FRG1 is a direct transcriptional regulator of nonsense-mediated mRNA decay genes.

FRG1 is the primary candidate gene for Fascioscapulohumeral Muscular Dystrophy. So far, its role has been reported in muscle development, vasculogenesis, angiogenesis, and tumorigenesis. Mechanistically studies suggest FRG1's role in RNA biogenesis which may have implications in multiple physiological processes and diseases, including tumorigenesis. Its probable role as hnRNP and association with NMD-related genes prompted us to look into FRG1's effect on NMD gene expression and the mechanism. Using microarray profiling in cell lines, we found that FRG1 altered the mRNA surveillance pathway and associated pathways, such as RNA transport and spliceosome machinery molecules. Multiple sequence alignment of core factors, namely, UPF1, UPF3B, and SMG1, showed conserved stretches of nucleotide sequence 'CTGGG'. Structural modeling followed by EMSA, ChIP-qPCR, and luciferase reporter assays showed 'CTGGG' as a FRG1 binding site. Analysis of the publicly available datasets showed that the expression of FRG1 correlates with NMD genes in different tissue types. We validated the effect of FRG1 on NMD gene transcription by qRT-PCR. Overall, FRG1 might be a transcriptional regulator of NMD genes.

Dengue Virus NS4b N-Terminus Disordered Region Interacts with NS3 Helicase C-Terminal Subdomain to Enhance Helicase Activity.

Dengue virus replicates its single-stranded RNA genome in membrane-bound complexes formed on the endoplasmic reticulum, where viral non-structural proteins (NS) and RNA co-localize. The NS proteins interact with one another and with the host proteins. The interaction of the viral helicase and protease, NS3, with the RNA-dependent RNA polymerase, NS5, and NS4b proteins is critical for replication. In vitro, NS3 helicase activity is enhanced by interaction with NS4b. We characterized the interaction between NS3 and NS4b and explained a possible mechanism for helicase activity modulation by NS4b. Our bacterial two-hybrid assay results showed that the N-terminal 57 residues region of NS4b is enough to interact with NS3. The molecular docking of the predicted NS4b structure onto the NS3 structure revealed that the N-terminal disordered region of NS4b wraps around the C-terminal subdomain (CTD) of the helicase. Further, NS3 helicase activity is enhanced upon interaction with NS4b. Molecular dynamics simulations on the NS4b-docked NS3 crystal structure and intrinsic tryptophan fluorescence studies suggest that the interaction results in NS3 CTD domain motions. Based on the interpretation of our results in light of the mechanism explained for NS3 helicase, NS4b-NS3 interaction modulating CTD dynamics is a plausible explanation for the helicase activity enhancement.

The Oncogene MYCN Modulates Glycolytic and Invasive Genes to Enhance Cell Viability and Migration in Human Retinoblastoma.

Retinoblastoma is usually initiated by biallelic RB1 gene inactivation. In addition, MYCN copy number alterations also contribute to RB pathogenesis. However, MYCN expression, its role in disease progression and correlation with RB histological risk factors are not well understood. We studied the expression of MYCN in enucleated RB patient specimens by immunohistochemistry. MYCN is overexpressed in RB compared to control retina. Our microarray gene expression analysis followed by qRT-PCR validation revealed that genes involved in glucose metabolism and migration are significantly downregulated in MYCN knockdown cells. Further, targeting MYCN in RB cells using small molecule compounds or shRNAs led to decreased cell survival and migration, increased apoptosis and cell cycle arrest, suggesting that MYCN inhibition can be a potential therapeutic strategy. We also noted that MYCN inhibition results in reduction in glucose uptake, lactate production, ROS levels and gelatinolytic activity of active-MMP9, explaining a possible mechanism of MYCN in RB. Taking clues from our findings, we tested a combination treatment of RB cells with carboplatin and MYCN inhibitors to find enhanced therapeutic efficacy compared to single drug treatment. Thus, MYCN inhibition can be a potential therapeutic strategy in combination with existing chemotherapy drugs to restrict tumor cell growth in RB.

Acidic pH-Induced Conformational Changes in Chikungunya Virus Fusion Protein E1: a Spring-Twisted Region in the Domain I-III Linker Acts as a Hinge Point for Swiveling Motion of Domains.

