HspB1 phosphorylation regulates its intramolecular dynamics and mechanosensitive molecular chaperone interaction with 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.

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

10.1101/325712 ◽

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.

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

Communications Biology ◽

10.1038/s42003-021-01864-1 ◽

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.

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Inhibition of acanthamoeba actomyosin-II ATPase activity and mechanochemical function by specific monoclonal antibodies.

The Journal of Cell Biology ◽

10.1083/jcb.99.3.1024 ◽

1984 ◽

Vol 99 (3) ◽

pp. 1024-1033 ◽

Cited By ~ 16

Author(s):

D P Kiehart ◽

T D Pollard

Keyword(s):

Monoclonal Antibodies ◽

Atpase Activity ◽

Conformational Changes ◽

Binding Sites ◽

Polyclonal Antibodies ◽

Myosin Ii ◽

Antigenic Site ◽

Actin Binding ◽

Filamentous Form ◽

Antibody Effects

Monoclonal and polyclonal antibodies that bind to myosin-II were tested for their ability to inhibit myosin ATPase activity, actomyosin ATPase activity, and contraction of cytoplasmic extracts. Numerous antibodies specifically inhibit the actin activated Mg++-ATPase activity of myosin-II in a dose-dependent fashion, but none blocked the ATPase activity of myosin alone. Control antibodies that do not bind to myosin-II and several specific antibodies that do bind have no effect on the actomyosin-II ATPase activity. In most cases, the saturation of a single antigenic site on the myosin-II heavy chain is sufficient for maximal inhibition of function. Numerous monoclonal antibodies also block the contraction of gelled extracts of Acanthamoeba cytoplasm. No polyclonal antibodies tested inhibited ATPase activity or gel contraction. As expected, most antibodies that block actin-activated ATPase activity also block gel contraction. Exceptions were three antibodies M2.2, -15, and -17, that appear to uncouple the ATPase activity from gel contraction: they block gel contraction without influencing ATPase activity. The mechanisms of inhibition of myosin function depends on the location of the antibody-binding sites. Those inhibitory antibodies that bind to the myosin-II heads presumably block actin binding or essential conformational changes in the myosin heads. A subset of the antibodies that bind to the proximal end of the myosin-II tail inhibit actomyosin-II ATPase activity and gel contraction. Although this part of the molecule is presumably some distance from the ATP and actin-binding sites, these antibody effects suggest that structural domains in this region are directly involved with or coupled to catalysis and energy transduction. A subset of the antibodies that bind to the tip of the myosin-II tail appear to inhibit ATPase activity and contraction through their inhibition of filament formation. They provide strong evidence for a substantial enhancement of the ATPase activity of myosin molecules in filamentous form and suggest that the myosin filaments may be required for cell motility.

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Phosphorylation-dependent Conformational Changes Induce a Switch in the Actin-binding Function of MARCKS

Journal of Biological Chemistry ◽

10.1074/jbc.274.51.36472 ◽

1999 ◽

Vol 274 (51) ◽

pp. 36472-36478 ◽

Cited By ~ 30

Author(s):

Michael R. Bubb ◽

Robert H. Lenox ◽

Arthur S. Edison

Keyword(s):

Conformational Changes ◽

Actin Binding ◽

Binding Function

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Deficiency in Nebulin Repeats of Sarcomeric Nebulette is Detrimental for Cardiomyocyte Tolerance to Exercise and Biomechanical Stress

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00732.2020 ◽

2021 ◽

Author(s):

Ramona M. Vejandla ◽

Buyan-Ochir Orgil ◽

Neely R. Alberson ◽

Ning Li ◽

Undral Munkhsaikhan ◽

...

