Insulin Growth Factor-2 Binding Protein 3 (IGF2BP3) Is a Glioblastoma-specific Marker That Activates Phosphatidylinositol 3-Kinase/Mitogen-activated Protein Kinase (PI3K/MAPK) Pathways by Modulating IGF-2

Glioblastoma is the most common and malignant form of primary astrocytoma. Upon investigation of the insulin-like growth factor (IGF) pathway, we found the IGF2BP3/IMP3 transcript and protein to be up-regulated in GBMs but not in lower grade astrocytomas (p<0.0001). IMP3 is an RNA binding protein known to bind to the 5'-untranslated region of IGF-2 mRNA, thereby activating its translation. Overexpression-and knockdown-based studies establish a role for IMP3 in promoting proliferation, anchorage-independent growth, invasion, and chemoresistance. IMP3 overexpressing B16F10 cells also showed increased tumor growth, angiogenesis, and metastasis, resulting in poor survival in a mouse model. Additionally, the infiltrating front, perivascular, and subpial regions in a majority of the GBMs stained positive for IMP3. Furthermore, two different murine glioma models were used to substantiate the above findings. In agreement with the translation activation functions of IMP3, we also found increased IGF-2 protein in the GBM tumor samples without a corresponding increase in its transcript levels. Also, in vitro IMP3 overexpression/knockdown modulated the IGF-2 protein levels without altering its transcript levels. Additionally, IGF-2 neutralization and supplementation studies established that the proproliferative effects of IMP3 were indeed mediated through IGF-2. Concordantly, PI3K and MAPK, the downstream effectors of IGF-2, are activated by IMP3 and are found to be essential for IMP3-induced cell proliferation. Thus, we have identified IMP3 as a GBM-specific proproliferative and proinvasive marker acting through IGF-2 resulting in the activation of oncogenic PI3K and MAPK pathways.

Nonspecific Interaction between DNA and Protein allows for Cooperativity: A Case Study with Mycobacterium DNA Binding Protein

Different DNA-binding proteins have different interaction modes with DNA. Sequence-specific DNA protein interaction has been mostly associated with regulatory processes inside a cell, and as such extensive studies have been made. Adequate data is also available on nonspecific DNA protein interaction, as an intermediate to protein's search for its cognate partner. Multidomain nonspecific DNA protein interaction involving physical sequestering of DNA has often been implicated to regulate gene expression indirectly. However, data available on this type of interaction is limited. One such interaction is the binding of DNA with mycobacterium DNA binding proteins. We have used the Langmuir-Blodgett technique to evaluate for the first time the kinetics and thermodynamics of Mycobacterium smegmatis Dps 1 binding to DNA. By immobilizing one of the interacting partners, we have shown that, when a kinetic bottleneck is applied, the binding mechanism showed cooperative binding (n = 2.72) at lower temperatures, but the degree of cooperativity gradually reduces (n = 1.38) as the temperature was increased We have also compared the kinetics and thermodynamics of sequence-specific and nonspecific DNA protein interactions under the same set of conditions.

Cloning, expression, purification, and biochemical characterisation of the FIC motif containing protein of Mycobacterium tuberculosis

