Identification of potential target genes associated with the reversion of androgen-dependent skeletal muscle atrophy.

Muscle wasting or atrophy is extensively associated with human systemic diseases including diabetes, cancer, and kidney failure. Accumulating evidence from transcriptional profiles has noted that a common set of genes, termed atrogenes, is modulated in atrophying muscles. However, the transcriptional changes that trigger the reversion or attenuation of muscle atrophy have not been characterized at the molecular level until now. Here, we applied cDNA microarrays to investigate the transcriptional response of androgen-sensitive Levator ani muscle (LA) during atrophy reversion. Most of the differentially expressed genes behaved as atrogenes and responded to castration-induced atrophy. However, seven genes (APLN, DUSP5, IGF1, PIK3IP1, KLHL38, PI15, and MKL1) did not respond to castration but instead responded exclusively to testosterone replacement. Considering that almost all proteins encoded by these genes are associated with the reversion of atrophy and may function as regulators of cell proliferation/growth, our results provide new perspectives on the existence of anti-atrogenes.

DNA damage response signaling does not trigger redistribution of SAMHD1 to nuclear foci.

SAMHD1 (Sterile alpha motif and histidine-aspartic acid (HD) domain containing protein 1) is a deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase (dNTPase) that restricts viral replication in infected cells. This protein is also involved in DNA repair by assisting in DNA end resection by homologous recombination (HR) after DNA double-strand break (DSB) induction with camptothecin (CPT) or etoposide (ETO). We showed that a monoclonal anti-SAMHD1 antibody produced against the full-length protein detected an unspecific 50 kDa protein that colocalized with dot-like structures after CPT treatment in HeLa cells. In contrast, a polyclonal anti-SAMHD1 antibody raised against the N-terminus of this protein specifically detected SAMHD1, as shown in Jurkat, HAP1KO and HEK293T SAMHD1-siRNA cell lysates compared with their respective controls. Our findings showed that SAMHD1 is not localized in dot-like structures under DSB induction in HeLa cells.

FBXO25 regulates MAPK signaling pathway through inhibition of ERK1/2 phosphorylation.

The FBXO25 mediates degradation of ELK-1 and thus inhibits transcriptional activation of immediate early genes (iEG). Here we show that FBXO25 regulates yet another node of this signaling pathway, by decreasing MAPK/ERK activity. We show that induction of FBXO25 reduced ERK1/2 phosphorylation independently of MEK1/2. Accordingly, in HAP1 FBXO25 knockout cells (FBXO25KO), we observed that upon PMA treatment ERK1/2 was more active than in parental cells. An increase in cell proliferation under receptor mediated activation of the ERK signaling pathway in FBXO25KO cells was also observed. Taken together we show that FBXO25 functions as a negative regulator of MAPK signaling though the reduction of ERK1/2 activation.

The F-box protein FBXO25 promotes the proteasome-dependent degradation of ELK-1 protein.

FBXO25 is one of the 69 known human F-box proteins that serve as specificity factors for a family of ubiquitin ligases composed of SKP1, Rbx1, Cullin1, and F-box protein (SCF1) that are involved in targeting proteins for degradation across the ubiquitin proteasome system. However, the substrates of most SCF E3 ligases remain unknown. Here, we applied an in chip ubiquitination screen using a human protein microarray to uncover putative substrates for the FBXO25 protein. Among several novel putative targets identified, the c-fos protooncogene regulator ELK-1 was characterized as the first endogenous substrate for SCF1(FBXO25) E3 ligase. FBXO25 interacted with and mediated the ubiquitination and proteasomal degradation of ELK-1 in HEK293T cells. In addition, FBXO25 overexpression suppressed induction of two ELK-1 target genes, c-fos and egr-1, in response to phorbol 12-myristate 13-acetate. Together, our findings show that FBXO25 mediates ELK-1 degradation through the ubiquitin proteasome system and thereby plays a role in regulating the activation of ELK-1 pathway in response to mitogens.

