Control Of Protein Degradation Research Articles

Integrated control of protein degradation in C. elegans muscle.

Protein degradation is a fundamental cellular process, the genomic control of which is incompletely understood. The advent of transgene-coded reporter proteins has enabled the development of C. elegans into a model for studying this problem. The regulation of muscle protein degradation is surprisingly complex, integrating multiple signals from hypodermis, intestine, neurons and muscle itself. Within the muscle, degradation is executed by separately regulated autophagy-lysosomal, ubiquitin-proteasome and calpain-mediated systems. The signal-transduction mechanisms, in some instances, involve modules previously identified for their roles in developmental processes, repurposed in terminally differentiated muscle to regulate the activities of pre-formed proteins. Here we review the genes, and mechanisms, which appear to coordinately control protein degradation within C. elegans muscle. We also consider these mechanisms in the context of development, physiology, pathophysiology and disease models.

The β2-Microglobulin–Free Heterodimerization of Rhesus Monkey MHC Class I A with Its Normally Spliced Variant Reduces the Ubiquitin-Dependent Degradation of MHC Class I A

The MHC class I (MHC I) molecules play a pivotal role in the regulation of immune responses by presenting antigenic peptides to CTLs and by regulating cytolytic activities of NK cells. In this article, we show that MHC I A in rhesus macaques can be alternatively spliced, generating a novel MHC I A isoform (termed "MHC I A-sv1") devoid of α(3) domain. Despite the absence of β2-microglobulin (β2m), the MHC I A-sv1 proteins reached the cell surface of K562-transfected cells as endoglycosidase H-sensitive glycoproteins that could form disulfide-bonded homodimers. Cycloheximide-based protein chase experiments showed that the MHC I A-sv1 proteins were more stable than the full-length MHC I A in transiently or stably transfected cell lines. Of particular interest, our studies demonstrated that MHC I A-sv1 could form β2m-free heterodimers with its full-length protein in mammalian cells. The formation of heterodimers was accompanied by a reduction in full-length MHC I A ubiquitination and consequent stabilization of the protein. Taken together, these results demonstrated that MHC I A-sv1 and MHC I A can form a novel heterodimeric complex as a result of the displacement of β2m and illustrated the relevance of regulated MHC I A protein degradation in the β2m-free heterodimerization-dependent control, which may have some implications for the MHC I A splice variant in the fine tuning of classical MHC I A/TCR and MHC I A/killer cell Ig-like receptor interactions.

The key role of ubiquitination and sumoylation in signaling and cancer: a research topic

EDITORIAL article Front. Oncol., 05 December 2012 | https://doi.org/10.3389/fonc.2012.00187

Therapeutic targeting of the endoplasmic reticulum in Alzheimer's disease.

The extensive prevalence of Alzheimer's disease (AD) places a tremendous burden physiologically, socially and economically upon those directly suffering and those caring for sufferers themselves. Considering the steady increases in numbers of patients diagnosed with Alzheimer's, the number of effective pharmacotherapeutic strategies to tackle the disease is still relatively few. As with many other neurodegenerative mechanisms, AD, is characterized by the continued presence and accumulation of cytotoxic protein aggregates, i.e. of beta-amyloid and the microtubule associated protein, tau. Therefore, one novel therapeutic avenue for the treatment of AD may be the actual targeting of factors that control protein synthesis, packaging and degradation. One of the prime cellular targets that, if effectively modulated, could accomplish this is the endoplasmic reticulum (ER). The ER can not only control cellular protein synthesis, trafficking and degradation but it is also closely associated with cytoprotective mechanisms, including calcium ion regulation and unfolded protein responses. This review will delineate some of the most important functional physiological features of the ER that, if effectively modulated, could result in beneficial amelioration or remediation of the negative cellular aspects of AD initiation and progression. While not a classical drug target, even with minimal levels of beneficial modulation, its multifactorial efficacy may amplify small effects resulting in significant therapeutic efficacy.