The role of FIC (Filamentation induced by cAMP)(2) domain containing proteins in the regulation of many vital pathways, mostly through the transfer of NMPs from NTPs to specific target proteins (NMPylation), in microorganisms, higher eukaryotes, and plants is emerging. The identity and function of FIC domain containing protein of the human pathogen, Mycobacterium tuberculosis, remains unknown. In this regard, M. tuberculosis fic gene (Mtfic) was cloned, overexpressed, and purified to homogeneity for its biochemical characterisation. It has the characteristic FIC motif, HPFREGNGRSTR (HPFxxGNGRxxR), spanning 144th to 155th residue. Neither the His-tagged nor the GST-tagged MtFic protein, overexpressed in Escherichia coil, nor expression of Mtfic in Mycobacterium smegmatis, yielded the protein in the soluble fraction. However, the maltose binding protein (MBP) tagged MtFic (MBP-MtFic) could be obtained partly in the soluble fraction. The cloned, overexpressed, and purified recombinant MBP-MtFic showed conversion of ATP, GTP, CTP, and UTP into AMP. GMP, CMP, and UMP, respectively. Sequence alignment with several FIC motif containing proteins, complemented with homology modeling on the FIC motif containing protein, VbhT of Bartonella schoenbuchensis as the template, showed conservation and interaction of residues constituting the FIC domain. Site-specific mutagenesis of the His144, or Glu148, or Asn150 of the FIC motif, or of Arg87 residue that constitutes the FIC domain, or complete deletion of the FIC motif, abolished the NTP to NMP conversion activity. The design of NMP formation assay using the recombinant, soluble MtFic would enable identification of its target substrate for NMPylation. (C) 2012 Elsevier Inc. All rights reserved.

Force and ATP hydrolysis dependent regulation of RecA nucleoprotein filament by single-stranded DNA binding protein

In Escherichia coli, the filament of RecA formed on single-stranded DNA (ssDNA) is essential for recombinational DNA repair. Although ssDNA-binding protein (SSB) plays a complicated role in RecA reactions in vivo, much of our understanding of the mechanism is based on RecA binding directly to ssDNA. Here we investigate the role of SSB in the regulation of RecA polymerization on ssDNA, based on the differential force responses of a single 576-nucleotide-long ssDNA associated with RecA and SSB. We find that SSB outcompetes higher concentrations of RecA, resulting in inhibition of RecA nucleation. In addition, we find that pre-formed RecA filaments de-polymerize at low force in an ATP hydrolysis- and SSB-dependent manner. At higher forces, re-polymerization takes place, which displaces SSB from ssDNA. These findings provide a physical picture of the competition between RecA and SSB under tension on the scale of the entire nucleoprotein SSB array, which have broad biological implications particularly with regard to competitive molecular binding.

Insulin like growth factor binding protein 4 promotes GBM progression and regulates key factors involved in EMT and invasion

Insulin like growth factor binding protein 4 (IGFBP4) regulates growth and development of tissues and organs by negatively regulating IGF signaling. Among most cancers, IGFBP4 has growth inhibitory role and reported as a down-regulated gene, except for renal cell carcinoma, wherein IGFBP4 promotes tumor progression. IGFBP4 expression has been shown to be higher in increasing grades of astrocytoma. However, the functional role of IGFBP4 in gliomas has not been explored. Surgical biopsies of 20 normal brain and 198 astrocytoma samples were analyzed for IGFBP4 expression by qRT-PCR. Highest expression of IGFBP4 mRNA was seen in GBM tumors compared to control brain tissues (median log2 of 2.035, p < 0.0001). Immunohistochemical analysis of 53 tissue samples revealed predominant nuclear staining of IGFBP4, seen maximally in GBMs when compared to DA and AA tumors (median LI = 29.12 +/- A 16.943, p < 0.001). Over expression of IGFBP4 in U343 glioma cells resulted in up-regulation of molecules involved in tumor growth, EMT and invasion such as pAkt, pErk, Vimentin, and N-cadherin and down-regulation of E-cadherin. Functionally, IGFBP4 over expression in these cells resulted in increased proliferation, migration and invasion as assessed by MTT, transwell migration, and Matrigel invasion assays. These findings were confirmed upon IGFBP4 knockdown in U251 glioma cells. Our data suggest a pro-tumorigenic role for IGFBP4 in glioma.

