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.

The mechanisms of type 2 diabetic skeletal muscle atrophy and the effects of commonly used hypoglycemic drugs: a review / 药学学报

Type 2 diabetes is a hypermetabolic disease characterized with disorders of glucose/lipid metabolism, absolute or relative lack of insulin, and can induce skeletal muscle atrophy. Hyperglycemia, hyperlipidemia, insulin resistance, and abnormal release of inflammatory factors can lead to abnormal signal transduction in skeletal muscle, thus make protein synthesis and degradation imbalance and eventually causing muscle atrophy. Under normal conditions, insulin-like growth factor 1 (IGF-1)/insulin can activate phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT). AKT not only increases protein synthesis through mammalian target protein of rapamycin (mTOR), but also phosphorylates forkhead box O (FoxO) transcription factor and then inhibits the transcription of several ubiquitin ligases (such as MAFbx/atrogin-1 and MuRF1), or autophagy related genes. The weakened IGF-1/PI3K/AKT pathway in type 2 diabetes is an important factor leading to skeletal muscle atrophy. Studies have shown that the commonly used anti-type 2 diabetic drugs have different effects in regulating the synthesis and degradation of skeletal muscle protein. Studies reported that drugs with effect of anti-diabetic muscle atrophy include thiazolidinediones, glucagon-like peptide analogs, glucose-sodium cotransporter 2 inhibitors, etc.; drugs that are still in controversial or even promote skeletal muscle atrophy include metformin, and some sulfonylurea or non-sulfonylurea insulin secretagogues. This article overviewed and analyzed the currently commonly used drugs for type 2 diabetes and summarized the related mechanisms, with the aim to provide references for the rational applications of drugs for type 2 diabetes.

Recombinant Expression and Purification of F-box and Leucine-rich Repeat Protein 5 (FBXL5) using a Prokaryotic Expression System

Background@#The F-box and Leucine-rich Repeat Protein 5 (FBXL5), a member of the E3 ligases, is considered to be the central iron sensor in mammals. The cryo-EM structure of FBXL5 in complex with IRP2 and SKP1 was reported by Wang et.al. in 2020. Surprisingly, a 2Fe-2S cluster seemed to be responsible for the iron-sensing capability of FBXL5. @*Objectives@#To further explore the mechanism of its regulation, it is important to study the interaction of FBXL5 with other proteins under regulated conditions so we attempted to express FBXL5 in the hopes of studying its interaction with IRPs in vitro. @*Methodology@#Plasmids were constructed to express FBXL5 in Escherichia coli expression hosts. Purification of an MBP-fused FBXL5 and GST-fused FBXL5 were performed using affinity chromatography. Peptide Mass Fingerprinting, Circular Dichroism spectroscopy, and SEC-MALS were employed to analyze the purified MBP-FBXL5. GST-FBXL5 was also used in a pull-down assay with Iron Regulatory Protein 1 (IRP1). @*Results@#We are successful in expressing and partially purifying full-length FBXL5 using E. coli with the aid of a protein tag, the maltose binding protein (MBP) tag. However, cleavage of the protein tag resulted in decreased stability of FBXL5 as shown in SEC-MALS data. CD spectroscopy showed consistent secondary structure of FBXL5. A preliminary pull-down assay of GST-FBXL5 with IRP1 showed that IRP1 displayed interaction with the recombinant GST-FBXL5. @*Conclusion@#FBXL5, a 78-kDa mammalian protein was overexpressed in a prokaryotic expression system made stable by a fusion protein. The interaction of GST-FBXL5 with IRP1 also shows that it is possible to study their interaction in vitro.

