Combined linkage and association studies show that HLA class II variants control levels of antibodies against Epstein-Barr virus antigens.

Over 95% of the adult population worldwide is infected with Epstein-Barr virus (EBV). EBV infection is associated with the development of several cancers, including Hodgkin lymphoma (HL). Elevated levels of anti-EBV antibodies have been associated with increased risk of HL. There is growing evidence that genetic factors control the levels of antibodies against EBV antigens. Here, we conducted linkage and association studies to search for genetic factors influencing either anti-viral capsid antigen (VCA) or anti-Epstein Barr nuclear antigen-1 (EBNA-1) IgG levels in a unique cohort of 424 individuals of European origin from 119 French families recruited through a Hodgkin lymphoma (HL) patient. No major locus controlling anti-VCA antibody levels was identified. However, we found that the HLA region influenced anti-EBNA-1 IgG titers. Refined association studies in this region identified a cluster of HLA class II variants associated with anti-EBNA-1 IgG titers (e.g. p = 5×10(-5) for rs9268403). The major allele of rs9268403 conferring a predisposition to high anti-EBNA-1 antibody levels was also associated with an increased risk of HL (p = 0.02). In summary, this study shows that HLA class II variants influenced anti-EBNA-1 IgG titers in a European population. It further shows the role of the same variants in the risk of HL.

Genome-wide association study identifies variants associated with progression of liver fibrosis from HCV infection.

BACKGROUND & AIMS: Polymorphisms in IL28B were shown to affect clearance of hepatitis C virus (HCV) infection in genome-wide association (GWA) studies. Only a fraction of patients with chronic HCV infection develop liver fibrosis, a process that might also be affected by genetic factors. We performed a 2-stage GWA study of liver fibrosis progression related to HCV infection. METHODS: We studied well-characterized HCV-infected patients of European descent who underwent liver biopsies before treatment. We defined various liver fibrosis phenotypes on the basis of METAVIR scores, with and without taking the duration of HCV infection into account. Our GWA analyses were conducted on a filtered primary cohort of 1161 patients using 780,650 single nucleotide polymorphisms (SNPs). We genotyped 96 SNPs with P values <5 × 10(-5) from an independent replication cohort of 962 patients. We then assessed the most interesting replicated SNPs using DNA samples collected from 219 patients who participated in separate GWA studies of HCV clearance. RESULTS: In the combined cohort of 2342 HCV-infected patients, the SNPs rs16851720 (in the total sample) and rs4374383 (in patients who received blood transfusions) were associated with fibrosis progression (P(combined) = 8.9 × 10(-9) and 1.1 × 10(-9), respectively). The SNP rs16851720 is located within RNF7, which encodes an antioxidant that protects against apoptosis. The SNP rs4374383, together with another replicated SNP, rs9380516 (P(combined) = 5.4 × 10(-7)), were linked to the functionally related genes MERTK and TULP1, which encode factors involved in phagocytosis of apoptotic cells by macrophages. CONCLUSIONS: Our GWA study identified several susceptibility loci for HCV-induced liver fibrosis; these were linked to genes that regulate apoptosis. Apoptotic control might therefore be involved in liver fibrosis.

IL28B alleles associated with poor hepatitis C virus (HCV) clearance protect against inflammation and fibrosis in patients infected with non-1 HCV genotypes.

