Role of deubiquitinase OTUD1 in acute lung injury in septic mice: relationship with TAK1-MAPK signaling pathway / 中华麻醉学杂志

Objective:To evaluate the role of deubiquitinase OTUD1 in acute lung injury in septic mice and the relationship with transforming growth factor-beta activated kinase 1(TAK1)-mitogen-activated protein kinase (MAPK) signaling pathway.Methods:Twenty male wild-type (WT) and 20 OTUD1 gene knockout (KO) C57BL/6N mice, aged 6-8 weeks, weighing 20-25 g, were divided into 4 groups ( n=10 each) using a random number table method: wild-type+ sham operation group (WT-Sham group), wild-type+ sepsis group (WT-SEP group), OTUD1-KO+ sham operation group (KO-Sham group) and OTUD1-KO+ SEP group (KO-SEP group). The acute lung injury was induced by cecal ligation and perforation (CLP) in anesthetized septic mice. Mice were sacrificed at 24 h after operation, blood samples were collected from the abdominal aorta, and lung tissues were collected. Blood gas analysis was performed using the i-STAT blood gas analyzer, PaO 2 and FiO 2 were recorded, and the oxygenation index (OI) was calculated. The morphology of lung tissues was examined with a light microscope for evaluation of lung injury, and lung injury scores were calculated. The wet to dry lung weight (W/D) ratio was measured, and the activities of MPO were measured. The concentrations of TNF-α and IL-6 in plasma were detected by enzyme-linked immunosorbent assay, and the expression of OTUD1, phosphorylated TAK1 (p-TAK1), phosphorylated p38 (p-p38), phosphorylated c-Jun amino-terminal kinase (p-JNK) and phosphorylated extracellular signal-regulated kinase (p-ERK) was detected using Western blot. Results:Compared with WT-Sham group, the PaO 2 and OI were significantly decreased, the lung injury score, W/D ratio, MPO activity, and plasma TNF-α and IL-6 concentrations were increased, and the expression of OTUD1, p-TAK1, p-p38, p-JNK and p-ERK protein in lung tissues was up-regulated in WT-SEP group ( P<0.05). Compared with WT-SEP group, the PaO 2 and OI were significantly decreased, the lung injury score, W/D ratio, MPO activity, and plasma TNF-α and IL-6 concentrations were increased, and the expression of OTUD1, p-TAK1, p-p38, p-JNK and p-ERK protein in lung tissues was up-regulated in KO-SEP group ( P<0.05). Conclusions:OTUD1 is involved in the endogenous protective mechanism against acute lung injury in septic mice, which may be related to the inhibition of TAK1-MAPK signaling pathway activation and decreased inflammatory responses.

Abrogation of USP7 is an alternative strategy to downregulate PD-L1 and sensitize gastric cancer cells to T cells killing

Targeting immune checkpoints such as programmed cell death protein 1 (PD-1) and programmed death ligand-1 (PD-L1) have been approved for treating melanoma, gastric cancer (GC) and bladder cancer with clinical benefit. Nevertheless, many patients failed to respond to anti-PD-1/PD-L1 treatment, so it is necessary to seek an alternative strategy for traditional PD-1/PD-L1 targeting immunotherapy. Here with the data from The Cancer Genome Atlas (TCGA) and our in-house tissue library, PD-L1 expression was found to be positively correlated with the expression of ubiquitin-specific processing protease 7 (USP7) in GC. Furthermore, USP7 directly interacted with PD-L1 in order to stabilize it, while abrogation of USP7 attenuated PD-L1/PD-1 interaction and sensitized cancer cells to T cell killing

Role of deubiquitinating enzymes in DNA double-strand break repair / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

DNA is the hereditary material in humans and almost all other organisms. It is essential for maintaining accurate transmission of genetic information. In the life cycle, DNA replication, cell division, or genome damage, including that caused by endogenous and exogenous agents, may cause DNA aberrations. Of all forms of DNA damage, DNA double-strand breaks (DSBs) are the most serious. If the repair function is defective, DNA damage may cause gene mutation, genome instability, and cell chromosome loss, which in turn can even lead to tumorigenesis. DNA damage can be repaired through multiple mechanisms. Homologous recombination (HR) and non-homologous end joining (NHEJ) are the two main repair mechanisms for DNA DSBs. Increasing amounts of evidence reveal that protein modifications play an essential role in DNA damage repair. Protein deubiquitination is a vital post-translational modification which removes ubiquitin molecules or polyubiquitinated chains from substrates in order to reverse the ubiquitination reaction. This review discusses the role of deubiquitinating enzymes (DUBs) in repairing DNA DSBs. Exploring the molecular mechanisms of DUB regulation in DSB repair will provide new insights to combat human diseases and develop novel therapeutic approaches.

