolipoprotein C-II

Publisher: American Chemical Society (ACS)

Date: 25-10-2002

DOI: 10.1021/BI026070V

Abstract: We have investigated the effect of disulfide cross-linking on amyloid formation by human apolipoprotein (apo) C-II. Three derivatives of apoC-II were generated by inserting a cysteine residue on either the N-terminus (C(N)-apoC-II), C-terminus (C(C)-apoC-II), or both termini (C(N)C(C)-apoC-II). Under reducing conditions, all derivatives formed amyloid with a fibrous ribbon morphology similar to that of wild-type apoC-II. Under oxidizing conditions, C(N)- and C(N)C(C)-apoC-II formed a highly tangled network of fibrils, suggesting that the addition of an N-terminal cysteine to apoC-II promotes interfibril disulfide cross-links. Fibrils formed by C(C)-apoC-II under oxidizing conditions were closely packed but less tangled than fibrils formed by the C(N) and C(N)C(C) derivatives. The frequency of closed ring structures was more than doubled for C(C)-apoC-II compared to wild-type apoC-II. The kinetics of fibril formation by all cysteine derivatives was markedly enhanced under oxidizing conditions, suggesting that disulfide cross-linking promotes amyloid formation. Substoichiometric levels of preformed C(N)- and C(C)-apoC-II dimers accelerate amyloid formation by wild-type apoC-II. These data suggest that the N- and C-termini of apoC-II are close together in the amyloid fibril such that covalent cross-linking of either the N or C end of apoC-II promotes nucleation and the "seeding" of fibril growth.

Tracking Mutant Huntingtin Aggregation Kinetics in Cells Reveals Three Major Populations That Include an Invariant Oligomer Pool

Publisher: Elsevier BV

Date: 07-2010

DOI: 10.1074/JBC.M109.084434

Hidden information on protein function in censuses of proteome foldedness

Publisher: Springer Science and Business Media LLC

Date: 14-04-2022

DOI: 10.1038/S41467-022-29661-2

Abstract: Methods that assay protein foldedness with proteomics have generated censuses of apparent protein folding stabilities in biological milieu. However, different censuses poorly correlate with each other. Here, we show that the reason for this is that methods targeting foldedness through monitoring amino acid sidechain reactivity also detect changes in conformation and ligand binding, which can be a substantial fraction of the data. We show that the reactivity of only one quarter of cysteine or methionine sidechains in proteins in a urea denaturation curve of mammalian cell lysate can be confidently explained by a two-state unfolding isotherm. Contrary to that expected from unfolding, up to one third of the cysteines decreased reactivity. These cysteines were enriched in proteins with functions relating to unfolded protein stress. One protein, chaperone HSPA8, displayed changes arising from ligand and cofactor binding. Unmasking this hidden information using the approaches outlined here should improve efforts to understand both folding and the remodeling of protein function directly in complex biological settings.

Flipping the switch: How cysteine oxidation directs tau amyloid conformations

Publisher: Elsevier BV

Date: 11-2021

DOI: 10.1016/J.JBC.2021.101309

Engineering Conformational Destabilization into Mouse Apolipoprotein E

Publisher: Elsevier BV

Date: 07-2005

DOI: 10.1074/JBC.M503910200

Sedimentation Analysis of Novel DNA Structures Formed by Homo-Oligonucleotides

Publisher: Elsevier BV

Date: 07-2001

DOI: 10.1016/S0006-3495(01)75706-2

Inclusion bodies formed by polyglutamine and poly(glycine-alanine) are enriched with distinct proteomes but converge in proteins that are risk factors for disease and involved in protein degradation

Publisher: Cold Spring Harbor Laboratory

Date: 04-05-2020

DOI: 10.1101/2020.05.04.076547

Abstract: Poly(glycine-alanine) (polyGA) is one of the dipolypeptides expressed in Motor Neuron Disease caused by C9ORF72 mutations and accumulates as inclusion bodies in the brain of patients. Superficially these inclusions are similar to those formed by polyglutamine (polyQ) in Huntington’s disease and both have been reported to form an amyloid-like structure suggesting they might aggregate via similar mechanisms to confer cellular dysfunction similarly. Here we investigated which endogenous proteins were enriched in these inclusions and whether aggregation-prone lengths of polyQ (Q 97 ), in context of Huntingtin exon 1, shared similar patterns to aggregation-prone lengths of polyGA (101 GA ). When co-expressed in the same cell, polyGA 101 and HttQ 97 inclusions adopted distinct phases with no overlap suggesting different endogenous proteins would be enriched. Proteomic analyses indeed yielded distinct sets of endogenous proteins recruited into the inclusion types. The proteosome, microtubules, TriC chaperones, and translational machinery were enriched in polyGA aggregates, whereas Dnaj chaperones, nuclear envelope and RNA splicing proteins were enriched in polyQ aggregates. Both structures revealed a synergy of degradation machinery including proteins in the polyQ aggregates that are risk factors for other neurodegenerative diseases involving protein aggregation when mutated, which suggests a convergence point in the pathomechanisms of these diseases.

Protein misfolding inside cells: The case of Huntingtin and Huntington's disease

Publisher: Wiley

Date: 11-2008

DOI: 10.1002/IUB.111

Abstract: Huntington's disease is one of the several neurodegenerative diseases caused by dominant mutations that expand the number of glutamine codons within an existing poly-glutamine (polyQ) repeat sequence of a gene. An expanded polyQ sequence in the huntingtin gene is known to cause the huntingtin protein to aggregate and form intracellular inclusions as disease progresses. However, the role that polyQ-induced aggregation plays in disease is yet to be fully determined. This review focuses on key questions remaining for how the expanded polyQ sequences affect the aggregation properties of the huntingtin protein and the corresponding effects on cellular machinery. The scope includes the technical challenges that remain for rigorously assessing the effects of aggregation on the cellular machinery.

Measuring proteostasis capacity using transiently transfected bait proteins by flow cytometry

Publisher: Springer Science and Business Media LLC

Date: 28-03-2018

DOI: 10.1038/PROTEX.2017.156

Immiscible inclusion bodies formed by polyglutamine and poly(glycine-alanine) are enriched with distinct proteomes but converge in proteins that are risk factors for disease and involved in protein de

Publisher: Public Library of Science (PLoS)

Date: 28-08-2020

DOI: 10.1371/JOURNAL.PONE.0233247

Arginine-rich C9ORF72 ALS Proteins Stall Ribosomes in a Manner Distinct From a Canonical Ribosome-Associated Quality Control Substrate

Publisher: Cold Spring Harbor Laboratory

Date: 10-02-2022

DOI: 10.1101/2022.02.09.479805

Abstract: Hexanucleotide expansion mutations in C9ORF72 are a cause of familial amyotrophic lateral sclerosis. We previously reported that long arginine-rich dipeptide repeats (DPR), mimicking abnormal proteins expressed from the hexanucleotide expansion, caused translation stalling when expressed in cell culture models. Whether this stalling provides a mechanism of pathogenicity remains to be determined. Here we explored the molecular features of DPR-induced stalling and examined whether known regulatory mechanisms of ribosome quality control (RQC) are involved to sense and resolve the stalls. We demonstrate that arginine-containing DPRs lead to stalling in a length dependent manner, with lengths longer than 40 repeats invoking severe translation arrest. Mutational screening of 40×Gly-Xxx DPRs shows that stalling is most pronounced where Xxx are positively charged amino acids (Arg or Lys). Through a genome-wide knockout screen we find that genes regulating stalling on polyadenosine mRNA coding for poly-Lys, a canonical RQC substrate, respond differently to the readthrough of arginine-rich DPRs. Indeed, we find evidence that DPR-mediated stalling has no natural regulatory responses even though the stalls may be sensed, as evidenced by an upregulation of RQC gene expression. These findings therefore implicate arginine-rich DPR-mediated stalled ribosomes as posing a particular danger to cellular health and viability.