UNLABELLED: Genetic polymorphisms near IL28B are associated with spontaneous and treatment-induced clearance of hepatitis C virus (HCV), two processes that require the appropriate activation of the host immune responses. Intrahepatic inflammation is believed to mirror such activation, but its relationship with IL28B polymorphisms has yet to be fully appreciated. We analyzed the association of IL28B polymorphisms with histological and follow-up features in 2335 chronically HCV-infected Caucasian patients. Assessable phenotypes before any antiviral treatment included necroinflammatory activity (n = 1,098), fibrosis (n = 1,527), fibrosis progression rate (n = 1,312), and hepatocellular carcinoma development (n = 1,915). Associations of alleles with the phenotypes were evaluated by univariate analysis and multivariate logistic regression, accounting for all relevant covariates. The rare G allele at IL28B marker rs8099917-previously shown to be at risk of treatment failure-was associated with lower activity (P = 0.04), lower fibrosis (P = 0.02) with a trend toward lower fibrosis progression rate (P = 0.06). When stratified according to HCV genotype, most significant associations were observed in patients infected with non-1 genotypes (P = 0.003 for activity, P = 0.001 for fibrosis, and P = 0.02 for fibrosis progression rate), where the odds ratio of having necroinflammation or rapid fibrosis progression for patients with IL28B genotypes TG or GG versus TT were 0.48 (95% confidence intervals 0.30-0.78) and 0.56 (0.35-0.92), respectively. IL28B polymorphisms were not predictive of the development of hepatocellular carcinoma. CONCLUSION: In chronic hepatitis C, IL28B variants associated with poor response to interferon therapy may predict slower fibrosis progression, especially in patients infected with non-1 HCV genotypes.

[Targeting of PP2A enzymes by viral proteins and cancer signalling]. / La famille des protéine phosphatases PP2A: une cible stratégique pour les virus et pour la transformation tumorale.

Protein phosphatase 2A (PP2A) is a large family of holoenzymes that comprises 1% of total cellular proteins and accounts for the majority of Ser/Thr phosphatase activity in eukaryotic cells. PP2A proteins are made of a core dimer, composed of a catalytic (C) subunit and a structural (A) subunit, in association with a third variable -regulatory (B) subunit. Although initially considered as a constitutive housekeeping enzyme, PP2A is indeed highly regulated by post-translational modifications of its catalytic subunit or by the identity of a regulatory type B subunit, which determines substrate specificity, subcellular localization and enzymatic activity of a defined holoenzyme. During the two last decades, multiple studies of structural and functional regulation of PP2A holoenzymes by viral proteins led to the identification of critical pathways for both viral biology and tumorigenesis. To date a dozen of different viruses (ADN/ARN or retrovirus) have been identified that encode viral proteins associated to PP2A. In this review, we analyze a biological strategy, used by various viruses based on the targeting of PP2A enzymes by viral proteins, in order to specifically deregulate cellular pathways of their hosts. The impact of such PP2A targeting for biomedical search, and in further therapeutic developments against cancer, will also be discussed.

PP2A targeting by viral proteins: a widespread biological strategy from DNA/RNA tumor viruses to HIV-1.

Protein phosphatase 2A (PP2A) is a large family of holoenzymes that comprises 1% of total cellular proteins and accounts for the majority of Ser/Thr phosphatase activity in eukaryotic cells. Although initially viewed as constitutive housekeeping enzymes, it is now well established that PP2A proteins represent a family of highly and sophistically regulated phosphatases. The past decade, multiple complementary studies have improved our knowledge about structural and functional regulation of PP2A holoenzymes. In this regard, after summarizing major cellular regulation, this review will mainly focus on discussing a particulate biological strategy, used by various viruses, which is based on the targeting of PP2A enzymes by viral proteins in order to specifically deregulate, for their own benefit, cellular pathways of their hosts. The impact of such PP2A targeting for research in human diseases, and in further therapeutic developments, is also discussed.

PP2A1 binding, cell transducing and apoptotic properties of Vpr(77-92): a new functional domain of HIV-1 Vpr proteins.

