ORCID Profile
0000-0001-7574-0044
Current Organisation
University of Wollongong
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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 | Biochemistry and Cell Biology not elsewhere classified | Structural Biology (incl. Macromolecular Modelling) | Protein Trafficking | Organic Chemistry | Characterisation of Biological Macromolecules | Analytical Spectrometry | Organic Chemical Synthesis | Nanochemistry and Supramolecular Chemistry | Structural Chemistry and Spectroscopy | Receptors and Membrane Biology | Biophysics | Enzymes | Animal Cell and Molecular Biology | Analytical Biochemistry | Cellular Interactions (Incl. Adhesion, Matrix, Cell Wall) | Animal Physiology—Cell | Medical Biochemistry: Proteins and Peptides (incl. Medical Proteomics) | Conservation And Biodiversity | Analytical Biochemistry | Cell Development, Proliferation and Death
ANZSRC Socio-Economic Objective (SEO)
Expanding Knowledge in the Biological Sciences | Neurodegenerative Disorders Related to Ageing | Biological sciences | Expanding Knowledge in the Medical and Health Sciences | Expanding Knowledge in the Chemical Sciences | Tourism not elsewhere classified | Infectious diseases | Clinical Health (Organs, Diseases and Abnormal Conditions) not elsewhere classified | Living resources (flora and fauna) | Inherited diseases (incl. gene therapy) | Chemical sciences | Physical sciences | Human Pharmaceutical Treatments (e.g. Antibiotics) | Reproductive system and disorders |
Related Links
Publications
Extracellular chaperones and proteostasis
Publisher: Annual Reviews
Date: 02-06-2013
DOI: 10.1146/ANNUREV-BIOCHEM-072711-163904
Abstract: There exists a family of currently untreatable, serious human diseases that arise from the inappropriate misfolding and aggregation of extracellular proteins. At present our understanding of mechanisms that operate to maintain proteostasis in extracellular body fluids is limited, but it has significantly advanced with the discovery of a small but growing family of constitutively secreted extracellular chaperones. The available evidence strongly suggests that these chaperones act as both sensors and disposal mediators of misfolded proteins in extracellular fluids, thereby normally protecting us from disease pathologies. It is critically important to further increase our understanding of the mechanisms that operate to effect extracellular proteostasis, as this is essential knowledge upon which to base the development of effective therapies for some of the world's most debilitating, costly, and intractable diseases.
Post-testicular sperm maturation and identification of an epididymal protein in the Japanese quail (Coturnix coturnix japonica)
Publisher: Bioscientifica
Date: 03-2014
DOI: 10.1530/REP-13-0566
Abstract: The role of the avian epididymis in post-testicular development and capacitation was examined to assess whether avian spermatozoa undergo any processes similar to those characteristic of mammalian sperm development. We found no evidence of a need for quail sperm to undergo capacitation and 90% of testicular sperm could bind to a perivitelline membrane and acrosome react. However, computer-assisted sperm analysis showed that 20% of testicular sperm from the quail were capable of movement and only about 12% of the motile sperm would have a curvilinear velocity greater than the mean for sperm from the distal epididymis. Nevertheless, epididymal transit was associated with increases in mean sperm velocity and the proportion of motile sperm. Together, these findings explain why earlier workers have achieved some fertilizations following inseminations of testicular spermatozoa and also demonstrate the need for some epididymal maturation of avian spermatozoa. Analysis of the electrophoretic profile of quail epididymal luminal proteins revealed that only one major protein (∼16 kDa) is secreted by the epididymis and it was virtually the only protein secreted by the ipsilateral epididymis following unilateral orchidectomy. Mass spectrometry showed that this protein is hemoglobin this finding was confirmed using anti-hemoglobin antibodies. It is suggested that hemoglobin may support sperm metabolism in the quail epididymis, aid in motility, and/or serve as an antioxidant.
The dissociated form of κ-casein is the precursor to its amyloid fibril formation
Publisher: Portland Press Ltd.
Date: 28-06-2010
DOI: 10.1042/BJ20091949
Abstract: Bovine milk κ-casein forms a self-associating oligomeric micelle-like species, in equilibrium with dissociated forms. In its native form, intra- and inter-molecular disulfide bonds lead to the formation of multimeric species ranging from monomers to decamers. When incubated under conditions of physiological pH and temperature, both reduced and non-reduced κ-casein form highly structured β-sheet amyloid fibrils. We investigated whether the precursor to κ-casein fibril formation is a dissociated state of the protein or its oligomeric micelle-like form. We show that reduced κ-casein is capable of forming fibrils well below its critical micelle concentration, i.e. at concentrations where only dissociated forms of the protein are present. Moreover, by regulating the degree of disulfide linkages, we were able to investigate how oligomerization of κ-casein influences its propensity for fibril formation under conditions of physiological pH and temperature. Thus, using fractions containing different proportions of multimeric species, we demonstrate that the propensity of the disulfide-linked multimers to form fibrils is inversely related to their size, with monomeric κ-casein being the most aggregation prone. We conclude that dissociated forms of κ-casein are the amyloidogenic precursors to fibril formation rather than oligomeric micelle-like species. The results highlight the role of oligomerization and natural binding partners in preventing amyloid fibril formation by disease-related proteins in vivo.
Model for amorphous aggregation processes
Publisher: American Physical Society (APS)
Date: 09-11-2009
Real-time single-molecule observation of chaperone-assisted protein folding
Publisher: American Association for the Advancement of Science (AAAS)
Date: 14-12-2022
Abstract: The ability of heat shock protein 70 (Hsp70) molecular chaperones to remodel the conformation of their clients is central to their biological function however, questions remain regarding the precise molecular mechanisms by which Hsp70 machinery interacts with the client and how this contributes toward efficient protein folding. Here, we used total internal reflection fluorescence (TIRF) microscopy and single-molecule fluorescence resonance energy transfer (smFRET) to temporally observe the conformational changes that occur to in idual firefly luciferase proteins as they are folded by the bacterial Hsp70 system. We observed multiple cycles of chaperone binding and release to an in idual client during refolding and determined that high rates of chaperone cycling improves refolding yield. Furthermore, we demonstrate that DnaJ remodels misfolded proteins via a conformational selection mechanism, whereas DnaK resolves misfolded states via mechanical unfolding. This study illustrates that the temporal observation of chaperone-assisted folding enables the elucidation of key mechanistic details inaccessible using other approaches.
New proteins identified in epididymal fluid from the platypus (Ornithorhynchus anatinus)
Publisher: CSIRO Publishing
Date: 2009
DOI: 10.1071/RD09091
Abstract: The platypus epididymal proteome is being studied because epididymal proteins are essential for male fertility in mammals and it is considered that knowledge of the epididymal proteome in an early mammal would be informative in assessing the convergence and ergence of proteins that are important in the function of the mammalian epididymis. Few of the epididymal proteins that have been identified in eutherian mammals were found in platypus caudal epididymal fluid, and the major epididymal proteins in the platypus (PXN-FBPL, SPARC and E-OR20) have never been identified in the epididymis of any other mammal.
De Novo Design, Synthesis, and Mechanistic Evaluation of Short Peptides That Mimic Heat Shock Protein 27 Activity
Publisher: American Chemical Society (ACS)
Date: 03-05-2021
Illuminating amyloid fibrils: Fluorescence-based single-molecule approaches
Publisher: Elsevier BV
Date: 2021
Single-molecule characterization of the interactions between extracellular chaperones and toxic alpha-synuclein oligomers
Publisher: Elsevier BV
Date: 06-2018
Proteostasis and the regulation of intra- and extracellular protein aggregation by ATP-independent molecular chaperones: Lens alpha-crystallins and milk caseins
Publisher: American Chemical Society (ACS)
Date: 14-02-2018
DOI: 10.1021/ACS.ACCOUNTS.7B00250
Abstract: Molecular chaperone proteins perform a ersity of roles inside and outside the cell. One of the most important is the stabilization of misfolding proteins to prevent their aggregation, a process that is potentially detrimental to cell viability. Diseases such as Alzheimer's, Parkinson's, and cataract are characterized by the accumulation of protein aggregates. In vivo, many proteins are metastable and therefore under mild destabilizing conditions have an inherent tendency to misfold, aggregate, and hence lose functionality. As a result, protein levels are tightly regulated inside and outside the cell. Protein homeostasis, or proteostasis, describes the network of biological pathways that ensures the proteome remains folded and functional. Proteostasis is a major factor in maintaining cell, tissue, and organismal viability. We have extensively investigated the structure and function of intra- and extracellular molecular chaperones that operate in an ATP-independent manner to stabilize proteins and prevent their misfolding and subsequent aggregation into amorphous particles or highly ordered amyloid fibrils. These types of chaperones are therefore crucial in maintaining proteostasis under normal and stress (e.g., elevated temperature) conditions. Despite their lack of sequence similarity, they exhibit many common features, i.e., extensive structural disorder, dynamism, malleability, heterogeneity, oligomerization, and similar mechanisms of chaperone action. In this Account, we concentrate on the chaperone roles of α-crystallins and caseins, the predominant proteins in the eye lens and milk, respectively. Intracellularly, the principal ATP-independent chaperones are the small heat-shock proteins (sHsps). In vivo, sHsps are the first line of defense in preventing intracellular protein aggregation. The lens proteins αA- and αB-crystallin are sHsps. They play a crucial role in maintaining solubility of the crystallins (including themselves) with age and hence in lens proteostasis and, ultimately, lens transparency. As there is little metabolic activity and no protein turnover in the lens, crystallins are very long lived proteins. Lens proteostasis is therefore very different to that in normal, metabolically active cells. Crystallins undergo extensive post-translational modification (PTM), including deamidation, racemization, phosphorylation, and truncation, which can alter their stability. Despite this, the lens remains transparent for tens of years, implying that lens proteostasis is intimately integrated with crystallin PTMs. Many PTMs do not significantly alter crystallin stability, solubility, and functionality, which thereby facilitates lens transparency. In the long term, however, extensive accumulation of crystallin PTMs leads to large-scale crystallin aggregation, lens opacification, and cataract formation. Extracellularly, various ATP-independent molecular chaperones exist that exhibit sHsp-like structural and functional features. For ex le, caseins, the major milk proteins, exhibit chaperone ability by inhibiting the amorphous and amyloid fibrillar aggregation of a ersity of destabilized proteins. Caseins maintain proteostasis within milk by preventing deleterious casein amyloid fibril formation via incorporation of thousands of in idual caseins into an amorphous structure known as the casein micelle. Hundreds of nanoclusters of calcium phosphate are sequestered within each casein micelle through interactions with short, highly phosphorylated casein sequences. This results in a stable biofluid that contains a high concentration of potentially amyloidogenic caseins and concentrations of calcium and phosphate that can be far in excess of the solubility of calcium phosphate. Casein micelle formation therefore performs vital roles in neonatal nutrition and calcium homeostasis in the mammary gland.