Model of biologically active apolipoprotein E bound to dipalmitoylphosphatidylcholine

Publisher: Elsevier BV

Date: 2006

DOI: 10.1074/JBC.M510851200

Macromolecular Crowding Accelerates Amyloid Formation by Human Apolipoprotein C-II

Publisher: Elsevier BV

Date: 03-2002

DOI: 10.1074/JBC.M110429200

Conformation Sensors that Distinguish Monomeric Proteins from Oligomers in Live Cells

Publisher: Elsevier BV

Date: 04-2010

DOI: 10.1016/J.CHEMBIOL.2010.03.011

Protein aggregation in cell biology: An aggregomics perspective of health and disease

Publisher: Elsevier BV

Date: 03-2020

DOI: 10.1016/J.SEMCDB.2018.05.003

Abstract: Maintaining protein homeostasis (proteostasis) is essential for cellular health and is governed by a network of quality control machinery comprising over 800 genes. When proteostasis becomes imbalanced, proteins can abnormally aggregate or become mislocalized. Inappropriate protein aggregation and proteostasis imbalance are two of the central pathological features of common neurodegenerative diseases including Alzheimer, Parkinson, Huntington, and motor neuron diseases. How aggregation contributes to the pathogenic mechanisms of disease remains incompletely understood. Here, we integrate some of the key and emerging ideas as to how protein aggregation relates to imbalanced proteostasis with an emphasis on Huntington disease as our area of main expertise. We propose the term "aggregomics" be coined in reference to how aggregation of particular proteins concomitantly influences the spatial organization and protein-protein interactions of the surrounding proteome. Meta-analysis of aggregated interactomes from various published datasets reveals chaperones and RNA-binding proteins are common components across various disease contexts. We conclude with an examination of therapeutic avenues targeting proteostasis mechanisms.

Putting huntingtin aggregation in view with windows into the cellular milieu

Publisher: Bentham Science Publishers Ltd.

Date: 03-2012

DOI: 10.2174/1568026611212220013

Abstract: Huntington's disease arises from CAG codon-repeat expansions in the Htt gene, which leads to a Htt gene product with an expanded polyglutamine (polyQ) sequence. The length of the polyQ expansion correlates with an increased tendency to form aggregates and clustering into micrometer-plus sized inclusion bodies in neurons and other cell types. Yet after nearly 20 years since the genetic basis for HD was identified, our knowledge of how polyQ-expanded Htt fragment aggregation relates to disease mechanisms remains fragmentary and controversial. Challenges remain in defining the aggregation process at the molecular level and how this process is influenced by, or influences cellular activities. Insight is further confounded by the term "aggregation" being used to describe a composite of distinct processes that may have opposing consequences to cell health and survival. This review discusses these issues in light of a historic summary of Htt aggregation in the cellular milieu and the intrinsic attributes of polyQ-expanded Htt that lead to aggregation. Finally, discussion centers on strategies forward to improve our knowledge for how aggregation relates to cellular dysfunction.

A Census of Hsp70-Mediated Proteome Solubility Changes upon Recovery from Heat Stress

Publisher: American Chemical Society (ACS)

Date: 07-04-2022

DOI: 10.1021/ACS.JPROTEOME.1C00920

Abstract: Eukaryotic cells respond to heat shock through several regulatory processes including upregulation of stress responsive chaperones and reversible shutdown of cellular activities through formation of protein assemblies. However, the underlying regulatory mechanisms of the recovery of these heat-induced protein assemblies remain largely elusive. Here, we measured the proteome abundance and solubility changes during recovery from heat shock in the mouse Neuro2a cell line. We found that prefoldins and translation machinery are rapidly down-regulated as the first step in the heat shock response. Analysis of proteome solubility reveals that a rapid mobilization of protein quality control machineries, along with changes in cellular energy metabolism, translational activity, and actin cytoskeleton are fundamental to the early stress responses. In contrast, longer term adaptation to stress involves renewal of core cellular components. Inhibition of the Hsp70 family, pivotal for the heat shock response, selectively and negatively affects the ribosomal machinery and delays the solubility recovery of many nuclear proteins. ProteomeXchange: PXD030069.

A thiol probe for measuring unfolded protein load and proteostasis in cells

Publisher: Springer Science and Business Media LLC

Date: 07-09-2017

DOI: 10.1038/S41467-017-00203-5

Abstract: When proteostasis becomes unbalanced, unfolded proteins can accumulate and aggregate. Here we report that the dye, tetraphenylethene maleimide (TPE-MI) can be used to measure cellular unfolded protein load. TPE-MI fluorescence is activated upon labelling free cysteine thiols, normally buried in the core of globular proteins that are exposed upon unfolding. Crucially TPE-MI does not become fluorescent when conjugated to soluble glutathione. We find that TPE-MI fluorescence is enhanced upon reaction with cellular proteomes under conditions promoting accumulation of unfolded proteins. TPE-MI reactivity can be used to track which proteins expose more cysteine residues under stress through proteomic analysis. We show that TPE-MI can report imbalances in proteostasis in induced pluripotent stem cell models of Huntington disease, as well as cells transfected with mutant Huntington exon 1 before the formation of visible aggregates. TPE-MI also detects protein damage following dihydroartemisinin treatment of the malaria parasites Plasmodium falciparum . TPE-MI therefore holds promise as a tool to probe proteostasis mechanisms in disease.

The structural basis for amyloid formation by plasma apolipoproteins: A review

Publisher: Springer Science and Business Media LLC

Date: 03-2002

DOI: 10.1007/S002490100172

Abstract: The formation of amyloid and other protein deposits in vivo is synonymous with many pathological conditions such as Alzheimer's disease, Creutzfeldt-Jakob disease and Parkinson's disease. Interestingly, many plasma apolipoproteins are also associated with amyloid deposits, including apolipoprotein (apo) A-I, apoA-II and apoE. Apolipoproteins share a number of structural and conformational properties, namely a large proportion of class A hipathic alpha-helices and limited conformational stability in the absence of lipid. Other proteins that form amyloid such as alpha-synuclein and serum amyloid A also contain hipathic alpha-helical domains similar to those found in apolipoproteins. In this review we develop a hypothesis to account for the widespread occurrence of apolipoproteins in amyloid deposits. We describe the conformational stability of human apoC-II and the stabilization of alpha-helical structure in the presence of phospholipid. We propose that lipid-free apoC-II forms partially folded intermediates prone to amyloid formation. Parameters that affect apolipoprotein lipid binding in vivo, such as protein and lipid oxidation or protein truncations and mutations, could promote apolipoprotein-related pathologies including those associated within amyloid deposits of atherosclerotic plaques.

