Abstract 1055: The 70 kDa Heat Shock Cognate Protein Plays A Novel Role In Myocardial Chemokine Expression And Cardiac Dysfunction Following Ischemia And Reperfusion Injury

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Ning Zou ◽  
Lihua Ao ◽  
Xiaoping Yang ◽  
Xin Su ◽  
David A Fullerton ◽  
...  
Keyword(s):  
Heat Shock ◽  
Inflammatory Response ◽  
Cardiac Dysfunction ◽  
Substrate Binding ◽  
Binding Domain ◽  
Chemokine Expression ◽  
Heat Shock Cognate Protein ◽  
Substrate Binding Domain ◽  
Cognate Protein

Myocardial ischemia and reperfusion (I/R) causes the release of cellular proteins including heat shock proteins (HSPs). The inducible HSP70 induces cytokine production in monocytes and dendritic cells through Toll-like receptor 4 (TLR4) signaling. In evaluation of the role of HSPs in myocardial inflammatory response in a mouse heart global I/R model, we found that the 70 kDa heat shock cognate protein (HSC70), but not HSP70, was released into the extracellular space and the coronary effluent during I/R. These observations prompted us to hypothesize that HSC70 plays a novel role in postischemic myocardial inflammatory response and cardiac dysfunction. Methods: We subjected mouse hearts to global I/R (20 min/60 min) or perfusion using the Langendorff method. We examined: the effect of HSC70 antibody on myocardial chemokine expression and cardiac functional recovery following I/R, the effect of recombinant HSC70 on myocardial chemokine expression and cardiac function and the role of TLR4 and the HSC70 substrate-binding domain in the effect of HSC70 on the heart. Results: In comparison with non-immune IgG, anti-HSC70 reduced myocardial expression of KC and MCP-1 mRNAs and proteins following I/R. Moreover, treatment with anti-HSC70 improved postischemic cardiac functional recovery (66±5.4% of baseline vs. 28±5.1% of baseline in hearts treated with non-immune IgG, p<0.01). Recombinant HSC70 induced myocardial expression of KC and MCP-1 mRNAs and proteins and caused cardiac dysfunction (72±2.6% of baseline vs. 98±3.9% of baseline in perfusion controls, p<0.001) in hearts with competent TLR4 (C3H/HeN). Interestingly, these effects of HSC70 were abrogated in hearts with defective TLR4 (C3H/HeJ). The potency of HSC70 was completely lost in the absence of its substrate-binding domain. Conclusions: Taken together, our studies demonstrate, for the first time, that HSC70 plays an important role in the induction of myocardial chemokines and cardiac dysfunction during I/R. The effect of HSC70 is dependent on TLR4 and requires the presence of the substrate-binding domain. The results suggest that the release of HSC70 into the extracellular space elicits an inflammatory response and causes mechanic dysfunction in the heart.

scholarly journals Critical role of extracellular heat shock cognate protein 70 in the myocardial inflammatory response and cardiac dysfunction after global ischemia-reperfusion

2008 ◽  
Vol 294 (6) ◽  
pp. H2805-H2813 ◽  
Author(s):  
Ning Zou ◽  
Lihua Ao ◽  
Joseph C. Cleveland ◽  
Xiaoping Yang ◽  
Xin Su ◽  
...  
Keyword(s):  
Heat Shock ◽  
Inflammatory Response ◽  
Cardiac Function ◽  
Cardiac Dysfunction ◽  
Ischemia Reperfusion ◽  
Substrate Binding ◽  
Critical Role ◽  
Heat Shock Cognate Protein ◽  
Substrate Binding Domain ◽  
Cognate Protein

Previous studies showed that Toll-like receptor 4 (TLR4) modulates the myocardial inflammatory response to ischemia-reperfusion injury, and we recently found that cytokines link TLR4 to postischemic cardiac dysfunction. Although TLR4 can be activated in cultured cells by endogenous agents including heat shock protein 70, how it is activated during myocardial ischemia-reperfusion is unknown. In the present study, we examined 1) whether heat shock cognate protein 70 (HSC70), which is constitutively expressed in the myocardium, is released during ischemia-reperfusion; 2) whether extracellular HSC70 induces the myocardial inflammatory response and modulates cardiac function; and 3) whether HSC70 exerts these effects via TLR4. We subjected isolated mouse hearts to global ischemia-reperfusion via the Langendorff technique. Immunoblotting and immunostaining detected the release of HSC70 from the myocardium during reperfusion. Treatment with an antibody specific to HSC70 suppressed myocardial cytokine expression and improved cardiac functional recovery after ischemia-reperfusion. Recombinant HSC70 induced NF-κB activation and cytokine expression and depressed myocardial contractility in a TLR4-dependent manner. These effects required the substrate-binding domain of HSC70. Fluorescence resonance energy transfer analysis of isolated macrophages demonstrated that extracellular HSC70 interacts with TLR4. Therefore, this study demonstrates for the first time that 1) the myocardium releases HSC70 during ischemia-reperfusion, 2) extracellular HSC70 contributes to the postischemic myocardial inflammatory response and to cardiac dysfunction, 3) HSC70 exerts these effects through a TLR4-dependent mechanism, and 4) the substrate-binding domain of HSC70 is required to induce these effects. Thus extracellular HSC70 plays a critical role in regulating the myocardial innate immune response and cardiac function after ischemia-reperfusion.

