scholarly journals TbPRMT6 Is a Type I Protein Arginine Methyltransferase That Contributes to Cytokinesis in Trypanosoma brucei

2010 ◽  
Vol 9 (6) ◽  
pp. 866-877 ◽  
Author(s):  
John C. Fisk ◽  
Cecilia Zurita-Lopez ◽  
Joyce Sayegh ◽  
Danielle L. Tomasello ◽  
Steven G. Clarke ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Protozoan Parasite ◽  
Arginine Methylation ◽  
Nuclear Pore ◽  
Type I ◽  
Content Type ◽  
Morphological Defects ◽  
Flagellar Proteins

ABSTRACT Arginine methylation is a widespread posttranslational modification of proteins catalyzed by a family of protein arginine methyltransferases (PRMTs). In Saccharomyces cerevisiae and mammals, this modification affects multiple cellular processes, such as chromatin remodeling leading to transcriptional regulation, RNA processing, DNA repair, and cell signaling. The protozoan parasite Trypanosoma brucei possesses five putative PRMTs in its genome. This is a large number of PRMTs relative to other unicellular eukaryotes, suggesting an important role for arginine methylation in trypanosomes. Here, we present the in vitro and in vivo characterization of a T. brucei enzyme homologous to human PRMT6, which we term TbPRMT6. Like human PRMT6, TbPRMT6 is a type I PRMT, catalyzing the production of monomethylarginine and asymmetric dimethylarginine residues. In in vitro methylation assays, TbPRMT6 utilizes bovine histones as a substrate, but it does not methylate several T. brucei glycine/arginine-rich proteins. As such, it exhibits a relatively narrow substrate specificity compared to other T. brucei PRMTs. Knockdown of TbPRMT6 in both procyclic form and bloodstream form T. brucei leads to a modest but reproducible effect on parasite growth in culture. Moreover, upon TbPRMT6 depletion, both PF and BF exhibit aberrant morphologies indicating defects in cell division, and these defects differ in the two life cycle stages. Mass spectrometry of TbPRMT6-associated proteins reveals histones, components of the nuclear pore complex, and flagellar proteins that may represent TbPRMT6 substrates contributing to the observed growth and morphological defects.

scholarly journals Hydroxylamine and Carboxymethoxylamine Can Inhibit Toxoplasma gondii Growth through an Aspartate Aminotransferase-Independent Pathway

2020 ◽  
Vol 64 (3) ◽  
Author(s):  
Jixu Li ◽  
Huanping Guo ◽  
Eloiza May Galon ◽  
Yang Gao ◽  
Seung-Hun Lee ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Drug Targets ◽  
Protozoan Parasite ◽  
Cell Types ◽  
Lytic Cycle ◽  
Type I ◽  
Content Type ◽  
Mechanism Of Inhibition

ABSTRACT Toxoplasma gondii is an obligate intracellular protozoan parasite and a successful parasitic pathogen in diverse organisms and host cell types. Hydroxylamine (HYD) and carboxymethoxylamine (CAR) have been reported as inhibitors of aspartate aminotransferases (AATs) and interfere with the proliferation in Plasmodium falciparum. Therefore, AATs are suggested as drug targets against Plasmodium. The T. gondii genome encodes only one predicted AAT in both T. gondii type I strain RH and type II strain PLK. However, the effects of HYD and CAR, as well as their relationship with AAT, on T. gondii remain unclear. In this study, we found that HYD and CAR impaired the lytic cycle of T. gondii in vitro, including the inhibition of invasion or reinvasion, intracellular replication, and egress. Importantly, HYD and CAR could control acute toxoplasmosis in vivo. Further studies showed that HYD and CAR could inhibit the transamination activity of rTgAAT in vitro. However, our results confirmed that deficiency of AAT in both RH and PLK did not reduce the virulence in mice, although the growth ability of the parasites was affected in vitro. HYD and CAR could still inhibit the growth of AAT-deficient parasites. These findings indicated that HYD and CAR inhibition of T. gondii growth and control of toxoplasmosis can occur in an AAT-independent pathway. Overall, further studies focusing on the elucidation of the mechanism of inhibition are warranted. Our study hints at new substrates of HYD and CAR as potential drug targets to inhibit T. gondii growth.