VLDL lipolysis products increase VLDL fluidity and convert apolipoprotein E4 into a more expanded conformation

Publisher: Elsevier BV

Date: 06-2010

DOI: 10.1194/JLR.M000406

Diagnostics for Amyloid Fibril Formation: Where to Begin?

Publisher: Humana Press

Date: 2011

DOI: 10.1007/978-1-60327-223-0_8

Abstract: Twenty-five proteins are known to form amyloid fibrils in vivo in association with disease (Westermark et al., Amyloid 12:1-4, 2005). However, the fundamental ability of a protein to form amyloid-like fibrils is far more widespread than in just the proteins associated with disease, and indeed this property can provide insight into the basic thermodynamics of folding and misfolding pathways. But how does one determine whether a protein has formed amyloid-like fibrils? In this chapter, we cover the basic steps toward defining the amyloid-like properties of a protein and how to measure the kinetics of fibrillization. We describe several basic tests for aggregation and the binding to two classic amyloid-reactive dyes, Congo Red, and thioflavin T, which are key indicators to the presence of fibrils.

Hidden information on protein function in censuses of proteome foldedness

Publisher: Cold Spring Harbor Laboratory

Date: 25-02-2021

DOI: 10.1101/2021.02.24.432609

Abstract: Methods that assay protein foldedness with proteomics have generated censuses of protein folding stabilities in biological milieu. Surprisingly, different censuses poorly correlate with each other. Here, we show that methods targeting foldedness through monitoring amino acid sidechain reactivity also detect changes in conformation and ligand binding. About one quarter of cysteine or methionine sidechains in proteins in mammalian cell lysate increase in reactivity upon chemical denaturant titration consistent with two-state unfolding. Paradoxically, up to one third decreased reactivity, which were enriched in proteins with functions relating to unfolded protein stress. One protein, chaperone HSPA8, displayed changes arising from ligand and cofactor binding. Unmasking this hidden information should improve efforts to understand both folding and the remodeling of protein function directly in complex biological settings. We show that proteome folding stability censuses are ill-defined because they earmark hidden information on conformation and ligand binding.

KineticMSI, an R-based framework for relative quantification of spatial isotopic incorporation in mass spectrometry imaging experiments [PREPRINT OR COMPARABLE]

Publisher: Cold Spring Harbor Laboratory

Date: 03-09-2022

DOI: 10.1101/2022.08.31.505954

Abstract: Kinetic mass spectrometry imaging (kMSI) integrates imaging-MS with stable isotope labelling to elucidate metabolic fluxes in a spatiotemporal manner. kMSI studies are h ered by high volumes of complex data and a lack of computational workflows for data analysis that additionally address replicated experiments. To meet these challenges, we developed KineticMSI, an open-source R-based tool for processing and analyzing kMSI datasets. KineticMSI includes statistical tools to quantify tracer incorporation across replicated treatment groups spatially in tissues. It allows users to make data-driven decisions by elucidating affected pathways associated with changes in metabolic turnover. We demonstrate a validation of our method by identifying metabolic changes in the hippoc us of a transgenic Huntington’s disease (HD) mouse model as compared to wild-type mice. We discovered significant changes in metabolism of neuronal cell body lipids (phosphatidylinositol and cardiolipins) in HD mice, previously masked by conventional statistical approaches that compare mean tracer incorporation across brain regions.

Tadpole-like conformations of huntingtin exon 1 with expanded polyglutamine engenders novel interactions in cells

Publisher: Cold Spring Harbor Laboratory

Date: 22-08-2017

DOI: 10.1101/179663

Abstract: Soluble huntingtin exon 1 (Httex1) with expanded polyglutamine (polyQ) engenders neurotoxicity in Huntington’s disease. To uncover the physical basis of this toxicity, we performed structural studies of soluble Httex1 for wild type and mutant polyQ lengths. Nuclear magnetic resonance experiments show evidence for conformational rigidity across the polyQ region. In contrast, hydrogen-deuterium exchange shows absence of backbone amide protection, suggesting negligible persistence of hydrogen bonds. The seemingly conflicting results are explained by all-atom simulations, which show that Httex1 adopts tadpole-like structures with a globular head encompassing the N-terminal hipathic and polyQ regions and the tail encompassing the C-terminal proline-rich region. The surface area of the globular domain increases monotonically with polyQ length. This stimulates sharp increases in gain-of-function interactions in cells for expanded polyQ, and one of these interactions is with the stress-granule protein Fus. Our results highlight plausible connections between Httex1 structure and routes to neurotoxicity.

Erratum: Corrigendum: Identifying polyglutamine protein species in situ that best predict neurodegeneration

Publisher: Springer Science and Business Media LLC

Date: 15-02-2012

DOI: 10.1038/NCHEMBIO0312-318A

Modulation of Apolipoprotein E Structure by Domain Interaction

Publisher: Elsevier BV

Date: 10-2005

DOI: 10.1074/JBC.M506044200

Suppression of apolipoprotein C-II amyloid formation by the extracellular chaperone, clusterin

Publisher: Wiley

Date: 26-05-2002

DOI: 10.1046/J.1432-1033.2002.02957.X

Abstract: The effect of the extracellular chaperone, clusterin, on amyloid fibril formation by lipid-free human apolipoprotein C-II (apoC-II) was investigated. Sub-stoichiometric levels of clusterin, derived from either plasma or semen, potently inhibit amyloid formation by apoC-II. Inhibition is dependent on apoC-II concentration, with more effective inhibition by clusterin observed at lower concentrations of apoC-II. The average sedimentation coefficient of apoC-II fibrils formed from apoC-II (0.3 mg.mL-1) is reduced by coincubation with clusterin (10 microg x mL(-1)). In contrast, addition of clusterin (0.1 mg x mL(-1)) to preformed apoC-II amyloid fibrils (0.3 mg x mL(-1)) does not affect the size distribution after 2 days. This sedimentation velocity data suggests that clusterin inhibits fibril growth but does not promote fibril dissociation. Electron micrographs indicate similar morphologies for amyloid fibrils formed in the presence or absence of clusterin. The substoichiometric nature of the inhibition suggests that clusterin interacts with transient amyloid nuclei leading to dissociation of the monomeric subunits. We propose a general role for clusterin in suppressing the growth of extracellular amyloid.

