Mahboob Ul Hussain

Micro-RNAs (miRNAs): genomic organisation, biogenesis and mode of action

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression in animals and in plants. In recent years, miRNAs have been shown to be important biological molecules for regulating various cellular functions. miRNAs function post-transcriptionally usually by base-pairing to the mRNA 3′-untranslated regions of the mRNAs and repress protein synthesis by mechanisms that are not fully understood. Various miRNA genes have been mapped in the genome of a number of organisms and the list continues to grow. Details regarding the genomic organisation, transcriptional regulation and post-transcriptional maturation of miRNAs are still emerging. In this review, information regarding the genomic organisation, biogenesis and regulation of expression of miRNAs is discussed.

Protein kinase C (PKC) mediated interaction between conexin43 (Cx43) and K(+)(ATP) channel subunit (Kir6.1) in cardiomyocyte mitochondria: Implications in cytoprotection against hypoxia induced cell apoptosis.

PURPOSE OF RESEARCH We have recently shown that adenosine-triphosphate-sensitive potassium [K(+)(ATP)] channel protein subunit, Kir6.1 is a phosphospecific interaction partner of the gap-junction protein connexin43 (Cx43). Since, both Cx43 and K(+)(ATP) are known to be involved in cell survival during hypoxia, we addressed the question, whether the interaction between Cx43 and K(+)(ATP) has a role in protecting cell against hypoxia-induced cell death. PRINCIPLE RESULTS We report here that the Kir6.1 protein interacts, in a phosphospecific manner with Cx43 in the mitochondria of cardiomyocytic cell line H9C2. The hypoxia for 12-h resulted in the appreciable increase in the phosphorylation at the serine 262 (S262) of the Cx43 with the concomitant increase in the Cx43 and Kir6.1 interaction. Moreover, the increased interaction was mediated by a signaling pathway involving PKC and more specifically by PKC epsilon. Functional implications of the association between the Cx43 and Kir6.1 were found to prevent mitochondria mediated hypoxia induced cell apoptosis. MAJOR CONCLUSIONS Our results demonstrate that PKC epsilon regulates the interaction between Cx43 and Kir6.1 in the cardiomyocyte mitochondria and this interaction prevents hypoxia induced cell death. Our results provide an interesting lead in developing effective strategies to protect cardiomyocytes from hypoxia/ischemia induced cell death.

MicroRNAs and human diseases: diagnostic and therapeutic potential.

MicroRNAs (miRNAs) are endogenous, non-coding small RNAs that regulate gene expression at the post-transcriptional level. Recent studies have shown that miRNAs are aberrantly expressed in various human diseases, ranging from cancer to cardiovascular hypertrophy. The expression profiles of the miRNAs clearly differentiate the normal from the pathological state and thus their potential as novel biomarkers in the diagnosis and prognosis of several human diseases is immense. Emerging data on the role of miRNAs in the pathogenesis of various human diseases have paved the way to test their ability to act as novel therapeutic tools. In the present review, we will explore the current knowledge about the role of miRNAs in various human diseases. In addition, we will focus on the emerging evidences demonstrating the potential of miRNAs as novel biomarkers and the strategies to use them as therapeutic tools.

Adenosine-triphosphate-sensitive K+ channel (Kir6.1): a novel phosphospecific interaction partner of connexin 43 (Cx43).

Connexin 43 (Cx43) is a phosphoprotein expressed in a wide variety of cells. Cx43 and adenosine-triphosphate-sensitive K(+)channels [K(+)(ATP)] are part of same signaling pathway that regulates cell survival during stress and ischemia preconditioning. Molecular mechanism for their coordinated role in ischemia/hypoxia preconditioning is not well known. Using pull down, co-immunoprecipitation assays and co-localization studies we provide evidence, for the first time that Kir6.1, a K(+)(ATP) channel protein component, can interact with Cx43. Further we show that the interaction was phospho-specific such that Cx43 phosphorylated at serine 262 (S262) interacted with Kir6.1 in preference to the unphosphorylated form of Cx43. Introduction of phospho-deficient mutation at serine 262 (S262A) in Cx43 completely abolished the interaction. Our data provide an interesting lead about a possible partnership between Cx43 and Kir6.1, which will help in better understanding their role in ischemia/hypoxia preconditioning.

Diversity of axin in signaling pathways and its relation to colorectal cancer

Colorectal cancer is one of the commonest cancers in western world, while majority of cases of CRC are sporadic, a significant minority occurs as a result of inherited genetic mutations. Various studies have demonstrated that tumorigenesis of colon arises as a result of accumulation of multiple genetic alterations targeting the single signal transduction pathway. Genetic alteration of axin has been implicated in different cancers including colorectal cancers. Axin being a multidomain protein interacts with multiple proteins and acts as major scaffold protein in signaling pathways like c-jun/SAPK, TGF-β, and Wnt. In different signaling pathways, axin utilizes different domains and hence exerts distinct roles.

Pathological implications of Cx43 down-regulation in human colon cancer.