BACKGROUND: The hallmark of HIV-1 pathogenesis is the progressive CD4(+) T cell depletion and high propensity of CD4(+) T cells to apoptosis. HIV-1 viral protein R (Vpr) is a major pro-apoptotic gene product. A first Vpr-mediated apoptotic mechanism that requires a physical interaction of HIV-1 Vpr(71-82) mitochondriotoxic domain containing the conserved sequence (71-)HFRIGCRHSRIG(-82) with the Adenine Nucleotide Translocator (ANT) has been characterized. The family of Ser/Thr protein phosphatase PP2A interacts with several viral proteins to regulate cell growth and apoptotic pathways. Previous studies based on yeast two hybrid assays and mutational experiments indicated that PP2A(1) is involved in the induction of G2 arrest by HIV-1 Vpr. PRINCIPAL FINDINGS: Experiments combining pull-down, cell penetration and apoptosis analyses in distinct human cells indicate that the PP2A(1) binding sequence from Vpr(77-92) is a new cell penetrating apoptotic sequence. We also found that the I84P mutation or the IIQ/VTR(83-85) and T89A substitutions in the Vpr(77-92) sequence prevent PP2A(1) binding, cell penetration and apoptosis. In addition the double R77A and R80A mutation known to inactivate the mitochondriotoxic Vpr(71-82) domain, has no effect on the biological properties of the Vpr(77-92) domain. CONCLUSION: Together our data provide evidence for the first time that the Vpr(77-92) sequence delineates a biological active domain of Vpr with PP2A(1) binding and pro-apoptotic capacities and, it is conceivable that this cell penetrating sequence may account for the Vpr internalization in uninfected cells. Finally, our data also implicate the existence of two partially overlapping pro-apoptotic domains in the Vpr C-terminal part, a redundancy that represents a new approach to address the question of biological relevance of HIV-1 Vpr. In this context, future studies will be required to determine the functional relevance of the Vpr(77-92) domain in full length Vpr protein and also in entire HIV provirus.

The combinatorial PP1-binding consensus Motif (R/K)x( (0,1))V/IxFxx(R/K)x(R/K) is a new apoptotic signature.

BACKGROUND: Previous studies established that PP1 is a target for Bcl-2 proteins and an important regulator of apoptosis. The two distinct functional PP1 consensus docking motifs, R/Kx((0,1))V/IxF and FxxR/KxR/K, involved in PP1 binding and cell death were previously characterized in the BH1 and BH3 domains of some Bcl-2 proteins. PRINCIPAL FINDINGS: In this study, we demonstrate that DPT-AIF(1), a peptide containing the AIF(562-571) sequence located in a c-terminal domain of AIF, is a new PP1 interacting and cell penetrating molecule. We also showed that DPT-AIF(1) provoked apoptosis in several human cell lines. Furthermore, DPT-APAF(1) a bi-partite cell penetrating peptide containing APAF-1(122-131), a non penetrating sequence from APAF-1 protein, linked to our previously described DPT-sh1 peptide shuttle, is also a PP1-interacting death molecule. Both AIF(562-571) and APAF-1(122-131) sequences contain a common R/Kx((0,1))V/IxFxxR/KxR/K motif, shared by several proteins involved in control of cell survival pathways. This motif combines the two distinct PP1c consensus docking motifs initially identified in some Bcl-2 proteins. Interestingly DPT-AIF(2) and DPT-APAF(2) that carry a F to A mutation within this combinatorial motif, no longer exhibited any PP1c binding or apoptotic effects. Moreover the F to A mutation in DPT-AIF(2) also suppressed cell penetration. CONCLUSION: These results indicate that the combinatorial PP1c docking motif R/Kx((0,1))V/IxFxxR/KxR/K, deduced from AIF(562-571) and APAF-1(122-131) sequences, is a new PP1c-dependent Apoptotic Signature. This motif is also a new tool for drug design that could be used to characterize potential anti-tumour molecules.

Mapping of protein phosphatase-6 association with its SAPS domain regulatory subunit using a model of helical repeats.

