Investigating the Chaperone Properties of a Novel Heat Shock Protein, Hsp70.c, from<i>Trypanosoma brucei</i>

Burger, Adélle; Ludewig, Michael H.; Boshoff, Aileen

doi:10.1155/2014/172582

Cited by 18 publications

(42 citation statements)

References 65 publications

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“…Despite this, TbHsp70.4 was purified by nickel affinity chromatography as full-length protein (Fig 1B). Recombinant Tbj2 was expressed in E. coli BL21 (DE3) cells, and subsequently purified using nickel affinity chromatography as previously described [27] (Fig S1A-B).…”

Section: Resultsmentioning

confidence: 99%

“…The Michaelis-Menten plots were generated from three independent batches of TbHsp70 and TbHsp70.4 (Fig 4). Both TbHsp70 and TbHsp70.4 exhibited intrinsic ATPase activity (Fig 4), though TbHsp70 was found to have a higher basal ATPase activity than TbHsp70.4 (Table S1) and the reported basal ATPase activity for TbHsp70.c (Table S1) [27]. However, the basal ATPase activities for the T. brucei Hsp70s were all found to be higher than those reported for human Hsp70 [42;44], bovine Hsc70 [45], and E. coli DnaK [46], but significantly lower than the values reported for Hsp70 homologues found in other protozoan parasites (Table S1).…”

Section: Resultsmentioning

confidence: 99%

“…Recombinant N-terminal His-tagged Tbj2 was purified under native conditions from E. coli BL21 (DE3) cells as previously described [27]. Samples were dialysed in DB or into the appropriate assay buffer for functional studies.…”

Section: Methodsmentioning

confidence: 99%

“…Tbj2 was identified to be an essential stress inducible Type I J-protein, as knockdown via RNAi revealed a severe growth defect and it was shown to reside in the parasite cytosol, [26]. Tbj2 was determined to possess holdase activity when chemically denatured rhodanese and thermally aggregated MDH were used as substrates [27].…”

Section: Introductionmentioning

confidence: 99%

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Trypanosoma bruceiJ protein 2 functionally cooperates with the cytosolic Hsp70.4 and Hsp70 proteins

Bentley

Boshoff

2019

Preprint

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30The etiological agent of African trypanosomiasis, Trypanosoma brucei, has been identified to 31 possess an expanded and diverse group of heat shock proteins, that have been implicated in 32 cytoprotection, differentiation, and subsequently progression and transmission of the disease. 33 Heat shock protein 70 is a highly conserved and ubiquitous molecular chaperone that is 34 important in maintaining protein homeostasis in the cell. Its function is regulated by a wide 35 range of co-chaperones; and inhibition of these functions and interactions with co-chaperones 36 are emerging as potential therapeutic targets for numerous diseases. This study sought to 37 biochemically characterize the cytosolic Hsp70 and Hsp70.4 proteins and to investigate if they 38 form a functional partnership with the Type I J-protein, Tbj2. The cytosolic localisation of the 39 proteins was confirmed by accessing the TrypTag endogenous tagging microscopy database. 40Expression of TbHsp70 was shown to be heat inducible, whilst TbHsp70.4 was constitutively 41 expressed. The basal ATPase activities of TbHsp70.4 and TbHsp70 were stimulated by Tbj2. 42It was further determined that Tbj2 forms a functional partnership with TbHsp70 and 43 TbHsp70.4 as the J-protein was shown to stimulate the ability of both proteins to mediate the 44 refolding of chemically denatured β-galactosidase. This study provides further insight into this 45 important class of proteins which may contribute to the development of new therapeutic 46 strategies to combat African Trypanosomiasis. 47 48 49 50 51 52 53 54 3 55 Introduction 56 African trypanosomiasis, a neglected tropical disease, afflicts humans as well as domestic and 57 wild animals, and has a detrimental impact on socioeconomic development in sub-Saharan 58 Africa [1]. There is a need for the development of more effective and safer chemotherapies to 59 treat the disease, due to existing drug toxicity, growing parasite resistance and the lack of a 60 vaccine [2]. The Hsp70/J-protein chaperone machinery has been implicated to play an integral 61 role in the development, differentiation, and survival of protozoan parasites, as they transition 62 through the various stages of their life cycle [3]. In Leishmania and Trypanosoma cruzi, heat 63 shock proteins have been shown to play an essential role in stress-induced stage differentiation 64 and are important for disease progression and transmission [4-5], making this protein family 65 an attractive chemotherapeutic target. The completion of the Trypanosoma brucei (T. brucei) 66 genome has expedited transcriptome and proteome analyses and revealed that the extracellular 67 parasite has an expanded and diverse Hsp70 and J-protein complement, with the parasite 68 possessing cytosolic Hsp70 members that display atypical Hsp70 features [6]. 69 70 The Hsp70 protein family is ubiquitous and plays an integral role in protein quality control and 71 maintaining protein homeostasis under normal and stressful conditions [7-8]. Evolution has 72 given rise to multiple ...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Trypanosoma bruceiJ protein 2 functionally cooperates with the cytosolic Hsp70.4 and Hsp70 proteins

