scholarly journals The Cross Talk between TbTim50 and PIP39, Two Aspartate-Based Protein Phosphatases, Maintains Cellular Homeostasis in Trypanosoma brucei

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Anuj Tripathi ◽  
Ujjal K. Singha ◽  
Victor Paromov ◽  
Salisha Hill ◽  
Siddharth Pratap ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Cross Talk ◽  
Developmental Regulation ◽  
Protein Phosphatases ◽  
Infectious Agent ◽  
Cellular Stress Response ◽  
Content Type ◽  
Mitochondrial Inner Membrane ◽  
Inner Membrane Protein ◽  
Metabolic Activities

Trypanosoma brucei, the infectious agent of African trypanosomiasis, must adapt to strikingly different host environments during its digenetic life cycle. Developmental regulation of mitochondrial activities is an essential part of these processes. We have shown previously that mitochondrial inner membrane protein translocase 50 in T. brucei (TbTim50) possesses a dually specific phosphatase activity and plays a role in the cellular stress response pathway. Using proteomics analysis, here we have elucidated a novel connection between TbTim50 and a protein phosphatase of the same family, PIP39, which is also a differentiation-related protein localized in glycosomes. We found that these two protein phosphatases cross talk via the AMPK pathway and modulate cellular metabolic activities under stress. Together, our results indicate the importance of a TbTim50 and PIP39 cascade for communication between mitochondria and other cellular parts in regulation of cell homeostasis in T. brucei.

scholarly journals Structural and Functional Association of Trypanosoma brucei MIX Protein with Cytochrome c Oxidase Complex

2008 ◽  
Vol 7 (11) ◽  
pp. 1994-2003 ◽  
Author(s):  
Alena Zíková ◽  
Aswini K. Panigrahi ◽  
Alessandro D. Uboldi ◽  
Rachel A. Dalley ◽  
Emanuela Handman ◽  
...  
Keyword(s):  
Cell Division ◽  
Trypanosoma Brucei ◽  
Leishmania Major ◽  
Affinity Purification ◽  
Major Effect ◽  
Multiprotein Complex ◽  
Complex Iv ◽  
Mitochondrial Inner Membrane ◽  
Single Allele ◽  
Inner Membrane Protein

ABSTRACT A mitochondrial inner membrane protein, designated MIX, seems to be essential for cell viability. The deletion of both alleles was not possible, and the deletion of a single allele led to a loss of virulence and aberrant mitochondrial segregation and cell division in Leishmania major. However, the mechanism by which MIX exerts its effect has not been determined. We show here that MIX is also expressed in the mitochondrion of Trypanosoma brucei, and using RNA interference, we found that its loss leads to a phenotype that is similar to that described for Leishmania. The loss of MIX also had a major effect on cytochrome c oxidase activity, on the mitochondrial membrane potential, and on the production of mitochondrial ATP by oxidative phosphorylation. Using a tandem affinity purification tag, we found that MIX is associated with a multiprotein complex that contains subunits of the mitochondrial cytochrome c oxidase complex (respiratory complex IV), the composition of which was characterized in detail. The specific function of MIX is unknown, but it appears to be important for the function of complex IV and for mitochondrial segregation and cell division in T. brucei.

scholarly journals Divergent Small Tim Homologues Are Associated with TbTim17 and Critical for the Biogenesis of TbTim17 Protein Complexes in Trypanosoma brucei

mSphere ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
Joseph T. Smith ◽  
Ujjal K. Singha ◽  
Smita Misra ◽  
Minu Chaudhuri
Keyword(s):  
Membrane Proteins ◽  
Trypanosoma Brucei ◽  
Mitochondrial Protein ◽  
Intermembrane Space ◽  
Inner Membrane ◽  
Content Type ◽  
Mitochondrial Inner Membrane ◽  
Mitochondrial Intermembrane Space ◽  
The Stability ◽  
Tim Proteins