The kinin B1 receptor regulates muscle-specific E3 ligases expression and is involved in skeletal muscle mass control.

Regulation of muscle mass depends on the balance between synthesis and degradation of proteins, which is under the control of different signalling pathways regulated by hormonal, neural and nutritional stimuli. Such stimuli are altered in several pathologies, including COPD (chronic obstructive pulmonary disease), diabetes, AIDS and cancer (cachexia), as well as in some conditions such as immobilization and aging (sarcopenia), leading to muscle atrophy, which represents a significant contribution to patient morbidity. The KKS (kallikrein-kinin system) is composed of the enzymes kallikreins, which generate active peptides called kinins that activate two G-protein-coupled receptors, namely B1 and B2, which are expressed in a variety of tissues. The local modulation of the KKS may account for its participation in different diseases, such as those of the cardiovascular, renal and central nervous systems, cancer and many inflammatory processes, including pain. Owing to such pleiotropic actions of the KKS by local modulatory events and the probable fine-tuning of associated signalling cascades involved in skeletal muscle catabolic disorders [for example, NF-κB (nuclear factor κB) and PI3K (phosphoinositide 3-kinase)/Akt pathways], we hypothesized that KKS might contribute to the modulation of intracellular responses in atrophying skeletal muscle. Our results show that kinin B1 receptor activation induced a decrease in the diameter of C2C12 myotubes, activation of NF-κB, a decrease in Akt phosphorylation levels, and an increase in the mRNA levels of the ubiquitin E3 ligases atrogin-1 and MuRF-1 (muscle RING-finger protein-1). In vivo, we observed an increase in kinin B1 receptor mRNA levels in an androgen-sensitive model of muscle atrophy. In the same model, inhibition of the kinin B1 receptor with a selective antagonist resulted in an impairment of atrogin-1 and MuRF-1 expression and IκB (inhibitor of NF-κB) phosphorylation. Moreover, knockout of the kinin B1 receptor in mice led to an impairment in MuRF-1 mRNA expression after induction of LA (levator ani) muscle atrophy. In conclusion, using pharmacological and gene-ablation tools, we have obtained evidence that the kinin B1 receptor plays a significant role in the regulation of skeletal muscle proteolysis in the LA muscle atrophy model.

Liquid Modernity and Imagistic Production: Reflections on Social Media and Free Associations.

Social media has profound impact on how we experience the world and interact with others. Rapidly advancing technology has created platforms that have become increasingly image-based and emotionally manipulative. Do the new patterns of communication change patients' mental processes? Is free association becoming more imagistic? Contemporary clinical settings invite new perspectives on the intersections between the social and individual realms, patients' modes of expression, and analysts' interpretations.

The E3 ubiquitin ligase SCF(Fbxo7) mediates proteasomal degradation of UXT isoform 2 (UXT-V2) to inhibit the NF-κB signaling pathway.

