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Intracellular and exosomal microRNAome profiling of human vascular smooth muscle cells during replicative senescence

Nguyen DDN, Zain SM, Kamarulzaman MH, Low TY, Chilian WM, Pan Y, et al.

Am J Physiol Heart Circ Physiol, 2021 10 01;321(4):H770-H783.
PMID: 34506226 DOI: 10.1152/ajpheart.00058.2021

Vascular aging is highly associated with cardiovascular morbidity and mortality. Although the senescence of vascular smooth muscle cells (VSMCs) has been well established as a major contributor to vascular aging, intracellular and exosomal microRNA (miRNA) signaling pathways in senescent VSMCs have not been fully elucidated. This study aimed to identify the differential expression of intracellular and exosomal miRNA in human VSMCs (hVSMCs) during replicative senescence. To achieve this aim, intracellular and exosomal miRNAs were isolated from hVSMCs and subsequently subjected to whole genome small RNA next-generation sequencing, bioinformatics analyses, and qPCR validation. Three significant findings were obtained. First, senescent hVSMC-derived exosomes tended to cluster together during replicative senescence and the molecular weight of the exosomal protein tumor susceptibility gene 101 (TSG-101) increased relative to the intracellular TSG-101, suggesting potential posttranslational modifications of exosomal TSG-101. Second, there was a significant decrease in both intracellular and exosomal hsa-miR-155-5p expression [n = 3, false discovery rate (FDR) < 0.05], potentially being a cell type-specific biomarker of hVSMCs during replicative senescence. Importantly, hsa-miR-155-5p was found to associate with cell-cycle arrest and elevated oxidative stress. Lastly, miRNAs from the intracellular pool, that is, hsa-miR-664a-3p, hsa-miR-664a-5p, hsa-miR-664b-3p, hsa-miR-4485-3p, hsa-miR-10527-5p, and hsa-miR-12136, and that from the exosomal pool, that is, hsa-miR-7704, were upregulated in hVSMCs during replicative senescence (n = 3, FDR < 0.05). Interestingly, these novel upregulated miRNAs were not functionally well annotated in hVSMCs to date. In conclusion, hVSMC-specific miRNA expression profiles during replicative senescence potentially provide valuable insights into the signaling pathways leading to vascular aging.NEW & NOTEWORTHY This is the first study on intracellular and exosomal miRNA profiling on human vascular smooth muscle cells during replicative senescence. Specific dysregulated sets of miRNAs were identified from human vascular smooth muscle cells. Hsa-miR-155-5p was significantly downregulated in both intracellular and exosomal hVSMCs, suggesting its crucial role in cellular senescence. Hsa-miR-155-5p might be the mediator in linking cellular senescence to vascular aging and atherosclerosis.

Matched MeSH terms: DNA-Binding Proteins/metabolism
Fulltext Nanoparticle-Encapsulated Camptothecin: Epigenetic Modulation in DNA Repair Mechanisms in Colon Cancer Cells

Farhana A, Koh AE, Tong JB, Alsrhani A, Kumar Subbiah S, Mok PL

Molecules, 2021 Sep 06;26(17).
PMID: 34500845 DOI: 10.3390/molecules26175414

Molecular crosstalk between the cellular epigenome and genome converge as a synergistic driver of oncogenic transformations. Besides other pathways, epigenetic regulatory circuits exert their effect towards cancer progression through the induction of DNA repair deficiencies. We explored this mechanism using a camptothecin encapsulated in β-cyclodextrin-EDTA-Fe3O4 nanoparticles (CPT-CEF)-treated HT29 cells model. We previously demonstrated that CPT-CEF treatment of HT29 cells effectively induces apoptosis and cell cycle arrest, stalling cancer progression. A comparative transcriptome analysis of CPT-CEF-treated versus untreated HT29 cells indicated that genes controlling mismatch repair, base excision repair, and homologues recombination were downregulated in these cancer cells. Our study demonstrated that treatment with CPT-CEF alleviated this repression. We observed that CPT-CEF exerts its effect by possibly affecting the DNA repair mechanism through epigenetic modulation involving genes of HMGB1, APEX1, and POLE3. Hence, we propose that CPT-CEF could be a DNA repair modulator that harnesses the cell's epigenomic plasticity to amend DNA repair deficiencies in cancer cells.

Matched MeSH terms: DNA-Binding Proteins/metabolism
Benchtop Isolation and Characterisation of Small Extracellular Vesicles from Human Mesenchymal Stem Cells

Tan KL, Chia WC, How CW, Tor YS, Show PL, Looi QHD, et al.

