The role of proteasome in muscle wasting of experimental arthritis

Abstract Background Rheumatoid arthritis is an autoimmune inflammatory disease that often leads patients to muscle impairment and physical disability. This study aimed to evaluate changes in the activity of proteasome system in skeletal muscles of mice with collagen-induced arthritis (CIA) and treated with etanercept or methotrexate. Methods Male DBA1/J mice were divided into four groups (n = 8 each): CIA-Vehicle (treated with saline), CIA-ETN (treated with etanercept, 5.5 mg/kg), CIA-MTX (treated with methotrexate, 35 mg/kg) and CO (healthy control group). Mice were treated two times a week for 6 weeks. Clinical score and hind paw edema were measured. Muscles were weighted after euthanasia and used to quantify proteasome activity, gene (MuRF-1, PMSα4, PSMβ5, PMSβ6, PSMβ7, PSMβ8, PSMβ9, and PSMβ10), and protein (PSMβ1, PSMβ5, PSMβ1i, PSMβ5i) expression of proteasome subunits. Results Both treatments slowed disease development, but only CIA-ETN maintained muscle weight compared to CIA-MTX and CIA-Vehicle groups. Etanercept treatment showed caspase-like activity of 26S proteasome similar to CO group, while CIA-Vehicle and CIA-MTX had higher activity compared to CO group (p: 0.0057). MuRF-1 mRNA expression was decreased after etanercept administration compared to CIA-Vehicle and CO groups (p: 0.002, p: 0.007, respectively). PSMβ8 and PSMβ9 mRNA levels were increased in CIA-Vehicle and CIA-MTX compared to CO group, while CIA-ETN presented no difference from CO. PMSβ6 mRNA expression was higher in CIA-Vehicle and CIA-MTX groups than in CO group. Protein levels of the PSMβ5 subunit were increased in CO group compared to CIA-Vehicle; after both etanercept and methotrexate treatments, PSMβ5 expression was higher than in CIA-Vehicle group and did not differ from CO group expression (p: 0.0025, p: 0.001, respectively). The inflammation-induced subunit β1 (LMP2) was enhanced after methotrexate treatment compared to CO group (p: 0.043). Conclusions The results of CIA-Vehicle show that arthritis increases muscle proteasome activation by enhanced caspase-like activity of 26S proteasome and increased PSMβ8 and PSMβ9 mRNA levels. Etanercept treatment was able to maintain the muscle weight and to modulate proteasome so that its activity and gene expression were compared to CO after TNF inhibition. The protein expression of inflammation-induced proteasome subunit was increased in muscle of CIA-MTX group but not following etanercept treatment. Thus, anti-TNF treatment may be an interesting approach to attenuate the arthritis-related muscle wasting.

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.

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

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.

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.

Advances in molecular function of p62 protein and its role in diseases / 生物工程学报

Sequestosome 1 (SQSTM1/p62) is a selective autophagy adaptor protein that plays an important role in the clearance of proteins to be degraded as well as in the maintenance of cellular proteostasis. p62 protein has multiple functional domains, which interact with several downstream proteins to precisely regulate multiple signaling pathways, thereby linking p62 to oxidative defense systems, inflammatory responses and nutrient sensing. Studies have shown that mutation or abnormal expression of p62 is closely related to the occurrence and development of various diseases, including neurodegenerative diseases, tumors, infectious diseases, genetic diseases and chronic diseases. This review summarizes the structural features and molecular functions of p62. Moreover, we systematically introduce its multiple functions in protein homeostasis and regulation of signaling pathways. Furthermore, the complexity and versatility of p62 in the occurrence and development of diseases are summarized, with the aim to provide a reference for understanding the function of p62 protein and facilitating related disease research.

