The present study aimed to investigate the effects of eccentric treadmill exercise on adaptive hypertrophy of skeletal muscle in rats. Thirty-two 3-month-old Sprague Dawley (SD) rats were selected and randomly assigned to one of the four groups based on their body weights: 2-week quiet control group (2C), 2-week downhill running exercise group (2E), 4-week quiet control group (4C), and 4-week downhill running exercise group (4E). The downhill running protocol for rats in the exercise groups involved slope of -16°, running speed of 16 m/min, training duration of 90 min, and 5 training sessions per week. Twenty-four hours after the final session of training, all the four groups of rats underwent an exhaustion treadmill exercise. After resting for 48 h, all the rats were euthanized and their gastrocnemius muscles were harvested for analysis. HE staining was used to measure the cross-sectional area (CSA) and diameter of muscle fibers. Transmission electron microscope was used to observe the ultrastructural changes in muscle fibers. Purithromycin surface labeling translation method was used to measure protein synthesis rate. Immunofluorescence double labeling was used to detect the colocalization levels of lysosomal-associated membrane protein 2 (Lamp2)-leucyl-tRNA synthetase (LARS) and Lamp2-mammalian target of rapamycin (mTOR). Western blot was used to measure the protein expression levels of myosin heavy chain (MHC) IIb and LARS, as well as the phosphorylation levels of mTOR, p70 ribosomal protein S6 kinase (p70S6K), and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1). The results showed that, compared with the 2C group rats, the 2E group rats showed significant increases in wet weight of gastrocnemius muscle, wet weight/body weight ratio, running distance, running time, pre- and post-exercise blood lactate levels, myofibrillar protein content, colocalization levels of Lamp2-LARS and Lamp2-mTOR, and LARS protein expression. Besides these above changes, compared with the 4C group, the 4E group further exhibited significantly increased fiber CSA, fiber diameter, protein synthesis rate, and phosphorylation levels of mTOR, p70S6K, and 4E-BP1. Compared with the quiet control groups, the exercise groups exhibited ultrastructural damage of rat gastrocnemius muscle, which was more pronounced in the 4E group. These findings suggest that eccentric treadmill exercise may promote mTOR translocation to lysosomal membrane, activating mTOR signaling via up-regulating LARS expression. This, in turn, increases protein synthesis rate through the mTOR-p70S6K-4E-BP1 signaling pathway, promoting protein deposition and inducing adaptive skeletal muscle hypertrophy. Although the ultrastructural changes of skeletal muscle are more pronounced, the relatively long training cycles during short-term exercise periods have a more significant effect on promoting gastrocnemius muscle protein synthesis and adaptive hypertrophy.
Animals ; Rats, Sprague-Dawley ; Physical Conditioning, Animal/physiology* ; Rats ; Muscle, Skeletal/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; Male ; Hypertrophy ; Adaptation, Physiological/physiology* ; Adaptor Proteins, Signal Transducing/metabolism* ; Ribosomal Protein S6 Kinases, 70-kDa/metabolism* ; Intracellular Signaling Peptides and Proteins

Ribosome is an intracellular ribonucleoprotein particle that serves as the site of protein biosynthesis. Ribosomal dysfunction caused by mutations in genes encoding ribosomal proteins (RPs) and ribosome biogenesis factors (RBFs) can lead to a spectrum of diseases, collectively known as ribosomopathy. Phase separation is a thermodynamic process that produces multiple phases from a homogeneous mixture. The formation of membraneless organelles and intracellular structures, including ribosomes and nucleoli, cannot occur without the involvement of phase separation. Here, ribosome structure, biogenesis, and their relationship with ribosomopathy are systematically reviewed. The tissue specificity of ribosomopathy and the role of phase separation in ribosomopathy are particularly discussed, which may offer some clues for understanding the mechanisms of ribosomopathy. Then, some new ideas for the prevention, diagnosis, and treatment of ribosomopathy are provided.
Humans ; Ribosomes/physiology* ; Ribosomal Proteins/metabolism* ; Mutation ; Animals ; Cell Nucleolus/metabolism* ; Protein Biosynthesis ; Phase Separation