Mol Biotechnol, 2021 Sep;63(9):780-791.
PMID: 34061307 DOI: 10.1007/s12033-021-00339-2

The objective of this study is to develop a simple protocol to isolate and characterise small extracellular vesicles (sEVs) from human umbilical cord-derived MSCs (hUC-MSCs). hUC-MSCs were characterised through analysis of morphology, immunophenotyping and multidifferentiation ability. SEVs were successfully isolated by ultrafiltration from the conditioned medium of hUC-MSCs. The sEVs' size distribution, intensity within a specific surface marker population were measured with zetasizer or nanoparticle tracking analysis. The expression of surface and internal markers of sEVs was also assessed by western blotting. Morphology of hUC-MSCs displayed as spindle-shaped, fibroblast-like adherent cells. Phenotypic analysis by flow cytometry revealed that hUC-MSCs expressed MSC surface marker, including CD90, CD73, CD105, CD44 and exhibited the capacity for osteogenic, adipogenic and chondrogenic differentiation. Populations of sEVs with CD9, CD63 and CD81 positive were detected with size distribution in the diameter of 63.2 to 162.5 nm. Typical sEVs biomarkers such as CD9, CD63, CD81, HSP70 and TSG101 were also detected with western blotting. Our study showed that sEVs from hUC-MSCs conditioned medium were successfully isolated and characterised. Downstream application of hUC-MSCs-sEVs will be further explored.

Matched MeSH terms: DNA-Binding Proteins/metabolism
Proteasomal degradation of p130 facilitate cell cycle deregulation and impairment of cellular differentiation in high-risk Human Papillomavirus 16 and 18 E7 transfected cells

Gandhi S, Nor Rashid N, Mohamad Razif MF, Othman S

Mol Biol Rep, 2021 Jun;48(6):5121-5133.
PMID: 34169395 DOI: 10.1007/s11033-021-06509-4

The High-Risk Human Papillomaviruses (HR-HPVs) 16 and 18 are known to cause cervical cancer, which is primarily attributed to E6 and E7 oncoproteins. In addition, recent studies have focused on the vital role of the p130 pocket protein as an oncosuppressor to limit the expression of E2F transcription factors required for cell cycle progression. In view of this, the current study was conducted to investigate the mechanism by which transfection with HPV16/18 E7 leads to the deregulation of the host cell cycle, altering the localisation of p130, and expression of differentiation genes in Human Keratinocytes (HaCaT) cells. Co-immunoprecipitation, Western blot analysis, immunofluorescence microscopy, flow cytometry, quantitative-Polymerase Chain Reaction (qPCR), and the inhibition of p130 by MG132 inhibitor were employed to investigate the loss of p130 and its disruption in HPV 16/18 E7-transfected HaCaT cells. The HPV16- and HPV18-transformed cells, known as CaSki and HeLa, respectively, were also used to complement the ectopic expressions of E7 in HaCaT cells. Normal keratinocytes displayed higher level of p130 expression than HPV-transformed cells. In addition, the immunofluorescence analysis revealed that both HPV 16/18 E7-transfected HaCaT and HPV-transformed cells exhibited higher level of cytoplasmic p130 compared to nuclear p130. A significant increase in the number of S/G2 phase cells in HPV-transformed cells was also recorded since E7 has been shown to stimulate proliferation through the deactivation of Retinoblastoma Protein (pRB)-dependent G1/S checkpoint. Furthermore, the findings recorded the down-regulation of keratinocyte differentiation markers, namely p130, keratin10, and involucrin. The proteasomal degradation of the exported p130 confirmed the cellular localisation pattern of p130, which was commonly observed in cancerous cells. The findings provide strong evidence that the localisation of nuclear p130 nuclear was disrupted by HPV16/18 E7 led to the deregulation of the cell cycle and the impairment of cellular differentiation ultimately lead to cellular transformation.

Matched MeSH terms: DNA-Binding Proteins/metabolism*
Lauric acid alleviates insulin resistance by improving mitochondrial biogenesis in THP-1 macrophages

Tham YY, Choo QC, Muhammad TST, Chew CH

Mol Biol Rep, 2020 Dec;47(12):9595-9607.
PMID: 33259010 DOI: 10.1007/s11033-020-06019-9