BACKGROUND: Helical repeat motifs are common among regulatory subunits for type-1 and type-2A protein Ser/Thr phosphatases. Yeast Sit4 is a distinctive type-2A phosphatase that has dedicated regulatory subunits named Sit4-Associated Proteins (SAPS). These subunits are conserved, and three human SAPS-related proteins are known to associate with PP6 phosphatase, the Sit4 human homologue. RESULTS: Here we show that endogenous SAPS subunit PP6R3 co-precipitates half of PP6 in cell extracts, and the SAPS region of PP6R3 is sufficient for binding PP6. The SAPS domain of recombinant GST-PP6R3 is relatively resistant to trypsin despite having many K and R residues, and the purified SAPS domain (residues 1-513) has a circular dichroic spectrum indicative of mostly alpha helical structure. We used sequence alignments and 3D-jury methods to develop alternative models for the SAPS domain, based on available structures of other helical repeat proteins. The models were used to select sites for charge-reversal substitutions in the SAPS domain of PP6R3 that were tested by co-precipitation of endogenous PP6c with FLAG-tagged PP6R3 from mammalian cells. Mutations that reduced binding with PP6 suggest that SAPS adopts a helical repeat similar to the structure of p115 golgin, but distinct from the PP2A-A subunit. These mutations did not cause perturbations in overall PP6R3 conformation, evidenced by no change in kinetics or preferential cleavage by chymotrypsin. CONCLUSION: The conserved SAPS domain in PP6R3 forms helical repeats similar to those in golgin p115 and negatively charged residues in interhelical loops are used to associate specifically with PP6. The results advance understanding of how distinctive helical repeat subunits uniquely distribute and differentially regulate closely related Ser/Thr phosphatases.

A PKA survival pathway inhibited by DPT-PKI, a new specific cell permeable PKA inhibitor, is induced by T. annulata in parasitized B-lymphocytes.

T. annulata, an intracellular pathogenic parasite of the Aplicomplexa protozoan family infects bovine B-lymphocytes and macrophages. Parasitized cells that become transformed survive and proliferate independently of exogenous growth factors. In the present study, we used the isogenic non parasitized BL3 and parasitized TBL3 B cell lines, as a model to evaluate the contribution of two-major PI3-K- and PKA-dependent anti-apoptotic pathways in the survival of T. annulata parasitized B lymphocytes. We found that T. annulata increases PKA activity, induces over-expression of the catalytic subunit and down-regulates the pro-survival phosphorylation state of Akt/PKB. Consistent with a role of PKA activation in survival, two pharmacological inhibitors H89 and KT5720 ablate PKA-dependent survival of parasitized cells. To specifically inhibit PKA pro-survival pathways we linked the DPTsh1 peptide shuttle sequence to PKI(5-24) and we generated DPT-PKI, a cell permeable PKI. DPT-PKI specifically inhibited PKA activity in bovine cell extracts and, as expected, also inhibited the PKA-dependent survival of T. annulata parasitized TBL3 cells. Thus, parasite-dependent constitutive activation of PKA in TBL3 cells generates an anti-apoptotic pathway that can protect T. annulata-infected B cells from apoptosis. These results also indicate that DPT-PKI could be a powerful tool to inhibit PKA pathways in other cell types.

Use of penetrating peptides interacting with PP1/PP2A proteins as a general approach for a drug phosphatase technology.

Protein phosphatase types 1 (PP1) and 2A (PP2A) represent two major families of serine/threonine protein phosphatases that have been implicated in the regulation of many cellular processes, including cell growth and apoptosis in mammalian cells. PP1 and PP2A proteins are composed of oligomeric complexes comprising a catalytic structure (PP1c or PP2AC) containing the enzymatic activity and at least one more interacting subunit. The binding of different subunits to a catalytic structure generates a broad variety of holoenzymes. We showed here that casein kinase 2alpha (Ck2alpha) and simian virus 40 small t antigen share a putative common beta-strand structure required for PP2A1 trimeric holoenzyme binding. We have also characterized DPT-sh1, a short basic peptide from Ck2alpha that interacted only in vitro with the PP2A-A subunit and behaves as a nontoxic penetrating shuttle in several cultivated human cell lines and chick embryos. In addition, DPT-sh1 specifically accumulated in human red cells infected with Plasmodium falciparum malaria parasites. We therefore designed bipartite peptides containing DPT-sh1 and PP1- or PP2A-interacting sequences. We found that DPT-5, a DPT-sh1-derived peptide containing a short sequence identified in CD28 antigen, interacts with PP2A-Balpha, and DPT-7, another DPT-sh1-derived peptide containing a short sequence identified in Bad as a PP1 catalytic consensus docking motif, induce apoptosis in cultivated cell lines. These results clearly indicate that the rational design of PP1/PP2A interacting peptides is a pertinent strategy to deregulate intracellular survival pathways.