Effect of Gandou Decoction on Mitophagy in Toxic Milk （TX） Mouse Model of Wilson Disease Based on Pink1/Parkin Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effects of Gandou decoction （GDD） on the mitophagy of hippocampal neurons in toxic milk （TX） mouse model of Wilson disease and explore the protective mechanism of GDD against neuron injury through the PTEN induced kinase 1 （Pink1） /E3 ubiquitin ligase （Parkin） pathway. MethodSixty mice were randomly divided into a blank group， a model group， a penicillamine group （0.09 g·kg-1）， and low- （5.5 g·kg-1）， medium- （11 g·kg-1）， and high-dose （22 g·kg-1） GDD groups， and treated correspondingly by gavage for 8 weeks. Morris water maze， traction test， and pole test were used for the evaluation of animal behaviors. Hematoxylin-eosin （HE） staining and transmission electron microscopy were used to observe cell apoptosis， ultrastructure， autophagy， and mitochondrial structure. The levels of superoxide dismutase （SOD）， reactive oxygen species （ROS）， and malondialdehyde （MDA） were detected by enzyme-linked immunosorbent assay （ELISA）. Real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of Pink1， Parkin， autophagy-associated protein Beclin-1， microtubule-associated protein 1 light chain 3Ⅱ （LC3Ⅱ）， and p62. Western blot was conducted to detect the protein expression of Pink1， Parkin， Beclin-1， LC3Ⅱ/Ⅰ， and p62. ResultCompared with the blank group， the model group showed prolonged escape latency， decreased times of platform crossing， lower score in the traction test， and longer pole climbing time （P<0.01）. Compared with the model group， the medium- and high-dose GDD groups and the penicillamine group showed shortened escape latencies， increased times of platform crossing， higher scores in the traction test， and shortened pole climbing time （P<0.01）. Compared with the blank group， the model group displayed severely damaged neurons and increased autophagosomes. Compared with the model group， the medium- and high-dose GDD groups and the penicillamine group showed improved neuron damage and reduced autophagosomes. The levels of ROS and MDA were higher and SOD was lower in the model group than those in the blank group （P<0.01）， while the levels of the above indicators were reversed by GDD intervention as compared with the model group （P<0.01）. Compared with the blank group， the model group exhibited up-regulated mRNA and protein expression of Pink1， Parkin， LC3Ⅱ， and Beclin-1 and down-regulated p62 （P<0.05）. Compared with the model group， the medium- and high-dose GDD groups showed reduced mRNA and protein expression of Pink1， Parkin， LC3Ⅱ， and Beclin-1 and increased p62 （P<0.05， P<0.01）. ConclusionGDD can significantly inhibit the excessive mitophagy in neurons of TX mice and protect neurons from damage. The mechanism may be related to the regulation of the Pink1/Parkin pathway.

Signaling pathways involved in inhibition of macrophage phagocytosis by TcpC of uropathogenic Escherichia coli / 中华微生物学和免疫学杂志

Objective:To investigate the signaling pathway of inhibiting macrophage phagocytosis of TIR domain-containing protein encoded by Escherichia coli (TcpC) N-terminal ubiquitin ligase active fragments of uropathogenic Escherichia coli. Methods:Bioinformatics software was used to analyze the amino acid sequences and the function of TcpC N-terminal ubiquitin ligase active fragments as well as the functional sites. PCR was performed to amplify tcpc-330, tcpc-450 and tcpc-510 genes and a prokaryotic expression system was constructed to express the target proteins. The recombinant proteins rTcpC-N110, rTcpC-N150 and rTcpC-N170 were purified by Ni-NTA affinity chromatography. LPS in the recombinant proteins was removed by Detoxi-gel chromatography. The expression of MyD88 at protein and mRNA levels in macrophages incubated with rTcpC-N110, rTcpC-N150, rTcpC-N170 or rTcpC-TIR was detected by Western blot and qRT-PCR. The activation of NF-κB signal pathway and the levels of proinflammatory factors in macrophages incubated with the above TcpC protein fragments were measured by Western blot and ELISA, respectively. Results:Cys12, Trp104 and Trp106 in the N-terminal fragment of TcpC were crucial amino acids in maintaining its ubiquitin ligase activity. The target recombinant proteins rTcpC-N110, rTcpC-N150 and rTcpC-N170 were successfully expressed and purified. After Detoxi-gel chromatography, rTcpC-N110, rTcpC-N150 and rTcpC-N170 extracts were undetectable for LPS. TcpC ubiquitin ligase fragments inhibited the expression of MyD88 at protein level, but not affect its expression at mRNA level in macrophages. LPS-induced phosphorylation of NF-κB signaling pathway-related proteins p50 and p65 was significantly inhibited in macrophages treated with TcpC ubiquitin ligase fragments. Moreover, LPS-induced production of pro-inflammatory factors was also significantly inhibited.Conclusions:The recombinant proteins rTcpC-N110, rTcpC-N150 and rTcpC-N170 could inhibit the expression of MyD88 at protein level and suppress the activation of NF-κB signaling pathway, suggesting that they were closely related to the inhibition of innate immune activity of macrophages.

