Hsp70 chaperone blocks α-synuclein oligomer formation via a novel engagement mechanism

Over-expression and aggregation of alpha-synuclein (ASyn) are linked to the onset and pathology of Parkinsons disease and related synucleinopathies. Elevated levels of the stress induced chaperone, Hsp70, protects against ASyn misfolding and ASyn-driven neurodegeneration in cell and animal models, yet there is minimal mechanistic understanding of this important protective pathway. It is generally assumed that Hsp70 binds to ASyn using its canonical and promiscuous substrate-binding cleft to limit aggregation. Here we report that this activity is due to a novel and unexpected mode of Hsp70 action, involving neither ATP nor the typical substrate-binding cleft. We use novel ASyn oligomerization assays to show that Hsp70 directly blocks ASyn oligomerization, an early event in ASyn misfolding. Using truncations, mutations and inhibitors, we confirmed that Hsp70 interacts with ASyn via an as yet unidentified, non-canonical interaction site in the C-terminal domain. Finally, a biological role for a non-canonical interaction was observed in H4 neuroglioma cells. Together, these findings suggest that new chemical approaches will be required to target Hsp70-ASyn interaction in synucleinopathies. Such approaches are likely to be more specific than targeting Hsp70 canonical actions. Additionally, these results raise the question of whether other misfolded proteins might also engage via the same non-canonical mechanism.

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Faculty Opinions recommendation of The cytoplasmic Hsp70 chaperone machinery subjects misfolded and endoplasmic reticulum import-incompetent proteins to degradation via the ubiquitin-proteasome system.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1047076.498127 ◽

2006 ◽

Author(s):

Cam Patterson

Keyword(s):

Endoplasmic Reticulum ◽

Ubiquitin Proteasome System ◽

Hsp70 Chaperone ◽

Ubiquitin Proteasome

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Faculty Opinions recommendation of Hsp90 breaks the deadlock of the hsp70 chaperone system.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.733133009.793562518 ◽

2019 ◽

Author(s):

Stuart Calderwood

Keyword(s):

Hsp70 Chaperone

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Review on the Application of Mixed-mode Chromatography for Separation of Structure Isoforms

Current Protein and Peptide Science ◽

10.2174/1389203718666171009111033 ◽

2018 ◽

Vol 20 (1) ◽

pp. 56-60 ◽

Cited By ~ 3

Author(s):

Tsutomu Arakawa

Keyword(s):

Ion Exchange ◽

Mixed Mode ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Charged State ◽

Partial Structure ◽

Reverse Phase ◽

Oligomer Formation ◽

Structure Rearrangement ◽

Disulfide Scrambling

Proteins often generate structure isoforms naturally or artificially due to, for example, different glycosylation, disulfide scrambling, partial structure rearrangement, oligomer formation or chemical modification. The isoform formations are normally accompanied by alterations in charged state or hydrophobicity. Thus, isoforms can be fractionated by reverse-phase, hydrophobic interaction or ion exchange chromatography. We have applied mixed-mode chromatography for fractionation of isoforms for several model proteins and observed that cation exchange Capto MMC and anion exchange Capto adhere columns are effective in separating conformational isoforms and self-associated oligomers.

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Disruption of the Complex between GAPDH and Hsp70 Sensitizes C6 Glioblastoma Cells to Hypoxic Stress

International Journal of Molecular Sciences ◽

10.3390/ijms22041520 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1520

Author(s):

Marina A. Mikeladze ◽

Elizaveta A. Dutysheva ◽

Victor G. Kartsev ◽

Boris A. Margulis ◽

Irina V. Guzhova ◽

...

