Comparison of Modules of Wild Type and Mutant Huntingtin and TP53 Protein Interaction Networks: Implications in Biological Processes and Functions

Disease-causing mutations usually change the interacting partners of mutant proteins. In this article, we propose that the biological consequences of mutation are directly related to the alteration of corresponding protein protein interaction networks (PPIN). Mutation of Huntingtin (HTT) which causes Huntington's disease (HD) and mutations to TP53 which is associated with different cancers are studied as two example cases. We construct the PPIN of wild type and mutant proteins separately and identify the structural modules of each of the networks. The functional role of these modules are then assessed by Gene Ontology (GO) enrichment analysis for biological processes (BPs). We find that a large number of significantly enriched (p<0.0001) GO terms in mutant PPIN were absent in the wild type PPIN indicating the gain of BPs due to mutation. Similarly some of the GO terms enriched in wild type PPIN cease to exist in the modules of mutant PPIN, representing the loss. GO terms common in modules of mutant and wild type networks indicate both loss and gain of BPs. We further assign relevant biological function(s) to each module by classifying the enriched GO terms associated with it. It turns out that most of these biological functions in HTT networks are already known to be altered in HD and those of TP53 networks are altered in cancers. We argue that gain of BPs, and the corresponding biological functions, are due to new interacting partners acquired by mutant proteins. The methodology we adopt here could be applied to genetic diseases where mutations alter the ability of the protein to interact with other proteins.Comment: 35 pages, 10 eps figures, (Supplementary material and Datasets are available on request

Inhibition of nucleoporin member Nup214 expression by miR-133b perturbs mitotic timing and leads to cell death

Background: Nucleoporins mediate nucleocytoplasmic exchange of macromolecules and several have been assigned active mitotic functions. Nucleoporins can participate in various mitotic functions like spindle assembly, kinetochore organisation and chromosome segregation- important for genome integrity. Pathways to genome integrity are frequently deregulated in cancer and many are regulated in part by microRNAs. Indeed, altered levels of numerous microRNAs have frequently been associated with tumorigenesis. Here, we unveil a microRNA-mediated regulation of the nucleoporin Nup214 and its downstream effect on genome integrity. Methods: Databases/bioinformatic tools such as miRBase, Oncomine and RNAhybrid predicted Nup214 as a miR-133b target. To validate this, we used luciferase reporter assays, Real-Time PCR and immuno-blotting. Flow cytometry and immuno-blots of mitotic markers were used to analyse cell cycle pattern upon thymidine synchronization and miR-133b treatment. Mitotic indices and chromosomal abnormalities were assessed by immuno-fluorescence for FITC-tagged phospho-H3 as well as video-microscopy for GFP-tagged histone H4. Annexin V/propidium iodide staining, caspase3/ PARP cleavage and colony formation assays were done to investigate cell death upon either miR-133b transfection or NUP214 knockdown by siRNA. UPCI:SCC084, HCT116, HeLa-H4-pEGFP and HEK293 (human oral squamous cell carcinoma, colorectal, cervical carcinomas and embryonic kidney cell lines, respectively) were used. miR-133b and NUP214 expressions were validated in cancer cell lines and tissues by Real-Time PCR. Results: Examination of head and neck tumour tissues and cancer cell lines revealed that Nup214 and miR-133b expressions are negatively correlated. In vitro, Nup214 was significantly downregulated by ectopic miR-133b. This downregulation elevated mitotic indices and delayed degradation of mitotic marker proteins cyclinB1 and cyclinA and dephosphorylation of H3. Moreover, this mitotic delay enhanced chromosomal abnormalities and apoptosis. Conclusions: We have identified NUP214, a member of the massive nuclear pore complex, as a novel miR-133b target. Thus, we have shown a hitherto unknown microRNA regulation of mitosis mediated by a member of the nucleoporin family. Based on observations, we also raise some hypotheses regarding transport-dependent/independent functions of Nup214 in this study. Our results hence attempt to explain why miR-133b is generally downregulated in tumours and lay out the potential for Nup214 as a therapeutic target in the treatment of cancer

Radiosensitivity and Induction of Apoptosis by High LET Carbon Ion Beam and Low LET Gamma Radiation: A Comparative Study

Cancer treatment with high LET heavy ion beam, especially, carbon ion beam ( 12 C), is becoming very popular over conventional radiotherapy like low LET gamma or X-ray. Combination of Poly(ADP-ribose) polymerase (PARP) inhibitor with xenotoxic drugs or conventional radiation (gamma or X-ray) is the newer approach for cancer therapy. The aim of our study was to compare the radiosensitivity and induction of apoptosis by high LET 12 C and low LET gamma radiation in HeLa and PARP-1 knocked down cells. We did comet assay to detect DNA breaks, clonogenic survival assay, and cell cycle analysis to measure recovery after DNA damage. We measured apoptotic parameters like nuclear fragmentation and caspase-3 activation. DNA damage, cell killing, and induction of apoptosis were significantly higher for 12 C than gamma radiation in HeLa. Cell killing and apoptosis were further elevated upon knocking down of PARP-1. Both 12 C and gamma induced G 2 /M arrest although the 12 C had greater effect. Unlike the gamma, 12 C irradiation affects DNA replication as detected by S-phase delay in cell cycle analysis. So, we conclude that high LET 12 C has greater potential over low LET gamma radiation in killing cells and radiosensitization upon PARP-1 inhibition was several folds greater for 12 C than gamma