ABSTRACT The small Tim proteins belong to a group of mitochondrial intermembrane space chaperones that aid in the import of mitochondrial inner membrane proteins with internal targeting signals. Trypanosoma brucei , the protozoan parasite that causes African trypanosomiasis, possesses multiple small Tim proteins that include homologues of T. brucei Tim9 (TbTim9) and Tim10 (TbTim10) and a unique small Tim that shares homology with both Tim8 and Tim13 (TbTim8/13). Here, we found that these three small TbTims are expressed as soluble mitochondrial intermembrane space proteins. Coimmunoprecipitation and mass spectrometry analysis showed that the small TbTims stably associated with each other and with TbTim17, the major component of the mitochondrial inner membrane translocase in T. brucei . Yeast two-hybrid analysis indicated direct interactions among the small TbTims; however, their interaction patterns appeared to be different from those of their counterparts in yeast and humans. Knockdown of the small TbTims reduced cell growth and decreased the steady-state level of TbTim17 and T. brucei ADP/ATP carrier (TbAAC), two polytopic mitochondrial inner membrane proteins. Knockdown of small TbTims also reduced the matured complexes of TbTim17 in mitochondria. Depletion of any of the small TbTims reduced TbTim17 import moderately but greatly hampered the stability of the TbTim17 complexes in T. brucei . Altogether, our results revealed that TbTim9, TbTim10, and TbTim8/13 interact with each other, associate with TbTim17, and play a crucial role in the integrity and maintenance of the levels of TbTim17 complexes. IMPORTANCE Trypanosoma brucei is the causative agent of African sleeping sickness. The parasite’s mitochondrion represents a useful source for potential chemotherapeutic targets. Similarly to yeast and humans, mitochondrial functions depend on the import of proteins that are encoded in the nucleus and made in the cytosol. Even though the machinery involved in this mitochondrial protein import process is becoming clearer in T. brucei , a comprehensive picture of protein complex composition and function is still lacking. In this study, we characterized three T. brucei small Tim proteins, TbTim9, TbTim10, and TbTim8/13. Although the parasite does not have the classical TIM22 complex that imports mitochondrial inner membrane proteins containing internal targeting signals in yeast or humans, we found that these small TbTims associate with TbTim17, the major subunit of the TbTIM complex in T. brucei , and play an essential role in the stability of the TbTim17 complexes. Therefore, these divergent proteins are critical for mitochondrial protein biogenesis in T. brucei .

scholarly journals Functional Complementation Analyses Reveal that the Single PRAT Family Protein of Trypanosoma brucei Is a Divergent Homolog of Tim17 in Saccharomyces cerevisiae

2015 ◽  
Vol 14 (3) ◽  
pp. 286-296 ◽  
Author(s):  
Ebony Weems ◽  
Ujjal K. Singha ◽  
VaNae Hamilton ◽  
Joseph T. Smith ◽  
Karin Waegemann ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Trypanosoma Brucei ◽  
Mitochondrial Proteins ◽  
Growth Defect ◽  
Heterologous Proteins ◽  
Homologous Proteins ◽  
Content Type ◽  
Mitochondrial Inner Membrane ◽  
Family Protein ◽  
Higher Eukaryotes

ABSTRACT Trypanosoma brucei , a parasitic protozoan that causes African trypanosomiasis, possesses a single member of the presequence and amino acid transporter (PRAT) protein family, which is referred to as TbTim17. In contrast, three homologous proteins, ScTim23, ScTim17, and ScTim22, are found in Saccharomyces cerevisiae and higher eukaryotes. Here, we show that TbTim17 cannot rescue Tim17, Tim23, or Tim22 mutants of S. cerevisiae . We expressed S. cerevisiae Tim23, Tim17, and Tim22 in T. brucei . These heterologous proteins were properly imported into mitochondria in the parasite. Further analysis revealed that although ScTim23 and ScTim17 were integrated into the mitochondrial inner membrane and assembled into a protein complex similar in size to TbTim17, only ScTim17 was stably associated with TbTim17. In contrast, ScTim22 existed as a protease-sensitive soluble protein in the T. brucei mitochondrion. In addition, the growth defect caused by TbTim17 knockdown in T. brucei was partially restored by the expression of ScTim17 but not by the expression of either ScTim23 or ScTim22, whereas the expression of TbTim17 fully complemented the growth defect caused by TbTim17 knockdown, as anticipated. Similar to the findings for cell growth, the defect in the import of mitochondrial proteins due to depletion of TbTim17 was in part restored by the expression of ScTim17 but was not complemented by the expression of either ScTim23 or ScTim22. Together, these results suggest that TbTim17 is divergent compared to ScTim23 but that its function is closer to that of ScTim17. In addition, ScTim22 could not be sorted properly in the T. brucei mitochondrion and thus failed to complement the function of TbTim17.

scholarly journals Characterization of the mitochondrial inner membrane protein translocator Tim17 from Trypanosoma brucei

2008 ◽  
Vol 159 (1) ◽  
pp. 30-43 ◽  
Author(s):  
Ujjal K. Singha ◽  
Emmanuel Peprah ◽  
Shuntae Williams ◽  
Robert Walker ◽  
Lipi Saha ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Trypanosoma Brucei ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Inner Membrane Protein

scholarly journals The Phosphohistidine Phosphatase SixA Targets a Phosphotransferase System

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Jane E. Schulte ◽  
Mark Goulian
Keyword(s):  
Escherichia Coli ◽  
Protein Phosphatases ◽  
Growth Defect ◽  
Phosphotransferase System ◽  
Protein Activity ◽  
Content Type ◽  
Bacterial Physiology ◽  
Phosphoryl Groups ◽  
Regulate Protein ◽  
Protein Components

