Molecular cloning and characterization of mitogen-activated protein kinase 2 in<i>Trypanosoma cruzi</i>

Bao, Yi; Weiss, Louis M.; Fen, Yan; Lisanti, Michael P.; Tanowitz, Herbert B.; Das, Bhaskar C.; Zheng, Renjian; Huang, Huan

doi:10.4161/cc.9.14.12372

Cited by 13 publications

(20 citation statements)

References 34 publications

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“…1C). These values are comparable to those from other studies using pTEX or pTEX-derived vectors to overexpress proteins in the cytosol (78,79), mitochondria (80), glycosomes (81), endosomal/lysosomal system and reservosomes (75,82), ER (68), flagellar pocket (83), or plasma membrane (69,(84)(85)(86) of T. cruzi. The protein was stable, with no indication of a C-terminal proteolytic cleavage event observed when expressed in L. tarentolae (21), and it appeared to be inserted into the rER and N-glycosylated normally (Fig.…”

Section: Discussionsupporting

confidence: 75%

“…Although it is difficult to exclude the possibility that the 7-fold overexpression of a single protein, such as TcOGNT2, inhibits metacyclogenesis by a nonspecific stress effect, it is notable that proliferation and TCT production were not affected ( Fig. 8 and 9) and that overexpression of other proteins failed to inhibit this process (58,68,69,(78)(79)(80)(81)(82)(84)(85)(86). The protein burden of 7-fold accumulation of a single enzyme is not anticipated to be great, and indeed, overexpression of a truncated N-terminal fragment of the TcOGNT2 homolog TbOGNT2 in Trypanosoma brucei did not affect Golgi replication (93).…”

Section: Discussionmentioning

confidence: 99%

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Golgi UDP-GlcNAc:Polypeptide O -α- N -Acetyl- d -Glucosaminyltransferase 2 (TcOGNT2) Regulates Trypomastigote Production and Function in Trypanosoma cruzi

et al. 2014

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e All life cycle stages of the protozoan parasite Trypanosoma cruzi are enveloped by mucin-like glycoproteins which, despite major changes in their polypeptide cores, are extensively and similarly O-glycosylated. O-Glycan biosynthesis is initiated by the addition of ␣GlcNAc to Thr in a reaction catalyzed by Golgi UDP-GlcNAc:polypeptide O-␣-N-acetyl-D-glucosaminyltransferases (pp␣GlcNAcTs), which are encoded by TcOGNT1 and TcOGNT2. We now directly show that TcOGNT2 is associated with the Golgi apparatus of the epimastigote stage and is markedly downregulated in both differentiated metacyclic trypomastigotes (MCTs) and cell culture-derived trypomastigotes (TCTs). The significance of downregulation was examined by forced continued expression of TcOGNT2, which resulted in a substantial increase of TcOGNT2 protein levels but only modestly increased pp␣GlcNAcT activity in extracts and altered cell surface glycosylation in TCTs. Constitutive TcOGNT2 overexpression had no discernible effect on proliferating epimastigotes but negatively affected production of both types of trypomastigotes. MCTs differentiated from epimastigotes at a low frequency, though they were apparently normal based on morphological and biochemical criteria. However, these MCTs exhibited an impaired ability to produce amastigotes and TCTs in cell culture monolayers, most likely due to a reduced infection frequency. Remarkably, inhibition of MCT production did not depend on TcOGNT2 catalytic activity, whereas TCT production was inhibited only by active TcOGNT2. These findings indicate that TcOGNT2 downregulation is important for proper differentiation of MCTs and functioning of TCTs and that TcOGNT2 regulates these functions by using both catalytic and noncatalytic mechanisms.

