scholarly journals Single-molecule fluorescence-based approach reveals novel mechanistic insights into human small heat shock protein chaperone function

2020 ◽  
pp. jbc.RA120.015419
Author(s):  
Caitlin L Johnston ◽  
Nicholas R Marzano ◽  
Bishnu P Paudel ◽  
George Wright ◽  
Justin L.P. Benesch ◽  
...  
Keyword(s):  
Heat Shock ◽  
Single Molecule ◽  
Small Heat Shock Protein ◽  
Vital Role ◽  
Misfolded Proteins ◽  
Single Molecule Fluorescence ◽  
Chaperone Function ◽  
Αb Crystallin ◽  
Client Proteins ◽  
Shock Proteins

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. The dynamic and polydisperse nature of sHsp oligomers has made studying them challenging using traditional biochemical approaches. Therefore, we have utilized a 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 dispersity 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.

scholarly journals Single-molecule fluorescence-based approach reveals novel mechanistic insights into small heat shock protein chaperone function

2020 ◽  
Author(s):  
Caitlin L. Johnston ◽  
Nicholas R. Marzano ◽  
Bishnu Paudel ◽  
George Wright ◽  
Justin L. P. Benesch ◽  
...  
Keyword(s):  
Heat Shock ◽  
Single Molecule ◽  
Protein Misfolding ◽  
Protein Complexes ◽  
Small Heat Shock Protein ◽  
Vital Role ◽  
Misfolded Proteins ◽  
Single Molecule Fluorescence ◽  
Chaperone Function ◽  
Αb Crystallin

AbstractSmall 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.

scholarly journals The Mitochondrial Small Heat Shock Protein HSP22 from Pea is a Thermosoluble Chaperone Prone to Co-Precipitate with Unfolding Client Proteins

2019 ◽  
Vol 21 (1) ◽  
pp. 97
Author(s):  
Marie-Hélène Avelange-Macherel ◽  
Aurélia Rolland ◽  
Marie-Pierre Hinault ◽  
Dimitri Tolleter ◽  
David Macherel
Keyword(s):  
Heat Shock ◽  
Recombinant Protein ◽  
Quaternary Structure ◽  
Small Heat Shock Protein ◽  
Effect Of Temperature ◽  
Molar Ratio ◽  
Client Proteins ◽  
Shock Proteins ◽  
Cellular Compartments

The small heat shock proteins (sHSPs) are molecular chaperones that share an alpha-crystallin domain but display a high diversity of sequence, expression, and localization. They are especially prominent in plants, populating most cellular compartments. In pea, mitochondrial HSP22 is induced by heat or oxidative stress in leaves but also strongly accumulates during seed development. The molecular function of HSP22 was addressed by studying the effect of temperature on its structural properties and chaperone effects using a recombinant or native protein. Overexpression of HSP22 significantly increased bacterial thermotolerance. The secondary structure of the recombinant protein was not affected by temperature in contrast with its quaternary structure. The purified protein formed large polydisperse oligomers that dissociated upon heating (42 °C) into smaller species (mainly monomers). The recombinant protein appeared thermosoluble but precipitated with thermosensitive proteins upon heat stress in assays either with single protein clients or within complex extracts. As shown by in vitro protection assays, HSP22 at high molar ratio could partly prevent the heat aggregation of rhodanese but not of malate dehydrogenase. HSP22 appears as a holdase that could possibly prevent the aggregation of some proteins while co-precipitating with others to facilitate their subsequent refolding by disaggregases or clearance by proteases.

Cytoskeletal disruption and small heat shock protein translocation immediately after lengthening contractions

2004 ◽  
Vol 286 (3) ◽  
pp. C713-C722 ◽  
Author(s):  
Timothy J. Koh ◽  
Joel Escobedo
Keyword(s):  
Heat Shock ◽  
Protein Translocation ◽  
Small Heat Shock Protein ◽  
Insoluble Fraction ◽  
Scaffolding Proteins ◽  
Lengthening Contractions ◽  
Small Hsps ◽  
Αb Crystallin ◽  
Shock Proteins ◽  
Cytoskeletal Elements

The purposes of this study were to determine whether, immediately after lengthening contractions, 1) levels of specific force-transmitting cytoskeletal elements are reduced in skeletal muscle cells and 2) cytosolic small heat shock proteins (HSPs) translocate to structures prone to disruption. Western blot analysis demonstrated decreased concentrations of z-disk proteins α-actinin and plectin and membrane scaffolding proteins dystrophin and β-spectrin in muscle exposed to lengthening contractions compared with contralateral control muscle. Lengthening contractions also resulted in immediate translocation of constitutively expressed HSP25 and αB-crystallin from the soluble to the insoluble fraction of muscle homogenates, and cryosections showed translocation from a diffuse, cytosolic localization to striations that corresponded to z-disks. Lengthening contraction-induced translocation of HSP25 and αB-crystallin was associated with phosphorylation of these small HSPs, which may trigger their protective activity. In summary, these findings demonstrate loss of z-disk and membrane scaffolding proteins immediately after lengthening contractions, and concomitant translocation of HSP25 and αB-crystallin to the z-disk, which may help to stabilize or repair cytoskeletal elements at this site.