Tandem Repeats in Genes, Proteins, and Disease

Publisher: Humana Press

Date: 2013

DOI: 10.1007/978-1-62703-438-8

Phospholipid Complexation and Association with Apolipoprotein C-II: Insights from Mass Spectrometry

Publisher: Elsevier BV

Date: 12-2003

DOI: 10.1016/S0006-3495(03)74795-X

Minimum Information about a Spinal Cord Injury Experiment: A Proposed Reporting Standard for Spinal Cord Injury Experiments

Publisher: Mary Ann Liebert Inc

Date: 08-2014

DOI: 10.1089/NEU.2014.3400

The Anti-Cancer IgM Monoclonal Antibody PAT-SM6 Binds with High Avidity to the Unfolded Protein Response Regulator GRP78

Publisher: Public Library of Science (PLoS)

Date: 19-09-2012

DOI: 10.1371/JOURNAL.PONE.0044927

Nascent mutant Huntingtin exon 1 chains do not stall on ribosomes during translation but aggregates do recruit machinery involved in ribosome quality control

Publisher: Cold Spring Harbor Laboratory

Date: 13-05-2020

DOI: 10.1101/2020.05.13.094060

Abstract: Mutations that cause Huntington’s Disease involve a polyglutamine (polyQ) sequence expansion beyond 35 repeats in exon 1 of Huntingtin. Intracellular inclusion bodies of mutant Huntingtin protein are a key feature of Huntington’s disease brain pathology. We previously showed that in cell culture the formation of inclusions involved the assembly of disordered structures of mHtt exon 1 fragments (Httex1) and they were enriched with translational machinery when first formed. We hypothesized that nascent mutant Httex1 chains co-aggregate during translation by phase separation into liquid-like disordered aggregates and then convert to more rigid, amyloid structures. Here we further examined the mechanisms of inclusion assembly in a human epithelial kidney (AD293) cell culture model and examined whether ribosome quality control machinery previously implicated in stalled ribosomes were involved. We found mHttex1 did not appear to stall translation of its own nascent chain and there was no recruitment of RNA into inclusions. However, proteins involved in translation or ribosome quality control were co-recruited into the inclusions (Ltn1 and Rack1) compared to a protein not anticipated to be involved (NACAD). Furthermore, we observed co-aggregation with other proteins previously identified in inclusions, including Upf-1 and chaperone-like proteins Sgta and Hspb1, which also suppressed aggregation at high co-expression levels. The newly formed inclusions contained immobile mHttex1 molecules which points to the disordered aggregates being mechanically rigid prior to amyloid formation.

NMR Structure of Human Apolipoprotein C-II in the Presence of Sodium Dodecyl Sulfate

Publisher: American Chemical Society (ACS)

Date: 10-04-2001

DOI: 10.1021/BI002821M

Abstract: The structure and protein-detergent interactions of apolipoprotein C-II (apoC-II) in the presence of SDS micelles have been investigated using circular dichroism and heteronuclear NMR techniques applied to (15)N-labeled protein. Micellar SDS, a commonly used mimetic of the lipoprotein surface, inhibits the aggregation of apoC-II and induces a stable structure containing approximately 60% alpha-helix as determined by circular dichroism. NMR reveals the first 12 residues of apoC-II to be structurally heterogeneous and largely disordered, with the rest of the protein forming a predominantly helical structure. Three regions of helical conformation, residues 16-36, 50-56, and 63-77, are well-defined by NMR-derived constraints, with the intervening regions showing more loosely defined helical conformation. The structure of apoC-II is compared to that determined for other apolipoproteins in a similar environment. Our results shed light on the lipid interactions of apoC-II and its mechanism of lipoprotein lipase activation.

SOD1 protein aggregates stimulate macropinocytosis in neurons to facilitate their propagation

Publisher: Springer Science and Business Media LLC

Date: 31-10-2015

DOI: 10.1186/S13024-015-0053-4

Apolipoprotein C-II39-62 activates lipoprotein lipase by direct lipid-independent binding

Publisher: American Chemical Society (ACS)

Date: 03-2000

DOI: 10.1021/BI992523T

Abstract: Apolipoprotein C-II (apoC-II) is an exchangeable plasma apolipoprotein and an endogenous activator of lipoprotein lipase (LpL). Genetic deficiencies of apoC-II and overexpression of apoC-II in transgenic mice are both associated with severe hyperlipidemia, indicating a complex role for apoC-II in the regulation of blood lipid levels. ApoC-II exerts no effect on the activity of LpL for soluble substrates, suggesting that activation occurs via the formation of a lipid-bound complex. We have synthesized a peptide corresponding to amino acid residues 39-62 of mature human apoC-II. This peptide does not bind to model lipid surfaces but retains the ability to activate LpL. Conjugation of the fluorophore 7-nitrobenz-2-oxa-1,3-diazole (NBD) to the N-terminal alpha-amino group of apoC-II39-62 facilitated determination of the affinity of the peptide for LpL using fluorescence anisotropy measurements. The dissociation constant describing this interaction was 0.23 microM, and was unchanged when LpL was lipid-bound. Competitive binding studies showed that apoC-II39-62 and full-length apoC-II exhibited the same affinity for LpL in aqueous solution, whereas the affinity for full-length apoC-II was increased at least 1 order of magnitude in the presence of lipid. We suggest that while the binding of apoC-II to the lipid surface promotes the formation of a high-affinity complex of apoC-II and LpL, activation occurs via direct helix-helix interactions between apoC-II39-62 and the loop covering the active site of LpL.

Arginine in C9ORF72 Dipolypeptides Mediates Promiscuous Proteome Binding and Multiple Modes of Toxicity

Publisher: Elsevier BV

Date: 04-2020

DOI: 10.1074/MCP.RA119.001888

Misfolded Polyglutamine, Polyalanine, and Superoxide Dismutase 1 Aggregate via Distinct Pathways in the Cell

Publisher: Elsevier BV

Date: 03-2014

DOI: 10.1074/JBC.M113.520189

The 2021 FASEB Virtual Science Research Conference on Protein Aggregation: Function, Dysfunction, and Disease, June 23–25, 2021

Publisher: Wiley

Date: 31-08-2021

DOI: 10.1096/FJ.202101291

Arginine-rich C9ORF72 ALS proteins stall ribosomes in a manner distinct from a canonical ribosome-associated quality control substrate

Publisher: Elsevier BV

Date: 2023

DOI: 10.1016/J.JBC.2022.102774

Conformational detection of prion protein with biarsenical labeling and FlAsH fluorescence

Publisher: Elsevier BV

Date: 03-2009

DOI: 10.1016/J.BBRC.2009.01.120

Abstract: Prion diseases are associated with the misfolding of the host-encoded cellular prion protein (PrP(C)) into a disease associated form (PrP(Sc)). Recombinant PrP can be refolded into either an alpha-helical rich conformation (alpha-PrP) resembling PrP(C) or a beta-sheet rich, protease resistant form similar to PrP(Sc). Here, we generated tetracysteine tagged recombinant PrP, folded this into alpha- or beta-PrP and determined the levels of FlAsH fluorescence. Insertion of the tetracysteine tag at three different sites within the 91-111 epitope readily distinguished beta-PrP from alpha-PrP upon FlAsH labeling. Labelling of tetracysteine tagged PrP in the alpha-helical form showed minimal fluorescence, whereas labeling of tagged PrP in the beta-sheet form showed high fluorescence indicating that this region is exposed upon conversion. This highlights a region of PrP that can be implicated in the development of diagnostics and is a novel, protease free mechanism for distinguishing PrP(Sc) from PrP(C). This technique may also be applied to any protein that undergoes conformational change and/or misfolding such as those involved in other neurodegenerative disorders including Alzheimer's, Huntington's and Parkinson's diseases.

Sub-micellar phospholipid accelerates amyloid formation by apolipoprotein C-II

Publisher: Wiley

Date: 10-04-2001

DOI: 10.1016/S0014-5793(01)02355-9

Abstract: Lipid-free human apolipoprotein C-II (apoC-II) forms amyloid fibrils with characteristic beta-structure. This conformation is distinct from the alpha-helical fold of lipid-bound apoC-II. We have investigated the effect of the short-chain phospholipid, dihexanoylphosphatidylcholine (DHPC) on amyloid formation by apoC-II. The alpha-helical content of apoC-II increases in the presence of micellar DHPC (16 mM) and amyloid formation is inhibited. However, at sub-micellar DHPC concentrations (below 8 mM) amyloid formation is accelerated 6 fold. These results suggest that in idual phospholipid molecules in vivo may exert significant effects on amyloid folding pathways.