Bentley

Boshoff

2019

Preprint

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“…The distribution of co-chaperones varies between species and between humans and parasites. For example, Plasmodium falciparum (the main agent of malaria) and Trypansoma brucei (which causes sleeping sickness in humans) possess a much bigger complement of Hsp40s than humans (Njunge et al, 2013;Burger et al, 2014). Some compounds that selectively target the interaction of Hsp40 with Hsp70 in malaria parasites have been shown to exhibit limited cytoxicity to human cells (Cockburn et al, 2011).…”

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confidence: 99%

Are Heat Shock Proteins Druggable Candidates?

Zininga¹,

Shonhai²

2014

American Journal of Biochemistry and Biotechnology

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The Hsp70/J-protein machinery of the African trypanosome, Trypanosoma brucei

Bentley

Jamabo

Boshoff

2019

Cell Stress and Chaperones

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The etiological agent of the neglected tropical disease African trypanosomiasis, Trypanosoma brucei, possesses an expanded and diverse repertoire of heat shock proteins, which have been implicated in cytoprotection, differentiation, as well as progression and transmission of the disease. Hsp70 plays a crucial role in proteostasis, and inhibition of its interactions with co-chaperones is emerging as a potential therapeutic target for numerous diseases. In light of genome annotations and the release of the genome sequence of the human infective subspecies, an updated and current in silico overview of the Hsp70/J-protein machinery in both T. brucei brucei and T. brucei gambiense was conducted. Functional, structural, and evolutionary analyses of the T. brucei Hsp70 and J-protein families were performed. The Hsp70 and J-proteins from humans and selected kinetoplastid parasites were used to assist in identifying proteins from T. brucei, as well as the prediction of potential Hsp70-J-protein partnerships. The Hsp70 and Jproteins were mined from numerous genome-wide proteomics studies, which included different lifecycle stages and subcellular localisations. In this study, 12 putative Hsp70 proteins and 67 putative J-proteins were identified to be encoded on the genomes of both T. brucei subspecies. Interestingly there are 6 type III J-proteins that possess tetratricopeptide repeat-containing (TPR) motifs. Overall, it is envisioned that the results of this study will provide a future context for studying the biology of the African trypanosome and evaluating Hsp70 and J-protein interactions as potential drug targets.

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Investigating the Chaperone Properties of a Novel Heat Shock Protein, Hsp70.c, fromTrypanosoma brucei

Cited by 18 publications

References 65 publications

Trypanosoma bruceiJ protein 2 functionally cooperates with the cytosolic Hsp70.4 and Hsp70 proteins

Trypanosoma bruceiJ protein 2 functionally cooperates with the cytosolic Hsp70.4 and Hsp70 proteins

Are Heat Shock Proteins Druggable Candidates?

The Hsp70/J-protein machinery of the African trypanosome, Trypanosoma brucei

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