Keyword(s):

Systolic Function ◽

Mechanical Strain ◽

Focal Adhesions ◽

Left Ventricular ◽

Signaling Network ◽

Treadmill Running ◽

Neonatal Rat ◽

Actin Binding ◽

Rat Cardiomyocytes ◽

Biomechanical Stress

Background: The actin-binding sarcomeric nebulette (NEBL) protein provides efficient contractile flexibility via interaction with desmin intermediate filaments. NEBL gene mutations affecting the nebulin repeat (NR) domain is known to induce cardiomyopathy. Objective: The study aimed to explore the roles of NEBL in exercise and biomechanical stress response. Methods: We ablated exon3 encoding the first NR of Nebl and created global Nebl3ex-/3ex- knockout mice. Cardiac function, structure and transcriptome was assessed before and after a 4-week treadmill regimen. A Nebl-based exercise signaling network was constructed using systems genetics methods. H9C2 and neonatal rat cardiomyocytes (NRCs) expressing wild-type or mutant NEBL underwent cyclic mechanical strain. Results: Nebl3ex-/3ex- mice demonstrated diastolic dysfunction with preserved systolic function at 6-months of age. After treadmill running, 4-month-old Nebl3ex-/3ex- mice developed concentric cardiac hypertrophy and left ventricular dilation compared to running Nebl+/+ and sedentary Nebl3ex-/3ex- mice. Disturbance of sarcomeric Z-disks and thin filaments architecture, disruption of intercalated disks and mitochondria were found in exercised Nebl3ex-/3ex- mice. A Nebl-based exercise signaling network included Csrp3, Des, Fbox32, Jup, Myh6, and Myh7. Disturbed expression of TM1, DES, JUP, b-catenin, MLP, α-actinin2 and vinculin proteins was demonstrated. In H9C2 cells, NEBL was recruited into focal adhesions at 24-hours post-strain and redistributed along with F-actin at 72-hours post-strain, suggesting time-dependent redistribution of NEBL in response to strain. NEBL mutations cause desmin disorganization in NRCs upon stretch. Conclusions: Upon stretch, NEBL deficiency causes disturbed sarcomere, Z-disks and desmin organization, and prevents NEBL redistribution to focal adhesions in cardiomyocytes, weakening cardiac tolerance to stress.

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Binding partner- and force-promoted changes in αE-catenin conformation probed by native cysteine labeling

Scientific Reports ◽

10.1038/s41598-019-51816-3 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Ksenia Terekhova ◽

Sabine Pokutta ◽

Yee S. Kee ◽

Jing Li ◽

Emad Tajkhorshid ◽

...

Keyword(s):

Conformational Changes ◽

Binding Protein ◽

Binding Domain ◽

Actin Binding ◽

Titration Calorimetry ◽

Allosteric Coupling ◽

Actin Binding Protein ◽

Binding Partners ◽

Actin Binding Domain ◽

Cysteine Labeling

Abstract Adherens Junctions (AJs) are cell-cell adhesion complexes that sense and propagate mechanical forces by coupling cadherins to the actin cytoskeleton via β-catenin and the F-actin binding protein αE-catenin. When subjected to mechanical force, the cadherin•catenin complex can tightly link to F-actin through αE-catenin, and also recruits the F-actin-binding protein vinculin. In this study, labeling of native cysteines combined with mass spectrometry revealed conformational changes in αE-catenin upon binding to the E-cadherin•β-catenin complex, vinculin and F-actin. A method to apply physiologically meaningful forces in solution revealed force-induced conformational changes in αE-catenin when bound to F-actin. Comparisons of wild-type αE-catenin and a mutant with enhanced vinculin affinity using cysteine labeling and isothermal titration calorimetry provide evidence for allosteric coupling of the N-terminal β-catenin-binding and the middle (M) vinculin-binding domain of αE-catenin. Cysteine labeling also revealed possible crosstalk between the actin-binding domain and the rest of the protein. The data provide insight into how binding partners and mechanical stress can regulate the conformation of full-length αE-catenin, and identify the M domain as a key transmitter of conformational changes.

