Biomedical subjects

K G Murthy

Publications and source records attributed to K G Murthy.

13 recordsLinked to original sources

PubMed2001

Diabetic endothelial dysfunction: the role of poly(ADP-ribose) polymerase activation.

Diabetic patients frequently suffer from retinopathy, nephropathy, neuropathy and accelerated atherosclerosis. The loss of endothelial function precedes these vascular alterations. Here we report that activation of poly(ADP-ribose) polymerase (PARP) is an important factor in the pathogenesis of endothelial dysfunction in diabetes. Destruction of islet cells with streptozotocin in mice induced hyperglycemia, intravascular oxidant production, DNA strand breakage, PARP activation and a selective loss of endothelium-dependent vasodilation. Treatment with a novel potent PARP inhibitor, starting after the time of islet destruction, maintained normal vascular responsiveness, despite the persistence of severe hyperglycemia. Endothelial cells incubated in high glucose exhibited production of reactive nitrogen and oxygen species, consequent single-strand DNA breakage, PARP activation and associated metabolic and functional impairment. Basal and high-glucose-induced nuclear factor-kappaB activation were suppressed in the PARP-deficient cells. Our results indicate that PARP may be a novel drug target for the therapy of diabetic endothelial dysfunction.

Animals↗

PubMed2001

Anti-inflammatory effects of inosine in human monocytes, neutrophils and epithelial cells in vitro.

Inosine is an endogenous purine, which has been recently shown to exert immunomodulatory, anti-inflammatory and anti-shock effects in rodent experimental systems. Some of these actions may be related to partial adenosine receptor agonistic effects. It has not been investigated previously whether inosine exerts similar immunomodulatory or anti-inflammatory effects in human cells or enzymes. Here we investigated the effects of inosine on the activation of human monocytes, neutrophils and epithelial cells in vitro. Furthermore, using a human inosine-5'-monophosphate dehydrogenase (IMPDH) enzyme, we examined the potential effects of inosine on the activity of IMPDH, an enzyme involved in the regulation of certain inflammatory/immune processes. Tumor necrosis factor alpha (TNF-alpha) production of bacterial lipopolysaccharide (LPS) stimulated whole blood was used as an indicator of human monocyte activation. The response was dose-dependently, partially suppressed in the presence of inosine. Inosine exerted a dose-dependent and, at the highest dose (3 mM), complete inhibition of the ability of human neutrophils activated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) to induce cytochrome C reduction in vitro. In the human colon cancer cell line HT-29, inosine dose-dependently attenuated the production of IL-8. Inosine failed to affect the activity of IMPDH. Taken together, we conclude that inosine exerts anti-inflammatory effects in many human cell types. Further studies need to establish whether inosine supplementation exerts anti-inflammatory effects in human beings.

Anti-Inflammatory Agents↗

PubMed2000

Phosphorylation of CPEB by Eg2 mediates the recruitment of CPSF into an active cytoplasmic polyadenylation complex.

The release of Xenopus oocytes from prophase I arrest is largely driven by the cytoplasmic polyadenylation-induced translation of dormant maternal mRNAs. Two cis elements, the CPE and the hexanucleotide AAUAAA, and their respective binding factors, CPEB and a cytoplasmic form of CPSF, control polyadenylation. The most proximal stimulus for polyadenylation is Eg2-catalyzed phosphorylation of CPEB serine 174. Here, we show that this phosphorylation event stimulates an interaction between CPEB and CPSF. This interaction is direct, does not require RNA tethering, and occurs through the 160 kDa subunit of CPSF. Eg2-stimulated and CPE-dependent polyadenylation is reconstituted in vitro using purified components. These results demonstrate that the molecular function of Eg2-phosphorylated CPEB is to recruit CPSF into an active cytoplasmic polyadenylation complex.

Animals↗

PubMed1998

Inhibition of poly(A) polymerase requires p34cdc2/cyclin B phosphorylation of multiple consensus and non-consensus sites.