Chikungunya virus (CHIKV), a mosquito-transmitted alphavirus, enters a cell through endocytosis, followed by viral and cell membrane fusion. The fusion protein, E1, undergoes an acid pH-induced pre- to postfusion conformation change during membrane fusion. As part of the conformation change, E1 dissociates from the receptor-binding protein, E2, and swivels its domains I and II over domain III to form an extended intermediate and then eventually to form a postfusion hairpin homotrimer. In this study, we tested if the domain I-III linker acts as a "hinge" for the swiveling motion of E1 domains. We found a conserved spring-twisted structure in the linker, stabilized by a salt bridge between a conserved arginine-aspartic acid pair, as a "hinge point" for domain swiveling. Molecular dynamics (MD) simulation of the CHIKV E1 or E2-E1 structure predicted that the spring-twisted region untwists at pH 5.5. Corroborating the prediction, introduction of a "cystine staple" at the hinge point, replacing the conserved arginine-aspartic acid pair with cysteine residues, resulted in loss of fusion activity of E1. MD simulation also predicted domain I-III swiveling at acidic pH. We tested if breaking the His 331-Lys 16 H bond between domains I and III, seen only in the prefusion conformation, is important for domain swiveling. When domains I and III are "stapled" by introducing a disulfide bond in between, E1 showed loss of fusion activity, implying that domain I and III dissociation is a critical acid pH-induced step in membrane fusion. However, replacement of His 331 with an acidic residue did not affect the pH threshold for fusion, suggesting His 331 is not an acid-sensing residue.IMPORTANCEAedes mosquito-transmitted viruses such as the Zika, dengue, and chikungunya viruses have spread globally. CHIKV, similar to many other enveloped viruses, enters cells in sequential steps: step 1 involves receptor binding followed by endocytosis, and step 2 involves viral-cell membrane fusion in the endocytic vesicle. The viral envelope surface protein, E1, performs membrane fusion. E1 is triggered to undergo conformational changes by acidic pH of the maturing endosome. Different domains of E1 rearrange during the pre- to postfusion conformation change. Using in silico analysis of the E1 structure and different biochemical experiments, we explained a structural mechanism of key conformational changes in E1 triggered by acidic pH. We noted two important structural changes in E1 at acidic pH. In the first, a spring-twisted region in a loop connecting two domains (I and III) untwists, bringing a swiveling motion of domains on each other. In the second, breaking of interactions between domains I and III and domain separation are required for membrane fusion. This knowledge will help devise new therapeutic strategies to block conformation changes in E1 and thus viral entry.

Conformational changes in Chikungunya virus E2 protein upon heparan sulfate receptor binding explain mechanism of E2-E1 dissociation during viral entry.

Receptor binding is the first step in viral cell entry. In enveloped virus cell entry, viral and host membrane fusion follows receptor binding. Viral surface receptor-binding protein associates with membrane fusion protein and masks its structure, to prevent pre-mature fusion activity. Dissociation of receptor-binding protein from fusion protein is an essential step before membrane fusion. Mechanism of receptor binding leading to dissociation of receptor binding and fusion protein is poorly understood in alphaviruses. Chikungunya virus (CHIKV), an alphavirus, re-emerged as a global pathogen in recent past. CHIKV surface envelope proteins, E2 and E1, function as receptor binding and fusion protein, respectively. Site of heparan sulfate (HS) receptor binding on E2-E1 heterodimer and its effect on E2-E1 heterodimer conformation is not known. Using molecular docking, we mapped HS binding to a positively charged pocket on E2 that is structurally conserved in alphaviruses. Based on our results from docking and sequence analysis, we identified a novel HS-binding sequence motif in E2. Purified E2 binds to heparin and HS specifically through charge interactions. Binding affinity of E2 to HS is comparable with other known HS-protein interactions (Kd ∼ 1.8 µM). Mutation of charged residues in the predicted HS-binding motif of E2 to alanine resulted in reduction of HS binding. Molecular dynamics (MD) simulations on E2, after docking HS, predicted allosteric domain movements. Fluorescence spectroscopy, far-UV circular dichroism spectroscopy, fluorescence resonance energy transfer experiments on HS-bound E2 corroborate our findings from MD simulations. We propose a mechanism where receptor-binding results in allosteric domain movements in E2, explaining E2-E1 dissociation.

Syncytial phenotype of C-terminally truncated herpes simplex virus type 1 gB is associated with diminished membrane interactions.

