Search

HERC2 gene encodes an E3 ubiquitin ligase involved in several cellular processes by regulating the ubiquitylation of different protein substrates. Biallelic pathogenic sequence variants in the HERC2 gene are associated with HERC2 Angelman-like syndrome. In pathogenic HERC2 variants, complete absence or marked reduction in HERC2 protein levels are observed. The most common pathological variant, c.1781C > T (p.Pro594Leu), encodes an unstable HERC2 protein. A better understanding of how pathologic HERC2 variants affect intracellular signalling may aid definition of potential new therapies for these disorders. For this purpose, we studied patient-derived cells with the HERC2 Pro594Leu variant. We observed alteration of mitogen-activated protein kinase signalling pathways, reflected by increased levels of C-RAF protein and p38 phosphorylation. HERC2 knockdown experiments reproduced the same effects in other human and mouse cells. Moreover, we demonstrated that HERC2 and RAF proteins form molecular complexes, pull-down and proteomic experiments showed that HERC2 regulates C-RAF ubiquitylation and we found out that the p38 activation due to HERC2 depletion occurs in a RAF/MKK3-dependent manner. The displayed cellular response was that patient-derived and other human cells with HERC2 deficiency showed higher resistance to oxidative stress with an increase in the master regulator of the antioxidant response NRF2 and its target genes. This resistance was independent of p53 and abolished by RAF or p38 inhibitors. Altogether, these findings identify the activation of C-RAF/MKK3/p38 signalling pathway in HERC2 Angelman-like syndrome and highlight the inhibition of RAF activity as a potential therapeutic option for individuals affected with these rare diseases. Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This study was funded by the following grants: JLR (Agencia Estatal de Investigación: PID2020-120344RB-I00/MCIN/AEI/10.13039/501100011033) and FV (PDC2021-121776-I00 and PID2020-117278 GB-I00 from MCIN/AEI/10.13039/501100011033 and FEDER “Una manera de hacer Europa” “NextGenerationEU”/PRTR. And Grant 202038-30 from “La Marató de TV3”). As well, this article is based upon work from COST Action ProteoCure CA20113, supported by COST (European Cooperation in Science and Technology). JSG and AMM received FPU Fellowships (FPU17/02413 and FPU18/06325, respectively) from the Spanish Ministry of Universities.

The ubiquitin-proteasome system is a post-translational regulatory pathway for controlling protein stability and activity that underlies many fundamental cellular processes, including cell cycle progression. Target proteins are tagged with ubiquitin molecules through the action of an enzymatic cascade composed of E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, and E3 ubiquitin ligases. One of the E3 ligases known to be responsible for the ubiquitination of cell cycle regulators in eukaryotes is the SKP1-CUL1-F-box complex (SCFC). In this work, we identified and studied the function of homologue proteins of the SCFC in the life cycle of Trypanosoma brucei, the causal agent of the African sleeping sickness. Depletion of trypanosomal SCFC components TbRBX1, TbSKP1, and TbCDC34 by RNAi resulted in decreased growth rate and contrasting cell cycle abnormalities for both procyclic (PCF) and bloodstream (BSF) forms. Depletion of TbRBX1 in PCF cells interfered with kinetoplast replication, whilst depletion of TbSKP1 arrested PCF and BSF cells in the G1/S transition. Silencing of TbCDC34 in BSF cells resulted in a block in cytokinesis and caused rapid clearance of parasites from infected mice. We also show that TbCDC34 is able to conjugate ubiquitin in vitro and in vivo, and that its activity is necessary for T. brucei infection progression in mice. This study reveals that different components of a putative SCFC have contrasting phenotypes once depleted from the cells, and that TbCDC34 is essential for trypanosome replication, making it a potential target for therapeutic intervention., This work was funded by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Exactas y Naturales (FCEyN), Universidad de Buenos Aires (UBA) and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), Argentina. (PICT 2005 N° 33631). Additional support was received from Boehringer-Ingelheim research fellowship. The Wellcome Trust Centre for Molecular Parasitology is supported by core funding from the Wellcome Trust [104111].

