P390 New features of molecular diagnostics of the regulation of molecular apoptotic pathway in ulcerative colitis

Background The incidence rate of ulcerative colitis (UC) in Russia is 5–30 cases per 100,000 per year. Molecular pathways of the UC development are not clear. The purpose of the work was to study the molecular interactions of the apoptotic pathway in patients with UC. Methods The clinical trial included 92 patients with UC. The clinical presentation of UC depends on the extent of involvement: distal colitis, extensive colitis; the severity of the disease. Criteria of the diagnosis of UC corresponded to ECCO Consensus. Mucosal specimens were graded according to grades 0, 1, 2, 3. The separation of proteins of colon mucosa was based on technologies of IEF, SDS-PAGE, 2DPAGE, by standard sets (MB-HIC C8 Kit, MB-IMAC Cu, MB-Wax Kit, Bruker, USA). The getting of mass-spectrogram was determined by matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry (MALDI-TOF-MS/MS, Ultraflex II, Bruker, USA). Statistical analysis was performed using the software Statistica 10.0 (Statsoft). Results HSP2 controls the apoptosis of colonocytes and immune response in damaged colon mucosa by Bcl-2 and IL-17, and is also responsible for the resistance to therapeutic strategies. Anti-apoptotic functions of HSP27 is possible through the interaction with DAXX7, the activation of Akt and the inhibition of the apoptosis. HSP47 interacts with collagen I, II, III, IV and V types, that contributes to the launch of autoimmune process in UC. Caspase 8 protects colonocytes from TNFα-induced cell death through necroptosis mechanism via the blockade of the expression of RIP3. Significant decrease of the expression of PPARγ promotes the activation of STAT and AP-1 signalling pathways, that promotes the increase of the synthesis of IL-2, -6, -8, -12, TNFα, matrix metalloproteinases, the activity of immune and inflammation processes in the colon mucosa. Results of qualitative proteomic analysis in patients with UC (Tab.) Conclusion Proteomic analysis has demonstrated the cascade with caspases 8, 9, 10, the release of cytochrome C from mitochondria, which interacts with Apaf-1, causing activation of caspase-9 in UC colon mucosa. TNF-α activates of initiator caspase-2, -8, and -10 in the apoptotic pathway. The NF-kB pathway induces cellular inhibitors of apoptosis, which function as specific caspase inhibitors in UC colonic mucosa.

cIAP1/2 inhibition synergizes with TNF inhibition in autoimmunity by down-regulating IL-17A and inducing Tregs

IL-17 and TNF-α are major effector cytokines in chronic inflammation. TNF-α inhibitors have revolutionized the treatment of rheumatoid arthritis (RA), although not all patients respond, and most relapse after treatment withdrawal. This may be due to a paradoxical exacerbation of TH17 responses by TNF-α inhibition. We examined the therapeutic potential of targeting cellular inhibitors of apoptosis 1 and 2 (cIAP1/2) in inflammation by its influence on human TH subsets and mice with collagen-induced arthritis. Inhibition of cIAP1/2 abrogated CD4+ IL-17A differentiation and IL-17 production. This was a direct effect on T cells, mediated by reducing NFATc1 expression. In mice, cIAP1/2 inhibition, when combined with etanercept, abrogated disease activity, which was associated with an increase in Tregs and was sustained after therapy retraction. We reveal an unexpected role for cIAP1/2 in regulating the balance between TH17 and Tregs and suggest that combined therapeutic inhibition could induce long-term remission in inflammatory diseases.

A complex containing lysine-acetylated actin inhibits the formin INF2

INF2 is a member of the formin family of actin assembly factors. Dominant mis-sense mutations in INF2 link to two diseases: focal segmental glomerulosclerosis (FSGS), a kidney disease; and Charcot-Marie-Tooth disease (CMTD), a neuropathy. All disease mutations map to the autoinhibitory Diaphanous Inhibitory Domain (DID). Curiously, purified INF2 is not autoinhibited, suggesting the existence of additional cellular inhibitors. We purified an INF2 inhibitor from mouse brain, and identified it as a complex between lysine-acetylated actin (KAc-actin) and cyclase-associated protein (CAP). Inhibition of INF2 by CAP/KAc-actin requires INF2 DID. Treatment of CAP/KAc-actin with histone deacetylase 6 (HDAC6) releases INF2 inhibition, while HDAC6 inhibitors block cellular INF2 activation. INF2 disease mutants are poorly inhibited by CAP/KAc-actin, suggesting that FSGS and CMTD result from reduced CAP/KAc-actin binding. This is the first demonstrated role for lysine-acetylated actin: regulation of an actin assembly factor by a novel mechanism, which we call facilitated auto-inhibition.

Eliminating hepatitis B by antagonizing cellular inhibitors of apoptosis

We have shown that cellular inhibitor of apoptosis proteins (cIAPs) impair clearance of hepatitis B virus (HBV) infection by preventing TNF-mediated killing/death of infected cells. A key question, with profound therapeutic implications, is whether this finding can be translated to the development of drugs that promote elimination of infected cells. Drug inhibitors of cIAPs were developed as cancer therapeutics to promote TNF-mediated tumor killing. These drugs are also known as Smac mimetics, because they mimic the action of the endogenous protein Smac/Diablo that antagonizes cIAP function. Here, we show using an immunocompetent mouse model of chronic HBV infection that birinapant and other Smac mimetics are able to rapidly reduce serum HBV DNA and serum HBV surface antigen, and they promote the elimination of hepatocytes containing HBV core antigen. The efficacy of Smac mimetics in treating HBV infection is dependent on their chemistry, host CD4+ T cells, and TNF. Birinapant enhances the ability of entecavir, an antiviral nucleoside analog, to reduce viral DNA production in HBV-infected animals. These results indicate that birinapant and other Smac mimetics may have efficacy in treating HBV infection and perhaps, other intracellular infections.