Muscle sparing in muscle RING finger 1 null mice: response to synthetic glucocorticoids

Skeletal muscle atrophy occurs under a variety of conditions and can result from alterations in both protein synthesis and protein degradation. The muscle-specific E3 ubiquitin ligases, MuRF1 and MAFbx, are excellent markers of muscle atrophy and increase under divergent atrophy-inducing conditions such as denervation and glucocorticoid treatment. While deletion of MuRF1 or MAFbx has been reported to spare muscle mass following 14 days of denervation, their role in other atrophy-inducing conditions is unclear. The goal of this study was to determine whether deletion of MuRF1 or MAFbx attenuates muscle atrophy after 2 weeks of treatment with the synthetic glucocorticoid dexamethasone (DEX). The response of the triceps surae (TS) and tibialis anterior (TA) muscles to 14 days of DEX treatment (3 mg kg(-1) day(-1)) was examined in 4 month-old male and female wild type (WT) and MuRF1 or MAFbx knock out (KO) mice. Following 14 days of DEX treatment, muscle wet weight was significantly decreased in the TS and TA of WT mice. Comparison of WT and KO mice following DEX treatment revealed significant sparing of mass in both sexes of the MuRF1 KO mice, but no muscle sparing in MAFbx KO mice. Further analysis of the MuRF1 KO mice showed significant sparing of fibre cross-sectional area and tension output in the gastrocnemius (GA) after DEX treatment. Muscle sparing in the MuRF1 KO mice was related to maintenance of protein synthesis, with no observed increases in protein degradation in either WT or MuRF1 KO mice. These results demonstrate that MuRF1 and MAFbx do not function similarly under all atrophy models, and that the primary role of MuRF1 may extend beyond controlling protein degradation via the ubiquitin proteasome system.

Small-Molecule Control of Protein Degradation Using Split Adaptors

Targeted intracellular degradation provides a method to study the biological function of proteins and has numerous applications in biotechnology. One promising approach uses adaptor proteins to target substrates with genetically encoded degradation tags for proteolysis. Here, we describe an engineered split-adaptor system, in which adaptor assembly and delivery of substrates to the ClpXP protease depends on a small molecule (rapamycin). This degradation system does not require modification of endogenous proteases, functions robustly over a wide range of adaptor concentrations, and does not require new synthesis of adaptors or proteases to initiate degradation. We demonstrate the efficacy of this system in E. coli by degrading tagged variants of LacI repressor and FtsA, an essential cell-division protein. In the latter case, addition of rapamycin causes pronounced filamentation because daughter cells cannot divide. Strikingly, washing rapamycin away reverses this phenotype. Our system is highly modular, with clearly defined interfaces for substrate binding, protease binding, and adaptor assembly, providing a clear path to extend this system to other degradation tags, proteases, or induction systems. Together, these new reagents should be useful in controlling protein degradation in bacteria.

Post-Transcriptional Fine-tuning of COP9 Signalosome Subunit Biosynthesis is Regulated by the c-Myc/Lin28B/let-7 Pathway

The COP9 signalosome (CSN) complex controls protein degradation via the ubiquitin (Ub) proteasome system (UPS) in eukaryotes. In mammalian cells, the multimeric CSN is composed of eight subunits (CSN1 – CSN8). It regulates cullin-RING Ub ligases (CRLs), which target essential regulatory proteins for ubiquitination and subsequent degradation. Thereby, the CSN cooperates with the UPS in a variety of essential cellular functions, including DNA repair, cell cycle and differentiation. Although functions of the CSN have been elucidated, mechanisms and regulatory principles of its de novo formation are completely unknown. Here, we show that there is a fundamental mechanism that allows a coordinated expression of all CSN subunits, a prerequisite for CSN assembly. CSN subunit mRNAs are targets of miRNAs of the let-7 family suppressing CSN subunit expression in human cells. Factors that reduce or block let-7 miRNAs induce the coordinated expression of CSN subunits. For instance, over-expression of CSN1 specifically traps let-7a-1 miRNA and elevates CSN subunit levels by two- to fourfold in a coordinated manner. CSN subunit expression is also increased by specific miRNA inhibitors or by interferon (IFN)-mediated induction of STAT1 and c-Myc reducing levels of let-7 miRNAs. Activation of STAT1 by IFNα or IFNγ induces c-Myc, which increases CSN subunit expression via the Lin28B/let-7 regulatory pathway. By contrast, a let-7a-1 mimic reduces CSN subunit expression. Our data show that let-7 miRNAs control the fine-tuning and coordinated expression of subunits for CSN de novo formation, presumably a general regulatory principle for other Zomes complexes as well.