Post-testicular sperm environment and fertility
Publisher: Elsevier BV
Date: 07-2004
Structural and functional aspects of hetero-oligomers formed by the small heat shock proteins αB-crystallin and HSP27
Publisher: Elsevier BV
Date: 05-2013
Small heat-shock proteins prevent α-synuclein aggregation via transient interactions and their efficacy is affected by the rate of aggregation
Publisher: Elsevier BV
Date: 10-2016
The interaction of αB-crystallin with mature α-synuclein amyloid fibrils inhibits their elongation
Publisher: Elsevier BV
Date: 03-2010
Bioprospecting keratinous materials
Publisher: Medknow
Date: 2010
The small heat shock proteins αB-crystallin (HSPB5) and Hsp27 (HSPB1) inhibit the intracellular aggregation of α-synuclein
Publisher: Springer Science and Business Media LLC
Date: 23-03-2017
The effect of small molecules in modulating the chaperone activity of αB-crystallin against ordered and disordered protein aggregation
Publisher: Wiley
Date: 23-01-2008
DOI: 10.1111/J.1742-4658.2008.06257.X
Abstract: Protein aggregation can proceed via disordered or ordered mechanisms, with the latter being associated with amyloid fibril formation, which has been linked to a number of debilitating conditions including Alzheimer's, Parkinson's and Creutzfeldt-Jakob diseases. Small heat-shock proteins (sHsps), such as alphaB-crystallin, act as chaperones to prevent protein aggregation and are thought to play a key role in the prevention of protein-misfolding diseases. In this study, we have explored the potential for small molecules such as arginine and guanidine to affect the chaperone activity of alphaB-crystallin against disordered (amorphous) and ordered (amyloid fibril) forms of protein aggregation. The effect of these additives is highly dependent upon the target protein undergoing aggregation. Importantly, our results show that the chaperone action of alphaB-crystallin against aggregation of the disease-related amyloid fibril forming protein alpha-synucleinA53T is enhanced in the presence of arginine and similar positively charged compounds (such as lysine and guanidine). Thus, our results suggest that target protein identity plays a critical role in governing the effect of small molecules on the chaperone action of sHsps. Significantly, small molecules that regulate the activity of sHsps may provide a mechanism to protect cells from the toxic protein aggregation that is associated with some protein-misfolding diseases.
Regional Differences in Heat Shock Protein 25 Expression in Brain and Spinal Cord Astrocytes of Wild-Type and SOD1G93A Mice
Publisher: MDPI AG
Date: 19-05-2021
Abstract: Heterogeneity of glia in different CNS regions may contribute to the selective vulnerability of neuronal populations in neurodegenerative conditions such as amyotrophic lateral sclerosis (ALS). Here, we explored regional variations in the expression of heat shock protein 25 in glia under conditions of acute and chronic stress. Hsp27 (Hsp27 murine orthologue: Hsp25) fulfils a number of cytoprotective functions and may therefore be a possible therapeutic target in ALS. We identified a subpopulation of astrocytes in primary murine mixed glial cultures that expressed Hsp25. Under basal conditions, the proportion of Hsp25-positive astrocytes was twice as high in spinal cord cultures than in cortical cultures. To explore the physiological role of the elevated Hsp25 expression in spinal cord astrocytes, we exposed cortical and spinal cord glia to acute stress, using heat stress and pro-inflammatory stimuli. Surprisingly, we observed no stress-induced increase in Hsp25 expression in either cortical or spinal cord astrocytes. Similarly, exposure to endogenous stress, as modelled in glial cultures from SOD1 G93A-ALS mice, did not increase Hsp25 expression above that observed in astrocytes from wild-type mice. In vivo, Hsp25 expression was greater under conditions of chronic stress present in the spinal cord of SOD1 G93A mice than in wild-type mice, although this increase in expression is likely to be due to the extensive gliosis that occurs in this model. Together, these results show that there are differences in the expression of Hsp25 in astrocytes in different regions of the central nervous system, but Hsp25 expression is not upregulated under acute or chronic stress conditions.
Carboxymethylated-κ-casein: A convenient tool for the identification of polyphenolic inhibitors of amyloid fibril formation
Publisher: Elsevier BV
Date: 2010
DOI: 10.1016/J.BMC.2009.10.063
Abstract: Reduced and carboxymethylated-kappa-casein (RCM-kappa-CN) is a milk-derived amyloidogenic protein that readily undergoes nucleation-dependent aggregation and amyloid fibril formation via a similar pathway to disease-specific amyloidogenic peptides like amyloid beta (Abeta), which is associated with Alzheimer's disease. In this study, a series of flavonoids, many known to be inhibitors of Abeta fibril formation, were screened for their ability to inhibit RCM-kappa-CN fibrilisation, and the results were compared with literature data on Abeta inhibition. Flavonoids that had a high degree of hydroxylation and molecular planarity gave good inhibition of RCM-kappa-CN fibril formation. IC(50) values were between 10- and 200-fold higher with RCM-kappa-CN than literature results for Abeta fibril inhibition, however, with few exceptions, they showed a similar trend in potency. The convenience and reproducibility of the RCM-kappa-CN assay make it an economic alternative first screen for Abeta inhibitory activity, especially for use with large compound libraries.
Dissociation from the oligomeric state is the rate-limiting step in fibril formation by κ-casein
Publisher: Elsevier BV
Date: 04-2008
Amyloid fibril formation by bovine milk αs2 -casein occurs under physiological conditions yet is prevented by its natural counterpart, αs1 -casein
Publisher: American Chemical Society (ACS)
Date: 27-02-2008
DOI: 10.1021/BI701278C
Abstract: The calcified proteinaceous deposits, or corpora amylacea, of bovine mammary tissue often comprise a network of amyloid fibrils, the origins of which have not been fully elucidated. Here, we demonstrate by transmission electron microscopy, dye binding assays, and X-ray fiber diffraction that bovine milk alpha s2-casein, a protein synthesized and secreted by mammary epithelial cells, readily forms fibrils in vitro. As a component of whole alpha s-casein, alpha s2-casein was separated from alpha s1-casein under nonreducing conditions via cation-exchange chromatography. Upon incubation at neutral pH and 37 degrees C, the spherical particles typical of alpha s2-casein rapidly converted to twisted, ribbon-like fibrils approximately 12 nm in diameter, which occasionally formed loop structures. Despite their irregular morphology, these fibrils possessed a beta-sheet core structure and the ability to bind amyloidophilic dyes such as thioflavin T. Fibril formation was optimal at pH 6.5-6.7 and was promoted by higher incubation temperatures. Interestingly, the protein appeared to be less prone to fibril formation upon disulfide bond reduction with dithiothreitol. Thus, alpha s2-casein is particularly susceptible to fibril formation under physiological conditions. However, our findings indicate that alpha s2-casein fibril formation is potently inhibited by its natural counterpart, alpha s1-casein, while is only partially inhibited by beta-casein. These findings highlight the inherent propensity of casein proteins to form amyloid fibrils and the importance of casein-casein interactions in preventing such fibril formation in vivo.
Proteomics Approaches for Biomarker and Drug Target Discovery in ALS and FTD
Publisher: Frontiers Media SA
Date: 11-06-2019
Real-time single-molecule observation of chaperone-assisted protein folding
Publisher: Cold Spring Harbor Laboratory
Date: 10-05-2022
DOI: 10.1101/2022.05.09.491114
Abstract: The ability of Hsp70 molecular chaperones to remodel the conformation of their clients is central to their biological function however, questions remain regarding the precise molecular mechanisms by which Hsp70 machinery interacts with the client and how this contributes towards efficient protein folding. Here, we used Total Internal Reflection Fluorescence (TIRF) microscopy and single-molecule Fluorescence Resonance Energy Transfer (smFRET) to temporally observe the conformational changes that occur to in idual firefly luciferase (Fluc) proteins as they are folded by the bacterial Hsp70 system. For the first time, we observed multiple cycles of chaperone binding-and-release to an in idual client during refolding and that high rates of chaperone cycling improves refolding yield. Furthermore, we demonstrate that DnaJ remodels misfolded proteins via a conformational selection mechanism whereas DnaK resolves misfolded states via mechanical unfolding. This study illustrates that the temporal observation of chaperone-assisted folding enables the elucidation of key mechanistic details inaccessible using other approaches.
