Activation of nucleotide-binding oligomerization domain 2 by muramyl dipeptide negatively regulates Toll-like receptor 9-mediated colonic inflammation through the induction of deubiquitinating enzyme A expression.

Mutations in nucleotide-binding oligomerization domain 2 (NOD2) are associated with Crohn's disease (CD). Although NOD2 activation contributes to the maintenance of intestinal homeostasis through the negative regulation of pro-inflammatory cytokine responses mediated by Toll-like receptors (TLRs), the effects of NOD2 activation on interferon (IFN)-α responses induced by TLR9 have been poorly defined. To explore the cross-talk between NOD2 and TLR9, human monocytes or dendritic cells (DCs) were stimulated with NOD2 and/or TLR9 ligands to measure IFN-α production. The severity of dextran sodium sulfate (DSS)-induced colitis was compared in mice treated with NOD2 and/or TLR9 ligands. Expression of IFN-α and IFN-stimulated genes (ISGs) was examined in the colonic mucosa of patients with inflammatory bowel disease (IBD). NOD2 activation reduced TLR9-induced IFN-α production by monocytes and DCs in a deubiquitinating enzyme A (DUBA)-dependent manner. Activation of DUBA induced by the co-stimulation of TLR9 and NOD2 inhibited Lys63-linked polyubiquitination of TRAF3 and suppressed TLR9-mediated IFN-α production. NOD2 activation in hematopoietic cells protected mice from TLR9-induced exacerbation of DSS-induced colitis by down-regulating IFN-α responses and up-regulating DUBA expression. Colonic mucosa of patients with active and remitted IBD phases was characterized by the enhanced and reduced expression of ISGs, respectively. Expression levels of IFN-α and IL-6 positively correlated in the active colonic mucosa of patients with ulcerative colitis and CD, whereas DUBA expression inversely correlated with that of IFN-α in patients with CD. Collectively, these data suggest that DUBA-dependent negative effect of NOD2 on TLR9-mediated IFN-α responses contributes to the maintenance of intestinal homeostasis.

Rainbow trout DUBA inhibits type I interferon signaling by deubiquitinating TRAF3.

Deubiquitinating enzyme A (DUBA), a member of the ovarian tumor (OTU) subfamily of deubiquitinases (DUBs), is recognized for its negative regulatory role in type I interferon (IFN) expression downstream of Toll-like receptor 3 (TLR3). However, its involvement in the TLR3 signaling pathway in fish remains largely unexplored. In this study, we investigated the regulatory role of DUBA (OmDUBA) in the TLR3 response in rainbow trout (Oncorhynchus mykiss). OmDUBA features a conserved OTU domain, and its expression increased in RTH-149 cells following stimulation with the TLR3 agonist poly(I:C). Gain- and loss-of-function experiments demonstrated that OmDUBA attenuated the activation of TANK-binding kinase 1 (TBK1), resulting in a subsequent reduction in type I IFN expression and IFN-stimulated response element (ISRE) activation in poly(I:C)-stimulated cells. OmDUBA interacted with TRAF3, a crucial mediator in TLR3-mediated type I IFN production. Under poly(I:C) stimulation, there was an augmentation in the K63-linked polyubiquitination of TRAF3, a process significantly inhibited upon OmDUBA overexpression. These findings suggest that OmDUBA may function similarly to its mammalian counterparts in downregulating the poly(I:C)-induced type I IFN response in rainbow trout by removing the K63-linked ubiquitin chain on TRAF3. Our study provides novel insights into the role of fish DUBA in antiviral immunity.

Regulation of type I IFN responses by deubiquitinating enzyme A in inflammatory bowel diseases.

The development of Inflammatory bowel disease (IBD) is driven by excessive production of pro-inflammatory cytokines including TNF-α, IL-12, and IL-23. This notion is supported by the remarkable clinical success of biologics targeting these cytokines. Recognition of cell wall components derived from intestinal bacteria by Toll-like receptors (TLRs) induces the production of these pro-inflammatory cytokines by macrophages and dendritic cells in human IBD and experimental colitis model. Although sensing of bacterial nucleic acids by endosomal TLRs, specifically TLR3, TLR7, and TLR9 leads to robust production of type I IFNs, it remains debatable whether TLR-mediated type I IFN responses are pathogenic or protective in IBD patients. Additionally, recent studies identified deubiquitinating enzyme A (DUBA) as a novel negative regulator of TLR-mediated type I IFN responses. In light of these observations and their potential applications, in this review, we summarize recent findings on the roles of type I IFN responses and DUBA-mediated negative regulation of these responses in human IBD and experimental colitis model.

Modulation of conformational equilibrium by phosphorylation underlies the activation of deubiquitinase A.

