Intrinsic structural dynamics dictate enzymatic activity and inhibition.

Enzymes are known to sample various conformations, many of which are critical for their biological function. However, structural characterizations of enzymes predominantly focus on the most populated conformation. As a result, single-point mutations often produce structures that are similar or essentially identical to those of the wild-type enzyme despite large changes in enzymatic activity. Here, we show for mutants of a histone deacetylase enzyme (HDAC8) that reduced enzymatic activities, reduced inhibitor affinities, and reduced residence times are all captured by the rate constants between intrinsically sampled conformations that, in turn, can be obtained independently by solution NMR spectroscopy. Thus, for the HDAC8 enzyme, the dynamic sampling of conformations dictates both enzymatic activity and inhibitor potency. Our analysis also dissects the functional role of the conformations sampled, where specific conformations distinct from those in available structures are responsible for substrate and inhibitor binding, catalysis, and product dissociation. Precise structures alone often do not adequately explain the effect of missense mutations on enzymatic activity and drug potency. Our findings not only assign functional roles to several conformational states of HDAC8 but they also underscore the paramount role of dynamics, which will have general implications for characterizing missense mutations and designing inhibitors.

An integrative characterization of proline cis and trans conformers in a disordered peptide.

Intrinsically disordered proteins (IDPs) often contain proline residues that undergo cis/trans isomerization. While molecular dynamics (MD) simulations have the potential to fully characterize the proline cis and trans subensembles, they are limited by the slow timescales of isomerization and force field inaccuracies. NMR spectroscopy can report on ensemble-averaged observables for both the cis-proline and trans-proline states, but a full atomistic characterization of these conformers is challenging. Given the importance of proline cis/trans isomerization for influencing the conformational sampling of disordered proteins, we employed a combination of all-atom MD simulations with enhanced sampling (metadynamics), NMR, and small-angle x-ray scattering (SAXS) to characterize the two subensembles of the ORF6 C-terminal region (ORF6CTR) from SARS-CoV-2 corresponding to the proline-57 (P57) cis and trans states. We performed MD simulations in three distinct force fields: AMBER03ws, AMBER99SB-disp, and CHARMM36m, which are all optimized for disordered proteins. Each simulation was run for an accumulated time of 180-220 µs until convergence was reached, as assessed by blocking analysis. A good agreement between the cis-P57 populations predicted from metadynamic simulations in AMBER03ws was observed with populations obtained from experimental NMR data. Moreover, we observed good agreement between the radius of gyration predicted from the metadynamic simulations in AMBER03ws and that measured using SAXS. Our findings suggest that both the cis-P57 and trans-P57 conformations of ORF6CTR are extremely dynamic and that interdisciplinary approaches combining both multiscale computations and experiments offer avenues to explore highly dynamic states that cannot be reliably characterized by either approach in isolation.

Picosecond Dynamics of a Small Molecule in Its Bound State with an Intrinsically Disordered Protein.

Intrinsically disordered proteins (IDPs) are highly dynamic biomolecules that rapidly interconvert among many structural conformations. These dynamic biomolecules are involved in cancers, neurodegeneration, cardiovascular illnesses, and viral infections. Despite their enormous therapeutic potential, IDPs have generally been considered undruggable because of their lack of classical long-lived binding pockets for small molecules. Currently, only a few instances are known where small molecules have been observed to interact with IDPs, and this situation is further exacerbated by the limited sensitivity of experimental techniques to detect such binding events. Here, using experimental nuclear magnetic resonance (NMR) spectroscopy 19F transverse spin-relaxation measurements, we discovered that a small molecule, 5-fluoroindole, interacts with the disordered domains of non-structural protein 5A from hepatitis C virus with a Kd of 260 ± 110 µM. Our analysis also allowed us to determine the rotational correlation times (τc) for the free and bound states of 5-fluoroindole. In the free state, we observed a rotational correlation time of 27.0 ± 1.3 ps, whereas in the bound state, τc only increased to 46 ± 10 ps. Our findings imply that it is possible for small molecules to engage with IDPs in exceptionally dynamic ways, in sharp contrast to the rigid binding modes typically exhibited when small molecules bind to well-defined binding pockets within structured proteins.

Expression analysis and function of mitochondrial genome-encoded microRNAs.

