The role of SUMOylation in the protective mechanisms of therapeutic hypothermia on neonatal hypoxic-ischemic encephalopathy / 中国新生儿科杂志

Objective:To study the role of SUMOylation in the process of therapeutic hypothermia on neural stem cells (NSCs) in neonatal hypoxic-ischemic encephalopathy.Methods:SUMOylation is an essential post-translational modification involving small ubiquitin-like modifiers (SUMOs). Primary-cultured NSCs from mice were assigned into four groups: control group, hypoxia group, hypothermia group and hypoxia+hypothermia group. Western Blot was used to detect the protein levels of SUMO2/3, hypoxia-inducible factor-1α (HIF-1α), peroxisome proliferator-activated receptor γ coactivator factor 1α (PGC-1α) and octamer binding transcription factor 4 (Oct4). The diameters of NSCs were compared. ELISA was used to detect lactate dehydrogenase (LDH) level. Apoptosis was examined using flow cytometry. Immunofluorescence method was used to measure the differentiation of NSCs into neuronal cells.Results:Compared with the control group, the levels of SUMO2/3, HIF-1αand PGC-1α in NSCs of the hypoxia group increased 33%, 126% and 140%, respectively ( P<0.05). Compared with the control group, the levels of SUMO2/3 and PGC-1α in NSCs of the hypothermia group increased 52% and 536%, respectively ( P<0.05). Compared with the hypoxia group, the levels of SUMO2/3, HIF-1α, PGC-1α and Oct4 in the hypoxia+hypothermia group increased 44%, 40%, 230% and 59%, respectively ( P<0.05). The diameters of NSCs in hypoxia group, hypothermia group and hypoxia+hypothermia group were smaller than control group, and hypoxia+hypothermia group smaller than hypoxia group ( P<0.05). No significant differences existed in LDH levels between hypothermia group and control group ( P>0.05). LDH level in hypoxia+hypothermia group were significantly lower than hypoxia group ( P<0.05). No significant differences existed in the cell death rates between hypothermia group and control group ( P>0.05). The cell death rate in hypoxia+hypothermia group was significantly lower than hypoxia group ( P<0.05). Compared with the control group, the expressions of Nestin in both hypoxia group and hypothermia group were increased, but neuron specific enolase (NSE) were decreased ( P<0.05). Compared with hypoxia group and hypothermia group, the level of Nestin in hypoxia+hypothermia group was further increased, while NSE was further decreased ( P<0.05). Conclusions:Therapeutic hypothermia may increase the tolerance of NSCs to hypoxia by enhancing SUMO modification of proteins, providing theoretical basis for the treatment of hypoxic-ischemic encephalopathy with therapeutic hypothermia.

Research progress on linear ubiquitin chain assembly complex and OTU deubiquitinase with linear linkage specificity in tumor / 药学实践杂志

Linear ubiquitination is an important post-translational modification that has been discovered in recent years. The linear ubiquitin chain is formed by the linkage of glycine residue of one ubiquitin protein to the methionine residue of another ubiquitin. This process is regulated by the linear ubiquitin chain assembly complex (LUBAC) and the OTU deubiquitinase with linear linkage specificity (OTULIN). Linear ubiquitination is involved in various biological processes, including immune response, inflammation, and cell apoptosis. Recent studies have shown that linear ubiquitination is closely related to the occurrence, development, and drug resistance of tumors by affecting signaling pathways such as NF-κB and Wnt/β-catenin. The research progress on the function of LUBAC and OTULIN in tumors was reviewed in this paper.

Dioscin Inhibits Ubiquitin-Proteasome System in Cancer Cells / 肿瘤防治研究

Objective To explore the antitumor small molecules targeting the ubiquitin–proteasome system (UPS) on the basis of active molecules from traditional Chinese medicine. Methods UbG76V-GFP stably expressing cell line was constructed to screen novel small molecule inhibitors targeting UPS. The fluorogenic substrates of Suc-LLVY-AMC, Z-LLE-AMC, and Boc-LRR-AMC were used to assess the effect of dioscin on the 20S proteasome hydrolase activity. The Ub-AMC substrate was used to evaluate the effect of dioscin on the intracellular deubiquitinating enzyme activity. Western blot was used to detect the effect of dioscin on intracellular ubiquitination levels. CCK-8 and colony formation assays were used to detect the inhibitory effect of dioscin on the tumor cell proliferation. Results Dioscin is a UPS inhibitor discovered through the UbG76V-GFP reporter system. It enhances intracellular ubiquitination and inhibits tumor cell proliferation and colony formation by targeting deubiquitinating enzymes. Conclusion Dioscin could significantly inhibit tumor cell proliferation by targeting ubiquitin–proteasome.

The dichotomous role of immunoproteasome in cancer: Friend or foe?

Immunoproteasome is a variant of proteasome with structural differences in 20S subunits optimizing them for the production of antigenic peptides with higher binding affinity to major histocompatibility complex (MHC)-I molecules. Apart from this primary function in antigen presentation, immunoproteasome is also responsible for the degradation of proteins, both unfolded proteins for the maintenance of protein homeostasis and tumor suppressor proteins contributing to tumor progression. The altered expression of immunoproteasome is frequently observed in cancers; however, its expression levels and effects vary among different cancer types exhibiting antagonistic roles in tumor development. This review focuses on the dichotomous role of immunoproteasome in different cancer types, as well as summarizes the current progression in immunoproteasome activators and inhibitors. Specifically targeting immunoproteasome may be a beneficial therapeutic intervention in cancer treatment and understanding the role of immunoproteasome in cancers will provide a significant therapeutic insight for the prevention and treatment of cancers.

