Translocation of Small Heat Shock Proteins to the Actin Cytoskeleton upon Proteasomal Inhibition

Verschuure, Pauline; Croes, Yvonne; IJssel, Paul R.L.A. van den; Quinlan, Roy A.; Jong, Wilfried W. de; Boelens, Wilbert C.

doi:10.1006/jmcc.2001.1493

Cited by 70 publications

(60 citation statements)

References 38 publications

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“…22 Furthermore, previous studies have shown that Hsp20 is translocated to actin filaments on stress, suggesting its cytoskeletal stabilizing function in cardiomyocytes. 23,32 Taken together, these properties of Hsp20 suggest that it may benefit the ischemic heart at multiple levels.…”

Section: Discussionmentioning

confidence: 99%

Novel Cardioprotective Role of a Small Heat-Shock Protein, Hsp20, Against Ischemia/Reperfusion Injury

et al. 2005

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Background-Heat-shock proteins (Hsps) have been shown to render cardioprotection from stress-induced injury; however, little is known about the role of another small heat-shock protein, Hsp20, which regulates activities of vasodilation and platelet aggregation, in cardioprotection against ischemia injury. We recently reported that increased expression of Hsp20 in cardiomyocytes was associated with improved contraction and protection against ␤-agonistinduced apoptosis. Methods and Results-To investigate whether overexpression of Hsp20 exerts protective effects in both ex vivo and in vivo ischemia/reperfusion (I/R) injury, we generated a transgenic (TG) mouse model with cardiac-specific overexpression of Hsp20 (10-fold). TG and wild-type (WT) hearts were then subjected to global no-flow I/R (45 minutes/120 minutes) using the Langendorff preparation. TG hearts exhibited improved recovery of contractile performance over the whole reperfusion period. This improvement was accompanied by a 2-fold decrease in lactate dehydrogenase released from the TG hearts. The extent of infarction and apoptotic cell death was also significantly decreased, which was associated with increased protein ratio of Bcl-2/Bax and reduced caspase-3 activity in TG hearts. Furthermore, in vivo experiments of 30-minute myocardial ischemia, via coronary artery occlusion, followed by 24-hour reperfusion, showed that the infarct region-to-risk region ratio was 8.1Ϯ1.1% in TG hearts (nϭ7), compared with 19.5Ϯ2.1% in WT hearts (nϭ11, PϽ0.001). Conclusions-Our data demonstrate that increased Hsp20 expression in the heart protects against I/R injury, resulting in improved recovery of cardiac function and reduced infarction. Thus, Hsp20 may constitute a new therapeutic target for ischemic heart diseases.

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Section: Discussionmentioning

confidence: 99%

Novel Cardioprotective Role of a Small Heat-Shock Protein, Hsp20, Against Ischemia/Reperfusion Injury

et al. 2005

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“…Numerous reports have shown that heat shock proteins may play multiple roles in cellular structure, metabolism, and adaptation to stress, thus protecting cells against various stimuli. 19,20 Interestingly, the onset of heart failure in rats, after coronary artery ligation, was associated with increases in myocardial HSP27, 21 whereas ischemic preconditioning caused translocation and phosphorylation of ␣B-crystallin in the isolated rat heart, suggesting its potential cardioprotective role. 22 Furthermore, ␣B-crystallin was shown to inhibit both the mitochondrial and death receptor pathways mediated cell apoptosis.…”

Section: Heat Shock Proteinsmentioning

confidence: 99%

Phosphoproteome Analysis of Cardiomyocytes Subjected to β-Adrenergic Stimulation

Chu

Egnaczyk

Zhao

et al. 2004

Circulation Research

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Abstract-Posttranslational modification of target substrates underlies biological processes through activation/inactivation of signaling cascades. To concurrently identify the phosphoprotein substrates associated with cardiac ␤-adrenergic signaling, the mouse myocyte phosphoproteome was analyzed using 2-D gel electrophoresis in combination with 32 P autoradiography. Phosphoprotein spots, detected by silver staining, were identified using MALDI-TOF mass spectrometry in conjunction with computer-assisted protein spot matching. Stimulation with isoproterenol (1 mol/L for 5 minutes) was associated with maximal increases in myocyte contractile parameters, and significant stimulation of the phosphorylation of troponin I (190Ϯ23%) and succinyl CoA synthetase (160Ϯ16%), whereas the phosphorylation of pyruvate dehydrogenase (48Ϯ10%), NADH-ubiquinone oxidoreductase (46Ϯ6%), heat shock protein 27 (18Ϯ3%), ␣B-crystallin (20Ϯ3%), and an unidentified 26-kDa protein (29Ϯ7%) was significantly decreased, compared with unstimulated cells (100%). After sustained (30 minutes) stimulation with isoproterenol, only the alterations in the phosphorylation levels of troponin I and NADH-ubiquinone oxidoreductase were maintained and de novo phosphorylation of a phosphoprotein (Ϸ20 kDa and pI 5.5) was observed. The tryptic peptide fragments of this phosphoprotein were sequenced using postsource decay mass spectrometry, and the protein was subsequently cloned and designated as p20, based on its high sequence homology with rat and human skeletal p20. The mouse cardiac p20 contains the conserved domain sequences for heat shock proteins, and the RRAS consensus sequence for cAMP-PKA substrates. LC-MS/MS phosphorylation mapping confirmed phosphorylation of Ser 16 in p20 on ␤-agonist stimulation. Adenoviral gene transfer of p20 was associated with significant increases in contractility and Ca transient peak in adult rat cardiomyocytes, suggesting an important role of p20 in cardiac function. These findings suggest that cardiomyocytes undergo significant posttranslational modification via phosphorylation in a multitude of proteins to dynamically fine-tune cardiac responses to ␤-adrenergic signaling. ␤-Adrenergic receptors and their signaling effectors are key mediators of neurohormonal influence over the cardiovascular system, modulating the strength, velocity, and frequency of cardiac contraction and relaxation. 1 At the subcellular level, the cardiac responses to ␤-adrenergic agonists are associated with phosphorylation of several downstream target phosphoproteins. Multiple studies have indicated that the most critical substrates for the positive inotropic and lusitropic ␤-agonist effects are phospholamban in the sarcoplasmic reticulum (SR) and troponin I (TnI) in the myofilaments, both phosphorylated through the cAMPdependent protein kinase A (PKA) pathway. 2-4 Phosphorylation of phospholamban relieves its inhibitory effects on SERCA2, with subsequent acceleration of SR Ca 2ϩ transport, and phosphorylation of TnI reduces the myofilament sensiti...