Lessons from Fruit Fly Models for PolyQ Neurodegenerative Disorders

Polyglutamine (polyQ) diseases, resulting from a dynamic expansion of glutamine repeats in a polypeptide, are a class of genetically inherited, late-onset neurodegenerative disorders which, despite wide expression of the mutated gene in brain and other tissues, affect defined subpopulations of neurons in a disease-specific manner. This presentation will review the advantages of modelling these neurodegenerative diseases in Drosophila, and the insights obtained. Studies with the fruit fly models have successfully identified a variety of genetic modifiers, and have helped to further our understanding of some of the molecular events that follow expression of the abnormal polyQ proteins. Expression of the mutant polyQ proteins causes, as a consequence of intra-cellular and inter-cellular networking, mis-regulation in the sensitive neurons at multiple points, such as during transcriptional regulation, post-transcriptional regulation, cell signalling, protein quality control (protein folding and degradation networks), and axonal transport, resulting ultimately in cell death. The diversity of genetic modifiers of polyQ toxicity, identified through extensive genetic screening in the fruit fly and in other models, clearly reflects a complex network effect of the presence of the mutated protein. Such a network effect poses a major challenge for therapeutic applications.

The Alg5 ortholog Wollknäuel is essential for correct epidermal differentiation during Drosophila late embryogenesis

The formation of an extracellular matrix (ECM) presupposes an ordered delivery of its components to ensure its stereotypic architecture. The Drosophila cuticle is an ECM produced by the epidermis at its apical site and is characterized by a layered organization. To understand the mechanisms of cuticle assembly during development, we have investigated early aspects of protein N-glycosylation, i.e. the attachment of a dolichol-linked oligosaccharide to distinct Asn sites of a protein known to be essential for sorting in the secretory pathway. Mutations in the Drosophila alg5 gene wollknäuel (wol) that codes for an enzyme initiating the glucosylation of the dolichol-linked oligosaccharide decrease, as expected, glucosylation and the amounts of N-glycosylated proteins such as the cuticle-organizing factor Knickkopf, without affecting their correct localization. At the same time, the polarity determinants Crumbs and atypical protein kinase C accumulate at the apical plasma membrane in wol deficient embryos. In part, these perturbations may also be caused by the unfolded protein response, which is commonly triggered by ER stress and downsizes transcription and translation in general. In any case, they are associated with the loss of cuticle layering and aberrant apical plasma membrane organization suggesting that glucosylation, either directly or indirectly through controlling protein degradation, is important for the efficient and balanced deployment of the biochemical functions of secreted and membrane-associated proteins during epidermal differentiation.

Quantitative Protein and mRNA Profiling Shows Selective Post-Transcriptional Control of Protein Expression by Vasopressin in Kidney Cells

Previous studies in yeast have supported the view that post-transcriptional regulation of protein abundances may be more important than previously believed. Here we ask the question: "In a physiological regulatory process (the response of mammalian kidney cells to the hormone vasopressin), what fraction of the expressed proteome undergoes a change in abundance and what fraction of the regulated proteins have corresponding changes in mRNA levels?" In humans and other mammals, vasopressin fulfills a vital homeostatic role (viz. regulation of renal water excretion) by regulating the water channel aquaporin-2 in collecting duct cells. To address the question posed, we utilized large-scale quantitative protein mass spectrometry (LC-MS/MS) employing stable isotopic labeling in cultured mpkCCD cells ('SILAC') coupled with transcriptomic profiling using oligonucleotide expression arrays (Affymetrix). Preliminary studies analyzing two nominally identical control samples by SILAC LC-MS/MS yielded a relative S.D. of 13% (for ratios), establishing the precision of the SILAC approach in our hands. We quantified nearly 3000 proteins with nontargeted SILAC LC-MS/MS, comparing vasopressin- versus vehicle-treated samples. Of these proteins 786 of them were quantified in each of 3 experiments, allowing statistical analysis and 188 of these showed significant vasopressin-induced changes in abundance, including aquaporin-2 (20-fold increase). Among the proteins with statistically significant abundance changes, a large fraction (at least one-third) was found to lack changes in the corresponding mRNA species (despite sufficient statistical power), indicating that post-transcriptional regulation of protein abundance plays an important role in the vasopressin response. Bioinformatic analysis of the regulated proteins (versus all transcripts) shows enrichment of glutathione S-transferase isoforms as well as proteins involved in organization of the actin cytoskeleton. The latter suggests that long-term regulatory processes may contribute to actomyosin-dependent trafficking of the water channel aquaporin-2. The results provide impetus for increased focus on translational regulation and regulation of protein degradation in physiological control in mammalian epithelial cells.