Effects of contusion and exhaustive exercise on murf1 and mafbx in the skeletal muscle of rats / Efectos de la contusión y del ejercicio exhaustivo sobre murf1 y mafbx en el músculo esquelético de ratas / Efeitos do trauma contuso e do exercício exaustivo no em murf1 e mafbx no músculo esquelético de ratos

ABSTRACT Objective To study the effects of contusion and exhaustive exercise on the expression of degradation-related factors MuRF1 and MAFbx in the skeletal muscle of rats and describe the repair mechanism of skeletal muscle injury. Methods Forty-two male SD rats were randomly divided into 7 groups. The rats in each group were killed at different time points (0h, 24h, 48h) after exhaustive exercise (E0, E24, E48) and contusion (D0, D24, D48), respectively, and in the resting state in control group (C). The right gastrocnemius muscles were resected and divided into two parts, one for the mRNAs of MuRF1 and MAFbx by real-time PCR, and the other for protein measurement by Western blotting. Results Compared with the control group, the MuRF1 mRNA and protein expression of the skeletal muscle in the E0 group was markedly increased (P <0.05) and followed by a downward trend in E24 the E48 groups. On the other hand, MuRF1 mRNA expression of the skeletal muscle in the D24 group was significantly upregulated (P <0.01), then decreased in the D48 group (P <0.01). Meanwhile, compared with the C group, MAFbx mRNA gene expression continued to be upregulated in D24 and D48 (P <0.05), but decreased in E24 and E48 (p<0.01). On the other hand, the NF-κB protein contents of the skeletal muscle in the D0, D24, and D48 groups, as well as in the E48 group, were markedly downregulated (P <0.05), and the one in E48 was also remarkably downregulated (P <0.05). Conclusion NF-κB may negatively regulate the process of protein degradation by the NF-κB / MuRF1 signal pathway. Level of evidence III; Therapeutic studies investigating the results of treatment.

RESUMEN Objetivo Estudiar los efectos de la contusión y del ejercicio exhaustivo sobre la expresión de los factores relacionados con la degradación MuRF1 y MAFbx en el músculo esquelético de ratas y describir el mecanismo de reparación de la lesión muscular esquelética. Métodos Cuarenta y dos ratas macho SD fueron divididas aleatoriamente en 7 grupos. Las ratas de cada grupo fueron sacrificadas en diferentes momentos (0h, 24h, 48h) después del ejercicio exhaustivo (E0, E24, E48) y de la contusión (D0, D24, D48), respectivamente, y en estado de reposo en el grupo de control (C). Se resecaron los músculos gastrocnemios derechos y se dividieron en dos partes, una para los ARNm de MuRF1 y MAFbx mediante PCR en tiempo real y la otra para la medición de proteínas mediante Western blot. Resultados En comparación con el grupo control, el ARNm de MuRF1 y la expresión proteica del músculo esquelético en el grupo E0 se incrementó notablemente (P <0,05) y fueron seguidos por una tendencia a la baja en los grupos E24 y E48. Por otra parte, la expresión del ARNm de MuRF1 del músculo esquelético en el grupo D24 fue significativamente regulada al alza (P <0,01), y luego disminuyó en el grupo D48 (P <0,01). Mientras tanto, en comparación con el grupo C, la expresión génica del ARNm de MAFbx permaneció regulada al alza en D24 y D48 (P <0,05), pero disminuyó en E24 y E48 (p<0,01). Por otro lado, el contenido de proteína NF-κB del músculo esquelético en los grupos D0, D24 y D48, así como en el grupo E48, se vio notablemente regulado a la baja (P <0,05), y el del grupo E48 también se vio notablemente regulado a la baja (P <0,05). Conclusión NF-κB puede regular negativamente el proceso de degradación de la proteína a través de la vía NF-κB / MuRF1. Nivel de evidencia III; Estudios terapéuticos que investigan los resultados del tratamiento.

RESUMO Objetivo Estudar os efeitos do trauma contuso e do exercício exaustivo na expressão dos fatores relacionados à degradação MuRF1 e MAFbx no músculo esquelético de ratos e descrever o mecanismo de reparo da lesão muscular esquelética. Métodos Quarenta e dois ratos SD machos foram divididos aleatoriamente em 7 grupos. Os ratos de cada grupo foram mortos em diferentes momentos (0h, 24h, 48h) após exercício exaustivo (E0, E24, E48) e trauma contuso (D0, D24, D48), respectivamente, e no estado de repouso no grupo controle (C). Os músculos gastrocnêmios direitos foram ressecados e divididos em duas partes, uma para os mRNAs de MuRF1 e MAFbx por PCR em tempo real e outra para a medição de proteínas a partir do Western blot. Resultados Em comparação com o grupo controle, o mRNA de MuRF1 e a expressão proteica do músculo esquelético no grupo E0 foram acentuadamente aumentados (P <0,05) e seguidos por uma tendência descendente nos grupos E24 e E48. Por outro lado, a expressão do mRNA de MuRF1 do músculo esquelético no grupo D24 foi significativamente regulada para cima (P <0,01), depois diminuiu no grupo D48 (P <0,01). Enquanto isso, em comparação com o grupo C, a expressão gênica do mRNA de MAFbx continuou regulada para cima em D24 e D48 (P <0,05), mas diminuiu em E24 e E48 (p<0,01). Por outro lado, os teores de proteína NF-κB do músculo esquelético nos grupos D0, D24 e D48, bem como no grupo E48, foram marcadamente regulados para baixo (P <0,05), e o do grupo E48 também foi notavelmente regulado para baixo (P <0,05). Conclusão NF-κB pode regular negativamente o processo de degradação da proteína pela via NF-κB / MuRF1. Nível de evidência III; Estudos terapêuticos que investigam os resultados do tratamento.