BACKGROUND: Ubiquitously eXpressed Transcript isoform 2 (UXTV2) is a prefoldin-like protein involved in NF-κB signaling, apoptosis, and the androgen and estrogen response. UXT-V2 is a cofactor in the NF-κB transcriptional enhanceosome, and its knockdown inhibits TNF-α -induced NF-κB activation. Fbxo7 is an F-box protein that interacts with SKP1, Cullin1 and RBX1 proteins to form an SCF(Fbxo7) E3 ubiquitin ligase complex. Fbxo7 negatively regulates NF-κB signaling through TRAF2 and cIAP1 ubiquitination. METHODS: We combine co-immunoprecipitation, ubiquitination in vitro and in vivo, cycloheximide chase assay, ubiquitin chain restriction analysis and microscopy to investigate interaction between Fbxo7 and overexpressed UXT-V2-HA. RESULTS: The Ubl domain of Fbxo7 contributes to interaction with UXTV2. This substrate is polyubiquitinated by SCF(Fbxo7) with K48 and K63 ubiquitin chain linkages in vitro and in vivo. This post-translational modification decreases UXT-V2 stability and promotes its proteasomal degradation. We further show that UXTV1, an alternatively spliced isoform of UXT, containing 12 additional amino acids at the N-terminus as compared to UXTV2, also interacts with and is ubiquitinated by Fbxo7. Moreover, FBXO7 knockdown promotes UXT-V2 accumulation, and the overexpression of Fbxo7-ΔF-box protects UXT-V2 from proteasomal degradation and enhances the responsiveness of NF-κB reporter. We find that UXT-V2 colocalizes with Fbxo7 in the cell nucleus. CONCLUSIONS: Together, our study reveals that SCF(Fbxo7) mediates the proteasomal degradation of UXT-V2 causing the inhibition of the NF-κB signaling pathway. GENERAL SIGNIFICANCE: Discovering new substrates of E3 ubiquitin-ligase SCF(Fbxo7) contributes to understand its function in different diseases such as cancer and Parkinson.

Time course of skeletal muscle loss and oxidative stress in rats with Walker 256 solid tumor.

Reactive oxygen species oxidize proteins and modulate the proteasomal system in muscle-wasting cancer cachexia. On day 5 (D5), day 10 (D10), and day 14 (D14) after tumor implantation, skeletal muscle was evaluated. Carbonylated proteins and thiobarbituric acid reactive substances were measured. Chemiluminescence was employed for lipid hydroperoxide estimation. Glutathione, superoxide dismutase, and total radical antioxidant capacity were evaluated. The proteasomal system was assessed by mRNA atrogin-1 expression. Increased muscle wasting, lipid hydroperoxide, and superoxide dismutase, and decreased glutathione levels and total radical antioxidant capacity, were found on D5 in accordance with increased mRNA atrogin-1 expression. All parameters were significantly modified in animals treated with α-tocopherol. The elevation in aldehylde levels and carbonylated proteins observed on D10 were reversed by α-tocopherol treatment. Oxidative stress may trigger signal transduction of the proteasomal system and cause protein oxidation. These pathways may be associated with the mechanism of muscle wasting that occurs in cancer cachexia.

Tumor necrosis factor receptor-associated factor 6 interaction with alpha-synuclein enhances cell death through the Nuclear Factor-kB pathway.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disease characterized by intracellular inclusions named Lewy bodies (LB), and alpha-synuclein (asyn) is the major component of these protein aggregates. The precise physiological and pathological roles of asyn are not fully understood. Nevertheless, asyn present in LB is ubiquitinated but fails to reach the 26S proteasome. The mutation A30 P is related to an aggressive and early-onset form of PD. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an E3 ubiquitin ligase, and it interacts and ubiquitinates the asyn in atypical chains (lysine K6, K27, K29, and K33). Methods: Here, we investigated the role of TRAF6 interaction with asyn and the involvement of nuclear factor κB (NF-κB), a key transcription factor in pro-inflammatory signaling pathway activation. RESULTS AND CONCLUSION: We demonstrated that TRAF6 binds to both WT and the mutant form A30 P asyn in an SH-SY5Y cell model. Additionally, the interaction between TRAF6 and WT asyn induced an increase in the activation of NF-κB, leading to changes in TNF, IL-1ß and IL-10 levels and culminating in reduced cell viability. Interestingly, the activation of NF-κB and gene regulation were not found in A30 P asyn. These data point to a novel role of TRAF6 in the pathophysiology of PD.

Arginine methylation analysis of the splicing-associated SR protein SFRS9/SRP30C.

