Analysis of Molecular Chaperone Activities Using In Vitro and In Vivo Approaches

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.

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Molecular Chaperone GRP94 Binds to the Fanconi Anemia Group C Protein and Regulates Its Intracellular Expression

Blood ◽

10.1182/blood.v91.11.4379 ◽

1998 ◽

Vol 91 (11) ◽

pp. 4379-4386 ◽

Cited By ~ 45

Author(s):

Taizo Hoshino ◽

Jianxiang Wang ◽

Marcel P. Devetten ◽

Nobuhisa Iwata ◽

Sachiko Kajigaya ◽

...

Keyword(s):

Fanconi Anemia ◽

Molecular Chaperone ◽

B Lymphocyte ◽

Complementation Group ◽

Splicing Mutation ◽

Cytoplasmic Proteins ◽

Intracellular Expression ◽

Anemia Group

Abstract The FAC protein encoded by the gene defective in Fanconi anemia (FA) complementation group C binds to at least three ubiquitous cytoplasmic proteins in vitro. We used here the complete coding sequence ofFAC in a yeast two-hybrid screen to identify interacting proteins. The molecular chaperone GRP94 was isolated twice from a B-lymphocyte cDNA library. Binding was confirmed by coimmunoprecipitation of FAC and GRP94 from cytosolic, but not nuclear, lysates of transfected COS-1 cells, as well as from mouse liver cytoplasmic extracts. Deletion mutants of FAC showed that residues 103-308 were required for interaction with GRP94, and a natural splicing mutation within the IVS-4 of FAC that removes residues 111-148 failed to bind GRP94. Ribozyme-mediated inactivation of GRP94 in the rat NRK cell line led to significantly reduced levels of immunoreactive FAC and concomitant hypersensitivity to mitomycin C, similar to the cellular phenotype of FA. Our results demonstrate that GRP94 interacts with FAC both in vitro and in vivo and regulates its intracellular level in a cell culture model. In addition, the pathogenicity of the IVS-4 splicing mutation in the FAC gene may be mediated in part by its inability to bind to GRP94.

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Inhibition of the heat shock protein 90 molecular chaperone in vitro and in vivo by novel, synthetic, potent resorcinylic pyrazole/isoxazole amide analogues

Molecular Cancer Therapeutics ◽

10.1158/1535-7163.mct-07-0149 ◽

2007 ◽

Vol 6 (4) ◽

pp. 1198-1211 ◽

Cited By ~ 85

Author(s):

Swee Y. Sharp ◽

Chrisostomos Prodromou ◽

Kathy Boxall ◽

Marissa V. Powers ◽

Joanna L. Holmes ◽

...

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Molecular Chaperone ◽

Heat Shock Protein 90

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Conformational processing of oncogenic v-Src kinase by the molecular chaperone Hsp90

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1424342112 ◽

2015 ◽

Vol 112 (25) ◽

pp. E3189-E3198 ◽

Cited By ~ 49

Author(s):

Edgar E. Boczek ◽

Lasse G. Reefschläger ◽

Marco Dehling ◽

Tobias J. Struller ◽

Elisabeth Häusler ◽

...

Keyword(s):

Molecular Dynamics ◽

High Activity ◽

Molecular Chaperone ◽

Src Kinase ◽

Hydrogen Deuterium Exchange ◽

Hydrogen Deuterium ◽

Client Proteins ◽

Dynamics Simulations

Hsp90 is a molecular chaperone involved in the activation of numerous client proteins, including many kinases. The most stringent kinase client is the oncogenic kinase v-Src. To elucidate how Hsp90 chaperones kinases, we reconstituted v-Src kinase chaperoning in vitro and show that its activation is ATP-dependent, with the cochaperone Cdc37 increasing the efficiency. Consistent with in vivo results, we find that Hsp90 does not influence the almost identical c-Src kinase. To explain these findings, we designed Src kinase chimeras that gradually transform c-Src into v-Src and show that their Hsp90 dependence correlates with compactness and folding cooperativity. Molecular dynamics simulations and hydrogen/deuterium exchange of Hsp90-dependent Src kinase variants further reveal increased transitions between inactive and active states and exposure of specific kinase regions. Thus, Hsp90 shifts an ensemble of conformations of v-Src toward high activity states that would otherwise be metastable and poorly populated.

