Inhibition of Natural Killer Cells through Engagement of CD81 by the Major Hepatitis C Virus Envelope Protein

The immune response against hepatitis C virus (HCV) is rarely effective at clearing the virus, resulting in ∼170 million chronic HCV infections worldwide. Here we report that ligation of an HCV receptor (CD81) inhibits natural killer (NK) cells. Cross-linking of CD81 by the major envelope protein of HCV (HCV-E2) or anti-CD81 antibodies blocks NK cell activation, cytokine production, cytotoxic granule release, and proliferation. This inhibitory effect was observed using both activated and resting NK cells. Conversely, on NK-like T cell clones, including those expressing NK cell inhibitory receptors, CD81 ligation delivered a costimulatory signal. Engagement of CD81 on NK cells blocks tyrosine phosphorylation through a mechanism which is distinct from the negative signaling pathways associated with NK cell inhibitory receptors for major histocompatibility complex class I. These results implicate HCV-E2–mediated inhibition of NK cells as an efficient HCV evasion strategy targeting the early antiviral activities of NK cells and allowing the virus to establish itself as a chronic infection

Mono-ADP-ribosylation of the G protein βγ subunit : characterisation of the enzymes involved

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ARTD10/PARP10 Induces ADP-Ribosylation of GAPDH and Recruits GAPDH into Cytosolic Membrane-Free Cell Bodies When Overexpressed in Mammalian Cells

Protein ADP-ribosylation is a reversible post-translational modification of cellular proteins that is catalysed by enzymes that transfer one (mono) or several (poly) units of ADP-ribose from β-NAD+ to a specific amino acid of the target protein. The most studied member of the ADP-ribosyltransferase family is PARP1 (also known as ADP-ribosyltransferase diphtheria toxin-like 1, ARTD1), which is directly activated by DNA strand breaks and is involved in DNA damage repair, chromatin remodelling and transcriptional regulation. Much less is known about the further 16 members of this family. Among these, ARTD10/PARP10 has been previously characterised as a mono-ADP-ribosyltransferase with a role in cell proliferation and in NF-kB signalling. In the present study, we identified the glycolytic enzyme GAPDH as an interactor and a novel cellular target for ARTD10/PARP10. Moreover, we detected the co-localisation of GAPDH and ARTD10/PARP10 in well-defined cytosolic bodies, which we show here to be membrane-free, rounded structures using immunogold labelling and electron microscopy. Using the cognitive binding module macro domain to visualise ADP-ribosylated proteins by immunofluorescence microscopy in cells over-expressing the ARTD10/PARP10 enzyme, we show that the staining of the ARTD10/PARP10-dependent cytosolic bodies was lost when the cells were treated with compounds that inhibit ARTD10/PARP10, either by directly inhibiting the enzyme or by reducing the cellular NAD+ levels. In parallel, the same treatment affected the co-localisation of GAPDH and ARTD10/PARP10, as GAPDH disappeared from the cytosolic cell bodies, which indicates that its presence there depends on the catalytic activity of ARTD10/PARP10. In line with this, in cells over-expressing the ARTD10/PARP10 catalytic domain alone, which we show here to form stress granules, GAPDH was recruited into stress granules. These data identify ARTD10/PARP10 as the enzyme that modifies and recruits GAPDH into cytosolic structures

Mono-ADP-ribosylation of the G Protein βγ Dimer Is Modulated by Hormones and Inhibited by Arf6*

Mono-ADP-ribosylation is a reversible post-translational modification that can modulate the functions of target proteins. We have previously demonstrated that the β subunit of heterotrimeric G proteins is endogenously mono-ADP-ribosylated, and once modified, the βγ dimer is inactive toward its effector enzymes. To better understand the physiological relevance of this post-translational modification, we have studied its hormonal regulation. Here, we report that Gβ subunit mono-ADP-ribosylation is differentially modulated by G protein-coupled receptors. In intact cells, hormone stimulation of the thrombin receptor induces Gβ subunit mono-ADP-ribosylation, which can affect G protein signaling. Conversely, hormone stimulation of the gonadotropin-releasing hormone receptor (GnRHR) inhibits Gβ subunit mono-ADP-ribosylation. We also provide the first demonstration that activation of the GnRHR can activate the ADP-ribosylation factor Arf6, which in turn inhibits Gβ subunit mono-ADP-ribosylation. Indeed, removal of Arf6 from purified plasma membranes results in loss of GnRHR-mediated inhibition of Gβ subunit mono-ADP-ribosylation, which is fully restored by re-addition of purified, myristoylated Arf6. We show that Arf6 acts as a competitive inhibitor of the endogenous ADP-ribosyltransferase and is itself modified by this enzyme. These data provide further understanding of the mechanisms that regulate endogenous ADP-ribosylation of the Gβ subunit, and they demonstrate a novel role for Arf6 in hormone regulation of Gβ subunit mono-ADP-ribosylation