Identification of heat shock factor binding protein in Plasmodium falciparum

Background: Heat shock factor binding protein (HSBP) was originally discovered in a yeast two-hybrid screen as an interacting partner of heat shock factor (HSF). It appears to be conserved in all eukaryotes studied so far, with yeast being the only exception. Cell biological analysis of HSBP in mammals suggests its role as a negative regulator of heat shock response as it appears to interact with HSF only during the recovery phase following exposure to heat stress. While the identification of HSF in the malaria parasite is still eluding biologists, this study for the first time, reports the presence of a homologue of HSBP in Plasmodium falciparum. Methods: PfHSBP was cloned and purified as his-tag fusion protein. CD (Circular dichroism) spectroscopy was performed to predict the secondary structure. Immunoblots and immunofluorescence approaches were used to study expression and localization of HSBP in P. falciparum. Cellular fractionation was performed to examine subcellular distribution of PfHSBP. Immunoprecipitation was carried out to identify HSBP interacting partner in P. falciparum. Results: PfHSBP is a conserved protein with a high helical content and has a propensity to form homo-oligomers. PfHSBP was cloned, expressed and purified. The in vivo protein expression profile shows maximal expression in trophozoites. The protein was found to exist in oligomeric form as trimer and hexamer. PfHSBP is predominantly localized in the parasite cytosol, however, upon heat shock, it translocates to the nucleus. This study also reports the interaction of PfHSBP with PfHSP70-1 in the cytoplasm of the parasite. Conclusions: This study emphasizes the structural and biochemical conservation of PfHSBP with its mammalian counterpart and highlights its potential role in regulation of heat shock response in the malaria parasite. Analysis of HSBP may be an important step towards identification of the transcription factor regulating the heat shock response in P. falciparum.

A Genetic Analysis of the Functional Interactions within Mycobacterium tuberculosis Single-Stranded DNA Binding Protein

Single-stranded DNA binding proteins (SSBs) are vital in all organisms. SSBs of Escherichia coli (EcoSSB) and Mycobacterium tuberculosis (MtuSSB) are homotetrameric. The N-terminal domains (NTD) of these SSBs (responsible for their tetramerization and DNA binding) are structurally well defined. However, their C-terminal domains (CTD) possess undefined structures. EcoSSB NTD consists of beta 1-beta 1'-beta 2-beta 3-alpha-beta 4-beta 45(1)-beta 45(2)-beta 5 secondary structure elements. MtuSSB NTD includes an additional beta-strand (beta 6) forming a novel hook-like structure. Recently, we observed that MtuSSB complemented an E. coli Delta ssb strain. However, a chimeric SSB (m beta 4-beta 5), wherein only the terminal part of NTD (beta 4-beta 5 region possessing L-45 loop) of EcoSSB was substituted with that from MtuSSB, failed to function in E. coli in spite of its normal DNA binding and oligomerization properties. Here, we designed new chimeras by transplanting selected regions of MtuSSB into EcoSSB to understand the functional significance of the various secondary structure elements within SSB. All chimeric SSBs formed homotetramers and showed normal DNA binding. The m beta 4-beta 6 construct obtained by substitution of the region downstream of beta 5 in m beta 4-beta 5 SSB with the corresponding region (beta 6) of MtuSSB complemented the E. coli strain indicating a functional interaction between the L-45 loop and the beta 6 strand of MtuSSB.

Serum proteomics of hepatitis C virus infection reveals retinol-binding protein 4 as a novel regulator

Persistent infection of hepatitis C virus (HCV) can lead to liver cirrhosis and hepatocellular carcinoma, which are currently diagnosed by invasive liver biopsy. Approximately 15-20% of cases of chronic liver diseases in India are caused by HCV infection. In North India, genotype 3 is predominant, whereas genotype 1 is predominant in southern parts of India. The aim of this study was to identify differentially regulated serum proteins in HCV-infected Indian patients (genotypes 1 and 3) using a two-dimensional electrophoresis approach. We identified eight differentially expressed proteins by MS. Expression levels of one of the highly upregulated proteins, retinol-binding protein 4 (RBP4), was validated by ELISA and Western blotting in two independent cohorts. We also confirmed our observation in the JFH1 infectious cell culture system. Interestingly, the HCV core protein enhanced RBP4 levels and partial knockdown of RBP4 had a positive impact on HCV replication, suggesting a possible role for this cellular protein in regulating HCV infection. Analysis of RBP4-interacting partners using a bioinformatic approach revealed novel insights into the possible involvement of RBP4 in HCV-induced pathogenesis. Taken together, this study provided information on the proteome profile of the HCV-infected Indian population, and revealed a link between HCV infection, RBP4 and insulin resistance.