Research advances on deubiquitinating enzymes involved in the development of hepatocellular carcinoma / 中国肿瘤临床

The ubiquitin-proteasome pathway is the primary regulator of intracellular protein degradation. In recent years, it has been found that multiple members of the deubiquitinating enzyme family play an important role in the development of liver cancer. The deubiquitinating enzymes remove ubiquitin chains of substrate proteins to maintain their stability; thus, they affect tumor progression via maintaining the stability of proteins involved in cell apoptosis, autophagy, cell cycle regulation, and DNA damage. Some small molecule inhibitors can exert an anti-tumor effect by inhibiting the activity of deubiquitinating enzymes. Here, we have reviewed research regarding deubiquitinating enzymes and their small molecule inhibitors in order to provide a new theoretical foundation for exploring novel and effective therapeutic targets for liver cancer.

Discovery of 1,2,3triazolo4,5-pyrimidine derivatives as highly potent, selective, and cellularly active USP28 inhibitors

Ubiquitin specific peptidase 28 (USP28) is closely associated to the occurrence and development of various malignancies, and thus has been validated as a promising therapeutic target for cancer therapy. To date, only few USP28 inhibitors with moderate inhibitory activity have been reported, highly potent and selective USP28 inhibitors with new chemotypes remain to be discovered for pathologically investigating the roles of deubiquitinase. In this current study, we reported the synthesis and biological evaluation of new [1,2,3]triazolo[4,5-]pyrimidine derivatives as potent USP28 inhibitors. Especially, compound potently inhibited USP28 (IC = 1.10 ± 0.02 μmol/L,  = 40 nmol/L), showing selectivity over USP7 and LSD1 (IC > 100 μmol/L). Compound was cellularly engaged to USP28 in gastric cancer cells. Compound reversibly bound to USP28 and directly affected its protein levels, thus inhibiting the proliferation, cell cycle at S phase, and epithelial-mesenchymal transition (EMT) progression in gastric cancer cell lines. Docking studies were performed to rationalize the potency of compound . Collectively, compound could serve as a new tool compound for the development of new USP28 inhibitors for exploring the roles of deubiquitinase in cancers.

A new gene inventory of the ubiquitin and ubiquitin-like conjugation pathways in Giardia intestinalis

BACKGROUND Ubiquitin (Ub) and Ub-like proteins (Ub-L) are critical regulators of complex cellular processes such as the cell cycle, DNA repair, transcription, chromatin remodeling, signal translation, and protein degradation. Giardia intestinalis possesses an experimentally proven Ub-conjugation system; however, a limited number of enzymes involved in this process were identified using basic local alignment search tool (BLAST). This is due to the limitations of BLAST's ability to identify homologous functional regions when similarity between the sequences dips to < 30%. In addition Ub-Ls and their conjugating enzymes have not been fully elucidated in Giardia. OBJETIVE To identify the enzymes involved in the Ub and Ub-Ls conjugation processes using intelligent systems based on the hidden Markov models (HMMs). METHODS We performed an HMM search of functional Pfam domains found in the key enzymes of these pathways in Giardia's proteome. Each open reading frame identified was analysed by sequence homology, domain architecture, and transcription levels. FINDINGS We identified 118 genes, 106 of which corresponded to the ubiquitination process (Ub, E1, E2, E3, and DUB enzymes). The E3 ligase group was the largest group with 82 members; 71 of which harbored a characteristic RING domain. Four Ub-Ls were identified and the conjugation enzymes for NEDD8 and URM1 were described for first time. The 3D model for Ub-Ls displayed the β-grasp fold typical. Furthermore, our sequence analysis for the corresponding activating enzymes detected the essential motifs required for conjugation. MAIN CONCLUSIONS Our findings highlight the complexity of Giardia's Ub-conjugation system, which is drastically different from that previously reported, and provides evidence for the presence of NEDDylation and URMylation enzymes in the genome and transcriptome of G. intestinalis.

The roles of deubiquitinating enzymes in cancer therapy / 药学学报

Ubiquitination and deubiquitination play important roles in the regulation of protein stability and function. Deubiquitinating enzymes (DUBs) are involved in the regulation of survival, migration and proliferation of cancer cells, by participating in a variety of signaling pathways. Most of the DUBs promote the malignant transformation and progression, while the others may function as tumor-suppressors. Given the central roles of DUBs in tumorigenesis and malignant progression, some of these enzymes have been regarded as promising anti-cancer targets. This paper reviews the recent advances in tumor-related DUBs and inhibitors.

Preliminary study of the enzyme ubiquitin carboxylterminal hydrolase 14 (UBP6) in Giardia intestinalis: structural bioinformatic analysis and transcriptional profile during encystation / Estudio preliminar de la enzima ubiquitina carboxiterminal hidrolasa 14 (UBP6) en Giardia intestinalis: análisis bioinformático de su estructura y perfil transcripcional durante la enquistación / Estudo preliminar da enzima ubiquitina carboxi-terminal hidrolasa 14 (UBP6) em Giardia intestinalis: análise bioinformática estrutural e perfil transcricional durante encistamento