OBJECTIVE: To investigate the molecular mechanism underlying the anti-hepatic fibrosis activity of ethyl acetate fraction Dicliptera chinensis (L.) Juss. (EDC) in human hepatic stellate cells (HSCs) in vitro and in a carbon tetrachloride (CCl₄)-induced hepatic fibrosis mouse model in vivo. METHODS: For in vitro study, HSCs were pre-treated with platelet-derived growth factor (10 ng/mL) for 2 h to ensure activation and treated with EDC for 24 h and 48 h, respectively. The effect of EDC on HSCs was assessed using cell counting kit-8 assay, EdU staining, transmission electron microscopy, immunofluorescence staining, and Western blot, respectively. For in vivo experiments, mice were intraperitoneally injected with CCl₄ (2 ° L/g, adjusted to a 25% concentration in olive oil), 3 times per week for 6 weeks, to develop a hepatic fibrosis model. Forty 8-week-old male C57BL/6 mice were divided into 4 groups using a random number table (n=10), including control, model, positive control and EDC treatment groups. Mice in the EDC and colchicine groups were intragastrically administered EDC (0.5 g/kg) or colchicine (0.2 mg/kg) once per day for 6 weeks. Mice in the control and model groups received an equal volume of saline. Biochemical assays and histological examinations were used to assess liver damage. Protein expression levels of α -smooth muscle actin (α -SMA) and microtubule-associated protein light chain 3B (LC3B) were measured by Western blot. RESULTS: EDC reduced pathological damage associated with liver fibrosis, downregulated the expression of α -SMA and upregulated the expression of LC3B (P<0.05), both in HSCs and the CCl₄-induced liver fibrosis mouse model. The intervention of bafilomycin A1 and rapamycin in HSCs strongly supported the notion that inhibition of autophagy enhanced α -SMA protein expression levels (P<0.01). The results also found that the levels of phosphoinositide (PI3K), p-PI3K, AKT, p-AKT, mammalian target of rapamycin (mTOR), p-mTOR, and p-p70S6K all decreased after EDC treatment (P<0.05). CONCLUSIONS EDC has anti-hepatic fibrosis activity by inducing autophagy and might be a potential drug to be further developed for human liver fibrosis therapy.
Acetates ; Animals ; Autophagy ; Carbon Tetrachloride ; Hepatic Stellate Cells ; Liver/pathology* ; Liver Cirrhosis/pathology* ; Male ; Mice ; Mice, Inbred C57BL ; Phosphatidylinositol 3-Kinases ; Proto-Oncogene Proteins c-akt/metabolism* ; Ribosomal Protein S6 Kinases, 70-kDa ; Signal Transduction ; TOR Serine-Threonine Kinases/metabolism*

In recent years, two novel proteins in the ribosomes of mycobacteria have been discovered by cryo-electron microscopy. The protein bS22 is located near the decoding center of the 30S subunit, and the protein bL37 is located near the peptidyl transferase center of the 50S subunit. Since these two proteins bind to conserved regions of the ribosome targeted by antibiotics, it is speculated that they might affect the binding of related drugs to these targets. Therefore, we knocked out the genes encoding these two proteins in wild-type Mycolicibacterium smegmatis mc²155 through homologous recombination, and then determined the growth curves of these mutants and their sensitivity to related antibiotics. The results showed that compared with the wild-type strain, the growth rate of these two mutants did not change significantly. However, mutant ΔbS22 showed increased sensitivity to capreomycin, kanamycin, amikacin, streptomycin, gentamicin, paromomycin, and hygromycin B, while mutant ΔbL37 showed increased sensitivity to linezolid. These changes in antibiotics sensitivity were restored by gene complementation. This study hints at the possibility of using ribosomal proteins bS22 and bL37 as targets for drug design.
Anti-Bacterial Agents/pharmacology* ; Cryoelectron Microscopy ; Mycobacterium/genetics* ; Ribosomal Proteins/genetics* ; Ribosomes/metabolism*

