https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/10 Fri, 03 Apr 2026 13:38:00 GMT 2026-04-03T13:38:00Z https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/1463 Title: Pf-Phospho: a machine learning-based phosphorylation sites prediction tool for Plasmodium proteins Authors: Gupta, Priya; Venkadesan, Sureshkumar; Mohanty, Debasisa Abstract: Even though several in silico tools are available for prediction of the phosphorylation sites for mammalian, yeast or plant proteins, currently no software is available for predicting phosphosites for Plasmodium proteins. However, the availability of significant amount of phospho-proteomics data during the last decade and advances in machine learning (ML) algorithms have opened up the opportunities for deciphering phosphorylation patterns of plasmodial system and developing ML-based phosphosite prediction tools for Plasmodium. We have developed Pf-Phospho, an ML-based method for prediction of phosphosites by training Random Forest classifiers using a large data set of 12 096 phosphosites of Plasmodium falciparum and Plasmodium bergei. Of the 12 096 known phosphosites, 75% of sites have been used for training/validation of the classifier, while remaining 25% have been used as completely unseen test data for blind testing. It is encouraging to note that Pf-Phospho can predict the kinase-independent phosphosites with 84% sensitivity, 75% specificity and 78% precision. In addition, it can also predict kinase-specific phosphosites for five plasmodial kinases-PfPKG, Plasmodium falciparum, PfPKA, PfPK7 and PbCDPK4 with high accuracy. Pf-Phospho (http://www.nii.ac.in/pfphospho.html) outperforms other widely used phosphosite prediction tools, which have been trained using mammalian phosphoproteome data. It also has been integrated with other widely used resources such as PlasmoDB, MPMP, Pfam and recently available ML-based predicted structures by AlphaFold2. Currently, Pf-phospho is the only bioinformatics resource available for ML-based prediction of phospho-signaling networks of Plasmodium and is a user-friendly platform for integrative analysis of phospho-signaling along with metabolic and protein-protein interaction networks. Sat, 01 Jan 2022 00:00:00 GMT https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/1463 2022-01-01T00:00:00Z https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/1393 Title: Single GDP-dissociation Inhibitor Protein regulates endocytic and secretory pathways in Leishmania Authors: Mukhopadhyay, Amitabha Abstract: The role of GDP dissociation inhibitor (GDI) protein in regulation of Rab cycle in Leishmania is not known. Here, we have cloned and characterized the functions of GDI homologue in vivo in Leishmania. Our results have shown that LdGDI:WT along with GDP removes the Rab5 from purified endosomes and inhibits the homotypic fusion between early endosomes. Whereas, LdGDI:R239A, a dominant negative mutant of GDI, under the same condition neither removes the Rab5 from endosome nor inhibits fusion. To determine the role of Ld-GDI in vivo, transgenic parasites overexpressing GFP-LdGDI:WT or GFP-LdGDI:R239A, are co-expressed with RFP-LdRab5:WT, RFP-LdRab7:WT or RFP-LdRab1:WT. Our results have shown that overexpression of GFP-LdGDI:WT extracts the RFP-LdRab5, RFP-LdRab7 or RFP-LdRab1 from their discrete endomembrane predominantly into cytosol. No change in the distribution of indicated Rabs is detected with overexpression of GFP-LdGDI:R239A. To determine the functional significance, we have used hemoglobin as an endocytic marker and gp63 as a marker for secretory pathway. We have found that overexpression of GFP-LdGDI:WT enhances the lysosomal targeting of internalized hemoglobin and the secretion of gp63 in the parasites possibly by triggering Rab cycle. This is the first demonstration of a single GDI ubiquitously regulating both endocytic and secretory pathways in Leishmania. Fri, 01 Jan 2016 00:00:00 GMT https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/1393 2016-01-01T00:00:00Z https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/1387 Title: 14-3-3γ Prevents Centrosome Amplification and Neoplastic Progression Authors: Mukhopadhyay, Amitabha Abstract: More than 80% of malignant tumors show centrosome amplification and clustering. Centrosome amplification results from aberrations in the centrosome duplication cycle, which is strictly coordinated with DNA-replication-cycle. However, the relationship between cell-cycle regulators and centrosome duplicating factors is not well understood. This report demonstrates that 14-3-3γ localizes to the centrosome and 14-3-3γ loss leads to centrosome amplification. Loss of 14-3-3γ results in the phosphorylation of NPM1 at Thr-199, causing early centriole disjunction and centrosome hyper-duplication. The centrosome amplification led to aneuploidy and increased tumor formation in mice. Importantly, an increase in passage of the 14-3-3γ-knockdown cells led to an increase in the number of cells containing clustered centrosomes leading to the generation of pseudo-bipolar spindles. The increase in pseudo-bipolar spindles was reversed and an increase in the number of multi-polar spindles was observed upon expression of a constitutively active 14-3-3-binding-defective-mutant of cdc25C (S216A) in the 14-3-3γ knockdown cells. The increase in multi-polar spindle formation was associated with decreased cell viability and a decrease in tumor growth. Our findings uncover the molecular basis of regulation of centrosome duplication by 14-3-3γ and inhibition of tumor growth by premature activation of the mitotic program and the disruption of centrosome clustering. Fri, 01 Jan 2016 00:00:00 GMT https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/1387 2016-01-01T00:00:00Z https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/1386 Title: GTPase Sar1 regulates the trafficking and secretion of the virulence factor gp63 in Leishmania Authors: Mukhopadhyay, Amitabha; Parashar, Smriti Abstract: Metalloprotease gp63 (Leishmania donovani gp63 (Ldgp63)) is a critical virulence factor secreted by Leishmania However, how newly synthesized Ldgp63 exits the endoplasmic reticulum (ER) and is secreted by this parasite is unknown. Here, we cloned, expressed, and characterized the GTPase LdSar1 and other COPII components like LdSec23, LdSec24, LdSec13, and LdSec31 from Leishmania to understand their role in ER exit of Ldgp63. Using dominant-positive (LdSar1:H74L) and dominant-negative (LdSar1:T34N) mutants of LdSar1, we found that GTP-bound LdSar1 specifically binds to LdSec23, which binds, in turn, with LdSec24(1-702) to form a prebudding complex. Moreover, LdSec13 specifically interacted with His6-LdSec31(1-603), and LdSec31 bound the prebudding complex via LdSec23. Interestingly, dileucine 594/595 and valine 597 residues present in the Ldgp63 C-terminal domain were critical for binding with LdSec24(703-966), and GFP-Ldgp63L594A/L595A or GFP-Ldgp63V597S mutants failed to exit from the ER. Moreover, Ldgp63-containing COPII vesicle budding from the ER was inhibited by LdSar1:T34N in an in vitro budding assay, indicating that GTP-bound LdSar1 is required for budding of Ldgp63-containing COPII vesicles. To directly demonstrate the function of LdSar1 in Ldgp63 trafficking, we coexpressed RFP-Ldgp63 along with LdSar1:WT-GFP or LdSar1:T34N-GFP and found that LdSar1:T34N overexpression blocks Ldgp63 trafficking and secretion in Leishmania Finally, we noted significantly compromised survival of LdSar1:T34N-GFP-overexpressing transgenic parasites in macrophages. Taken together, these results indicated that Ldgp63 interacts with the COPII complex via LdSec24 for Ldgp63 ER exit and subsequent secretion. Sun, 01 Jan 2017 00:00:00 GMT https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/1386 2017-01-01T00:00:00Z