Site-directed mutations in the C-terminal extension of human αB-crystallin affect chaperone function and block amyloid fibril formation
Publisher: Public Library of Science (PLoS)
Date: 17-10-2007
Crystallin proteins and amyloid fibrils
Publisher: Springer Science and Business Media LLC
Date: 22-09-2009
DOI: 10.1007/S00018-008-8327-4
Abstract: Improper protein folding (misfolding) can lead to the formation of disordered (amorphous) or ordered (amyloid fibril) aggregates. The major lens protein, alpha-crystallin, is a member of the small heat-shock protein (sHsp) family of intracellular molecular chaperone proteins that prevent protein aggregation. Whilst the chaperone activity of sHsps against amorphously aggregating proteins has been well studied, its action against fibril-forming proteins has received less attention despite the presence of sHsps in deposits found in fibril-associated diseases (e.g. Alzheimer's and Parkinson's). In this review, the literature on the interaction of alphaB-crystallin and other sHsps with fibril-forming proteins is summarized. In particular, the ability of sHsps to prevent fibril formation, their mechanisms of action and the possible in vivo consequences of such associations are discussed. Finally, the fibril-forming propensity of the crystallin proteins and its implications for cataract formation are described along with the potential use of fibrillar crystallin proteins as bionanomaterials.
Stress in native grasses under ecologically relevant heat waves
Publisher: Public Library of Science (PLoS)
Date: 11-10-2018
Preventing α-synuclein aggregation: The role of the small heat-shock molecular chaperone proteins
Publisher: Elsevier BV
Date: 09-2014
DOI: 10.1016/J.BBADIS.2014.06.024
Abstract: Protein homeostasis, or proteostasis, is the process of maintaining the conformational and functional integrity of the proteome. The failure of proteostasis can result in the accumulation of non-native proteins leading to their aggregation and deposition in cells and in tissues. The amyloid fibrillar aggregation of the protein α-synuclein into Lewy bodies and Lewy neuritis is associated with neurodegenerative diseases classified as α-synucleinopathies, which include Parkinson's disease and dementia with Lewy bodies. The small heat-shock proteins (sHsps) are molecular chaperones that are one of the cell's first lines of defence against protein aggregation. They act to stabilise partially folded protein intermediates, in an ATP-independent manner, to maintain cellular proteostasis under stress conditions. Thus, the sHsps appear ideally suited to protect against α-synuclein aggregation, yet these fail to do so in the context of the α-synucleinopathies. This review discusses how sHsps interact with α-synuclein to prevent its aggregation and, in doing so, highlights the multi-faceted nature of the mechanisms used by sHsps to prevent the fibrillar aggregation of proteins. It also examines what factors may contribute to α-synuclein escaping the sHsp chaperones in the context of the α-synucleinopathies.
Analysis of the mechanism by which calcium negatively regulates the tyrosine phosphorylation cascade associated with sperm capacitation
Publisher: The Company of Biologists
Date: 15-01-2004
DOI: 10.1242/JCS.00842
Abstract: The capacitation of mammalian spermatozoa involves the activation of a cAMP-mediated signal transduction pathway that drives tyrosine phosphorylation via mechanisms that are unique to this cell type. Controversy surrounds the impact of extracellular calcium on this process, with positive and negative effects being recorded in independent publications. We clearly demonstrate that the presence of calcium in the external medium decreases tyrosine phosphorylation in both human and mouse spermatozoa. Under these conditions, a rise in intracellular pH was recorded, however, this event was not responsible for the observed changes in phosphotyrosine expression. Rather, the impact of calcium on tyrosine phosphorylation in these cells was associated with an unexpected change in the intracellular availability of ATP. Thus, the ATP content of both human and mouse spermatozoa fell significantly when these cells were incubated in the presence of external calcium. Furthermore, the removal of glucose, or addition of 2-deoxyglucose, decreased ATP levels within human spermatozoon populations and induced a corresponding decline in phosphotyrosine expression. In contrast, the mitochondrial inhibitor rotenone had no effect on either ATP levels or tyrosine phosphorylation. Addition of the affinity-labeling probe 8-N3 ATP confirmed our prediction that spermatozoa have many calcium-dependent ATPases. Moreover, addition of the ATPase inhibitor thapsigargin, increased intracellular calcium levels, decreased ATP and suppressed tyrosine phosphorylation. Based on these findings, the present study indicates that extracellular calcium suppresses tyrosine phosphorylation by decreasing the availability of intracellular ATP, and not by activating tyrosine phosphatases or inhibiting tyrosine kinases as has been previously suggested.
Native disulphide-linked dimers facilitate amyloid fibril formation by bovine milk αS2 -casein
Publisher: Elsevier BV
Date: 03-2021
Neurodegenerative disease-associated protein aggregates are poor inducers of the heat shock response in neuronal cells
Publisher: The Company of Biologists
Date: 2020
DOI: 10.1242/JCS.243709
Abstract: Protein aggregates that result in inclusions formation are a pathological hallmark common to many neurodegenerative diseases, including amyotrophic lateral sclerosis, Parkinson's disease and Huntington's disease. Under conditions of cellular stress, activation of the heat shock response (HSR) results in an increase in the levels of molecular chaperones and is a first line of cellular defence against inclusion formation. It remains to be established whether neurodegenerative disease-associated proteins and inclusions are themselves capable of inducing an HSR in neuronal cells. To address this, we generated a neuroblastoma cell line that expresses a fluorescent reporter protein under conditions of heat shock transcription factor 1-mediated HSR induction. We show that the HSR is not induced by exogenous treatment with aggregated forms of recombinant α-synuclein or the G93A mutant of superoxide dismutase-1 (SOD1G93A) nor intracellular expression of SOD1G93A or a pathogenic form of polyQ-expanded huntingtin (Htt72Q). These results suggest that pathogenic proteins evade detection or impair induction of the HSR in neuronal cells. A failure of protein aggregation to induce an HSR may contribute to the development of inclusion pathology in neurodegenerative diseases.
Small heat-shock proteins: Important players in regulating cellular proteostasis
Publisher: Springer Science and Business Media LLC
Date: 29-10-2015
DOI: 10.1007/S00018-014-1754-5
Abstract: Small heat-shock proteins (sHsps) are a erse family of intra-cellular molecular chaperone proteins that play a critical role in mitigating and preventing protein aggregation under stress conditions such as elevated temperature, oxidation and infection. In doing so, they assist in the maintenance of protein homeostasis (proteostasis) thereby avoiding the deleterious effects that result from loss of protein function and/or protein aggregation. The chaperone properties of sHsps are therefore employed extensively in many tissues to prevent the development of diseases associated with protein aggregation. Significant progress has been made of late in understanding the structure and chaperone mechanism of sHsps. In this review, we discuss some of these advances, with a focus on mammalian sHsp hetero-oligomerisation, the mechanism by which sHsps act as molecular chaperones to prevent both amorphous and fibrillar protein aggregation, and the role of post-translational modifications in sHsp chaperone function, particularly in the context of disease.
Small heat-shock proteins interact with a flanking domain to suppress polyglutamine aggregation
Publisher: Proceedings of the National Academy of Sciences
Date: 19-05-2010
Abstract: Small heat-shock proteins (sHsps) are molecular chaperones that play an important protective role against cellular protein misfolding by interacting with partially unfolded proteins on their off-folding pathway, preventing their aggregation. Polyglutamine (polyQ) repeat expansion leads to the formation of fibrillar protein aggregates and neuronal cell death in nine diseases, including Huntington disease and the spinocerebellar ataxias (SCAs). There is evidence that sHsps have a role in suppression of polyQ-induced neurodegeneration for ex le, the sHsp alphaB-crystallin (αB-c) has been identified as a suppressor of SCA3 toxicity in a Drosophila model. However, the molecular mechanism for this suppression is unknown. In this study we tested the ability of αB-c to suppress the aggregation of a polyQ protein. We found that αB-c does not inhibit the formation of SDS-insoluble polyQ fibrils. We further tested the effect of αB-c on the aggregation of ataxin-3, a polyQ protein that aggregates via a two-stage aggregation mechanism. The first stage involves association of the N-terminal Josephin domain followed by polyQ-mediated interactions and the formation of SDS-resistant mature fibrils. Our data show that αB-c potently inhibits the first stage of ataxin-3 aggregation however, the second polyQ-dependent stage can still proceed. By using NMR spectroscopy, we have determined that αB-c interacts with an extensive region on the surface of the Josephin domain. These data provide an ex le of a domain/region flanking an amyloidogenic sequence that has a critical role in modulating aggregation of a polypeptide and plays a role in the interaction with molecular chaperones to prevent this aggregation.
The small heat shock protein Hsp27 binds α-synuclein fibrils, preventing elongation and cytotoxicity
Publisher: Elsevier BV
Date: 03-2018
The heat shock response in neurons and astroglia and its role in neurodegenerative diseases
Publisher: Springer Science and Business Media LLC
Date: 18-09-2017
The Monomeric α-Crystallin Domain of the Small Heat-shock Proteins αB-crystallin and Hsp27 Binds Amyloid Fibril Ends: sHSP α-crystallin domain binds amyloid fibril ends
Publisher: Elsevier BV
Date: 08-2022
DOI: 10.1016/J.JMB.2022.167711
Abstract: Small heat-shock proteins (sHSPs) are ubiquitously expressed molecular chaperones present in all kingdoms of life that inhibit protein misfolding and aggregation. Despite their importance in proteostasis, the structure-function relationships of sHSPs remain elusive. Human sHSPs are characterised by a central, highly conserved α-crystallin domain (ACD) and variable-length N- and C-terminal regions. The ACD forms antiparallel homodimers via an extended β-strand, creating a shared β-sheet at the dimer interface. The N- and C-terminal regions mediate formation of higher order oligomers that are thought to act as storage forms for chaperone-active dimers. We investigated the interactions of the ACD of two human sHSPs, αB-crystallin (αB-C) and Hsp27, with apolipoprotein C-II amyloid fibrils using analytical ultracentrifugation and nuclear magnetic resonance spectroscopy. The ACD was found to interact transiently with amyloid fibrils to inhibit fibril elongation and naturally occurring fibril end-to-end joining. This interaction was sensitive to the concentration of fibril ends indicating a 'fibril-capping' interaction. Furthermore, resonances arising from the ACD monomer were attenuated to a greater extent than those of the ACD dimer in the presence of fibrils, suggesting that the monomer may bind fibrils. This hypothesis was supported by mutagenesis studies in which disulfide cross-linked ACD dimers formed by both αB-C and Hsp27 were less effective at inhibiting amyloid fibril elongation and fibril end-to-end joining than ACD constructs lacking disulfide cross-linking. Our results indicate that sHSP monomers inhibit amyloid fibril elongation, highlighting the importance of the dynamic oligomeric nature of sHSPs for client binding.