OBJECTIVE: To investigate the effects and mechanisms of Kindlin-2 on uterus development and reproductive capacity in female mice. METHODS: Cdh16-Cre tool mice and Kindlin-2^flox/flox mice were used to construct the mouse model of uterus specific knockout of Kindlin-2, and the effects of Kindlin-2 deletion on uterine development and reproduction capacity of female mice were observed. High expression and knockdown of Kindlin-2 in endometrial cancer cell lines HEC-1 and Ish were used to detect the regulation of mammalian target of rapamycin (mTOR) signaling pathway. In addition, uterine proteins of the female mice with specific knockout of Kindlin-2 and female mice in the control group were extracted to detect the protein levels of key molecules of mTOR signaling pathway and Hippo signaling pathway. RESULTS: The mouse model of uterine specific knockout of Kindlin-2 was successfully constructed. The knockout efficiency of Kindlin-2 in mouse uterus was identified and verified by mouse tail polymerase chain reaction (PCR), Western blot protein identification, immunohistochemical staining (IHC) and other methods. Compared with the control group, the female mice with uterus specific deletion of Kindlin-2 lost weight, seriously impaired reproductive ability, and the number of newborn mice decreased, but the proportion of the female mice and male mice in the newborn mice did not change. Hematoxylin eosin staining (HE) experiment showed that the endometrium of Kindlin-2 knockout group was incomplete and the thickness of uterine wall became thinner. In terms of mechanism, the deletion of Kindlin-2 in endo-metrial cancer cell lines HEC-1 and Ish could downregulate the protein levels of mTOR, phosphorylated mTOR, adenosine monophosphate-activated protein kinase (AMPK), phosphorylated AMPK and phosphorylated ribosomal protein S6 (S6), and the mTOR signal pathway was inhibited. It was found that the specific deletion of Kindlin-2 could upregulate the protein levels of Mps one binding 1 (MOB1) and phosphorylated Yes-associated protein (YAP) in the uterus of the female mice, and the Hippo signal pathway was activated. CONCLUSION Kindlin-2 inhibits the development of uterus by inhibiting mTOR signal pathway and activating Hippo signal pathway, thereby inhibiting the fertility of female mice.
AMP-Activated Protein Kinases/metabolism* ; Adenosine Monophosphate/metabolism* ; Animals ; Cadherins/metabolism* ; Cytoskeletal Proteins/metabolism* ; Endometrium/metabolism* ; Eosine Yellowish-(YS)/metabolism* ; Female ; Hematoxylin/metabolism* ; Hippo Signaling Pathway ; Male ; Mammals/metabolism* ; Mice ; Muscle Proteins ; Ribosomal Protein S6/metabolism* ; Sirolimus/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; YAP-Signaling Proteins

LIN28 is an RNA binding protein with important roles in early embryo development, stem cell differentiation/reprogramming, tumorigenesis and metabolism. Previous studies have focused mainly on its role in the cytosol where it interacts with Let-7 microRNA precursors or mRNAs, and few have addressed LIN28's role within the nucleus. Here, we show that LIN28 displays dynamic temporal and spatial expression during murine embryo development. Maternal LIN28 expression drops upon exit from the 2-cell stage, and zygotic LIN28 protein is induced at the forming nucleolus during 4-cell to blastocyst stage development, to become dominantly expressed in the cytosol after implantation. In cultured pluripotent stem cells (PSCs), loss of LIN28 led to nucleolar stress and activation of a 2-cell/4-cell-like transcriptional program characterized by the expression of endogenous retrovirus genes. Mechanistically, LIN28 binds to small nucleolar RNAs and rRNA to maintain nucleolar integrity, and its loss leads to nucleolar phase separation defects, ribosomal stress and activation of P53 which in turn binds to and activates 2C transcription factor Dux. LIN28 also resides in a complex containing the nucleolar factor Nucleolin (NCL) and the transcriptional repressor TRIM28, and LIN28 loss leads to reduced occupancy of the NCL/TRIM28 complex on the Dux and rDNA loci, and thus de-repressed Dux and reduced rRNA expression. Lin28 knockout cells with nucleolar stress are more likely to assume a slowly cycling, translationally inert and anabolically inactive state, which is a part of previously unappreciated 2C-like transcriptional program. These findings elucidate novel roles for nucleolar LIN28 in PSCs, and a new mechanism linking 2C program and nucleolar functions in PSCs and early embryo development.
Animals ; Cell Differentiation ; Embryo, Mammalian/metabolism* ; Embryonic Development ; Mice ; Pluripotent Stem Cells/metabolism* ; RNA, Messenger/genetics* ; RNA, Ribosomal ; RNA-Binding Proteins/metabolism* ; Transcription Factors/metabolism* ; Zygote/metabolism*