The human SFRS9/SRp30c belongs to the SR family of splicing regulators. Despite evidence that members of this protein family may be targeted by arginine methylation, this has yet to be experimentally addressed. In this study, we found that SFRS9 is a target for PRMT1-mediated arginine methylation in vitro, and that it is immunoprecipitated from HEK-293 lysates by antibodies that recognize both mono- and dimethylated arginines. We further observed that upon treatment with the methylation inhibitor Adox, the fluorescent EGFP-SFRS9 re-localizes to dot-like structures in the cell nucleus. In subsequent confocal analyses, we found that EGFP-SFRS9 localizes to nucleoli in Adox-treated cells. Our findings indicate the importance of arginine methylation for the subnuclear localization of SFRS9.

FBXO25, an F-box protein homologue of atrogin-1, is not induced in atrophying muscle.

Atrogin-1/MAFbx/FBXO32 is a muscle-specific ubiquitin-ligase (E3) that is dramatically increased in atrophying muscle. Here, we have investigated the functional relationship between atrogin-1 and FBXO25 which shares 65% amino acid identity. Using a RT-PCR, we demonstrated that FBXO25 is highly expressed in brain, kidney, and intestine, whereas atrogin-1 expression is largely restricted to striate muscle. FBXO25 was shown here to contain a functional F-box domain that binds to Skp1 and thereby to Roc1 and Cul1, the major components of SCF-type E3s. In addition, the productive SCF complex containing FBXO25 showed ubiquitin ligase activity. We investigated the differential expression of atrogin-1 and FBXO25 in fasted and dexamethasone-treated mice and also in rats with streptozotocin-induced diabetes. Although the atrogin-1 was strongly induced in muscle in all three models, no changes were observed in the expression of FBXO25. Therefore, here we have shown that FBXO25 is a novel F-box protein analogous to atrogin-1, which is not involved in muscle atrophy. Further functional studies should elucidate the exact role of FBXO25 in the ubiquitin-proteasome pathway.

The intracellular distribution and secretion of endopeptidases 24.15 (EC 3.4.24.15) and 24.16 (EC 3.4.24.16).

Endopeptidase 24.15 (EC 3.4.24.15; EP24.15) and endopeptidase 24.16 (EC 3.4.24.16; EP24.16) are enzymes involved in general peptide metabolism in mammalian cells and tissues. This review will focus on morphological and biochemical aspects related to the subcellular distribution and secretion of these homologous enzymes in the central nervous system. These are important issues for a better understanding of the functions of EP24.15 and EP24.16 within neuroendocrine systems.

Angiotensin II-independent angiotensin-(1-7) formation in rat hippocampus: involvement of thimet oligopeptidase.

The involvement and relevance of the renin-angiotensin system have been established clearly in cardiovascular diseases, and renin-angiotensin system involvement has also been investigated extensively in the central nervous system. Angiotensin II acts classically by binding to the AT1 and AT2 receptors. However, other pathways within the renin-angiotensin system have been described more recently, such as one in which angiotensin-(1-7) (Ang-(1-7)) binds to the receptor Mas. In the central nervous system specifically, it has been reported that this heptapeptide is involved in learning and memory processes that occur in central limbic regions, such as the hippocampus. Therefore, this prompted us to investigate the possible role of the Ang-(1-7)-receptor Mas pathway in epileptic seizures, which are also known to recruit limbic areas. In the present study, we show that Ang-(1-7) is the main metabolite of angiotensin I in rat hippocampi, and, strikingly, that thimet oligopeptidase is the main enzyme involved in the generation of Ang-(1-7). Furthermore, elevations in the levels of thimet oligopeptidase, Ang-(1-7), and of receptor Mas transcripts are observed in chronically stimulated epileptic rats, which suggest that the thimet oligopeptidase-Ang-(1-7)-receptor Mas axis may have a functional relevance in the pathophysiology of these animals. In summary, our data, which describe a new preferential biochemical pathway for the generation of Ang-(1-7) in the central nervous system and an increase in the levels of various elements of the related thimet oligopeptidase-Ang-(1-7)-receptor Mas pathway, unveil potential new roles of the renin-angiotensin system in central nervous system pathophysiology.