Deubiquitinases deconjugate ubiquitin modifications from target proteins and are involved in many cellular processes in eukaryotes. The functions of deubiquitinases are regulated by post-translational modifications, mainly phosphorylation and ubiquitination. Post-translational modifications can result in subtle changes in structural and dynamic properties, which are difficult to identify but functionally important. In this work, we used NMR spectroscopy to characterize the conformational properties of the human deubiquitinase A (DUBA), a negative regulator of type I interferon. DUBA activity is regulated by phosphorylation at a single serine residue, Ser-177. We found that the catalytic rate constant of DUBA is enhanced by phosphorylation. By comparing NMR and enzyme kinetics data among different forms of DUBA with low and high activities, we concluded that a two-state equilibrium that was present only in phosphorylated DUBA is important for DUBA activity. Our results highlight the importance of defining conformational dynamics in understanding the mechanism of DUBA activation.

The deubiquitinase OTUD5 regulates Ku80 stability and non-homologous end joining.

The ability of cells to repair DNA double-strand breaks (DSBs) is important for maintaining genome stability and eliminating oncogenic DNA lesions. Two distinct and complementary pathways, non-homologous end joining (NHEJ) and homologous recombination (HR), are employed by mammalian cells to repair DNA DSBs. Each pathway is tightly controlled in response to increased DSBs. The Ku heterodimer has been shown to play a regulatory role in NHEJ repair. Ku80 ubiquitination contributes to the selection of a DSB repair pathway by causing the removal of Ku heterodimers from DSB sites. However, whether Ku80 deubiquitination also plays a role in regulating DSB repair is unknown. To address this question, we performed a comprehensive study of the deubiquitinase specific for Ku80, and our study showed that the deubiquitinase OTUD5 serves as an important regulator of NHEJ repair by increasing the stability of Ku80. Further studies revealed that OTUD5 depletion impaired NHEJ repair, and hence reduced overall DSB repair. Furthermore, OTUD5-depleted cells displayed excess end resection; as a result, HR repair was facilitated by OTUD5 depletion during the S/G2 phase. In summary, our study demonstrates that OTUD5 is a specific deubiquitinase for Ku80 and establishes OTUD5 as an important and positive regulator of NHEJ repair.

microRNA-210 participates in regulating RIG-I signaling pathway via targeting DUBA in miiuy croaker after poly(I:C) stimulation.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that participate in the regulation of various biological processes. A series of microRNAs have been shown to be important regulators of both innate and adaptive immune responses, including RIG-I signaling pathway. In this study, we evaluated the regulation role of miR-210 in the RLRs signaling pathway of miiuy croaker. Upon poly(I:C) stimulation, the expression of miR-210 in both miiuy croaker spleen tissues and macrophages were significantly upregulated. By means of the dual luciferase reporter assay, a direct interaction between miR-210 and the 3-untranslated region (UTR) of Deubiquitinating enzyme A (DUBA) was confirmed, and we found that miR-210 could reduce the luciferase levels of wild-type DUBA 3'UTR, whereas mutant-type led to a complete abrogation of the negative effect. Furthermore, the negative regulatory effects of pre-miR-210 on DUBA have been indicated in a dose- and time-dependent manners. As DUBA is an important regulator involved in the RLRs signaling pathway and could bind with and regulate TRAF3, we also examined the expression patterns of DUBA and TRAF3 in vivo and in vitro. We found that the expression of both DUBA and TRAF3 were significantly changed upon poly(I:C) stimulation in miiuy croaker. The expression patterns between miR-210 and DUBA showed a negative correlation, which indicated that miR-210 can target and downregulate the expression of DUBA. Overall, these results will enrich the knowledge of immune response related miRNAs in miiuy croaker, which will be useful for better understanding the complicated regulatory networks in fish species.

Design, Synthesis, and Evaluation of Linker-Duocarmycin Payloads: Toward Selection of HER2-Targeting Antibody-Drug Conjugate SYD985.

