scholarly journals Phosphorylation of HspB1 regulates its mechanosensitive molecular chaperone interaction with native filamin C

2018 ◽  
Author(s):  
Miranda P. Collier ◽  
T. Reid Alderson ◽  
Carin P. de Villiers ◽  
Daisy Nicholls ◽  
Heidi Y. Gastall ◽  
...  
Keyword(s):  
Mechanical Stress ◽  
Molecular Chaperone ◽  
Striated Muscle ◽  
Native Mass Spectrometry ◽  
Cellular Stress Response ◽  
Binding Affinities ◽  
Filamin A ◽  
Heterogeneous Structures ◽  
Shock Proteins ◽  
Filamin C

AbstractSmall heat-shock proteins (sHsps; HspBs) are molecular chaperones involved in the cellular stress response and a range of basal functions. Despite a multitude of targets, sHsp interactions are not well understood due their heterogeneous structures and weak binding affinities. The most widely expressed human sHsp, HspB1, is prevalent in striated muscle, where the actin cross-linker filamin C (FLNC, γ-filamin, ABP-L) is a putative binding partner. Musculoskeletal HspB1 is phosphorylated in response to a variety of cues, including mechanical stress, which promotes oligomer disassembly and association with myoarchitectural elements. Here, we report the up-regulation and interaction of both proteins in the hearts of a mouse model of heart failure, with HspB1 being phosphorylated and FLNC increasingly associated with the sarcomeric Z-disc. We used a combination of structural approaches to reveal that phosphorylation of HspB1 results in increased availability of the residues surrounding the phosphosite, facilitating their interaction with folded FLNC domains equivalent to a force-sensing region in the paralog filamin A. By employing native mass spectrometry, we show that domains 18 to 21 of FLNC are extensible under conditions mimicking force, with phosphorylated HspB1 stabilising an intermediate from further unfolding. These findings report on conformations accessible during the cycles of mechanical extension central to filamin function, and are consistent with an interaction between the chaperone and a native target that is strengthened upon the application of force. This may represent a new mode of molecular chaperone activity, allowing HspB1 to protect FLNC from over-extension during mechanical stress.

scholarly journals HspB1 phosphorylation regulates its intramolecular dynamics and mechanosensitive molecular chaperone interaction with filamin C

2019 ◽  
Vol 5 (5) ◽  
pp. eaav8421 ◽  
Author(s):  
Miranda P. Collier ◽  
T. Reid Alderson ◽  
Carin P. de Villiers ◽  
Daisy Nicholls ◽  
Heidi Y. Gastall ◽  
...  
Keyword(s):  
Mechanical Stress ◽  
Conformational Changes ◽  
Molecular Chaperone ◽  
Striated Muscle ◽  
Biomechanical Properties ◽  
Actin Binding ◽  
Intramolecular Dynamics ◽  
Protection Mechanism ◽  
Biomechanical Stress ◽  
Filamin C

Mechanical force–induced conformational changes in proteins underpin a variety of physiological functions, typified in muscle contractile machinery. Mutations in the actin-binding protein filamin C (FLNC) are linked to musculoskeletal pathologies characterized by altered biomechanical properties and sometimes aggregates. HspB1, an abundant molecular chaperone, is prevalent in striated muscle where it is phosphorylated in response to cues including mechanical stress. We report the interaction and up-regulation of both proteins in three mouse models of biomechanical stress, with HspB1 being phosphorylated and FLNC being localized to load-bearing sites. We show how phosphorylation leads to increased exposure of the residues surrounding the HspB1 phosphosite, facilitating their binding to a compact multidomain region of FLNC proposed to have mechanosensing functions. Steered unfolding of FLNC reveals that its extension trajectory is modulated by the phosphorylated region of HspB1. This may represent a posttranslationally regulated chaperone-client protection mechanism targeting over-extension during mechanical stress.

scholarly journals Myopathy associated LDB3 mutation causes Z-disc disassembly and protein aggregation through PKCα and TSC2-mTOR downregulation