Conformational dynamics and self-association of intrinsically disordered Huntingtin exon 1 in cells

Publisher: Royal Society of Chemistry (RSC)

Date: 2017

DOI: 10.1039/C6CP08167C

Abstract: In-cell temperature jump experiments induce monomer collapse, misfolding and self-association of the Huntingtin exon 1 protein.

Tracking protein aggregation and mislocalization in cells with flow cytometry.

Publisher: Springer Science and Business Media LLC

Date: 18-03-2012

DOI: 10.1038/NMETH.1930

Abstract: We applied pulse-shape analysis (PulSA) to monitor protein localization changes in mammalian cells by flow cytometry. PulSA enabled high-throughput tracking of protein aggregation, translocation from the cytoplasm to the nucleus and trafficking from the plasma membrane to the Golgi as well as stress-granule formation. Combining PulSA with tetracysteine-based oligomer sensors in a cell model of Huntington's disease enabled further separation of cells enriched with monomers, oligomers and inclusion bodies.

Human-specific gene ARHGAP11B promotes basal progenitor amplification and neocortex expansion

Publisher: American Association for the Advancement of Science (AAAS)

Date: 27-03-2015

DOI: 10.1126/SCIENCE.AAA1975

Abstract: Humans are much smarter than mice—key to this is the relative thickness of the human brain's neocortex. Florio et al. combed through genes expressed in the progenitor cells that build the neocortex and zeroed in on one gene found in humans but not in mice. The gene, which seems to differentiate humans from chimpanzees, drives proliferation of the key progenitor cells. Mice expressing this human gene during development built more elaborate brains. Science , this issue p. 1465

Pulse Shape Analysis (PulSA) to Track Protein Translocalization in Cells by Flow Cytometry: Applications for Polyglutamine Aggregation

Publisher: Humana Press

Date: 2013

DOI: 10.1007/978-1-62703-438-8_6

Abstract: Pulse shape analysis (PulSA) is a flow cytometry-based method that can be used to study protein localization patterns in cells. Ex les for its use include tracking the formation of inclusion bodies of polyglutamine-expanded proteins and other aggregating proteins. The method can also be used for phenomena relating to protein movements in cells such as translocation from the cytoplasm to the nucleus, trafficking from the plasma membrane to the Golgi, and stress granule formation. An attractive feature is its capacity to quantify these parameters in whole-cell populations very quickly and in high throughput. We describe the basic experimental details for performing PulSA using expression of GFP-tagged proteins, endogenous proteins labelled immunofluorescently, and organelle dyes.

Simultaneous Binding of the Anti-Cancer IgM Monoclonal Antibody PAT-SM6 to Low Density Lipoproteins and GRP78

Publisher: Public Library of Science (PLoS)

Date: 19-04-2013

DOI: 10.1371/JOURNAL.PONE.0061239

Size Analysis of Polyglutamine Protein Aggregates Using Fluorescence Detection in an Analytical Ultracentrifuge

Publisher: Humana Press

Date: 2013

DOI: 10.1007/978-1-62703-438-8_4

Abstract: Defining the aggregation process of proteins formed by poly-amino acid repeats in cells remains a challenging task due to a lack of robust techniques for their isolation and quantitation. Sedimentation velocity methodology using fluorescence detected analytical ultracentrifugation is one approach that can offer significant insight into aggregation formation and kinetics. While this technique has traditionally been used with purified proteins, it is now possible for substantial information to be collected with studies using cell lysates expressing a GFP-tagged protein of interest. In this chapter, we describe protocols for s le preparation and setting up the fluorescence detection system in an analytical ultracentrifuge to perform sedimentation velocity experiments on cell lysates containing aggregates formed by poly-amino acid repeat proteins.

Cultural Influence on Nurses’ Pain Observations Related to Dementia: An Integrative Review

Publisher: Elsevier BV

Date: 08-2023

DOI: 10.1016/J.PMN.2023.01.006

Insight on the Molecular Envelope of Lipid-Bound Apolipoprotein E from Electron Paramagnetic Resonance Spectroscopy

Publisher: Elsevier BV

Date: 02-2009

DOI: 10.1016/J.JMB.2008.12.040

Arginine valency inC9ORF72dipolypeptides mediates promiscuous proteome binding that stalls ribosomes, disable actin cytoskeleton assembly and impairs arginine methylation of endogenous proteins

Publisher: Cold Spring Harbor Laboratory

Date: 28-08-2019

DOI: 10.1101/749127

Abstract: C9ORF72 -associated Motor Neuron Disease patients feature abnormal expression of 5 dipeptide repeat (DPR) polymers. Here we used quantitative proteomics in a Neuro2a cell model to demonstrate that the valency of Arg in the most toxic DPRS, PR and GR, drives promiscuous binding to the proteome, compared to a relative sparse binding of the more inert AP and GA. Notable targets included ribosomal proteins, translation initiation factors and translation elongation factors. PR and GR comprising more than 10 repeats robustly stalled the ribosome suggesting high-valency Arg electrostatically jams the ribosome exit tunnel during synthesis. Poly-GR also bound to arginine methylases and induced hypomethylation of endogenous proteins, with a profound destabilization of the actin cytoskeleton. Our findings point to arginine in GR and PR polymers as multivalent toxins to translation as well as arginine methylation with concomitant downstream effects on widespread biological processes including ribosome biogenesis, mRNA splicing and cytoskeleton assembly. The major genetic cause of MND are mutations in an intron of the C9ORF72 gene that lead to the expansion in the length of a hexanucleotide repeat sequence, and subsequent non-AUG mediated translation of the intron into 5 different DPRs. The two DPRs containing Arg are potently toxic in animal and cell models. Our research shows that the valency of Arg mediates widespread proteome binding especially affecting machinery involved in Arg-methylation, cytoskeleton and translation. We suggest the mechanisms for toxicity are multipronged and involve electrostatic jamming of ribosomes during translation, acting as substrate mimetics for arginine methylase activity that renders the endogenous proteome hypomethylated and impairing actin cytoskeleton assembly. These mechanisms explain pathologic signatures previous reported in human brain pathology.

Polyglutamine Aggregation in Huntington and Related Diseases

Publisher: Springer New York

Date: 2012

DOI: 10.1007/978-1-4614-5434-2_8

Abstract: Polyglutamine (polyQ)-expansions in different proteins cause nine neurodegenerative diseases. While polyQ aggregation is a key pathological hallmark of these diseases, how aggregation relates to pathogenesis remains contentious. In this chapter, we review what is known about the aggregation process and how cells respond and interact with the polyQ-expanded proteins. We cover detailed biophysical and structural studies to uncover the intrinsic features of polyQ aggregates and concomitant effects in the cellular environment. We also examine the functional consequences ofpolyQ aggregation and how cells may attempt to intervene and guide the aggregation process.