HERC1 is a ubiquitin ligase protein, which, when mutated, induces several malformations and intellectual disability in humans. The animal model of HERC1 mutation is the mouse tambaleante characterized by: (1) overproduction of the protein; (2) cerebellar Purkinje cells death by autophagy; (3) dysregulation of autophagy in spinal cord motor neurons, and CA3 and neocortical pyramidal neurons; (4) impairment of associative learning, linked to altered spinogenesis and absence of LTP in the lateral amygdala; and, (5) motor impairment due to delayed action potential transmission, decrease synaptic transmission efciency and altered myelination in the peripheral nervous system. To investigate the putative role of HERC1 in the presynaptic dynamics we have performed a series of experiments in cultured tambaleante hippocampal neurons by using transmission electron microscopy, FM1-43 destaining and immunocytochemistry. Our results show: (1) a decrease in the number of synaptic vesicles; (2) reduced active zones; (3) less clathrin immunoreactivity and less presynaptic endings over the hippocampal main dendritic trees; which contrast with (4) a greater number of endosomes and autophagosomes in the presynaptic endings of the tambaleante neurons relative to control ones. Altogether these results show an important role of HERC1 in the regulation of presynaptic membrane dynamics. Junta de Andalucía BIO-209 MINECO-DGICYT BFU2015-64536-R Junta de Andalucía BIO-122 MINECO-DGICYT BFU2015-64536-R MINECO-AEI/FEDER, UE, BFU2016-80295-R

The E3 ubiquitin ligase RNF8 plays critical roles in maintaining genomic stability by promoting the repair of DNA double-strand breaks (DSBs) through ubiquitin signaling. Abnormal activation of Notch signaling and defective repair of DSBs promote breast cancer risk. Here, we found that low expression of the full-length RNF8 correlated with poor prognosis for breast cancer patients. Our data revealed that in addition to its role in the repair of DSBs, RNF8 regulated Notch1 signaling and cell-fate determination of mammary luminal progenitors. Mechanistically, RNF8 acted as a negative regulator of Notch signaling by ubiquitylating the active NOTCH1 protein (N1ICD), leading to its degradation. Consistent with abnormal activation of Notch signaling and impaired repair of DSBs in Rnf8-mutant mammary epithelial cells, we observed increased risk of mammary tumorigenesis in mouse models for RNF8 deficiency. Notably, deficiency of RNF8 sensitized breast cancer cells to combination of pharmacological inhibitors of Notch signaling and poly(ADP-ribose) polymerase (PARP), suggesting implications for treatment of breast cancer associated with impaired RNF8 expression or function.

The 26S proteasome is the central proteolytic machinery of the ubiquitin proteasome system (UPS), which is involved in the degradation of ubiquitinated protein substrates. Recently, UPS inhibition has been shown to be a key factor in fatty liver graft preservation during organ cold storage using University of Wisconsin solution (UW) and Institute Georges Lopez (IGL-1) solutions. However, the merits of IGL-1 and histidine-tryptophan-ketoglutarate (HTK) solutions for fatty liver preservation have not been compared. Fatty liver grafts from obese Zücker rats were preserved for 24 h at 4 ◦C. Aspartate aminotransferase and alanine aminotransferase (AST/ALT), glutamate dehydrogenase (GLDH), ATP, adenosine monophosphate protein kinase (AMPK), e-NOS, proteasome activity and liver polyubiquitinated proteins were determined. IGL-1 solution prevented ATP breakdown during cold-storage preservation of steatotic livers to a greater extent than HTK solution. There were concomitant increases in AMPK activation, e-NOS (endothelial NOS (NO synthase)) expression and UPS inhibition. UPS activity is closely related to the composition of the solution used to preserve the organ. IGL-1 solution provided significantly better protection against ischemia-reperfusion for cold-stored fatty liver grafts than HTK solution. The effect is exerted through the activation of the protective AMPK signaling pathway, an increase in e-NOS expression and a dysregulation of the UPS., This work was supported by Instituto de Salud Carlos III (ISCIII) through the FIS project PI12/0056 co-funded by FEDER from Regional Development European Funds (European Union).

