Identification of a novel Sorcin isoform with a different C-terminal but intact dimerization property.

Sorcin (Sri), a member of penta EF-hand protein family plays a diverse role in maintaining calcium homeostasis, cell cycle and vesicular trafficking. Sri is highly conserved amongst mammals and consists of N-terminal glycine rich domain and C-terminal calcium binding domain that mediates its dimerization and interacts with different compounds. In the present study, with the help of combination of computational and molecular biology techniques, we have identified a novel isoform (Sri-N) in mouse which differs only in the C-terminal domain with that of Sri reported earlier. The novel isoform contains a new last exon that is different from the one present in the reported transcript (Sri). The presence of the novel isoform was further validated in different tissues by RT-PCR and DNA sequencing. The transcript was conceptually translated and subjected to in-silico analysis using different bioinformatics tools. The novel transcript variant encodes for a longer protein isoform without any change in the sub-cellular localization as predicted by PSORT-II online tool. Molecular modelling was performed to compare the structural changes in Sri-N and Sri isoforms. The structural characterization of the novel isoform using MD simulation depicted its overall stability under the physiological conditions. The molecular docking of proteins with various chemotherapeutic drugs revealed that their binding affinity is more for Sri-N as compared to that for the previously reported transcript Sri.

Biophysical insight into the binding mechanism of doxofylline to bovine serum albumin: An in vitro and in silico approach.

Insight into the mechanistic binding of bovine serum albumin (BSA) with doxofylline can layout pivotal enlightenment with relevance to pharmacokinetics and pharmacodynamics properties. Herein, many spectroscopic techniques and computational methods had been employed to interpret the structural and binding dynamics of BSA-doxofylline interaction. Doxofylline quenched the intrinsic fluorescence of BSA by static quenching. The stoichiometry and the binding constant of the BSA-doxofylline complex were 1:1 and in the order of 103 M-1. It was also concluded that the binding process was spontaneous and exothermic, primarily based on the thermodynamic study. Circular dichroism and three-dimensional excitation-emission matrix fluorescence results concluded pronounced conformational and microenvironmental changes in BSA structure on binding with doxofylline. The influence of metal ions and vitamins on the binding affinity of the BSA-doxofylline system were also explored. The in vitro findings were further supported by in silico analysis. With a score value of -6.25 kcal/mol, molecular docking showed strong interactions. Molecular dynamics simulation interpretation also suggested the stable binding with lower deviation in the values of RMSD and RMSF obtained by uninterrupted long simulation run. These studies will propose the optimum potency of distribution of the doxofylline into the bloodstream for asthma treatment.

NSAIDs Induced Regulation of Alternatively Spliced Transcript Isoforms: Possible Role in Cancer and Alzheimer Disease.

BACKGROUND: Alternative splicing is one of the post transcriptional modifications through which multiple mRNA isoforms are produced from any gene, also known as splice variants. These are expressed in tissue and developmental stage specific manner that are important during the development. Most human genes undergo alternative splicing, thus contributing to the diversity of proteins. However, many abnormal splicing processes may result in human diseases. Non-steroidal antiinflammatory drugs (NSAIDs) are medications that act as analgesics, anti-pyretics and antiinflammatory by affecting Cox genes and their products. Usually NSAIDs cause gastrotoxicity however, isozyme-specific NSAIDs exhibit a comparatively reduced gastrotoxic effect. Such NSAIDs have a broader range of application particularly as chemo-preventive drugs. It is known that changes at the active site of an enzyme may illicit a diverse range of responses. Such changes might explain the underlying reason as to why patients appear to respond differently to different NSAIDs. METHODS: An extensive literature search has been carried out using Pubmed and web of science databases considering the papers in last 10 years mainly on alternative splicing and NSAIDs. CONCLUSION: We have reviewed in detail the insight into the action of NSAIDs targeting specific isoforms of different genes. In future, the complete understanding of NSAIDs associated genes and their expression studies may be helpful in generating drugs with increased specificity.

Identification of differentially expressed three novel transcript variants of mouse ARNT gene.

The aryl hydrocarbon receptor nuclear translocator (ARNT/HIF1-ß) is an obligatory transcriptional partner of the aryl hydrocarbon receptor (AHR) and hypoxia-inducible factor-1α (HIF-1α). It has a basic helix-loop-helix domain that belongs to period-ARNT-single-minded (PAS) protein family. PAS proteins act as heterodimeric transcription factors with ARNT being master dimerization partner. The ARNT-HIF-1α complex is an important transcriptional regulator of the hypoxic response of the tumor cells. Previous studies have reported two transcript variants of the gene produced by alternative splicing in mouse. One transcript variant contains all 22 exons while the other variant lacks exon-E5. In our study, using combinatorial approach comprising bioinformatics tools and molecular biology techniques involving RT-PCR, semi-nested PCR, sequencing and qPCR, we have identified three novel transcript variants of Arnt gene in mouse. All three new transcripts arise as a result of alternative splicing of newly identified exons with exon-E2, replacing reported exon-E1. These transcripts encode for three protein isofoms having different N-termini. The expression of these transcripts was found to be different in different tissues of adult mice. In silico analysis of the upstream region of the new exons revealed three distinct promoter regions designated as PA, PB and PC present upstream of newly identified exons. These promoters possess potential signature sequences for common as well as different transcription factors suggesting complex regulation of Arnt gene. In silico post translational studies of the conceptually translated amino acid sequences of these transcripts show similarity in some of the properties while differ in others. The diversity at N-termini of protein isoforms suggests the possibility of forming different complexes in different tissues and may also be important for unique interactions with partner molecules.