Neuroprotective effects of the PPARß/δ antagonist GSK0660 in in vitro and in vivo Parkinson's disease models

BACKGROUND: The underlying mechanism of Parkinson's disease are still unidentified, but excitotoxicity, oxidative stress, and neuroinflammation are considered key actors. Proliferator activated receptors (PPARs) are transcription factors involved in the control of numerous pathways. Specifically, PPARß/δ is recognized as an oxidative stress sensor, and we have previously reported that it plays a detrimental role in neurodegeneration. METHODS: Basing on this concept, in this work, we tested the potential effects of a specific PPARß/δ antagonist (GSK0660) in an in vitro model of Parkinson's disease. Specifically, live-cell imaging, gene expression, Western blot, proteasome analyses, mitochondrial and bioenergetic studies were performed. Since we obtained promising results, we tested this antagonist in a 6-hydroxydopamine hemilesioned mouse model. In the animal model, behavioral tests, histological analysis, immunofluorescence and western blot of substantia nigra and striatum upon GSK0660 were assayed. RESULTS: Our findings suggested that PPARß/δ antagonist has neuroprotective potential due to neurotrophic support, anti-apoptotic and anti-oxidative effects paralleled to an amelioration of mitochondria and proteasome activity. These findings are strongly supported also by the siRNA results demonstrating that by silencing PPARß/δ a significative rescue of the dopaminergic neurons was obtained, thus indicating an involvement of PPARß/δ in PD's pathogenesis. Interestingly, in the animal model, GSK0660 treatment confirmed neuroprotective effects observed in the in vitro studies. Neuroprotective effects were highlighted by the behavioural performance and apomorphine rotation tests amelioration and the reduction of dopaminergic neuronal loss. These data were also confirmed by imaging and western blotting, indeed, the tested compound decreased astrogliosis and activated microglia, concomitant with an upregulation of neuroprotective pathways. CONCLUSIONS: In summary, PPARß/δ antagonist displayed neuroprotective activities against 6-hydroxydopamine detrimental effects both in vitro and in vivo models of Parkinson's disease, suggesting that it may represent a novel therapeutic approach for this disorder.

Mieloma múltiple en Chile. Uso de drogas noveles en dos centros nacionales

Background: The incorporation of novel drugs, such as proteasome inhibitors and immunomodulators, improved considerably the survival of patients with multiple myeloma. Aim: To evaluate the effect on survival of proteasome inhibitors and immunomodulators in patients with multiple myeloma in two national hospitals. Material and Methods: Review of clinical records from two hospitals of Santiago. Epidemiological, clinical, laboratory and therapeutic data was obtained from 144 patients with multiple myeloma diagnosed between 2002 and 2016. Results: Information was retrieved from 78 patients at one center and from 66 at the other center. The mean age at diagnosis was 58 and 62 years, the proportion of males was 53% and 52%, and presentation at stage III was 34% and 46%, respectively. The use of novel drugs, mainly bortezomib, was 90% in one of the centers and 3% in the other one. The use of autologous stem-cell transplantation was 47% and 3% respectively. The median overall survival of patients from the centers with and without access to novel drugs was 117 and 71 months respectively (p < 0.05). The five-year overall survival was 93 and 43% respectively (p < 0.05). Conclusions: The use of novel drugs, especially bortezomib, and autologous stem-cell transplantation significantly improved the survival of multiple myeloma patients treated in national hospitals. It is necessary to include them as a first line treatment.

Screening of the ubiquitin-proteasome system activators for anti-Alzheimer's disease by the high-content fluorescence imaging system / 中国天然药物