The voltage-dependent potassium channel Kv1.3 plays essential physiological functions in the immune system. Kv1.3, regulating the membrane potential, facilitates downstream Ca2+ -dependent pathways and becomes concentrated in specific membrane microdomains that serve as signaling platforms. Increased and/or delocalized expression of the channel is observed at the onset of several autoimmune diseases. In this work, we show that adenosine (ADO), which is a potent endogenous modulator, stimulates PKC, thereby causing immunosuppression. PKC activation triggers down-regulation of Kv1.3 by inducing a clathrin-mediated endocytic event that targets the channel to lysosomal-degradative compartments. Therefore, the abundance of Kv1.3 at the cell surface decreases, which is clearly compatible with an effective anti-inflammatory response. This mechanism requires ubiquitination of Kv1.3, catalyzed by the E3 ubiquitin-ligase Nedd4-2. Postsynaptic density protein 95 (PSD-95), a member of the MAGUK family, recruits Kv1.3 into lipid-raft microdomains and protects the channel against ubiquitination and endocytosis. Therefore, the Kv1.3/PSD-95 association fine-tunes the anti-inflammatory response in leukocytes. Because Kv1.3 is a promising multi-therapeutic target against human pathologies, our results have physiological relevance. In addition, this work elucidates the ADO-dependent PKC-mediated molecular mechanism that triggers immunomodulation by targeting Kv1.3 in leukocytes.

The reduction of oxidative stress is suggested to be one of the main mechanisms to explain the benefits of subnormothermic perfusion against ischemic liver damage. In this study we investigated the early cellular mechanisms induced in isolated ratliversafter15minperfusionattemperaturesrangingfromnormothermia(37 ∘ C) to subnormothermia (26 ∘ Cand22 ∘ C). Subnormothermic perfusion was found to maintain hepatic viability. Perfusion at 22 ∘ C raised reduced glutathione levels and the activity of glutathione reductase; however, lipid and protein oxidation still occurred as determined by malondialdehyde, 4-hydroxynonenal-protein adducts, and advanced oxidation protein products. In livers perfused at 22 ∘ C the lysosomal and ubiquitin proteasome system (UPS) were both activated. The 26S chymotrypsin-like ( 훽 5) proteasome activity was significantly increased in the 26 ∘ C (46%) and 22 ∘ C (42%) groups. The increased proteasome activity may be due to increased Rpt6 Ser120 phosphorylation, which is known to enhance 26S proteasome activity. Together, our results indicate that the early events produced by subnormothermic perfusion in the liver can induce oxidative stress concomitantly with antioxidant glutathione preservation and enhanced function of the lysosomal and UPS systems. Thus, a brief hypothermia could trigger antioxidant mechanisms and may be functioning as a preconditioning stimulus.

Ubiquitination is a relevant cell regulatory mechanism to determine protein fate and function. Most data has focused on the role of ubiquitin as a tag molecule to target substrates to proteasome degradation, and on its impact in the control of cell cycle, protein homeostasis and cancer. Only recently, systematic assays have pointed to the relevance of the ubiquitin pathway in the development and differentiation of tissues and organs, and its implication in hereditary diseases. Moreover, although the activity and composition of ubiquitin ligases has been largely addressed, the role of the deubiquitinating enzymes (DUBs) in specific tissues, such as the retina, remains mainly unknown. In this work, we undertook a systematic analysis of the transcriptional levels of DUB genes in the adult mouse retina by RT-qPCR and analyzed the expression pattern by in situ hybridization and fluorescent immunohistochemistry, thus providing a unique spatial reference map of retinal DUB expression. We also performed a systematic phylogenetic analysis to understand the origin and the presence/absence of DUB genes in the genomes of diverse animal taxa that represent most of the known animal diversity. The expression landscape obtained supports the potential subfunctionalization of paralogs in those families that expanded in vertebrates. Overall, our results constitute a reference framework for further characterization of the DUB roles in the retina and suggest new candidates for inherited retinal disorders.