MicroRNAs (miRNAs) play a significant role in nuclear and mitochondrial anterograde and retrograde signaling. Most of the miRNAs found inside mitochondria are encoded in the nuclear genome, with a few mitochondrial genome-encoded non-coding RNAs having been reported. In this study, we have identified 13 mitochondrial genome-encoded microRNAs (mitomiRs), which were differentially expressed in breast cancer cell lines (MCF-7, MDA-MB-468 and MDA-MB-231), non-malignant breast epithelial cell line (MCF-10A), and normal and breast cancer tissue specimens. We found that mitochondrial DNA (mtDNA) depletion and inhibition of mitochondrial transcription led to reduced expression of mitomiRs in breast cancer cells. MitomiRs physically interacted with Ago2, an RNA-induced silencing complex (RISC) protein, in the cytoplasm and inside mitochondria. MitomiRs regulate the expression of both nuclear and mitochondrial transcripts in breast cancer cells. We showed that mitomiR-5 targets the PPARGC1A gene and regulates mtDNA copy number in breast cancer cells. MitomiRs identified in the present study may be a promising tool for expression and functional analysis in patients with a defective mitochondrial phenotype, including cancer and metabolic syndromes. This article has an associated First Person interview with the first author of the paper.

Exposure of environmental trace elements in prostate cancer patients: A multiple metal analysis.

Prostate cancer (PCa) is the second leading cause of cancer-related deaths among men. To elucidate the connection between trace elements (arsenic: As, cadmium: Cd, lead: Pb, chromium: Cr, and nickel: Ni) and the risk of PCa, we analyzed trace element levels in the serum, urine, and tissues of PCa patients, while also examining their smoking status. We correlated these levels with their smoking habits. Notably, levels of Cd (P ≤ 0.05) and As (P ≤ 0.01) were significantly higher in the tumor tissue than in adjacent tissues. No significant differences were observed in the levels of Pb, Cr and Ni. Additionally, urinary Cd levels in 70% and arsenic levels in 2.3% of the PCa cohort were markedly higher than the CDC-reported cutoff (Cd ≤ 0.185 µg/L & As ≤100 µg/L). None displayed elevated levels of urinary Pb, Cr, and Ni. Conversely, in serum samples, the concentration of arsenic exceeded the CDC-determined limit (As ≤1.0 µg/L) in 31.69% of PCa patients. However, only 7.04% of patients had higher serum Cd levels than the CDC standard values (Cd ≤ 0.315 µg/L), while all PCa patients exceeded the Cr CDC limit (Cr ≤ 0.16 µg/L) and the Ni CDC limit (Ni ≤ 0.2 µg/L). On the contrary, no significant differences were observed in serum Pb (Pb ≤ 35.0 µg/L). Our findings establish a positive link between Cd and arsenic tissue concentrations and the risk of PCa. Subsequent studies are essential to determine whether elevated trace element levels pose a risk for the development of prostate carcinogenesis. Interestingly, among the PCa cohort comprising smokers, notably higher Cd levels were observed only in tumor tissues (P ≤ 0.01) and urine (P ≤ 0.05) compared to other elements or in other specimens.

Bioinformatic Analysis of miR-200b/429 and Hub Gene Network in Cervical Cancer.

The miR-200b/429 located at 1p36 is a highly conserved miRNA cluster emerging as a critical regulator of cervical cancer. Using publicly available miRNA expression data from TCGA and GEO followed by independent validation, we aimed to identify the association between miR-200b/429 expression and cervical cancer. miR-200b/429 cluster was significantly overexpressed in cancer samples compared to normal samples. miR-200b/429 expression did not correlate with patient survival; however, its overexpression correlated with histological type. Protein-protein interaction analysis of 90 target genes of miR-200b/429 identified EZH2, FLT1, IGF2, IRS1, JUN, KDR, SOX2, MYB, ZEB1, and TIMP2 as the top ten hub genes. PI3K-AKT and MAPK signaling pathways emerged as major target pathways of miR-200b/429 and their hub genes. Kaplan-Meier survival analysis showed the expression of seven miR-200b/429 target genes (EZH2, FLT1, IGF2, IRS1, JUN, SOX2, and TIMP2) to influence the overall survival of patients. The miR-200a-3p and miR-200b-5p could help predict cervical cancer with metastatic potential. The cancer hallmark enrichment analysis showed hub genes to promote growth, sustained proliferation, resistance to apoptosis, induction of angiogenesis, activation of invasion, and metastasis, enabling replicative immortality, evading immune destruction, and tumor-promoting inflammation. The drug-gene interaction analysis identified 182 potential drugs to interact with 27 target genes of miR-200b/429 with paclitaxel, doxorubicin, dabrafenib, bortezomib, docetaxel, ABT-199, eribulin, vorinostat, etoposide, and mitoxantrone emerging as the top ten best candidate drugs. Taken together, miR-200b/429 and associated hub genes can be helpful for prognostic application and clinical management of cervical cancer.