Speckle type POZ adaptor protein (SPOP) and its role in cancer / La proteína adaptadora Speckle-type POZ (SPOP) y su papel en cáncer

Abstract Proteasomal degradation is an essential regulatory mechanism for cellular homeostasis maintenance. The speckle-type POZ adaptor protein (SPOP) is part of the ubiquitin ligase E3 cullin-3 RING-box1 complex, responsible for the ubiquitination and proteasomal degradation of biomolecules involved in cell cycle control, proliferation, response to DNA damage, epigenetic control, and hormone signaling, among others. Changes in SPOP have been associated with the development of different types of cancer, since it can act as a tumor suppressor mainly in prostate, breast, colorectal, lung cancer and liver cancer, due to point mutations and/or reduced expression, or as an oncogene in kidney cancer by protein overexpression. In endometrial cancer it has a dual role, since it can act as a tumor suppressor or as an oncogene. SPOP is a potential prognostic biomarker and a promising therapeutic target.

Resumen La degradación proteosómica es un mecanismo de regulación esencial para el mantenimiento de la homeostasis celular. La proteína adaptadora Speckle-type POZ (SPOP) hace parte del complejo ubiquitin ligasa E3 cullin-3 RING-box1, encargado de la ubiquitinación y degradación proteosomal de biomoléculas involucradas en el control del ciclo celular, proliferación, respuesta al daño de ADN, control epigenético, señalización hormonal, entre otros. Las alteraciones en SPOP han sido asociadas al desarrollo de diferentes tipos de cáncer, ya que puede actuar como supresor tumoral principalmente en cáncer de próstata, mama, colorrectal y pulmón, debido a mutaciones puntuales y/o expresión reducida o como oncogén en cáncer riñón por sobreexpresión de la proteína. En cáncer endometrial tiene un rol dual, ya que puede actuar como supresor tumoral o como oncogén. SPOP es considerado como un potencial biomarcador pronóstico y un objetivo terapéutico prometedor.

mTOR-targeted cancer therapy: great target but disappointing clinical outcomes, why? / 医学前沿

The mammalian target of rapamycin (mTOR) critically regulates several essential biological functions, such as cell growth, metabolism, survival, and immune response by forming two important complexes, namely, mTOR complex 1 (mTORC1) and complex 2 (mTORC2). mTOR signaling is often dysregulated in cancers and has been considered an attractive cancer therapeutic target. Great efforts have been made to develop efficacious mTOR inhibitors, particularly mTOR kinase inhibitors, which suppress mTORC1 and mTORC2; however, major success has not been achieved. With the strong scientific rationale, the intriguing question is why cancers are insensitive or not responsive to mTOR-targeted cancer therapy in clinics. Beyond early findings on induced activation of PI3K/Akt, MEK/ERK, and Mnk/eIF4E survival signaling pathways that compromise the efficacy of rapalog-based cancer therapy, recent findings on the essential role of GSK3 in mediating cancer cell response to mTOR inhibitors and mTORC1 inhibition-induced upregulation of PD-L1 in cancer cells may provide some explanations. These new findings may also offer us the opportunity to rationally utilize mTOR inhibitors in cancer therapy. Further elucidation of the biology of complicated mTOR networks may bring us the hope to develop effective therapeutic strategies with mTOR inhibitors against cancer.