scholarly journals Engineering of a Polydisperse Small Heat-Shock Protein Reveals Conserved Motifs of Oligomer Plasticity

2018 ◽  
Author(s):  
Sanjay Mishra ◽  
Shane A. Chandler ◽  
Dewight Williams ◽  
Derek P. Claxton ◽  
Hanane A. Koteiche ◽  
...  
Keyword(s):  
Heat Shock ◽  
Small Heat Shock Protein ◽  
Native Mass Spectrometry ◽  
General Mechanism ◽  
Modular Architecture ◽  
Subunit Exchange ◽  
Functional Regulation ◽  
Client Proteins ◽  
Shock Proteins ◽  
Functional Analyses

AbstractSmall heat-shock proteins (sHSP) are molecular chaperones that bind and sequester partially and globally unfolded states of their client proteins. Of paramount importance to their physiological roles is the assembly into large oligomers, which for mammalian sHSP are polydisperse and undergo subunit exchange. The flexibility and dynamic nature of these oligomers mediates functional regulation by phosphorylation and underpins the deleterious effects of disease-linked mutations. Previously, we discovered that the archaeal Hsp16.5, which natively forms ordered and symmetric 24-subunit oligomers, can be engineered to transition to an ordered and symmetric 48-subunit oligomer by insertion of a peptide from human HspB1 (Hsp27) at the junction of the N-terminal and α-crystallin domains. Here, we carried out a detailed analysis of the determinants of Hsp16.5 oligomeric plasticity by altering the sequence and length of the inserted peptide. Utilizing light scattering, blue native gel electrophoresis, native mass spectrometry and electron microscopy, we uncovered the existence of an array of oligomeric states (30 to 38 subunits) that can be populated as a consequence of different insertions. These oligomers are intermediate states on the assembly pathway of the 48-subunit oligomer as two of the variants can concurrently form 24-subunit or 30-38 subunit polydisperse oligomers. Polydisperse Hsp16.5 oligomers displayed higher affinity to a model client protein consistent with a general mechanism for recognition and binding that involves increased access of the hydrophobic N-terminal region. Our findings, which integrate structural and functional analyses from evolutionarily-distant sHSP, support a model wherein the modular architecture of these proteins encodes motifs of oligomer polydispersity, dissociation and expansion to achieve functional diversity and regulation.

scholarly journals Quaternary dynamics and plasticity underlie small heat shock protein chaperone function

2010 ◽  
Vol 107 (5) ◽  
pp. 2007-2012 ◽  
Author(s):  
Florian Stengel ◽  
Andrew J. Baldwin ◽  
Alexander J. Painter ◽  
Nomalie Jaya ◽  
Eman Basha ◽  
...  
Keyword(s):  
Heat Shock ◽  
Structural Biology ◽  
Protein Function ◽  
Quaternary Structure ◽  
Small Heat Shock Protein ◽  
Cellular Protein ◽  
Protein Homeostasis ◽  
Chaperone Function ◽  
Shock Proteins ◽  
Unique Mass

Small Heat Shock Proteins (sHSPs) are a diverse family of molecular chaperones that prevent protein aggregation by binding clients destabilized during cellular stress. Here we probe the architecture and dynamics of complexes formed between an oligomeric sHSP and client by employing unique mass spectrometry strategies. We observe over 300 different stoichiometries of interaction, demonstrating that an ensemble of structures underlies the protection these chaperones confer to unfolding clients. This astonishing heterogeneity not only makes the system quite distinct in behavior to ATP-dependent chaperones, but also renders it intractable by conventional structural biology approaches. We find that thermally regulated quaternary dynamics of the sHSP establish and maintain the plasticity of the system. This extends the paradigm that intrinsic dynamics are crucial to protein function to include equilibrium fluctuations in quaternary structure, and suggests they are integral to the sHSPs’ role in the cellular protein homeostasis network.

scholarly journals The Archaeal Small Heat Shock Protein Hsp17.6 Protects Proteins from Oxidative Inactivation

2021 ◽  
Vol 22 (5) ◽  
pp. 2591
Author(s):  
Pengfei Ma ◽  
Jie Li ◽  
Lei Qi ◽  
Xiuzhu Dong
Keyword(s):  
Heat Shock ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Small Heat Shock Protein ◽  
Phase Cell ◽  
Molecular Weights ◽  
Oxidative Inactivation ◽  
Methionine Residues ◽  
Shock Proteins ◽  
And Function