Single-molecule fluorescence-based approach reveals novel mechanistic insights into small heat shock protein chaperone function
Publisher: Cold Spring Harbor Laboratory
Date: 17-02-2020
DOI: 10.1101/2020.02.16.951632
Abstract: Small heat shock proteins (sHsps) are a family of ubiquitous intracellular molecular chaperones that are up-regulated under stress conditions and play a vital role in protein homeostasis (proteostasis). It is commonly accepted that these chaperones work by trapping misfolded proteins to prevent their aggregation, however fundamental questions regarding the molecular mechanism by which sHsps interact with misfolded proteins remain unanswered. Traditionally, it has been difficult to study sHsp function due to the dynamic and heterogenous nature of the species formed between sHsps and aggregation-prone proteins. Single-molecule techniques have emerged as a powerful tool to study dynamic protein complexes and we have therefore developed a novel single-molecule fluorescence-based approach to observe the chaperone action of human αB-crystallin (αBc, HSPB5). Using this approach we have, for the first time, determined the stoichiometries of complexes formed between αBc and a model client protein, chloride intracellular channel 1 (CLIC1). By examining the polydispersity and stoichiometries of these complexes over time, and in response to different concentrations of αBc, we have uncovered unique and important insights into a two-step mechanism by which αBc interacts with misfolded client proteins to prevent their aggregation. Understanding this fundamental mechanism of sHsp action is crucial to understanding how these molecular chaperone function to protect the cell from protein misfolding and their overall role in the cellular proteostasis network.
An α-cyanostilbene derivative for the enhanced detection and imaging of amyloid fibril aggregates
Publisher: Cold Spring Harbor Laboratory
Date: 27-06-2020
DOI: 10.1101/2020.06.25.172627
Abstract: The aggregation of proteins into amyloid fibrils has been implicated in the pathogenesis of a variety of neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease. Benzothiazole dyes such as Thioflavin T (ThT) are well characterised and widely used fluorescent probes for monitoring amyloid fibril formation. However, existing dyes lack sensitivity and specificity to oligomeric intermediates formed during fibril formation. In this work we describe the use of an α-cyanostilbene derivative with aggregation-induced emission properties (called ASCP) as a fluorescent probe for the detection of amyloid fibrils. Similar to ThT, ASCP is fluorogenic in the presence of amyloid fibrils and upon binding and excitation at 460 nm produces a red-shifted emission with a large Stokes shift of 145 nm. ASCP has a higher binding affinity to fibrillar α-synuclein than ThT and likely shares the same binding sites to amyloid fibrils. Importantly, ASCP was found to also be fluorogenic in the presence of amorphous aggregates and can detect oligomeric species formed early during aggregation. Moreover, ASCP can be used to visualise fibrils via Total Internal Reflection Fluorescence (TIRF) microscopy and, due to its large Stokes shift, simultaneously monitor the fluorescence emission of other labelled proteins following excitation with the same laser used to excite ASCP. Consequently, ASCP possesses enhanced and unique spectral characteristics compared to ThT that make it a promising alternative for the in vitro study of amyloid fibrils and the mechanisms by which they form.
Monitoring Early-Stage Protein Aggregation by an Aggregation-Induced Emission Fluorogen
Publisher: American Chemical Society (ACS)
Date: 23-08-2017
DOI: 10.1021/ACS.ANALCHEM.7B02090
Abstract: Highly ordered protein aggregates, termed amyloid fibrils, are associated with a broad range of diseases, many of which are neurodegenerative, for ex le, Alzheimer's and Parkinson's. The transition from soluble, functional protein into insoluble amyloid fibril occurs via a complex process involving the initial generation of highly dynamic early stage aggregates or prefibrillar species. Amyloid probes, for ex le, thioflavin T and Congo red, have been used for decades as the gold standard for detecting amyloid fibrils in solution and tissue sections. However, these well-established dyes do not detect the presence of prefibrillar species formed during the early stages of protein aggregation. Prefibillar species have been proposed to play a key role in the cytotoxicity of amyloid fibrils and the pathogenesis of neurodegenerative diseases. Herein, we report a novel fluorescent dye (bis(triphenylphosphonium) tetraphenylethene (TPE-TPP)) with aggregation-induced emission characteristics for monitoring the aggregation process of amyloid fibrils. An increase in TPE-TPP fluorescence intensity is observed only with ordered protein aggregation, such as amyloid fibril formation, and not with stable molten globules states or amorphously aggregating species. Importantly, TPE-TPP can detect the presence of prefibrillar species formed early during fibril formation. TPE-TPP exhibits a distinctive spectral shift in the presence of prefibrillar species, indicating a unique structural feature of these intermediates. Using fluorescence polarization, which reflects the mobility of the emitting entity, the specific oligomeric pathways undertaken by various proteins during fibrillation could be discerned. Furthermore, we demonstrate the broad applicability of TPE-TPP to monitor amyloid fibril aggregation, including under erse conditions such as at acidic pH and elevated temperature, or in the presence of amyloid inhibitors.
Rapid flow cytometric measurement of protein inclusions and nuclear trafficking
Publisher: Springer Science and Business Media LLC
Date: 12-08-2016
DOI: 10.1038/SREP31138
Abstract: Proteinaceous cytoplasmic inclusions are an indicator of dysfunction in normal cellular proteostasis and a hallmark of many neurodegenerative diseases. We describe a simple and rapid new flow cytometry-based method to enumerate, characterise and, if desired, physically recover protein inclusions from cells. This technique can analyse and resolve a broad variety of inclusions differing in both size and protein composition, making it applicable to essentially any model of intracellular protein aggregation. The method also allows rapid quantification of the nuclear trafficking of fluorescently labelled molecules.
Assessment of metal concentrations in the SOD1G93A mouse model of amyotrophic lateral sclerosis and its potential role in muscular denervation, with particular focus on muscle tissue
Publisher: Elsevier BV
Date: 04-2018
DOI: 10.1016/J.MCN.2018.03.001
Abstract: Amyotrophic lateral sclerosis (ALS) is among the most common of the motor neuron diseases, and arguably the most devastating. During the course of this fatal neurodegenerative disorder, motor neurons undergo progressive degeneration. The currently best-understood animal models of ALS are based on the over-expression of mutant isoforms of Cu/Zn superoxide dismutase 1 (SOD1) these indicate that there is a perturbation in metal homeostasis with disease progression. Copper metabolism in particular is affected in the central nervous system (CNS) and muscle tissue. This present study assessed previously published and newly gathered concentrations of transition metals (Cu, Zn, Fe and Se) in CNS (brain and spinal cord) and non-CNS (liver, intestine, heart and muscle) tissues from transgenic mice over-expressing the G93A mutant SOD1 isoform (SOD1 Cu accumulates in non-CNS tissues at pre-symptomatic stages in SOD1 It is hypothesized that the observed Cu accumulations may represent a pathologic feature of ALS, which may actively contribute to axonal retraction leading to muscular denervation, and possibly significantly contributing to disease pathology. Therefore, it is proposed that the toxic-gain-of-function and dying-back hypotheses to explain the molecular drivers of ALS may not be separate, in idual processes rather our data suggests that they are parallel processes.
αB-Crystallin inhibits the cell toxicity associated with amyloid fibril formation by κ-casein and the amyloid-β peptide
Publisher: Springer Science and Business Media LLC
Date: 15-07-2010
Compartmentalization of prion isoforms within the reproductive tract of the ram
Publisher: Oxford University Press (OUP)
Date: 09-2004
The contribution of proteomics to understanding epididymal maturation of mammalian spermatozoa
Publisher: Informa UK Limited
Date: 12-07-2012
DOI: 10.3109/19396368.2012.663233
Abstract: The acquisition of the ability of the male gamete to fertilize an ovum is the result of numerous and sequential steps of differentiation of spermatozoa that occur as they transit from the testis to the end of the epididymal tubule. The post gonadal sperm modifications are mostly related to motility, egg binding, and penetration processes. As the activity of the epididymis and its luminal fluid composition are believed to be directly related to 'sperm maturation', a review on epididymal proteins is presented. Comparative studies have shown that the epididymal activities are species specific. Nevertheless, for all mammalian species studied, similarities exist in the sequential proteomic changes of the luminal composition of the epididymal tubule and proteins on the sperm surface. The potential roles of these modifications are discussed.
SOD1 protein aggregates stimulate macropinocytosis in neurons to facilitate their propagation
Publisher: Springer Science and Business Media LLC
Date: 31-10-2015
Molecular dynamics analysis of apolipoprotein-D - lipid hydroperoxide interactions: Mechanism for selective oxidation of Met-93
Publisher: Public Library of Science (PLoS)
Date: 30-03-2012
Small heat shock proteins: multifaceted proteins with important implications for life
Publisher: Springer Science and Business Media LLC
Date: 13-02-2019
NMR spectroscopy of 14-3-3ζ reveals a flexible C-terminal extension: differentiation of the chaperone and phosphoserine-binding activities of 14-3-3ζ
Publisher: Portland Press Ltd.