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Pankaj Pathak ◽  
Yotam Blech-Hermoni ◽  
Kalpana Subedi ◽  
Jessica Mpamugo ◽  
Charissa Obeng-Nyarko ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Aggregation ◽  
Mechanical Stress ◽  
Striated Muscle ◽  
Stress Signaling ◽  
Lim Protein ◽  
Underlying Mechanisms ◽  
Lim Proteins ◽  
Bearing Structure ◽  
Filamin C

AbstractMechanical stress induced by contractions constantly threatens the integrity of muscle Z-disc, a crucial force-bearing structure in striated muscle. The PDZ-LIM proteins have been proposed to function as adaptors in transducing mechanical signals to preserve the Z-disc structure, however the underlying mechanisms remain poorly understood. Here, we show that LDB3, a well-characterized striated muscle PDZ-LIM protein, modulates mechanical stress signaling through interactions with the mechanosensing domain in filamin C, its chaperone HSPA8, and PKCα in the Z-disc of skeletal muscle. Studies of Ldb3Ala165Val/+ mice indicate that the myopathy-associated LDB3 p.Ala165Val mutation triggers early aggregation of filamin C and its chaperones at muscle Z-disc before aggregation of the mutant protein. The mutation causes protein aggregation and eventually Z-disc myofibrillar disruption by impairing PKCα and TSC2-mTOR, two important signaling pathways regulating protein stability and disposal of damaged cytoskeletal components at a major mechanosensor hub in the Z-disc of skeletal muscle.

scholarly journals The Effect of Oxidized Dopamine on the Structure and Molecular Chaperone Function of the Small Heat-Shock Proteins, αB-Crystallin and Hsp27

2021 ◽  
Vol 22 (7) ◽  
pp. 3700
Author(s):  
Junna Hayashi ◽  
Jennifer Ton ◽  
Sparsh Negi ◽  
Daniel E. K. M. Stephens ◽  
Dean L. Pountney ◽  
...  
Keyword(s):  
Heat Shock ◽  
Protein Aggregation ◽  
Molecular Chaperone ◽  
Cross Linking ◽  
Αb Crystallin ◽  
The Cross ◽  
Shock Proteins ◽  
And Function

Oxidation of the neurotransmitter, dopamine (DA), is a pathological hallmark of Parkinson’s disease (PD). Oxidized DA forms adducts with proteins which can alter their functionality. αB-crystallin and Hsp27 are intracellular, small heat-shock molecular chaperone proteins (sHsps) which form the first line of defense to prevent protein aggregation under conditions of cellular stress. In vitro, the effects of oxidized DA on the structure and function of αB-crystallin and Hsp27 were investigated. Oxidized DA promoted the cross-linking of αB-crystallin and Hsp27 to form well-defined dimer, trimer, tetramer, etc., species, as monitored by SDS-PAGE. Lysine residues were involved in the cross-links. The secondary structure of the sHsps was not altered significantly upon cross-linking with oxidized DA but their oligomeric size was increased. When modified with a molar equivalent of DA, sHsp chaperone functionality was largely retained in preventing both amorphous and amyloid fibrillar aggregation, including fibril formation of mutant (A53T) α-synuclein, a protein whose aggregation is associated with autosomal PD. In the main, higher levels of sHsp modification with DA led to a reduction in chaperone effectiveness. In vivo, DA is sequestered into acidic vesicles to prevent its oxidation and, intracellularly, oxidation is minimized by mM levels of the antioxidant, glutathione. In vitro, acidic pH and glutathione prevented the formation of oxidized DA-induced cross-linking of the sHsps. Oxidized DA-modified αB-crystallin and Hsp27 were not cytotoxic. In a cellular context, retention of significant chaperone functionality by mildly oxidized DA-modified sHsps would contribute to proteostasis by preventing protein aggregation (particularly of α-synuclein) that is associated with PD.

scholarly journals Relative interfacial cleavage energetics of protein complexes revealed by surface collisions

2019 ◽  
Vol 116 (17) ◽  
pp. 8143-8148 ◽  
Author(s):  
Sophie R. Harvey ◽  
Justin T. Seffernick ◽  
Royston S. Quintyn ◽  
Yang Song ◽  
Yue Ju ◽  
...  
Keyword(s):  
Computational Models ◽  
Protein Complexes ◽  
Specific Binding ◽  
Native Mass Spectrometry ◽  
Binding Affinities ◽  
Training Set ◽  
Subunit Interactions ◽  
Binding Partners ◽  
Relative Binding