We showed previously that p34(cdc2)/cyclin B (MPF) hyperphosphorylates poly(A) polymerase (PAP) during M-phase of the cell cycle, causing repression of its enzymatic activity. Mutation of three cyclin-dependent kinase (cdk) consensus sites in the PAP C-terminal regulatory domain prevented complete phosphorylation and MPF-mediated repression. Here we show that PAP also contains four nearby non-consensus cdk sites that are phosphorylated by MPF. Remarkably, full phosphorylation of all these cdk sites was required for repression of PAP activity, and partial phosphorylation had no detectable effect. The consensus sites were phosphorylated in vitro at a 10-fold lower concentration of MPF than the non-consensus sites. Consistent with this, during meiotic maturation of Xenopus oocytes, consensus sites were phosphorylated prior to the non-consensus sites at metaphase of meiosis I, and remained so throughout maturation, while the non-consensus sites did not become fully phosphorylated until after 12 h of metaphase II arrest. We propose that PAP's multiple cdk sites, and their differential sensitivity to MPF, provide a mechanism to link repression specifically to late M-phase. We discuss the possibility that this reflects a general means to control the timing of cdk-dependent regulatory events during the cell cycle.

Amino Acid Sequence↗

PubMed1998

High mobility group protein-1 (HMG-1) is a unique activator of p53.

The binding of p53 protein to DNA is stimulated by its interaction with covalent as well as noncovalent modifiers. We report the identification of a factor from HeLa nuclear extracts that activates p53 DNA binding. This factor was purified to homogeneity and identified as the high mobility group protein, HMG-1. HMG-1 belongs to a family of highly conserved chromatin-associated nucleoproteins that bend DNA and facilitate the binding of various transcription factors to their cognate DNA sequences. We demonstrate that recombinant His-tagged HMG-1 enhances p53 DNA binding in vitro and also that HMG-1 and p53 can interact directly in vitro. Unexpectedly, HMG-1 also stimulates DNA binding by p53Delta30, a carboxy-terminally deleted form of the protein that is considered to be constitutively active, suggesting that HMG-1 stimulates p53 by a mechanism that is distinct from other known activators of p53. Finally, using transient transfection assays we show that HMG-1 can increase p53 and p53Delta30-mediated transactivation in vivo. HMG-1 promotes the assembly of higher order p53 nucleoprotein structures, and these data, along with the fact that HMG-1 is capable of bending DNA, suggest that HMG-1 may activate p53 DNA binding by a novel mechanism involving a structural change in the target DNA.

DNA↗

PubMed1997

Transcription factor TFIID recruits factor CPSF for formation of 3' end of mRNA.

Initiation of transcription by RNA polymerase II from a promoter region on DNA requires the assembly of several initiation factors to form a preinitiation complex. Assembly of this complex is initiated by the binding of the transcription factor TFIID, composed of the TATA-box binding protein (TBP) and TBP-associated factors (TAF[II]s), to the promoter. We have now characterized an immunopurified TFIID complex which we unexpectedly find contains the cleavage-polyadenylation specificity factor (CPSF), one of the factors required for formation of the 3' end of messenger RNA. CPSF is brought to the preinitiation complex by TFIID, but after transcription starts, CPSF dissociates from TFIID and becomes associated with the elongating polymerase. We also show that overexpression of recombinant TBP in HeLa cells decreases polyadenylation without affecting the correct initiation of transcription of the reporter gene. This indicates that, owing to incomplete assembly of TFIID on recombinant TBP, CPSF is not brought to the promoter and therefore polyadenylation becomes less efficient. Our observations have thus revealed a link between transcription initiation and elongation by RNA polymerase II and processing of the 3' end of mRNA.

Amino Acid Sequence↗

PubMed1997

Identification of redox/repair protein Ref-1 as a potent activator of p53.

p53 can be isolated from cells in a form that is inert for binding to DNA but that can be stimulated dramatically by phosphorylation, antibody binding, or short single strands of DNA. This suggests that upon genotoxic stress, cells can convert latent p53 to one that is active for DNA binding. Surprisingly, we observed that latent p53 is as effective in activating transcription in vitro as is active p53. We found that HeLa nuclear extracts can stimulate DNA binding by latent p53 and have purified from them a p53-stimulating protein that we have determined to be the product of the Ref-1 gene. Interestingly, Ref-1 is a dual function protein that can both regulate the redox state of a number of proteins and function as a DNA repair (A/P) endonuclease. We observed that oxidized forms of full-length and carboxy-terminally truncated p53 (p53 delta30), which are inactive for DNA binding, are both stimulated by the Ref-1 protein. However, in the presence of reducing agent, Ref-1 is an extremely potent stimulator of full-length p53 but not p53 delta30. These and additional data indicate that Ref-1 protein stimulates p53 by both redox-dependent and -independent means and imply a key role for it in p53 regulation. Importantly, we have also determined that Ref-1 can stimulate p53 transactivation in vivo. This is the first example of a noncovalent protein modifier of p53 function identified in cells.

