Srimonti Sarkar | Bose Institute (original) (raw)

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Papers by Srimonti Sarkar

Parasites & Vectors, 2015

Background: Giardia lamblia switches its lifecycle between trophozoite and cyst forms and the pro... more Background: Giardia lamblia switches its lifecycle between trophozoite and cyst forms and the proteasome plays a pivotal role in this switching event. Compared to most model eukaryotes, the proteasome of this parasite has already been documented to have certain variations. This study was undertaken to characterize the ubiquitin receptor, GlRpn10, of the 19S regulatory particle of the Giardia proteasome and determine its cellular localization in trophozoites, encysting trophozoites and cysts. Method: Sequence alignment and domain architecture analyses were performed to characterize GlRpn10. In vitro ubiquitin binding assay, functional complementation and biochemical studies verified the protein's ability to function as ubiquitin receptor in the context of the yeast proteasome. Immunofluorescence localization was performed with antibody against GlRpn10 to determine its distribution in trophozoites, encysting trophozoites and cysts. Real-time PCR and Western blotting were performed to monitor the expression pattern of GlRpn10 during encystation. Result: GlRpn10 contained a functional ubiquitin interacting motif, which was capable of binding to ubiquitin. Although it contained a truncated VWA domain, it was still capable of partially complementing the function of the yeast Rpn10 orthologue. Apart from localizing to the nucleus and cytosol, GlRpn10 was also present at flagellar pores of trophozoites and this localization was microtubule-dependent. Although there was no change in the cellular levels of GlRpn10 during encystation, its selective distribution at the flagellar pores was absent.

Molecular biology of the cell, 2004

The multivesicular body (MVB) sorting pathway provides a mechanism for delivering transmembrane p... more The multivesicular body (MVB) sorting pathway provides a mechanism for delivering transmembrane proteins into the lumen of the lysosome/vacuole. Recent studies demonstrated that ubiquitin modification acts in cis as a signal for the sorting of cargoes into this pathway. Here, we present results from a genetic selection designed to identify mutants that missort MVB cargoes. This selection identified a point mutation in ubiquitin ligase Rsp5 (Rsp5-326). At the permissive temperature, this mutant is specifically defective for ubiquitination and sorting of the ubiquitin-dependent MVB cargo precursor carboxypeptidase S (pCPS), but not ligand-induced ubiquitination of Ste2. A previous study implicated Tul1 as the ubiquitin ligase responsible for MVB sorting of pCPS. However, we detected no defect in either the sorting or ubiquitination of pCPS in tul1 mutants. We had previously shown that Fab1 phosphatidylinositol 3-phosphate 5-kinase is also required for MVB sorting of pCPS, but not Ste2...

Journal of atherosclerosis and thrombosis, Jan 31, 2010

Cigarette smoking is a major risk for developing atherosclerosis; however, the underlying mechani... more Cigarette smoking is a major risk for developing atherosclerosis; however, the underlying mechanism is poorly understood. This paucity of knowledge is largely attributed to the lack of an animal model; therefore, our efforts were targeted towards establishing cigarette smoke (CS)-induced atherosclerosis in guinea pig. To understand the mechanism, we investigated apoptosis, an event implicated in atherosclerosis, in the aorta of CS-exposed animals. Since a major deleterious effect of CS is oxidative stress, we also examined the effect of vitamin C, an antioxidant, on CS- induced atherosclerosis. Guinea pigs on a diet with or without vitamin C supplement were exposed to CS for different time periods. Aortal sections from these animals were examined for atherosclerotic changes by staining with H&E and Oil red O. Atherogenic changes were observed in sections obtained from CS-exposed guinea pigs only. TUNEL assay showed the occurrence of apoptosis in CS-exposed guinea pig aorta. Our resu...

BMC Genomics, 2010

Background: Phosphatidylinositol 3-phosphate is involved in regulation of several key cellular pr... more Background: Phosphatidylinositol 3-phosphate is involved in regulation of several key cellular processes, mainly endocytosis, signaling, nuclear processes, cytoskeletal remodelling, cell survival, membrane trafficking, phagosome maturation and autophagy. In most cases effector proteins bind to this lipid, using either FYVE or PX domain. These two domains are distributed amongst varied life forms such as virus, protists, fungi, viridiplantae and metazoa. As

Proceedings of the National Academy of Sciences, 1999

Nuclear tRNA aminoacylation was proposed to provide a proofreading step in Xenopus oocytes, ensur... more Nuclear tRNA aminoacylation was proposed to provide a proofreading step in Xenopus oocytes, ensuring nuclear export of functional tRNAs [Lund, E. & Dahlberg, J. E. (1998) Science 282, 2082-2085].