scholarly journals Indirubin Analogues Inhibit Trypanosoma brucei Glycogen Synthase Kinase 3 Short and T. brucei Growth

2019 ◽  
Vol 63 (6) ◽  
Author(s):  
Antonia Efstathiou ◽  
Nicolas Gaboriaud-Kolar ◽  
Vassilios Myrianthopoulos ◽  
Konstantina Vougogiannopoulou ◽  
Ines Subota ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Glycogen Synthase ◽  
Intracellular Localization ◽  
Protozoan Parasite ◽  
Glycogen Synthase Kinase ◽  
Glycogen Synthase Kinase 3 ◽  
Content Type ◽  
Antitrypanosomal Activity ◽  
Half Maximal Effective Concentration

ABSTRACT The protozoan parasite Trypanosoma brucei is the causative agent of human African trypanosomiasis (HAT). The disease is fatal if it remains untreated, whereas most drug treatments are inadequate due to high toxicity, difficulties in administration, and low central nervous system penetration. T. brucei glycogen synthase kinase 3 short (TbGSK3s) is essential for parasite survival and thus represents a potential drug target that could be exploited for HAT treatment. Indirubins, effective leishmanicidals, provide a versatile scaffold for the development of potent GSK3 inhibitors. Herein, we report on the screening of 69 indirubin analogues against T. brucei bloodstream forms. Of these, 32 compounds had potent antitrypanosomal activity (half-maximal effective concentration = 0.050 to 3.2 μM) and good selectivity for the analogues over human HepG2 cells (range, 7.4- to over 641-fold). The majority of analogues were potent inhibitors of TbGSK3s, and correlation studies for an indirubin subset, namely, the 6-bromosubstituted 3′-oxime bearing an extra bulky substituent on the 3′ oxime [(6-BIO-3′-bulky)-substituted indirubins], revealed a positive correlation between kinase inhibition and antitrypanosomal activity. Insights into this indirubin-TbGSK3s interaction were provided by structure-activity relationship studies. Comparison between 6-BIO-3′-bulky-substituted indirubin-treated parasites and parasites silenced for TbGSK3s by RNA interference suggested that the above-described compounds may target TbGSK3s in vivo. To further understand the molecular basis of the growth arrest brought about by the inhibition or ablation of TbGSK3s, we investigated the intracellular localization of TbGSK3s. TbGSK3s was present in cytoskeletal structures, including the flagellum and basal body area. Overall, these results give insights into the mode of action of 6-BIO-3′-bulky-substituted indirubins that are promising hits for antitrypanosomal drug discovery.

scholarly journals Assessment of Mycobacterium tuberculosis Pantothenate Kinase Vulnerability through Target Knockdown and Mechanistically Diverse Inhibitors

2014 ◽  
Vol 58 (6) ◽  
pp. 3312-3326 ◽  
Author(s):  
B. K. Kishore Reddy ◽  
Sudhir Landge ◽  
Sudha Ravishankar ◽  
Vikas Patil ◽  
Vikas Shinde ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Present Report ◽  
Kinetic Studies ◽  
Antimycobacterial Activity ◽  
Type I ◽  
Pantothenate Kinase ◽  
Content Type ◽  
Rate Limiting Step

ABSTRACTPantothenate kinase (PanK) catalyzes the phosphorylation of pantothenate, the first committed and rate-limiting step toward coenzyme A (CoA) biosynthesis. In our earlier reports, we had established that the type I isoform encoded by thecoaAgene is an essential pantothenate kinase inMycobacterium tuberculosis, and this vital information was then exploited to screen large libraries for identification of mechanistically different classes of PanK inhibitors. The present report summarizes the synthesis and expansion efforts to understand the structure-activity relationships leading to the optimization of enzyme inhibition along with antimycobacterial activity. Additionally, we report the progression of two distinct classes of inhibitors, the triazoles, which are ATP competitors, and the biaryl acetic acids, with a mixed mode of inhibition. Cocrystallization studies provided evidence of these inhibitors binding to the enzyme. This was further substantiated with the biaryl acids having MIC against the wild-typeM. tuberculosisstrain and the subsequent establishment of a target link with an upshift in MIC in a strain overexpressing PanK. On the other hand, the ATP competitors had cellular activity only in aM. tuberculosisknockdown strain with reduced PanK expression levels. Additionally,in vitroandin vivosurvival kinetic studies performed with aM. tuberculosisPanK (MtPanK) knockdown strain indicated that the target levels have to be significantly reduced to bring in growth inhibition. The dual approaches employed here thus established the poor vulnerability of PanK inM. tuberculosis.