Frequent Mutation of BAP1 in Metastasizing Uveal Melanomas

Metastasis is a defining feature of malignant tumors and is the most common cause of cancer-related death, yet the genetics of metastasis are poorly understood. We used exome capture coupled with massively parallel sequencing to search for metastasis-related mutations in highly metastatic uveal melanomas of the eye. Inactivating somatic mutations were identified in the gene encoding BRCA1-associated protein 1 (BAP1) on chromosome 3p21.1 in 26 of 31 (84%) metastasizing tumors, including 15 mutations causing premature protein termination and 5 affecting its ubiquitin carboxyl-terminal hydrolase domain. One tumor harbored a frameshift mutation that was germline in origin, thus representing a susceptibility allele. These findings implicate loss of BAP1 in uveal melanoma metastasis and suggest that the BAP1 pathway may be a valuable therapeutic target.

Transcriptome changes in grapevine (Vitis vinifera L.) cv. Malbec leaves induced by ultraviolet-B radiation.

BackgroundUltraviolet-B radiation (UV-B, 280-315 nm) is a natural component of sunlight, which has numerous regulatory effects on plant physiology. The nature of the response to UV-B is dependent on fluence rate, dose, duration and wavelength of the UV-B treatment. Some reports have analyzed the changes in gene expression caused by UV-B light on several plant species using microarray technology. However, there is no information on the transcriptome response triggered by UV-B in grapevine. In this paper we investigate the gene expression responses of leaves from in vitro cultured Vitis vinifera cv. Malbec plants subjected to the same dose of biologically effective UV-B radiation (4.75 kJ m-2 d-1) administered at two different fluence rates (16 h at ≅ 8.25 μW cm-2, 4 h at ≅ 33 μW cm-2) using a new custom made GrapeGen Affymetrix GeneChip®.ResultsThe number of genes modulated by high fluence rate UV-B doubled the number of genes modulated by low fluence UV-B. Their functional analyses revealed several functional categories commonly regulated by both UV-B treatments as well as categories more specifically modulated depending on UV-B fluence rate. General protective responses, namely the induction of pathways regulating synthesis of UV-B absorbing compounds such as the Phenylpropanoid pathway, the induction of different antioxidant defense systems and the activation of pathways commonly associated with pathogen defense and abiotic stress responses seem to play critical roles in grapevine responses against UV-B radiation. Furthermore, high fluence rate UV-B seemed to specifically modulate additional pathways and processes in order to protect grapevine plantlets against UV-B-induced oxidative stress, stop the cell cycle progression, and control protein degradation. On the other hand, low fluence rate UV-B regulated the expression of specific responses in the metabolism of auxin and abscisic acid as well as in the modification of cell walls that could be involved in UV-B acclimation-like processes.ConclusionOur results show the UV-B radiation effects on the leaf transcriptome of grapevine (Vitis vinifera cv. Malbec) plantlets. Functional categories commonly modulated under both UV-B treatments as well as transcripts specifically regulated in an UV-B-intensity dependent way were identified. While high fluence rate UV-B had regulatory effects mainly on defense or general multiple-stress responses pathways, low fluence rate UV-B promoted the expression of genes that could be involved in UV-B protection or the amelioration of the UV-B-induced damage. This study also provides an extensive list of genes regulating multiple metabolic pathways involved in the response of grapevine to UV-B that can be used for future researches.

A Single ClpS Monomer Is Sufficient to Direct the Activity of the ClpA Hexamer

ClpS is an adaptor protein that interacts with ClpA and promotes degradation of proteins with N-end rule degradation motifs (N-degrons) by ClpAP while blocking degradation of substrates with other motifs. Although monomeric ClpS forms a 1:1 complex with an isolated N-domain of ClpA, only one molecule of ClpS binds with high affinity to ClpA hexamers (ClpA(6)). One or two additional molecules per hexamer bind with lower affinity. Tightly bound ClpS dissociates slowly from ClpA(6) with a t((1/2)) of approximately 3 min at 37 degrees C. Maximum activation of degradation of the N-end rule substrate, LR-GFP(Venus), occurs with a single ClpS bound per ClpA(6); one ClpS is also sufficient to inhibit degradation of proteins without N-degrons. ClpS competitively inhibits degradation of unfolded substrates that interact with ClpA N-domains and is a non-competitive inhibitor with substrates that depend on internal binding sites in ClpA. ClpS inhibition of substrate binding is dependent on the order of addition. When added first, ClpS blocks binding of both high and low affinity substrates; however, when substrates first form committed complexes with ClpA(6), ClpS cannot displace them or block their degradation by ClpP. We propose that the first molecule of ClpS binds to the N-domain and to an additional functional binding site, sterically blocking binding of non-N-end rule substrates as well as additional ClpS molecules to ClpA(6). Limiting ClpS-mediated substrate delivery to one per ClpA(6) avoids congestion at the axial channel and allows facile transfer of proteins to the unfolding and translocation apparatus.