An Unusual Case of Hyperthyroidism.

INTRODUCTION: Thyroid gland dysfunction greatly alters the hemodynamics of the body resulting in major changes in cardiac output, blood pressure and pulmonary vascular resistance amongst others. Hyperthyroidism is associated with an increased morbidity and mortality from cardiovascular disease. Thyrotoxicosis is commonly associated with exacerbation of underlying coronary heart disease, with atrial fibrillation and systolic dysfunction. It is less well appreciated that hyperthyroidism is also associated with pulmonary arterial hypertension (PAH) and right heart failure. MATERIALS: History -We present a 46 years old female, Presented to our hospital with complaints of Breathlessness on exertion since 3 months gradually progressed from MMRC grade 1 to grade 4 over the period of 2 months without any diurnal/postural variation Cough with expectoration since 3 weeks associated with weight loss. RESULT: Examination-Patient is severely malnourished with BMI 11.6 kg/m2 . Bilateral multiple cervical lymph nodes palpable, 6-8 in number discrete, mobile, soft consistency, measuring 1 cm in size changes. Thyroid is symmetrically enlarged, soft in consistency, moving with deglutition, Systemic examination-Apex beat palpable at 5th intercostal space 2 cm lateral to the MCL with normal character Parasternal heave grade 3+ Palpable P2+ A high pitched, rumbling, pansystolic murmur of grade 3, non radiating heard best with the diaphragm of stethoscope with patient in supine position. Unique features-Both thyroid lobes appear enlarged in size and show homogeneously increased radiotracer uptake. CONCLUSION: IMPRESSION- Well-visualized thyroid gland with increased trapping function. In the given clinical context scan findings favour hyperthyroid status-Graves'disease. Take home message-Hyperthyroidism is a reversible cause of pulmonary hypertension.

Study of Correlation of Bone Mineral Density with Severity of Liver Cirrhosis.

INTRODUCTION: Liver cirrhosis is a common ailment that is widely prevalent in our country and across the world. There are several manifestations of this disease. Metabolic bone disease also has an association with cirrhosis. The present study was designed to study the correlation between bone mineral density (BMD) and the severity of liver cirrhosis. MATERIALS AND METHODS: This was a case-control study. A total of 35 diagnosed cases of liver cirrhosis and 35 age and sex-matched controls were included in the study. BMD was measured by dual-energy X-ray absorptiometry (DEXA) at the hip joint and lumbar spine. Child-Turcotte-Pugh (CTP) score was used for assessing the severity of liver cirrhosis. RESULTS: Out of the 35 cases of cirrhosis, 25 had either osteopenia or osteoporosis. The mean T-score at the hip joint in cases was -1.47 ± 1.62 and in controls, it was -0.56 ± 1.67 (p < 0.001). The mean T-score detected in the lumbar spine was -1.33 ± 1.66 and in controls -0.41 ± 1.67 (p < 0.001). There was a significant inverse correlation between CTP scores and BMD. CONCLUSION: The present study revealed that abnormal BMD is highly prevalent in patients with liver cirrhosis. There is also a significant relationship between the severity of cirrhosis and BMD.

Implications of critical node-dependent unidirectional cross-talk of Plasmodium SUMO pathway proteins.