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“…Constitutive expression of two sHSPs, the ␣-crystallins of the vertebrate lens, plays a critical role in the acquisition and maintenance of lens optical properties (7)(8)(9). In the heart, ␣B-crystallin and heat-shock protein 27 are involved in stress tolerance (10). Inherited mutations in the ␣-crystallins have been linked to pathogenic conditions such as cataract and desmin-related myopathy (11,12).…”

mentioning

confidence: 99%

Mechanism of Chaperone Function in Small Heat-shock Proteins

Sathish

Stein

Yang

et al. 2003

Journal of Biological Chemistry

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To further develop the mechanistic understanding of small heat-shock protein (sHSP) chaperone activity, we investigate the nature of the intermediate states recognized by ␣-crystallin and the conformations that are stably bound. The model substrates consist of a set of well characterized, destabilized T4 Lysozyme (T4L) mutants that have been shown to differentially bind ␣-crystallin in a manner that reflects their free-energy of unfolding. A new approach for the detection of complex formation is introduced based on the conformational sensitivity of the fluorescent probe bimane, site-specifically introduced in T4L. Emission spectra of bimanelabeled T4L reveal two distinct patterns of intensity changes upon binding that depend on the molar ratio of ␣-crystallin to T4L. This directly demonstrates the twomode nature of the binding process by the ␣-crystallins. Biphasic binding isotherms, obtained and analyzed over a wide range of T4L concentrations, demonstrate a substantially quenched bimane fluorescence in the low affinity-bound T4L that is similar to the quenching level observed due to denaturant unfolding. Furthermore, the pattern of intensity changes that occur upon binding of a T4L variant, bimane-labeled at an alternative solvent-exposed site, establishes a direct correlation between the quenching level observed in binding and unfolding. The results can be interpreted in terms of a model where ␣-crystallin binds at least two conformationally distinct non-native states of T4L, one of which is substantially unfolded and is bound with low affinity. A high affinity binding mode to compact states may be relevant to chaperone function in the lens, where protein damage is unlikely to cause global unfolding.The cellular response to increased temperature and other forms of stress involves the expression of multiple superfamilies of heat-shock proteins (HSPs) 1 (1). Many HSPs are molecular chaperones that recognize and bind protein non-native states thereby suppressing aggregation and facilitating refolding and/or subsequent degradation (2, 3). Because the recognition process, the mechanism of substrate release, and the downstream fate of the bound substrate seem to be distinct in each superfamily, HSPs have evolved distinct roles in the context of the life cycle of proteins as well as during stress (2). Small heat shock proteins (sHSPs) are a ubiquitous superfamily of heat-shock proteins characterized by the relatively small mass of the polypeptide chain and the presence of a conserved protein module in their C termini, the ␣-crystallin domain (4, 5). They assemble into oligomeric structures with varying degrees of order and substantial divergence in size and symmetry across the evolutionary spectrum. Ten sHSPs have been identified in the human genome (6). Constitutive expression of two sHSPs, the ␣-crystallins of the vertebrate lens, plays a critical role in the acquisition and maintenance of lens optical properties (7-9). In the heart, ␣B-crystallin and heat-shock protein 27 are involved in stress tolerance (10). In...

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Translocation of Small Heat Shock Proteins to the Actin Cytoskeleton upon Proteasomal Inhibition

Cited by 70 publications

References 38 publications

Novel Cardioprotective Role of a Small Heat-Shock Protein, Hsp20, Against Ischemia/Reperfusion Injury

Novel Cardioprotective Role of a Small Heat-Shock Protein, Hsp20, Against Ischemia/Reperfusion Injury

Phosphoproteome Analysis of Cardiomyocytes Subjected to β-Adrenergic Stimulation

Mechanism of Chaperone Function in Small Heat-shock Proteins

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