Objective: The expression patterns of ribosomal large subunit protein 23a (RPL23a) in mouse testes and GC-1 cells were analyzed to investigate the potential relationship between RPL23a expression and spermatogonia apoptosis upon exposure to X-ray. Methods: Male mice and GC-1 cells were irradiated with X-ray, terminal dUTP nick end-labelling (TUNEL) was performed to detect apoptotic spermatogonia Results: Ionizing radiation (IR) increased spermatogonia apoptosis, the expression of RPL11, MDM2 and p53, and decreased RPL23a expression in mice spermatogonia Conclusion These results suggested that IR reduced RPL23a expression, leading to weakened the RPL23a-RPL11 interactions, which may have activated p53, resulting in spermatogonia apoptosis. These results provide insights into environmental and clinical risks of radiotherapy following exposure to IR in male fertility. The graphical abstract was available in the web of www.besjournal.com.
Animals ; Apoptosis/genetics* ; Gene Expression Regulation ; Male ; Mice ; Ribosomal Proteins/metabolism* ; Signal Transduction ; Spermatogonia/radiation effects*

Objective To identify the expression of ribosomal protein S9(RPS9)in multiple myeloma(MM)and explore its effect on the biological characteristics of myeloma cells and the corresponding mechanisms. Methods Bone marrow mononuclear cells were harvested in 10 healthy volunteers(CON group)and bone marrow CD138 +cells from 30 MM patients(CD138+group).Quantitative polymerase chain reaction(qPCR)was performed to detect RPS9 expression at mRNA level.In three cases from CON group and 11 cases from CD138+group,Western blot was performed to detect RPS9 at protein level.GSE19784 dataset was employed to detect the relationships of RPS9 expression with the overall survival rate,nuclear factor-κB(NF-κB),small ubiquitin-like modifier(SUMO),and ubiquitin pathway.After the RPS9 knock-down vector was constructed,flow cytometry was performed to detect the infection efficiency and qPCR and Western blot to detect the knock-down efficiency.RPMI8226 was divided into CON group and RPS9-short hairpin RNA(shRNA)group,in which annexin V allophycocyanin/propidium iodide(PI)double staining was performed to detect the change of apoptosis,CCK8 to detect the proliferation change,and PI staining to detect cell cycle change.After sentrin-specific protease 1(SENP1)overexpression vector was constructed,Western blot was performed to detect the phosphorylation of P65 and inhibitory subunit-κBα(IκBα)from NF-κB pathway in CON,RPS9-shRNA,and RPS9-shRNA-SENP1 cells;in addition,annexin V/PI double staining was also performed to detect the apoptosis in these three cells. Results The relative expression of RPS9 in CON group and CD138+group was(1.00±0.12)and(5.45±0.71),respectively(t=4.291,P=0.0036).Western blot showed RPS9 expression was high in most myeloma CD138+cells.The high expression of RPS9 was associated with both extramedullary invasion and overall survival in GSE19784 dataset.After RPMI8226 was infected with CON or RPS9-shRNA lentivirus for 48 hours,flow cytometry confirmed that the infection efficiencies were above 90% in both groups.qPCR and Western blot confirmed that RPS9 expression was inhibited at both mRNA and protein levels.After RPMI8226 CON and RPS9-shRNA infected with virus for 48 hours,the proportion of annexin V-positive cells in CON and RPS9-shRNA cells was(3.47±0.37)% and(18.60±64.00)%(t=9.015,P=0.0008).The proliferation index significantly differed between CON group and RPS9-shRNA group at 72 hours(t=6.846,P=0.0024).When CON and RPS9-shRNA were infected with virus for 48 hours,the proportion of G2 phase cells was(29.28±3.42)% and(10.43±1.43)%,respectively(t=9.329,P=0.0007).The RPS9 expression was positively correlated with SENP1 in GSE19784 dataset and negatively correlated with IκBα coding gene NFKBIA.Western blot further confirmed that RPS9 knockdown inhibited the expression of SENP1,inhibited the phosphorylation of NF-κB subunit P65 and inhibitor IκBα,and promoted the expression of IκBα.Overexpression of SENP1 not only impeded this effect but also reduced RPS9-induced apoptosis. Conclusions RPS9 is highly expressed in MM CD138+cells and is associated with overall survival and extramedullary infiltration.Inhibition of RPS9 can promote apoptosis,cell cycle arrest,and proliferation of myeloma cells.RPS9 can affect the activation of NF-κB pathway and cell apoptosis through SENP1,suggesting that SENP1 may be a key factor in the biological effect of RPS9.
Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Cysteine Endopeptidases ; metabolism ; Humans ; Multiple Myeloma ; metabolism ; Ribosomal Proteins ; metabolism ; Signal Transduction