The endoparasitic pathogen, Plasmodium falciparum (Pf), modulates protein-protein interactions to employ post-translational modifications like SUMOylation to establish successful infections. The interaction between E1 and E2 (Ubc9) enzymes governs species specificity in the Plasmodium SUMOylation pathway. Here, we demonstrate that a unidirectional cross-species interaction exists between Pf-SUMO and human E2, whereas Hs-SUMO1 failed to interact with Pf-E2. Biochemical and biophysical analyses revealed that surface-accessible aspartates of Pf-SUMO determine the efficacy and specificity of SUMO-Ubc9 interactions. Furthermore, we demonstrate that critical residues of the Pf-Ubc9 N terminus are responsible for diminished Hs-SUMO1 and Pf-Ubc9 interaction. Mutating these residues to corresponding Hs-Ubc9 residues restores electrostatic, π-π, and hydrophobic interactions and allows efficient cross-species interactions. We suggest that, in comparison with human counterparts, Plasmodium SUMO and Ubc9 proteins have acquired critical changes on their surfaces as nodes, which Plasmodium can use to exploit the host SUMOylation machinery.

Metastatic suppression by DOC2B is mediated by inhibition of epithelial-mesenchymal transition and induction of senescence.

Senescence induction and epithelial-mesenchymal transition (EMT) events are the opposite sides of the spectrum of cancer phenotypes. The key molecules involved in these processes may get influenced or altered by genetic and epigenetic changes during tumor progression. Double C2-like domain beta (DOC2B), an intracellular vesicle trafficking protein of the double C2 protein family, plays a critical role in exocytosis, neurotransmitter release, and intracellular vesicle trafficking. DOC2B is repressed by DNA promoter hypermethylation and functions as a tumor growth regulator in cervical cancer. To date, the molecular mechanisms of DOC2B in cervical cancer progression and metastasis is elusive. Herein, the biological functions and molecular mechanisms regulated by DOC2B and its impact on senescence and EMT are described. DOC2B inhibition promotes proliferation, growth, and migration by relieving G0/G1-S arrest, actin remodeling, and anoikis resistance in Cal27 cells. It enhanced tumor growth and liver metastasis in nude mice with the concomitant increase in metastasis-associated CD55 and CD61 expression. Inhibition of EMT and promotion of senescence by DOC2B is a calcium-dependent process and accompanied by calcium-mediated interaction between DOC2B and CDH1. In addition, we have identified several EMT and senescence regulators as targets of DOC2B. We show that DOC2B may act as a metastatic suppressor by inhibiting EMT through induction of senescence via DOC2B-calcium-EMT-senescence axis.

Prevalence of Comorbidities in Survivors and Non-Survivors of Severe COVID-19 at a Dedicated COVID Care Centre.

Coronavirus disease 2019 (COVID-19) has resulted in considerable morbidity and mortality worldwide since December 2019. Diseases such as diabetes, hypertension, cardiovascular and pulmonary diseases are common comorbidities in COVID-19 patients and have been correlated with increased disease severity. Comorbidities lead the COVID-19 patient into a vicious infectious circle and are substantially associated with significant morbidity and mortality. This study was aimed to estimate prevalence of comorbidities in severe category of COVID survivors and non survivors. More than 90% patients with multiple comorbidities admitted to ICU did not survive compared to those with one or two comorbidities. Diabetes followed by hypertension was the most common comorbidity in these patients. Thus comorbid individuals must adopt vigilant preventive measures including vaccination and they require intensive management for better outcome.

ZNF471 modulates EMT and functions as methylation regulated tumor suppressor with diagnostic and prognostic significance in cervical cancer.

Cervical cancer (CC) is a leading cause of cancer-related death among women in developing countries. However, the underlying mechanisms and molecular targets for therapy remain to be fully understood. We investigated the epigenetic regulation, biological functions, and clinical utility of zinc-finger protein 471 (ZNF471) in CC. Analysis of cervical tissues and five independent public datasets of CC showed significant hypermethylation of the ZNF471 gene promoter. In CC cell lines, promoter DNA methylation was inversely correlated with ZNF471 expression. The sensitivity and specificity of the ZNF471 hypermethylation for squamous intraepithelial lesion (SIL) vs tumor and normal vs tumor was above 85% with AUC of 0.937. High methylation and low ZNF471 expression predicted poor overall and recurrence-free survival. We identified -686 to +114 bp as ZNF471 promoter, regulated by methylation using transient transfection and luciferase assays. The promoter CpG site methylation of ZNF471 was significantly different among cancer types and tumor grades. Gal4-based heterologous luciferase reporter gene assays revealed that ZNF471 acts as a transcriptional repressor. The retroviral mediated overexpression of ZNF471 in SiHa and CaSki cells inhibited growth, proliferation, cell migration, invasion; delayed cell cycle progression in vitro by increasing cell doubling time; and reduced tumor growth in vivo in nude mice. ZNF471 overexpression inhibited key members of epithelial-mesenchymal transition (EMT), Wnt, and PI3K-AKT signaling pathways. ZNF471 inhibited EMT by directly targeting vimentin as analyzed by bioinformatic analysis, ChIP-PCR, and western blotting. Thus, ZNF471 CpG specific promoter methylation may determine the prognosis of CC and could function as a potential tumor suppressor by targeting EMT signaling.