Degradation of proteins by PROTACs and other strategies

Blocking the biological functions of scaffold proteins and aggregated proteins is a challenging goal. PROTAC proteolysis-targeting chimaera (PROTAC) technology may be the solution, considering its ability to selectively degrade target proteins. Recent progress in the PROTAC strategy include identification of the structure of the first ternary eutectic complex, extra-terminal domain-4-PROTAC-Von-Hippel-Lindau (BRD4-PROTAC-VHL), and PROTAC ARV-110 has entered clinical trials for the treatment of prostate cancer in 2019. These discoveries strongly proved the value of the PROTAC strategy. In this perspective, we summarized recent meaningful research of PROTAC, including the types of degradation proteins, preliminary biological data in vitro and in vivo, and new E3 ubiquitin ligases. Importantly, the molecular design, optimization strategy and clinical application of candidate molecules are highlighted in detail. Future perspectives for development of advanced PROTAC in medical fields have also been discussed systematically.

Mechanism of Effect of Ubiquitination Modification Mediated by E3 Ubiquitin Ligase in Inflammatory Bowel Disease / 胃肠病学

Ubiquitination is the important type of post-translational modification of proteins, and its main effects include protein degradation and functional regulation. Studies have shown that abnormal ubiquitination is involved in the development and progress of inflammatory bowel disease (IBD), which makes some ubiquitinases and their antibodies having the potential to be important markers for clinical diagnosis and evaluation of severity of Crohn's disease (CD). This article reviewed the research on mechanism of effect of ubiquitination modification mediated by E3 ubiquitin ligase in IBD.

Effects of electroacupuncture on skeletal muscle and blood glucose in rats with diabetic amyotrophy / 中国针灸

OBJECTIVE@#To explore the effects of electroacupuncture (EA) on skeletal muscle and blood glucose in rats with diabetic amyotrophy.@*METHODS@#Among 40 SD rats, 10 rats were randomly selected into the control group and received no treatment. The remaining 30 rats were treated with intraperitoneal injection of streptozotocin (STZ, 60 mg/kg) to establish diabetes mellitus (DM) model, and then the rats were treated with vascular ligation at right posterior limb to establish amyotrophy model. The rats with diabetic amyotrophy were randomly divided into a model group and an EA group, 10 rats in each group (10 rats were excluded due to unsuccessful model establishment and death). The rats in the EA group was treated with EA at right-side "Yishu (EX-B 3)" "Shenshu (BL 23)" "Zusanli (ST 36)" and "Sanyinjiao (SP 6)", disperse-dense wave, 2 Hz/ 15 Hz, 20 minutes each time, once a day for 3 weeks. Before and after EA treatment, the blood sample was collected from inner canthus and the "glucose oxidase-peroxidase" method was used to detect fasting blood glucose level; ELISA method was used to detect insulin content. At the end of the treatment, HE staining method was used to observe the morphology of ischemic skeletal muscle in the right hindlimb; the real-time PCR method was used to detect the mRNA expression of muscle atrophy F-box (MAFbx), muscle ring finger-1 (MuRF1) and forkhead box O3a (FOXO3a) in the ischemic skeletal muscle tissue of right hindlimb.@*RESULTS@#Before the treatment, the body mass in the model group and EA group was lower than that in the control group (<0.01); after the treatment, the body mass in the control group was increased, while the body mass in the model group and EA group was decreased (<0.01). Compared with the control group, the fasting blood glucose was significantly increased and insulin content was significantly decreased in the model group (<0.01); compared with the model group, the fasting blood glucose was significantly decreased and the insulin content was significantly increased in the EA group after treatment (<0.01). The muscle fibers of the model group were obviously broken, the number of the nuclei decreased, and the nuclei shrinked or even dissolved; the morphology of the muscle tissue of the EA group after intervention was improved compared with the model group. Compared with the control group, the cross-sectional area of ischemic skeletal muscle cells in the right hindlimb in the model group was decreased (<0.01); compared with the model group, the cross-sectional area of ischemic skeletal muscle cells in the right hindlimb was increased in EA group (<0.05). Compared with the control group, the levels of MAFbx, MuRF1 and FOXO3a mRNA in the right hindlimb ischemic skeletal muscle in the model group were increased significantly (<0.01, <0.05); compared with the model group, the levels of MAFbx, MuRF1 and FOXO3a mRNA in the EA group were decreased significantly (<0.05, <0.01).@*CONCLUSION@#EA may play a role in the treatment of diabetic amyotrophy by inducing FOXO3a to reduce the transcription of MAFbx and MuRF1.