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Molecular Chaperones in the Yeast Endoplasmic Reticulum Maintain the Solubility of Proteins for Retrotranslocation and Degradation

The Journal of Cell Biology ◽

10.1083/jcb.153.5.1061 ◽

2001 ◽

Vol 153 (5) ◽

pp. 1061-1070 ◽

Cited By ~ 240

Author(s):

Shuh-ichi Nishikawa ◽

Sheara W. Fewell ◽

Yoshihito Kato ◽

Jeffrey L. Brodsky ◽

Toshiya Endo

Keyword(s):

Endoplasmic Reticulum ◽

Molecular Chaperone ◽

Molecular Chaperones ◽

Elevated Temperatures ◽

Carboxypeptidase Y ◽

Mutant Form ◽

Genes Encoding

Endoplasmic reticulum (ER)-associated degradation (ERAD) is the process by which aberrant proteins in the ER lumen are exported back to the cytosol and degraded by the proteasome. Although ER molecular chaperones are required for ERAD, their specific role(s) in this process have been ill defined. To understand how one group of interacting lumenal chaperones facilitates ERAD, the fates of pro–α-factor and a mutant form of carboxypeptidase Y were examined both in vivo and in vitro. We found that these ERAD substrates are stabilized and aggregate in the ER at elevated temperatures when BiP, the lumenal Hsp70 molecular chaperone, is mutated, or when the genes encoding the J domain–containing proteins Jem1p and Scj1p are deleted. In contrast, deletion of JEM1 and SCJ1 had little effect on the ERAD of a membrane protein. These results suggest that one role of the BiP, Jem1p, and Scj1p chaperones is to maintain lumenal ERAD substrates in a retrotranslocation-competent state.

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Heat-shock protein gp96/grp94 is an essential chaperone for the platelet glycoprotein Ib-IX-V complex

Blood ◽

10.1182/blood-2011-01-330464 ◽

2011 ◽

Vol 117 (26) ◽

pp. 7136-7144 ◽

Cited By ~ 46

Author(s):

Matthew Staron ◽

Shuang Wu ◽

Feng Hong ◽

Aleksandra Stojanovic ◽

Xiaoping Du ◽

...

Keyword(s):

Molecular Chaperone ◽

Bleeding Time ◽

Platelet Glycoprotein ◽

Glycoprotein Ib ◽

Giant Platelets ◽

Prolonged Bleeding Time ◽

Heat Shock Protein Gp96 ◽

Concomitant Reduction

The platelet glycoprotein Ib-IX-V complex (GPIb-IX-IV) is the receptor for VWF and is responsible for VWF-mediated platelet activation and aggregation. Loss of the GPIb-IX-V complex is pathogenic for Bernard-soulier Syndrome (BSS), which is characterized by macrothrombocytopenia and impaired platelet function. It remains unclear how the GPIb-IX-V complex is assembled and whether there is a role for a specific molecular chaperone in the process. In the present study, we report that the assembly of the GPIb-IX-V complex depends critically on a molecular chaperone in the endoplasmic reticulum (ER): gp96 (also known as grp94 and HSP90b1). gp96/grp94 deletion in the murine hematopoietic system results in thrombocytopenia, prolonged bleeding time, and giant platelets that are clinically indistinguishable from human BSS. Loss of gp96/grp94 in vivo and in vitro leads to the concomitant reduction in GPIb-IX complex expression due to ER-associated degradation. We further demonstrate that gp96/grp94 binds selectively to the GPIX subunit, but not to gpIbα or gpIbβ. Therefore, we identify the platelet GPIX subunit of the GPIb-IX-V complex as an obligate and novel client of gp96/grp94.

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DNAJB4 molecular chaperone distinguishes WT from mutant E-cadherin, determining their fate in vitro and in vivo

Human Molecular Genetics ◽

10.1093/hmg/ddt602 ◽

2013 ◽

Vol 23 (8) ◽

pp. 2094-2105 ◽

Cited By ~ 12

Author(s):

Joana Simões-Correia ◽

Diana I. Silva ◽

Soraia Melo ◽

Joana Figueiredo ◽

Joana Caldeira ◽

...