Study of Endothelial Dysfunction by Flow Mediated Vasodilation in Individuals with Asymptomatic Hyperuricemia.

INTRODUCTION: Asymptomatic hyperuricemia is a condition where the serum uric acid levels are elevated but the individual does not have any sign or symptoms of gout or renal stones. The relationship of hyperuricemia with hypertension, diabetes and chronic kidney disease is established. There are studies which show an association of hyperuricemia with endothelial dysfunction thus increasing the cardiovascular risk in these individuals. We therefore undertook this study to observe endothelial dysfunction in individuals of asymptomatic hyperuricemia. METHODOLOGY: This was a case control study where 40 individuals with asymptomatic hyperuricemia and 40 age and sex matched healthy controls with normal serum uric acid levels were included. Endothelial function was studied by flow mediated vasodilation in the brachial artery. RESULTS: The mean age of cases was 45.03±16.44 years while that of control subjects was 44.70±14.31. There were 22 females and 18 males among the cases while there were 24 females and 16 males among the controls. The mean serum uric acid level in cases was 7.27±1.13 mg% while that of controls was 4.52±1.05 mg%. The FMD was 5.57±1.39% in cases while it was 7.73±1.56% in controls and this difference was statistically significant. CONCLUSION: The present study showed that significant endothelial dysfunction is present in individuals of asymptomatic hyperuricemia in comparison to healthy age and sex matched controls.

Study of Association of Metabolic Syndrome and Risk Factors of Nephrolithiasis.

BACKGROUND AND AIM: The increasing incidence of nephrolithiasis in recent decades is coinciding with rising epidemic of obesity, metabolic syndrome, and type 2 diabetes. This temporal concordance suggests that a link might exist between these metabolic abnormalities and urinary stone disease. Therefore, the present study was aimed to investigate the association between presence of risk factors of nephrolithiasis and metabolic syndrome. METHODS: In a hospital-based, case control study, hundred patients of metabolic syndrome diagnosed according to IDF criteria and hundred age and matched controls were studied for presence of risk factors of nephrolithiasis. RESULTS: Patients with metabolic syndrome had significantly higher uricosuri a,hypercalciuria,oxaluria and hypocitraturia. The prevalence of risk factors of nephrolithiasis was also higher in patients with metabolic syndrome. The most prevalent was low urinary pH in 40% patients with mean pH of 5.8±1.6. Amongst other factors, 33% had hyperuricemia, 29% had hypercalciuria, 15% had oxaluria 13% had hypocitraturia and 10% had hyperuricosuria. Significant correlation was observed between risk factors of nephrolithiasis and components of metabolic syndrome. CONCLUSION: The present study provides an evidence of association between risk factors of nephrolithiasis and metabolic syndrome and suggests that nephrolithiasis may be a systemic disorder representing the interaction of multiple metabolic derangements. Determining common modifiable risk factors for the development of kidney stones might uncover new preventive strategies.

Intra-residue methyl-methyl correlations for valine and leucine residues in large proteins from a 3D-HMBC-HMQC experiment.

Methyl-TROSY based NMR experiments have over the last two decades become one of the most important means to characterise dynamics and functional mechanisms of large proteins and macromolecular machines in solution. The chemical shift assignment of methyl groups in large proteins is, however, still not trivial and it is typically performed using backbone-dependent experiments in a 'divide and conquer' approach, mutations, structure-based assignments or a combination of these. Structure-based assignment of methyl groups is an emerging strategy, which reduces the time and cost required as well as providing a method that is independent of a backbone assignment. One crucial step in available structure-based assignment protocols is linking the two prochiral methyl groups of leucine and valine residues. This has previously been achieved by recording NOESY spectra with short mixing times or by comparing NOESY spectra. Herein, we present a method based on through-bond scalar coupling transfers, a 3D-HMBC-HMQC experiment, to link the intra-residue methyl groups of leucine and valine. It is shown that the HMBC-HMQC method has several advantages over solely using NOESY spectra since a unique intra-residue cross-peak is observed. Moreover, overlap in the methyl-TROSY HMQC spectrum can easily be identified with the HMBC-HMQC experiment, thereby removing possible ambiguities in the assignment.