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.

- gene silencing enhances H9 T lymphocyte-mediated killing of human laryngeal squamous cancer Hep-2 cells / 南方医科大学学报

OBJECTIVE@#To investigate the effect of sputum ubiquitin ligase (Cbl-b) gene known-down on the cytotoxicity of H9 T lymphocytes against human laryngeal squamous cancer Hep-2 cells and explore the underlying mechanism.@*METHODS@#CD4 T lymphocytes isolated from 12 patients with laryngeal squamous carcinoma and 12 healthy individuals were examined for Cbl-b mRNA expressions using RT-PCR. H9 T lymphocytes cultured in 96-well plates were transfected with Cbl-b siRNA via liposomes followed by treatment with an anti-IL-2 monoclonal antibody, with H9 T lymphocytes transfected with a scrambled sequence as the negative control. The expressions of Cbl-b mRNA and protein in the cells were detected using real-time fluorescent quantitative PCR and Western blotting, respectively. The killing effect of the treated T lymphocytes against Hep-2 cells was assessed using the cell counting kit (CCK-8). The positive expression rates of CD69 and CD25 on the surface of H9 T lymphocytes were determined using flow cytometry, and the levels of interleukin-2 (IL-2) and interferon-gamma (INF-γ) in the culture supernatants of H9 T lymphocytes were detected with ELISA.@*RESULTS@#The CD4 T lymphocytes from patients with laryngeal squamous carcinoma showed significantly increased Cbl-b mRNA level compared with those from healthy individuals ( < 0.05). Transfection of H9 T lymphocytes with Cbl-b siRNA significantly reduced the expression levels of Cbl-b mRNA and protein ( < 0.05), which were not significantly affected by subsequent treatment of the cells with the anti-IL-2 antibody (>0.05). At different target-effector ratios, the Cbl-b siRNA-transfected cells showed significantly higher Hep-2 cell killing rates and higher positivity rates of CD69 and CD25 expressions than the blank and negative control cells and the cells with both Cbl-b siRNA transfection and anti-IL-2 treatment ( < 0.05). Cbl-b silencing in H9 T lymphocytes resulted in significantly increased levels of IL-2 and INF-γ in the supernatant as compared with those in the blank and negative control groups ( < 0.05).@*CONCLUSIONS@#Cbl-b gene silencing effectively enhances the killing effect of H9 T lymphocytes against Hep-2 cells probably as the result of enhanced IL-2 secretion and T lymphocyte activation.

Expression of MITOL in the cerebrospinal fluid of patients with postherpetic neuralgia and its clinical sig-nificance / 实用医学杂志

Objective To explore the expression of mitochondrial ubiquitin ligase(MITOL)in the cere-brospinal fluid from patients with postherpetic neuralgia(PHN). Methods A total of 45 patients who were diag-nosed with PHN in our hospital from August 2015 to December 2016 were enrolled in this study,and 38 gender-and age-matched patients with lumbar disc herniation were recruited as IDH group.The MITOL was measured by the ELISA and Western blot methods.The correlation between the expression of MITOL in cerebrospinal fluids and clinical characteristics of patients with PHN was evaluated.Results The expression of MITOL was significantly in-creased in cerebrospinal fluids of patients with PHN compared with IDH,and it was associated with age and the pain intensity(P<0.05).Logistic regression analysis showed age(OR,2.717)and the pain intensity(OR,3.042) were risk factors for PHN. Conclusions The expression of MITOL is significantly increased in the cerebrospinal fluid of PHN,which may be associated with age and pain intensity.