scholarly journals A New NonpolarN-Hydroxy Imidazoline Lead Compound with Improved Activity in a Murine Model of Late-Stage Trypanosoma brucei brucei Infection Is Not Cross-Resistant with Diamidines

2014 ◽  
Vol 59 (2) ◽  
pp. 890-904 ◽  
Author(s):  
Carlos H. Ríos Martínez ◽  
Florence Miller ◽  
Kayathiri Ganeshamoorthy ◽  
Fabienne Glacial ◽  
Marcel Kaiser ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Late Stage ◽  
Parent Compound ◽  
Central Nervous System Infection ◽  
Sleeping Sickness ◽  
Intrinsic Activity ◽  
Cross Resistance ◽  
Content Type

ABSTRACTTreatment of late-stage sleeping sickness requires drugs that can cross the blood-brain barrier (BBB) to reach the parasites located in the brain. We report here the synthesis and evaluation of four newN-hydroxy and 12 newN-alkoxy derivatives of bisimidazoline leads as potential agents for the treatment of late-stage sleeping sickness. These compounds, which have reduced basicity compared to the parent leads (i.e., are less ionized at physiological pH), were evaluatedin vitroagainstTrypanosoma brucei rhodesienseandin vivoin murine models of first- and second-stage sleeping sickness. Resistance profile, physicochemical parameters,in vitroBBB permeability, and microsomal stability also were determined. TheN-hydroxy imidazoline analogues were the most effectivein vivo, with 4-((1-hydroxy-4,5-dihydro-1H-imidazol-2-yl)amino)-N-(4-((1-hydroxy-4,5-dihydro-1H-imidazol-2-yl)amino)phenyl)benzamide (14d) showing 100% cures in the first-stage disease, while 15d, 16d, and 17d appeared to slightly improve survival. In addition, 14d showed weak activity in the chronic model of central nervous system infection in mice. No evidence of reduction of this compound with hepatic microsomes and mitochondria was foundin vitro, suggesting thatN-hydroxy imidazolines are metabolically stable and have intrinsic activity againstT. brucei. In contrast to its unsubstituted parent compound, the uptake of 14d inT. bruceiwas independent of known drug transporters (i.e.,T. bruceiAT1/P2 and HAPT), indicating a lower predisposition to cross-resistance with other diamidines and arsenical drugs. Hence, theN-hydroxy bisimidazolines (14d in particular) represent a new class of promising antitrypanosomal agents.

scholarly journals Interferon-Stimulated Gene (ISG)-Expression Screening Reveals the Specific Antibunyaviral Activity of ISG20

2018 ◽  
Vol 92 (13) ◽  
Author(s):  
Junjie Feng ◽  
Arthur Wickenhagen ◽  
Matthew L. Turnbull ◽  
Veronica V. Rezelj ◽  
Felix Kreher ◽  
...  
Keyword(s):  
Viral Infections ◽  
Rhesus Macaques ◽  
Type I ◽  
Emerging Viruses ◽  
Content Type ◽  
Expression Screening ◽  
Interferon Stimulated Genes ◽  
Life Threatening