Differential expression of novel MicroRNAs from developing fetal heart of Gallus gallus domesticus implies a role in cardiac development.

Heart development is a complex process regulated by multi-layered genetic as well epigenetic regulators many of which are still unknown. Besides their critical role during cardiac development, these molecular regulators emerge as key modulators of cardiovascular pathologies, where fetal cardiac genes' re-expression is witnessed. MicroRNAs have recently emerged as a crucial part of signalling cascade in both development and diseases. We aimed to identify, validate, and perform functional annotation of putative novel miRNAs using chicken as a cardiac development model system. Novel miRNAs were obtained through deep sequencing of small RNAs extracted from chicken embryonic cardiac tissue of different developmental stages. After filtering out real pre-miRNAs, their expression analysis, potential target gene's prediction and functional annotations were performed. Expression analysis revealed that miRNAs were differentially expressed during different developmental stages of chicken heart. The expression of selected putative novel miRNAs was further validated by real-time PCR. Our analysis indicated the presence of novel cardiac miRNAs that might be regulating critical cardiac development events such as cardiac cell growth, differentiation, cardiac action potential generation and signal transduction.

Enumeration of deregulated miRNAs in liquid and tissue biopsies of cervical cancer.

OBJECTIVE: The altered miRNAs expression in cervical cancer tissue can be a critical player during tumorigenesis, may contribute to tumor cell heterogeneity and may determine distinct phenotypes within the tumor. Recent studies have highlighted the role of circulating miRNAs as a minimally-invasive biomarker and its potential as biosignature to complement routine tissue-based procedures. METHODS: In order to determine whether miRNAs in serum can indicate changes in cervical tissue specimens, we performed small RNA sequencing and selected miRNAs were validated using qRT-PCR in serum and tissue specimens (n = 115). Further, luciferase assay were performed to investigate the interactions between hsa-miR-409-3p and hsa-miR-454-3p binding sites on 3'UTR region of MTF2 and ST18 respectively. RESULTS: We have identified a total of 14 differentially expressed miRNAs common in serum and tissue specimens. Among them, hsa-miR-17-5p, hsa-miR-32-5p and hsa-miR-454-3p were upregulated while, hsa-miR-409-3p was downregulated in serum and tissue of cervical cancer subjects. Our in-silico small RNA sequencing data analysis identified isomiRs and classified miRNA into clusters and subtypes (exonic, intronic and intergenic) with respect to the expression status in serum and tissue specimens. Expression level of hsa-miR-409-3p and hsa-miR-454-3p were inversely correlated with their target genes MTF2 and ST18 levels respectively in human cervical cancer specimens. Luciferase assay demonstrated that hsa-miR-409-3p and hsa-miR-454-3p functionally interacts with 3'-UTR of MTF2 and ST18 respectively to decrease their activity. CONCLUSION: Our results support the significant role of circulating miRNAs in disease dissemination and their potential utility as biosignatures of clinical relevance.

DNA methylation regulated microRNAs in human cervical cancer.