Journal of Cell Science, 2002

Current Microbiology, 2011

The morphology of the endomembrane system of Giardia lamblia appears to be significantly differen... more The morphology of the endomembrane system of Giardia lamblia appears to be significantly different from higher eukaryotes. Therefore, the molecular mechanisms controlling vesicular trafficking are also likely to be altered. Since FYVE domain is a known regulator of endosomal trafficking, the authors used BLAST search to identify FYVE domain(s) in G. lamblia. A 990 amino acid long putative FYVE domain-containing ORF was identified, which contains all the conserved sequence elements in the ligand binding pocket. Phylogenetic analysis reveals that this domain is significantly diverged. The authors have shown that the corresponding gene is expressed in G. lamblia trophozoites and cysts. In spite of this phylogenetic divergence, in vitro biochemical assay indicates that this domain preferentially binds to phosphatidylinositol 3-phosphate {PtdIns(3)P}and in vivo expression of the GFP-tagged G. lamblia FYVE domain in S. cerevisiae, displays its selective localization to PtdIns(3)P-enriched endosomes. This is the first study to characterize a PtdIns(3)P effector protein in this early-diverged eukaryote.

BMC Genomics, 2010

Background: Phosphatidylinositol 3-phosphate is involved in regulation of several key cellular pr... more Background: Phosphatidylinositol 3-phosphate is involved in regulation of several key cellular processes, mainly endocytosis, signaling, nuclear processes, cytoskeletal remodelling, cell survival, membrane trafficking, phagosome maturation and autophagy. In most cases effector proteins bind to this lipid, using either FYVE or PX domain. These two domains are distributed amongst varied life forms such as virus, protists, fungi, viridiplantae and metazoa. As the binding ligand is identical for both domains, the goal of this study was to understand if there is any selectivity for either of these domains in different taxa. Further, to understand the different cellular functions that these domains may be involved in, we analyzed the taxonomic distribution of additional domains that associate with FYVE and PX. Results: There is selectivity for either FYVE or PX in individual genomes where both domains are present. Fungi and metazoa encode more PX, whereas streptophytes in viridiplantae encode more FYVE. Excess of FYVE in streptophytes results from proteins containing RCC1and DZC domains and FYVE domains in these proteins have a non-canonical ligand-binding site. Within a taxonomic group the selected domain associates with a higher number of other domains and is thus expected to discharge a larger number of cellular functions. Also, while certain associated domains are present in all taxonomic groups, most of them are unique to a specific group indicating that while certain common functions are discharged by these domains in all taxonomic groups, some functions appear to be group specific. Conclusions: Although both FYVE and PX bind to PtdIns(3)P, genomes of different taxa show distinct selectivity of encoding either of the two. Higher numbers of taxonomic group specific domains co-occur with the more abundant domain (FYVE/PX) indicating that group-specific rare domain architectures might have emerged to accomplish certain group-specific functions.

Parasites & Vectors, 2015

Background: Giardia lamblia switches its lifecycle between trophozoite and cyst forms and the pro... more Background: Giardia lamblia switches its lifecycle between trophozoite and cyst forms and the proteasome plays a pivotal role in this switching event. Compared to most model eukaryotes, the proteasome of this parasite has already been documented to have certain variations. This study was undertaken to characterize the ubiquitin receptor, GlRpn10, of the 19S regulatory particle of the Giardia proteasome and determine its cellular localization in trophozoites, encysting trophozoites and cysts. Method: Sequence alignment and domain architecture analyses were performed to characterize GlRpn10. In vitro ubiquitin binding assay, functional complementation and biochemical studies verified the protein's ability to function as ubiquitin receptor in the context of the yeast proteasome. Immunofluorescence localization was performed with antibody against GlRpn10 to determine its distribution in trophozoites, encysting trophozoites and cysts. Real-time PCR and Western blotting were performed to monitor the expression pattern of GlRpn10 during encystation. Result: GlRpn10 contained a functional ubiquitin interacting motif, which was capable of binding to ubiquitin. Although it contained a truncated VWA domain, it was still capable of partially complementing the function of the yeast Rpn10 orthologue. Apart from localizing to the nucleus and cytosol, GlRpn10 was also present at flagellar pores of trophozoites and this localization was microtubule-dependent. Although there was no change in the cellular levels of GlRpn10 during encystation, its selective distribution at the flagellar pores was absent.

Molecular biology of the cell, 2004

The multivesicular body (MVB) sorting pathway provides a mechanism for delivering transmembrane p... more The multivesicular body (MVB) sorting pathway provides a mechanism for delivering transmembrane proteins into the lumen of the lysosome/vacuole. Recent studies demonstrated that ubiquitin modification acts in cis as a signal for the sorting of cargoes into this pathway. Here, we present results from a genetic selection designed to identify mutants that missort MVB cargoes. This selection identified a point mutation in ubiquitin ligase Rsp5 (Rsp5-326). At the permissive temperature, this mutant is specifically defective for ubiquitination and sorting of the ubiquitin-dependent MVB cargo precursor carboxypeptidase S (pCPS), but not ligand-induced ubiquitination of Ste2. A previous study implicated Tul1 as the ubiquitin ligase responsible for MVB sorting of pCPS. However, we detected no defect in either the sorting or ubiquitination of pCPS in tul1 mutants. We had previously shown that Fab1 phosphatidylinositol 3-phosphate 5-kinase is also required for MVB sorting of pCPS, but not Ste2...