ABSTRACTSixA, a well-conserved protein found in proteobacteria, actinobacteria, and cyanobacteria, is the only reported example of a bacterial phosphohistidine phosphatase. A single protein target of SixA has been reported to date: theEscherichia colihistidine kinase ArcB. The present work analyzes an ArcB-independent growth defect of asixAdeletion inE. coli. A screen for suppressors, analysis of various mutants, and phosphorylation assays indicate that SixA modulates phosphorylation of the nitrogen-related phosphotransferase system (PTSNtr). The PTSNtris a widely conserved bacterial pathway that regulates diverse metabolic processes through the phosphorylation states of its protein components, EINtr, NPr, and EIIANtr, which receive phosphoryl groups on histidine residues. However, a mechanism for dephosphorylating this system has not been reported. The results presented here suggest a model in which SixA removes phosphoryl groups from the PTSNtrby acting on NPr. This work uncovers a new role for the phosphohistidine phosphatase SixA and, through factors that affect SixA expression or activity, may point to additional inputs that regulate the PTSNtr.IMPORTANCEOne common means to regulate protein activity is through phosphorylation. Protein phosphatases exist to reverse this process, returning the protein to the unphosphorylated form. The vast majority of protein phosphatases that have been identified target phosphoserine, phosphotheronine, and phosphotyrosine. A widely conserved phosphohistidine phosphatase was identified inEscherichia coli20 years ago but remains relatively understudied. The present work shows that this phosphatase modulates the nitrogen-related phosphotransferase system, a pathway that is regulated by nitrogen and carbon metabolism and affects diverse aspects of bacterial physiology. Until now, there was no known mechanism for removing phosphoryl groups from this pathway.

scholarly journals Variable Virulence of Biotype 3Vibrio vulnificusdue to MARTX Toxin Effector Domain Composition

mSphere ◽  
2017 ◽  
Vol 2 (4) ◽  
Author(s):  
Byoung Sik Kim ◽  
Hannah E. Gavin ◽  
Karla J. F. Satchell
Keyword(s):  
Food Chain ◽  
Vibrio Vulnificus ◽  
Infectious Agent ◽  
High Morbidity ◽  
Effector Domain ◽  
Content Type ◽  
Human Food ◽  
Effector Domains ◽  
Human Food Chain ◽  
Natural Hosts

ABSTRACTVibrio vulnificusis an environmental organism that causes septic human infections characterized by high morbidity and mortality. The annual incidence and global distribution of this pathogen are increasing as ocean waters warm. Clinical strains exhibit variations in the primary virulence toxin, suggesting a potential for the emergence of new strains with altered virulence properties. A clonal outbreak of tilapia-associated wound infections in Israel serves as a natural experiment for the sudden emergence of a newV. vulnificusstrain. The effector domain content of the multifunctional autoprocessing RTX (MARTX) toxin of the outbreak-associated biotype 3 (BT3) strains was previously shown to harbor a modification generated by recombination. The modification introduced an actin-induced adenylate cyclase effector domain (ExoY) and an effector domain that disrupts the Golgi organelle (DmX). Here, we report that the exchange of these effector domains for a putative progenitor biotype 1 toxin arrangement produces a toxin that slows the lysis kinetics of targeted epithelial cells but increases cellular rounding phenotypes in response to bacteria. In addition, replacing the biotype 3 toxin variant with the putative progenitor biotype 1 variant renders the resulting strain significantly more virulent in mice. This suggests that the exchange of MARTX effector domains during the emergence of BT3 generated a toxin with reduced toxin potency, resulting in decreased virulence of this outbreak-associated strain. We posit that selection for reduced virulence may serve as a route for this lethal infectious agent to enter the human food chain by allowing it to persist in natural hosts.IMPORTANCEVibrio vulnificusis a serious infection linked to climate change. The virulence capacity of these bacteria can vary by gene exchange, resulting in new variants of the primary virulence toxin. In this study, we tested whether the emergence of an epidemic strain ofV. vulnificuswith a novel toxin variant correlated with a change in virulence. We found that restoring the biotype 3 toxin variant to the putative progenitor-type toxin resulted in dramatically increased virulence, revealing that the emergence of the biotype 3 strain could be linked to virulence reduction. This reduced virulence, previously found also in the biotype 1 strain, suggests that reduced virulence may stimulate outbreaks, as strains have greater capacity to enter the human food chain through reduced impact to environmental hosts.

scholarly journals The Nuclear Export Receptors TbMex67 and TbMtr2 Are Required for Ribosome Biogenesis in Trypanosoma brucei

