Selective Hsp70-Dependent Docking of Hsp104 to Protein Aggregates Protects the Cell from the Toxicity of the Disaggregase

Chamera, Tomasz; Kłosowska, Agnieszka; Janta, Anna; Wyszkowski, Hubert; Obuchowski, Igor; Gumowski, Krzysztof; Liberek, Krzysztof

doi:10.1016/j.jmb.2019.04.014

Cited by 15 publications

(17 citation statements)

References 59 publications

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“…To gain more insight into the functional differences between the JDPs at the initial step of substrate processing, we employed biolayer interferometry (BLI). This technique analyzes changes of thickness of a protein layer at the surface of an optical sensor and, therefore, allows to observe the time-resolved binding of chaperones to a protein that had been immobilized and heat-aggregated on the sensor ( 44 ). The Sis1–Ssa1 system showed delayed binding to aggregated luciferase, reaching the thickness of ∼7 nm ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In previous studies, it has been shown that the efficacy of disaggregation depends strongly on how effectively Hsp104 binds to the substrate, which in turn largely relies on the Hsp104 interaction with Hsp70 ( 9 , 44 , 49 ). Knowing that Sis1–Ssa1 remodels aggregates in a way that results in more Hsp70 bound to the aggregate surface ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…BLI. Aggregate-binding experiments were performed as previously described (44), using the BLItz and Octet K2 instruments (ForteBio). The Ni-NTA biosensor (ForteBio Dip and Read) was initially hydrated with the buffer A; next, it was immersed for 10 min in the buffer A with 6-M urea and 8.2 μM His-tagged luciferase.…”

Section: Methodsmentioning

confidence: 99%

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Class-specific interactions between Sis1 J-domain protein and Hsp70 chaperone potentiate disaggregation of misfolded proteins

Wyszkowski

Janta

Sztangierska

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Protein homeostasis is constantly being challenged with protein misfolding that leads to aggregation. Hsp70 is one of the versatile chaperones that interact with misfolded proteins and actively support their folding. Multifunctional Hsp70s are harnessed to specific roles by J-domain proteins (JDPs, also known as Hsp40s). Interaction with the J-domain of these cochaperones stimulates ATP hydrolysis in Hsp70, which stabilizes substrate binding. In eukaryotes, two classes of JDPs, Class A and Class B, engage Hsp70 in the reactivation of aggregated proteins. In most species, excluding metazoans, protein recovery also relies on an Hsp100 disaggregase. Although intensely studied, many mechanistic details of how the two JDP classes regulate protein disaggregation are still unknown. Here, we explore functional differences between the yeast Class A (Ydj1) and Class B (Sis1) JDPs at the individual stages of protein disaggregation. With real-time biochemical tools, we show that Ydj1 alone is superior to Sis1 in aggregate binding, yet it is Sis1 that recruits more Ssa1 molecules to the substrate. This advantage of Sis1 depends on its ability to bind to the EEVD motif of Hsp70, a quality specific to most of Class B JDPs. This second interaction also conditions the Hsp70-induced aggregate modification that boosts its subsequent dissolution by the Hsp104 disaggregase. Our results suggest that the Sis1-mediated chaperone assembly at the aggregate surface potentiates the entropic pulling, driven polypeptide disentanglement, while Ydj1 binding favors the refolding of the solubilized proteins. Such subspecialization of the JDPs across protein reactivation improves the robustness and efficiency of the disaggregation machinery.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Class-specific interactions between Sis1 J-domain protein and Hsp70 chaperone potentiate disaggregation of misfolded proteins

Wyszkowski

Janta

Sztangierska

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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“…Toxicity of ClpB in bacteria and its orthologue Hsp104 in yeast has been observed for "hyperactive" protein variants with mutations within the coiled-coil middle domain (55,59,60). However, unlike the DBeQ-treated ClpB, the toxic hyperactive variants display elevated ATPase and disaggregase activities.…”