Antibody-drug conjugates (ADCs) that are currently on the market or in clinical trials are predominantly based on two drug classes: auristatins and maytansinoids. Both are tubulin binders and block the cell in its progression through mitosis. We set out to develop a new class of linker-drugs based on duocarmycins, potent DNA-alkylating agents that are composed of a DNA-alkylating and a DNA-binding moiety and that bind into the minor groove of DNA. Linker-drugs were evaluated as ADCs by conjugation to the anti-HER2 antibody trastuzumab via reduced interchain disulfides. Duocarmycin 3b, bearing an imidazo[1,2-a]pyridine-based DNA-binding unit, was selected as the drug moiety, notably because of its rapid degradation in plasma. The drug was incorporated into the linker-drugs in its inactive prodrug form, seco-duocarmycin 3a. Linker attachment to the hydroxyl group in the DNA-alkylating moiety was favored over linking to the DNA-binding moiety, as the first approach gave more consistent results for in vitro cytotoxicity and generated ADCs with excellent human plasma stability. Linker-drug 2 was eventually selected based on the properties of the corresponding trastuzumab conjugate, SYD983, which had an average drug-to-antibody ratio (DAR) of about 2. SYD983 showed subnanomolar potencies against multiple human cancer cell lines, was highly efficacious in a BT-474 xenograft model, and had a long half-life in cynomolgus monkeys, in line with high stability in monkey and human plasma. Studies comparing ADCs with a different average DAR showed that a higher average DAR leads to increased efficacy but also to somewhat less favorable physicochemical and toxicological properties. Fractionation of SYD983 with hydrophobic interaction chromatography resulted in SYD985, consisting of about 95% DAR2 and DAR4 species in an approximate 2:1 ratio and having an average DAR of about 2.8. SYD985 combines several favorable properties from the unfractionated ADCs with an improved homogeneity. It was selected for further development and recently entered clinical Phase I evaluation.

The Deubiquitinating Enzyme OTUD5 Sustains Inflammatory Cytokine Response in Inflammatory Bowel Disease.

BACKGROUND AND AIMS: The inflammatory bowel disease [IBD]-associated immune response is marked by excessive production of a variety of inflammatory cytokines, which are supposed to sustain and amplify the pathological process. OTUD5 is a deubiquitinating enzyme, which regulates cytokine production by both innate and adaptive immune cells. Here, we investigated the expression and role of OTUD5 in IBD. METHODS: OTUD5 expression was evaluated in mucosal samples of patients with Crohn's disease [CD], patients with ulcerative colitis [UC], and controls, as well as in mice with trinitrobenzene-sulphonic acid [TNBS]-induced colitis by real-time polymerase chain reaction, western blotting, immunohistochemistry, and immunofluorescence. Moreover, OTUD5 was assessed in lamina propria mononuclear cells [LPMC] stimulated with inflammatory cytokines. TNF-α, IL-6, and IL-10 were evaluated in LPMCs of IBD patients and in colitic mice transfected with a specific OTUD5 antisense oligonucleotide [AS]. RESULTS: OTUD5 protein, but not RNA, expression was increased in inflamed ileal and colonic mucosal samples of patients with CD and patients with UC as compared with controls. In IBD, OTUD5-expressing cells were abundant in both epithelial and lamina propria compartments, and non-CD3+, HLA-DR+ LPMC were one of the major sources of the protein. OTUD5 expression was enhanced by IFN-γ through a p38/MAPK-dependent mechanism, and the AS-induced knockdown of OTUD5 in LPMCs of IBD patients and colitic mice reduced TNF-α. CONCLUSIONS: Our data show that OTUD5 is overexpressed in both CD and UC and suggest the involvement of such a protein in the amplification of the aberrant cytokine response in IBD.

Study of meat quality and flavour in different cuts of Duroc-Bamei binary hybrid pigs.

BACKGROUND: Meat quality and flavour are important criteria for judging fresh pork and processed products. However, there have been few studies on meat quality and volatile flavour substances of different parts of binary hybrid pigs. OBJECTIVE: To study the differences in meat quality and volatile flavour substances between different cuts of pork, which could provide the basis for consumer decision-making when purchasing pork. METHODS: Twenty Du-Ba binary hybrid pigs (first filial [F1] generation) bred from Duroc and Bamei pigs were used. This study systematically compared and analysed the basic nutritional components, amino acid composition, fatty acid composition and flavour profiles of longissimus dorsi, rib muscle and tendon meat of four Du-Ba binary hybrid pigs; all assays were repeated in triplicate. RESULTS: Crude protein, calcium and phosphorus content in tendon meat were higher than that in the longissimus dorsi. The intramuscular fat content of the rib muscle was higher than that in the longissimus dorsi and tendon meat (p < 0.05). The amino acid content was highest in the tendon meat. The levels of essential amino acids and flavour-associated amino acids per kilogram of longissimus dorsi were higher than those in the rib muscle and tendon meat. Moreover, the content of aspartic acid, serine and cystine were higher in the longissimus dorsi than in the other two parts. The type of saturated fatty acids and the type and content of unsaturated fatty acids in tendon meat were higher than in the longissimus dorsi and rib muscle. The total content of volatile flavour compounds was higher in the longissimus dorsi than in the rib muscle and tendon meat. CONCLUSION: The rib muscle contains high deposits of fat, and tendon meat has a relatively high nutritional value, while the longissimus dorsi has a stronger flavour.