Nascent mutant Huntingtin exon 1 chains do not stall on ribosomes during translation but aggregates do recruit machinery involved in ribosome quality control and RNA

Publisher: Public Library of Science (PLoS)

Date: 31-07-2020

DOI: 10.1371/JOURNAL.PONE.0233583

Apolipoprotein E4 Forms a Molten Globule

Publisher: Elsevier BV

Date: 12-2002

DOI: 10.1074/JBC.M204898200

Crystal structure of the TIR domain from the flax disease resistance protein L6

Publisher: Worldwide Protein Data Bank

Date: 27-04-2011

DOI: 10.2210/PDB3OZI/PDB

The molecular chaperone, alpha-crystallin, inhibits amyloid formation by apolipoprotein C-II

Publisher: Elsevier BV

Date: 09-2001

DOI: 10.1074/JBC.M105285200

A biosensor to gauge protein homeostasis resilience differences in the nucleus compared to cytosol of mammalian cells

Publisher: Cold Spring Harbor Laboratory

Date: 19-04-2021

DOI: 10.1101/2021.04.19.440383

Abstract: An extensive network of chaperones and other proteins maintain protein homeostasis and guard against inappropriate protein aggregation that is a hallmark of neurodegenerative diseases. Using a fluorescence resonance energy-based biosensor that simultaneously reports on intact cellular chaperone holdase activity and detrimental aggregation propensity, we investigated the buffering capacity of the systems managing protein homeostasis in the nucleus of the human cell line HEK293 compared to the cytosol. We found that the nucleus showed lower net holdase activity and reduced capacity to suppress protein aggregation, suggesting that the nuclear quality control resources are less effective compared to those in the cytosol. Aggregation of mutant huntingtin exon 1 protein (Httex1) in the cytosol appeared to deplete cytosolic chaperone supply by depleting holdase activity. Unexpectedly, the same stress increased holdase activity in the nucleus suggesting that proteostasis stress can trigger a rebalance of chaperone supply in different subcellular compartments. Collectively the findings suggest the cytosol has more capacity to manage imbalances in proteome foldedness than the nucleus, but chaperone supply can be redirected into the nucleus under conditions of proteostasis stress caused by cytosolic protein aggregation.

A Biosensor of Src Family Kinase Conformation by Exposable Tetracysteine Useful for Cell-Based Screening

Publisher: American Chemical Society (ACS)

Date: 15-05-2014

DOI: 10.1021/CB500242Q

Abstract: We developed a new approach to distinguish distinct protein conformations in live cells. The method, exposable tetracysteine (XTC), involved placing an engineered tetracysteine motif into a target protein that has conditional access to biarsenical dye binding by conformational state. XTC was used to distinguish open and closed regulatory conformations of Src family kinases. Substituting just four residues with cysteines in the conserved SH2 domain of three Src-family kinases (c-Src, Lck, Lyn) enabled open and closed conformations to be monitored on the basis of binding differences to biarsenical dyes FlAsH or ReAsH. Fusion of the kinases with a fluorescent protein tracked the kinase presence, and the XTC approach enabled simultaneous assessment of regulatory state. The c-Src XTC biosensor was applied in a boutique screen of kinase inhibitors, which revealed six compounds to induce conformational closure. The XTC approach demonstrates new potential for assays targeting conformational changes in key proteins in disease and biology.

Fibrillar Amyloid Protein Present in Atheroma Activates CD36 Signal Transduction

Publisher: Elsevier BV

Date: 03-2004

DOI: 10.1074/JBC.M311735200

Identifying polyglutamine protein species in situ that best predict neurodegeneration

Publisher: Springer Science and Business Media LLC

Date: 30-10-2011

DOI: 10.1038/NCHEMBIO.694

Walking the tightrope: Proteostasis and neurodegenerative disease

Publisher: Wiley

Date: 08-03-2016

DOI: 10.1111/JNC.13575

Abstract: A characteristic of many neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS), is the aggregation of specific proteins into protein inclusions and/or plaques in degenerating brains. While much of the aggregated protein consists of disease specific proteins, such as amyloid-β, α-synuclein, or superoxide dismutase1 (SOD1), many other proteins are known to aggregate in these disorders. Although the role of protein aggregates in the pathogenesis of neurodegenerative diseases remains unknown, the ubiquitous association of misfolded and aggregated proteins indicates that significant dysfunction in protein homeostasis (proteostasis) occurs in these diseases. Proteostasis is the concept that the integrity of the proteome is in fine balance and requires proteins in a specific conformation, concentration, and location to be functional. In this review, we discuss the role of specific mechanisms, both inside and outside cells, which maintain proteostasis, including molecular chaperones, protein degradation pathways, and the active formation of inclusions, in neurodegenerative diseases associated with protein aggregation. A characteristic of many neurodegenerative diseases is the aggregation of specific proteins, which alone provides strong evidence that protein homeostasis is disrupted in these disease states. Proteostasis is the maintenance of the proteome in the correct conformation, concentration, and location by functional pathways such as molecular chaperones and protein degradation machinery. Here, we discuss the potential roles of quality control pathways, both inside and outside cells, in the loss of proteostasis during aging and disease.

Disease-Associated Polyglutamine Stretches in Monomeric Huntingtin Adopt a Compact Structure

Publisher: Elsevier BV

Date: 08-2012

DOI: 10.1016/J.JMB.2012.01.034

Sedimentation velocity analysis of amyloid oligomers and fibrils using fluorescence detection

Publisher: Elsevier BV

Date: 05-2011

DOI: 10.1016/J.YMETH.2010.10.004

Abstract: The assembly of proteins into large fibrillar aggregates, known as amyloid fibrils, is associated with a number of common and debilitating diseases. In some cases, proteins deposit extracellularly, while in others the aggregation is intracellular. A common feature of these diseases is the presence of aggregates of different sizes, including mature fibrils, small oligomeric precursors, and other less well understood structural forms such as amorphous aggregates. These various species possess distinct biochemical, biophysical, and pathological properties. Here, we detail a number of techniques that can be employed to examine amyloid fibrils and oligomers using a fluorescence-detection system (FDS) coupled with the analytical ultracentrifuge. Sedimentation velocity analysis using fluorescence detection is a particularly useful method for resolving the complex heterogeneity present in amyloid systems and can be used to characterize aggregation in exceptional detail. Furthermore, the fluorescence detection module provides a number of particularly attractive features for the analysis of aggregating proteins. It expands the practical range of concentrations of aggregating proteins under study, which is useful for greater insight into the aggregation process. It also enables the assessment of aggregation behavior in complex biological solutions, such as cell lysates, and the assessment of processes that regulate in-cell or extracellular aggregation kinetics. Four methods of fluorescent detection that are compatible with the current generation of FDS instrumentation are described: (1) Detection of soluble amyloid fibrils using a covalently bound fluorophore. (2) Detection of amyloid fibrils using an extrinsic dye that emits fluorescence when bound to fibrils. (3) Detection of fluorescently-labeled lipids and their interaction with oligomeric amyloid intermediates. (4) Detection of green fluorescence protein (GFP) constructs and their interactions within mammalian cell lysates.

Sedimentation Velocity Analysis of Flexible Macromolecules: Self-Association and Tangling of Amyloid Fibrils

Publisher: Elsevier BV

Date: 04-2003

DOI: 10.1016/S0006-3495(03)75061-9

SerpinB2 (PAI-2) Modulates Proteostasis via Binding Misfolded Proteins and Promotion of Cytoprotective Inclusion Formation.