Testosterone represses ubiquitin ligases atrogin-1 and Murf-1 expression in an androgen-sensitive rat skeletal muscle in vivo.

Skeletal muscle atrophy induced by denervation and metabolic diseases has been associated with increased ubiquitin ligase expression. In the present study, we evaluate the influence of androgens on muscle ubiquitin ligases atrogin-1/MAFbx/FBXO32 and Murf-1/Trim63 expression and its correlation with maintenance of muscle mass by using the testosterone-dependent fast-twitch levator ani muscle (LA) from normal or castrated adult male Wistar rats. Gene expression was determined by qRT-PCR and/or immunoblotting. Castration induced progressive loss of LA mass (30% of control, 90 days) and an exponential decrease of LA cytoplasm-to-nucleus ratio (nuclear domain; 22% of control after 60 days). Testosterone deprivation induced a 31-fold increase in LA atrogin-1 mRNA and an 18-fold increase in Murf-1 mRNA detected after 2 and 7 days of castration, respectively. Acute (24 h) testosterone administration fully repressed atrogin-1 and Murf-1 mRNA expression to control levels. Atrogin-1 protein was also increased by castration up to 170% after 30 days. Testosterone administration for 7 days restored atrogin-1 protein to control levels. In addition to the well known stimulus of protein synthesis, our results show that testosterone maintains muscle mass by repressing ubiquitin ligases, indicating that inhibition of ubiquitin-proteasome catabolic system is critical for trophic action of androgens in skeletal muscle. Besides, since neither castration nor androgen treatment had any effect on weight or ubiquitin ligases mRNA levels of extensor digitorum longus muscle, a fast-twitch muscle with low androgen sensitivity, our study shows that perineal muscle LA is a suitable in vivo model to evaluate regulation of muscle proteolysis, closely resembling human muscle responsiveness to androgens.

Atrogin-1 and MuRF1 regulate cardiac MyBP-C levels via different mechanisms.

AIMS: Familial hypertrophic cardiomyopathy (FHC) is frequently caused by cardiac myosin-binding protein C (cMyBP-C) gene mutations, which should result in C-terminal truncated mutants. However, truncated mutants were not detected in myocardial tissue of FHC patients and were rapidly degraded by the ubiquitin-proteasome system (UPS) after gene transfer in cardiac myocytes. Since the diversity and specificity of UPS regulation lie in E3 ubiquitin ligases, we investigated whether the muscle-specific E3 ligases atrogin-1 or muscle ring finger protein-1 (MuRF1) mediate degradation of truncated cMyBP-C. METHODS AND RESULTS: Human wild-type (WT) and truncated (M7t, resulting from a human mutation) cMyBP-C species were co-immunoprecipitated with atrogin-1 after adenoviral overexpression in cardiac myocytes, and WT-cMyBP-C was identified as an interaction partner of MuRF1 by yeast two-hybrid screens. Overexpression of atrogin-1 in cardiac myocytes decreased the protein level of M7t-cMyBP-C by 80% and left WT-cMyBP-C level unaffected. This was rescued by proteasome inhibition. In contrast, overexpression of MuRF1 in cardiac myocytes not only reduced the protein level of WT- and M7t-cMyBP-C by >60%, but also the level of myosin heavy chains (MHCs) by >40%, which were not rescued by proteasome inhibition. Both exogenous cMyBP-C and endogenous MHC mRNA levels were markedly reduced by MuRF1 overexpression. Similar to cardiac myocytes, MuRF1-overexpressing (TG) mice exhibited 40% lower levels of MHC mRNAs and proteins. Protein levels of cMyBP-C were 29% higher in MuRF1 knockout and 34% lower in TG than in WT, without a corresponding change in mRNA levels. CONCLUSION: These data suggest that atrogin-1 specifically targets truncated M7t-cMyBP-C, but not WT-cMyBP-C, for proteasomal degradation and that MuRF1 indirectly reduces cMyBP-C levels by regulating the transcription of MHC.