Date: 13-07-2011
DOI: 10.1042/BJ20102178
Abstract: Intracellular 14-3-3 proteins bind to many proteins, via a specific phosphoserine motif, regulating erse cellular tasks including cell signalling and disease progression. The 14-3-3ζ isoform is a molecular chaperone, preventing the stress-induced aggregation of target proteins in a manner comparable with that of the unrelated sHsps (small heat-shock proteins). 1H-NMR spectroscopy revealed the presence of a flexible and unstructured C-terminal extension, 12 amino acids in length, which protrudes from the domain core of 14-3-3ζ and is similar in structure and length to the C-terminal extension of mammalian sHsps. The extension stabilizes 14-3-3ζ, but has no direct role in chaperone action. Lys49 is an important functional residue within the ligand-binding groove of 14-3-3ζ with K49E 14-3-3ζ exhibiting markedly reduced binding to phosphorylated and non-phosphorylated ligands. The R18 peptide binds to the binding groove of 14-3-3ζ with high affinity and also reduces the interaction of 14-3-3ζ ligands. However, neither the K49E mutation nor the presence of the R18 peptide affected the chaperone activity of 14-3-3ζ, implying that the C-terminal extension and binding groove of 14-3-3ζ do not mediate interaction with target proteins during chaperone action. Other region(s) in 14-3-3ζ are most likely to be involved, i.e. the protein's chaperone and phosphoserine-binding activities are functionally and structurally separated.
AR-12 inhibits multiple chaperones concomitant with stimulating autophagosome formation collectively preventing virus replication
Publisher: Wiley
Date: 06-06-2016
DOI: 10.1002/JCP.25431
Proteostasis in the Male and Female Germline: A New Outlook on the Maintenance of Reproductive Health
Publisher: Frontiers Media SA
Date: 16-04-2021
DOI: 10.3389/FCELL.2021.660626
Abstract: For fully differentiated, long lived cells the maintenance of protein homeostasis (proteostasis) becomes a crucial determinant of cellular function and viability. Neurons are the most well-known ex le of this phenomenon where the majority of these cells must survive the entire course of life. However, male and female germ cells are also uniquely dependent on the maintenance of proteostasis to achieve successful fertilization. Oocytes, also long-lived cells, are subjected to prolonged periods of arrest and are largely reliant on the translation of stored mRNAs, accumulated during the growth period, to support meiotic maturation and subsequent embryogenesis. Conversely, sperm cells, while relatively ephemeral, are completely reliant on proteostasis due to the absence of both transcription and translation. Despite these remarkable, cell-specific features there has been little focus on understanding protein homeostasis in reproductive cells and how/whether proteostasis is “reset” during embryogenesis. Here, we seek to capture the momentum of this growing field by highlighting novel findings regarding germline proteostasis and how this knowledge can be used to promote reproductive health. In this review we capture proteostasis in the context of both somatic cell and germline aging and discuss the influence of oxidative stress on protein function. In particular, we highlight the contributions of proteostasis changes to oocyte aging and encourage a focus in this area that may complement the extensive analyses of DNA damage and aneuploidy that have long occupied the oocyte aging field. Moreover, we discuss the influence of common non-enzymatic protein modifications on the stability of proteins in the male germline, how these changes affect sperm function, and how they may be prevented to preserve fertility. Through this review we aim to bring to light a new trajectory for our field and highlight the potential to harness the germ cell’s natural proteostasis mechanisms to improve reproductive health. This manuscript will be of interest to those in the fields of proteostasis, aging, male and female gamete reproductive biology, embryogenesis, and life course health.
Mimicking phosphorylation of αB-crystallin affects its chaperone activity
Publisher: Portland Press Ltd.
Date: 11-12-2007
DOI: 10.1042/BJ20060981
Abstract: αB-crystallin is a member of the sHsp (small heat-shock protein) family that prevents misfolded target proteins from aggregating and precipitating. Phosphorylation at three serine residues (Ser19, Ser45 and Ser59) is a major post-translational modification that occurs to αB-crystallin. In the present study, we produced recombi-nant proteins designed to mimic phosphorylation of αB-crystallin by incorporating a negative charge at these sites. We employed these mimics to undertake a mechanistic and structural invest-igation of the effect of phosphorylation on the chaperone activity of αB-crystallin to protect against two types of protein misfolding, i.e. amorphous aggregation and amyloid fibril assembly. We show that mimicking phosphorylation of αB-crystallin results in more efficient chaperone activity against both heat-induced and reduc-tion-induced amorphous aggregation of target proteins. Mimick-ing phosphorylation increased the chaperone activity of αB-crystallin against one amyloid-forming target protein (κ-casein), but decreased it against another (ccβ-Trp peptide). We observed that both target protein identity and solution (buffer) conditions are critical factors in determining the relative chaperone ability of wild-type and phosphorylated αB-crystallins. The present study provides evidence for the regulation of the chaperone activity of αB-crystallin by phosphorylation and indicates that this may play an important role in alleviating the pathogenic effects associated with protein conformational diseases.
Using single-molecule approaches to understand the molecular mechanisms of heat-shock protein chaperone function
Publisher: Elsevier BV
Date: 10-2018
DOI: 10.1016/J.JMB.2018.05.021
Abstract: The heat-shock proteins (Hsp) are a family of molecular chaperones, which collectively form a network that is critical for the maintenance of protein homeostasis. Traditional ensemble-based measurements have provided a wealth of knowledge on the function of in idual Hsps and the Hsp network however, such techniques are limited in their ability to resolve the heterogeneous, dynamic and transient interactions that molecular chaperones make with their client proteins. Single-molecule techniques have emerged as a powerful tool to study dynamic biological systems, as they enable rare and transient populations to be identified that would usually be masked in ensemble measurements. Thus, single-molecule techniques are particularly amenable for the study of Hsps and have begun to be used to reveal novel mechanistic details of their function. In this review, we discuss the current understanding of the chaperone action of Hsps and how gaps in the field can be addressed using single-molecule methods. Specifically, this review focuses on the ATP-independent small Hsps and the broader Hsp network and describes how these dynamic systems are amenable to single-molecule techniques.
Avoiding the oligomeric state: αB-crystallin inhibits fragmentation and induces dissociation of apolipoprotein C-II amyloid fibrils
Publisher: Wiley
Date: 16-11-2013
DOI: 10.1096/FJ.12-220657
Abstract: The in vivo aggregation of proteins into amyloid fibrils suggests that cellular mechanisms that normally prevent or reverse this aggregation have failed. The small heat-shock molecular chaperone protein αB-crystallin (αB-c) inhibits amyloid formation and colocalizes with amyloid plaques however, the physiological reason for this localization remains unexplored. Here, using apolipoprotein C-II (apoC-II) as a model fibril-forming system, we show that αB-c binds directly to mature amyloid fibrils (Kd 5.4 ± 0.5 μM). In doing so, αB-c stabilized the fibrils from dilution-induced fragmentation, halted elongation of partially formed fibrils, and promoted the dissociation of mature fibrils into soluble monomers. Moreover, in the absence of dilution, the association of αB-c with apoC-II fibrils induced a 14-fold increase in average aggregate size, resulting in large fibrillar tangles reminiscent of protein inclusions. We propose that the binding of αB-c to fibrils prevents fragmentation and mediates the lateral association of fibrils into large inclusions. We further postulate that transient interactions of apoC-II with αB-c induce a fibril-incompetent monomeric apoC-II form, preventing oligomerization and promoting fibril dissociation. This work reveals previously unrecognized mechanisms of αB-c chaperone action in amyloid assembly and fibril dynamics, and provides a rationale for the in vivo colocalization of small heat-shock proteins with amyloid deposits.-Binger, K. J., Ecroyd, H., Yang, S., Carver, J. A., Howlett, G. J., Griffin, M. D. W. Avoiding the oligomeric state: αB-crystallin inhibits fragmentation and induces dissociation of apolipoprotein C-II amyloid fibrils.
The small heat-shock protein αB-crystallin uses different mechanisms of chaperone action to prevent the amorphous versus fibrillar aggregation of α-lactalbumin
Publisher: Portland Press Ltd.
Date: 21-11-2012
DOI: 10.1042/BJ20121187
Abstract: Stress conditions can destabilize proteins, promoting them to unfold and adopt intermediately folded states. Partially folded protein intermediates are unstable and prone to aggregation down off-folding pathways leading to the formation of either amorphous or amyloid fibril aggregates. The sHsp (small heat-shock protein) αB-crystallin acts as a molecular chaperone to prevent both amorphous and fibrillar protein aggregation however, the precise molecular mechanisms behind its chaperone action are incompletely understood. To investigate whether the chaperone activity of αB-crystallin is dependent upon the form of aggregation (amorphous compared with fibrillar), bovine α-lactalbumin was developed as a model target protein that could be induced to aggregate down either off-folding pathway using comparable buffer conditions. Thus when α-lactalbumin was reduced it aggregated amorphously, whereas a reduced and carboxymethylated form aggregated to form amyloid fibrils. Using this model, αB-crystallin was shown to be a more efficient chaperone against amorphously aggregating α-lactalbumin than when it aggregated to form fibrils. Moreover, αB-crystallin forms high molecular mass complexes with α-lactalbumin to prevent its amorphous aggregation, but prevents fibril formation via weak transient interactions. Thus, the conformational stability of the protein intermediate, which is a precursor to aggregation, plays a critical role in modulating the chaperone mechanism of αB-crystallin.
Formation and dissociation of sperm bundles in monotremes
Publisher: Oxford University Press (OUP)
Date: 24-08-2016
DOI: 10.1095/BIOLREPROD.116.140491
Abstract: Because monotremes are the earliest offshoot of the mammalian lineage, the platypus and short-beaked echidna were studied as model animals to assess the origin and biological significance of adaptations considered unique to therian mammals: epididymal sperm maturation and subsequent capacitation. We show that spermatozoa from both species assemble into bundles of approximately 100 cells during passage through the epididymis and that an epididymal protein-secreted protein, acidic, cysteine-rich (osteonectin SPARC)-is involved in bundle formation. The bundles persisted during incubation in vitro for at least 1 h under conditions that capacitate therian spermatozoa, and then underwent a time-dependent dissociation to release spermatozoa capable of fertilization. Only after this dissociation could the spermatozoa bind to the perivitelline membrane of a hen's egg, display an altered form of motility reminiscent of hyperactivation, and be induced to undergo an acrosome reaction. It is concluded that the development of sperm bundles in the monotreme epididymis mandates that they require a time-dependent process to be capable of fertilizing an ovum. However, because this functional end point was achieved without overt changes in protein tyrosine phosphorylation (a hallmark of capacitation in therians), it is concluded that the process in monotremes is distinctly different from capacitation in therian mammals.