This paper presents a combined approach with two aims. The first is to analyze the reported sequence of the enzyme ubiquitin carboxyl-terminal hydrolase 14 of Giardia intestinalis (UBP6) through computational methods to find components related with its hypothetical function. The second is to determine if the protein-coding gene is expressed in G. intestinalis and, if such is the case, also determine its transcription pattern along the life cycle of the parasite. It was established that the protein belongs to the family of Cys-dependent deubiquitinases and more specifically to ubiquitin specific proteases (USPs). Moreover, the catalytic center with the complete triad as well as typical features of the USP motif were also identified. Since the computational findings suggest that the enzyme could be functional, reverse transcription coupled to PCR was used as a first approach to establish if in fact the coding gene is expressed in the parasite. Interestingly, it was found not only that the gene is expressed, but also that there is a transcription variation along the life cycle of the parasite. These two findings are the starting point for further studies since they tentatively suggest that this enzyme could be involved in the protein turnover that occurs during parasite encystation. Although preliminary, this study is the first report concerning the study of a specific deubiquitinating enzyme in the parasite G. intestinalis.

En este trabajo se presenta una estrategia combinada que buscaba, primero, analizar por métodos computacionales la secuencia de la enzima ubiquitina carboxilo-terminal hidrolasa 14 de Giardia intestinalis (UBP6) reportada para buscar componentes relacionados con su función hipotética y segundo, determinar si el gen que codifica para la proteína se expresa en G. intestinalis y si lo hace, cómo es su patrón de transcripción a lo largo del ciclo de vida del parásito. Se encontró que la proteína pertenece a la familia de deubiquitinasas dependientes de cisteína y más específicamente a las proteasas específicas para ubiquitina (USPs por ubiquitin specific proteases). También se identificaron el centro catalítico con la triada completa así como características típicas del motivo USP. Teniendo en cuenta que los resultados computacionales sugieren que la enzima puede ser funcional, se usó la técnica de transcripción reversa acoplada a PCR como un primer acercamiento para establecer si el gen codificante se expresa en el parásito. De manera interesante, se determinó no solo que el gen se expresa sino que existe una variación de su transcripción a lo largo del ciclo de vida del parásito. Estos hallazgos son el punto de partida para posteriores estudios ya que sugieren de manera preliminar que esta enzima podría estar involucrada en el recambio de proteínas que ocurre en el parásito durante el proceso de enquistación. Aunque preliminar, este estudio es el primer reporte acerca de una enzima deubiquitinadora específica en el parásito G. intestinalis.

Este artigo apresenta uma abordagem combinada com dois objetivos. A primeira é analisar a sequência informou da enzima ubiquitina carboxil-terminal hidrolase 14 de Giardia intestinalis (UBP6) através de métodos computacionais para encontrar os componentes relacionados com a sua função hipotética. A segunda é para determinar se o gene de codificação da proteína é expressa em G. intestinalis e, se for o caso, também determinar o seu padrão de transcrição ao longo do ciclo de vida do parasita. Foi estabelecido que a proteína pertence à família de deubiquitinases Cys-dependentes e mais especificamente para proteases específicas de ubiquitina (USPs por ubiquitin specific proteases). Além disso, o centro catalítico com a tríade completo, bem como as características típicas do motivo USP também foram identificados. Uma vez que os resultados computacionais sugerem que a enzima poderia ser funcional, a transcrição reversa acoplada a PCR foi utilizado como uma primeira abordagem para determinar se, de facto, o gene codificante é expressa no parasita. Curiosamente, verificou-se não só que o gene é expresso, mas também que há uma variação de transcrição ao longo do ciclo de vida do parasita. Estes dois elementos são o ponto de partida para estudos posteriores, uma vez que tentativas sugerem que esta enzima pode estar envolvida no refill de proteínas que ocorre durante o parasita encistamento. Embora preliminares, este estudo é o primeiro relatório relativo ao estudo de uma enzima deubiquitinadora específica no parasita intestinalis.

Conservation and developmental expression of ubiquitin isopeptidases in Schistosoma mansoni

Several genes related to the ubiquitin (Ub)-proteasome pathway, including those coding for proteasome subunits and conjugation enzymes, are differentially expressed during the Schistosoma mansoni life cycle. Although deubiquitinating enzymes have been reported to be negative regulators of protein ubiquitination and shown to play an important role in Ub-dependent processes, little is known about their role in S. mansoni . In this study, we analysed the Ub carboxyl-terminal hydrolase (UCHs) proteins found in the database of the parasite’s genome. An in silico ana- lysis (GeneDB and MEROPS) identified three different UCH family members in the genome, Sm UCH-L3, Sm UCH-L5 and Sm BAP-1 and a phylogenetic analysis confirmed the evolutionary conservation of the proteins. We performed quantitative reverse transcription-polymerase chain reaction and observed a differential expression profile for all of the investigated transcripts between the cercariae and adult worm stages. These results were corroborated by low rates of Z-Arg-Leu-Arg-Gly-Gly-AMC hydrolysis in a crude extract obtained from cercariae in parallel with high Ub conjugate levels in the same extracts. We suggest that the accumulation of ubiquitinated proteins in the cercaria and early schistosomulum stages is related to a decrease in 26S proteasome activity. Taken together, our data suggest that UCH family members contribute to regulating the activity of the Ub-proteasome system during the life cycle of this parasite.