Glioblastoma-derived Macrophage Colony-stimulating Factor (MCSF) Induces Microglial Release of Insulin-like Growth Factor-binding Protein 1 (IGFBP1) to Promote Angiogenesis

Glioblastoma (grade IV glioma/GBM) is the most common primary adult malignant brain tumor with poor prognosis. To characterize molecular determinants of tumor-stroma interaction in GBM, we profiled 48 serum cytokines and identified macrophage colony-stimulating factor (MCSF) as one of the elevated cytokines in sera from GBM patients. Both MCSF transcript and protein were up-regulated in GBM tissue samples through a spleen tyrosine kinase (SYK)-dependent activation of the PI3K-NF kappa B pathway. Ectopic overexpression and silencing experiments revealed that glioma-secreted MCSF has no role in autocrine functions and M2 polarization of macrophages. In contrast, silencing expression of MCSF in glioma cells prevented tube formation of human umbilical vein endothelial cells elicited by the supernatant from monocytes/microglial cells treated with conditioned medium from glioma cells. Quantitative proteomics based on stable isotope labeling by amino acids in cell culture showed that glioma-derived MCSF induces changes in microglial secretome and identified insulin-like growth factor-binding protein 1 (IGFBP1) as one of the MCSF-regulated proteins secreted by microglia. Silencing IGFBP1 expression in microglial cells or its neutralization by an antibody reduced the ability of supernatants derived from microglial cells treated with glioma cell-conditioned medium to induce angiogenesis. In conclusion, this study shows up-regulation of MCSF in GBM via a SYK-PI3K-NF kappa B-dependent mechanism and identifies IGFBP1 released by microglial cells as a novel mediator of MCSF-induced angiogenesis, of potential interest for developing targeted therapy to prevent GBM progression.

HuR Displaces Polypyrimidine Tract Binding Protein To Facilitate La Binding to the 3 ` Untranslated Region and Enhances Hepatitis C Virus Replication

HuR is a ubiquitous, RNA binding protein that influences the stability and translation of several cellular mRNAs. Here, we report a novel role for HuR, as a regulator of proteins assembling at the 3' untranslated region (UTR) of viral RNA in the context of hepatitis C virus (HCV) infection. HuR relocalizes from the nucleus to the cytoplasm upon HCV infection, interacts with the viral polymerase (NS5B), and gets redistributed into compartments of viral RNA synthesis. Depletion in HuR levels leads to a significant reduction in viral RNA synthesis. We further demonstrate that the interaction of HuR with the 3' UTR of the viral RNA affects the interaction of two host proteins, La and polypyrimidine tract binding protein (PTB), at this site. HuR interacts with La and facilitates La binding to the 3' UTR, enhancing La-mediated circularization of the HCV genome and thus viral replication. In addition, it competes with PTB for association with the 3' UTR, which might stimulate viral replication. Results suggest that HuR influences the formation of a cellular/viral ribonucleoprotein complex, which is important for efficient initiation of viral RNA replication. Our study unravels a novel strategy of regulation of HCV replication through an interplay of host and viral proteins, orchestrated by HuR. IMPORTANCE Hepatitis C virus (HCV) is highly dependent on various host factors for efficient replication of the viral RNA. Here, we have shown how a host factor (HuR) migrates from the nucleus to the cytoplasm and gets recruited in the protein complex assembling at the 3' untranslated region (UTR) of HCV RNA. At the 3' UTR, it facilitates circularization of the viral genome through interaction with another host factor, La, which is critical for replication. Also, it competes with the host protein PTB, which is a negative regulator of viral replication. Results demonstrate a unique strategy of regulation of HCV replication by a host protein through alteration of its subcellular localization and interacting partners. The study has advanced our knowledge of the molecular mechanism of HCV replication and unraveled the complex interplay between the host factors and viral RNA that could be targeted for therapeutic interventions.