OBJECTIVE: To investigate the effect of rosuvastatin on homocysteine (Hcy) induced mousevascular smooth muscle cells(VSMCs) dedifferentiation and endoplasmic reticulum stress(ERS). METHODS: VSMCs were co-cultured with Hcy and different concentration of rosuvastatin (0.1, 1.0 and 10 μmol/L). Cytoskeleton remodeling, VSMCs phenotype markers (smooth muscle actin-α, calponin and osteopontin) and ERS marker mRNAs (Herpud1, XBP1s and GRP78) were detected at predicted time. Tunicamycin was used to induce, respectively 4-phenylbutyrate(4-PBA) inhibition, ERS in VSMCs and cellular migration, proliferation and expression of phenotype proteins were analyzed. Mammalian target of rapamycin(mTOR)-P70S6 kinase (P70S6K) signaling agonist phosphatidic acid and inhibitor rapamycin were used in Rsv treated VSMCs. And then mTOR signaling and ERS associated mRNAs were detected. RESULTS: Compared with Hcy group, Hcy+ Rsv group (1.0 and 10 μmol/L) showed enhanced α-SMA and calponin expression (<0.01), suppressed ERS mRNA levels (<0.01) and promoted polarity of cytoskeleton. Compared with Hcy group, Hcy+Rsv group and Hcy+4-PBA group showed suppressed proliferation, migration and enhanced contractile protein expression (<0.01); while tunicamycin could reverse the effect of Rsv on Hcy treated cells. Furthermore, alleviated mTOR-P70S6K phosphorylation and ERS (<0.01)were observed in Hcy+Rsv group and Hcy+rapamycin group, compared with Hcy group; while phosphatidic acid inhibited the effect of Rsv on mTOR signaling activation and ERS mRNA levels (<0.01). CONCLUSIONS Rosuvastatin could inhibit Hcy induced VSMCs dedifferentiation suppressing ERS, which might be regulated by mTOR-P70S6K signaling.
Actins ; metabolism ; Animals ; Calcium-Binding Proteins ; metabolism ; Cell Dedifferentiation ; drug effects ; Cells, Cultured ; Endoplasmic Reticulum Stress ; drug effects ; Heat-Shock Proteins ; metabolism ; Homocysteine ; Membrane Proteins ; metabolism ; Mice ; Microfilament Proteins ; metabolism ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Rosuvastatin Calcium ; pharmacology ; TOR Serine-Threonine Kinases ; metabolism ; X-Box Binding Protein 1 ; metabolism

Protozoan viruses may influence the function and pathogenicity of the protozoa. Trichomonas vaginalis is a parasitic protozoan that could contain a double stranded RNA (dsRNA) virus, T. vaginalis virus (TVV). However, there are few reports on the properties of the virus. To further determine variations in protein expression of T. vaginalis, we detected 2 strains of T. vaginalis; the virus-infected (V⁺) and uninfected (V⁻) isolates to examine differentially expressed proteins upon TVV infection. Using a stable isotope N-terminal labeling strategy (iTRAQ) on soluble fractions to analyze proteomes, we identified 293 proteins, of which 50 were altered in V⁺ compared with V⁻ isolates. The results showed that the expression of 29 proteins was increased, and 21 proteins decreased in V⁺ isolates. These differentially expressed proteins can be classified into 4 categories: ribosomal proteins, metabolic enzymes, heat shock proteins, and putative uncharacterized proteins. Quantitative PCR was used to detect 4 metabolic processes proteins: glycogen phosphorylase, malate dehydrogenase, triosephosphate isomerase, and glucose-6-phosphate isomerase, which were differentially expressed in V⁺ and V⁻ isolates. Our findings suggest that mRNA levels of these genes were consistent with protein expression levels. This study was the first which analyzed protein expression variations upon TVV infection. These observations will provide a basis for future studies concerning the possible roles of these proteins in host-parasite interactions.
Glucose-6-Phosphate Isomerase ; Glycogen Phosphorylase ; Heat-Shock Proteins ; Host-Parasite Interactions ; Malate Dehydrogenase ; Metabolism ; Polymerase Chain Reaction ; Proteome ; Reticuloendotheliosis virus ; Ribosomal Proteins ; RNA, Double-Stranded ; RNA, Messenger ; Trichomonas vaginalis* ; Trichomonas* ; Triose-Phosphate Isomerase ; Virulence