Salvianolic acid B exerts its anti-tumor immunity by targeting USP2 and reducing the PD-L1 level / 药学学报

With the development of small-molecule immunotherapy drugs, its combination with the programmed cell death ligand 1/programmed cell death protein 1 (PD-L1/PD-1) antibodies would provide a new opportunity for cancer treatment. Therefore, targeting PD-L1/PD-1 axis by small-molecule drug is an attractive approach to enhance antitumor immunity and considered as the next generation of tumor immunotherapy. In the present study, we investigated the anti-tumor role of salvianolic acid B (SAB) by regulating the PD-L1 level in tumors. Changes of total PD-L1 and membrane PD-L1 levels were determined by Western blot, flow cytometry and PD-1/PD-L1 interaction assays. The expression of mRNA level of PD-L1 was detected by real-time PCR. The cytotoxicity of activated peripheral blood mononuclear cell (PBMC) cells toward co-cultured tumor cells was measured by cell impedance assay and crystal violet experiment. Surface plasma resonance technique was used to analyze the direct interaction between SAB and ubiquitin carboxyl-terminal hydrolase 2 (USP2). The antitumor effect of SAB in vivo was examined by C57BL/6 mice bearing MC38 xenograft tumor (all animal experiments were conducted in accordance with the Animal Ethics Committee of the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences). Western blot and flow cytometry assay showed that SAB can significantly downregulate the abundance of PD-L1 in RKO and PC3 cells in dose- and time-dependent manner. PD-1/PD-L1 binding assay revealed that SAB reduces the binding of tumor cells to recombinant PD-1 protein. Mechanism studies revealed that SAB can bind directly to USP2 protein and inhibit its activity, thus promote the ubiquitin-proteasome pathway degradation of PD-L1 proteins. In addition, Cell impedance and crystal violet staining indicated that SAB enhances the killing activity of co-cultured PBMC cells toward tumor cells. MC38 tumor transplanted mouse experiments revealed that SAB treatment displayed significant suppression in the growth of MC38 tumor xenografts in C57BL/6 mice with an inhibition rate of 63.2% at 20 mg·kg-1. Our results demonstrate that SAB exerts its anti-tumor activity by direct binding and inhibiting the activity of USP2 and reducing the PD-L1 level. Our study provides an important material basis and scientific basis for the potential application of SAB in tumor immunotherapy drug targeting USP2-PD-L1 axis.

Advances and prospects in targeted protein degradation / 药学实践杂志

Targeted protein degradation (TPD) techniques eliminate pathogenic proteins by hijacking the intracellular proteolysis machinery which includes the ubiquitin-proteasome system (UPS) and the lysosomal degradation pathway, holding promise to overcome the limitations of traditional inhibitors and further broaden the target space including many “undruggable” targets, and provide new targeted treatments for drug discovery. In this review, recent advances in a variety of promising TPD strategies were summarized, such as proteolysis targeting chimera (PROTAC), molecular glue, lysosome-targeting chimaera (LYTAC), autophagosome-tethering compound (ATTEC), autophagy-targeting chimera AUTAC and AUTOTAC, particularly. The representative case studies, potential applications and challenges were analyzed.

Research advance on ubiquitin-specific protease 14 as novel carcinogenesis biomarker / 环境与职业医学

Hazardous environmental factors as well as occupational factors can lead to elevated incidence of diseases including tumors, and specific molecular biomarkers are needed to guide the diagnosis and treatment of diseases. In recent years, ubiquitin-specific protease 14 (USP14) has gradually attracted the attention of researchers. USP14 is widely expressed in various organs of human body and regulates the stability and degradation of important proteins in various signaling pathways. Studies have shown that its abnormal expression is highly correlated with tumors, neurodegenerative diseases, autophagy, immune response, and viral infections, and is involved in the regulation of various classic signaling pathways. It has been shown to play a key role in the development of various human diseases and can be used as a diagnostic and prognostic molecular biomarker and therapeutic target in the development of tumors. This paper reviewed the current status of research on the structure and regulation of USP14 and its function in physiological and pathological processes, with the aim of providing a reference for research on diseases or injuries caused by environmental and occupational factors.

Identification and study of regulatory axis that miRNA-224-5p targets NEDD4L gene in clear cell renal cell carcinoma / 肿瘤研究与临床