Progress in research on the novel cancer therapeutic target Cdc20 / 药学学报

The anaphase promoting complex (APC) regulates cell cycle progression by forming two functionally distinct E3 ubiquitin ligase complexes, APCCdc20 activated by cell division cycle protein 20 (Cdc20) and APCCdh1 activated by Cdc20 homologue 1 (Cdh1), respectively. Cdc20 and Cdh1 have different functions in the occurrence and development of the tumor. Cdc20 is a cancer promoter while Cdh1 suppresses tumorigenesis. Emerging evidence has begun to reveal that Cdc20 has positive functions in tumorigenesis, the overexpression of Cdc20 has been observed in many cancers. Currently, Cdc20 inhibitors, mostly non-specific inhibitors except apcin, not only block the combination between Cdc20 and APC, also block the combination between Cdh1 and APC, which leads to a poor selectivity. In this paper, the Cdc20 role in the development and process of cancers and its inhibitors are reviewed.

Role of RNF87 in predicting the prognosis of human hepatocellular carcinoma patients / 中国现代普通外科进展

Objective:To investigate the level and prognostic significance of RNF87 in human hepatocellular carcinoma.Methods:Detected the expression of RNF87 in 98 HCC tissues by immunohistochemistry and Western Blot.According to the clinical data of the patients,we analyzed the relationship between RNF87 level and the prognosis of the HCC patients.Results:The level of RNF87 in HCC tissues is down-regulated,compared with the adjacent tissues.And the expression of RNF87 was significantly related to the prognosis of HCC patients.Besides,the lower level of RNF87 was also obviously related with microvascular invasion.Conclusions:The down-regulated level of RNF87 may be one of the risk factors of human hepatocellular carcinoma progression;RNF87 maybe one of potential tumor suppressors;the level of RNF87 can be used as an indicator to predict the prognosis of HCC patients.

Loss of Ovarian Function Results in Increased Loss of Skeletal Muscle in Arthritic Rats / Perda da função ovariana resulta em perda de músculo esquelético aumentada em ratos artríticos

Objective We studied the effects of loss of ovarian function (ovariectomy) onmuscle mass of gastrocnemius and themRNA levels of IGF-1, atrogin-1, MuRF-1, andmyostatin in an experimental model of rheumatoid arthritis in rats. Methods We randomly allocated 24 female Wistar rats (9 weeks, 195.3±17.4 grams) into four groups: control (CT-Sham; n = 6); rheumatoid arthritis (RA; n = 6); ovariectomy without rheumatoid arthritis (OV; n = 6); ovariectomy with rheumatoid arthritis (RAOV; n = 6). We performed the ovariectomy (OV and RAOV) or Sham (CTSham or RA) procedures at the same time, fifteen days before the rheumatoid arthritis induction. The RA and RAOV groups were immunized and then were injected with Met- BSA in the tibiotarsal joint. After 15 days of intra-articular injections the animals were euthanized. We evaluated the external manifestations of rheumatoid arthritis (perimeter joint) as well as animal weight, and food intake throughout the study. We also analyzed the cross-sectional areas (CSA) of gastrocnemius muscle fibers in 200 fibers (H&E method). In the gastrocnemius muscle, we analyzed mRNA expression by quantitative real time PCR followed by the Livak method (ΔΔCT). Results The rheumatoid arthritis induced reduction in CSA of gastrocnemius muscle fibers. The RAOV group showed a lower CSA of gastrocnemius muscle fibers compared to RA and CT-Sham groups. Skeletal muscle IGF-1 mRNA increased in arthritics and ovariectomized rats. The increased IGF-1 mRNA was higher in OV groups than in the RA and RAOV groups. Antrogin-1 mRNA also increased in the gastrocnemius muscle of arthritic and ovariectomized rats. However, the increased atrogin-1 mRNA was higher in RAOV groups than in the RA and OV groups. Gastrocnemius muscle MuRF-1 mRNA increased in the OVand RAOVgroups, but not in the RA and Shamgroups. However, the RAOV group showed higher MuRF-1 mRNA than the OV group. The myostatin gene expression was similar in all groups. Conclusion Loss of ovarian function results in increased loss of skeletal musclerelated ubiquitin ligases atrogin-1 and MuRF-1 in arthritic rats.