Brain-derived neurotrophic factor (BDNF) regulates synaptic strengthening and memory consolidation, and altered BDNF expression is implicated in a number of neuropsychiatric and neurodegenerative disorders. BDNF potentiates N-methyl-D-aspartate receptor function through activation of Fyn and ERK1/2. STriatal-Enriched protein tyrosine Phosphatase (STEP) is also implicated in many of the same disorders as BDNF but, in contrast to BDNF, STEP opposes the development of synaptic strengthening. STEP-mediated dephosphorylation of the NMDA receptor subunit GluN2B promotes internalization of GluN2B-containing NMDA receptors, while dephosphorylation of the kinases Fyn, Pyk2, and ERK1/2 leads to their inactivation. Thus, STEP and BDNF have opposing functions. In this study, we demonstrate that manipulation of BDNF expression has a reciprocal effect on STEP61 levels. Reduced BDNF signaling leads to elevation of STEP61 both in BDNF(+/-) mice and after acute BDNF knockdown in cortical cultures. Moreover, a newly identified STEP inhibitor reverses the biochemical and motor abnormalities in BDNF(+/-) mice. In contrast, increased BDNF signaling upon treatment with a tropomyosin receptor kinase B agonist results in degradation of STEP61 and a subsequent increase in the tyrosine phosphorylation of STEP substrates in cultured neurons and in mouse frontal cortex. These findings indicate that BDNF-tropomyosin receptor kinase B signaling leads to degradation of STEP61 , while decreased BDNF expression results in increased STEP61 activity. A better understanding of the opposing interaction between STEP and BDNF in normal cognitive functions and in neuropsychiatric disorders will hopefully lead to better therapeutic strategies. Altered expression of BDNF and STEP61 has been implicated in several neurological disorders. BDNF and STEP61 are known to regulate synaptic strengthening, but in opposite directions. Here, we report that reduced BDNF signaling leads to elevation of STEP61 both in BDNF(+/-) mice and after acute BDNF knockdown in cortical cultures. In contrast, activation of TrkB receptor results in the degradation of STEP61 and reverses hyperlocomotor activity in BDNF(+/-) mice. Moreover, inhibition of STEP61 by TC-2153 is sufficient to enhance the Tyr phosphorylation of STEP substrates and also reverses hyperlocomotion in BDNF(+/-) mice. These findings give us a better understanding of the regulation of STEP61 by BDNF in normal cognitive functions and in neuropsychiatric disorders.

UEV proteins are enzymatically inactive variants of the E2 ubiquitin-conjugating enzymes that regulate noncanonical elongation of ubiquitin chains. In Saccharomyces cerevisiae, UEV is part of the RAD6-mediated error-free DNA repair pathway. In mammalian cells, UEV proteins can modulate c-FOS transcription and the G2-M transition of the cell cycle. Here we show that the UEV genes from phylogenetically distant organisms present a remarkable conservation in their exon–intron structure. We also show that the human UEV1 gene is fused with the previously unknown gene Kua. In Caenorhabditis elegans and Drosophila melanogaster, Kua and UEV are in separated loci, and are expressed as independent transcripts and proteins. In humans, Kua and UEV1 are adjacent genes, expressed either as separate transcripts encoding independent Kua and UEV1 proteins, or as a hybrid Kua–UEV transcript, encoding a two-domain protein. Kua proteins represent a novel class of conserved proteins with juxtamembrane histidine-rich motifs. Experiments with epitope-tagged proteins show that UEV1A is a nuclear protein, whereas both Kua and Kua–UEV localize to cytoplasmic structures, indicating that the Kua domain determines the cytoplasmic localization of Kua–UEV. Therefore, the addition of a Kua domain to UEV in the fused Kua–UEV protein confers new biological properties to this regulator of variant polyubiquitination./n/n[Kua cDNAs isolated by RT-PCR and described in this paper have been deposited in the GenBank data library under accession nos. AF1155120 (H. sapiens) and AF152361 (D. melanogaster). Genomic clones containing UEV genes: S. cerevisiae, YGL087c (accession no. Z72609); S. pombe, c338 (accession no. AL023781); P. falciparum, MAL3P2 (accession no. AL034558); A. thaliana, F26F24 (accession no. AC005292); C. elegans, F39B2 (accession no. Z92834); D. melanogaster, AC014908; and H. sapiens, 1185N5 (accession no. AL034423). Accession numbers for Kua cDNAs in GenBank dbEST: M. musculus, AA7853; T. cruzi, AI612534. Other Kua-containing sequences: A. thaliana genomic clones F10M23 (accession no. AL035440), F19K23 (accession no. AC000375), and T20K9 (accession no. AC004786).