Objective:To screen key genes of renal clear cell carcinoma based on bioinformatics methods, identify possible microRNA (miRNA)-mRNA action axis, and explore the expression of related genes in clear cell renal cell carcinoma tissues and cells.Methods:Gene expression profiles of GSE40435 and GSE71302 datasets were obtained from the Gene Expression Omnibus (GEO) database. TCGA-KIRC datasets were obtained from The Cancer Genome Atlas (TCGA) database. R software was used to identify the differentially expressed mRNA and miRNA, and the functional enrichment analysis was performed. STRING database and Cytoscape software were used to perform the protein interaction analysis. The prognosis-related differentially expressed miRNA was evaluated by the Oncomir database. The potential targeted genes regulated by miRNA were determined by using TargetScan and miRDB targeted gene prediction tools. The tissue samples and clinicopathological features of 34 patients with clear cell renal cell carcinoma in the First Hospital of Shanxi Medical University from June to December 2021 were collected, and normal renal cell line 293T and clear cell renal cell carcinoma cell line 786O were selected. The real-time fluorescence quantitative polymerase chain reaction (qRT-PCR), was used to detect the relative expression of genes; Western blotting and immunohistochemical staining were used to detect the expression levels of the targeted proteins. The dual luciferase reporter gene assay was carried out to verify the targeting relationship between genes.Results:A total of 1 351 differentially expressed mRNA and 50 differentially expressed miRNA were screened and identified. The result of functional enrichment analysis suggested that the fatty acid metabolism pathway and xenobiotic metabolism pathway were suppressed in clear cell renal cell carcinoma, while the apoptosis and immune response pathways were activated. Protein interaction analysis suggested that the signal transduction and protein ubiquitination pathways might play a key role in clear cell renal cell carcinoma. The screening results showed that miRNA-224-5p (miR-224-5p) was most closely associated with clear cell renal cell carcinoma progression and was highly expressed in tumor tissues, and its prognosis-related target gene was NEDD4L. The relative expression of NEDD4L mRNA in clear cell renal cell carcinoma tissues and paraneoplastic tissues were 0.138±0.103 and 1.000±0.026 ( t = 46.23, P < 0.05), and the relative expression of miR-224-5p was 1.000±0.043 and 0.129±0.108 ( t = 45.28, P < 0.05). The differences of NEDD4L mRNA and miR-224-5p expressions in different grades and stages of clear cell renal cell carcinoma tissues were statistically significant (all P < 0.05). The expression of NEDD4L protein was decreased in clear cell renal cell carcinoma. The relative expression of NEDD4L gene in 293T and 786O cells were 1.000±0.125 and 0.210±0.044 ( t = 17.52, P < 0.05); the relative expressions of miR-224-5p gene were 0.209±0.049 and 1.000±0.234 ( t = 10.61, P < 0.05). The relative expressions of NEDD4L mRNA in miRNA mimic group and negative control group were 0.236±0.062 and 1.000±0.024, and the difference was statistically significant ( t = 43.56, P < 0.05). NEDD4L protein expression was reduced in the miRNA mimic group. Dual luciferase reporter gene assay suggested that NEDD4L was a direct target gene of miR-224-5p. Conclusions:In clear cell renal cell carcinoma, miR-224-5p targets and regulates NEDD4L expression, and this mechanism may be related to carcinogenesis and progression of clear cell renal cell carcinoma.

Research progress in the role of E3 ubiquitin ligases in viral infection / 中华微生物学和免疫学杂志

Ubiquitination modifications are a kind of post-translational modifications of proteins widely found in eukaryotes and involved in a variety of biological activities. E3 ubiquitin ligases are an important component of the ubiquitin system, with the function of specific recognition of substrate proteins and mediation of different types of ubiquitination modifications. They can regulate the function and life time of substrate proteins. Recent studies have shown that E3 ubiquitin ligases are widely involved in the regulation of the host innate immune response and can directly or indirectly influence viral infection. Moreover, viruses are able to encode or hijack E3 ubiquitin ligases in their long-term evolution, allowing them to play an important role in viral infection and replication cycle. This paper reviewed the progress in the mechanisms of E3 ubiquitin ligases in innate immune responses and viral infection in recent years.

Progress in the role of ubiquitin-proteasome system in hepatitis B virus X protein-mediated HBV infection and hepatocarcinogenesis / 中华微生物学和免疫学杂志

Chronic hepatitis B virus (HBV) infection is one of the leading causes of hepatocellular carcinoma (HCC). Ubiquitin-proteasome system (UPS) mediates the post-translational modification and degradation of protein in eukaryotic cells. Recent studies have revealed that UPS plays an important role in HBV infection and hepatocarcinogenesis. Targeting HBx to intervene in the progression of HBV infection or HBV-related HCC has become a research hotspot both domestically and internationally in recent years. This article reviewed the progress in HBx promoting HBV infection and hepatocarcinogenesis through UPS.

Relationship and associated mechanism of serum fibroblast growth factor 21 and sarcopenia in hemodialysis patients / 中华肾脏病杂志