Ubiquitin-proteasome system (UPS) plays an important role in neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). The discovery of UPS activators for anti-neurodegenerative diseases is becoming increasingly important. In this study, we aimed to identify potential UPS activators using the high-throughput screening method with the high-content fluorescence imaging system and validate the neuroprotective effect in the cell models of AD. At first, stable YFP-CL1 HT22 cells were successfully constructed by transfecting the YFP-CL1 plasmid into HT22 cells, together with G418 screening. The degradation activity of the test compounds via UPS was monitored by detecting the YFP fluorescence intensity reflected by the ubiquitin-proteasome degradation signal CL1. By employing the high-content fluorescence imaging system, together with stable YFP-CL1 HT22 cells, the UPS activators were successfully screened from our established TCM library. The representative images were captured and analyzed, and quantification of the YFP fluorescence intensity was performed by flow cytometry. Then, the neuroprotective effect of the UPS activators was investigated in pEGFP-N1-APP (APP), pRK5-EGFP-Tau P301L (Tau P301L), or pRK5-EGFP-Tau (Tau) transiently transfected HT22 cells using fluorescence imaging, flow cytometry, and Western blot. In conclusion, our study established a high-content fluorescence imaging system coupled with stable YFP-CL1 HT22 cells for the high-throughput screening of the UPS activators. Three compounds, namely salvianolic acid A (SAA), salvianolic acid B (SAB), and ellagic acid (EA), were identified to significantly decrease YFP fluorescence intensity, which suggested that these three compounds are UPS activators. The identified UPS activators were demonstrated to clear AD-related proteins, including APP, Tau, and Tau P301L. Therefore, these findings provide a novel insight into the discovery and development of anti-AD drugs.

Effects of oxidative stress, PSMB5, TFEB, and lysosomes on sodium arsenite-induced liver injury in rats / 环境与职业医学

Background Liver damage presented in endemic arsenic poisoning is usually serious. Studies have shown that oxidative stress, proteasome beta 5 subunit (PSMB5), regulatory transcription factor EB (TFEB), and lysosomes are associated with liver injury, but their specific links to arsenic-induced liver injury remain unclear. Objective Using a sodium arsenite (NaAsO2)-induced rat liver injury model established earlier by the research group, the expressions of PSMB5, TFEB, and lysosomal associated membrane protein 1 (LAMP1) in liver tissues were detected. Methods Twenty-four SPF Wistar rats were randomly divided into control group, and low, medium, and high dose groups, with 6 rats in each group, half male and half female. The exposure concentrations were 0, 25, 50, and 100 mg·L−1 NaAsO2 solutions for 24 weeks. At the end of the experiment, liver was dissected after rats were anesthetized. The levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT), total bile acid (TBA), and catalase (CAT) in liver tissues were detected by chemical colorimetry, and the levels of lipid peroxide (LPO), 4-hydroxynonenal (4-HNE), LAMP1, and cathepsin D (CTSD) in liver tissues were detected by enzyme-linked immunosorbent assay (ELISA); the transcriptional expression levels of PSMB5 and TFEB in liver tissues were detected by real-time fluorescence quantitative PCR (RT-qPCR), and the protein expressions of PSMB5, TFEB, and phosphorylated TFEB (p-TFEB) in liver tissues were detected by immunohistochemistry. Results The results of chemical colorimetry and ELISA showed that compared with the control group, the liver homogenate levels of ALP, TBA, and LAMP1 of each arsenic-exposed group, the ALT and LPO in the medium and high concentration groups, the 4-HNE and CTSD in the high concentration group were increased, while the CAT activity of each arsenic-exposed group was decreased (P<0.05). The results of real-time fluorescence quantitative PCR showed that the transcription levels of PSMB5 and TFEB in the liver tissues of each arsenic-exposed group were decreased compared with those of the control group (P<0.05). The results of immunohistochemistry showed that compared with the control group, the expression of PSMB5 of each arsenic-exposed group were decreased, the expression of TFEB in the medium and high concentration groups was decreased, while the expression of p-TFEB of each arsenic-exposed group was increased (P<0.05). The expression of TFEB protein gradually decreased in the nucleus, while the expression of p-TFEB protein gradually increased in the cytoplasm, but no expression of p-TFEB was found in the nucleus. The results of Pearson correlation analysis showed that PSMB5 in liver tissues was positively correlated with CAT (r=0.818, P＜0.05), and negatively correlated with 4-HNE and p-TFEB (r=−0.582, r=−0.899; P＜0.05); TFEB was negatively correlated with CTSD and LAMP1 (r=−0.457, r=−0.564; P＜0.05); CTSD was positively correlated with ALT and ALP (r=0.529, r=0.485; P＜0.05). Conclusion Long-term exposure to NaAsO2 can induce oxidative stress, inhibit the expression of PSMB5 and TFEB, promote the accumulation of p-TFEB in the cytoplasm, decrease the nuclear entry of active TFEB, damage the lysosome, and cause liver damage.