Background: Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the dysfunctional Cl - channel in Cystic Fibrosis, undergoes complex biosynthesis at the endoplasmic reticulum involving several molecular chaperones including Hsp70 and many co-chaperones. Bcl-2associated athanogenes (BAGs) constitute a protein family sharing an Hsc70-binding domain. BAG-1 possesses an ubiquitin-like domain (Ub-LD) responsible for proteasomal association and for promoting substrate release from Hsc70/Hsp70 in vitro by accelerating the chaperone ATP/ADP exchange rate. Methods: Herein, we studied the in vivo effect of BAG-1 on the turnover and processing of wild type (wt)- and F508del-CFTR, the most frequent mutation in CF patients. Results: Results show that BAG-1 associates with both wt- and F508del-CFTR (in higher yields with the latter) through its Ub-LD and independently of Hsc70. Moreover, the immature form of F508del-CFTR (but not of wt-CFTR) is stabilized by BAG-1 overexpression, albeit in a cell-type specific way, without detectable maturation. Data also show that BAG-1 and the proteasome inhibitor ALLN are not additive on stabilizing F508del-CFTR and this effect depends on BAG-1 Ub-LD. Moreover, under BAG-1 overexpression, a reduction in ubiquitinylated-CFTR occurs suggesting that BAG-1 competes with Ub. Conclusion: Overall, data are compatible with a mechanism in which BAG-1 stabilizes F508del-CFTR by direct binding, probably competing out ubiquitin to partially avoid its proteasomal degradation.

Huntington's disease (HD) inclusions are stained with anti-ubiquitin and anti-proteasome antibodies. This, together with proteasome activity studies on transfected cells, suggest that an impairment of the ubiquitin-proteasome system (UPS) may be key in HD pathogenesis. To test whether proteasome activity is impaired in vivo, we performed enzymatic assays for the three peptidase activities of the proteasome in brain extracts from the HD94 conditional mouse model of HD. We found no inhibition of any of the activities, suggesting that if UPS impairment happens in vivo, it is not at the level of the proteasome catalytic core. Intriguingly, the chymotrypsin- and trypsin-like activities increased selectively in the affected and aggregate-containing regions: cortex and striatum. Western blot analysis revealed no difference in total proteasome content whereas an increase in the interferon-inducible subunits of the immunoproteasome, LMP2 and LMP7, was observed. These subunits confer to the proteasome catalytic properties that are optimal for MHC-I peptide presentation. Immunohistochemistry in control mouse brain revealed LMP2 and LMP7 mainly in neurons. Accordingly, their increase in HD94 mice predominantly took place in neurons, and 5% of the ubiquitin-positive cortical aggregates were also LMP2-positive. Ultrastructural analysis of neurons with high level of immunoproteasome subunits revealed signs of neurodegeneration like nuclear indentation or fragmentation and dark cell appearance. The neuronal induction of LMP2 and LMP7 and the associated signs of neurodegeneration were also found in HD postmortem brains. Our results indicate that LMP2 and LMP7 participate in normal neuronal physiology and suggest a role in HD neurodegeneration., This work was supported by grants from Comunidad Autónoma de Madrid, Asociación Española de Corea de Huntington (ACHE), Fundación “La Caixa”, Spanish Comisión Interministerial de Ciencia y Tecnología, and by an institutional grant from Fundación Ramón Areces. E.M.-A. was the recipient of predoctoral fellowships from ACHE and Fundación Ferrer. M.D.-H. is the recipient of a postdoctoral fellowship from Comunidad Autónoma de Madrid.

