Inflammatory responses mediated by NOD2 rely on RIP2 kinase and ubiquitin ligase XIAP for the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and cytokine ...production. Herein, we demonstrate that selective XIAP antagonism blocks NOD2-mediated inflammatory signaling and cytokine production by interfering with XIAP-RIP2 binding, which removes XIAP from its ubiquitination substrate RIP2. We also establish that the kinase activity of RIP2 is dispensable for NOD2 signaling. Rather, the conformation of the RIP2 kinase domain functions to regulate binding to the XIAP-BIR2 domain. Effective RIP2 kinase inhibitors block NOD2 signaling by disrupting RIP2-XIAP interaction. Finally, we identify NOD2 signaling and XIAP-dependent ubiquitination sites on RIP2 and show that mutating these lysine residues adversely affects NOD2 pathway signaling. Overall, these results reveal a critical role for the XIAP-RIP2 interaction in NOD2 inflammatory signaling and provide a molecular basis for the design of innovative therapeutic strategies based on XIAP antagonists and RIP2 kinase inhibitors.
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•NOD2 requires RIP2 kinase and E3 XIAP for the activation of inflammatory signaling•XIAP antagonists block NOD2 signaling by interfering with XIAP binding to RIP2•The kinase activity of RIP2 is dispensable for NOD2 signaling•RIP2 kinase inhibitors block NOD2 signaling by disrupting RIP2-XIAP interaction
Goncharov et al. demonstrate that the kinase activity of RIP2 is dispensable for NOD2 signaling. Instead, effective RIP2 kinase inhibitors block NOD2 signaling by disrupting RIP2-XIAP interaction in a similar fashion as XIAP antagonists. These findings provide a novel therapeutic strategy for targeting the NOD2 pathway.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The inhibitor of apoptosis (IAP) proteins are a family of anti-apoptotic regulators found in viruses and metazoans. c-IAP1 and c-IAP2 are recruited to tumor necrosis factor receptor 1 ...(TNFR1)-associated complexes where they can regulate receptor-mediated signaling. Both c-IAP1 and c-IAP2 have been implicated in TNFα-stimulated NF-κB activation. However, individual c-IAP1 and c-IAP2 gene knock-outs in mice did not reveal changes in TNF signaling pathways, and the phenotype of a combined deficiency of c-IAPs has yet to be reported. Here we investigate the role of c-IAP1 and c-IAP2 in TNFα-stimulated activation of NF-κB. We demonstrate that TNFα-induced NF-κB activation is severely diminished in the absence of both c-IAP proteins. In addition, combined absence of c-IAP1 and c-IAP2 rendered cells sensitive to TNFα-induced cell death. Using cells with genetic ablation of c-IAP1 or cells where the c-IAP proteins were eliminated using IAP antagonists, we show that TNFα-induced RIP1 ubiquitination is abrogated in the absence of c-IAPs. Furthermore, we reconstitute the ubiquitination process with purified components in vitro and demonstrate that c-IAP1, in collaboration with the ubiquitin conjugating enzyme (E2) enzyme UbcH5a, mediates polymerization of Lys-63-linked chains on RIP1. Therefore, c-IAP1 and c-IAP2 are required for TNFα-stimulated RIP1 ubiquitination and NF-κB activation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The expansion of multiple drug resistant (MDR) strains of
presents an immense threat for public health. Annually, this microorganism causes thousands of lethal nosocomial infections worldwide. ...Currently, it has been shown that certain strains of lactic acid bacteria (LAB) can efficiently inhibit growth of
and the formation of its biofilms; however, the active principle of such action remains unknown. In the current article, the growth inhibition of MDR
by two LAB-
LR1 and
F-is demonstrated, and the nature of this inhibition studied at the level of exoproteome. This article shows that the exoproteomes of studied LAB contains both classically and non-classically secreted proteins. While for
LR1 the substantial portion of classically secreted proteins was presented by cell-wall-degrading enzymes, for
F only one out of four classically secreted proteins was presented by cell-wall hydrolase. Non-classically secreted proteins of both LAB were primarily metabolic enzymes, for some of which a possible moonlighting functioning was proposed. These results contribute to knowledge regarding antagonistic interaction between LAB and pathogenic and opportunistic microorganisms and set new perspectives for the use of LAB to control the spread of these microorganisms.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Torin-2, a synthetic compound, is a highly selective inhibitor of both TORC1 and TORC2 (target of rapamycin) complexes as an alternative to the well-known immunosuppressor, geroprotector, and ...potential anti-cancer natural compound rapamycin. Torin-2 is effective at hundreds of times lower concentrations and prevents some negative side effects of rapamycin. Moreover, it inhibits the rapamycin-resistant TORC2 complex. In this work, we evaluated transcriptomic changes in
heads induced with lifetime diets containing Torin-2 and suggested possible neuroprotective mechanisms of Torin-2. The analysis included
of three ages (2, 4, and 6 weeks old), separately for males and females. Torin-2, taken at the lowest concentration being tested (0.5 μM per 1 L of nutrient paste), had a slight positive effect on the lifespan of
