Optimal Prevention of Client-Protein Aggregation on the Surface of the Hsp70 Chaperone: A Computational Study

Abstract Plasmodium falciparum heat shock protein 70 (PfHsp70-1) is thought to play an essential role in parasite survival and virulence in the human host, making it a potential antimalarial drug target. A malate dehydrogenase based aggregation suppression assay was adapted for the screening of small molecule modulators of Hsp70. A number of small molecules of natural (marine prenylated alkaloids and terrestrial plant naphthoquinones) and related synthetic origin were screened for their effects on the protein aggregation suppression activity of purified recombinant PfHsp70-1. Five compounds (malonganenone A-C, lapachol and bromo-β-lapachona) were found to inhibit the chaperone activity of PfHsp70-1 in a concentration dependent manner, with lapachol preferentially inhibiting PfHsp70-1 compared to another control Hsp70. Using growth inhibition assays on P. falciparum infected erythrocytes, all of the compounds, except for malonganenone B, were found to inhibit parasite growth with IC50 values in the low micromolar range. Overall, this study has identified two novel classes of small molecule inhibitors of PfHsp70-1, one representing a new class of antiplasmodial compounds (malonganenones). In addition to demonstrating the validity of PfHsp70-1 as a possible drug target, the compounds reported in this study will be potentially useful as molecular probes for fundamental studies on Hsp70 chaperone function.

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Promiscuous binding by Hsp70 results in conformational heterogeneity and fuzzy chaperone-substrate ensembles

eLife ◽

10.7554/elife.28030 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 42

Author(s):

Rina Rosenzweig ◽

Ashok Sekhar ◽

Jayashree Nagesh ◽

Lewis E Kay

Keyword(s):

Structural Information ◽

Structural Heterogeneity ◽

Solution Nmr ◽

Client Protein ◽

Biochemical Processes ◽

Nmr Study ◽

Methyl Trosy ◽

Hsp70 Chaperone ◽

Kinetic Traps ◽

Conformational Free Energy

The Hsp70 chaperone system is integrated into a myriad of biochemical processes that are critical for cellular proteostasis. Although detailed pictures of Hsp70 bound with peptides have emerged, correspondingly detailed structural information on complexes with folding-competent substrates remains lacking. Here we report a methyl-TROSY based solution NMR study showing that the Escherichia coli version of Hsp70, DnaK, binds to as many as four distinct sites on a small 53-residue client protein, hTRF1. A fraction of hTRF1 chains are also bound to two DnaK molecules simultaneously, resulting in a mixture of DnaK-substrate sub-ensembles that are structurally heterogeneous. The interactions of Hsp70 with a client protein at different sites results in a fuzzy chaperone-substrate ensemble and suggests a mechanism for Hsp70 function whereby the structural heterogeneity of released substrate molecules enables them to circumvent kinetic traps in their conformational free energy landscape and fold efficiently to the native state.

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Hsp70 chaperone: a master player in protein homeostasis

F1000Research ◽

10.12688/f1000research.15528.1 ◽

2018 ◽

Vol 7 ◽

pp. 1497 ◽

Cited By ~ 36

Author(s):

María Rosario Fernández-Fernández ◽

José María Valpuesta

Keyword(s):

Structural Information ◽

Protein Complexes ◽

Protein Homeostasis ◽

Client Protein ◽

P Protein ◽

Hsp70 Chaperone ◽

Cellular Context ◽

Client Proteins ◽

Functional Mechanisms

Protein homeostasis (proteostasis) is an essential pillar for correct cellular function. Impairments in proteostasis are encountered both in aging and in several human disease conditions. Molecular chaperones are important players for proteostasis; in particular, heat shock protein 70 (Hsp70) has an essential role in protein folding, disaggregation, and degradation. We have recently proposed a model for Hsp70 functioning as a “multiple socket”. In the model, Hsp70 provides a physical platform for the binding of client proteins, other chaperones, and cochaperones. The final fate of the client protein is dictated by the set of Hsp70 interactions that occur in a given cellular context. Obtaining structural information of the different Hsp70-based protein complexes will provide valuable knowledge to understand the functional mechanisms behind the master role of Hsp70 in proteostasis. We additionally evaluate some of the challenges for attaining high-resolution structures of such complexes.

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Targeting DNA topoisomerases or checkpoint kinases results in an overload of chaperone systems, triggering aggregation of a metastable subproteome

10.1101/2021.06.04.447142 ◽

2021 ◽

Author(s):

Wouter Huiting ◽

Suzanne L Dekker ◽

Joris van der Lienden ◽

Rafaella Mergener ◽

Gabriel V Furtado ◽

...

Keyword(s):

Dna Damage ◽

High Risk ◽

Protein Aggregation ◽

Dna Damage Response ◽

Molecular Chaperone ◽

Dna Topoisomerases ◽

Checkpoint Kinases ◽

Proteotoxic Stress ◽

Damage Response ◽

Hsp70 Chaperone

A loss of the checkpoint kinase ATM leads to impairments in the DNA damage response, and in humans causes cerebellar neurodegeneration, and a high risk to cancer. A loss of ATM is also associated with increased protein aggregation. The relevance and characteristics of this aggregation are still incompletely understood. Moreover, it is unclear to what extent other genotoxic conditions can trigger protein aggregation as well. Here, we show that targeting ATM, but also ATR or DNA topoisomerases result in a similar, widespread aggregation of a metastable, disease-associated subfraction of the proteome. Aggregation-prone model substrates, including Huntingtin exon1 containing an expanded polyglutamine repeat, aggregate faster under these conditions. This increased aggregation results from an overload of chaperone systems, which lowers the cell-intrinsic threshold for proteins to aggregate. In line with this, we find that inhibition of the HSP70 chaperone system further exacerbates the increased protein aggregation. Moreover, we identify the molecular chaperone HSPB5 as a potent suppressor of it. Our findings reveal that various genotoxic conditions trigger widespread protein aggregation in a manner that is highly reminiscent of the aggregation occurring in situations of proteotoxic stress and in proteinopathies.