ABSTRACT Bunyaviruses pose a significant threat to human health, prosperity, and food security. In response to viral infections, interferons (IFNs) upregulate the expression of hundreds of interferon-stimulated genes (ISGs), whose cumulative action can potently inhibit the replication of bunyaviruses. We used a flow cytometry-based method to screen the ability of ∼500 unique ISGs from humans and rhesus macaques to inhibit the replication of Bunyamwera orthobunyavirus (BUNV), the prototype of both the Peribunyaviridae family and the Bunyavirales order. Candidates possessing antibunyaviral activity were further examined using a panel of divergent bunyaviruses. Interestingly, one candidate, ISG20, exhibited potent antibunyaviral activity against most viruses examined from the Peribunyaviridae , Hantaviridae , and Nairoviridae families, whereas phleboviruses ( Phenuiviridae ) largely escaped inhibition. Similar to the case against other viruses known to be targeted by ISG20, the antibunyaviral activity of ISG20 is dependent upon its functional RNase activity. Through use of an infectious virus-like particle (VLP) assay (based on the BUNV minigenome system), we confirmed that gene expression from all 3 viral segments is strongly inhibited by ISG20. Using in vitro evolution, we generated a substantially ISG20-resistant BUNV and mapped the determinants of ISG20 sensitivity/resistance. Taking all the data together, we report that ISG20 is a broad and potent antibunyaviral factor but that some bunyaviruses are remarkably ISG20 resistant. Thus, ISG20 sensitivity/resistance may influence the pathogenesis of bunyaviruses, many of which are emerging viruses of clinical or veterinary significance. IMPORTANCE There are hundreds of bunyaviruses, many of which cause life-threatening acute diseases in humans and livestock. The interferon (IFN) system is a key component of innate immunity, and type I IFNs limit bunyaviral propagation both in vitro and in vivo . Type I IFN signaling results in the upregulation of hundreds of IFN-stimulated genes (ISGs), whose concerted action generates an “antiviral state.” Although IFNs are critical in limiting bunyaviral replication and pathogenesis, much is still unknown about which ISGs inhibit bunyaviruses. Using ISG-expression screening, we examined the ability of ∼500 unique ISGs to inhibit Bunyamwera orthobunyavirus (BUNV), the prototypical bunyavirus. Using this approach, we identified ISG20, an interferon-stimulated exonuclease, as a potent inhibitor of BUNV. Interestingly, ISG20 possesses highly selective antibunyaviral activity, with multiple bunyaviruses being potently inhibited while some largely escape inhibition. We speculate that the ability of some bunyaviruses to escape ISG20 may influence their pathogenesis.

scholarly journals Toll-Like Receptor 9 Signaling in Dendritic Cells Regulates Neutrophil Recruitment to Inflammatory Foci following Leishmania infantum Infection

2015 ◽  
Vol 83 (12) ◽  
pp. 4604-4616 ◽  
Author(s):  
Laís Sacramento ◽  
Silvia C. Trevelin ◽  
Manuela S. Nascimento ◽  
Djalma S. Lima-Jùnior ◽  
Diego L. Costa ◽  
...  
Keyword(s):  
Leishmania Infantum ◽  
Critical Role ◽  
Costimulatory Molecule ◽  
Protozoan Parasite ◽  
Neutrophil Recruitment ◽  
Content Type ◽  
Target Organs ◽  
Myd88 Signaling

Leishmania infantumis a protozoan parasite that causes visceral leishmaniasis (VL). This infection triggers dendritic cell (DC) activation through the recognition of microbial products by Toll-like receptors (TLRs). Among the TLRs, TLR9 is required for DC activation by differentLeishmaniaspecies. We demonstrated that TLR9 is upregulatedin vitroandin vivoduring infection. We show that C57BL/6 mice deficient in TLR9 expression (TLR9−/−mice) are more susceptible to infection and display higher parasite numbers in the spleen and liver. The increased susceptibility of TLR9−/−mice was due to the impaired recruitment of neutrophils to the infection foci associated with reduced levels of neutrophil chemoattractants released by DCs in the target organs. Moreover, both Th1 and Th17 cells were also committed in TLR9−/−mice. TLR9-dependent neutrophil recruitment is mediated via the MyD88 signaling pathway but is TIR domain-containing adapter-inducing interferon beta (TRIF) independent. Furthermore,L. infantumfailed to activate both plasmacytoid and myeloid DCs from TLR9−/−mice, which presented reduced surface costimulatory molecule expression and chemokine release. Interestingly, neutrophil chemotaxis was affected bothin vitroandin vivowhen DCs were derived from TLR9−/−mice. Our results suggest that TLR9 plays a critical role in neutrophil recruitment during the protective response againstL. infantuminfection that could be associated with DC activation.