Mitochondrial dysfunction plays a crucial role in the central pathogenesis of insulin resistance and type 2 diabetes mellitus. Macrophages play important roles in the pathogenesis of insulin resistance. Lauric acid is a 12-carbon medium chain fatty acid (MCFA) found abundantly in coconut oil or palm kernel oil and it comes with multiple beneficial effects. This research objective was to uncover the effects of the lauric acid on glucose uptake, mitochondrial function and mitochondrial biogenesis in insulin-resistant macrophages. THP-1 monocytes were differentiated into macrophages and induce insulin resistance, before they were treated with increasing doses of lauric acid (5 μM, 10 μM, 20 μM, and 50 μM). Glucose uptake assay, cellular ROS and ATP production assays, mitochondrial content and membrane potential assay were carried out to analyse the effects of lauric acid on insulin resistance and mitochondrial biogenesis in the macrophages. Quantitative RT-PCR (qRT-PCR) and western blot analysis were also performed to determine the expression of the key regulators. Insulin-resistant macrophages showed lower glucose uptake, GLUT-1 and GLUT-3 expression, and increased hallmarks of mitochondrial dysfunction. Interestingly, lauric acid treatment upregulated glucose uptake, GLUT-1 and GLUT-3 expressions. The treatment also restored the mitochondrial biogenesis in the insulin-resistant macrophages by improving ATP production, oxygen consumption, mitochondrial content and potential, while it promoted the expression of mitochondrial biogenesis regulator genes such as TFAM, PGC-1α and PPAR-γ. We show here that lauric acid has the potential to improve insulin sensitivity and mitochondrial dysregulation in insulin-resistant macrophages.

Matched MeSH terms: DNA-Binding Proteins/metabolism
Fulltext Novel discovery of Averrhoa bilimbi ethanolic leaf extract in the stimulation of brown fat differentiation program in combating diet-induced obesity

Lau WK, Noruddin NAA, Ariffin AH, Mahmud MZ, Noor MHM, Amanah A, et al.

BMC Complement Altern Med, 2019 Sep 05;19(1):243.
PMID: 31488120 DOI: 10.1186/s12906-019-2640-3

BACKGROUND: Brown adipocytes are known to promote energy expenditure and limit weight gain to combat obesity. Averrhoa bilimbi, locally called belimbing buluh (DBB), is mainly used as an ethnomedicine in the treatment of metabolic disorders including diabetes mellitus, hypertension and obesity. The present study aims to investigate the browning activity on white adipocytes by A. bilimbi leaf extract and to evaluate the potential mechanisms.
METHODS: Ethanolic leaf extract of A. bilimbi was exposed to Myf5 lineage precursor cells to stimulate adipocyte differentiation. Protein expressions of brown adipocyte markers were determined through high content screening analysis and validated through western blotting. Mito Stress Test assay was conducted to evaluate the cellular oxygen consumption rate upon A. bilimbi treatment.
RESULTS: A. bilimbi ethanolic leaf extract exhibited an adipogenesis effect similar to a PPARgamma agonist. It also demonstrated brown adipocyte differentiation in myoblastic Myf5-positive precursor cells. Expression of UCP1 and PRDM16 were induced. The basal metabolic rate and respiratory capacity of mitochondria were increased upon A. bilimbi treatment.
CONCLUSIONS: The findings suggest that Averrhoa bilimbi ethanolic leaf extract induces adipocyte browning through PRDM16 activation and enhances mitochondria activity due to UCP1 up-regulation.

Matched MeSH terms: DNA-Binding Proteins/metabolism
Fulltext RSAD2 and AIM2 Modulate Coxsackievirus A16 and Enterovirus A71 Replication in Neuronal Cells in Different Ways That May Be Associated with Their 5' Nontranslated Regions

Yogarajah T, Ong KC, Perera D, Wong KT

J Virol, 2018 03 15;92(6).
PMID: 29263272 DOI: 10.1128/JVI.01914-17

Coxsackievirus A16 (CV-A16) and enterovirus A71 (EV-A71) are closely related enteroviruses that cause the same hand, foot, and mouth disease (HFMD), but neurological complications occur only very rarely in CV-A16 compared to EV-A71 infections. To elucidate host responses that may be able to explain these differences, we performed transcriptomic analysis and real-time quantitative PCR (RT-qPCR) in CV-A16-infected neuroblastoma cells (SK-N-SH), and the results showed that the radical S-adenosylmethionine domain containing 2 (RSAD2) was the highest upregulated gene in the antimicrobial pathway. Increased RSAD2 expression was correlated with reduced viral replication, while RSAD2 knockdown cells were correlated with increased replication. EV-A71 replication showed no apparent correlation to RSAD2 expressions. Absent in melanoma 2 (AIM2), which is associated with pyroptotic cell death, was upregulated in EV-A71-infected neurons but not in CV-A16 infection, suggesting that the AIM2 inflammasome played a significant role in suppressing EV-A71 replication. Chimeric viruses derived from CV-A16 and EV-A71 but containing swapped 5' nontranslated regions (5' NTRs) showed that RSAD2 expression/viral replication and AIM2 expression/viral replication patterns may be linked to the 5' NTRs of parental viruses. Differences in secondary structure of internal ribosomal entry sites within the 5' NTR may be responsible for these findings. Overall, our results suggest that CV-A16 and EV-A71 elicit different host responses to infection, which may help explain the apparent lower incidence of CV-A16-associated neurovirulence in HFMD outbreaks compared to EV-A71 infection.IMPORTANCE Although coxsackievirus A16 (CV-A16) and enterovirus A17 (EV-A71) both cause hand, foot, and mouth disease, EV-A71 has emerged as a leading cause of nonpolio, enteroviral fatal encephalomyelitis among young children. The significance of our research is in the identification of the possible differing and novel mechanisms of CV-A16 and EV-A71 inhibition in neuronal cells that may impact viral neuropathogenesis. We further showed that viral 5' NTRs may play significant roles in eliciting different host response mechanisms.

