Biomedical subjects

M Heiland

Publications and source records attributed to M Heiland.

23 records · Page 2Linked to original sources

PubMed1999

Identification and characterization of a histone binding site of the non-structural protein 3 of hepatitis C virus.

BACKGROUND: Chronic hepatitis resulting from the hepatitis C virus (HCV) infection leads to cirrhosis in at least half the infected patients and increases the risk of hepatocellular carcinoma. There are indications that this pathogenic effect may result from the disturbance of intracellular signal cascades caused by the interaction with viral antigens. Although a great amount of data has been accumulated about functional regions in HCV proteins, relatively little is known about their intracellular targets. Previously, we have demonstrated that the full-length non-structural protein 3 of HCV (NS3) (Borowski P, Heiland M, Feucht H, Laufs R. Characterisation of non-structural protein 3 of hepatitis C virus as modulator of protein phosphorylation mediated by PKA and PKC. Evidences for action on the level of substrate and enzyme. Arch Virol 1999a; 144) and its NH2- and COOH-terminal truncated form (Borowski P, Heiland M, Oehlmann K, Becker B, Kornetzky L, Feucht HH, Laufs R. Non-structural protein 3 of hepatitis C virus inhibits phosphorylation mediated by cAMP-dependent protein kinase. Eur J Biochem 1996;237:611-618) associate to stable complexes with core histones H2B and H4. The changes of the properties of histones as substrate for cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) were found as a direct consequence of the interaction. OBJECTIVE: In the present study we further these observations, localize the histone binding domain of NS3 and investigate the mechanisms by which NS3 affects the functions of the histones in vitro. STUDY DESIGN: HCV protein exhibiting the mentioned histone binding activity was produced in a bacterial expression system, purified and binding to histones was biochemically characterized. The region of NS3 involved in the interaction with histones was defined by proteolytic fragmentation, microsequencing and a specific histone binding assay. Furthermore, a functional test to quantify the interaction of histones with DNA was established and the binding of DNA to histone as a function of NS3 concentration was analysed by means of graphical methods. RESULTS: The investigated fragment of HCV polyprotein consisting of amino acid residues 1189-1525 (HCV-polyprotein-(1189-1525)) displayed significant histone binding activity. The binding occurred at a molar ratio 1:1 of histone to HCV-polyprotein-(1189-1525) and was mediated by a linear stretch of amino acids located between the residues 1343 and 1379 of the HCV polyprotein. To demonstrate that HCV-polyprotein-(1189-1525) affects the binding of DNA to histones we used two independent methods: overlay assay and binding assay on Sepharose beads. Graphic analysis of the binding kinetics revealed an uncompetitive type of inhibition. CONCLUSIONS: Our results provide the first evidence that NS3 binds and affects the functions of core histones. The mechanism by which the NS3 interferes with the histone functions involves conformational changes of histone molecule.

Binding Sites↗

PubMed1998

Purification of catalytic domain of rat spleen p72syk kinase and its phosphorylation and activation by protein kinase C.

The catalytic domain of p72(syk) kinase (CDp72(syk)) was purified from a 30000 g particulate fraction of rat spleen. The purification procedure employed sequential chromatography on columns of DEAE-Sephacel and Superdex-200, and elution from HA-Ultrogel by chloride. The analysis of the final CDp72(syk) preparation by SDS/PAGE revealed a major silver-stained 40 kDa protein. The kinase was identified by covalent modification of its ATP-binding site with [14C]5'-fluorosulphonylbenzoyladenosine and by immunoblotting with a polyclonal antibody against the 'linker' region of p72(syk). By using poly(Glu4, Tyr1) as a substrate, the specific activity of the enzyme was determined as 18.5 nmol Pi/min per mg. Casein, histones H1 and H2B and myelin basic protein were efficiently phosphorylated by CDp72(syk). The kinase exhibited a limited ability to phosphorylate random polymers containing tyrosine residues. CDp72(syk) autophosphorylation activity was associated with an activation of the kinase towards exogenous substrates. The extent of activation was dependent on the substrates added. CDp72(syk) was phosphorylated by protein kinase C (PKC) on serine and threonine residues. With a newly developed assay method, we demonstrated that the PKC-mediated phosphorylation had a strong activating effect on the tyrosine kinase activity of CDp72(syk). Studies extended to conventional PKC isoforms revealed an isoform-dependent manner (alpha > betaI = betaII > gamma) of CDp72(syk) phosphorylation. The different phosphorylation efficiencies of the PKC isoforms closely correlated with the ability to enhance the tyrosine kinase activity.