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Section: Discussionsupporting

confidence: 75%

Section: Discussionmentioning

confidence: 99%

Golgi UDP-GlcNAc:Polypeptide O -α- N -Acetyl- d -Glucosaminyltransferase 2 (TcOGNT2) Regulates Trypomastigote Production and Function in Trypanosoma cruzi

et al. 2014

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“…SLPI is an important modulator of inflammatory responses responsible for cardiac remodeling 35,36 and was observed to be upregulated in the acute stage (six-fold increase), which waned as the infection evolved into the chronic stage. 25 In the present study, we also observed overexpression of SLPI gene, however, we were unable to demonstrate this increase by immunoblotting (data not shown). Interestingly, both our group 27 and Garg et al 37 observed that both in vitro and in vivo, among the most repressed genes includes those for oxidative phosphorylation complexes I and IV.…”

contrasting

confidence: 61%

“…We evaluated the functional status of TP in these cells by stimulating with 50 nM IBOP, a thromboxane-mimetic agent and measuring the activation of ERK pathway by immunoblotting using anti phospho ERK antibody. 25 Both WT-EC and TPα-EC expressed high levels of phospho ERK when stimulated with IBOP indicating an intact TP signaling pathways in these cells. However, we could not detect ERK activation in TP null ECs stimulated with IBOP (Fig.…”

Section: Discussionmentioning

confidence: 91%

Microarray analysis of the mammalian thromboxane receptor-Trypanosoma cruziinteraction

Tanowitz

Mukhopadhyay

Ashton³

et al. 2011

Cell Cycle

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“…Also, the deletion of TbMAPK2 affected differentiation (96), and it was reported that TbMAPK4 confers resistance to temperature stress (97). Recently, the TcMAPK2 was also characterized and, although it had high homology with ERK2 from lower eukaryotes, presented significant differences from mammalian homologs (98). In trypomastigotes, these MAPK2s were located mostly along the flagellum, whereas in intracellular amastigotes they were concentrated in the plasma membrane.…”

Section: Protein Kinases Phosphatases and Other Proteins Related Tomentioning

confidence: 99%

Quantitative Proteomic and Phosphoproteomic Analysis of Trypanosoma cruzi Amastigogenesis

Queiroz

Charneau

Mandacaru

et al. 2014

Molecular & Cellular Proteomics

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Chagas disease is a tropical neglected disease endemic in Latin America caused by the protozoan Trypanosoma cruzi. The parasite has four major life stages: epimastigote, metacyclic trypomastigote, bloodstream trypomastigote, and amastigote. The differentiation from infective trypomastigotes into replicative amastigotes, called amastigogenesis, takes place in vivo inside mammalian host cells after a period of incubation in an acidic phagolysosome. This differentiation process can be mimicked in vitro by incubating tissue-culture-derived trypomastigotes in acidic DMEM. Here we used this well-established differentiation protocol to perform a comprehensive quantitative proteomic and phosphoproteomic analysis of T. cruzi amastigogenesis. Samples from fully differentiated forms and two biologically relevant intermediate time points were Lys-C/trypsin digested, iTRAQ-labeled, and multiplexed. Subsequently, phosphopeptides were enriched using a TiO 2 matrix. Non-phosphorylated peptides were fractionated via hydrophilic interaction liquid chromatography prior to LC-MS/MS analysis. LC-MS/MS and bioinformatics procedures were used for protein and phosphopeptide quantitation, identification, and phosphorylation site assignment. We were able to identify regulated proteins and pathways involved in coordinating amastigogenesis. We also observed that a significant proportion of the regulated proteins were membrane proteins. Modulated phosphorylation events coordinated by protein kinases and phosphatases that are part of the signaling cascade induced by incubation in acidic medium were also evinced. To our knowledge, this work is the most comprehensive quantitative proteomics study of T. cruzi amastigogenesis, and these data will serve as a trustworthy basis for future studies, and possibly for new potential drug targets. Molecular & Cellular Proteomics 13:

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Molecular cloning and characterization of mitogen-activated protein kinase 2 inTrypanosoma cruzi

Cited by 13 publications

References 34 publications

Golgi UDP-GlcNAc:Polypeptide O -α- N -Acetyl- d -Glucosaminyltransferase 2 (TcOGNT2) Regulates Trypomastigote Production and Function in Trypanosoma cruzi

Golgi UDP-GlcNAc:Polypeptide O -α- N -Acetyl- d -Glucosaminyltransferase 2 (TcOGNT2) Regulates Trypomastigote Production and Function in Trypanosoma cruzi

Microarray analysis of the mammalian thromboxane receptor-Trypanosoma cruziinteraction

Quantitative Proteomic and Phosphoproteomic Analysis of Trypanosoma cruzi Amastigogenesis

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