To fulfill their biological functions, proteins must interact with their specific binding partners and often function as large assemblies composed of multiple proteins or proteins plus other biomolecules. Structural characterization of these complexes, including identification of all binding partners, their relative binding affinities, and complex topology, is integral for understanding function. Understanding how proteins assemble and how subunits in a complex interact is a cornerstone of structural biology. Here we report a native mass spectrometry (MS)-based method to characterize subunit interactions in globular protein complexes. We demonstrate that dissociation of protein complexes by surface collisions, at the lower end of the typical surface-induced dissociation (SID) collision energy range, consistently cleaves the weakest protein:protein interfaces, producing products that are reflective of the known structure. We present here combined results for multiple complexes as a training set, two validation cases, and four computational models. We show that SID appearance energies can be predicted from structures via a computationally derived expression containing three terms (number of residues in a given interface, unsatisfied hydrogen bonds, and a rigidity factor).

scholarly journals Thermally induced aggregation and its suppression of silkworm small heat shock protein sHSP19.9

2017 ◽  
Vol 46 (1) ◽  
pp. 57-64
Author(s):  
MT Hossain ◽  
Y Aso
Keyword(s):  
Ionic Strength ◽  
Heat Shock ◽  
Molecular Chaperone ◽  
Reaction Medium ◽  
Small Heat Shock Protein ◽  
Time Dependent ◽  
Thermally Induced ◽  
Shock Proteins ◽  
Low Ionic Strength ◽  
Induced Aggregation

About ten genes responsible for small heat-shock proteins (sHSP) have been isolated from silkworm. sHSP19.9 is one of the important member among them. Heat-induced stability of the sHSP was investigated at 60ºC with 20 mM HEPES buffer pH 7.7 containing 10 mM NaCl (low-ionic strength). Along with it probable suppression of the aggregation was also examined. At the mentioned reaction medium, sHSP19.9 was observed to be aggregated on the concentration- and time-dependent manners. It was successfully suppressed with dithiothreitol (DTT), higher-ionic strengths, Cysteine residues modifications and molecular chaperone: sHSP20.8.Bang. J. Anim. Sci. 2017. 46 (1): 57-64

scholarly journals Environmental Particulate Air Pollution Exposure and the Oxidative Stress Responses: A Brief Review of the Impact on the Organism and Animal Models of Research

2021 ◽  
Author(s):  
Pauline Brendler Goettems Fiorin ◽  
Mirna Stela Ludwig ◽  
Matias Nunes Frizzo ◽  
Thiago Gomes Heck
Keyword(s):  
Oxidative Stress ◽  
Particulate Matter ◽  
Stress Responses ◽  
Cellular Stress ◽  
Size Composition ◽  
Solid Particles ◽  
Cellular Stress Response ◽  
Liquid Droplets ◽  
Shock Proteins ◽  
Ultrafine Particulate Matter

Particulate matter (PM) is a mixture of solid particles and liquid droplets found in the air, and it is one of the most harmful air pollutants. When inhaled, it affects the pulmonary system, cardiovascular systems, and other tissues. The size, composition, and deposition of PM, mainly related to fine and ultrafine particulate matter, are factors that determine the harmful effects of exposure to particles. Among the main effects is the inducer of ROS production, and consequently oxidative tissue damage in target organs and other responses, mediated by inflammatory cytokines and cellular stress response. The main pathway through which particles are potent mediators of oxidative stress is the damage caused to DNA and lipid molecules, whereas the pro-inflammatory response involves an immune response against PM, which in turn, it is related to cell stress responses observed by heat shock proteins (HSPs) expression and release. Thus, the ability of an organism to respond to PM inhalation requires anti-oxidative, anti-inflammatory, and cellular stress defenses that can be impaired in susceptible subjects as people with chronic diseases as diabetes and obesity. In this chapter, we discuss the mechanistic aspects of PM effects on health and present some animal research models in particle inhalation studies.

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