Production of the R2 subunit of ribonucleotide reductase from herpes simplex virus with prokaryotic and eukaryotic expression systems: higher activity of R2 produced by eukaryotic cells related to higher iron-binding capacity

Lamarche, Nathalie; Matton, Gilles; Massie, Bernard; Fontecave, Marc; Atta, Mohamed; Dumas, F; Gaudreau, Pierrette; Langelier, Yves

doi:10.1042/bj3200129

Cited by 19 publications

(20 citation statements)

References 46 publications

(60 reference statements)

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“…Conditions for cytosolic and nuclear protein fraction extraction have been described previously [36]. Protein content was analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting [37]. mAbs against caspase-8 (1C12) and phospho-p42/44 ERK1/2 (Thr202/ Tyr204; E10), and polyclonal antibodies against p42/44 ERK1/2, Akt, phospho-Akt (Thr308), JNK and phospho-JNK (Thr183/Tyr185), were from Cell Signaling Technology.…”

Section: Protein Extraction and Immunoblot Analysismentioning

confidence: 99%

The ribonucleotide reductase R1 subunits of herpes simplex virus types 1 and 2 protect cells against TNFα- and FasL-induced apoptosis by interacting with caspase-8

et al. 2010

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We previously reported that HSV-2 R1, the R1 subunit (ICP10; UL39) of herpes simplex virus type-2 ribonucleotide reductase, protects cells against apoptosis induced by the death receptor (DR) ligands tumor necrosis factor-alpha-(TNFa) and Fas ligand (FasL) by interrupting DR-mediated signaling at, or upstream of, caspase-8 activation. Further investigation of the molecular mechanism underlying HSV-2 R1 protection showed that extracellularregulated kinase 1/2 (ERK1/2), phosphatidylinositol 3-kinase (PI3-K)/Akt, NF-jB and JNK survival pathways do not play a major role in this antiapoptotic function. Interaction studies revealed that HSV-2 R1 interacted constitutively with caspase-8. The HSV-2 R1 deletion mutant R1(1-834)-GFP and Epstein-Barr virus (EBV) R1, which did not protect against apoptosis induced by DR ligands, did not interact with caspase-8, indicating that interaction is required for protection. HSV-2 R1 impaired caspase-8 activation induced by caspase-8 over-expression, suggesting that interaction between the two proteins prevents caspase-8 dimerization/activation. HSV-2 R1 bound to caspase-8 directly through its prodomain but did not interact with either its caspase domain or Fas-associated death domain protein (FADD). Interaction between HSV-2 R1 and caspase-8 disrupted FADD-caspase-8 binding. We further demonstrated that individually expressed HSV-1 R1 (ICP6) shares, with HSV-2 R1, the ability to bind caspase-8 and to protect cells against DR-induced apoptosis. Finally, as the long-lived Fas protein remained stable during the early period of infection, experiments with the HSV-1 UL39 deletion mutant ICP6D showed that HSV-1 R1 could be essential for the protection of HSV-1-infected cells against FasL.Keywords FasL Á ICP6 Á ICP10 Á Viral inhibitor of apoptosis Á Caspase-8Electronic supplementary material The online version of this article

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Section: Protein Extraction and Immunoblot Analysismentioning

confidence: 99%

The ribonucleotide reductase R1 subunits of herpes simplex virus types 1 and 2 protect cells against TNFα- and FasL-induced apoptosis by interacting with caspase-8

et al. 2010

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“…From the molar extinction coefficient for the Fe2+-bathophenanthroline complex (e535 = 22,000 M -l.cm -1 [18]) a final concentration of the released iron of 88% and 31% from mouse and HSV-2 R2, respectively, was determined (100% refer to four iron molecules per R2). The low iron content in HSV-2 R2 (31%) estimated from the Fe2+-bathophenanthroline complex may be explained by the 3-fold lower affinity of the virus protein for iron, produced in a procaryotic expression system with the pET-R2 vector [19]. After removing the inhibitor by gel filtration, mouse R2 could be reconstituted using the anaerobic iron/oxygen procedure (see Materials and Methods).…”

Section: Reaction Of the Iron Center In Protein R2 With P-propoxyphenolmentioning

confidence: 99%

Reduction of the tyrosyl radical and the iron center in protein R2 of ribonucleotide reductase from mouse, herpes simplex virus and E. coli by p‐alkoxyphenols

et al. 1995

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The rate of reduction of the tyrosyl radical in the small subunit of ribonucleotide reductase (protein R2) from E. coli, mouse, and herpes simplex virus (HSV-2) by a series of p-alkoxyphenols with different alkyl chains, have been studied by stopped-flow UV-vis and stopped-flow EPR spectroscopy. The reduction and release of iron in R2 by the inhibitors was followed using bathophenanthroline as chelator of Fe 2+. p-Alkoxyphenols reduce the mouse R2 tyrosyl radical 1-2 orders of magnitude faster than the HSV-2 and E. coli radical. In contrast to E. coli, the iron center in R2 from mouse and HSV-2 is reduced by the inhibitors. For mouse R2, the rate of reduction of the tyrosyl radical increases in parallel with increasing alkyl chain length of the inhibitor, an observation which may be important for the design of new antiproliferative drugs.

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“…Cytoplasmic extracts were obtained by lysis in IP buffer. The protein content was analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting (43). Primary antibodies for immunoblotting included anti-FADD (A66-2; BD Biosciences), anti-␤-actin (AC15; Abcam), anti-RIP1 (G322-2; BD Biosciences), anti-FLAG (M2; Sigma), anti-myc (9E10; Upstate), anti-HSV-1 infected cell polypeptide 0 (ICP0) (5H7; Abcam), anti-c-FLIP (NF6; Alexis Biochemicals), and anti-TRIF (AL227; Alexis Biochemicals).…”

Section: Methodsmentioning

confidence: 99%

The Ribonucleotide Reductase R1 Subunits of Herpes Simplex Virus 1 and 2 Protect Cells against Poly(I · C)-Induced Apoptosis

Dufour

Bertrand

Pearson

et al. 2011

J Virol

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Production of the R2 subunit of ribonucleotide reductase from herpes simplex virus with prokaryotic and eukaryotic expression systems: higher activity of R2 produced by eukaryotic cells related to higher iron-binding capacity

Cited by 19 publications

References 46 publications

The ribonucleotide reductase R1 subunits of herpes simplex virus types 1 and 2 protect cells against TNFα- and FasL-induced apoptosis by interacting with caspase-8

The ribonucleotide reductase R1 subunits of herpes simplex virus types 1 and 2 protect cells against TNFα- and FasL-induced apoptosis by interacting with caspase-8

Reduction of the tyrosyl radical and the iron center in protein R2 of ribonucleotide reductase from mouse, herpes simplex virus and E. coli by p‐alkoxyphenols

The Ribonucleotide Reductase R1 Subunits of Herpes Simplex Virus 1 and 2 Protect Cells against Poly(I · C)-Induced Apoptosis

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