Matched MeSH terms: DNA-Binding Proteins/metabolism*
Sex-specific differences in cardiovascular and metabolic hormones with integrated signalling in the paraventricular nucleus of the hypothalamus

Loewen SP, Paterson AR, Loh SY, Rogers MF, Hindmarch CCT, Murphy D, et al.

Exp Physiol, 2017 11 01;102(11):1373-1379.
PMID: 28762571 DOI: 10.1113/EP086436

NEW FINDINGS: What is the topic of this review? We describe roles of crucial signalling molecules in the paraventricular nucleus of the hypothalamus and highlight recent data suggesting sex-specific changes in the expression of crucial signalling molecules and their receptors, which may underlie sex differences in both cardiovascular and metabolic function. What advances does it highlight? This review highlights the integrative capacity of the paraventricular nucleus in mediating cardiovascular and metabolic effects by integrating information from multiple signalling molecules. It also proposes that these signalling molecules have sex-specific differential gene expression, indicating the importance of considering these differences in our ongoing search to understand the female-male differences in the regulation of crucial autonomic systems. Many traditional cardiovascular hormones have been implicated in metabolic function. Conversely, many hormones traditionally involved in metabolic regulation have an effect on cardiovascular function. Many of these signalling molecules exert such effects through specific actions in the paraventricular nucleus, an integrative autonomic control centre located in the hypothalamus. Here, we focus on four cardiovascular/metabolic peptide hormones that signal within the paraventricular nucleus, namely angiotensin II, orexin, adiponectin and nesfatin-1. Each of these hormones has specific electrophysiological effects on paraventricular nucleus neurons that can be related to its physiological actions. In addition, we introduce preliminary transcriptomic data indicating that the genes for some of these hormones and their receptors have sex-specific differential expression.

Matched MeSH terms: DNA-Binding Proteins/metabolism*
Fulltext AIM2 Inflammasome-Mediated Pyroptosis in Enterovirus A71-Infected Neuronal Cells Restricts Viral Replication

Yogarajah T, Ong KC, Perera D, Wong KT

Sci Rep, 2017 07 19;7(1):5845.
PMID: 28724943 DOI: 10.1038/s41598-017-05589-2

Encephalomyelitis is a well-known complication of hand, foot, and mouth disease (HFMD) due to Enterovirus 71 (EV71) infection. Viral RNA/antigens could be detected in the central nervous system (CNS) neurons in fatal encephalomyelitis but the mechanisms of neuronal cell death is not clearly understood. We investigated the role of absent in melanoma 2 (AIM2) inflammasome in neuronal cell death, and its relationship to viral replication. Our transcriptomic analysis, RT-qPCR, Western blot, immunofluorescence and flow cytometry studies consistently showed AIM2 gene up-regulation and protein expression in EV-A71-infected SK-N-SH cells. Downstream AIM2-induced genes, CARD16, caspase-1 and IL-1β were also up-regulated and caspase-1 was activated to form cleaved caspase-1 p20 subunits. As evidenced by 7-AAD positivity, pyroptosis was confirmed in infected cells. Overall, these findings have a strong correlation with decreases in viral titers, copy numbers and proteins, and reduced proportions of infected cells. AIM2 and viral antigens were detected by immunohistochemistry in infected neurons in inflamed areas of the CNS in EV-A71 encephalomyelitis. In infected AIM2-knockdown cells, AIM2 and related downstream gene expressions, and pyroptosis were suppressed, resulting in significantly increased virus infection. These results support the notion that AIM2 inflammasome-mediated pyroptosis is an important mechanism of neuronal cell death and it could play an important role in limiting EV-A71 replication.