Functional association of human Ki-1/57 with pre-mRNA splicing events.

The cytoplasmic and nuclear protein Ki-1/57 was first identified in malignant cells from Hodgkin's lymphoma. Despite studies showing its phosphorylation, arginine methylation, and interaction with several regulatory proteins, the functional role of Ki-1/57 in human cells remains to be determined. Here, we investigated the relationship of Ki-1/57 with RNA functions. Through immunoprecipitation assays, we verified the association of Ki-1/57 with the endogenous splicing proteins hnRNPQ and SFRS9 in HeLa cell extracts. We also found that recombinant Ki-1/57 was able to bind to a poly-U RNA probe in electrophoretic mobility shift assays. In a classic splicing test, we showed that Ki-1/57 can modify the splicing site selection of the adenoviral E1A minigene in a dose-dependent manner. Further confocal and fluorescence microscopy analysis revealed the localization of enhanced green fluorescent proteinKi-1/57 to nuclear bodies involved in RNA processing and or small nuclear ribonucleoprotein assembly, depending on the cellular methylation status and its N-terminal region. In summary, our findings suggest that Ki-1/57 is probably involved in cellular events related to RNA functions, such as pre-mRNA splicing.

FBXO25-associated nuclear domains: a novel subnuclear structure.

Skp1, Cul1, Rbx1, and the FBXO25 protein form a functional ubiquitin ligase complex. Here, we investigate the cellular distribution of FBXO25 and its colocalization with some nuclear proteins by using immunochemical and biochemical approaches. FBXO25 was monitored with affinity-purified antibodies raised against the recombinant fragment spanning residues 2-62 of the FBXO25 sequence. FBXO25 protein was expressed in all mouse tissues tested except striated muscle, as indicated by immunoblot analysis. Confocal analysis revealed that the endogenous FBXO25 was partially concentrated in a novel dot-like nuclear domain that is distinct from clastosomes and other well-characterized structures. These nuclear compartments contain a high concentration of ubiquitin conjugates and at least two other components of the ubiquitin-proteasome system: 20S proteasome and Skp1. We propose to name these compartments FBXO25-associated nuclear domains. Interestingly, inhibition of transcription by actinomycin D or heat-shock treatment drastically affected the nuclear organization of FBXO25-containing structures, indicating that they are dynamic compartments influenced by the transcriptional activity of the cell. Also, we present evidences that an FBXO25-dependent ubiquitin ligase activity prevents aggregation of recombinant polyglutamine-containing huntingtin protein in the nucleus of human embryonic kidney 293 cells, suggesting that this protein can be a target for the nuclear FBXO25 mediated ubiquitination.

The N-terminal region of eukaryotic translation initiation factor 5A signals to nuclear localization of the protein.

The eukaryotic translation initiation factor 5A (eIF5A) is a ubiquitous protein of eukaryotic and archaeal organisms which undergoes hypusination, a unique post-translational modification. We have generated a polyclonal antibody against murine eIF5A, which in immunocytochemical assays in B16-F10 cells revealed that the endogenous protein is preferentially localized to the nuclear region. We therefore analyzed possible structural features present in eIF5A proteins that could be responsible for that characteristic. Multiple sequence alignment analysis of eIF5A proteins from different eukaryotic and archaeal organisms showed that the former sequences have an extended N-terminal segment. We have then performed in silico prediction analyses and constructed different truncated forms of murine eIF5A to verify any possible role that the N-terminal extension might have in determining the subcellular localization of the eIF5A in eukaryotic organisms. Our results indicate that the N-terminal extension of the eukaryotic eIF5A contributes in signaling this protein to nuclear localization, despite of bearing no structural similarity with classical nuclear localization signals.