Under a Creative Commons license.-- et al., Extracellular signal-regulated kinase 5 (ERK5), also known as big mitogen-activated protein kinase (MAPK) 1, is implicated in a wide range of biologic processes, which include proliferation or vascularization. Here, we show that ERK5 is degraded through the ubiquitin-proteasome system, in a process mediated by the tumor suppressor von Hippel-Lindau (VHL) gene, through a prolyl hydroxylation-dependent mechanism. Our conclusions derive from transient transfection assays in Cos7 cells, as well as the study of endogenous ERK5 in different experimental systems such as MCF7, HMEC, or Caki-2 cell lines. In fact, the specific knockdown of ERK5 in pVHL-negative cell lines promotes a decrease in proliferation and migration, supporting the role of this MAPK in cellular transformation. Furthermore, in a short series of fresh samples from human clear cell renal cell carcinoma, high levels of ERK5 correlate with more aggressive and metastatic stages of the disease. Therefore, our results provide new biochemical data suggesting that ERK5 is a novel target of the tumor suppressor VHL, opening a new field of research on the role of ERK5 in renal carcinomas., This work was supported by grants from Fundación Leticia Castillejo Castillo and Ministerio de Economía y Competitividad (SAF2009-07329 and SAF2012-30862) and grant JCCM PPII10-0141-040 to R.S.P., grant FIS PI080432 and a grant from Fundación Para la Investigación en Urología to A.S.S.-S., grant FISCAM PI2007-38 to J.M.G.-B., and grant FIS PS09/00868 to A.E.-O. R.S.P. and A.P. Research Institutes and the work carried out in their laboratories receive support from the European Community through the Regional Development Funding Program (FEDER).

We have used a method previously described by Reeves and Candido (1) to partially release histone deacetylase from cell nuclei together with putative regulators of the enzyme. Histone deacetylase released from testis cell nuclei and its putative regulators were separated by gel filtration in Sepharose 6B. A peak of low molecular weight contains a heat-stable factor that stimulate histone deacetylase in vitro. Many of the properties of the activator coincide with those of the protein HMG-20 (ubiquitin). Ubiquitin isolated from testis cell nuclei stimulated histone deacetylase in vitro. It has been suggested that HMG-17 partially inhibits histone deacetylase in Fried cell nuclei (2). In our system, HMG-17 shows no inhibitory effect on histone deacetylase activity.

12 páginas, 5 figuras, 2 tablas., Lafora progressive myoclonus epilepsy (Lafora disease, LD) is a fatal rare autosomal recessive neurodegenerative disorder characterized by the accumulation of insoluble ubiquitinated polyglucosan inclusions in the cytoplasm of neurons, which is most commonly associated with mutations in two genes: EPM2A, encoding the glucan phosphatase laforin, and EPM2B, encoding the E3-ubiquitin ligase malin. The present study analyzes possible inflammatory responses in the mouse lines Epm2a-/- (laforin knock-out) and Epm2b-/- (malin knock-out) with disease progression. Increased numbers of reactive astrocytes (expressing the GFAP marker) and microglia (expressing the Iba1 marker) together with increased expression of genes encoding cytokines and mediators of the inflammatory response occur in both mouse lines although with marked genotype differences. C3ar1 and CxCl10 mRNAs are significantly increased in Epm2a-/- mice aged 12 months when compared with age-matched controls, whereas C3ar1, C4b, Ccl4, CxCl10, Il1b, Il6, Tnfα and Il10ra mRNAs are significantly up-regulated in Epm2b-/- at the same age. This is accompanied by increased protein levels of IL1-β, IL6, TNFα and Cox2 particularly in Epm2b-/- mice. The severity of inflammatory changes correlates with more severe clinical symptoms previously described in Epm2b-/- mice. These findings show for the first time increased innate inflammatory responses in a neurodegenerative disease with polyglucosan intraneuronal deposits which increase with disease progression, in a way similar to what is seen in neurodegenerative diseases with abnormal protein aggregates. These findings also point to the possibility of using anti-inflammatory agents to mitigate the degenerative process in LD., This study was funded by the Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III – Fondos FEDER, a way to build Europe FIS grants PI14/00757 and PI14/00328 to IF, and grants from the Spanish Ministry of Economy and Competiveness SAF2014-54604-C3-1-R and from the Generalitat Valenciana (PrometeoII/2014/029) to PS. We thank T. Yohannan for editorial assistance.

