Cytosolic and mitochondrial Hsp90 in cytokinesis, mitochondrial DNA replication, and drug action in Trypanosoma brucei

2021 ◽  
Author(s):  
Kirsten J. Meyer ◽  
Theresa A. Shapiro
Keyword(s):  
Mitochondrial Dna ◽  
Trypanosoma Brucei ◽  
Cell Biology ◽  
Drug Targets ◽  
Heat Shock Protein 90 ◽  
Cell Cycle Regulators ◽  
Hsp90 Inhibitors ◽  
African Trypanosomes ◽  
Hsp90 Activity

Trypanosoma brucei subspecies cause African sleeping sickness in humans, an infection that is commonly fatal if not treated, and available therapies are limited. Previous studies have shown that heat shock protein 90 (Hsp90) inhibitors have potent and vivid activity against bloodstream form trypanosomes. Hsp90s are phylogenetically conserved and essential catalysts that function at the crux of cell biology, where they ensure the proper folding of proteins and their assembly into multicomponent complexes. To assess the specificity of Hsp90 inhibitors and further define the role of Hsp90s in African trypanosomes, we used RNAi to knockdown cytosolic and mitochondrial Hsp90s (HSP83 and HSP84, respectively). Loss of either protein led to cell death but the phenotypes were distinctly different. Depletion of cytosolic HSP83 closely mimicked the consequences of chemically depleting Hsp90 activity with inhibitor 17-AAG. In these cells cytokinesis was severely disrupted and segregation of the kinetoplast (the massive mitochondrial DNA structure unique to this family of eukaryotic pathogens) was impaired, leading to cells with abnormal kDNA structures. Quite differently, knockdown of mitochondrial HSP84 did not impair cytokinesis but halted the initiation of new kDNA synthesis, generating cells without kDNA. These findings highlight the central role for Hsp90s in chaperoning cell cycle regulators in trypanosomes, reveal their unique function in kinetoplast replication, and reinforce their specificity and value as drug targets.

scholarly journals Amino Acid Transporters as Disease Modifiers and Drug Targets

2018 ◽  
Vol 23 (4) ◽  
pp. 303-320 ◽  
Author(s):  
Stefan Bröer
Keyword(s):  
Amino Acid ◽  
Amino Acid Transport ◽  
Neurological Disorders ◽  
Cell Biology ◽  
Drug Targets ◽  
Therapeutic Strategy ◽  
Amino Acid Transporters ◽  
Acid Transport ◽  
Membrane Protein Expression

Amino acids perform a variety of functions in cells and organisms, particularly in the synthesis of proteins, as energy metabolites, neurotransmitters, and precursors for many other molecules. Amino acid transport plays a key role in all these functions. Inhibition of amino acid transport is pursued as a therapeutic strategy in several areas, such as diabetes and related metabolic disorders, neurological disorders, cancer, and stem cell biology. The role of amino acid transporters in these disorders and processes is well established, but the implementation of amino acid transporters as drug targets is still in its infancy. This is at least in part due to the underdeveloped pharmacology of this group of membrane proteins. Recent advances in structural biology, membrane protein expression, and inhibitor screening methodology will see an increased number of improved and selective inhibitors of amino acid transporters that can serve as tool compounds for further studies.

scholarly journals Gambogic acid identifies an isoform-specific druggable pocket in the middle domain of Hsp90β

2016 ◽  
Vol 113 (33) ◽  
pp. E4801-E4809 ◽  
Author(s):  
Kendrick H. Yim ◽  
Thomas L. Prince ◽  
Shiwei Qu ◽  
Fang Bai ◽  
Patricia A. Jennings ◽  
...  
Keyword(s):  
Molecular Chaperone ◽  
Drug Targets ◽  
Heat Shock Protein 90 ◽  
Hsp90 Inhibitor ◽  
Gambogic Acid ◽  
Disease States ◽  
Middle Domain ◽  
Health And Disease ◽  
A Site

