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BackgroundOver the recent years, a number of genomes have been successfully sequenced and this was followed by genome annotation projects to help understand the biological capabilities of newly sequenced genomes. To improve the annotation of Plasmodium falciparum proteins, we earlier developed parasite specific matrices (PfSSM) and demonstrated their (Smat80 and PfFSmat60) better performance over standard matrices (BLOSUM and PAM). Here we extend that study to nine apicomplexan species other than P. falciparum and develop a web application ApicoAlign for improving the annotation of apicomplexan proteins.ResultsThe SMAT80 and PfFSmat60 matrices perform better for apicomplexan proteins compared to BLOSUM in detecting the orthologs and improving the alignment of these proteins with their potential orthologs respectively. Database searches against non-redundant (nr) database have shown that SMAT80 gives superior performance compared to BLOSUM series in terms of E-values, bit scores, percent identity, alignment length and mismatches for most of the apicomplexan proteins studied here. Using these matrices, we were able to find orthologs for rhomboid proteases of P. berghei, P. falciparum &P. vivax and large subunit of U2 snRNP auxiliary factor of Cryptosporidium parvum in Arabidopsis thaliana. We also show improved pairwise alignments of proteins from Apicomplexa viz. Cryptosporidium parvum and P. falciparum with their orthologs from other species using the PfFSmat60 matrix.ConclusionsThe SMAT80 and PfFSmat60 substitution matrices perform better for apicomplexan proteins compared to BLOSUM series. Since they can be helpful in improving the annotation of apicomplexan genomes and their functional characterization, we have developed a web server ApicoAlign for finding orthologs and aligning apicomplexan proteins.

Malaria remains a worldwide concern in terms of morbidity and mortality. Limited understanding of the Plasmodium proteome makes it challenging to control malaria. Understanding of the expression and functions of different Plasmodium proteins will help in knowing this organism's virulence properties, besides facilitating the drug development process. In this study, we characterize the lipid binding and biophysical properties of the putative Plasmodium falciparum acyl-CoA binding proteins (PfACBPs), which may have intriguing functions in different stages of P. falciparum life cycle. While the PfACBPs can bind to long-chain fatty acyl-CoAs with high affinity, their affinity for shortchain fatty acyl-CoAs is weak. Base-stacking, electrostatic, and hydrophobic interactions between the aromatic rings, charged groups or residues, and hydrophobic chains or residues are responsible for acyl-CoA binding to PfACBPs. PfACBPs can also bind to phospholipids. PfACBPs cannot bind to the fatty acids and unphosphorylated fatty acid esters. PfACBPs are globular− helical proteins that contain a conserved acyl-CoA binding region. They exist in folded or unfolded conformations without attaining any intermediate state. In a systematic high-throughput in silico screening, mefloquine is identified as a potential ligand of PfACBPs. Binding affinities of mefloquine are much higher than those of fatty acyl-CoAs for all PfACBPs. Mefloquine binds to the acyl-CoA binding pocket of PfACBPs, thereby engaging many of the critical residues. Thus, mefloquine acts as a competitive inhibitor against fatty acyl-CoA binding to PfACBPs, leading to the prevention of P. falciparum growth and proliferation. Taken together, our study characterizes the functions of annotated PfACBPs and highlights the mechanistic details of their inactivation by mefloquine.

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Background A number of apicomplexan genomes have been sequenced successfully in recent years and this would help in understanding the biology of apicomplexan parasites. The members of the phylum Apicomplexa are important protozoan parasites (Plasmodium, Toxoplasma and Cryptosporidium etc) that cause some of the deadly diseases in humans and animals. In our earlier studies, we have shown that the standard BLOSUM matrices are not suitable for compositionally biased apicomplexan proteins. So we developed a novel series (SMAT and PfFSmat60) of substitution matrices which performed better in comparison to standard BLOSUM matrices and developed ApicoAlign, a sequence search and alignment tool for apicomplexan proteins. In this study, we demonstrate the higher specificity of these matrices and make an attempt to improve the annotation of apicomplexan kinases and proteases. Results The ROC curves proved that SMAT80 performs best for apicomplexan proteins followed by compositionally adjusted BLOSUM62 (PSI-BLAST searches), BLOSUM90 and BLOSUM62 matrices in terms of detecting true positives. The poor E-values and/or bit scores given by SMAT80 matrix for the experimentally identified coccidia-specific oocyst wall proteins against hematozoan (non-coccidian) parasites further supported the higher specificity of the same. SMAT80 uniquely detected (missed by BLOSUM) orthologs for 1374 apicomplexan hypothetical proteins against SwissProt database and predicted 70 kinases and 17 proteases. Further analysis confirmed the conservation of functional residues of kinase domain in one of the SMAT80 detected kinases. Similarly, one of the SMAT80 detected proteases was predicted to be a rhomboid protease. Conclusions The parasite specific substitution matrices have higher specificity for apicomplexan proteins and are helpful in detecting the orthologs missed by BLOSUM matrices and thereby improve the annotation of apicomplexan proteins which are hypothetical or with unknown function.