Ubiquitin-specific peptidase 22 (USP22) regulates the level and function of hepatitis B virus X protein / 中华微生物学和免疫学杂志

Objective:To investigate the effects of the interaction between ubiquitin-specific peptidase 22 (USP22) and hepatitis B virus X protein (HBx) on the protein level and the biological function of HBx.Methods:The interactions between HBx and USP22 were analyzed by GST pull-down, co-immunoprecipitation assay and confocal laser scanning assay. USP22 recombinant plasmids or specific siRNA were transiently co-transfected with HBx plasmids. Western blot were used to detect the protein level of HBx. The half-life and degradation pathway of HBx in the transfected cells treated with cycloheximide (CHX) or proteasome inhibitor MG132 were detected. In vivo ubiquitination assay was used to detect the ubiquitination of HBx with USP22 overexpression. Moreover, dual-luciferase reporter assay and colony formation assay were used to analyze the effects of USP22 on the biological function of HBx. Results:USP22 could interact with HBx in vivo and in vitro. USP22 significantly increased the stability of HBx and inhibited the proteasome-mediated degradation of HBx protein by reducing the ubiquitination of HBx, thereby enhancing the biological function of HBx. Conclusions:USP22 inhibited HBx protein degradation through ubiquitin-dependent proteasome pathway, thus enhancing the stability and biological function of HBx.

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.

Mechanism of Liu Junzitang in Treatment of Muscle Atrophy in Mice with Lung Cancer Cachexia Through Regulating STAT3/Ubiquitin Proteasome Pathway / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Liu Junzitang in preventing and treating muscle atrophy in mice with lung cancer cachexia based on the signal transducer and activator of transcription 3（STAT3）/ubiquitin proteasome pathway in vivo. MethodForty C57BL/6 mice aged six weeks were randomly divided into a blank group， a model group， a Liu Junzitang group， an inhibitor group （stattic group），and a Liu Junzitang + inhibitor group （combination group）， with eight mice in each group. The cachectic muscle atrophy model was induced by subcutaneous inoculation of Lewis lung cancer cell line under the right anterior armpit in mice except those in the blank group. On the 8th day after subcutaneous inoculation， the mice in the corresponding groups received Liu Junzitang （9.56 g·kg-1·d-1） by gavage and intraperitoneal injection of stattic ［25 mg·kg-1·（2 d）-1］. After three weeks of drug intervention， the body weight and gastrocnemius muscle weight were recorded. Hematoxylin-eosin （HE） staining was used to observe the pathological changes and cross-sectional area of gastrocnemius muscle fibers in mice. Western blot was used to detect the expression of phosphorylated-STAT3 （p-STAT3）， STAT3， muscle atrophy F-box （MAFbx）， and muscle RING finger protein 1 （MuRF1） in the gastrocnemius muscle. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of STAT3， MAFbx， and MuRF1 in the gastrocnemius muscle. ResultCompared with the blank group， the model group showed lightened body and the gastrocnemius muscle， reduced cross-sectional area of gastrocnemius muscle fibers， and increased protein expression of p-STAT3， STAT3， MAFbx， and MuRF1 and mRNA expression of STAT3， MuRF1， and MAFbx in the gastrocnemius muscle （P<0.05）. Compared with the model group， the Liu Junzitang group showed increased body weight， gastrocnemius muscle weight， and cross-sectional area of gastrocnemius muscle fibers （P<0.05）， and decreased protein expression of p-STAT3， STAT3， MuRF1， MAFbx， and mRNA expression of STAT3 and MAFbx in gastrocnemius muscle （P<0.05）. Compared with the model group， the inhibitor group showed increased body weight and cross-sectional area of gastrocnemius muscle fibers （P<0.05）， and reduced protein expression of p-STAT3， STAT3， MuRF1， MAFbx， and mRNA expression of STAT3 and MAFbx in gastrocnemius muscle （P<0.05）. Compared with the model group， the combination group showed increased body weight and gastrocnemius muscle weight （P<0.05），and decreased protein expression of p-STAT3， STAT3， MuRF1， and mRNA expression of STAT3 and MAFbx in the gastrocnemius muscle （P<0.05）. Compared with the Liu Junzitang group， the stattic group and the combination group showed reduced expression of p-STAT3 protein in the gastrocnemius muscle （P<0.05）. ConclusionLiu Junzitang can prevent and treat muscle atrophy in mice with lung cancer cachexia， and its mechanism may be associated with the protein and mRNA expression related to the STAT3-mediated ubiquitin proteasome pathway.