Matched MeSH terms: DNA-Binding Proteins/metabolism*
Fulltext Cellular Homeostasis and Antioxidant Response in Epithelial HT29 Cells on Titania Nanotube Arrays Surface

Smn Mydin RB, Sreekantan S, Hazan R, Farid Wajidi MF, Mat I

Oxid Med Cell Longev, 2017;2017:3708048.
PMID: 28337249 DOI: 10.1155/2017/3708048

Cell growth and proliferative activities on titania nanotube arrays (TNA) have raised alerts on genotoxicity risk. Present toxicogenomic approach focused on epithelial HT29 cells with TNA surface. Fledgling cell-TNA interaction has triggered G0/G1 cell cycle arrests and initiates DNA damage surveillance checkpoint, which possibly indicated the cellular stress stimuli. A profound gene regulation was observed to be involved in cellular growth and survival signals such as p53 and AKT expressions. Interestingly, the activation of redox regulator pathways (antioxidant defense) was observed through the cascade interactions of GADD45, MYC, CHECK1, and ATR genes. These mechanisms furnish to protect DNA during cellular division from an oxidative challenge, set in motion with XRRC5 and RAD50 genes for DNA damage and repair activities. The cell fate decision on TNA-nanoenvironment has been reported to possibly regulate proliferative activities via expression of p27 and BCL2 tumor suppressor proteins, cogent with SKP2 and BCL2 oncogenic proteins suppression. Findings suggested that epithelial HT29 cells on the surface of TNA may have a positive regulation via cell-homeostasis mechanisms: a careful circadian orchestration between cell proliferation, survival, and death. This nanomolecular knowledge could be beneficial for advanced medical applications such as in nanomedicine and nanotherapeutics.

Matched MeSH terms: DNA-Binding Proteins/metabolism
Fulltext Aberrant Inflammasome Activation Characterizes Tuberculosis-Associated Immune Reconstitution Inflammatory Syndrome

Tan HY, Yong YK, Shankar EM, Paukovics G, Ellegård R, Larsson M, et al.

J Immunol, 2016 05 15;196(10):4052-63.
PMID: 27076678 DOI: 10.4049/jimmunol.1502203

Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) complicates combination antiretroviral therapy (cART) in up to 25% of patients with HIV/TB coinfection. Monocytes and IL-18, a signature cytokine of inflammasome activation, are implicated in TB-IRIS pathogenesis. In this study, we investigated inflammasome activation both pre- and post-cART in TB-IRIS patients. HIV/TB patients exhibited higher proportions of monocytes expressing activated caspase-1 (casp1) pre-cART, compared with HIV patients without TB, and patients who developed TB-IRIS exhibited the greatest increase in casp1 expression. CD64(+) monocytes were a marker of increased casp1 expression. Furthermore, IL-1β, another marker of inflammasome activation, was also elevated during TB-IRIS. TB-IRIS patients also exhibited greater upregulation of NLRP3 and AIM2 inflammasome mRNA, compared with controls. Analysis of plasma mitochondrial DNA levels showed that TB-IRIS patients experienced greater cell death, especially pre-cART. Plasma NO levels were lower both pre- and post-cART in TB-IRIS patients, providing evidence of inadequate inflammasome regulation. Plasma IL-18 levels pre-cART correlated inversely with NO levels but positively with monocyte casp1 expression and mitochondrial DNA levels, and expression of IL-18Rα on CD4(+) T cells and NK cells was higher in TB-IRIS patients, providing evidence that IL-18 is a marker of inflammasome activation. We propose that inflammasome activation in monocytes/macrophages of HIV/TB patients increases with ineffective T cell-dependent activation of monocytes/macrophages, priming them for an excessive inflammatory response after cART is commenced, which is greatest in patients with TB-IRIS.

Matched MeSH terms: DNA-Binding Proteins/metabolism
Human topoisomerase II alpha as a prognostic biomarker in cancer chemotherapy

Ali Y, Abd Hamid S

Tumour Biol., 2016 Jan;37(1):47-55.
PMID: 26482620 DOI: 10.1007/s13277-015-4270-9

Topoisomerases are nuclear enzymes that regulate topology of DNA by facilitating the temporary cleavage and ligation cycle of DNA. Among all forms of topoisomerases, TOP-IIA is extensively associated with cell proliferation and therefore is an important therapeutic target in diseases that involved cellular proliferation such as cancers. Nearly half of present-day antitumor regimens contain at least one prescription that act as a topoisomerase inhibitor. Generally, tumor cells show divergent expression of TOP-IIA compared to normal cells. The remarkable expression of TOP-IIA in various carcinomas provides a significant biomarker toward understanding the nature of malignancy. TOP-IIA expression and amplification studies help in diagnosing cancer and to observe the disease progression, overall survival (OS) of patients, and response to therapy. This review highlights the research output and analysis in exploring the standing of TOP-IIA in various carcinomas. As some reports show contradiction within the same field of interest, the outline of that may help to induce researchers for further investigation and clarification. To the best of our knowledge, this is the first overview briefly summarizing the prognostic feature of TOP-IIA in various types of cancer.