Tissue protein hypercatabolism (TPH) is a most important feature in cancer cachexia, particularly with regard to the skeletal muscle. The rat ascites hepatoma Yoshida AH-130 is a very suitable model system for studying the mechanisms involved in the processes that lead to tissue depletion, since it induces in the host a rapid and progressive muscle waste mainly due to TPH (Tessitore, L., G. Bonelli, and F. M. Baccino. 1987. Biochem. J. 241:153-159). Detectable plasma levels of tumor necrosis factor-alpha associated with marked perturbations in the hormonal homeostasis have been shown to concur in forcing metabolism into a catabolic setting (Tessitore, L., P. Costelli, and F. M. Baccino. 1993. Br. J. Cancer. 67:15-23). The present study was directed to investigate if beta 2-adrenergic agonists, which are known to favor skeletal muscle hypertrophy, could effectively antagonize the enhanced muscle protein breakdown in this cancer cachexia model. One such agent, i.e., clenbuterol, indeed largely prevented skeletal muscle waste in AH-130-bearing rats by restoring protein degradative rates close to control values. This normalization of protein breakdown rates was achieved through a decrease of the hyperactivation of the ATP-ubiquitin-dependent proteolytic pathway, as previously demonstrated in our laboratory (Llovera, M., C. García-Martínez, N. Agell, M. Marzábal, F. J. López-Soriano, and J. M. Argilés. 1994. FEBS (Fed. Eur. Biochem. Soc.) Lett. 338:311-318). By contrast, the drug did not exert any measurable effect on various parenchymal organs, nor did it modify the plasma level of corticosterone and insulin, which were increased and decreased, respectively, in the tumor hosts. The present data give new insights into the mechanisms by which clenbuterol exerts its preventive effect on muscle protein waste and seem to warrant the implementation of experimental protocols involving the use of clenbuterol or alike drugs in the treatment of pathological states involving TPH, particularly in skeletal muscle and heart, such as in the present model of cancer cachexia.

This work was supported by funds from the MICINN (AGL2009-12427 and AGL2010-17324), the Catalonian Government (2009SGR-00402) and the “Xarxa de Referència d’R + D + I en Aqüicultura” and, the European Union through the project LIFECYCLE (FP7-222719). Background: The proteolytic enzymes involved in normal protein turnover in fish muscle are also responsible for post-mortem softening of the flesh and are therefore potential determinants of product quality. The main enzyme systems involved are calpains, cathepsins, and the ubiquitin-proteasome (UbP). In this study on Sparus aurata (Sa), the coding sequences of cathepsins (SaCTSB and SaCTSDb) and UbP family members (SaN3 and SaUb) were cloned from fast skeletal muscle, and their expression patterns were examined during ontogeny and in a fasting/re-feeding experiment. Results: The amino acid sequences identified shared 66-100% overall identity with their orthologues in other vertebrates, with well conserved characteristic functional domains and catalytic residues. SaCTSDb showed phylogenetic, sequence and tissue distribution differences with respect to its paralogue SaCTSDa, previously identified in the ovary. Expression of gilthead sea bream cathepsins (B, L, Da, Db) and UbP members (N3, Ub, MuRF1 and MAFbx) in fast skeletal muscle was determined at three different life-history stages and in response to fasting and re-feeding in juveniles. Most of the proteolytic genes analysed were significantly up-regulated during fasting, and down-regulated with re-feeding and, between the fingerling (15 g) and juvenile/adult stages (~50/500 g), consistent with a decrease in muscle proteolysis in both later contexts. In contrast, SaCTSDa and SaMuRF1 expression was relatively stable with ontogeny and SaUb had higher expression in fingerlings and adults than juveniles. Conclusions: The data obtained in the present study suggest that cathepsins and UbP genes in gilthead sea bream are co-ordinately regulated during ontogeny to control muscle growth, and indicate that feeding regimes can modulate their expression, providing a potential dietary method of influencing post-mortem fillet tenderisation, and hence, product quality. Publisher PDF