Phosphomimics destabilize Hsp27 oligomeric assemblies and enhance chaperone activity
Publisher: Elsevier BV
Date: 02-2015
DOI: 10.1016/J.CHEMBIOL.2015.01.001
Abstract: Serine phosphorylation of the mammalian small heat-shock protein Hsp27 at residues 15, 78, and 82 is thought to regulate its structure and chaperone function however, the site-specific impact has not been established. We used mass spectrometry to assess the combinatorial effect of mutations that mimic phosphorylation upon the oligomeric state of Hsp27. Comprehensive dimerization yielded a relatively uncrowded spectrum, composed solely of even-sized oligomers. Modification at one or two serines decreased the average oligomeric size, while the triple mutant was predominantly a dimer. These changes were reflected in a greater propensity for oligomers to dissociate upon increased modification. The ability of Hsp27 to prevent amorphous or fibrillar aggregation of target proteins was enhanced and correlated with the amount of dissociated species present. We propose that, in vivo, phosphorylation promotes oligomer dissociation, thereby enhancing chaperone activity. Our data support a model in which dimers are the chaperone-active component of Hsp27.
N- And C-terminal regions of aB-crystallin and Hsp27 mediate inhibition of amyloid nucleation, fibril binding, and fibril disaggregation
Publisher: Elsevier BV
Date: 07-2020
Letter to the Editor: A response to Horne and Lucey (2017)
Publisher: American Dairy Science Association
Date: 07-2017
Single-molecule fluorescence-based approach reveals novel mechanistic insights into human small heat shock protein chaperone function
Publisher: Elsevier BV
Date: 2021
Protease-activated alpha-2-macroglobulin can inhibit amyloid formation via two distinct mechanisms
Publisher: Wiley
Date: 23-01-2013
Neurodegenerative disease-associated protein aggregates are poor inducers of the heat shock response in neuronal-like cells
Publisher: Cold Spring Harbor Laboratory
Date: 06-01-2020
DOI: 10.1101/2020.01.06.896654
Abstract: Protein aggregation that results in the formation of inclusions is strongly correlated with neuronal death and is a pathological hallmark common to many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Huntington’s disease. Cells are thought to dramatically up-regulate the levels of heat shock proteins during periods of cellular stress via induction of the heat shock response (HSR). Heat shock proteins are well-characterised molecular chaperones that interact with aggregation-prone proteins to either stabilise, refold, or traffic protein for degradation. The reason why heat shock proteins are unable to maintain the solubility of particular proteins in neurodegenerative disease is unknown. We sought to determine whether neurodegenerative disease-associated protein aggregates can induce the HSR. Here, we generated a neuroblastoma cell line that expresses a fluorescent reporter under conditions of HSR induction, for ex le heat shock. Using these cells, we show that the HSR is not induced by exogenous treatment with aggregated forms of Parkinson’s disease-associated α -synuclein or the ALS-associated G93A mutant of superoxide dismutase-1 (SOD1 G93A ). Furthermore, flow cytometric analysis revealed that intracellular expression of SOD1 G93A or a pathogenic form of polyQ-expanded huntingtin (Htt 72Q ), similarly, results in no or low induction of the HSR. In contrast, expression of a non-pathogenic but aggregation-prone form of firefly luciferase (Fluc) did induce an HSR in a significantly greater proportion of cells. Finally, we show that HSR induction is dependent on the intracellular levels of the aggregation-prone proteins, but the pathogenic proteins (SOD1 G93A and Htt 72Q ) elicit a significantly lower HSR compared to the non-pathogenic proteins (Fluc). These results suggest that pathogenic proteins either evade detection or impair induction of the HSR in neuronal-like cells. Therefore, defective HSR induction may facilitate the initiation of protein aggregation leading to inclusion formation in neurodegenerative diseases.
Longitudinal assessment of metal concentrations and copper isotope ratios in the G93A SOD1 mouse model of amyotrophic lateral sclerosis
Publisher: Oxford University Press (OUP)
Date: 2017
DOI: 10.1039/C6MT00270F
Abstract: Amyotrophic lateral sclerosis (ALS) is a motor neuron disease, which involves progressive motor neuron degeneration in the central nervous system (CNS). The G93A SOD1 mouse model simulates one of the most common causes of familial ALS through the overexpression of a mutated form of the human gene encoding copper/zinc superoxide dismutase (SOD1). Transition metals, particularly Cu and Zn, have been shown to behave abnormally in the disease context and have been hypothesized to contribute to and potentially trigger the disease. In this study, concentrations of Cu, Zn and Fe, as well as Cu isotope ratios were assessed in keystone tissues of ALS, including the brain, spinal cord, muscle and whole blood, from transgenic mutant SOD1
Functional amyloid protection in the eye lens: Retention of α-crystallin molecular chaperone activity after modification into amyloid fibrils
Publisher: MDPI AG
Date: 12-09-2017
DOI: 10.3390/BIOM7030067
The development of signal transduction pathways during epididymal maturation is calcium dependent
Publisher: Elsevier BV
Date: 04-2004
Evaluating the effect of phosphorylation on the structure and dynamics of Hsp27 dimers by means of ion mobility mass spectrometry
Publisher: American Chemical Society (ACS)
Date: 04-12-2017
DOI: 10.1021/ACS.ANALCHEM.7B03328
Abstract: The quaternary structure and dynamics of the human small heat-shock protein Hsp27 are linked to its molecular chaperone function and influenced by post-translational modifications, including phosphorylation. Phosphorylation of Hsp27 promotes oligomer dissociation and can enhance chaperone activity. This study explored the impact of phosphorylation on the quaternary structure and dynamics of Hsp27. Using mutations that mimic phosphorylation, and ion mobility mass spectrometry, we show that successive substitutions result in an increase in the conformational heterogeneity of Hsp27 dimers. In contrast, we did not detect any changes in the structure of an Hsp27 12-mer, representative of larger Hsp27 oligomers. Our data suggest that oligomer dissociation and increased flexibility of the dimer contribute to the enhanced chaperone activity of phosphorylated Hsp27. Thus, post-translational modifications such as phosphorylation play a crucial role in modulating both the tertiary and quaternary structure of Hsp27, which is pivotal to its function as a key component of the proteostasis network in cells. Our data demonstrate the utility of ion mobility mass spectrometry for probing the structure and dynamics of heterogeneous proteins.
Exploiting flow cytometry for the unbiased quantification of protein inclusions in Caenorhabditis elegans
Publisher: Wiley
Date: 02-03-2022
DOI: 10.1111/JNC.15591
Abstract: The aggregation of proteins into inclusions or plaques is a prominent hallmark of a erse range of pathologies including neurodegenerative diseases. The quantification of such inclusions in Caenorhabditis elegans models of aggregation is usually achieved by fluorescence microscopy or other techniques involving biochemical fractionation of worm lysates. Here, we describe a simple and rapid flow cytometry‐based approach that allows fluorescently tagged inclusions to be enumerated in whole worm lysate in a quantitative and unbiased fashion. We demonstrate that this technique is applicable to multiple C. elegans models of aggregation and importantly, can be used to monitor the dynamics of inclusion formation in response to heat shock and during ageing. This includes the characterisation of physicochemical properties of inclusions, such as their apparent size, which may reveal how aggregate formation is distinct in different tissues or at different stages of pathology or ageing. This new method can be used as a powerful technique for the medium‐ to high‐throughput quantification of inclusions in future studies of genetic or chemical modulators of aggregation in C. elegans . image
The effect of milk constituents and crowding agents on amyloid fibril formation by κ-casein
Publisher: American Chemical Society (ACS)
Date: 04-02-2016
Abstract: When not incorporated into the casein micelle, κ-casein, a major milk protein, rapidly forms amyloid fibrils at physiological pH and temperature. In this study, the effects of milk components (calcium, lactose, lipids, and heparan sulfate) and crowding agents on reduced and carboxymethylated (RCM) κ-casein fibril formation was investigated using far-UV circular dichroism spectroscopy, thioflavin T binding assays, and transmission electron microscopy. Longer-chain phosphatidylcholine lipids, which form the lining of milk ducts and milk fat globules, enhanced RCM κ-casein fibril formation irrespective of whether the lipids were in a monomeric or micellar state, whereas shorter-chain phospholipids and triglycerides had little effect. Heparan sulfate, a component of the milk fat globule membrane and catalyst of amyloid deposition in extracellular tissue, had little effect on the kinetics of RCM κ-casein fibril formation. Major nutritional components such as calcium and lactose also had no significant effect. Macromolecular crowding enhances protein-protein interactions, but in contrast to other fibril-forming species, the extent of RCM κ-casein fibril formation was reduced by the presence of a variety of crowding agents. These data are consistent with a mechanism of κ-casein fibril formation in which the rate-determining step is dissociation from the oligomer to give the highly amyloidogenic monomer. We conclude that the interaction of κ-casein with membrane-associated phospholipids along its secretory pathway may contribute to the development of amyloid deposits in mammary tissue. However, the formation of spherical oligomers such as casein micelles is favored over amyloid fibrils in the crowded environment of milk, within which the occurrence of amyloid fibrils is low.