Connexin 43 is an important gap junction protein in vertebrates and is known for its tumor suppressive properties. Cx43 is abundantly expressed in the human intestinal epithelial cells and muscularis mucosae. To explore the role of Cx43 in the genesis of human colon cancer, we performed the expression analysis of Cx43 in 80 cases of histopathologically confirmed and clinically diagnosed human colon cancer samples and adjacent control tissue and assessed correlations with clinicopathological variables. Western blotting using anti-Cx43 antibody indicated that the expression of Cx43 was significantly down regulated (75%) in the cancer samples as compared to the adjacent control samples. Moreover, immunohistochemical analysis of the tissue samples confirmed the down regulation of the Cx43 in the intestinal epithelial cells. Cx43 down regulation showed significant association (p<0.05) with the histological type and tumor invasion properties of the cancer. Our data demonstrated that loss of Cx43 may be an important event in colon carcinogenesis and tumor progression, providing significant insights about the tumor suppressive properties of the Cx43 and its potential as a diagnostic marker for colon cancer.

Impact of molecular alterations of BRAF in the pathogenesis of thyroid cancer.

BRAF alterations represent a novel indicator of the progression and aggressiveness of thyroid carcinogenesis. So, the main aim of the study was to elucidate the involvement of BRAF gene mutations and its expression in Kashmiri (North India) patients and investigate their association with clinico-pathological characteristics. Mutational analysis of BRAF gene was performed by polymerase chain reaction followed by DNA sequencing, whereas analysis of BRAF protein expression was done by western blotting. Overall mutations in BRAF was found to be 25% (15 of 60) and all of them were transversions (T>A) affecting codon 600 (valine to glutamine), restricted only to papillary thyroid cancer and well-differentiated grade. Patients with well-differentiated disease and in particular elevated thyroid-stimulating hormone levels were significantly associated with BRAF mutations (P < 0.05). Overall, 90% (54 of 60) of thyroid cancer cases showed increased expression of BRAF and non-smokers being significantly associated with BRAF over-expression. Totally, 86.7% (13 of 15) of BRAF mutation-positive patients were having over-expression of BRAF protein, whereas 91.2% (41 of 45) of patients with wild-type BRAF status were having over-expressed BRAF protein (P > 0.05). We conclude that both mutational events as well as over-expression of BRAF gene is highly implicated in pathogenesis of thyroid cancer and the BRAF protein over-expression is independent of the BRAF mutational status of thyroid cancer patients.

The XRCC1 Arg399Gln Gene Polymorphism and Risk of Colorectal Cancer: a Study in Kashmir

BACKGROUND The DNA repair gene XRCC1 Arg399Gln gene polymorphism has been found to be implicated in the development of various cancers, including colorectal cancer (CRC), in different populations. We aimed to determine any association of this polymorphism with the risk of CRC in Kashmir. MATERIALS AND METHODS A total of 120 confirmed cases of CRC and 146 healthy cancer free controls from the Kashmiri population were included in this study. Genotyping was carried out by the polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) method. RESULTS Genotype frequencies of XRCC1 Arg399Gln observed in controls were 34.2%, 42.5% and 23.3% for GG (Arg/Arg), GA (Arg/Gln), AA( Gln/Gln), respectively, and 28.3%, 66.7% and 5% in cases, with an odds ratio (OR)=5.7 and 95% confidence interval (CI) =2.3-14.1 (p=0.0001). No significant association of Arg399Gln SNP with any clinicopathological parameters of CRC was found. CONCLUSIONS We found the protective role of 399Gln allele against risk to the development of CRC. The XRCC1 heterozygote status appears to be a strong risk factor for CRC development in the Kashmiri population.

Rapamycin inhibition of baculovirus recombinant (BVr) ribosomal protein S6 kinase (S6K1) is mediated by an event other than phosphorylation

BackgroundRibosomal protein S6 kinase 1(S6K1) is an evolutionary conserved kinase that is activated in response to growth factors and viral stimuli to influence cellular growth and proliferation. This downstream effector of target of rapamycin (TOR) signaling cascade is known to be directly activated by TOR- kinase mediated hydrophobic motif (HM) phosphorylation at Threonine 412 (T412). Selective loss of this phosphorylation by inactivation of TOR kinase or activation/recruitment of a phosphatase has accordingly been implicated in mediating inhibition by rapamycin.FindingsWe present evidence that baculovirus driven expression of S6K1 in insect cells (Sf9) fails to activate the enzyme and instead renders it modestly active representing 4-6 folds less activity than its fully active mammalian counterpart. Contrary to the contention that viral infection activates TOR signaling pathway, we report that BVr enzyme fails to exhibit putative TOR dependent phosphorylation at the HM and the resultant phosphorylation at the activation loop (AL) of the enzyme, correlating with the level of activity observed. Surprisingly, the BVr enzyme continued to exhibit sensitivity to rapamycin that remained unaffected by mutations compromised for TOR phosphorylation (T412A) or deletions compromised for TOR binding (ΔNH 2-46/ΔCT104).ConclusionsThese data together with the ability of the BVr enzyme to resist inactivation by phosphatases indicate that inhibition by rapamycin is not mediated by any phosphorylation event in general and TOR dependent phosphorylation in particular.

Transcriptional Regulation of the Connexin Gene

As described above, alternate splicing and differential promoter usage can give rise to various transcripts of a particular connexin gene. Some of these transcripts are ubiquitously expressed, while some are tissue specific. The transcription of connexin genes is controlled by of various transcription factors and other biological substances. The transcription factors play an important role in regulating the expression of connexin gene. The types of transcription factor(s) determine whether a particular connexin is expressed ubiquitously or in cell type-specific manner. In the following sections, the role of various transcription factors and other signalling molecules that regulate the ubiquitous or tissue-specific expression of connexin genes will be discussed.