males (+4% on the average) and no positive effect on females. At the same time, RNA-Seq analysis revealed interesting and previously undiscussed effects of Torin-2, which differed between sexes as well as in flies of different ages. Among the cellular pathways mostly altered by Torin-2 at the gene expression level, we identified immune response, protein folding (heat shock proteins), histone modification, actin cytoskeleton organization, phototransduction and sexual behavior. Additionally, we revealed that Torin-2 predominantly reduced the expression of
gene responsible for the conversion of L-serine to D-serine and thus regulating activity of NMDA receptor. Via western blot analysis, we showed than in old males Torin-2 tends to increase the ratio of the active phosphorylated form of ERK, the lowest node of the MAPK cascade, which may play a significant role in neuroprotection. Thus, the complex effect of Torin-2 may be due to the interplay of the immune system, hormonal background, and metabolism. Our work is of interest for further research in the field of NMDA-mediated neurodegeneration.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
A synthetic procedure for the synthesis of azacrown ethers with a combination of pendant arms has been developed and the synthesized ligand, characterized by various techniques, was studied. The ...prepared benzoazacrown ether with hybrid pendant arms and its complexes with copper and lead cations were studied in terms of biomedical applications. Similarly to a fully acetate analog, the new one binds both cations with close stability constants, despite the decrease in both constants. The calculated geometry of the complexes correlate with the data from X-ray absorption and NMR spectroscopy. Coordination of both cations differs due to the difference between the ionic radii. However, these chelation modes provide effective shielding of cations in both cases, that was shown by the stability of their complexes in the biologically relevant media towards transchelation and transmetallation.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Molecular heterogeneity in prostate cancer (PCa) is one of the key reasons underlying the differing likelihoods of recurrence after surgical treatment in individual patients of the same clinical ...category. In this study, we performed RNA-Seq profiling of 58 localized PCa and 43 locally advanced PCa tissue samples obtained as a result of radical prostatectomy on a cohort of Russian patients. Based on bioinformatics analysis, we examined features of the transcriptome profiles within the high-risk group, including within the most commonly represented molecular subtype, TMPRSS2-ERG. The most significantly affected biological processes in the samples were also identified, so that they may be further studied in the search for new potential therapeutic targets for the categories of PCa under consideration. The highest predictive potential was found with the
,
,
,
,
,
, and
genes. We also reviewed the main transcriptome changes in the groups at intermediate risk of PCa-Gleason Score 7 (groups 2 and 3 according to the ISUP classification)-on the basis of which the
,
, and
genes were identified as promising additional prognostic markers, the statistical significance of which was confirmed using qPCR validation.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Radical prostatectomy is the gold standard treatment for prostate cancer (PCa); however, it does not always completely cure PCa, and patients often experience a recurrence of the disease. In ...addition, the clinical and pathological parameters used to assess the prognosis and choose further tactics for treating a patient are insufficiently informative and need to be supplemented with new markers. In this study, we performed RNA-Seq of PCa tissue samples, aimed at identifying potential prognostic markers at the level of gene expression and miRNAs associated with one of the key signs of cancer aggressiveness-lymphatic dissemination. The relative expression of candidate markers was validated by quantitative PCR, including an independent sample of patients based on archival material. Statistically significant results, derived from an independent set of samples, were confirmed for miR-148a-3p and miR-615-3p, as well as for the
,
, and
genes. Considering the obtained validation data, we also analyzed the predictive value of models based on various combinations of identified markers using algorithms based on machine learning. The highest predictive potential was shown for the "CST2 + OCLN + pT" model (AUC = 0.863) based on the CatBoost Classifier algorithm.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Flax (Linum usitatissimum L.) is a crop plant used for fiber and oil production. Although potentially high-yielding flax varieties have been developed, environmental stresses markedly decrease flax ...production. Among biotic stresses, Fusarium oxysporum f. sp. lini is recognized as one of the most devastating flax pathogens. It causes wilt disease that is one of the major limiting factors for flax production worldwide. Breeding and cultivation of flax varieties resistant to F. oxysporum is the most effective method for controlling wilt disease. Although the mechanisms of flax response to Fusarium have been actively studied, data on the plant response to infection and resistance gene candidates are currently very limited.