Section: Dbeq-mediated Modulation Of the Allostery Withinmentioning

confidence: 99%

Repurposing p97 inhibitors for chemical modulation of the bacterial ClpB–DnaK bichaperone system

Glaza

Ranaweera

Shiva

et al. 2021

Journal of Biological Chemistry

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The ClpB/DnaK bi-chaperone system reactivates aggregated cellular proteins and is essential for survival of bacteria, fungi, protozoa, and plants under stress. AAA+ ATPase ClpB is a promising target for the development of antimicrobials, because a loss of its activity is detrimental for survival of many pathogens and no apparent ClpB orthologs are found in metazoans. We investigated ClpB activity in the presence of several compounds that were previously described as inhibitor leads for the human AAA+ ATPase p97, an anti-tumor target. We discovered that N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ), the least potent among the tested p97 inhibitors, binds to ClpB with a Kd~60 μM and inhibits the casein-activated, but not the basal ATPase activity of ClpB with an IC50~5 μM. The remaining p97 ligands, which displayed a higher affinity towards p97, did not affect the ClpB ATPase. DBeQ also interacted with DnaK with a Kd~100 μM, did not affect the DnaK ATPase, but inhibited the DnaK chaperone activity in vitro. DBeQ inhibited the reactivation of aggregated proteins by the ClpB/DnaK bi-chaperone system in vitro with an IC50~5 μM and suppressed the growth of cultured E. coli. The DBeQ-induced loss of E. coli proliferation was exacerbated by heat shock, but was nearly eliminated in a ClpB-deficient E. coli strain, which demonstrates a significant selectivity of DBeQ towards ClpB in cells. Our results provide chemical validation of ClpB as a target for developing novel antimicrobials. We identified DBeQ as a promising lead compound for structural optimization aimed at selective targeting of ClpB and/or DnaK.

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“…Interestingly, targeting ClpB with DBeQ in E. coli cells produced toxicity that transcended a loss-of-function phenotype observed in the clpB -null strain. Toxicity of ClpB in bacteria and Hsp104 in yeast has been observed before for “hyperactive” protein variants with mutations within the middle domain [ 76 , 77 , 78 ]. Whether an analogous toxic gain of function occurs in the DBeQ-treated ClpB remains to be investigated.…”

Section: Clpb As a Druggable Targetmentioning

confidence: 93%

Hsp100 Molecular Chaperone ClpB and Its Role in Virulence of Bacterial Pathogens

Kędzierska‐Mieszkowska

Żółkiewski

2021

IJMS

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This review focuses on the molecular chaperone ClpB that belongs to the Hsp100/Clp subfamily of the AAA+ ATPases and its biological function in selected bacterial pathogens, causing a variety of human infectious diseases, including zoonoses. It has been established that ClpB disaggregates and reactivates aggregated cellular proteins. It has been postulated that ClpB’s protein disaggregation activity supports the survival of pathogenic bacteria under host-induced stresses (e.g., high temperature and oxidative stress), which allows them to rapidly adapt to the human host and establish infection. Interestingly, ClpB may also perform other functions in pathogenic bacteria, which are required for their virulence. Since ClpB is not found in human cells, this chaperone emerges as an attractive target for novel antimicrobial therapies in combating bacterial infections.

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Selective Hsp70-Dependent Docking of Hsp104 to Protein Aggregates Protects the Cell from the Toxicity of the Disaggregase

Cited by 15 publications

References 59 publications

Class-specific interactions between Sis1 J-domain protein and Hsp70 chaperone potentiate disaggregation of misfolded proteins

Class-specific interactions between Sis1 J-domain protein and Hsp70 chaperone potentiate disaggregation of misfolded proteins

Repurposing p97 inhibitors for chemical modulation of the bacterial ClpB–DnaK bichaperone system

Hsp100 Molecular Chaperone ClpB and Its Role in Virulence of Bacterial Pathogens

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