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Constance Rink ◽  
Martin Ciganda ◽  
Noreen Williams
Keyword(s):  
Trypanosoma Brucei ◽  
Nuclear Export ◽  
Ribosome Biogenesis ◽  
Biological Process ◽  
Large Subunit ◽  
Critical Role ◽  
Ribosomal Subunits ◽  
Content Type ◽  
Protein Components ◽  
Export Receptors

ABSTRACT Ribosomal maturation is a complex and highly conserved biological process involving migration of a continuously changing RNP across multiple cellular compartments. A critical point in this process is the translocation of individual ribosomal subunits (60S and 40S) from the nucleus to the cytoplasm, and a number of export factors participate in this process. In this study, we characterize the functional role of the auxiliary export receptors TbMex67 and TbMtr2 in ribosome biogenesis in the parasite Trypanosoma brucei. We demonstrate that depletion of each of these proteins dramatically impacts the steady-state levels of other proteins involved in ribosome biogenesis, including the trypanosome-specific factors P34 and P37. In addition, we observe that the loss of TbMex67 or TbMtr2 leads to aberrant ribosome formation, rRNA processing, and polysome formation. Although the TbMex67-TbMtr2 heterodimer is structurally distinct from Mex67-Mtr2 complexes previously studied, our data show that they retain a conserved function in ribosome biogenesis. IMPORTANCE The nuclear export of ribosomal subunits (60S and 40S) depends in part on the activity of the essential auxiliary export receptors TbMtr2 and TbMex67. When these proteins are individually depleted from the medically and agriculturally significant parasite Trypanosoma brucei, distinct alterations in the processing of the rRNAs of the large subunit (60S) are observed as well as aberrations in the assembly of functional ribosomes (polysomes). We also established that TbMex67 and TbMtr2 interact directly or indirectly with the protein components of the 5S RNP, including the trypanosome-specific P34/P37. The critical role that TbMex67 and TbMtr2 play in this essential biological process together with their parasite-specific interactions may provide new therapeutic targets against this important parasite.

Abstract 15552: Acute Estrogen-GPER1 Stimulation Preserves Mitochondrial Inner Membrane Protein (Mitofilin) From Degradation caused by Ischemia/Reperfusion Stress

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Tanoya L. C Harris ◽  
Bjorn Olde ◽  
Fredrik Leeb-Lundberg ◽  
Jean C. Bopassa
Keyword(s):  
Electron Microscopy ◽  
Spatial Organization ◽  
Ischemia Reperfusion ◽  
Estrogen Treatment ◽  
Mitochondrial Inner Membrane ◽  
Mitochondrial Integrity ◽  
Inner Membrane Protein ◽  
Estrogen Effect ◽  
And Function ◽  
High Resolution Microscopy

Introduction: We recently found that acute estrogen treatment delays the mitochondrial permeability transition pore opening and reduces ROS production after ischemia/reperfusion, suggesting that estrogen promotes mitochondrial integrity. As mitochondrial inner membrane protein (mitofilin) has been found to control mitochondrial cristae morphology and function, we investigated whether estrogen effect on mitochondrial integrity after ischemia/reperfusion involved regulation of mitofilin via G-Protein Coupled Estrogen Receptor1 (GPER1) activation. Methods: Isolated hearts from male WT (C57BL/6NCrL), and GPER1-/- mice were perfused using Langendorff technique, with and without estrogen (40 nM). Hearts were subjected to 20 min global ischemia followed by 10 min reperfusion. Mitochondria were isolated, and 2D-DIGE followed by mass spectrometry was performed. Mitofilin expression was confirmed by Western blot analysis in mitochondrial fractions. Mitofilin distribution in cardiomyocytes, and its spatial organization in single mitochondria were visualized using high resolution microscopy. Electron microscopy was used to observe the state of mitochondrial cristae morphology. Results: Analysis revealded 52 unique proteins of interest, in which mitofilin was identified. Immunoblot analysis confirmed an increased in mitofilin level with estrogen treatment as compared to control in WT but not in GPER1-/-. We found, as observed in non-ischemic myocytes, that mitofilin in estrogen-treated cardiomyocytes was distributed in the peri-membrane and T-tubules, while only peri-membrane mitofilin was more visible in control group. High resolution microscopy showed a better spatial organization of mitofilin in single mitochondria with estrogen treatment compared to control, in which mitofilin was almost absent. Electron microscopy revealded that mitochondrial morphology was preserved with estrogen treatment, as cristae were well organized compared to control, in which cristae were disrupted. Conclusion: These data indicate that estrogen up-regulates mitofilin expression during ischemia/reperfusion. Estrogen effect on mitofilin may contribute to improved mitochondrial integrity and function.

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