Because of their importance in maintaining protein homeostasis, molecular chaperones, including heat-shock protein 90 (Hsp90), represent attractive drug targets. Although a number of Hsp90 inhibitors are in preclinical/clinical development, none strongly differentiate between constitutively expressed Hsp90β and stress-induced Hsp90α, the two cytosolic paralogs of this molecular chaperone. Thus, the importance of inhibiting one or the other paralog in different disease states remains unknown. We show that the natural product, gambogic acid (GBA), binds selectively to a site in the middle domain of Hsp90β, identifying GBA as an Hsp90β-specific Hsp90 inhibitor. Furthermore, using computational and medicinal chemistry, we identified a GBA analog, referred to as DAP-19, which binds potently and selectively to Hsp90β. Because of its unprecedented selectivity for Hsp90β among all Hsp90 paralogs, GBA thus provides a new chemical tool to study the unique biological role of this abundantly expressed molecular chaperone in health and disease.

scholarly journals A Mitochondrial DNA Primase Is Essential for Cell Growth and Kinetoplast DNA Replication in Trypanosoma brucei

2010 ◽  
Vol 30 (6) ◽  
pp. 1319-1328 ◽  
Author(s):  
Jane C. Hines ◽  
Dan S. Ray
Keyword(s):  
Mitochondrial Dna ◽  
Dna Replication ◽  
Cell Growth ◽  
Trypanosoma Brucei ◽  
Dna Polymerase ◽  
Rnase H ◽  
Kinetoplast Dna ◽  
African Trypanosomes ◽  
Dna Primase

ABSTRACT Kinetoplast DNA in African trypanosomes contains a novel form of mitochondrial DNA consisting of thousands of minicircles and dozens of maxicircles topologically interlocked to form a two-dimensional sheet. The replication of this unusual form of mitochondrial DNA has been studied for more than 30 years, and although a large number of kinetoplast replication genes and proteins have been identified, in vitro replication of these DNAs has not been possible since a kinetoplast DNA primase has not been available. We describe here a Trypanosoma brucei DNA primase gene, PRI1, that encodes a 70-kDa protein that localizes to the kinetoplast and is essential for both cell growth and kinetoplast DNA replication. The expression of PRI1 mRNA is cyclic and reaches maximum levels at a time corresponding to duplication of the kinetoplast DNA. A 3′-hydroxyl-terminated oligoriboadenylate is synthesized on a poly(dT) template by a recombinant form of the PRI1 protein and is subsequently elongated by DNA polymerase and added dATP. Poly(dA) synthesis is dependent on both PRI1 protein and ATP and is inhibited by RNase H treatment of the product of PRI1 synthesis.

scholarly journals Inhibitors of HSP90 in melanoma

APOPTOSIS ◽  
2019 ◽  
Vol 25 (1-2) ◽  
pp. 12-28 ◽  
Author(s):  
Aleksandra Mielczarek-Lewandowska ◽  
Mariusz L. Hartman ◽  
Malgorzata Czyz
Keyword(s):  
Current Knowledge ◽  
Heat Shock Protein 90 ◽  
Response To Therapy ◽  
Hsp90 Inhibitors ◽  
Chemical Structures ◽  
Client Proteins ◽  
Melanoma Treatment ◽  
Hsp90 Protein ◽  
Potential Therapeutics

Abstract HSP90 (heat shock protein 90) is an ATP-dependent molecular chaperone involved in a proper folding and maturation of hundreds of proteins. HSP90 is abundantly expressed in cancer, including melanoma. HSP90 client proteins are the key oncoproteins of several signaling pathways controlling melanoma development, progression and response to therapy. A number of natural and synthetic compounds of different chemical structures and binding sites within HSP90 have been identified as selective HSP90 inhibitors. The majority of HSP90-targeting agents affect N-terminal ATPase activity of HSP90. In contrast to N-terminal inhibitors, agents interacting with the middle and C-terminal domains of HSP90 do not induce HSP70-dependent cytoprotective response. Several inhibitors of HSP90 were tested against melanoma in pre-clinical studies and clinical trials, providing evidence that these agents can be considered either as single or complementary therapeutic strategy. This review summarizes current knowledge on the role of HSP90 protein in cancer with focus on melanoma, and provides an overview of structurally different HSP90 inhibitors that are considered as potential therapeutics for melanoma treatment.

scholarly journals The genetics of African trypanosomes : vector-borne diseases : trypanosomosis