DNAJB chaperones inhibit aggregation of destabilised proteins via a C-terminal region distinct from that used to prevent amyloid formation
Publisher: Cold Spring Harbor Laboratory
Date: 08-10-2020
DOI: 10.1101/2020.10.08.326280
Abstract: Disturbances to protein homeostasis (proteostasis) can lead to protein aggregation and inclusion formation, processes associated with a variety of neurodegenerative disorders. DNAJBs are molecular chaperones previously identified as potent suppressors of disease-related protein aggregation. In this work, we over-expressed a destabilised isoform of firefly luciferase (R188Q/R261Q Fluc Fluc DM ) in cells to assess the capacity of DNAJBs to inhibit inclusion formation. Co-expression of all DNAJBs tested significantly inhibited the intracellular aggregation of Fluc DM . Moreover, we show that DNAJBs suppress aggregation by supporting the Hsp70-dependent degradation of Fluc DM via the proteasome. The serine-rich stretch in DNAJB6 and DNAJB8, essential for preventing fibrillar aggregation, is not involved in the suppression of Fluc DM inclusion formation. Conversely, deletion of the C-terminal TTK-LKS region in DNAJB8, a region not required to suppress polyQ aggregation, abolished its ability to inhibit inclusion formation by Fluc DM . Thus, our data suggest that DNAJB6 and DNAJB8 possess two distinct domains involved in the inhibition of protein aggregation, one responsible for binding to β-hairpins that form during amyloid formation and another that mediates the degradation of destabilised client proteins via the proteasome. Specialised DNAJB molecular chaperones are potent suppressors of protein aggregation and interact with different types of client proteins via distinct C-terminal regions
Polymorphism in casein protein aggregation and amyloid fibril formation
Publisher: Elsevier
Date: 2013
Using bicistronic constructs to evaluate the chaperone activities of heat shock proteins in cells
Publisher: Springer Science and Business Media LLC
Date: 24-05-2017
DOI: 10.1038/S41598-017-02459-9
Abstract: Heat shock proteins (Hsps) are molecular chaperones that prevent the aggregation of client proteins by facilitating their refolding, or trafficking them for degradation. The chaperone activities of Hsps are dependent on dynamic protein-protein interactions, including their oligomerisation into large multi-subunit complexes. Thus, tagging Hsps with fluorescent proteins can interfere with their chaperone activity. To overcome this limitation, we have exploited bicistronic constructs for the concurrent expression of a non-tagged Hsp and fluorescent reporter from a single mRNA in cells. We used the Hsp-encoding bicistronic constructs in a cell-based model of protein aggregation, using a destabilised (mutant) form of firefly luciferase (mFluc) that forms inclusion bodies in cells. Expression of Hsp40, Hsp70, or Hsp40 and Hsp70 in cells expressing mFluc decreased the formation of inclusion bodies by 25–46% compared to controls. Moreover, there was a concentration-dependent decrease in the proportion of cells with inclusions when Hsp70, or Hsp40 and Hsp70 were co-expressed with mFluc in cells. The Hsp-encoding bicistronic constructs enable transfection efficiencies and concentration-dependent effects of Hsp expression to be determined using fluorescence based techniques, without the need to tag the Hsp with a fluorescent protein.
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.
Multi-kinase inhibitors can associate with heat shock proteins through their NH2-termini by which they suppress chaperone function
Publisher: Impact Journals, LLC
Date: 12-02-2016
Role of the epididymis in sperm competition
Publisher: Medknow
Date: 07-2007
DOI: 10.1111/J.1745-7262.2007.00284.X
Abstract: Although it is generally understood that the testes recruited kidney ducts for reproductive function during the evolution of vertebrates, little is understood of the biological significance of the adaptation. In the context of the evolution of the mammalian epididymis, this report provides evidence that a major role of the epididymis is to enhance a male's chance of achieving paternity in a competitive mating system. A unique ex le of sperm cooperation in monotremes is used as evidence that the epididymis produces sperm competition proteins to form groups of 100 sperm into bundles that have a forward motility nearly thrice that of in idual spermatozoa. As it required 3-h incubation in vitro under capacitation conditions to release motile sperm from the bundles, it is suggested that the monotremes provide an ex le of capacitation that is quite different from capacitation in higher mammals. It is suggested that variation between species in the intensity of sperm competition could explain the variation that occurs between species in the amount of post-testicular sperm maturation and storage in the epididymis, an explanation of why the human epididymis does not play as important a role in reproduction as the epididymis of most mammals.
Enhanced molecular chaperone activity of the small heat-shock protein αB-crystallin following covalent immobilization onto a solid-phase support
Publisher: Wiley
Date: 10-01-2011
DOI: 10.1002/BIP.21584
Abstract: The well-characterized small heat-shock protein, alphaB-crystallin, acts as a molecular chaperone by interacting with unfolding proteins to prevent their aggregation and precipitation. Structural perturbation (e.g., partial unfolding) enhances the in vitro chaperone activity of alphaB-crystallin. Proteins often undergo structural perturbations at the surface of a synthetic material, which may alter their biological activity. This study investigated the activity of alphaB-crystallin when covalently bound to a support surface alphaB-crystallin was immobilized onto a range of solid material surfaces, and its characteristics and chaperone activity were assessed. Immobilization was achieved via a plasma-deposited thin polymeric interlayer containing aldehyde surface groups and reductive amination, leading to the covalent binding of alphaB-crystallin lysine residues to the surface aldehyde groups via Schiff-base linkages. Immobilized alphaB-crystallin was characterized by X-ray photoelectron spectroscopy, atomic force microscopy, and quartz crystal microgravimetry, which showed that 300 ng cm(-2) (dry mass) of oligomeric alphaB-crystallin was bound to the surface. Immobilized alphaB-crystallin exhibited a significant enhancement (up to 5000-fold, when compared with the equivalent activity of alphaB-crystallin in solution) of its chaperone activity against various proteins undergoing both amorphous and amyloid fibril forms of aggregation. The enhanced molecular chaperone activity of immobilized alphaB-crystallin has potential applications in preventing protein misfolding, including against amyloid disease processes, such as dialysis-related amyloidosis, and for biodiagnostic detection of misfolded proteins.
Testicular descent, sperm maturation and capacitation. Lessons from our most distant relatives, the monotremes
Publisher: CSIRO Publishing
Date: 2009
DOI: 10.1071/RD09081
Abstract: The present review examines whether monotremes may help to resolve three questions relating to sperm production in mammals: why the testes descend into a scrotum in most mammals, why spermatozoa are infertile when they leave the testes and require a period of maturation in the specific milieu provided by the epididymides, and why ejaculated spermatozoa cannot immediately fertilise an ovum until they undergo capacitation within the female reproductive tract. Comparisons of monotremes with other mammals indicate that there is a need for considerable work on monotremes. It is hypothesised that testicular descent should be related to epididymal differentiation. Spermatozoa and ova from both groups share many of the proteins that are thought to be involved in gamete interaction, and although epididymal sperm maturation is significant it is probably less complex in monotremes than in other mammals. However, the monotreme epididymis is unique in forming spermatozoa into bundles of 100 with greatly enhanced motility compared with in idual spermatozoa. Bundle formation involves a highly organised interaction with epididymal proteins, and the bundles persist during incubation in vitro, except in specialised medium, in which spermatozoa separate after 2–3 h incubation. It is suggested that this represents an early form of capacitation.
Binding of the molecular chaperone αb-crystallin to Aβ amyloid fibrils inhibits fibril elongation
Publisher: Elsevier BV
Date: 10-2011
Unraveling the mysteries of protein folding and misfolding
Publisher: Wiley
Date: 12-2008
DOI: 10.1002/IUB.117
Abstract: This mini-review focuses on the processes and consequences of protein folding and misfolding. The latter process often leads to protein aggregation and precipitation with the aggregates adopting either highly ordered (amyloid fibril) or disordered (amorphous) forms. In particular, the amyloid fibril is discussed because this form has gained considerable notoriety due to its close links to a variety of debilitating diseases including Alzheimer's, Parkinson's, Huntington's, and Creutzfeldt-Jakob diseases, and type-II diabetes. In each of these diseases a different protein forms fibrils, yet the fibrils formed have a very similar structure. The mechanism by which fibrils form, fibril structure, and the cytotoxicity associated with fibril formation are discussed. The generic nature of amyloid fibril structure suggests that a common target may be accessible to treat amyloid fibril-associated diseases. As such, the ability of some molecules, for ex le, the small heat-shock family of molecular chaperone proteins, to inhibit fibril formation is of interest due to their therapeutic potential.
Numerical investigation of transient, low-power metal vapour discharges occurring in near limit ignitions of flammable gas
Publisher: IOP Publishing
Date: 15-11-2018
Casein structures in the context of unfolded proteins
Publisher: Elsevier BV
Date: 07-2015
The influence of the N-terminal region proximal to the core domain on the assembly and chaperone activity of αB-crystallin
Publisher: Springer Science and Business Media LLC
Date: 08-03-2018
The small heat shock proteins αb-crystallin and Hsp27 suppress SOD1 aggregation in vitro
Publisher: Springer Science and Business Media LLC
Date: 21-09-2013
Invited review: Caseins and the casein micelle: Their biological functions, structures, and behavior in foods1
Publisher: American Dairy Science Association
Date: 10-2013
Abstract: A typical casein micelle contains thousands of casein molecules, most of which form thermodynamically stable complexes with nanoclusters of amorphous calcium phosphate. Like many other unfolded proteins, caseins have an actual or potential tendency to assemble into toxic amyloid fibrils, particularly at the high concentrations found in milk. Fibrils do not form in milk because an alternative aggregation pathway is followed that results in formation of the casein micelle. As a result of forming micelles, nutritious milk can be secreted and stored without causing either pathological calcification or amyloidosis of the mother's mammary tissue. The ability to sequester nanoclusters of amorphous calcium phosphate in a stable complex is not unique to caseins. It has been demonstrated using a number of noncasein secreted phosphoproteins and may be of general physiological importance in preventing calcification of other biofluids and soft tissues. Thus, competent noncasein phosphoproteins have similar patterns of phosphorylation and the same type of flexible, unfolded conformation as caseins. The ability to suppress amyloid fibril formation by forming an alternative amorphous aggregate is also not unique to caseins and underlies the action of molecular chaperones such as the small heat-shock proteins. The open structure of the protein matrix of casein micelles is fragile and easily perturbed by changes in its environment. Perturbations can cause the polypeptide chains to segregate into regions of greater and lesser density. As a result, the reliable determination of the native structure of casein micelles continues to be extremely challenging. The biological functions of caseins, such as their chaperone activity, are determined by their composition and flexible conformation and by how the casein polypeptide chains interact with each other. These same properties determine how caseins behave in the manufacture of many dairy products and how they can be used as functional ingredients in other foods.