The transcriptomes of two resistant and two susceptible flax cultivars with respect to Fusarium wilt, as well as two resistant BC
F
populations, which were grown under control conditions or inoculated with F. oxysporum, were sequenced using the Illumina platform. Genes showing changes in expression under F. oxysporum infection were identified in both resistant and susceptible flax genotypes. We observed the predominant overexpression of numerous genes that are involved in defense response. This was more pronounced in resistant cultivars. In susceptible cultivars, significant downregulation of genes involved in cell wall organization or biogenesis was observed in response to F. oxysporum. In the resistant genotypes, upregulation of genes related to NAD(P)H oxidase activity was detected. Upregulation of a number of genes, including that encoding beta-1,3-glucanase, was significantly greater in the cultivars and BC
F
populations resistant to Fusarium wilt than in susceptible cultivars in response to F. oxysporum infection.
Using high-throughput sequencing, we identified genes involved in the early defense response of L. usitatissimum against the fungus F. oxysporum. In response to F. oxysporum infection, we detected changes in the expression of pathogenesis-related protein-encoding genes and genes involved in ROS production or related to cell wall biogenesis. Furthermore, we identified genes that were upregulated specifically in flax genotypes resistant to Fusarium wilt. We suggest that the identified genes in resistant cultivars and BC
F
populations showing induced expression in response to F. oxysporum infection are the most promising resistance gene candidates.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Following radical surgery, patients may suffer a relapse. It is important to identify such patients so that therapy tactics can be modified appropriately. Existing stratification schemes do not ...display the probability of recurrence with enough precision since locally advanced prostate cancer (PCa) is classified as high-risk but is not ranked in greater detail. Between 40 and 50% of PCa cases belong to the TMPRSS2-ERG subtype that is a sufficiently homogeneous group for high-precision prognostic marker search to be possible. This study includes two independent cohorts and is based on high throughput sequencing and qPCR data. As a result, we have been able to suggest a perspective-trained model involving a deep neural network based on both qPCR data for mRNA and miRNA and clinicopathological criteria that can be used for recurrence risk forecasts in patients with TMPRSS2-ERG-positive, locally advanced PCa (the model uses ALDH3A2 + ODF2 + QSOX2 + hsa-miR-503-5p + ISUP + pT, with an AUC = 0.944). In addition to the prognostic model’s use of identified differentially expressed genes and miRNAs, miRNA–target pairs were found that correlate with the prognosis and can be presented as an interactome network.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Ubiquitin ligases are critical components of the ubiquitination process that determine substrate specificity and, in collaboration with E2 ubiquitin‐conjugating enzymes, regulate the nature of ...polyubiquitin chains assembled on their substrates. Cellular inhibitor of apoptosis (c‐IAP1 and c‐IAP2) proteins are recruited to TNFR1‐associated signalling complexes where they regulate receptor‐stimulated NF‐κB activation through their RING domain ubiquitin ligase activity. Using a directed yeast two‐hybrid screen, we found several novel and previously identified E2 partners of IAP RING domains. Among these, the UbcH5 family of E2 enzymes are critical regulators of the stability of c‐IAP1 protein following destabilizing stimuli such as TWEAK or CD40 signalling or IAP antagonists. We demonstrate that c‐IAP1 and UbcH5 family promote K11‐linked polyubiquitination of receptor‐interacting protein 1 (RIP1) in vitro and in vivo. We further show that TNFα‐stimulated NF‐κB activation involves endogenous K11‐linked ubiquitination of RIP1 within the TNFR1 signalling complex that is c‐IAP1 and UbcH5 dependent. Lastly, NF‐κB essential modifier efficiently binds K11‐linked ubiquitin chains, suggesting that this ubiquitin linkage may have a signalling role in the activation of proliferative cellular pathways.
Canonical Lys48‐ and Lys63‐linked as well as non‐canonical linear polyubiquitin chains are involved in receptor‐mediated NF‐κB activation. This study identifies c‐IAP1‐generated Lys11‐linked chains, so far implicated only in cell‐cycle control and proteasome targeting, as an additional signal in this pathway.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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