Matched MeSH terms: DNA-Binding Proteins/metabolism*
Metabolic Control of Type 2 Diabetes by Targeting the GLUT4 Glucose Transporter: Intervention Approaches

Alam F, Islam MA, Khalil MI, Gan SH

Curr Pharm Des, 2016;22(20):3034-49.
PMID: 26951104 DOI: 10.2174/1381612822666160307145801

Type 2 diabetes mellitus (T2DM), the most common form of diabetes, is characterized by insulin resistance in the hepatic and peripheral tissues. Glucose transporter 4 (GLUT4) plays a major role in the pathophysiology of T2DM. Its defective expression or translocation to the peripheral cell plasma membrane in T2DM patients hinders the entrance of glucose into the cell for energy production. In addition to suitable drugs, an appropriate diet and/or exercise can be implemented to target the increase in GLUT4 expression, GLUT4 concentrations and GLUT4 translocation to the cell surface when managing the glucose metabolism of T2DM patients. In this review, we discussed successful intervention strategies that were individually administered or coupled with diet and/or exercise and affected the expression and translocation of GLUT4 in T2DM while reducing the excess glucose load from the blood. Additionally, some potentially good synthetic and natural compounds, which can activate the insulin-independent GLUT4 signaling pathways for the efficient management of T2DM, are highlighted as possible targets or emerging alternative sources for future anti-diabetic drug development.

Matched MeSH terms: DNA-Binding Proteins/metabolism*
Fulltext The nucleoprotein of influenza A virus induces p53 signaling and apoptosis via attenuation of host ubiquitin ligase RNF43

Nailwal H, Sharma S, Mayank AK, Lal SK

Cell Death Dis, 2015 May 21;6:e1768.
PMID: 25996295 DOI: 10.1038/cddis.2015.131

The interplay between influenza virus and host factors to support the viral life cycle is well documented. Influenza A virus (IAV) proteins interact with an array of cellular proteins and hijack host pathways which are at the helm of cellular responses to facilitate virus invasion. The multifaceted nature of the ubiquitination pathway for protein regulation makes it a vulnerable target of many viruses including IAV. To this end we conducted a yeast two-hybrid screen to search for cellular ubiquitin ligases important for influenza virus replication. We identified host protein, RING finger protein 43 (RNF43), a RING-type E3 ubiquitin ligase, as a novel interactor of nucleoprotein (NP) of IAV and an essential partner to induce NP-driven p53-mediated apoptosis in IAV-infected cells. In this study, we demonstrate that IAV leads to attenuation of RNF43 transcripts and hence its respective protein levels in the cellular milieu whereas in RNF43 depleted cells, viral replication was escalated several folds. Moreover, RNF43 polyubiquitinates p53 which further leads to its destabilization resulting in a decrease in induction of the p53 apoptotic pathway, a hitherto unknown process targeted by NP for p53 stabilization and accumulation. Collectively, these results conclude that NP targets RNF43 to modulate p53 ubiquitination levels and hence causes p53 stabilization which is conducive to an enhanced apoptosis level in the host cells. In conclusion, our study unravels a novel strategy adopted by IAV for utilizing the much conserved ubiquitin proteasomal pathway.

Matched MeSH terms: DNA-Binding Proteins/metabolism*
Fulltext Sumoylation controls the timing of Tup1-mediated transcriptional deactivation

Ng CH, Akhter A, Yurko N, Burgener JM, Rosonina E, Manley JL

Nat Commun, 2015 Mar 13;6:6610.
PMID: 25766875 DOI: 10.1038/ncomms7610

The small ubiquitin-like modifier (SUMO) is implicated in various cellular activities, including transcriptional regulation. We previously showed that the yeast activator Gcn4 becomes sumoylated during activation, facilitating its eventual promoter eviction and transcriptional shut off. Here we show that the corepressor Tup1 is sumoylated, at two specific lysines, under various stress conditions. Mutation of these sites has no effect on Tup1 recruitment or RNAP II promoter occupancy immediately following induction. However, Tup1 levels subsequently decrease, while RNAP II and transcription increase in Tup1 mutant cells. Consistent with this, a Tup1 mutant displaying increased sumoylation led to reduced transcription. We also show that coordinated sumoylation of Gcn4 and Tup1 enhances Gcn4 promoter eviction and that multiple Tup1-interacting proteins become sumoylated after stress. Together, our studies provide evidence that coordinated sumoylation of Gcn4, Tup1 and likely other factors dampens activated transcription by stabilizing Tup1 binding and stimulating Gcn4 and RNAP II removal.