P.168 Role of chaperonin-containing TCP complex (CCT) and heat shock cognate protein 70 (Hsc70) in the HCV RNA replication

2006 ◽  
Vol 36 ◽  
pp. S112-S113
Author(s):  
T. Suzuki ◽  
Y. Inoue ◽  
H. Aizaki ◽  
M. Matsuda ◽  
M. Shirakura ◽  
...  
Keyword(s):  
Heat Shock ◽  
Rna Replication ◽  
Hcv Rna ◽  
Heat Shock Cognate Protein ◽  
Cognate Protein

scholarly journals Insights into the effect of the J-domain on the substrate binding domain (SBD) of the 70 kDa heat shock protein, Hsp70, from a chimeric human J-SBD polypeptide

2018 ◽  
Author(s):  
Ana O. Tiroli-Cepeda ◽  
Thiago V. Seraphim ◽  
Júlio C. Borges ◽  
Carlos H. I. Ramos
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Substrate Binding ◽  
Binding Domain ◽  
Disulfonic Acid ◽  
Natural Interaction ◽  
C Terminus ◽  
J Domain ◽  
Chimeric Polypeptide ◽  
Substrate Binding Domain

AbstractDnaJ/Hsp40 chaperones deliver unfolded proteins and stimulate the ATPase activity of DnaK/Hsp70 via their J-domain, a crucial event for the function that this system has in assisting protein folding. The interaction between Hsp40 and Hsp70 is transient and thus difficult to study, since mixing the binding partners can lead to quick dissociation due to their low affinity, creating a challenge for detailed analysis. As a consequence, knowledge of many important aspects of the mechanism of interaction is still lacking, for instance, the effect that J-domain binding has on Hsp70. In this study, we investigated whether it would be possible to gain understanding of this interaction by engineering a chimeric polypeptide where the J-domain of Hsp40 was covalently attached to the substrate binding domain (SBD) of Hsp70 by a flexible linker. The rationale for this is that an increase in the proximity between the interacting partners in this engineered chimera will promote the natural interaction and facilitate the characterization of the protein– protein interaction, which is a requirement to gain further understanding of many biological processes. The resulting chimera, termed J-SBD, was properly folded and had properties not present in the SBD alone. J-SBD behaved primarily as a monomer in all conditions tested and exhibited chaperone activity, as shown by aggregation protection and substrate binding assays, which revealed decreased binding to bis-ANS, a probe for hydrophobic patches. Collectively, our results suggest that Hsp40 binding to Hsp70 via the J-domain shifts the Hsp70 equilibrium towards the monomer state to expose hydrophobic sites prone to substrate accommodation.AbbreviationsBis-ANS (4,4’-Dianilino-1,1’-Binaphthyl-5,5’-Disulfonic Acid; CD, circular dichroism; Hsp, heat shock protein; J-SBD, chimeric polypeptide in which the J-domain of Hsp40 (at the N-terminus) is covalently attached to the substrate binding domain of Hsp70 (at the C-terminus) by a flexible linker; SBD: substrate binding domain of Hsp70.

scholarly journals The β6/β7 region of the Hsp70 substrate-binding domain mediates heat-shock response and prion propagation

2017 ◽  
Vol 75 (8) ◽  
pp. 1445-1459 ◽  
Author(s):  
Linan Xu ◽  
Weibin Gong ◽  
Sarah A. Cusack ◽  
Huiwen Wu ◽  
Harriët M. Loovers ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Response ◽  
Substrate Binding ◽  
Binding Domain ◽  
Shock Response ◽  
Prion Propagation ◽  
Substrate Binding Domain

scholarly journals Necroptosis-blocking compound NBC1 targets heat shock protein 70 to inhibit MLKL polymerization and necroptosis

2020 ◽  
Vol 117 (12) ◽  
pp. 6521-6530 ◽  
Author(s):  
Andrea N. Johnston ◽  
Yuyong Ma ◽  
Hua Liu ◽  
Shuzhen Liu ◽  
Sarah Hanna-Addams ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Substrate Binding ◽  
Kinase Domain ◽  
Binding Domain ◽  
Compound Libraries ◽  
Cell Death Pathway ◽  
Molecule Inhibitor ◽  
Substrate Binding Domain

Necroptosis is a regulated necrotic cell death pathway involved in development and disease. Its signaling cascade results in the formation of disulfide bond-dependent amyloid-like polymers of mixed lineage kinase domain-like protein (MLKL), which mediate proinflammatory cell membrane disruption. We screened compound libraries provided by the National Cancer Institute and identified a small-molecule inhibitor of necroptosis named necroptosis-blocking compound 1 (NBC1). Biotin-labeled NBC1 specifically conjugates to heat shock protein Hsp70. NBC1 and PES-Cl, a known Hsp70 substrate-binding inhibitor, block the formation of MLKL polymers, but not MLKL tetramers in necroptosis-induced cells. In vitro,recombinant Hsp70 interacts with the N-terminal domain (NTD) of MLKL and promotes NTD polymerization, which has been shown to mediate the cell killing activity. Furthermore, the substrate-binding domain (SBD) of Hsp70 is sufficient to promote MLKL polymerization. NBC1 covalently conjugates cysteine 574 and cysteine 603 of the SBD to block its function. In addition, an SBD mutant with both cysteines mutated to serines loses its ability to promote MLKL polymerization. Interestingly, knockdown of Hsp70 in cells leads to MLKL destabilization, suggesting that MLKL might also be a client protein of Hsp70. In summary, using NBC1, an inhibitor of necroptosis, we identified Hsp70 as a molecular chaperone performing dual functions in necroptosis. It stabilizes MLKL protein under normal condition and promotes MLKL polymerization through its substrate-binding domain during necroptosis.

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