Julien Guergnon

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)‐κB 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‐κB activation, without inducing apoptosis.

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 Vpr77–92: 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 Vpr71-82 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 PP2A1 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 PP2A1 binding sequence from Vpr77–92 is a new cell penetrating apoptotic sequence. We also found that the I84P mutation or the IIQ/VTR83–85 and T89A substitutions in the Vpr77–92 sequence prevent PP2A1 binding, cell penetration and apoptosis. In addition the double R77A and R80A mutation known to inactivate the mitochondriotoxic Vpr71–82 domain, has no effect on the biological properties of the Vpr77–92 domain. Conclusion Together our data provide evidence for the first time that the Vpr77–92 sequence delineates a biological active domain of Vpr with PP2A1 binding and pro-apopototic 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 Vpr77–92 domain in full length Vpr protein and also in entire HIV provirus.

Use of micro-rotation imaging to study JNK-mediated cell survival in Theileria parva-infected B-lymphocytes.

Lymphocytes infected with the protozoan parasite Theileria parva are transformed to permanently proliferating cells, an event underlying the pathology of the disease. However, the molecular signalling mediating this process is complex and poorly understood. Here, we show that down-regulation of JNK signalling by transient over expression of a dominant-negative mutant of JNK (JNK-APF) significantly increases Annexin-V-phycoerythrin (V-PE) labelling on infected B cell populations observed using flow cytometry. To establish whether this increase was specifically due to apoptosis, we used a novel single-cell imaging method: micro-rotation (MR)-imaging, designed to allow high-resolution 3-dimensional imaging of single cells in suspension. With this method we visualized subcellular patterns of V-PE uptake and chromatin organization in lymphocytes co-transfected with JNK-APF and GFP-tagged histone-H2B. This single-cell approach allowed us to clearly reveal characteristic apoptotic phenotypes, whose patterns reflected progressive states of programmed cell death due to JNK down-regulation. Our results strongly suggest a role for JNK in the survival of Theileria-infected B cells, and demonstrate the powerful utility of a new and unique 3-dimensional imaging method for living cells in suspension.

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-AIF1, a peptide containing the AIF562–571 sequence located in a c-terminal domain of AIF, is a new PP1 interacting and cell penetrating molecule. We also showed that DPT-AIF1 provoked apoptosis in several human cell lines. Furthermore, DPT-APAF1 a bi-partite cell penetrating peptide containing APAF-1122–131, 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 AIF562–571 and APAF-1122–131 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-AIF2 and DPT-APAF2 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-AIF2 also suppressed cell penetration. Conclusion These results indicate that the combinatorial PP1c docking motif R/Kx(0,1)V/IxFxxR/KxR/K, deduced from AIF562–571 and APAF-1122–131 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.