A unique uracil-DNA binding protein of the uracil DNA glycosylase superfamily

Uracil DNA glycosylases (UDGs) are an important group of DNA repair enzymes, which pioneer the base excision repair pathway by recognizing and excising uracil from DNA. Based on two short conserved sequences (motifs A and B), UDGs have been classified into six families. Here we report a novel UDG, UdgX, from Mycobacterium smegmatis and other organisms. UdgX specifically recognizes uracil in DNA, forms a tight complex stable to sodium dodecyl sulphate, 2-mercaptoethanol, urea and heat treatment, and shows no detectable uracil excision. UdgX shares highest homology to family 4 UDGs possessing Fe-S cluster. UdgX possesses a conserved sequence, KRRIH, which forms a flexible loop playing an important role in its activity. Mutations of H in the KRRIH sequence to S, G, A or Q lead to gain of uracil excision activity in MsmUdgX, establishing it as a novel member of the UDG superfamily. Our observations suggest that UdgX marks the uracil-DNA for its repair by a RecA dependent process. Finally, we observed that the tight binding activity of UdgX is useful in detecting uracils in the genomes.

Functional roles of N-terminal and C-terminal domains in the overall activity of a novel single-stranded DNA binding protein of Deinococcus radiodurans

Single-stranded DNA binding protein (Ssb) of Deinococcus radiodurans comprises N- and C-terminal oligonucleotide/oligosaccharide binding (OB) folds connected by a beta hairpin connector. To assign functional roles to the individual OB folds, we generated three Ssb variants: Ssb(N) (N-terminal without connector), Ssb(NC) (N-terminal with connector) and Ssb(C) (C-terminal), each harboring one OB fold. Both Ssb(N) and Ssb(NC) displayed weak single-stranded DNA (ssDNA) binding activity, compared to the full-length Ssb (Ssb(FL)). The level of ssDNA binding activity displayed by SsbC was intermediate between Ssb(FL) and Ssb(N). Ssb(C) and Ssb(FL) predominantly existed as homo-dimers while Ssb(NC)/Ssb(N) formed different oligomeric forms. In vitro, Ssb(NC) or Ssb(N) formed a binary complex with Ssb(C) that displayed enhanced ssDNA binding activity. Unlike Ssb(FL), Ssb variants were able to differentially modulate topoisomerase-I activity, but failed to stimulate Deinococcal RecA-promoted DNA strand exchange. The results suggest that the C-terminal OB fold is primarily responsible for ssDNA binding. The N-terminal OB fold binds weakly to ssDNA but is involved in multimerization. (C) 2015 The Authors. Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. This is an open access article under the CC BY-NC-ND license.

Nucleotide sequence and expression inE. coli of the complete P4 type VP4 from a G2 serotype human rotavirus

The complete sequence of a P4 type VP4 gene from a G2 serotype human rotavirus, IS2, isolated in India has been determined. Although the IS2 VP4 is highly homologous to the other P4 type alleles, it contained acidic amino acid substitutions at several positions that make it acidic among the P4 type alleles that are basic. Moreover, comparative sequence analysis revealed unusual polymorphism in members of the P4 type at amino acid position 393 which is highly conserved in members of other VP4 types. To date, expression of complete VP4 inE. coli has not been achieved. In this study we present successful expression inE. coli of the complete VP4 as well as VP8* and VP5* cleavage subunits in soluble form as fusion proteins of the maltose-binding protein (MBP) and their purification by single-step affinity chromatography. The hemagglutinating activity exhibited by the recombinant protein was specifically inhibited by the antiserum raised against it. Availability of pure VP4 proteins should facilitate development of polyclonal and monoclonal antibodies (MAbs) for P serotyping of rotaviruses.