Ixazomib-Based Regimen Followed by Autologous Hematopoietic Stem Cell Transplantation in the Treatment of POEMS Syndrome / 中国实验血液学杂志

OBJECTIVE@#To investigate the safety and efficacy of a new proteasome inhibitor Ixazomib followed by autologous hematopoietic stem cell transplantation (AHSCT) in the treatment of POEMS syndrome.@*METHODS@#The clinical manifestations, diagnosis and treatment process and follow-up results of 4 patients with POEMS syndrome who were treated with Ixazomib-based regimen combined with AHSCT in Wuhan No.1 Hospital from February 2018 to July 2020 were analyzed retrospectively. All patients were male, aged from 37-54 years old, with varying degrees of peripheral neuropathy, organ enlargement (liver, spleen or lymph nodes), circulatory overload (peripheral edema and/or pleural effusion), osteosclerosis, endocrine diseases (thyroid, gonads, etc.), skin changes (pigmentation, hemangioma, white nails, etc.), M protein, papilledema and other clinical manifestations and characteristics at the time of initial treatment. Two patients were pathologically diagnosed as hyaline vascular Castleman disease by lymph node biopsy. Three patients underwent lumbar puncture examinations and all showed elevated cerebrospinal fluid protein. All patients received at least 2 cycles of sequential AHSCT after induction chemotherapy based on ixazomib. The follow-up time was 10-28 months, and the median follow-up time was 16 months.@*RESULTS@#All cases survived. The complications were controllable during the treatment. Moreover, the clinical symptoms related to the disease were improved to a certain extent after the treatment. The levels of vascular endothelial growth factor (VEGF) showed a gradual decline.@*CONCLUSION@#Ixazomib combined with AHSCT is safe and effective in the treatment of POEMS syndrome.

Experimental study of proteasome inhibitor MG132 up-regulates Wnt/β-catenin signaling pathway to improve osteoporosis / 中国骨伤