The cytoplasmic domain of glycoprotein B (gB) from herpes simplex virus type 1 (HSV-1) is an important regulator of membrane fusion. C-terminal truncations of the cytoplasmic domain lead to either hyperfusion or fusion-null phenotypes. Currently, neither the structure of the cytoplasmic domain nor its mechanism of fusion regulation is known. Here we show, for the first time, that the full-length cytoplasmic domain of HSV-1 gB associates stably with lipid membranes, preferentially binding to membranes containing anionic head groups. This interaction involves a large increase in helical content. However, the truncated cytoplasmic domains associated with the hyperfusion phenotype show a small increase in helical structure and a diminished association with lipid membranes, whereas the one associated with the fusion-null phenotype shows no increase in helical structure and only a minimal association with lipid membranes. We hypothesize that stable binding to lipid membranes is an important part of the mechanism by which the cytoplasmic domain negatively regulates membrane fusion. Moreover, our experiments with truncated cytoplasmic domains point to two specific regions that are critical for membrane interactions. Taken together, our work provides several important new insights into the architecture of the cytoplasmic domain of HSV-1 gB and its interaction with lipid membranes.

Interaction of mammalian Hsp22 with lipid membranes.

Hsp22/HspB8 is a member of the small heat-shock protein family, whose function is not yet completely understood. Our immunolocalization studies in a human neuroblastoma cell line, SK-N-SH, using confocal microscopy show that a significant fraction of Hsp22 is localized to the plasma membrane. We therefore investigated its interactions with lipid vesicles in vitro. Intrinsic tryptophan fluorescence is quenched in the presence of lipid vesicles derived from either bovine brain lipid extract or purified lipids. Time-resolved fluorescence studies show a decrease in the lifetimes of the tryptophan residues. Both of these results indicate burial of some tryptophan residues of Hsp22 upon interaction with lipid vesicles. Membrane interactions also lead to increase in fluorescence polarization of Hsp22. Gel-filtration chromatography shows that Hsp22 binds stably with lipid vesicles; the extent of binding depends on the nature of the lipid. Hsp22 binds more strongly to vesicles made of lipids containing a phosphatidic acid, phosphatidylinositol or phosphatidylserine headgroup (known to be present in the inner leaflet of plasma membrane) compared with lipid vesicles made of a phosphatidylcholine head-group alone. Far-UV CD spectra reveal conformational changes upon binding to the lipid vesicles or in membrane-mimetic solvent, trifluoroethanol. Thus our fluorescence, CD and gel-filtration studies show that Hsp22 interacts with membrane and this interaction leads to stable binding and conformational changes. The present study therefore clearly demonstrates that Hsp22 exhibits potential membrane interaction that may play an important role in its cellular functions.

Mammalian Hsp22 is a heat-inducible small heat-shock protein with chaperone-like activity.

A newly identified 22 kDa protein that interacts with Hsp27 (heat-shock protein 27) was shown to possess the characteristic alpha-crystallin domain, hence named Hsp22, and categorized as a member of the sHsp (small Hsp) family. Independent studies from different laboratories reported the protein with different names such as Hsp22, H11 kinase, E2IG1 and HspB8. We have identified, on the basis of the nucleotide sequence analysis, putative heat-shock factor 1 binding sites upstream of the Hsp22 translation start site. We demonstrate that indeed Hsp22 is heat-inducible. We show, in vitro, chaperone-like activity of Hsp22 in preventing dithiothreitol-induced aggregation of insulin and thermal aggregation of citrate synthase. We have cloned rat Hsp22, overexpressed and purified the protein to homogeneity and studied its structural and functional aspects. We find that Hsp22 fragments on storage. MS analysis of fragments suggests that the fragmentation might be due to the presence of labile peptide bonds. We have established conditions to improve its stability. Far-UV CD indicates a randomly coiled structure for Hsp22. Quaternary structure analyses by glycerol density-gradient centrifugation and gel filtration chromatography show that Hsp22 exists as a monomer in vitro, unlike other members of the sHsp family. Hsp22 exhibits significantly exposed hydrophobic surfaces as reported by bis-8-anilinonaphthalene-l-sulphonic acid fluorescence. We find that the chaperone-like activity is temperature dependent. Thus Hsp22 appears to be a true member of the sHsp family, which exists as a monomer in vitro and exhibits chaperone-like activity.