Characterization of the Molecular-Chaperone Function of the Heat-Shock-Cognate-70-Interacting Protein

McKusick–Kaufman syndrome (MKKS) is a recessively inherited human genetic disease characterized by several developmental anomalies. Mutations in the MKKS gene also cause Bardet–Biedl syndrome (BBS), a genetically heterogeneous disorder with pleiotropic symptoms. However, little is known about how MKKS mutations lead to disease. Here, we show that disease-causing mutants of MKKS are rapidly degraded via the ubiquitin–proteasome pathway in a manner dependent on HSC70 interacting protein (CHIP), a chaperone-dependent ubiquitin ligase. Although wild-type MKKS quickly shuttles between the centrosome and cytosol in living cells, the rapidly degraded mutants often fail to localize to the centrosome. Inhibition of proteasome functions causes MKKS mutants to form insoluble structures at the centrosome. CHIP and partner chaperones, including heat-shock protein (HSP)70/heat-shock cognate 70 and HSP90, strongly recognize MKKS mutants. Modest knockdown of CHIP by RNA interference moderately inhibited the degradation of MKKS mutants. These results indicate that the MKKS mutants have an abnormal conformation and that chaperone-dependent degradation mediated by CHIP is a key feature of MKKS/BBS diseases.

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Exploring Novel Functions of the Small GTPase Ypt1p under Heat-Shock by Characterizing a Temperature-Sensitive Mutant Yeast Strain, ypt1-G80D

International Journal of Molecular Sciences ◽

10.3390/ijms20010132 ◽

2019 ◽

Vol 20 (1) ◽

pp. 132 ◽

Cited By ~ 1

Author(s):

Chang Ho Kang ◽

Joung Hun Park ◽

Eun Seon Lee ◽

Seol Ki Paeng ◽

Ho Byoung Chae ◽

...

Keyword(s):

Heat Stress ◽

Heat Shock ◽

Molecular Chaperone ◽

Stress Sensitivity ◽

Small Gtpase ◽

Stress Conditions ◽

Temperature Sensitive ◽

Heat Sensitivity ◽

Chemical Chaperone ◽

Chaperone Function

In our previous study, we found that Ypt1p, a Rab family small GTPase protein, exhibits a stress-driven structural and functional switch from a GTPase to a molecular chaperone, and mediates thermo tolerance in Saccharomyces cerevisiae. In the current study, we focused on the temperature-sensitive ypt1-G80D mutant, and found that the mutant cells are highly sensitive to heat-shock, due to a deficiency in the chaperone function of Ypt1pG80D. This defect results from an inability of the protein to form high molecular weight polymers, even though it retains almost normal GTPase function. The heat-stress sensitivity of ypt1-G80D cells was partially recovered by treatment with 4-phenylbutyric acid, a chemical chaperone. These findings indicate that loss of the chaperone function of Ypt1pG80D underlies the heat sensitivity of ypt1-G80D cells. We also compared the proteomes of YPT1 (wild-type) and ypt1-G80D cells to investigate Ypt1p-controlled proteins under heat-stress conditions. Our findings suggest that Ypt1p controls an abundance of proteins involved in metabolism, protein synthesis, cellular energy generation, stress response, and DNA regulation. Finally, we suggest that Ypt1p essentially regulates fundamental cellular processes under heat-stress conditions by acting as a molecular chaperone.

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Cloning and molecular characterization of heat shock cognate 70 from tiger shrimp (Penaeus monodon)

Cell Stress and Chaperones ◽

10.1379/csc-47r.1 ◽

2004 ◽

Vol 9 (4) ◽

pp. 332 ◽

Cited By ~ 54

Author(s):

Wan-Yu Lo ◽

Kuan-Fu Liu ◽

I-Chiu Liao ◽

Yen-Ling Song

Keyword(s):

Heat Shock ◽

Molecular Characterization ◽

Penaeus Monodon ◽

Tiger Shrimp ◽

Heat Shock Cognate 70

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Characterization of the Relationship between the Chaperone and Lipid-Binding Functions of the 70-Kda Heat-Shock Protein, HspA1A

10.20944/preprints202005.0150.v1 ◽

2020 ◽

Author(s):

Larissa Smulders ◽

Amanda Daniels ◽

Caroline Plescia ◽

Devon Burger ◽

Robert V. Stahelin ◽

...

Keyword(s):

Radiation Therapy ◽

Plasma Membrane ◽

Heat Shock ◽

Secondary Structure ◽

Molecular Chaperone ◽

Inorganic Phosphate ◽

Lipid Binding ◽

Cellular Survival ◽

The Relationship

HspA1A is a molecular chaperone that plays indispensable roles in cellular survival. HspA1A also translocates to the plasma membrane (PM) of stressed and cancer cells. This translocation results in the cell-surface presentation of HspA1A rendering these tumors radiation insensitive. Thus, a putative therapeutic would be to inhibit HspA1A’s PM translocation. However, to specifically stop the PM translocation of HspA1A, which is lipid-driven, it is imperative to characterize the lipid-binding regions of HspA1A and the relationship between the chaperone and lipid-binding functions of HspA1A, which remain unknown. To elucidate this relationship, we determined the effect of binding to phosphatidylserine (PS) on the secondary structure and chaperone functions of HspA1A. Circular dichroism revealed that binding to PS had minimal alterations on HspA1A’s secondary structure. Measuring the release of inorganic phosphate revealed that PS-binding had no effect on the ATPase activity of HspA1A. In contrast, PS-binding showed subtle but consistent increases in the refolding activities of HspA1A. These observations strongly support the notion that the chaperone and lipid-binding activities of HspA1A are dependent but the regions mediating these functions do not overlap. These findings provide the basis for future interventions to inhibit HspA1A’s PM-translocation in tumor cells, making them sensitive to radiation therapy.

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