Using succinic anhydride, a succinylated derivative of anti-urease IgG having 49 +/- 6% modification was prepared and its physicochemical and immunological properties were studied. IgG undergoes substantial changes in its native conformation on succinylation, which was mainly attributed to electrostatic destabilization of the native protein conformation. The modified IgG exhibited a decrease in its cross-reactivity with urease. This decrease is attributed to the conformational change in IgG upon succinylation and/or is due to the disruption of the lysine residues in the antigen-binding site of IgG upon succinylation, which may be involved in binding the antigen. IgG was able to bind to the specific antigen although its conformation was partially modified. Therefore, partial modification of the conformation of the antigen-binding site of IgG is permissible in order to bind to the antigen.

Although HPV infection is the major cause of tumorigenesis in cervical cancer; interestingly it has been noted that HPV infection does not always lead to cervical cancer. This suggests that there could be some unknown factor that directly or indirectly interacts with these HPV oncoproteins leading to tumorigenesis. Our study aims to understand whether any correlation exists between the Piwi homologs with HPV oncoproteins in cervical cancer. To begin with, the expression pattern of Piwi proteins were evaluated in cultured cervical cancer (C33A, SiHa, and CaSki) cell lines. We observed that all the Piwi variants were differentially expressed in all the selected cervical cancer cell lines with PiwiL1 showing the highest expression in CaSki cells, suggesting a positive correlation with HPV oncoproteins. On investigating this correlation further, we found that PiwiL1 was co‐expressed and physically interacted with HPV oncoproteins E6 and E7. Not only E6 and E7 physically interact with PiwiL1, but also accentuated its expression when over‐expressed in HaCaT cells. Since PiwiL1 protein is known for its overexpression in many cancers, we wanted to know how this molecule is regulated in cancer. For this, we have carried out an in silico analysis of the PiwiL1 promoter to find the possible binding TFs using multiple online tools such as Alibhabha, AllegenPromo, and TFsitescan. We further shortlisted the TFs based on their function in cancer, interestingly we found both p53 and E2F in the list. Further analysis of these selected factors suggested that PiwiL1 promoter activity was significantly upregulated in the presence of E2F, whereas it was downregulated with p53. The binding of these transcription factors and their differential regulation of PiwiL1 promoter could be one of the reasons for its over‐expressed status in cancer. In addition to this, it is also known that the Piwi‐piRNA complex act as guiding signals for factors that play a significant role in tumorigenesis. Therefore, we have constructed a pipeline to predict the piRNAs from a small RNA Sequencing Data (SRP119662) obtained from the NCBI database, we identified 2274 piRNAs from both control and tumor samples, out of this, only 9 piRNAs were found to be significantly differentially expressed compared to control. Even though in silico analysis predicted 6 piRNAs to be significantly upregulated and 3 piRNAs to be significantly downregulated, the qPCR analysis was not entirely in accordance to the prediction. Out of the 3 down‐regulated piRNAs, hsa_piR_007863 showed elevated levels in CaSki in comparison to HaCaT while hsa_piR_002320 which was predicted to be upregulated in tumor showed lower expression. Our preliminary data suggest that PiwiL1 protein and associated piRNAs may have an important role in HPV‐associated tumorigenesis, whose exact molecular mechanism needs to be further evaluated.

Star-PAP is a non-canonical poly(A) polymerase that selects mRNA targets for polyadenylation. Yet, genome-wide direct Star-PAP targets or the mechanism of specific mRNA recognition is still vague. Here, we employ HITS-CLIP to map the cellular Star-PAP binding landscape and the mechanism of global Star-PAP mRNA association. We show a transcriptome-wide association of Star-PAP that is diminished on Star-PAP depletion. Consistent with its role in the 3′-UTR processing, we observed a high association of Star-PAP at the 3′-UTR region. Strikingly, there is an enrichment of Star-PAP at the coding region exons (CDS) in 42% of target mRNAs. We demonstrate that Star-PAP binding de-stabilises these mRNAs indicating a new role of Star-PAP in mRNA metabolism. Comparison with earlier microarray data reveals that while UTR-associated transcripts are down-regulated, CDS-associated mRNAs are largely up-regulated on Star-PAP depletion. Strikingly, the knockdown of a Star-PAP coregulator RBM10 resulted in a global loss of Star-PAP association on target mRNAs. Consistently, RBM10 depletion compromises 3′-end processing of a set of Star-PAP target mRNAs, while regulating stability/turnover of a different set of mRNAs. Our results establish a global profile of Star-PAP mRNA association and a novel role of Star-PAP in the mRNA metabolism that requires RBM10-mRNA association in the cell.