Journal of atherosclerosis and thrombosis, Jan 31, 2010

Cigarette smoking is a major risk for developing atherosclerosis; however, the underlying mechani... more Cigarette smoking is a major risk for developing atherosclerosis; however, the underlying mechanism is poorly understood. This paucity of knowledge is largely attributed to the lack of an animal model; therefore, our efforts were targeted towards establishing cigarette smoke (CS)-induced atherosclerosis in guinea pig. To understand the mechanism, we investigated apoptosis, an event implicated in atherosclerosis, in the aorta of CS-exposed animals. Since a major deleterious effect of CS is oxidative stress, we also examined the effect of vitamin C, an antioxidant, on CS- induced atherosclerosis. Guinea pigs on a diet with or without vitamin C supplement were exposed to CS for different time periods. Aortal sections from these animals were examined for atherosclerotic changes by staining with H&E and Oil red O. Atherogenic changes were observed in sections obtained from CS-exposed guinea pigs only. TUNEL assay showed the occurrence of apoptosis in CS-exposed guinea pig aorta. Our resu...

BMC Genomics, 2010

Background: Phosphatidylinositol 3-phosphate is involved in regulation of several key cellular pr... more Background: Phosphatidylinositol 3-phosphate is involved in regulation of several key cellular processes, mainly endocytosis, signaling, nuclear processes, cytoskeletal remodelling, cell survival, membrane trafficking, phagosome maturation and autophagy. In most cases effector proteins bind to this lipid, using either FYVE or PX domain. These two domains are distributed amongst varied life forms such as virus, protists, fungi, viridiplantae and metazoa. As

Proceedings of the National Academy of Sciences, 1999

Nuclear tRNA aminoacylation was proposed to provide a proofreading step in Xenopus oocytes, ensur... more Nuclear tRNA aminoacylation was proposed to provide a proofreading step in Xenopus oocytes, ensuring nuclear export of functional tRNAs [Lund, E. & Dahlberg, J. E. (1998) Science 282, 2082-2085].

Journal of Cell Science, 2002

Current Microbiology, 2011

The morphology of the endomembrane system of Giardia lamblia appears to be significantly differen... more The morphology of the endomembrane system of Giardia lamblia appears to be significantly different from higher eukaryotes. Therefore, the molecular mechanisms controlling vesicular trafficking are also likely to be altered. Since FYVE domain is a known regulator of endosomal trafficking, the authors used BLAST search to identify FYVE domain(s) in G. lamblia. A 990 amino acid long putative FYVE domain-containing ORF was identified, which contains all the conserved sequence elements in the ligand binding pocket. Phylogenetic analysis reveals that this domain is significantly diverged. The authors have shown that the corresponding gene is expressed in G. lamblia trophozoites and cysts. In spite of this phylogenetic divergence, in vitro biochemical assay indicates that this domain preferentially binds to phosphatidylinositol 3-phosphate {PtdIns(3)P}and in vivo expression of the GFP-tagged G. lamblia FYVE domain in S. cerevisiae, displays its selective localization to PtdIns(3)P-enriched endosomes. This is the first study to characterize a PtdIns(3)P effector protein in this early-diverged eukaryote.

BMC Genomics, 2010

Background: Phosphatidylinositol 3-phosphate is involved in regulation of several key cellular pr... more Background: Phosphatidylinositol 3-phosphate is involved in regulation of several key cellular processes, mainly endocytosis, signaling, nuclear processes, cytoskeletal remodelling, cell survival, membrane trafficking, phagosome maturation and autophagy. In most cases effector proteins bind to this lipid, using either FYVE or PX domain. These two domains are distributed amongst varied life forms such as virus, protists, fungi, viridiplantae and metazoa. As the binding ligand is identical for both domains, the goal of this study was to understand if there is any selectivity for either of these domains in different taxa. Further, to understand the different cellular functions that these domains may be involved in, we analyzed the taxonomic distribution of additional domains that associate with FYVE and PX. Results: There is selectivity for either FYVE or PX in individual genomes where both domains are present. Fungi and metazoa encode more PX, whereas streptophytes in viridiplantae encode more FYVE. Excess of FYVE in streptophytes results from proteins containing RCC1and DZC domains and FYVE domains in these proteins have a non-canonical ligand-binding site. Within a taxonomic group the selected domain associates with a higher number of other domains and is thus expected to discharge a larger number of cellular functions. Also, while certain associated domains are present in all taxonomic groups, most of them are unique to a specific group indicating that while certain common functions are discharged by these domains in all taxonomic groups, some functions appear to be group specific. Conclusions: Although both FYVE and PX bind to PtdIns(3)P, genomes of different taxa show distinct selectivity of encoding either of the two. Higher numbers of taxonomic group specific domains co-occur with the more abundant domain (FYVE/PX) indicating that group-specific rare domain architectures might have emerged to accomplish certain group-specific functions.