Objetivo Foram estudados os efeitos da perda da função ovariana (ovariectomia) sobre músculo esquelético e os níveis de RNAm de IGF-1, atrogina-1, MuRF-1, e de miostatina em modelo experimental de artrite reumatóide em ratos. Métodos 24 ratos Wistar (9 semanas, 195,3±17,4 gramas) foram distribuídos aleatoriamente em quatro grupos: controle (CT-Sham, n = 6); artrite reumatóide (RA, n = 6); ovariectomia sem artrite reumatóide (OV; n = 6); ovariectomia com artrite reumatóide (RAOV; n = 6). Os procedimentos da ovariectomia (OV e RAOV) ou simulação da ovariectomia (CT-Shamou RA) foramrealizados aomesmo tempo, quinze dias antes da indução da artrite reumatóide. Os grupos RA e RAOV foramimunizados e, em seguida, foram injetados com Met-BSA na articulação tibiotársica. Após 15 dias das injeções intra-articulares, os animais foram eutanasiados. Foram avaliadas as manifestações externas da artrite reumatóide (perimetria articular), bem como o peso dos animais e a ingestão de alimentos ao longo do estudo. Além disso, as áreas de secção transversa (CSA) do músculo gastrocnêmio foram analisadas em 200 fibras (método H & E). No músculo gastrocnêmio, a expressão de RNAm foi analisada por PCR quantitativo em tempo real, seguido pelo método Livak (ΔΔCT). Resultados A artrite reumatoide reduziu a CSA das fibras do músculo gastrocnêmio. O grupo RAOV mostrou uma CSA menor nas fibras do músculo gastrocnêmio em comparação com os grupos RA e CT-Sham. O RNAm do IGF-1 do músculo esquelético aumentou nos ratos artríticos e ovariectomizados. O RNAm do IGF-1 foi maior nos grupos OV do que nos grupos RA e RAOV. A expressão de antrogina-1 também aumentou no músculo gastrocnêmio dos ratos artríticos e ovariectomizados. No entanto, o aumento do RNAm da atrogina-1 foi maior no grupo RAOV do que nos grupos RA e OV. O RNAm da MuRF-1 aumentou nos grupos OV e RAOV, mas não nos grupos RA e CT-Sham. Porém, o grupo RAOV apresentou maior expressão gênica de MuRF-1 do que o grupo OV. A expressão do gene da miostatina foi semelhante em todos os grupos. Conclusão A perda de função ovariana resulta em perda de músculo esquelético associado às ubiquitina-ligases atrogina-1 e MuRF-1 em ratos artríticos.

Identification and Molecular Characterization of Parkin in Clonorchis sinensis

Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to Zn2+ were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochondrial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis.