Objective:To investigate the relationship between serum fibroblast growth factor 21 (FGF21) and sarcopenia in hemodialysis (HD) patients, and to explore the relationship between FGF21 and signal pathways related to skeletal muscle metabolism in uremic state at the cellular level.Methods:The data of the HD patients from the blood purification center of the Third Affiliated Hospital of Soochow University were collected in this prospective observational study between January 2018 and December 2019. Serum FGF21 concentration was detected by enzyme-linked immunosorbent assay (ELISA). Meanwhile, the skeletal muscle indexes (SMI) at the fourth thoracic vertebra (T4) and the first lumbar vertebra (L1) were assessed by chest CT. According to the T4 SMI and L1 SMI, the patients were divided into sarcopenia group and non-sarcopenia group. The relationship between serum FGF21 and sarcopenia was analyzed. The C2C12 mouse myoblasts were cultured in vitro, which were intervened with healthy human serum, healthy human serum+different concentrations of FGF21, uremic serum, uremic serum+different concentrations of FGF21. The expressions of muscle ring finger protein-1 (MURF1), muscle atrophy F-box (Atrogin-1), myogenic differentiation (MyoD) and myogenin (MyoG) were detected by Western blotting. Results:A total of 118 HD patients with age of (52.64±15.29) years were enrolled in the study, including 64 males (54.2%) and 54 females (45.8%). The images at T4 and L1 level assessed by chest CT could be acquired from 118 patients and 82 patients, respectively. According to the lowest sex-specific quartile ( P25) of T4 SMI (male < 59.92 cm 2/m 2, female < 46.75 cm 2/m 2) and the lowest sex-specific quartile ( P25) of L1 SMI (male < 29.02 cm 2/m 2, female < 24.50 cm 2/m 2), patients were divided into sarcopenia group and non-sarcopenia group, and there were 29(24.58%) and 20(24.39%) patients in the sarcopenia group, respectively. When the patients were divided into two groups according to the sex-specific lowest quartile of T4 SMI, although the serum FGF21 level in the sarcopenia group was higher than that in the non-sarcopenia group, there was no statistical significance between the two groups [448.52(183.96, 1 684.08) ng/L vs. 273.65 (152.83, 535.54) ng/L, Z=-1.741, P=0.082]. When the patients were divided into two groups according to the sex-specific lowest quartile of L1 SMI, the serum FGF21 level in the sarcopenia group was significantly higher than that in the non-sarcopenia group [460.95(188.91, 1 276.38) ng/L vs. 239.10(133.25, 466.36) ng/L, Z=-2.170, P=0.030]. Binary logistic regression analysis showed that higher serum FGF21 was an independent influencing factor for sarcopenia in HD patients regardless of whether the patients were divided into two groups according to the sex-specific lowest quartile of T4 SMI or the sex-specific lowest quartile of L1 SMI (T4 SMI grouping: OR=4.085, 95% CI 1.778-9.388, P=0.001; L1 SMI grouping: OR=7.327, 95% CI 1.841-29.160, P=0.005). At T4 and L1 levels, the area under the receiver operating characteristic curve of FGF21 in predicting sarcopenia in HD patients was 0.636(95% CI 0.494-0.779, P=0.036) and 0.684(95% CI 0.535-0.833, P=0.018), respectively. Cell experiment showed that compared with the uremic serum group, the expressions of MURF1 and Atrogin-1 in myotube cells were increased, while the expressions of MyoD and MyoG were significantly decreased in uremic serum+FGF21 group (both P < 0.05). Conclusions:Higher serum FGF21 is associated with an increased risk of sarcopenia in HD patients. FGF21 may increase the expression of ubiquitin proteasome system, reduce the synthesis and differentiation of skeletal muscle protein, and promote the occurrence of muscle atrophy in uremic patients

Role of PIAS-regulated SUMOylation of PPARγ in endogenous protective mechanism against endotoxin-induced acute lung injury in mice / 中华麻醉学杂志

Objective:To evaluate the role of small ubiquitin-associated modifier (SUMO) E3 ligase (PIAS)-regulated SUMOylation of peroxisome proliferator-activated receptor γ (PPARγ) in the endogenous protective mechanism against endotoxin-induced acute lung injury (ALI) in mice.Methods:Experiment Ⅰ Twenty-four clean-grade wild type male C57BL/6 mice, aged 6-8 weeks, weighing 18-22 g, were divided into 4 groups ( n=6 each) using a random number table method: control group (C group), ALI group, ALI+ PPARγ inducer TZD group (ALI+ T group) and ALI+ TZD+ SUMOylation inhibitor anacardic acid group (ALI+ T+ A group). Lipopolysaccharide (LPS) 15 mg/kg was injected into the tail vein to develop the ALI model. In ALI+ T+ A group, anacardic acid 5 mg/kg was intraperitoneally injected at 1 h before LPS administration. In ALI+ T group and ALI+ T+ A group, TZD 50 mg/kg was intraperitoneally injected at 30 min before LPS administration. The mice were sacrificed at 12 h after LPS administration, and the lung tissues were obtained to examine the pathological changes which were scored and to determine the wet/dry (W/D) weight ratio, and expression of PIAS1, PIAS2, PIAS3 and PIASy protein and mRNA (by Western blot or polymerase chain reaction). Experiment Ⅱ Mouse alveolar macrophages (MH-S cells) were cultured in vitro and divided into 4 groups ( n=5 each) using a random number table method: control group (C group), LPS group, LPS+ PIAS2 siRNA group (L+ P group) and LPS+ Con siRNA group (L+ C group). Cells were routinely cultured in group C. Cells were stimulated with 10 μg/ml LPS to develop the model of endotoxin challenge. PIAS2 siRNA 50 nmol/L and Con siRNA 50 nmol/L were transfected at 48 h before LPS was added in L+ P group and L+ C group, respectively. The cells were collected at 24 h of incubation with LPS to determine the cell viability, levels of M1 and M2 alveolar macrophages (by flow cytometry), expression of PIAS2 and PPARγ (by Western blot), co-expression of PPARγ-SUMO1 (by immunoprecipitation) and expression of tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) mRNA (by polymerase chain reaction). The ratio of M1/M2 was calculated. Results:Experiment Ⅰ Compared with C group, the lung injury scores and W/D ratio were significantly increased, and the expression of PIAS2 protein and mRNA was up-regulated in the other three groups ( P<0.05). Compared with ALI group, the lung injury scores and W/D ratio were significantly decreased, and the expression of PIAS2 protein and mRNA was up-regulated in ALI+ T group and ALI+ T+ A group ( P<0.05). Compared with ALI+ T group, the lung injury scores and W/D ratio were significantly increased, and the expression of PIAS2 protein and mRNA was down-regulated in ALI+ T+ A group ( P<0.05). There was no significant difference in the expression of PIAS1, PIAS3 and PIASy protein and mRNA in lung tissues among the four groups ( P>0.05). Experiment Ⅱ Compared with C group, the cell viability was significantly decreased, the expression of PPARγ and co-expression of PPARγ-SUMO1 was up-regulated, the levels of M1 and M2 macrophages and M1/M2 ratio were increased, the expression of TNF-α mRNA was up-regulated, and the expression of IL-10 mRNA was down-regulated in the other three groups, and PIAS2 expression was significantly up-regulated in L group and L+ C group ( P<0.05). Compared with L group, the cell viability was significantly decreased, the expression of PIAS2 and PPARγ and PPARγ-SUMO1 co-expression were down-regulated, the M1 macrophage level and M1/M2 ratio were increased, TNF-α mRNA expression was up-regulated, and the expression of IL-10 mRNA was down-regulated in L+ P group ( P<0.05), and no significant change was found in the parameters mentioned above in L+ C group ( P>0.05). Compared with L+ C group, the cell viability was significantly decreased, the expression of PIAS2 and PPARγ and co-expression of PPARγ-SUMO1 were down-regulated, the level of M1 alveolar macrophages and M1/M2 ratio were increased, the expression of TNF-α mRNA was down-regulated, and the expression of IL-10 mRNA was up-regulated in L+ P group ( P<0.05). Conclusions:PIAS2-regulated SUMOylation of PPARγ is the endogenous protective mechanism against endotoxin-induced ALI in mice, which may be related to inhibition of macrophage polarization into M1 type and alleviation of inflammatory responses.