Carbon-Oxygen Lyases↗

PubMed1996

Cell-cycle related regulation of poly(A) polymerase by phosphorylation.

The poly(A) tail found on almost all eukaryotic messenger RNAs is important in enhancing translation initiation and determining mRNA stability. Control of poly(A)-tail synthesis thus has the potential to be a key regulatory step in gene expression and is indeed known to be important during early development in many organisms. To study a possible basis for such regulation, we examined phosphorylation of poly(A) polymerase (PAP) by p34(cdc2)/cyclin B (maturation/mitosis-promoting factor, MPF). We show here that PAP can be phosphorylated in vivo and in vitro by MPF. Consistent with this, PAP becomes hyperphosphorylated both during meiotic maturation of Xenopus laevis oocytes and in HeLa cells arrested at M phase, times in the cell-cycle when MPF is known to be active. We show further that hyperphosphorylation by MPF dramatically reduces the activity of purified PAP, and that PAP isolated from mitotic HeLa cells is similarly inhibited by hyperphosphorylation. This repression probably contributes to the well established reductions in poly(A)+ RNA and/or protein synthesis known to occur in M-phase cells.

Animals↗

PubMed1996

Interaction between the U1 snRNP-A protein and the 160-kD subunit of cleavage-polyadenylation specificity factor increases polyadenylation efficiency in vitro.

We have previously shown that the U1 snRNP-A protein (U1A) interacts with elements in SV40 late polyadenylation signal and that this association increases polyadenylation efficiency. It was postulated that this interaction occurs to facilitate protein-protein association between components of the U1 snRNP and proteins of the polyadenylation complex. We have now used GST fusion protein experiments, coimmunoprecipitations and Far Western blot analyses to demonstrate direct binding between U1A and the 160-kD subunit of cleavage-polyadenylation specificity factor (CPSF). In addition, Western blot analyses of fractions from various stages of CPSF purification indicated that U1A copurified with CPSF to a point but could be separated in the highly purified fractions. These data suggest that U1A protein is not an integral component of CPSF but may be able to interact and affect its activity. In this regard, the addition of purified, recombinant U1A to polyadenylation reactions containing CPSF, poly(A) polymerase, and a precleaved RNA substrate resulted in concentration-dependent increases in both the level of polyadenylation and poly(A) tail length. In agreement with the increase in polyadenylation efficiency caused by U1A, recombinant U1A stabilized the interaction of CPSF with the AAUAAA-containing substrate RNA in electrophoretic mobility shift experiments. These findings suggest that, in addition to its function in splicing, U1A plays a more global role in RNA processing through effects on polyadenylation.

Glutathione Transferase↗

PubMed1995

The 160-kD subunit of human cleavage-polyadenylation specificity factor coordinates pre-mRNA 3'-end formation.

Cleavage-polyadenylation specificity factor (CPSF) is a multisubunit protein that plays a central role in 3' processing of mammalian pre-mRNAs. CPSF recognizes the AAUAAA signal in the pre-mRNA and interacts with other proteins to facilitate both RNA cleavage and poly(A) synthesis. Here we describe the isolation of cDNAs encoding the largest subunit of CPSF (160K) as well as characterization of the protein product. Antibodies raised against the recombinant protein inhibit polyadenylation in vitro, which can be restored by purified CPSF. Extending previous studies, which suggested that 160K contacts the pre-mRNA, we show that purified recombinant 160K can, by itself, bind preferentially to AAUAAA-containing RNAs. While the sequence of 160K reveals similarities to the RNP1 and RNP2 motifs found in many RNA-binding proteins, no clear match to a known RNA-binding domain was found, and RNA recognition is therefore likely mediated by a highly diverged or novel structure. We also show that 160K binds specifically to both the 77K (suppressor of forked) subunit of the cleavage factor CstF and to poly(A) polymerase (PAP). These results provide explanations for previously observed cooperative interactions between CPSF and CstF, which are responsible for poly(A) site specification, and between CPSF and PAP, which are necessary for synthesis of the poly(A) tail. Also supporting a direct role for 160K in these interactions is the fact that 160K by itself retains partial ability to cooperate with CstF in binding pre-mRNA and, unexpectedly, inhibits PAP activity in in vitro assays. We discuss the significance of these multiple functions and also a possible evolutionary link between yeast and mammalian polyadenylation suggested by the properties and sequence of 160K.