The functional roles of the unstructured N- and C-terminal regions in αB-crystallin and other mammalian small heat-shock proteins
Publisher: Springer Science and Business Media LLC
Date: 08-04-2017
The growing world of small heat shock proteins: from structure to functions
Publisher: Springer Science and Business Media LLC
Date: 31-03-2017
Monotremes provide a key to understanding the evolutionary significance of epididymal sperm maturation
Publisher: Wiley
Date: 25-03-2011
DOI: 10.2164/JANDROL.110.012716
Abstract: It has been widely accepted that mammalian spermatozoa are infertile when they leave the testes and require a period of maturation in both the epididymis and the female reproductive tract before acquiring the ability to fertilize an oocyte. However, the necessity for such a complex process of posttesticular sperm maturation appears to be unique to mammals because it is well established that these processes do not directly influence the fertilizing ability of the spermatozoa of birds, reptiles, and other lower vertebrates. Because of their key evolutionary position and form of reproduction, we contend that monotremes (platypus and echidna) provide a unique model for resolving why these processes are necessary. In the present review, we examine evidence that the epididymal maturation of monotreme spermatozoa is far less complex than in other mammals. However, a unique feature of the monotreme epididymis lies in its ability to promote the formation of elaborate sperm bundles that serve to greatly enhance the cells' motility. It is suggested that this intriguing cooperative strategy used by monotreme sperm represents an early form of epididymal maturation that appears to have been elaborated upon during the evolution of higher mammals, possibly as an adaptation for sperm competition.
The epididymal soluble prion protein forms a high-molecular-mass complex in association with hydrophobic proteins
Publisher: Portland Press Ltd.
Date: 08-11-2005
DOI: 10.1042/BJ20050459
Abstract: We have shown previously that a ‘soluble’ form of PrP (prion protein), not associated with membranous vesicles, exists in the male reproductive fluid [Ecroyd, Sarradin, Dacheux and Gatti (2004) Biol. Reprod. 71, 993–1001]. Attempts to purify this ‘soluble’ PrP indicated that it behaves like a high-molecular-mass complex of more than 350 kDa and always co-purified with the same set of proteins. The main associated proteins were sequenced by MS and were found to match to clusterin (apolipoprotein J), BPI (bacterial permeability-increasing protein), carboxylesterase-like urinary excreted protein (cauxin), β-mannosidase and β-galactosidase. Immunoblotting and enzymatic assay confirmed the presence of clusterin and a cauxin-like protein and showed that a 17 kDa hydrophobic epididymal protein was also associated with this complex. These associated proteins were not separated by a high ionic strength treatment but were by 2-mercaptoethanol, probably due to its action on reducing disulphide bonds that maintain the interaction of components of the complex. Our results suggest that the associated PrP retains its GPI (glycosylphosphatidylinositol) anchor, in contrast with brain-derived PrP, and that it is resistant to cleavage by phosphatidylinositol-specific phospholipase C. Based on these results, the identity of the associated proteins and the overall biochemical properties of this protein ensemble, we suggest that ‘soluble’ PrP can form protein complexes that are maintained by hydrophobic interactions, in a similar manner to lipoprotein vesicles or micellar complexes.
Characterisation of Amyloid Fibril Formation by Small Heat-shock Chaperone Proteins Human αA-, αB- and R120G αB-Crystallins
Publisher: Elsevier BV
Date: 09-2007
DOI: 10.1016/J.JMB.2007.06.060
Abstract: AlphaB-Crystallin is a ubiquitous small heat-shock protein (sHsp) renowned for its chaperone ability to prevent target protein aggregation. It is stress-inducible and its up-regulation is associated with a number of disorders, including those linked to the deposition of misfolded proteins, such as Alzheimer's and Parkinson's diseases. We have characterised the formation of amyloid fibrils by human alphaB-crystallin in detail, and also that of alphaA-crystallin and the disease-related mutant R120G alphaB-crystallin. We find that the last 12 amino acid residues of the C-terminal region of alphaB-crystallin are predicted from their physico-chemical properties to have a very low propensity to aggregate. (1)H NMR spectroscopy reveals that this hydrophilic C-terminal region is flexible both in its solution state and in amyloid fibrils, where it protrudes from the fibrillar core. We demonstrate, in addition, that the equilibrium between different protofilament assemblies can be manipulated and controlled in vitro to select for particular alphaB-crystallin amyloid morphologies. Overall, this study suggests that there could be a fine balance in vivo between the native functional sHsp state and the formation of amyloid fibrils.
Exploiting flow cytometry for the unbiased quantification of protein inclusions inCaenorhabditis elegans
Publisher: Cold Spring Harbor Laboratory
Date: 31-08-2021
DOI: 10.1101/2021.08.29.458141
Abstract: The aggregation of proteins into inclusions or plaques is a prominent hallmark of a erse range of pathologies including neurodegenerative diseases. The quantification of such inclusions in Caenorhabditis elegans models of aggregation is usually achieved by fluorescence microscopy or other techniques involving biochemical fractionation of worm lysates. Here, we describe a simple and rapid flow cytometry-based approach that allows fluorescently-tagged inclusions to be enumerated in whole worm lysate in a quantitative and unbiased fashion. We demonstrate that this technique is applicable to multiple C. elegans models of aggregation and importantly, can be used to monitor the dynamics of inclusion formation in response to heat shock and during aging. This includes the characterisation of physicochemical properties of inclusions, such as their size, which may reveal how aggregate formation is distinct in different tissues or at different stages of pathology or aging. This new method can be used as a powerful technique for the medium- to high-throughput quantification of inclusions in future studies of genetic or chemical modulators of aggregation in C. elegans .
An automated chromatography procedure optimized for analysis of stable Cu isotopes from biological materials
Publisher: Royal Society of Chemistry (RSC)
Date: 2016
DOI: 10.1039/C6JA00120C
Abstract: An automated ion-exchange chromatography method is presented for the separation of copper from biological s les for stable, isotope analysis.
Endogenous redox activity in mouse spermatozoa and its role in regulating the tyrosine phosphorylation events associated with sperm capacitation
Publisher: Oxford University Press (OUP)
Date: 07-2003
High tolerance of repeated heatwaves in Australian native plants
Publisher: Wiley
Date: 29-01-2019
DOI: 10.1111/AEC.12700
Tyrosine Phosphorylation of HSP-90 during Mammalian Sperm Capacitation
Publisher: Oxford University Press (OUP)
Date: 12-2003
Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations
Publisher: Walter de Gruyter GmbH
Date: 18-01-2016
Abstract: Living systems protect themselves from aberrant proteins by a network of chaperones. We have tested in vitro the effects of different concentrations, ranging from 0 to 16 μ m , of two molecular chaperones, namely αB-crystallin and clusterin, and an engineered monomeric variant of transthyretin (M-TTR), on the morphology and cytotoxicity of preformed toxic oligomers of HypF-N, which represent a useful model of misfolded protein aggregates. Using atomic force microscopy imaging and static light scattering analysis, all were found to bind HypF-N oligomers and increase the size of the aggregates, to an extent that correlates with chaperone concentration. SDS-PAGE profiles have shown that the large aggregates were predominantly composed of the HypF-N protein. ANS fluorescence measurements show that the chaperone-induced clustering of HypF-N oligomers does not change the overall solvent exposure of hydrophobic residues on the surface of the oligomers. αB-crystallin, clusterin and M-TTR can diminish the cytotoxic effects of the HypF-N oligomers at all chaperone concentration, as demonstrated by MTT reduction and Ca 2+ influx measurements. The observation that the protective effect is primarily at all concentrations of chaperones, both when the increase in HypF-N aggregate size is minimal and large, emphasizes the efficiency and versatility of these protein molecules.
An α-Cyanostilbene Derivative for the Enhanced Detection and Imaging of Amyloid Fibril Aggregates
Publisher: American Chemical Society (ACS)
Date: 23-11-2020
An epididymal form of cauxin, a carboxylesterase-like enzyme, is present and active in mammalian male reproductive fluids
Publisher: Oxford University Press (OUP)
Date: 02-2006
DOI: 10.1095/BIOLREPROD.105.046847
Abstract: Mass spectrometric analysis of a prion protein (PrP)-containing complex isolated from ram cauda epididymal fluid revealed a protein that showed homology to a carboxylesterase-like protein previously identified in cat urine (cauxin). Using anti-cauxin antibodies, immunoreactive bands were detected in corpus and cauda epididymal fluid from all mammals tested (ram, boar, mouse, and cat). In the ram, the protein was also present in seminal fluid but not found to be associated with sperm. The bands reacting with the anti-cauxin antibody coincided with those having esterase activity in a zymographic assay and its levels paralleled the esterase activity of native epididymal fluids. A partial nucleotide sequence of 1143 bp, corresponding to 380 amino acids, was obtained by RT-PCR lification from total RNA from the corpus epididymis (zone 6). The deduced protein sequence shows a high degree of homology (up to 90%) with the different cauxin proteins found in databases but only up to 60% with other known carboxylesterases. By PCR, strong mRNA expression was found in the corpus and cauda epididymis, while the testis, kidney, and caput epididymis had low expression. No mRNA was detected in the lung, heart, or liver. These data demonstrate that an epididymal form of the cauxin enzyme is secreted into mammalian epididymal fluid. In the ram, it is associated with a high molecular-weight PrP-associated complex and may be responsible for the majority of the esterase activity in the cauda epididymal fluid of this species.
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