Publisher: Public Library of Science (PLoS)

Date: 17-06-2015

DOI: 10.1371/JOURNAL.PONE.0130136

Huntingtin Inclusions Trigger Cellular Quiescence, Deactivate Apoptosis, and Lead to Delayed Necrosis

Publisher: Elsevier BV

Date: 05-2017

DOI: 10.1016/J.CELREP.2017.04.029

Abstract: Competing models exist in the literature for the relationship between mutant Huntingtin exon 1 (Httex1) inclusion formation and toxicity. In one, inclusions are adaptive by sequestering the proteotoxicity of soluble Httex1. In the other, inclusions compromise cellular activity as a result of proteome co-aggregation. Using a biosensor of Httex1 conformation in mammalian cell models, we discovered a mechanism that reconciles these competing models. Newly formed inclusions were composed of disordered Httex1 and ribonucleoproteins. As inclusions matured, Httex1 reconfigured into amyloid, and other glutamine-rich and prion domain-containing proteins were recruited. Soluble Httex1 caused a hyperpolarized mitochondrial membrane potential, increased reactive oxygen species, and promoted apoptosis. Inclusion formation triggered a collapsed mitochondrial potential, cellular quiescence, and deactivated apoptosis. We propose a revised model where sequestration of soluble Httex1 inclusions can remove the trigger for apoptosis but also co-aggregate other proteins, which curtails cellular metabolism and leads to a slow death by necrosis.

Illuminating chaperone activity

Publisher: Springer Science and Business Media LLC

Date: 22-03-2017

DOI: 10.1038/NCHEMBIO.2332

Tyrosine 416 Is Phosphorylated in the Closed, Repressed Conformation of c-Src

Publisher: Public Library of Science (PLoS)

Date: 26-07-2013

DOI: 10.1371/JOURNAL.PONE.0071035

Distinct partitioning of ALS associated TDP-43, FUS and SOD1 mutants into cellular inclusions

Publisher: Springer Science and Business Media LLC

Date: 21-08-2015

DOI: 10.1038/SREP13416

Abstract: Amyotrophic lateral sclerosis is a rapidly progressing neurodegenerative disease associated with protein misfolding and aggregation. Most cases are characterized by TDP-43 positive inclusions, while a minority of familial ALS cases are instead FUS and SOD1 positive respectively. Cells can generate inclusions of variable type including previously characterized aggresomes, IPOD or JUNQ structures depending on the misfolded protein. SOD1 invariably forms JUNQ inclusions but it remains unclear whether other ALS protein aggregates arise as one of these previously described inclusion types or form unique structures. Here we show that FUS variably partitioned to IPOD, JUNQ or alternate structures, contain a mobile fraction, were not microtubule dependent and initially did not contain ubiquitin. TDP-43 inclusions formed in a microtubule independent manner, did not contain a mobile fraction but variably colocalized to JUNQ inclusions and another alternate structure. We conclude that the RNA binding proteins TDP-43 and FUS do not consistently fit the currently characterised inclusion models suggesting that cells have a larger repertoire for generating inclusions than currently thought and imply that toxicity in ALS does not stem from a particular aggregation process or aggregate structure.

The allosteric mechanism induced by protein kinase A (PKA) phosphorylation of dematin (band 4.9).

Publisher: Elsevier BV

Date: 07-2015

DOI: 10.1074/JBC.A112.438861

Protein painting reveals pervasive remodeling of conserved proteostasis machinery in response to pharmacological stimuli

Publisher: Springer Science and Business Media LLC

Date: 28-11-2022

DOI: 10.1038/S41540-022-00256-3

Abstract: The correct spatio-temporal organization of the proteome is essential for cellular homeostasis. However, a detailed mechanistic understanding of this organization and how it is altered in response to external stimuli in the intact cellular environment is as-yet unrealized. ‘Protein painting methods provide a means to address this gap in knowledge by monitoring the conformational status of proteins within cells at the proteome-wide scale. Here, we demonstrate the ability of a protein painting method employing tetraphenylethene maleimide (TPE-MI) to reveal proteome network remodeling in whole cells in response to a cohort of commonly used pharmacological stimuli of varying specificity. We report specific, albeit heterogeneous, responses to in idual stimuli that coalesce on a conserved set of core cellular machineries. This work expands our understanding of proteome conformational remodeling in response to cellular stimuli, and provides a blueprint for assessing how these conformational changes may contribute to disorders characterized by proteostasis imbalance.

The Asian Biophysics Association—supporting biophysics in the greater Asia region

Publisher: Springer Science and Business Media LLC

Date: 17-05-2019

DOI: 10.1007/S12551-019-00552-Z

Tadpole-like conformations of huntingtin exon 1 are characterized by conformational heterogeneity that persists regardless of polyglutamine length.

Publisher: Elsevier BV

Date: 05-2018

DOI: 10.1016/J.JMB.2018.03.031

Decision letter: A first order phase transition mechanism underlies protein aggregation in mammalian cells

Publisher: eLife Sciences Publications, Ltd

Date: 29-08-2018

DOI: 10.7554/ELIFE.39695.018

Structural insights into the multifunctionality of rabies virus P3 protein

Publisher: Proceedings of the National Academy of Sciences

Date: 29-03-2023

DOI: 10.1073/PNAS.2217066120

Abstract: Viruses form extensive interfaces with host proteins to modulate the biology of the infected cell, frequently via multifunctional viral proteins. These proteins are conventionally considered as assemblies of independent functional modules, where the presence or absence of modules determines the overall composite phenotype. However, this model cannot account for functions observed in specific viral proteins. For ex le, rabies virus (RABV) P3 protein is a truncated form of the pathogenicity factor P protein, but displays a unique phenotype with functions not seen in longer isoforms, indicating that changes beyond the simple complement of functional modules define the functions of P3. Here, we report structural and cellular analyses of P3 derived from the pathogenic RABV strain Nishigahara (Nish) and an attenuated derivative strain (Ni-CE). We identify a network of intraprotomer interactions involving the globular C-terminal domain and intrinsically disordered regions (IDRs) of the N-terminal region that characterize the fully functional Nish P3 to fluctuate between open and closed states, whereas the defective Ni-CE P3 is predominantly open. This conformational difference appears to be due to the single mutation N226H in Ni-CE P3. We find that Nish P3, but not Ni-CE or N226H P3, undergoes liquid–liquid phase separation and this property correlates with the capacity of P3 to interact with different cellular membrane-less organelles, including those associated with immune evasion and pathogenesis. Our analyses propose that discrete functions of a critical multifunctional viral protein depend on the conformational arrangements of distant in idual domains and IDRs, in addition to their independent functions.

Amino-terminal Domain Stability Mediates Apolipoprotein E Aggregation into Neurotoxic Fibrils

Publisher: Elsevier BV

Date: 09-2006

DOI: 10.1016/J.JMB.2006.06.080

Abstract: The three isoforms of apolipoprotein (apo) E are strongly associated with different risks for Alzheimer's disease: apoE4>apoE3>apoE2. Here, we show at physiological salt concentrations and pH that native tetramers of apoE form soluble aggregates in vitro that bind the amyloid dyes thioflavin T and Congo red. However, unlike classic amyloid fibrils, the aggregates adopt an irregular protofilament-like morphology and are seemingly highly alpha-helical. The aggregates formed at substantially different rates (apoE4>apoE3>apoE2) and were significantly more toxic to cultured neuronal cells than the tetramer. Since the three isoforms have large differences in conformational stability that can influence aggregation and amyloid pathways, we tested the effects of mutations that increased or decreased stability. Decreasing the conformational stability of the amino-terminal domain of apoE increased aggregation rates and vice versa. Our findings provide a new perspective for an isoform-specific pathogenic role for apoE aggregation in which differences in the conformational stability of the amino-terminal domain mediate neurodegeneration.