Regulation of miRNA gene expression by DNA promoter methylation may represent a key mechanism to drive cervical cancer progression. In order to understand the impact of DNA promoter methylation on miRNAs at various stages of cervical carcinogenesis, we performed DNA methylation microarray on Normal Cervical Epithelium (NCE), Cervical Intraepithelial Neoplasia (CIN I-III) and Squamous Cell Carcinoma (SCC) tissues to identify differentially methylated miRNAs followed by validation by bisulfite sequencing. Further, expression of miRNAs was analyzed by qRT-PCR in clinical tissues and cervical cancer cell lines. Transcriptional activity was determined by luciferase assay. We identified a total of 69 hypermethylated and hypomethylated miRNA promoters encompassing 78 CpG islands in all except Y chromosome, among the three groups. The candidate DNA promoters of miR-424 were significantly hypermethylated and miR-200b and miR-34c were significantly hypomethylated in SCC compared to NCE (P < 0.05). Expression of miR-424, miR-200b, and miR-34c were inversely correlated with promoter DNA methylation in tissue samples. Treatment of cell lines with 5-aza-2'-deoxycytidine showed differential expression in all three miRNAs. We observed a decrease in miRNA promoter activity following in vitro SssI methylase treatment of miR-424, miR-200b, and miR-34c. Luciferase assay demonstrated that miR-200b and miR-424 functionally interacts with 3'-UTR of HIPK3 and RBBP6 respectively and decreased their activity in presence of miR-200b and miR-424 mimics transfected in SiHa cells. Taken together, we have identified deregulation of miRNAs by aberrant DNA promoter methylation, leading to its transcriptional silencing during cervical carcinogenesis, which can be potential targets for diagnosis and therapy.

Structure, dynamics, and biochemical characterization of ADF/cofilin Twinstar from Drosophilamelanogaster.

BACKGROUND: Twinstar is an ADF/cofilin family protein, which is expressed by the tsr gene in Drosophila melanogaster. Twinstar is one of the main regulators of actin cytoskeleton remodelling and is essential for vital cellular processes like cytokinesis and endocytosis. METHODS: We have characterized the structure and dynamics of Twinstar by solution NMR spectroscopy, the interaction of Twinstar with rabbit muscle actin by ITC, and biochemical activities of Twinstar through different biochemical assays using fluorescence spectroscopy and ultra-centrifugation. RESULTS: The solution structure of Twinstar shows characteristic ADF-H fold with well-formed G/F-site and F-site for interaction with actin. The structure possesses an extended F-loop, which is rigid at the base, but flexible towards its apical region. Twinstar shares similar dynamics for the G/F-site with C. elegans homologs, UNC-60A and UNC-60B. However, the dynamics of its F-loop are different from its C. elegans homologs. Twinstar shows strong affinity for ADP-G-Actin and ATP-G-Actin with Kds of ~7.6 nM and ~0.4 µM, respectively. It shows mild F-actin depolymerizing activity and stable interaction with F-actin with a Kd of ~5.0 µM. It inhibits the rate of the nucleotide exchange in a dose dependent manner. CONCLUSION: On the basis of structure, dynamics, and biochemical activity, Twinstar can be taken to execute its biochemical role by facilitating directional growth and maintenance of length of actin filaments. GENERAL SIGNIFICANCE: This study characterizes the structure, backbone dynamics, and biochemical activities of Twinstar of Drosophila, which provides an insight into the regulation of actin dynamics in the member of phylum insecta.

Solution structure and dynamics of glia maturation factor from Caenorhabditis elegans.

BACKGROUND: The GMF class of the ADF-H domain family proteins regulate actin dynamics by binding to the Arp2/3 complex and F-actin through their Site-1 and Site-2, respectively. CeGMF of C. elegans is analogous to GMFγ of human and mouse and is 138 amino acids in length. METHODS: We have characterized the solution structure and dynamics of CeGMF by solution NMR spectroscopy and its thermal stability by DSC. RESULTS: The solution structure of CeGMF shows canonical ADF-H fold with two additional ß-strands in the ß4-ß5 loop region. The Site-1 of CeGMF is well formed and residues of all three regions of Site-1 show dynamic flexibility. However, the ß4-ß5 loop of Site-2 is less inclined towards the C-terminal, as the latter is truncated by four residues in comparison to GMF isoforms of human and mouse. Regions of Site-2 show motions on ns-ps timescale, but dynamic flexibility of ß4-ß5 loop is low in comparison to corresponding F-loop region of ADF/cofilin UNC-60B. A general difference in packing of α3 and α1 between GMF and ADF/cofilins was noticed. Additionally, thermal stability of CeGMF was significantly higher than its ADF/cofilin homologs. CONCLUSION: We have presented the first solution structure of GMF from C. elegans, which highlights the structural differences between the Site-2 of CeGMF and mammalian GMF isoforms. Further, we have seen the differences in structure, dynamics, and thermal stability of GMF and ADF/cofilin. GENERAL SIGNIFICANCE: This study provides a useful insight to structural and dynamics factors that define the specificity of GMF towards Arp2/3 complex.