scholarly journals Structural Characterization of Acidic M17 Leucine Aminopeptidases from the TriTryps and Evaluation of Their Role in Nutrient Starvation in Trypanosoma brucei

mSphere ◽  
2017 ◽  
Vol 2 (4) ◽  
Author(s):  
Jennifer Timm ◽  
Maria Valente ◽  
Daniel García-Caballero ◽  
Keith S. Wilson ◽  
Dolores González-Pacanowska
Keyword(s):  
Homologous Recombination ◽  
Host Cell ◽  
Trypanosoma Brucei ◽  
Cell Invasion ◽  
Essential Amino Acids ◽  
Host Cell Invasion ◽  
Content Type ◽  
Hydrolysis Of

ABSTRACT Leucine aminopeptidases (LAPs) catalyze the hydrolysis of the N-terminal amino acid of peptides and are considered potential drug targets. They are involved in multiple functions ranging from host cell invasion and provision of essential amino acids to site-specific homologous recombination and transcription regulation. In kinetoplastid parasites, there are at least three distinct LAPs. The availability of the crystal structures provides important information for drug design. Here we report the structure of the acidic LAPs from three kinetoplastids in complex with different inhibitors and explore their role in Trypanosoma brucei survival under various nutrient conditions. Importantly, the acidic LAP is dispensable for growth both in vitro and in vivo, an observation that questions its use as a specific drug target. While LAP-A is not essential, leucine depletion and subcellular localization studies performed under starvation conditions suggest a possible function of LAP-A in the response to nutrient restriction. Leucine aminopeptidase (LAP) is found in all kingdoms of life and catalyzes the metal-dependent hydrolysis of the N-terminal amino acid residue of peptide or amino acyl substrates. LAPs have been shown to participate in the N-terminal processing of certain proteins in mammalian cells and in homologous recombination and transcription regulation in bacteria, while in parasites, they are involved in host cell invasion and provision of essential amino acids for growth. The enzyme is essential for survival in Plasmodium falciparum, where its drug target potential has been suggested. We report here the X-ray structures of three kinetoplastid acidic LAPs (LAP-As from Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major) which were solved in the metal-free and unliganded forms, as well as in a number of ligand complexes, providing insight into ligand binding, metal ion requirements, and oligomeric state. In addition, we analyzed mutant cells defective in LAP-A in Trypanosoma brucei, strongly suggesting that the enzyme is not required for the growth of this parasite either in vitro or in vivo. In procyclic cells, LAP-A was equally distributed throughout the cytoplasm, yet upon starvation, it relocalizes in particles that concentrate in the perinuclear region. Overexpression of the enzyme conferred a growth advantage when parasites were grown in leucine-deficient medium. Overall, the results suggest that in T. brucei, LAP-A may participate in protein degradation associated with nutrient depletion. IMPORTANCE Leucine aminopeptidases (LAPs) catalyze the hydrolysis of the N-terminal amino acid of peptides and are considered potential drug targets. They are involved in multiple functions ranging from host cell invasion and provision of essential amino acids to site-specific homologous recombination and transcription regulation. In kinetoplastid parasites, there are at least three distinct LAPs. The availability of the crystal structures provides important information for drug design. Here we report the structure of the acidic LAPs from three kinetoplastids in complex with different inhibitors and explore their role in Trypanosoma brucei survival under various nutrient conditions. Importantly, the acidic LAP is dispensable for growth both in vitro and in vivo, an observation that questions its use as a specific drug target. While LAP-A is not essential, leucine depletion and subcellular localization studies performed under starvation conditions suggest a possible function of LAP-A in the response to nutrient restriction.

scholarly journals Broadening the Spectrum of β-Lactam Antibiotics through Inhibition of Signal Peptidase Type I

2012 ◽  
Vol 56 (9) ◽  
pp. 4662-4670 ◽  
Author(s):  
Alex G. Therien ◽  
Joann L. Huber ◽  
Kenneth E. Wilson ◽  
Patrick Beaulieu ◽  
Alexandre Caron ◽  
...  
Keyword(s):  
Treatment Options ◽  
Signal Peptidase ◽  
Type I ◽  
New Agents ◽  
Content Type ◽  
Cyclic Depsipeptide ◽  
Serious Infections ◽  
Synthetic Derivative