Animals↗

PubMed1997

Nonstructural protein 3 of hepatitis C virus blocks the distribution of the free catalytic subunit of cyclic AMP-dependent protein kinase.

Chronic hepatitis resulting from hepatitis C virus (HCV) infection develops into cirrhosis in at least half of infected patients and increases the risk of hepatocellular carcinoma. The pathogenic effects of a number of viruses result from the disturbance of intracellular signal cascades caused by viral antigens. Therefore, we investigated the interaction of nonstructural protein 3 (NS3) of HCV with the cyclic AMP-dependent signal pathway. We found a similarity between the HCV sequence Arg-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg localized in NS3 and the general consensus sequence of protein kinase A (PKA). Consequently, the catalytic (C) subunit of PKA bound to a bacterially expressed fragment of HCV polyprotein containing amino acid residues 1189 to 1525. When this fragment was introduced into cells, it inhibited the translocation of the C subunit into the nucleus after stimulation with forskolin. The result of this inhibition was significantly reduced histone phosphorylation. Therefore, the presence of NS3 in the cytoplasm of infected cells may affect a wide range of PKA functions and contribute to the pathogenesis of the diseases caused by HCV.

Biological Transport↗

PubMed1996

Non-structural protein 3 of hepatitis C virus inhibits phosphorylation mediated by cAMP-dependent protein kinase.

Inspection of the amino acid sequence of the non-structural region of the hepatitis C virus (HCV) gene product reveals a sequence of 14 amino acids, Arg1487-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg1500 , located in the non-structural protein, NS3. This sequence is highly similar to the inhibitory site of the heat-stable inhibitor of cAMP-dependent protein kinase (PKA) and to the autophosphorylation site in the hinge region of the PKA type II regulatory domain. A synthetic peptide that corresponds to the HCV sequence above and a set of shorter analogues act as competitive inhibitors of PKA. A 43.5-kDa fragment of NS3 that consists of residues 1189-1525 of the HCV polyprotein inhibits PKA in a similar range to the investigated synthetic peptides. In contrast to the short peptides, which show competitive inhibition, HCV-polyprotein-(1189-1525) influences PKA in a mixed-inhibition-type manner. A possible mechanism explaining these differences is the formation of complexes that consist of the protein substrate, the enzyme and the HCV-polyprotein-(1189-1525). Binding studies with PKA and the non-hydrolysable ATP analogue [14C]fluorosulfonylbenzoyladenosine and [3H]cAMP do not reveal any influence of the short HCV-derived peptides or HCV-polyprotein-(1189-1525) upon the affinity of PKA for these nucleotides. The complex interactions of the NS3 fragments could influence one of the most important signal pathways of the cell and, therefore, could possibly provide new pathological mechanisms for HCV infections of liver.

Amino Acid Sequence↗

PubMed1996

Characterization of the C-terminal domain of ras-GTPase-activating protein (ras-GAP) as substrate for epidermal growth factor receptor and p60c-src kinase.

We describe in vitro tyrosine phosphorylation of the C-terminal 334 amino acids of ras-GTPase-activating protein (ras-GAP)1 that contains the activity domain for ras interaction. To date, there have been no other phosphorylation sites determined than the reported in N-terminal domain of ras-GAP Tyr-460, which is considered to be the major phosphorylation site of ras-GAP. In our assays some differences of the kinetic parameters were observed when the reaction was catalyzed by EGF-R compared to p60c-src. Enzyme specific regulation of activity is associated with autophosphorylation which leads to reduced (in case of EGF-R) or increased (in case of p60c-src) phosphorylation of the C-terminal 334 amino acids of ras-GAP (GAP334). Because of the characteristics of these investigated reactions the phosphorylation of GAP334 seems to be-independent from the presence of SH2 or SH3 domains-triggered off by complex mechanisms different from those regulating the phosphorylation at Tyr-460.

Electrophoresis, Polyacrylamide Gel↗