Amino Acid Sequence↗

PubMed1994

Poly(A) polymerase contains multiple functional domains.

Poly(A) polymerase (PAP) contains regions of similarity with several known protein domains. Through site-directed mutagenesis, we provide evidence that PAP contains a functional ribonucleoprotein-type RNA binding domain (RBD) that is responsible for primer binding, making it the only known polymerase to contain such a domain. The RBD is adjacent to, and probably overlaps with, an apparent catalytic region responsible for polymerization. Despite the presence of sequence similarities, this catalytic domain appears to be distinct from the conserved polymerase module found in a large number of RNA-dependent polymerases. PAP contains two nuclear localization signals (NLSs) in its C terminus, each by itself similar to the consensus bipartite NLS found in many nuclear proteins. Mutagenesis experiments indicate that both signals, which are separated by nearly 140 residues, play important roles in directing PAP exclusively to the nucleus. Surprisingly, basic amino acids in the N-terminal-most NLS are also essential for AAUAAA-dependent polyadenylation but not for nonspecific poly(A) synthesis, suggesting that this region of PAP is involved in interactions both with nuclear targeting proteins and with nuclear polyadenylation factors. The serine/threonine-rich C terminus is multiply phosphorylated, including at sites affected by mutations in either NLS.

Amino Acid Sequence↗

PubMed1992

Characterization of the multisubunit cleavage-polyadenylation specificity factor from calf thymus.

Cleavage-polyadenylation specificity factor (CPSF) is one of five separable factors known to be required for 3' cleavage and polyadenylation of mRNA precursors in vitro. Previous studies have shown that the cleavage and poly(A) addition reactions can be uncoupled in vitro and have suggested that CPSF may be the only factor essential for both of these subreactions. Here we report the purification of CPSF to near homogeneity from calf thymus and show that the purified factor contains three polypeptides of 165, 105, and 70 kDa. These polypeptides cosediment precisely with CPSF activity, which has a sedimentation coefficient of 11.5 S. Consistent with previous reports from our laboratory, purified CPSF does not contain a detectable RNA component, indicating that it is a multisubunit protein and not a small nuclear ribonucleoprotein. Extensively purified bovine CPSF can function with human poly(A) polymerase to bring about AAUAAA-dependent poly(A) addition or with human cleavage factors to catalyze accurate 3' cleavage of a pre-mRNA substrate. UV cross-linking and gel retention analyses demonstrate that highly purified CPSF interacts with one of these cleavage factors, the multisubunit cleavage-stimulation factor, to facilitate stable binding of both to an AAUAAA-containing pre-mRNA. Likewise, evidence is presented indicating that poly(A) polymerase and CPSF can interact directly.

Animals↗

PubMed1991

A nuclear micrococcal-sensitive, ATP-dependent exoribonuclease degrades uncapped but not capped RNA substrates.

We have developed an assay for an exoribonuclease present in HeLa cell nuclear extracts that degrades capped but not uncapped RNA substrates, and used it to partially purify and characterize such an activity. Capped and uncapped transcripts of different sizes (37-317 nt) were incubated with fractionated nuclear extracts, and in all cases the capped RNAs were stable while their uncapped counterparts were completely degraded. No changes in activity were detected when cap analogs were included in reaction mixtures, suggesting that the stability of capped RNAs was not due to a cap binding protein. The exoribonuclease was shown to be specific for RNA, and to function processively with either substrates containing 5'-hydroxyl or 5'-phosphorylated ends. The products were predominantly 5'-mononucleotides, and no detectable intermediates were observed at any reaction time points. Sedimentation analysis suggests that the native size of the nuclease is 7.4S or approximately 150 kDa. Interestingly, a nucleoside triphosphate was found to be necessary for specific and complete degradation of the uncapped RNAs. Finally, micrococcal nuclease (MN) pretreatment of the partially purified enzyme inhibited its activity. As several controls indicated that this was not due to non-specific effects of MN, this finding suggests that the exoribonuclease contains an essential RNA component.

Adenosine Triphosphate↗