Protein painting reveals pervasive remodeling of conserved proteostasis machinery in response to pharmacological stimuli

Publisher: Cold Spring Harbor Laboratory

Date: 15-05-2022

DOI: 10.1101/2022.05.14.491969

Abstract: Accurate spatio-temporal organization of the proteome is essential for cellular homeostasis. However, a detailed mechanistic understanding of this organization and how it is altered in response to external stimuli in the intact cellular environment is as-yet unrealized. To address this need, ‘protein painting’ methods have emerged as a way to gain insight into the conformational status of proteins within cells at the proteome-wide scale. For ex le, tetraphenylethene maleimide (TPE-MI) has previously been used to quantify the engagement of quality control machinery with client proteins in cell lysates. Here, we showcase the ability of TPE-MI to additionally reveal proteome network remodeling in whole cells in response to a cohort of commonly used pharmacological stimuli of varying specificity. We report specific, albeit heterogeneous, responses to in idual stimuli that coalesce on a conserved set of core cellular machineries. This work expands our understanding of proteome conformational remodeling in response to cellular stimuli, and provides a blueprint for assessing how these conformational changes may contribute to disorders characterized by proteostasis imbalance.

Modest Declines in Proteome Quality Impair Hematopoietic Stem Cell Self-Renewal

Publisher: Elsevier BV

Date: 2020

DOI: 10.1016/J.CELREP.2019.12.003

The Circularization of Amyloid Fibrils Formed by Apolipoprotein C-II

Publisher: Elsevier BV

Date: 12-2003

DOI: 10.1016/S0006-3495(03)74812-7

AMP-Activated Protein Kinase β-Subunit Requires Internal Motion for Optimal Carbohydrate Binding

Publisher: Elsevier BV

Date: 2012

DOI: 10.1016/J.BPJ.2011.12.012

Sequence grammar underlying unfolding and phase separation of globular proteins

Publisher: Cold Spring Harbor Laboratory

Date: 20-08-2021

DOI: 10.1101/2021.08.20.457073

Abstract: Aberrant phase separation of globular proteins is associated with many diseases. Here, we use a model protein system to understand how unfolded states of globular proteins drive phase separation and the formation of unfolded protein deposits (UPODs). For UPODs to form, the concentrations of unfolded molecules must be above a threshold value. Additionally, unfolded molecules must possess appropriate sequence grammars to drive phase separation. While UPODs recruit molecular chaperones, their compositional profiles are also influenced by synergistic physicochemical interactions governed by the sequence grammars of unfolded proteins and sequence features of cellular proteins. Overall, we find that the driving forces for phase separation and the compositional profiles of UPODs are governed by the sequence grammar of unfolded proteins. Our studies highlight the need for uncovering the sequence grammars of unfolded proteins that drive UPOD formation and lead to gain-of-function interactions whereby proteins are aberrantly recruited into UPODs. Unfolded states of globular proteins phase separate to form UPODs in cells The fraction of unfolded molecules and the sticker grammar govern phase separation Hydrophobic residues act as stickers that engage in intermolecular interactions Sticker grammar also influences gain-of-function recruitment into aberrant UPODs

Variability of Extreme Hydro-Climate Parameters in the North-Eastern Region of United Arab Emirates

Publisher: Elsevier BV

Date: 2016

DOI: 10.1016/J.PROENG.2016.07.563

Widespread remodelling of proteome solubility in response to different protein homeostasis stresses

Publisher: Cold Spring Harbor Laboratory

Date: 03-07-2019

DOI: 10.1101/692103

Abstract: The accumulation of protein deposits in neurodegenerative diseases involves the presence of a metastable subproteome vulnerable to aggregation. To investigate this subproteome and the mechanisms that regulates it, we measured the proteome solubility of the Neuro2a cell line under protein homeostasis stresses induced by Huntington Disease proteotoxicity Hsp70, Hsp90, proteasome and ER-mediated folding inhibition and oxidative stress. We found one-quarter of the proteome extensively changed solubility. Remarkably, almost all the increases in insolubility were counteracted by increases in solubility of other proteins. Each stress directed a highly specific pattern of change, which reflected the remodelling of protein complexes involved in adaptation to perturbation, most notably stress granule proteins, which responded differently to different stresses. These results indicate that the robustness of protein homeostasis relies on the absence of proteins highly vulnerable to aggregation and on large changes in aggregation state of regulatory mechanisms that restore protein solubility upon specific perturbations.

Structural and functional analysis of a plant resistance protein TIR domain reveals interfaces for self-association, signaling, and autoregulation.

Publisher: Elsevier BV

Date: 03-2011

DOI: 10.1016/J.CHOM.2011.02.009

University Of Helsinki

Location: Finland

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Griffith University

Location: Australia

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University Of Ruhuna

Location: Sri Lanka

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University Of Sri Jayewardenepura

Location: Sri Lanka

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Gladstone Institutes

Location: United States of America

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University Of Melbourne

Location: Australia

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Biophysical Identification Of Natural Human Antibody Targets

Start Date: 2010

End Date: 2012

Funder: Australian Research Council

Pathogenic And Adaptive Molecular Interactions With Mutant Huntingtin Exon 1

Start Date: 2016

End Date: 2019

Funder: National Health and Medical Research Council

Single Molecule Imaging Laboratory

Start Date: 2015

End Date: 2015

Funder: Australian Research Council

Surveillance Of The Mechanisms Controlling Proteome Foldedness

Start Date: 2017

End Date: 2019

Funder: Australian Research Council

Defining The Capacity Of Cells To Keep The Proteome Folded Over Space And Time

Start Date: 2017

End Date: 2020

Funder: International Human Frontier Science Program Organization

Proteostasis Mechanics Of Neurodegenerative Diseases

Start Date: 2019

End Date: 2023

Funder: National Health and Medical Research Council

How The Organization Of The Cell Membrane Determines T Lymphocytes Activation

Start Date: 2014

End Date: 2017

Funder: National Health and Medical Research Council

Altered Nuclear Trafficking And Nuclear Body Dynamics As Drivers Of Ataxin-1 Toxicity

Start Date: 2017

End Date: 2019

Funder: National Health and Medical Research Council

Discovery Projects - Grant ID: DP120102763

Start Date: 2012

End Date: 12-2014

Amount: $345,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP100100392

Start Date: 02-2010

End Date: 02-2013

Amount: $300,000.00

Funder: Australian Research Council

ARC Future Fellowships - Grant ID: FT120100039

Start Date: 12-2012

End Date: 12-2016

Amount: $707,628.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP170103093

Start Date: 2017

End Date: 12-2020

Amount: $466,500.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP230101050

Start Date: 2023

End Date: 12-2025

Amount: $543,594.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE150100163

Start Date: 2015

End Date: 12-2016

Amount: $560,000.00

Funder: Australian Research Council