Keyword(s):

Molecular Chaperone ◽

E Cadherin

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DUK114, the Drosophila orthologue of bovine brain calpain activator protein, is a molecular chaperone

Biochemical Journal ◽

10.1042/bj20040668 ◽

2004 ◽

Vol 383 (1) ◽

pp. 165-170 ◽

Cited By ~ 26

Author(s):

Attila FARKAS ◽

Gábor NARDAI ◽

Peter CSERMELY ◽

Peter TOMPA ◽

Peter FRIEDRICH

Keyword(s):

Heat Shock ◽

Molecular Chaperone ◽

Molecular Chaperones ◽

Citrate Synthase ◽

Sequence Similarity ◽

Heat Shock Protein 90 ◽

Chaperone Activity ◽

In Vitro Assays

UK114, the goat liver tumour antigen, is a member of a widely distributed family of conserved low-molecular-mass proteins (YER057c/YjgF/UK114), the function of which is ill understood. To the various orthologues diverse functions have been ascribed, such as translation inhibition, regulation of purine repressor or calpain activation. Owing to a limited sequence similarity to Hsp90 (heat-shock protein 90), they have also been proposed to be molecular chaperones; however, this has never been tested. In the present paper, we report the cloning and characterization of the Drosophila orthologue, DUK114. In brief, DUK114 had no effect that would have qualified it as a calpain activator. In contrast, it proved to be a very potent molecular chaperone in in vitro assays. In a heat-aggregation test, it significantly decelerated the formation of citrate synthase aggregates. In a reverse assay, the recovery of the enzyme from urea- and heat-induced denatured states was accelerated almost 3-fold. On a molar basis, the chaperone activity of the 15-kDa DUK114 is comparable with that of Hsp90, the almost 6-times-larger archetypal molecular chaperone. In similar assays, DUK114 was ineffective with Drosophila calpain A or calpain B. To test for its chaperone activity in vivo, DUK114 was transfected into Schneider (S2) cells; after heat shock, the number of viable non-transfected cells started to increase after a lag time; in the presence of DUK114, cell proliferation started at once. Our work is the first experimental evidence that DUK114, and possibly other members of this family, are molecular chaperones.

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Molecular Chaperone GRP94 Binds to the Fanconi Anemia Group C Protein and Regulates Its Intracellular Expression

Blood ◽

10.1182/blood.v91.11.4379.411k14_4379_4386 ◽

1998 ◽

Vol 91 (11) ◽

pp. 4379-4386 ◽

Cited By ~ 5

Author(s):

Taizo Hoshino ◽

Jianxiang Wang ◽

Marcel P. Devetten ◽

Nobuhisa Iwata ◽

Sachiko Kajigaya ◽

...

Keyword(s):

Fanconi Anemia ◽

Molecular Chaperone ◽

B Lymphocyte ◽

Complementation Group ◽

Splicing Mutation ◽

Cytoplasmic Proteins ◽

Intracellular Expression ◽

Anemia Group

The FAC protein encoded by the gene defective in Fanconi anemia (FA) complementation group C binds to at least three ubiquitous cytoplasmic proteins in vitro. We used here the complete coding sequence ofFAC in a yeast two-hybrid screen to identify interacting proteins. The molecular chaperone GRP94 was isolated twice from a B-lymphocyte cDNA library. Binding was confirmed by coimmunoprecipitation of FAC and GRP94 from cytosolic, but not nuclear, lysates of transfected COS-1 cells, as well as from mouse liver cytoplasmic extracts. Deletion mutants of FAC showed that residues 103-308 were required for interaction with GRP94, and a natural splicing mutation within the IVS-4 of FAC that removes residues 111-148 failed to bind GRP94. Ribozyme-mediated inactivation of GRP94 in the rat NRK cell line led to significantly reduced levels of immunoreactive FAC and concomitant hypersensitivity to mitomycin C, similar to the cellular phenotype of FA. Our results demonstrate that GRP94 interacts with FAC both in vitro and in vivo and regulates its intracellular level in a cell culture model. In addition, the pathogenicity of the IVS-4 splicing mutation in the FAC gene may be mediated in part by its inability to bind to GRP94.

Download Full-text