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Substrate Binding Drives Large-Scale Conformational Changes in the Hsp90 Molecular Chaperone

Molecular Cell ◽

10.1016/j.molcel.2011.01.029 ◽

2011 ◽

Vol 42 (1) ◽

pp. 96-105 ◽

Cited By ~ 116

Author(s):

Timothy O. Street ◽

Laura A. Lavery ◽

David A. Agard

Keyword(s):

Conformational Changes ◽

Molecular Chaperone ◽

Large Scale ◽

Substrate Binding

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Nebulin Interacts with CapZ and Regulates Thin Filament Architecture within the Z-Disc

Molecular Biology of the Cell ◽

10.1091/mbc.e07-07-0690 ◽

2008 ◽

Vol 19 (5) ◽

pp. 1837-1847 ◽

Cited By ~ 53

Author(s):

Christopher T. Pappas ◽

Nandini Bhattacharya ◽

John A. Cooper ◽

Carol C. Gregorio

Keyword(s):

Actin Filaments ◽

Thin Filament ◽

Actin Polymerization ◽

Striated Muscle ◽

Solid Phase ◽

Molecular Model ◽

Direct Interaction ◽

Tryptophan Fluorescence ◽

Actin Binding

The barbed ends of actin filaments in striated muscle are anchored within the Z-disc and capped by CapZ; this protein blocks actin polymerization and depolymerization in vitro. The mature lengths of the thin filaments are likely specified by the giant “molecular ruler” nebulin, which spans the length of the thin filament. Here, we report that CapZ specifically interacts with the C terminus of nebulin (modules 160–164) in blot overlay, solid-phase binding, tryptophan fluorescence, and SPOTs membrane assays. Binding of nebulin modules 160–164 to CapZ does not affect the ability of CapZ to cap actin filaments in vitro, consistent with our observation that neither of the two C-terminal actin binding regions of CapZ is necessary for its interaction with nebulin. Knockdown of nebulin in chick skeletal myotubes using small interfering RNA results in a reduction of assembled CapZ, and, strikingly, a loss of the uniform alignment of the barbed ends of the actin filaments. These data suggest that nebulin restricts the position of thin filament barbed ends to the Z-disc via a direct interaction with CapZ. We propose a novel molecular model of Z-disc architecture in which nebulin interacts with CapZ from a thin filament of an adjacent sarcomere, thus providing a structural link between sarcomeres.

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Analysis of myofibrillar structure and assembly using fluorescently labeled contractile proteins.

The Journal of Cell Biology ◽

10.1083/jcb.98.3.825 ◽

1984 ◽

Vol 98 (3) ◽

pp. 825-833 ◽

Cited By ~ 65

Author(s):

J W Sanger ◽

B Mittal ◽

J M Sanger

Keyword(s):

Actin Filaments ◽

Striated Muscle ◽

Contractile Proteins ◽

Actin Binding ◽

Thin Filaments ◽

In Vitro System ◽

Self Association ◽

Cross Bridges

To study how contractile proteins become organized into sarcomeric units in striated muscle, we have exposed glycerinated myofibrils to fluorescently labeled actin, alpha-actinin, and tropomyosin. In this in vitro system, alpha-actinin bound to the Z-bands and the binding could not be saturated by prior addition of excess unlabeled alpha-actinin. Conditions known to prevent self-association of alpha-actinin, however, blocked the binding of fluorescently labeled alpha-actinin to Z-bands. When tropomyosin was removed from the myofibrils, alpha-actinin then added to the thin filaments as well as the Z-bands. Actin bound in a doublet pattern to the regions of the myosin filaments where there were free cross-bridges i.e., in that part of the A-band free of interdigitating native thin filaments but not in the center of the A-band which lacks cross-bridges. In the presence of 0.1-0.2 mM ATP, no actin binding occurred. When unlabeled alpha-actinin was added first to myofibrils and then labeled actin was added fluorescence occurred not in a doublet pattern but along the entire length of the myofibril. Tropomyosin did not bind to myofibrils unless the existing tropomyosin was first removed, in which case it added to the thin filaments in the l-band. Tropomyosin did bind, however, to the exogenously added tropomyosin-free actin that localizes as a doublet in the A-band. These results indicate that the alpha-actinin present in Z-bands of myofibrils is fully complexed with actin, but can bind exogenous alpha-actinin and, if actin is added subsequently, the exogenous alpha-actinin in the Z-band will bind the newly formed fluorescent actin filaments. Myofibrillar actin filaments did not increase in length when G-actin was present under polymerizing conditions, nor did they bind any added tropomyosin. These observations are discussed in terms of the structure and in vivo assembly of myofibrils.

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