Vinay Murtadak

Suche


B 9 Viral regulators of the cellular ripoptosome protein complex

Principal investigator
Michael Stürzl

Mentor
Alexander Steinkasser

Viral regulators of the cellular ripoptosome protein complex

Apoptosis and necrosis are two common forms of programmed of cell death. A new mode of cell death has been recently described, termed necroptosis which shows the morphological features of necrosis but like apoptosis runs through a programmed process. Under physiological conditions, TNF receptor ligation results in the formation of TNFR complex 1 and TNFR complex 2 which play a role in cell survival and apoptosis respectively. TNFR complex 2 also regulates necroptosis upon recruitment of the two kinases, receptor interacting protein 1 (RIP1) and receptor interacting protein 3 (RIP 3) resulting in formation of a complex termed ripoptosome which is the central regulator of necroptosis. It was found that a protein termed cFLIP which exists in two isoforms (long and short) also plays a crucial role in necroptosis. cFLIPlong blocks necroptosis, while cFLIPshort promotes necroptosis.
Interestingly, there are certain poxviruses and herpesviruses including the Kaposi's sarcoma-associated herpesvirus (KSHV) which express a protein, called vFLIP, which shares structural similarities to cFLIPshort. We have shown that vFLIP is predominantly expressed in Kaposi´s sarcoma (KS) tissues, is resident in the cytoplasm and nucleus of infected cells, is a potent activator of NF-kB and via the latter activity contributes to reactive oxygen species detoxification via the induction of manganese superoxide dismutase. Experiments by our co-operators suggest that vFLIP expression alone induces necroptosis and organ pathology in mice generated to express KSHV-vFLIP specifically in epithelial cells of the gut. These preliminary findings obtained in vFLIP expressing transgenic mice and sporadic reports of a putative infectious etiology of colorectal cancer (CRC) suggested that vFLIPs or respective functional homologs of this protein may play a role in the development of colitis and/or CRC.
The overall goal of the project is to elucidate the functional role of vFLIPs in the regulation of necroptosis and in the formation of the ripoptosome complex and to determine the putative impact of this effect on the pathogenesis of KS, inflammatory bowels disease and colon cancer. A specific focus will be on the KSHV-vFLIP. In this framework, different cellular interacting partners of KSHV-vFLIP will be determined by immunoprecipitation (IP) assays using in vitro overexpression systems. We will also analyze the putative expression of vFLIP like molecules in CRC and colitis tissues. To this goal, degenerate primer sets based on the sequences of all presently known viral FLIPs will be generated and subjected to PCR on RNA and DNA extracted from CRC, colitis tissue and normal colon tissues of the respective patients. Finally, the impact of the results obtained above will be validated at the clinical level using serologic analyses and expression studies. It is hoped that this project will contribute to the understanding of the role of vFLIPs in the host pathogen interactions.

Figure: Schematic representation of TNF-R mediated formation of signaling complexes. Ligand binding to TNF-R allows assembly of different complexes regulating cell survival or different modes of cell death. The TNFR complex 1 involves the molecules TRADD, TRAF2/5, cIAP1/2 and the polyubiquitinated RIP1 and stimulates NF-κB pathway promoting cell survival. TNFR complex 2 also known as DISC (death inducing signaling complex) promotes apoptosis and is composed of molecules FADD, TRADD, caspase-8, as well as cFLIP (cellular FLICE-inhibitory protein), an inhibitor of caspase-8. Upon stimulus, RIP1 is deubiquitinated and can translocate to TNFR complex 2. Caspase 8 inactivates RIP1 and RIP3 by proteolytic cleavage and initiates apoptosis. The heterodimer of caspase 8 and cFLIPlong allows limited activation of caspase 8 and is insufficient to induce apoptosis but still sufficient to prevent RIP3 dependent necroptosis. In contrast, cFLIPshort blocks caspase activity completely that promotes necroptosis by suppressing RIP1 inactivation. This results in autophosphorylation of RIP1 and RIP3 and finally to induction of necroptosis through MLKL phosphorylation. KSHV-vFLIP shares structural similarities to cFLIPshort. In this framework, it is interesting to investigate role of vFLIP in the regulation of necroptosis and in the formation of the ripoptosome complex.

 

Publications

 

Presentations

October 2016 8th Annual Retreat, Erlangen School of Molecular Communication, Schloss Schney, Lichtenfels, Germany
”Viral regulators of the cellular ripoptosome protein complex”
Talk
     
October 2015 2nd International SFB 796 Conference: Mechanisms of microbial host cell manipulation: From plants to humans, Erlangen, Germany
Regulation of cell death by Kaposi's sarcoma-associated herpesvirus
Poster
     
July 2015 7th Annual Retreat, Erlangen School of Molecular Communication, Schloss Hirschberg, Beilngries, Germany
”Role of Kaposi's sarcoma-associated herpesvirus-encoded FLIP gene in necroptosis”
Poster
     
July 2014 6th Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
“Role of KSHV-encoded viral FLICE inhibitory protein in the regulation of necroptosis”
Talk
     
July 2013 5th Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
Role of KSHV-encoded viral FLICE inhibitory protein in the regulation of necroptosis
Talk
     

 

Awards

Best Talk Award

Third place

8th Annual Retreat, Erlangen School of Molecular Communication, Schloss Schney, Lichtenfels, Germany, October 2016
“Viral regulators of the cellular ripoptosome protein complex“