Effect of selective cerebral mild hypothermia on SUMO2/3 modification of Drp1 in a rat model of cerebral ischemia-reperfusion / 中华麻醉学杂志

Objective:To evaluate the effect of selective cerebral mild hypothermia on small ubiquitin-like modifier 2/3 (SUMO2/3) modification of dynamin-related protein 1 (Drp1) in a rat model of cerebral ischemia-reperfusion (I/R).Methods:Sixty clean-grade healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 240-260 g, were divided into 4 groups ( n=15 each) using a random number table method: sham operation group (S group), cerebral I/R group (I/R group), selective cerebral mild hypothermia group (HT group) and normal temperature group (NT group). The operation was performed under the monitoring of cerebral temperature and rectal temperature.Only the cervical blood vessels were exposed in S group, while focal cerebral I/R was induced by 2 h middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion in anesthetized animals in the other three groups.In HT group and NT group, 4 and 37 ℃ normal saline was perfused through the left internal carotid artery at a rate of 80 ml·kg -1·h -1 for 15 min, respectively. Modified neurological severity score (mNSS) was assessed at 24 h of reperfusion. Then the rats were sacrificed under deep anesthesia, brains were removed, brain tissues were obtained for determination of the percentage of cerebral infarct size (by TTC staining), and the ischemic penumbra tissues in the cerebral cortex were removed for examination of the ultra-structural changes of mitochondria (with a transmission electron microscope) and for determination of the SUMO2/3 modification of Drp1 (by CO-IP), expression of total Drp1 (T-Drp1) and total cytochrome c (T-Cytc) (by Western blot), and expression of mitochondrial outer membrane Drp1 (M-Drp1) and cytoplasmic Cytc (C-Cytc) (by Western blot) after isolation of mitochondria and cytoplasm. Results:Compared with S group, the mNSS and percentage of cerebral infarct size were significantly increased, the expression of M-Drp1, T-Drp1, C-Cytc and T-Cytc was up-regulated, and SUMO2/3 modification of Drp1 in ischemic penumbra area was increased ( P<0.05), the fragmentation of mitochondria was aggravated, and cristae rupture and vacuolation were obvious in the other three groups. Compared with I/R group, the mNSS and percentage of cerebral infarct size were significantly decreased, the expression of M-Drp1, T-Drp1, C-Cytc and T-Cytc was down-regulated, SUMO2/3 modification of Drp1 was increased ( P<0.05), the fragmentation of mitochondria was significantly attenuated, and cristae rupture and vacuolation were weakened in HT group. There were no significant differences in these detection parameters between NT group and I/R group ( P>0.05). Conclusions:The mechanism by which selective cerebral mild hypothermia alleviates the cerebral I/R injury is related to increased SUMO2/3 modification of Drp1, decreased binding of Drp1 to mitochondrial outer membrane, and reduced mitochondrial excessive fission in rats.

Study on the inhibitory effect of UBE2T on radiosensitivity of lung adenocarcinoma / 中华放射肿瘤学杂志