OBJECTIVE@#To explore the mechanism of proteasome inhibitor MG132 in improving osteoporosis.@*METHODS@#Total of 32 female SD rats, weighing 220 to 250 g and 8 weeks old, were selected. They were randomly divided into 4 groups(n=8). Rats of group A and group B were cut off ovaris on both sides to make model of osteoporosis, and then they were given proteasome inhibitors MG132 and dimethyl sufoxide (DMSO) respectively. Group C was a sham group and rats were given MG132. Group D was a normal group and rats were given MG132 too. The rats were killed in batches at 6 and 12 weeks after administration, and the femoral neck tissues were obtained. Relevant data were analyzed, such as pathomorphological observation, micro-CT analysis, detection of 20S proteasome activity in tissues, and expression of Wnt and β-catenin.@*RESULTS@#Morphological observation showed that the trabecular were slightly thinner, reticulated, and occasionally interrupted in group A, while the trabecular were obviously thinner and discontinuous in group B. And the trabecular were intact and arranged reticulated in group C and D. The analysis results of bone mineral density(BMD), bone surface(BS), bone volume/total volume(BV/TV) and trabecular thickness(Tb.Th) showed that group B was worse than other groups in all parameters at different time points(P<0.05), and group A was worse than group C and group D in BS(P<0.05), there was no significant difference in all parameters between group C and group D. RFU value of 20S proteasome in group B was significantly higher than that in other groups(P<0.05). According to the results of Western blot, the gray values of Wnt protein and β-catenin protein in group A were significantly higher than those in other groups (P<0.05).@*CONCLUSION@#MG-132, a ubiquitin proteasome inhibitor, can regulate Wnt/β-catenin signaling pathway by inhibiting the degradation of β-catenin protein, and delaying the occurrence and development of osteoporosis.

Research progress of small molecule proteasome activators / 药学学报

Proteasome controls the degradation of proteins closely related to life activities and plays a key role in the maintenance of protein homeostasis. Proteasome activities decrease with aging, followed by the overwhelming production of damaged proteins which far exceed the protein consumption. Accumulation of these proteins leads to various diseases including neurodegenerative diseases. Therefore, inducing toxic protein degradation is considered as a promising solution for the treatment of these diseases, while increasing the activity of proteasome is considered as an important strategy. However, the research in this field is still in the preliminary stage, and this review will focus on the discussion of the research progress of various small molecule proteasome activators, including research methods, pharmacological effects, structure-activity relationships and the existing problems.

Research on antitumor effects of small molecule inhibitors of deubiquitinases: new progress and new ideas / 药学学报

The abnormality of ubiquitin proteasome pathway is an important factor leading to the imbalance of protein homeostasis. In this process, the deubiquitinase responsible for removing the ubiquitin chain of protein substrate is very important. Its abnormal activity or expression can cause the functional changes of key oncogenic/tumor suppressor proteins, which directly or indirectly lead to the occurrence, development and malignant evolution of tumors. Based on this, the discovery and research of small molecule inhibitors targeting deubiquitinases have become a hot field of anti-tumor candidate drugs. This review will focus on the regulatory effect and mechanism of ubiquitin proteasome pathway, especially deubiquitinase on tumor, introduce the application of deubiquitinase small molecule inhibitors in tumor treatment, and discuss the research status and latest progress of small molecule inhibitors, so as to provide ideas for the research of new anti-tumor strategies based on deubiquitinase.

Research progress on proteasome subunits in regulating occurrence and development of hepatocellular carcinoma / 浙江大学学报·医学版

Proteasome is the eukaryotic organelle responsible for degradation of short-lived proteins and involved in maintaining cellular protein homeostasis. It has been reported that during the occurrence and development of hepatocellular carcinoma (HCC), the regulatory particle subunits of proteasome regulate a series of tumor-related proteins, and proliferation, survival-associated signaling molecules, including PTEN gene, P53, Bcl-2, Bcl-2 interacting mediator of cell death (Bim), cyclin-dependent kinase 4(CDK4), transforming growth factor β receptor (TGFBR), E2F1, growth factor receptor-bound protein 2 (GRB2) . Meanwhile, these subunits regulate some tumor-associated pathway protein, such as signal transducer and activator of transcription 3 (STAT3) and protein kinase B (AKT), inducing their malfunction to promote the occurrence, proliferation, invasion and metastasis of HCC. The core particle subunits are more to perform the degradation of HCC-related proteins, so inhibitors targeting the core particle show a good anti-tumor effect. This review summarizes the current research progress on the regulation and mechanism of proteasome subunits in promoting the occurrence and development .