ABSTRACTThe resistance of methicillin-resistantStaphylococcus aureus(MRSA) to all β-lactam classes limits treatment options for serious infections involving this organism. Our goal is to discover new agents that restore the activity of β-lactams against MRSA, an approach that has led to the discovery of two classes of natural product antibiotics, a cyclic depsipeptide (krisynomycin) and a lipoglycopeptide (actinocarbasin), which potentiate the activity of imipenem against MRSA strain COL. We report here that these imipenem synergists are inhibitors of the bacterial type I signal peptidase SpsB, a serine protease that is required for the secretion of proteins that are exported through the Sec and Tat systems. A synthetic derivative of actinocarbasin, M131, synergized with imipenem bothin vitroandin vivowith potent efficacy. Thein vitroactivity of M131 extends to clinical isolates of MRSA but not to a methicillin-sensitive strain. Synergy is restricted to β-lactam antibiotics and is not observed with other antibiotic classes. We propose that the SpsB inhibitors synergize with β-lactams by preventing the signal peptidase-mediated secretion of proteins required for β-lactam resistance. Combinations of SpsB inhibitors and β-lactams may expand the utility of these widely prescribed antibiotics to treat MRSA infections, analogous to β-lactamase inhibitors which restored the utility of this antibiotic class for the treatment of resistant Gram-negative infections.

scholarly journals RNase III Domain of KREPB9 and KREPB10 Association with Editosomes in Trypanosoma brucei

mSphere ◽  
2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Jason Carnes ◽  
Suzanne M. McDermott ◽  
Kenneth Stuart
Keyword(s):  
Life Cycle ◽  
Trypanosoma Brucei ◽  
Protozoan Parasite ◽  
Tsetse Fly ◽  
Accessory Proteins ◽  
Rnase Iii ◽  
Parasite Life Cycle ◽  
Null Cell ◽  
Content Type

ABSTRACT Editosomes are the multiprotein complexes that catalyze the insertion and deletion of uridines to create translatable mRNAs in the mitochondria of kinetoplastids. Recognition and cleavage of a broad diversity of RNA substrates in vivo require three functionally distinct RNase III-type endonucleases, as well as five additional editosome proteins that contain noncatalytic RNase III domains. RNase III domains have recently been identified in the editosome accessory proteins KREPB9 and KREPB10, suggesting a role related to editing endonuclease function. In this report, we definitively show that KREPB9 and KREPB10 are not essential in either bloodstream-form parasites (BF) or procyclic-form parasites (PF) by creating null or conditional null cell lines. While preedited and edited transcripts are largely unaffected by the loss of KREPB9 in both PF and BF, loss of KREPB10 produces distinct responses in BF and PF. BF cells lacking KREPB10 also lack edited CYb, while PF cells have increased edited A6, RPS12, ND3, and COII after loss of KREPB10. We also demonstrate that mutation of the RNase III domain of either KREPB9 or KREPB10 results in decreased association with ~20S editosomes. Editosome interactions with KREPB9 and KREPB10 are therefore mediated by the noncatalytic RNase III domain, consistent with a role in endonuclease specialization in Trypanosoma brucei. IMPORTANCE Trypanosoma brucei is a protozoan parasite that causes African sleeping sickness. U insertion/deletion RNA editing in T. brucei generates mature mitochondrial mRNAs. Editing is essential for survival in mammalian hosts and tsetse fly vectors and is differentially regulated during the parasite life cycle. Three multiprotein “editosomes,” typified by exclusive RNase III endonucleases that act at distinct sites, catalyze editing. Here, we show that editosome accessory proteins KREPB9 and KREPB10 are not essential for mammalian blood- or insect-form parasite survival but have specific and differential effects on edited RNA abundance in different stages. We also characterize KREPB9 and KREPB10 noncatalytic RNase III domains and show they are essential for editosome association, potentially via dimerization with RNase III domains in other editosome proteins. This work enhances the understanding of distinct editosome and accessory protein functions, and thus differential editing, during the parasite life cycle and highlights the importance of RNase III domain interactions to editosome architecture.

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