2009 ◽  
Vol 76 (1) ◽  
Author(s):  
A. Tait
Keyword(s):  
Trypanosoma Brucei ◽  
Sequence Data ◽  
Fundamental Property ◽  
Genetic Exchange ◽  
Sexual Cycle ◽  
Current Evidence ◽  
African Trypanosomes ◽  
Vector Borne Diseases ◽  
Vector Borne

The occurrence of genetic exchange is not only a fundamental property of an organism but its occurrence provides us with methods of analysis that can be used to dissect the basis of important traits. This paper will review the current evidence for a sexual cycle in Trypanosoma brucei and illustrate how genetic analysis can be used as a tool to identify genes of relevance to the disease, its treatment and transmission. The role of this process in the generation of diversity in the field will be illustrated by considering populations of the three sub-species of T. brucei and how the availability of genome sequence data has been exploited to study whether genetic exchange occurs in the trypanosomes infecting livestock. Finally, the use of population genetics as a methodology to identify genes under selection will be discussed and this will be illustrated as an approach to validating markers for drug resistance with examples from other parasitic protozoa.

The Role of Mitochondria in Stem Cell Biology

2007 ◽  
Vol 27 (1-3) ◽  
pp. 165-171 ◽  
Author(s):  
Claudia Nesti ◽  
Livia Pasquali ◽  
Francesca Vaglini ◽  
Gabriele Siciliano ◽  
Luigi Murri
Keyword(s):  
Stem Cells ◽  
Mitochondrial Dna ◽  
Stem Cell ◽  
Current Literature ◽  
Cell Biology ◽  
Mitochondrial Metabolism ◽  
Stem Cell Biology ◽  
Therapeutic Tool ◽  
Dna Mutations

This mini-review summarizes the current literature on the role of mitochondrial DNA mutations and mitochondrial metabolism in stem cell biology. The possible uses of stem cells as a therapeutic tool in mitochondrial disorders are also reported.

scholarly journals Interaction of Hsp90 with Ribosomal Proteins Protects from Ubiquitination and Proteasome-dependent Degradation

2006 ◽  
Vol 17 (2) ◽  
pp. 824-833 ◽  
Author(s):  
Tae-Sung Kim ◽  
Chang-Young Jang ◽  
Hag Dong Kim ◽  
Jae Yung Lee ◽  
Byung-Yoon Ahn ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Molecular Chaperone ◽  
Ribosomal Proteins ◽  
Heat Shock Protein 90 ◽  
Small Subunit ◽  
Hsp90 Inhibitors ◽  
Ribosomal Protein S6 ◽  
Hsp90 Activity ◽  
Ribosomal Protein S3 ◽  
The Stability

Heat-shock protein 90 (Hsp90) is a molecular chaperone that plays a key role in the conformational maturation of various transcription factors and protein kinases in signal transduction. Multifunctional ribosomal protein S3 (rpS3), a component of the ribosomal small subunit, is involved in DNA repair and apoptosis. Our data show that Hsp90 binds directly to rpS3 and the functional consequence of Hsp90-rpS3 interaction results in the prevention of the ubiquitination and the proteasome-dependent degradation of rpS3, subsequently retaining the function and the biogenesis of the ribosome. Interference of Hsp90 activity by Hsp90 inhibitors appears to dissociate rpS3 from Hsp90, associate the protein with Hsp70, and induce the degradation of free forms of rpS3. Furthermore, ribosomal protein S6 (rpS6) also interacted with Hsp90 and exhibited a similar effect upon treatment with Hsp90 inhibitors. Therefore, we conclude that Hsp90 regulates the function of ribosomes by maintaining the stability of 40S ribosomal proteins such as rpS3 and rpS6.

scholarly journals Depletion of the thioredoxin homologue tryparedoxin impairs antioxidative defence in African trypanosomes

2007 ◽  
Vol 402 (1) ◽  
pp. 43-49 ◽  
Author(s):  
Marcelo A. Comini ◽  
R. Luise Krauth-Siegel ◽  
Leopold Flohé
Keyword(s):  
Oxidative Stress ◽  
Rna Interference ◽  
Trypanosoma Brucei ◽  
Growth Phase ◽  
Essential Role ◽  
Biological Significance ◽  
Tsetse Fly ◽  
African Trypanosomes ◽  
Antioxidative Defence