Identification and Molecular Characterization of Parkin in Clonorchis sinensis

Clonorchis sinensis habitating in the bile duct of mammals causes clonorchiasis endemic in East Asian countries. Parkin is a RING-between-RING protein and has E3-ubiquitin ligase activity catalyzing ubiquitination and degradation of substrate proteins. A cDNA clone of C. sinensis was predicted to encode a polypeptide homologous to parkin (CsParkin) including 5 domains (Ubl, RING0, RING1, IBR, and RING2). The cysteine and histidine residues binding to Zn2+ were all conserved and participated in formation of tertiary structural RINGs. Conserved residues were also an E2-binding site in RING1 domain and a catalytic cysteine residue in the RING2 domain. Native CsParkin was determined to have an estimated molecular weight of 45.7 kDa from C. sinensis adults by immunoblotting. CsParkin revealed E3-ubiquitin ligase activity and higher expression in metacercariae than in adults. CsParkin was localized in the locomotive and male reproductive organs of C. sinensis adults, and extensively in metacercariae. Parkin has been found to participate in regulating mitochondrial function and energy metabolism in mammalian cells. From these results, it is suggested that CsParkin play roles in energy metabolism of the locomotive organs, and possibly in protein metabolism of the reproductive organs of C. sinensis.

La vía ubiquitina-proteasoma ¿destruir o construir? ese es el dilema / Ubiquitin-proteasome pathway to built or to destroy? that is the question

Introducción: la construcción de nuevas células es un proceso complejo y para lograr que sea unidireccional e irreversible la célula utiliza el mecanismo de destruir proteínas que se oponen al paso de una etapa a la siguiente. Objetivo: demostrar que la destrucción de proteínas contribuye a la reproducción celular. Método: se analizaron más de 500 artículos de los últimos 5 años publicados en revistas nacionales y de circulación internacional, disponibles en las bases de datos HINARI, PubMed y Perii y localizados mediante el sitio infomed. Desarrollo: primero, se hizo una exposición sobre la vía ubiquitina-proteasoma. Después, se analizó la participación en el ciclo celular de los dos grandes complejos con actividad de ubiquitina-ligasa que son los encargados de marcar las proteínas que deben ser destruidas. Estos complejos actúan consecutiva y coordinadamente, y sus acciones determinan el progreso en un solo sentido del ciclo de vida de la célula. Conclusiones: la destrucción selectiva de proteínas mediante la vía ubiquitina proteasoma permite la formación de nuevas células y con ello la perpetuación de la vida.

Introduction: the generation of new cells is an extremely complex process. To become this process unidirectional and irreversible cells destroy proteins which actions are oppose to the transition from one phase to the next one. Objective: to show that protein destruction is essential for cell reproduction. Material and Methods: more than 500 papers published during the last five years in national and international journals were analyzed. These articles are available in HINARI, PubMed, and Perii databases and were localized through infomed. Development: first, the ubiquitin-proteasome pathway is presented. Next, the contribution of the complexes SCF an anaphase promoting complex to the progression of the cell cycle is analysed. These complexes act consecutively and coordinately and their actions determine de progression of the cell´s life. Conclusions: the selective destroy of specific proteins by mean of ubiquitin-proteasome pathway, allow new cells formation, and ensure the continuity of life.

Molecular Mechanisms of Inhibiting the Muscle Atrophy by Electroacupuncture / 日本温泉気候物理医学会雑誌

 Electroacupuncture (EA) is an acupuncture technique that is stimulated by acupuncture needles with low-frequency microcurrent. The aim of this study is to elucidate the effect of EA and it's molecular mechanism on muscle atrophy by using an animal model: hindlimb-suspended (HS) mice in the disuse muscle atrophy model. To compare the effects of EA in HS mice and HS mice treated with EA (EA/HS), soleus muscle mass and soleus myofiber diameter were measured. We then used real-time quantitative RT-PCR to analyze the expression of myostatin and ubiquitin ligase genes in atrophic muscles of HS mice and in muscles of EA/HS mice. We found that EA/HS mice maintained a soleus muscle mass that was not significantly different from that of wild mice (WT), whereas HS mice had significantly reduced muscle mass. Also, the diameters of myofibers in EA/HS mice, which were not significantly different from wild values, were significantly larger than those in HS mice. Repeated EA treatment suppressed gene expression of myostatin and ubiquitin ligase genes in skeletal muscle of EA/HS mice but induced expression of these genes in HS mice. These findings suggest the molecular mechanism by EA: suppression of myostatin and ubiquitin ligase gene may be a key reaction of inhibiting the disuse muscle atrophy.