Keyword(s):

Cell Death ◽

Hypoxic Stress ◽

C6 Cells ◽

Tumor Resistance ◽

Glioblastoma Cells ◽

Enzyme Expression ◽

Hsp70 Chaperone ◽

Therapeutic Tools ◽

Enzyme Molecules

Hypoxia, which commonly accompanies tumor growth, depending on its strength may cause the enhancement of tumorigenicity of cancer cells or their death. One of the proteins targeted by hypoxia is glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and we demonstrated here that hypoxia mimicked by treating C6 rat glioblastoma cells with cobalt chloride caused an up-regulation of the enzyme expression, while further elevation of hypoxic stress caused the enzyme aggregation concomitantly with cell death. Reduction or elevation of GAPDH performed with the aid of specific shRNAs resulted in the augmentation of the tumorigenicity of C6 cells or their sensitization to hypoxic stress. Another hypoxia-regulated protein, Hsp70 chaperone, was shown to prevent the aggregation of oxidized GAPDH and to reduce hypoxia-mediated cell death. In order to release the enzyme molecules from the chaperone, we employed its inhibitor, derivative of colchicine. The compound was found to substantially increase aggregation of GAPDH and to sensitize C6 cells to hypoxia both in vitro and in animals bearing tumors with distinct levels of the enzyme expression. In conclusion, blocking the chaperonic activity of Hsp70 and its interaction with GAPDH may become a promising strategy to overcome tumor resistance to multiple environmental stresses and enhance existing therapeutic tools.

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Kinetic and structural parameters governing Fic-mediated adenylylation/AMPylation of the Hsp70 chaperone, BiP/GRP78

Cell Stress and Chaperones ◽

10.1007/s12192-021-01208-2 ◽

2021 ◽

Author(s):

Anwesha Sanyal ◽

Erica A. Zbornik ◽

Ben G. Watson ◽

Charles Christoffer ◽

Jia Ma ◽

...

Keyword(s):

Structural Parameters ◽

Hsp70 Chaperone

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Prevention of High Glucose-Mediated EMT by Inhibition of Hsp70 Chaperone

International Journal of Molecular Sciences ◽

10.3390/ijms22136902 ◽

2021 ◽

Vol 22 (13) ◽

pp. 6902

Author(s):

Alina D. Nikotina ◽

Snezhana A. Vladimirova ◽

Elena Y. Komarova ◽

Dmitry Alexeev ◽

Sergey Efremov ◽

...

Keyword(s):

Colon Cancer ◽

Transcription Factors ◽

Cell Motility ◽

Cancer Cells ◽

High Glucose ◽

Binding Capacity ◽

Colon Cancer Cells ◽

Mesenchymal Transition ◽

Hsp70 Chaperone ◽

Migration Capacity

Hyperglycemia may contribute to the progression of carcinomas by triggering epithelial-to-mesenchymal transition (EMT). Some proteostasis systems are involved in metastasis; in this paper, we sought to explore the mechanism of Hsp70 chaperone in EMT. We showed that knockdown of Hsp70 reduced cell migration capacity concomitantly with levels of mRNA of the Slug, Snail, and Twist markers of EMT, in colon cancer cells incubated in high glucose medium. Conversely, treatment of cells with Hsp70 inducer U-133 were found to elevate cell motility, along with the other EMT markers. To prove that inhibiting Hsp70 may reduce EMT efficiency, we treated cells with a CL-43 inhibitor of the HSF1 transcription factor, which lowered Hsp70 and HSF1 content in the control and induced EMT in carcinoma cells. Importantly, CL-43 reduced migration capacity, EMT-linked transcription factors, and increased content of epithelial marker E-cadherin in colon cancer cells of three lines, including one derived from a clinical sample. To prove that Hsp70 chaperone should be targeted when inhibiting the EMT pathway, we treated cancer cells with 2-phenylethynesulfonamide (PES) and demonstrated that the compound inhibited substrate-binding capacity of Hsp70. Furthermore, PES suppressed EMT features, cell motility, and expression of specific transcription factors. In conclusion, the Hsp70 chaperone machine efficiently protects mechanisms of the EMT, and the safe inhibitors of the chaperone are needed to hamper metastasis at its initial stage.

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