Objective:To investigate the effect of ubiquitin binding enzyme 2T (UBE2T) on the radiosensitivity of lung adenocarcinoma and unravel its possible mechanism.Methods:A total of 45 patients pathologically diagnosed with different stages of lung adenocarcinoma and treated with radiotherapy in the Second Affiliated Hospital of Zunyi Medical University from March, 2019 to December, 2021 were enrolled, and the efficacy was evaluated according to response evaluation criteria in solid tumors (RECIST1.1). All patients were divided into radiosensitive group ( n=25) and radioresistant group ( n=20). Radiosensitive group was complete remission (CR)+partial remission (PR), and radioresistant group was stable disease (SD) + progression disease (PD). Immunohistochemistry (IHC) was used to calculate the score based on the staining intensity and the number of positive cells. Chi-square test was combined to analyze the correlation between the expression level of UBE2T in paraffin specimens of lung adenocarcinoma patients and the radiosensitivity of patients. Lentivirus UBE2T-interfered (UBE2Tsh) A549 and UBE2T-overexpressed SPC-A-1 lung adenocarcinoma cells and their respective controls were constructed for irradiation and colony formation assay. The survivor fraction curve was fitted by single-hit multi-target model. The DNA double-strand break (DSB) marker γH2AX foci were detected by immunofluorescence (IF). The expression levels of UBE2T, γH 2AX and Rad51 proteins were detected by Western blot. Cell cycle and apoptosis rate of A549 were determined by flow cytometry. Binary variables were statistically analyzed by Fisher's exact probability method and measurement data were assessed by t-test. Results:High-expression level of UBE2T was correlated with the radiosensitivity of lung adenocarcinoma patients ( P<0.05). UBE2Tsh improved the radiosensitivity of A549 lung adenocarcinoma cells, and the sensitizing enhancement ratio (SER) was 1.795. UBE2T overexpression decreased the radiosensitivity of SPC-A-1 lung adenocarcinoma cells with an SER of 0.293. γH2AX foci number per cell were significantly increased in UBE2Tsh A549 cells after irradiation ( P<0.01) . Compared with the control group, the expression level of γH2AX protein was up-regulated ( P<0.01)and that of Rad51 protein was down-regulated in UBE2Tsh A549 cells after radiation ( P<0.001). Compared with the control group, the expression level of γH2AX protein was down-regulated ( P<0.05) and that of Rad51 protein was up-regulated in UBE2T overexpressed SPC-A-1 cells ( P<0.001). The proportion of UBE2Tsh A549 cells in G 2 phase was decreased ( P<0.01) and cell apoptosis was increased ( P<0.001). Conclusions:UBE2T might promote the radioresistance of lung adenocarcinoma cells by enhancing DNA DSB repair induced by radiotherapy, inducing cell cycle G 2 phase arrest, and reducing cell apoptosis.

Research progress of tripartite motif-containing protein 28 in tumors / 中国医师杂志

Tripartite motif-containing protein 28 is a kind of macromolecular protein with E3 ubiquitin ligase, which belongs to an important member of the TRIM protein family. As a new molecular biomarker, it has attracted wide attention. TRIM28 is highly expressed in many kinds of malignant tumors, which is closely related to clinicopathological features, and is also involved in biological behaviors such as proliferation, apoptosis, migration and invasion of tumor cells. TRIM28 may be a potential marker and therapeutic target for clinical diagnosis and prognosis of tumors. This study reviews the structure and biological function of TRIM28, its relationship with malignant tumors and the molecular mechanism of signal transduction pathway.

Elevated phosphorylated ubiquitin level elicits neuro-degeneration by aggregating the decline of ubiquitin-dependent proteasomal degradation / 中国药理学与毒理学杂志

OBJECTIVE To determine the roles of phosphorylated ubiquitin(pUb)on ubiquitin-dependent proteasomal(UPS)degradation activity,and the roles of pUb on neurodegeneration.METHODS We use PTEN induced kinase 1(PINK1)to phosphorylate ubiquitin.The Ub/S65A cannot be phosphorylated by PINK1,and was used to antagonize the roles of pUb.The Ub/S65E was used to mimic the roles of pUb.The roles of pUb on UPS degradation activity were determined by immunoflu-orescence,Western blot and TIRF microscope at cellular and protein level.The roles of pUb on neurodegeneration were determined by behavior tests,immunofluorescence,Golgi staining,TEM,Western blot and proteomics sacle in mouse.RESULTS The level of soluble PINK1(sPINK1)and pUb increased in the neurons of aged mouse brain,and in the cells upon the administration of MG132,a proteasome inhibitor.The elevation of sPINK1 and pUb was accompanied by protein aggregation upon aging or the proteasomal inhibition.The pink1 knockout alleviated proteasomal inhibition induced protein aggregation and association of ubiquitinated proteins with proteasome.The over-expression of sPINK1 increased pUb level in hippocampal neuron,which chronically induced protein aggregation,mitochondrial damage and damage the structure of neuronal spines.Such neuronal injury lead to cognitive impairment of mice.The roles of sPINK1 was reversed by co-expression with Ub/S65A,and was mimic by over-expression with Ub/S65E.CONCLUSION The phosphorylation of ubiquitin aggravates UPS degrada-tion,and accelerates neuronal degeneration upon the decline of proteasomal degradation in aging and age-related neuronal diseases.

F-box motif encoding genes as targets for the development of stress-tolerant traits in Saccharomyces cerevisiae

Stress tolerance is a useful trait actively sought by the bioprocess industry for biofactories dealing with bioconversion of varied raw materials or carbon sources. Fungal or yeast species are useful in bioconversion and are sustainable bioresources for biochemicals and biofuel production. Genetic manipulation strategies are in practice to enhance the tolerance against stress agents for the improved bioconversion process. In this review, we highlight the importance of the F-box motif encoding genes and their interactions in imparting the stress tolerance phenotype to the yeast species. The F-box motif proteins constitute a part of the SCF-E3 ligase complex and are involved in the recruitment, and ubiquitination, followed by degradation of the substrate proteins by the 26S proteasome. It highlights the current scenario on the F-box motif encoding genes and their interaction partners as targets for the stress tolerance phenotype in the yeast and plant species and their utility in the bioconversion processes.