The Role of Intrinsically Unstructured Proteins in Neurodegenerative Diseases

The number and importance of intrinsically disordered proteins (IUP), known to be involved in various human disorders, are growing rapidly. To test for the generalized implications of intrinsic disorders in proteins involved in Neurodegenerative diseases, disorder prediction tools have been applied to three datasets comprising of proteins involved in Huntington Disease (HD), Parkinson's disease (PD), Alzheimer's disease (AD). Results show, in general, proteins in disease datasets possess significantly enhanced intrinsic unstructuredness. Most of these disordered proteins in the disease datasets are found to be involved in neuronal activities, signal transduction, apoptosis, intracellular traffic, cell differentiation etc. Also these proteins are found to have more number of interactors and hence as the proportion of disorderedness (i.e., the length of the unfolded stretch) increased, the size of the interaction network simultaneously increased. All these observations reflect that, “Moonlighting” i.e. the contextual acquisition of different structural conformations (transient), eventually may allow these disordered proteins to act as network “hubs” and thus they may have crucial influences in the pathogenecity of neurodegenerative diseases

Regulation of miR-146a by RelA/NFkB and p53 in STHdhQ111/HdhQ111 Cells, a Cell Model of Huntington's Disease

Huntington's disease (HD) is caused by the expansion of N-terminal polymorphic poly Q stretch of the protein huntingtin (HTT). Deregulated microRNAs and loss of function of transcription factors recruited to mutant HTT aggregates could cause characteristic transcriptional deregulation associated with HD. We observed earlier that expressions of miR-125b, miR-146a and miR-150 are decreased in STHdhQ111/HdhQ111 cells, a model for HD in comparison to those of wild type STHdhQ7/HdhQ7 cells. In the present manuscript, we show by luciferase reporter assays and real time PCR that decreased miR-146a expression in STHdhQ111/HdhQ111 cells is due to decreased expression and activity of p65 subunit of NFkB (RelA/NFkB). By reporter luciferase assay, RT-PCR and western blot analysis, we also show that both miR-150 and miR-125b target p53. This partially explains the up regulation of p53 observed in HD. Elevated p53 interacts with RelA/NFkB, reduces its expression and activity and decreases the expression of miR-146a, while knocking down p53 increases RelA/NFkB and miR-146a expressions. We also demonstrate that expression of p53 is increased and levels of RelA/NFkB, miR-146a, miR-150 and miR-125b are decreased in striatum of R6/2 mice, a mouse model of HD and in cell models of HD. In a cell model, this effect could be reversed by exogenous expression of chaperone like proteins HYPK and Hsp70. We conclude that (i) miR-125b and miR-150 target p53, which in turn regulates RelA/NFkB and miR-146a expressions; (ii) reduced miR-125b and miR-150 expressions, increased p53 level and decreased RelA/NFkB and miR-146a expressions originate from mutant HTT (iii) p53 directly or indirectly regulates the expression of miR-146a. Our observation of interplay between transcription factors and miRNAs using HD cell model provides an important platform upon which further work is to be done to establish if such regulation plays any role in HD pathogenesis

Chaperone protein HYPK interacts with the first 17 amino acid region of Huntingtin and modulates mutant HTT-mediated aggregation and cytotoxicity

Huntington's disease is a polyglutamine expansion disorder, characterized by mutant HTT-mediated aggregate formation and cytotoxicity. Many reports suggests roles of N-terminal 17 amino acid domain of HTT (HTT-N17) towards subcellular localization, aggregate formation and subsequent pathogenicity induced by N-terminal HTT harboring polyQ stretch in pathogenic range. HYPK is a HTT-interacting chaperone which can reduce N-terminal mutant HTT-mediated aggregate formation and cytotoxicity in neuronal cell lines. However, how HYPK interacts with N-terminal fragment of HTT remained unknown. Here we report that specific interaction of HYPK with HTT-N17 is crucial for the chaperone activity of HYPK. Deletion of HTT-N17 leads to formation of tinier, SDS-soluble nuclear aggregates formed by N-terminal mutant HTT. The increased cytotoxicity imparted by these tiny aggregates might be contributed due to loss of interaction with HYPK. (C) 2014 Elsevier Inc. All rights reserved

Distribution of microRNA co-targets exhibit universality across a wide class of species

MicroRNAs (miRNAs) are small non-coding RNAs which regulate gene expression by binding to the $3'$ UTR of the corresponding messenger RNAs. We construct miRNA co-target networks for a wide class of species (22 in total) using a target prediction database, MicroCosm Targets. For each species, miRNA pairs having one or more common target genes are connected and the number of co-targets are assigned as the weight of these links. We show that the link-weight distributions of all the species collapse remarkably onto each other when scaled suitably —the scale-factor turns out to be a measure of complexity of the species. A simple model, where targets are chosen randomly by miRNAs, could provide the correct scaling function and suggest that the increase of species complexity is related to the increase of the fraction of genes typically targeted by their miRNAs