Small heat shock proteins (sHsps) are widely distributed among various types of organisms and function in preventing the irreversible aggregation of thermal denaturing proteins. Here, we report that Hsp17.6 from Methanolobus psychrophilus exhibited protection of proteins from oxidation inactivation. The overexpression of Hsp17.6 in Escherichia coli markedly increased the stationary phase cell density and survivability in HClO and H2O2. Treatments with 0.2 mM HClO or 10 mM H2O2 reduced malate dehydrogenase (MDH) activity to 57% and 77%, whereas the addition of Hsp17.6 recovered the activity to 70–90% and 86–100%, respectively. A similar effect for superoxide dismutase oxidation was determined for Hsp17.6. Non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis assays determined that the Hsp17.6 addition decreased H2O2-caused disulfide-linking protein contents and HClO-induced degradation of MDH; meanwhile, Hsp17.6 protein appeared to be oxidized with increased molecular weights. Mass spectrometry identified oxygen atoms introduced into the larger Hsp17.6 molecules, mainly at the aspartate and methionine residues. Substitution of some aspartate residues reduced Hsp17.6 in alleviating H2O2- and HClO-caused MDH inactivation and in enhancing the E. coli survivability in H2O2 and HClO, suggesting that the archaeal Hsp17.6 oxidation protection might depend on an “oxidant sink” effect, i.e., to consume the oxidants in environments via aspartate oxidation

scholarly journals Induction and phosphorylation of the small heat shock proteins HspB1/Hsp25 and HspB5/αB-crystallin in the rat retina upon optic nerve injury

2015 ◽  
Vol 21 (1) ◽  
pp. 167-178 ◽  
Author(s):  
Thomas Schmidt ◽  
Dietmar Fischer ◽  
Anastasia Andreadaki ◽  
Britta Bartelt-Kirbach ◽  
Nikola Golenhofen
Keyword(s):  
Optic Nerve ◽  
Heat Shock ◽  
Heat Shock Proteins ◽  
Nerve Injury ◽  
Small Heat Shock Proteins ◽  
Optic Nerve Injury ◽  
Rat Retina ◽  
Αb Crystallin ◽  
Shock Proteins

scholarly journals Heat shock factor 2 is required for maintaining proteostasis against febrile-range thermal stress and polyglutamine aggregation

2011 ◽  
Vol 22 (19) ◽  
pp. 3571-3583 ◽  
Author(s):  
Toyohide Shinkawa ◽  
Ke Tan ◽  
Mitsuaki Fujimoto ◽  
Naoki Hayashida ◽  
Kaoru Yamamoto ◽  
...  
Keyword(s):  
Thermal Stress ◽  
Heat Shock ◽  
Protein Misfolding ◽  
Reproductive Organs ◽  
Heat Shock Factors ◽  
Mild Heat ◽  
Promising Therapeutic Target ◽  
Αb Crystallin ◽  
Shock Proteins ◽  
Polyglutamine Protein

Heat shock response is characterized by the induction of heat shock proteins (HSPs), which facilitate protein folding, and non-HSP proteins with diverse functions, including protein degradation, and is regulated by heat shock factors (HSFs). HSF1 is a master regulator of HSP expression during heat shock in mammals, as is HSF3 in avians. HSF2 plays roles in development of the brain and reproductive organs. However, the fundamental roles of HSF2 in vertebrate cells have not been identified. Here we find that vertebrate HSF2 is activated during heat shock in the physiological range. HSF2 deficiency reduces threshold for chicken HSF3 or mouse HSF1 activation, resulting in increased HSP expression during mild heat shock. HSF2-null cells are more sensitive to sustained mild heat shock than wild-type cells, associated with the accumulation of ubiquitylated misfolded proteins. Furthermore, loss of HSF2 function increases the accumulation of aggregated polyglutamine protein and shortens the lifespan of R6/2 Huntington's disease mice, partly through αB-crystallin expression. These results identify HSF2 as a major regulator of proteostasis capacity against febrile-range thermal stress and suggest that HSF2 could be a promising therapeutic target for protein-misfolding diseases.

scholarly journals hsp26 of Saccharomyces cerevisiae is related to the superfamily of small heat shock proteins but is without a demonstrable function.

1989 ◽  
Vol 9 (11) ◽  
pp. 5265-5271 ◽  
Author(s):  
R E Susek ◽  
S L Lindquist
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Mutational Analysis ◽  
Small Heat Shock Protein ◽  
Major Effect ◽  
Selfish Dna ◽  
Dna Elements ◽  
Cloned Gene ◽  
Shock Proteins

Analysis of the cloned gene confirms that hsp26 of Saccharomyces cerevisiae is a member of the small heat shock protein superfamily. Previous mutational analysis failed to demonstrate any function for the protein. Further experiments presented here demonstrate that hsp26 has no obvious regulatory role and no major effect on thermotolerance. It is possible that the small heat shock protein genes originated as primitive viral or selfish DNA elements.

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