In trypanosomes, the thioredoxin-type protein TXN (tryparedoxin) is a multi-purpose oxidoreductase that is involved in the detoxification of hydroperoxides, the synthesis of DNA precursors and the replication of the kinetoplastid DNA. African trypanosomes possess two isoforms that are localized in the cytosol and in the mitochondrion of the parasites respectively. Here we report on the biological significance of the cTXN (cytosolic TXN) of Trypanosoma brucei for hydroperoxide detoxification. Depending on the growth phase, the concentration of the protein is 3–7-fold higher in the parasite form infecting mammals (50–100 μM) than in the form hosted by the tsetse fly (7–34 μM). Depletion of the mRNA in bloodstream trypanosomes by RNA interference revealed the indispensability of the protein. Proliferation and viability of cultured trypanosomes were impaired when TXN was lowered to 1 μM for more than 48 h. Although the levels of glutathione, glutathionylspermidine and trypanothione were increased 2–3.5-fold, the sensitivity against exogenously generated H2O2 was significantly enhanced. The results prove the essential role of the cTXN and its pivotal function in the parasite defence against oxidative stress.

scholarly journals Trypanosoma brucei Pex13.2 Is an Accessory Peroxin That Functions in the Import of Peroxisome Targeting Sequence Type 2 Proteins and Localizes to Subdomains of the Glycosome

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Logan P. Crowe ◽  
Christina L. Wilkinson ◽  
Kathleen R. Nicholson ◽  
Meredith T. Morris
Keyword(s):  
Trypanosoma Brucei ◽  
Drug Targets ◽  
Protein Import ◽  
Specific Protein ◽  
Soluble Receptors ◽  
Two Dimensional Gel Electrophoresis ◽  
Content Type ◽  
Superresolution Microscopy

ABSTRACT Kinetoplastid parasites, including Trypanosoma brucei, Trypanosoma cruzi, and Leishmania, harbor unique organelles known as glycosomes, which are evolutionarily related to peroxisomes. Glycosome/peroxisome biogenesis is mediated by proteins called peroxins that facilitate organelle formation, proliferation, and degradation and import of proteins housed therein. Import of matrix proteins occurs via one of two pathways that are dictated by their peroxisome targeting sequence (PTS). In PTS1 import, a C-terminal tripeptide sequence, most commonly SKL, is recognized by the soluble receptor Pex5. In PTS2 import, a less conserved N-terminal sequence is recognized by Pex7. The soluble receptors deliver their cargo to the import channel consisting minimally of Pex13 and Pex14. While much of the import process is conserved, kinetoplastids are the only organisms to have two Pex13s, Pex13.1 and Pex13.2. It is unclear why trypanosomes require two Pex13s when one is sufficient for most eukaryotes. To interrogate the role of Pex13.2, we have employed biochemical approaches to partially resolve the composition of the Pex13/Pex14 import complexes in T. brucei and characterized glycosome morphology and protein import in Pex13.2-deficient parasites. Here, we show that Pex13.2 is an integral glycosome membrane protein that interacts with Pex13.1 and Pex14. The N terminus of Pex13.2 faces the cytoplasmic side of the membrane, where it can facilitate interactions required for protein import. Two-dimensional gel electrophoresis revealed three glycosome membrane complexes containing combinations of Pex13.1, Pex13.2, and Pex14. The silencing of Pex13.2 resulted in parasites with fewer, larger glycosomes and disrupted glycosome protein import, suggesting the protein is involved in glycosome biogenesis as well as protein import. Furthermore, superresolution microscopy demonstrated that Pex13.2 localizes to discrete foci in the glycosome periphery, indicating that the glycosome periphery is not homogenous. IMPORTANCE Trypanosoma brucei causes human African trypanosomiasis and a wasting disease called Nagana in livestock. Current treatments are expensive, toxic, and difficult to administer. Because of this, the search for new drug targets is essential. T. brucei has glycosomes that are essential to parasite survival; however, our ability to target them in drug development is hindered by our lack of understanding about how these organelles are formed and maintained. This work forwards our understanding of how the parasite-specific protein Pex13.2 functions in glycosome protein import and lays the foundation for future studies focused on blocking Pex13.2 function, which would be lethal to bloodstream-form parasites that reside in the mammalian bloodstream.

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