Effects of SKP1 gene interference on apoptosis and regulation mechanism of ubiquitin-proteasome system in Parkinson's disease cell model / 中华行为医学与脑科学杂志

Objective:To investigate the effect of interfering S-phase kinase associated protein 1 (SKP1) gene on apoptosis in Parkinson's disease(PD) cell model induced by 1-methyl-4-phenyl-pyridine ion (MPP+ ) and the mechanism of ubiquitin proteasome system degradation of α-synuclein (α-syn) influence.Methods:SH-SY5Y cells were divided into control group, MPP+ group, SKP1 interference group, and SKP1 interference+ MG132(UPS inhibitor) group.The cells in the control group were cultured normally. The cells in the latter three groups were incubated with MPP+ (0.5 mmol/L) for 24 h as PD model cells.The cells in SKP1 interference group were transfected with lentivirus SKP1-siRNA, and the cells in SKP1 interference+ MG132 group were transfected with lentivirus SKP1 siRNA and added with MG132 (0.5 μmol/L) for 24 h. The protein levels and mRNA levels of SKP1, microtubule-associated protein light chain 3 (LC3), lysosome-associated membrane protein (LAMP), α-syn, ubiquitin activating enzyme E1 (UBE1), parkin, and p27 in cells were detected by Western blot and RT-PCR.Flow cytometry was used to detect cell apoptosis and cycle level, and CCK-8 method was used to detect cell proliferation level.Co-immunoprecipitation method was used to explore the interaction between SKP1 and p27. SPSS 23.0 software was used for statistical analysis. One-way ANOVA was used for comparison among groups, and LSD test was used for further pairwise comparison.Results:RT-PCR and Western blot results showed that the mRNA levels and protein levels of autophagy related proteins and ubiquitin related proteins LC3, LAMP2, α-syn, UBE1, parkin and p27 in the four groups were statistically significant(mRNA: F=99.155, 43.028, 138.464, 28.200, 22.009, 28.147, all P<0.05; F=245.517, 157.634, 315.920, 2 336.472, 477.429, 2 350.201, all P<0.05). The mRNA and protein levels of LC3, Lamp2, α-syn and p27 in SKP1 interference group were lower than those in MPP+ group (all P<0.05), while the mRNA and protein levels of UBE1 and parkin were higher than those in MPP+ group (all P<0.05). The mRNA and protein levels of LC3, α-syn and p27 in SKP1 interference+ MG132 group were higher than those in SKP1 interference group (all P<0.05), and the mRNA and protein levels of UBE1 and parkin were lower than those in SKP1 interference group (all P<0.05). The results of flow cytometry and CCK-8 method showed that the apoptosis rate and cell inhibition rate among the four groups were significantly different( F=2 749.420, 171.508, both P<0.05). The apoptosis rate of SKP1 interference group was lower than that of MPP+ group ((8.22±0.25)%, (15.30±0.21)%, P<0.05), while the cell inhibition rate of SKP1 interference group was lower than that of MPP+ group((26.31±3.73)%, (55.05±3.84)%, P<0.05). The apoptosis rate of SKP1 interference+ MG132 group ((9.49±0.07)%) was higher than that of SKP1 interference group, and the cell inhibition rate ((36.06±2.85)%) was higher than that of SKP1 interference group (both P<0.05). The results of immunoprecipitation method showed that P27 decreased after SKP1 immunoprecipitation. Conclusion:After SKP1 gene was interfered, the autophagy function of PD cells decreased, which may be related to parkin promoting α-syn ubiquitination, activating UBE1/ Parkin-mediated UPS pathway to degrade α-syn, and mediating P27 to inhibit apoptosis.

Ubiquitin-specific proteases 41 promotes the malignant phenotype and aoxorubicin-resistance by stabilizing RACK1 in triple negative breast cancer / 中华内分泌外科杂志

Objective:To investigate the expression of USP41 in triple-negative breast cancer (TNBC) and its correlation with malignant phenotype and adriamycin sensitivity.Methods:The expression of USP41 in TNBC resistant cell lines and clinical tissue samples was detected by Western blot and qPCR. Subsequently, the high expression of USP41 molecule was determined, and the role and possible mechanism of USP41 in the malignant phenotype and adriamycin resistance of TNBC were evaluated by cell biological methods such as CCK8, colony formation assay, transwell, Western blot, and CoIP-MS.Results:USP41 expression was significantly higher in triple-negative breast cancer samples than in adjacent non-cancerous tissues. USP41 expression was nearly 40-fold higher in the doxorubicin-resistant cell line MDA-MB-231/DXR, with an IC50 value of 6.86 μM. Interference with USP41 significantly increased the sensitivity of MDA-MB-231/DXR cells to doxorubicin. Interference with USP41 significantly inhibited cell proliferation, colony formation and migration of cells, with a decrease in the number of clones of 30%-80% and a decrease in the number of migrating cells of more than 70%, and the difference was statistically significant. In addition, USP41 knockdown improved the sensitivity of MDA-MB-231 cells to doxorubicin, with an IC50 decrease from 5.49 μM to 2.36 μM and 2.56 μM. CO-IP results showed that USP41 could directly interact with RACK1, and the expression of RACK1 was significantly higher in cancer tissues than in adjacent non-cancerous tissues. Interference with RACK1 inhibited MDA-MB-231 cell proliferation, with IC50 decreasing from 9.87 μM to 4.67 μM and 4.36 μM. Colony formation capacity decreased by more than 30% and the difference was statistically significant. USP41 knockdown decreased cell migration by more than 70% compared to control.Conclusion:High expression of USP41 is associated with malignant surface and adriamycin resistance in TNBC, and RACK1 may be a key molecule in the role of USP41.