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Novel Methodologies for the Computational Study of Protein Aggregation

Biophysical Journal ◽

10.1016/j.bpj.2014.11.2709 ◽

2015 ◽

Vol 108 (2) ◽

pp. 495a

Author(s):

David Shorthouse ◽

Thomas Gallagher ◽

Mark Sansom

Keyword(s):

Protein Aggregation ◽

Computational Study

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Auto-regulatory J-domain interactions control Hsp70 recruitment to the DnaJB8 chaperone

10.1101/2020.01.09.899237 ◽

2020 ◽

Author(s):

Bryan D. Ryder ◽

Irina Matlahov ◽

Sofia Bali ◽

Jaime Vaquer-Alicea ◽

Patrick C.A. van der Wel ◽

...

Keyword(s):

Substrate Specificity ◽

Protein Aggregation ◽

Regulatory Element ◽

Structure And Function ◽

High Substrate ◽

Evolutionarily Conserved ◽

Hsp70 Chaperone ◽

Domain Interactions ◽

J Domain ◽

And Function

AbstractThe Hsp40/Hsp70 chaperone families combine versatile folding capacity with high substrate specificity, which is mainly facilitated by Hsp40s. The structure and function of many Hsp40s remain poorly understood, particularly oligomeric Hsp40s that suppress protein aggregation. Here, we used a combination of biochemical and structural approaches to shed new light on the domain interactions of the Hsp40 DnaJB8, and how they regulate recruitment of partner Hsp70s. We identify an interaction between the J-Domain (JD) and C-terminal domain (CTD) of DnaJB8 that sequesters the JD surface, preventing Hsp70 interaction. We propose a new model for DnaJB8-Hsp70 regulation, whereby the JD-CTD interaction of DnaJB8 acts as a reversible autoinhibitory switch that can control the binding of Hsp70. These findings suggest that the evolutionarily conserved CTD of DnaJB8 is a regulatory element of chaperone activity in the proteostasis network.

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An innovative synergistic grid approach to the computational study of protein aggregation mechanisms

Journal of Molecular Modeling ◽

10.1007/s00894-014-2226-4 ◽

2014 ◽

Vol 20 (7) ◽

Cited By ~ 22

Author(s):

Noelia Faginas-Lago ◽

Margarita Albertí ◽

Alessandro Costantini ◽

Antonio Laganà ◽

Andrea Lombardi ◽

...

Keyword(s):

Protein Aggregation ◽

Computational Study ◽

Grid Approach

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Regulatory inter-domain interactions influence Hsp70 recruitment to the DnaJB8 chaperone

Nature Communications ◽

10.1038/s41467-021-21147-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Bryan D. Ryder ◽

Irina Matlahov ◽

Sofia Bali ◽

Jaime Vaquer-Alicea ◽

Patrick C. A. van der Wel ◽

...

Keyword(s):

Substrate Specificity ◽

Protein Aggregation ◽

Regulatory Element ◽

Structure And Function ◽

Evolutionarily Conserved ◽

Hsp70 Chaperone ◽

Domain Interactions ◽

J Domain ◽

And Function ◽

Shed Light

AbstractThe Hsp40/Hsp70 chaperone families combine versatile folding capacity with high substrate specificity, which is mainly facilitated by Hsp40s. The structure and function of many Hsp40s remain poorly understood, particularly oligomeric Hsp40s that suppress protein aggregation. Here, we used a combination of biochemical and structural approaches to shed light on the domain interactions of the Hsp40 DnaJB8, and how they may influence recruitment of partner Hsp70s. We identify an interaction between the J-Domain (JD) and C-terminal domain (CTD) of DnaJB8 that sequesters the JD surface, preventing Hsp70 interaction. We propose a model for DnaJB8-Hsp70 recruitment, whereby the JD-CTD interaction of DnaJB8 acts as a reversible switch that can control the binding of Hsp70. These findings suggest that the evolutionarily conserved CTD of DnaJB8 is a regulatory element of chaperone activity in the proteostasis network.

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An Ultra-Structural Study of Human Melanoma in Vivo and Vitro

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100091275 ◽

1976 ◽

Vol 34 ◽

pp. 258-259

Author(s):

James R. Gaylor ◽

Fredda Schafer ◽

Robert E. Nordquist

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Apparatus ◽

Protein Aggregation ◽

Structural Study ◽

Human Melanoma ◽

Protein Matrix ◽

Early Form ◽

Endoplasmic Reticulum Protein ◽

Mitochondrial Origin

Several theories on the origin of the melanosome exist. These include the Golgi origin theory, in which a tyrosinase-rich protein is "packaged" by the Golgi apparatus, thus forming the early form of the melanosome. A second theory postulates a mitochondrial origin of melanosomes. Its author contends that the melanosome is a modified mitochondria which acquires melanin during its development. A third theory states that a pre-melanosome is formed in the smooth or rough endoplasmic reticulum. Protein aggregation is suggested by one author as a possible source of the melanosome. This fourth theory postulates that the melanosome originates when the protein products of several genetic loci aggregate in the cytoplasm of the melanocyte. It is this protein matrix on which the melanin is deposited. It was with these theories in mind that this project was undertaken.

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