Serine/threonine protein phosphatases PP1 and PP2A are key players in apoptosis.

The reversible phosphorylation of proteins controlled by protein kinases and protein phosphatases is a major mechanism that regulates a wide variety of cellular processes. In contrast to C. elegans, recent studies in mammalian cells have highlighted a major role of serine/threonine protein phosphorylation in apoptosis. To illustrate the importance of dephosphorylation processes in apoptosis, this review will focus on recent studies suggesting that the interaction of the serine/threonine protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) with certain regulators of the Bcl-2 family is critically involved in the control of apoptosis.

Rafts: a simple way to control apoptosis by subcellular redistribution.

Apoptosis is an essential feature of development and homeostasis in higher organisms. Lipid rafts are subdomains of the plasma membrane that contain high concentrations of cholesterol and sphingolipids. In response to intra or extracellular stimuli, lipid rafts can include or exclude proteins to variable extents. This favors specific protein-protein interactions and modulates the activity of signaling cascades. Recently, a number of proteins involved in apoptotic signals have been located in lipid rafts. Among these proteins is included Bad, a pro-apoptotic molecule belonging to the Bcl-2 family. Bad is attached to lipid rafts in proliferating cells while associated to mitochondria in apoptotic cells, suggesting that the interaction of Bad with rafts is a dynamic process involved in the control of apoptosis. In this review, we briefly summarize the structure of rafts and illustrate their contribution to the control of apoptosis.

Apoptosis of Theileria-infected lymphocytes induced upon parasite death involves activation of caspases 9 and 3.

The intracellular parasite Theileria parva (T. parva) can infect bovine B and T-lymphocytes. T. parva-infected cells become transformed, and they survive and proliferate independently of exogenous growth factors. In vivo the uncontrolled cellular proliferation associated with lymphocyte transformation underlies the pathogenesis of the disease called East Coast Fever. The transformed state of parasitised cells can be reversed upon elimination of the parasite by specific theilericide drugs. In this study we found that elimination of the parasite by buparvaquone induces apoptosis of transformed B and CD8(+) T-lymphocytes. Apoptosis is accompanied by the activation of caspase 9 and caspase 3 and processing of poly(ADP ribose) polymerase and is inhibited by Z-VAD a general caspase inhibitor. Based on these observations, we propose that the lack of activation of a caspase 9 > caspase 3 > poly(ADP ribose) polymerase pathway is important and protects T. parva-transformed cells from spontaneous apoptosis.

A tumour necrosis factor alpha autocrine loop contributes to proliferation and nuclear factor-kappaB activation of Theileria parva-transformed B cells.

Theileria infection of bovine leucocytes induces uncontrolled proliferation and a transformed phenotype comparable to tumour cells. Infected cells have many characteristics of activated leucocytes and use autocrine loops to augment proliferation. We have shown previously that, in infected B cells, PI3-K controls a granulocyte-macrophage colony-stimulating factor (GM-CSF) autocrine loop to increase both proliferation and activation of the activator protein 1 (AP-1) transcription factor. We show here that the same infected B cells also use a tumour necrosis factor (TNF) alpha autocrine loop that again contributes to proliferation and augments nuclear factor (NF)-kappaB activation. Interestingly, both pharmacological inhibition of TNF synthesis and neutralizing anti-TNF antibodies lead to a reduction in proliferation and a 50% drop in NF-kappaB activation, without inducing apoptosis.