Matched MeSH terms: DNA-Binding Proteins/metabolism
Critical role of ARID3B in the expression of pro-apoptotic p53-target genes and apoptosis

Pratama E, Tian X, Lestari W, Iseki S, Ichwan SJ, Ikeda MA

Biochem Biophys Res Commun, 2015 Dec;468(1-2):248-54.
PMID: 26519881 DOI: 10.1016/j.bbrc.2015.10.121

ARID3A and ARID3B are transcriptional targets of p53. Recently, it has been reported that ARID3A plays a critical role in the transcriptional activation of pro-arrest p21 in response to DNA damage. However, the role of ARID3B in the p53 regulatory pathway remains poorly understood. Here we show that ARID3A and ARID3B specifically bind to putative ARID3-binding sites in p53 target genes in vitro and in vivo. ARID3B and, to a lesser extent, ARID3A silencing blocked transcriptional activation of pro-apoptotic p53 target genes, such as PUMA, PIG3, and p53. Furthermore, ectopic ARID3B, to a lesser extent, ARID3A expression activated the pro-apoptotic gene expression, and only ARID3B induced apoptosis. Finally, ARID3B but not ARID3A silencing blocked apoptosis induction following DNA damage. These results indicated that, although ARID3B and ARID3A share overlapping functions, ARID3B play a key role in the expression of pro-apoptotic p53-target genes and apoptosis.

Matched MeSH terms: DNA-Binding Proteins/metabolism
Analysis of Hereditary Nonpolyposis Colorectal Cancer in Malay Cohorts using Immunohistochemical Screening

Wan Juhari WK, Wan Abdul Rahman WF, Mohd Sidek AS, Abu Hassan MR, Ahmad Amin Noordin KB, Zakaria AD, et al.

Asian Pac J Cancer Prev, 2015;16(9):3767-71.
PMID: 25987035

BACKGROUND: Lynch syndrome (LS) is an inherited predisposition to colorectal, endometrial (uterine) and other cancers. Although most cancers are not inherited, about 5 percent (%) of people who have colorectal or endometrial cancer have the Lynch syndrome. It involves the alteration of mismatch repair (MMR) genes; MLH1, MSH2, MSH6 or PMS2. In this study, we analyzed the expression of MMR proteins in colorectal cancer in a Malay cohort by immunohistochemistry.
MATERIALS AND METHODS: A total of 17 patients were selected fulfilling one of the Bethesda criteria: colorectal cancer diagnosed in a patient aged less than 50 years old, having synchronous and metachronous colorectal cancer or with a strong family history. Immunohistochemical staining was performed on paraffin embedded tumour tissue samples using four antibodies: MLH1, MSH2, MSH6 and PMS2.
RESULTS: Twelve out of 17 patients (70.6%) were noted to have a family history. A total of 41% (n=7) of the patients had abnormal immunohistochemical staining with one or more of the four antibodies. Loss of expression were noted in 13 tumour tissues with a negative staining score <4. Of 13 tumour tissues, four showed loss expression of MLH1. For PMS2, loss of expression were noted in five cases. Both MSH2 and MSH6 showed loss of expression in two tumour tissues respectively.
CONCLUSIONS: Revised Bethesda criteria and immunohistochemical analysis constituted a convenient approach and is recommended to be a first-line screening for Lynch syndrome in Malay cohorts.

Matched MeSH terms: DNA-Binding Proteins/metabolism
Fulltext Transcription Factor CREB3L1 Regulates Endoplasmic Reticulum Stress Response Genes in the Osmotically Challenged Rat Hypothalamus

Greenwood M, Greenwood MP, Paton JF, Murphy D

PLoS One, 2015;10(4):e0124956.
PMID: 25915053 DOI: 10.1371/journal.pone.0124956

Arginine vasopressin (AVP) is synthesised in magnocellular neurons (MCNs) of supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus. In response to the hyperosmotic stressors of dehydration (complete fluid deprivation, DH) or salt loading (drinking 2% salt solution, SL), AVP synthesis increases in MCNs, which over-burdens the protein folding machinery in the endoplasmic reticulum (ER). ER stress and the unfolded protein response (UPR) are signaling pathways that improve ER function in response to the accumulation of misfold/unfold protein. We asked whether an ER stress response was activated in the SON and PVN of DH and SL rats. We observed increased mRNA expression for the immunoglobulin heavy chain binding protein (BiP), activating transcription factor 4 (Atf4), C/EBP-homologous protein (Chop), and cAMP responsive element binding protein 3 like 1 (Creb3l1) in both SON and PVN of DH and SL rats. Although we found no changes in the splicing pattern of X box-binding protein 1 (Xbp1), an increase in the level of the unspliced form of Xbp1 (Xbp1U) was observed in DH and SL rats. CREB3L1, a novel ER stress inducer, has been shown to be activated by ER stress to regulate the expression of target genes. We have previously shown that CREB3L1 is a transcriptional regulator of the AVP gene; however, a role for CREB3L1 in the response to ER stress has yet to be investigated in MCNs. Here, we used lentiviral vectors to introduce a dominant negative form of CREB3L1 (CREB3L1DN) in the rat SON. Expression of CREB3L1DN in the SON decreased Chop and Xbp1U mRNA levels, but not BiP and Atf4 transcript expression. CREB3L1 is thus implicated as a transcriptional mediator of the ER stress response in the osmotically stimulated SON.

Matched MeSH terms: DNA-Binding Proteins/metabolism
Fulltext The use of protein-DNA, chromatin immunoprecipitation, and transcriptome arrays to describe transcriptional circuits in the dehydrated male rat hypothalamus

Qiu J, Kleineidam A, Gouraud S, Yao ST, Greenwood M, Hoe SZ, et al.

Endocrinology, 2014 Nov;155(11):4380-90.
PMID: 25144923 DOI: 10.1210/en.2014-1448

The supraoptic nucleus (SON) of the hypothalamus is responsible for maintaining osmotic stability in mammals through its elaboration of the antidiuretic hormone arginine vasopressin. Upon dehydration, the SON undergoes a function-related plasticity, which includes remodeling of morphology, electrical properties, and biosynthetic activity. This process occurs alongside alterations in steady state transcript levels, which might be mediated by changes in the activity of transcription factors. In order to identify which transcription factors might be involved in changing patterns of gene expression, an Affymetrix protein-DNA array analysis was carried out. Nuclear extracts of SON from dehydrated and control male rats were analyzed for binding to the 345 consensus DNA transcription factor binding sequences of the array. Statistical analysis revealed significant changes in binding to 26 consensus elements, of which EMSA confirmed increased binding to signal transducer and activator of transcription (Stat) 1/Stat3, cellular Myelocytomatosis virus-like cellular proto-oncogene (c-Myc)-Myc-associated factor X (Max), and pre-B cell leukemia transcription factor 1 sequences after dehydration. Focusing on c-Myc and Max, we used quantitative PCR to confirm previous transcriptomic analysis that had suggested an increase in c-Myc, but not Max, mRNA levels in the SON after dehydration, and we demonstrated c-Myc- and Max-like immunoreactivities in SON arginine vasopressin-expressing cells. Finally, by comparing new data obtained from Roche-NimbleGen chromatin immunoprecipitation arrays with previously published transcriptomic data, we have identified putative c-Myc target genes whose expression changes in the SON after dehydration. These include known c-Myc targets, such as the Slc7a5 gene, which encodes the L-type amino acid transporter 1, ribosomal protein L24, histone deactylase 2, and the Rat sarcoma proto-oncogene (Ras)-related nuclear GTPase.

Matched MeSH terms: DNA-Binding Proteins/metabolism
Fulltext Combined protein- and nucleic acid-level effects of rs1143679 (R77H), a lupus-predisposing variant within ITGAM

Maiti AK, Kim-Howard X, Motghare P, Pradhan V, Chua KH, Sun C, et al.

Hum Mol Genet, 2014 Aug 1;23(15):4161-76.
PMID: 24608226 DOI: 10.1093/hmg/ddu106

Integrin alpha M (ITGAM; CD11b) is a component of the macrophage-1 antigen complex, which mediates leukocyte adhesion, migration and phagocytosis as part of the immune system. We previously identified a missense polymorphism, rs1143679 (R77H), strongly associated with systemic lupus erythematosus (SLE). However, the molecular mechanisms of this variant are incompletely understood. A meta-analysis of published and novel data on 28 439 individuals with European, African, Hispanic and Asian ancestries reinforces genetic association between rs1143679 and SLE [Pmeta = 3.60 × 10(-90), odds ratio (OR) = 1.76]. Since rs1143679 is in the most active region of chromatin regulation and transcription factor binding in ITGAM, we quantitated ITGAM RNA and surface protein levels in monocytes from patients with each rs1143679 genotype. We observed that transcript levels significantly decreased for the risk allele ('A') relative to the non-risk allele ('G'), in a dose-dependent fashion: ('AA' < 'AG' < 'GG'). CD11b protein levels in patients' monocytes were directly correlated with RNA levels. Strikingly, heterozygous individuals express much lower (average 10- to 15-fold reduction) amounts of the 'A' transcript than 'G' transcript. We found that the non-risk sequence surrounding rs1143679 exhibits transcriptional enhancer activity in vivo and binds to Ku70/80, NFKB1 and EBF1 in vitro, functions that are significantly reduced with the risk allele. Mutant CD11b protein shows significantly reduced binding to fibrinogen and vitronectin, relative to non-risk, both in purified protein and in cellular models. This two-pronged contribution (nucleic acid- and protein-level) of the rs1143679 risk allele to decreasing ITGAM activity provides insight into the molecular mechanisms of its potent association with SLE.

Matched MeSH terms: DNA-Binding Proteins/metabolism

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