L-Arginine stimulates proliferation and prevents endotoxin-induced death of intestinal cells.

This study tested the hypothesis that l-arginine (Arg) may stimulate cell proliferation and prevent lipopolysaccharide (LPS)-induced death of intestinal cells. Intestinal porcine epithelial cells (IPEC-1) were cultured for 4 days in Arg-free Dulbecco’s modified Eagle’s-F12 Ham medium (DMEM-F12) containing 10, 100 or 350 μM Arg and 0 or 20 ng/ml LPS. Cell numbers, protein concentrations, protein synthesis and degradation, as well as mammalian target of rapamycin (mTOR) and Toll-like receptor 4 (TLR4) signaling pathways were determined. Without LPS, IPEC-1 cells exhibited time- and Arg-dependent growth curves. LPS treatment increased cell death and reduced protein concentrations in IPEC-1 cells. Addition of 100 and 350 μM Arg to culture medium dose-dependently attenuated LPS-induced cell death and reduction of protein concentrations, in comparison with the basal medium containing 10 μM Arg. Furthermore, supplementation of 100 and 350 μM Arg increased protein synthesis and reduced protein degradation in both control and LPS-treated IPEC-1 cells. Consistent with the data on cell growth and protein turnover, addition of 100 or 350 μM Arg to culture medium increased relative protein levels for phosphorylated mTOR and phosphorylated ribosomal protein S6 kinase-1, while reducing the relative levels of TLR4 and phosphorylated levels of nuclear factor-κB in LPS-treated IPEC-1 cells. These results demonstrate a protective effect of Arg against LPS-induced enterocyte damage through mechanisms involving mTOR and TLR4 signaling pathways, as well as intracellular protein turnover.

Bisphenol, npcRNAs and Utero-Ovarian Feed-Back Control of Breast Cancer Chemosensitivity

In the February 2009 issue of Environmental Health Perspectives, La Pensee et al. (2009) postulated that bisphenol A (BPA), at nanomolecular doses, confers chemo resistance in estrogen receptor (ER)-α–positive and –negative breast cancer cells. Certainly, drug resistance is well-known to be an important complication in a variety of cancer chemotherapy options. Several molecular mechanisms have been suggested to explain the onset of drug resistance. Determining the exact mechanism in a particular case is challenging both at the clinical and preclinical research levels because the genome-wide and proteomic approaches to mechanistic studies are still at a developing stage (Zhang and Liu 2007). With regard to cisplatin, doxorubicin, and vinblastine cytotoxicity, there are proposed mechanisms of cytotoxicity in breast cancer cells and cell line s other than ER or receptor-mediated molecular signals. These mechanisms involve not only apoptosis pathways but also other regulatory, functional, and structural mechanisms of phenotypic expression in breast cancer models that could interfere with androgen receptor-mediated transcriptional activities (Aube et al. 2008). In addition, after the invention of microarray systems, more focus has been placed on the large number of human transcripts that have been described but do not code for proteins, such as nonprotein coding RNAs (Mallardo et al. 2008). These may include subfractions of small (microRNAs, small nucleolar RNAs) and long RNAs (anti-sense RNA, double-stranded RNA, and long RNA species) that function as regulators of other mRNAs at the transcriptional and post-transcriptional levels and control protein ubiquitination and degradation, with possible other roles yet to be elucidated. Various species of nonprotein-coding RNAs (npcRNAs) have been found to be differentially expressed in diverse types of cancer, including breast cancer subtypes (Mallardo et al. 2008). Because BPA is a highly reactive chemical, it would not be surprising if it interacts with some of these npc RNAs that could mediate ER response. Recent reports from other laboratories have tended to support a role of npcRNAs in BPA-mediated mechanisms involved in breast cancer and a possible physiochemical interaction of BPA with estrogen and non-estrogen-mediated chemosensitivity-inducing pathway elements. For example, Hong et al. (2006) used expression micro array technology to predict hormone-responsive activities in response to estrogen and endocrine disruptors. According to these authors, the expression levels of only 555 genes (7.42%) among the 7,636 genes spotted on microarray chips were enhanced by > 2-fold after treatment with estradiol (E2), suggesting that direct or rapid response to E2 is widespread at the mRNA levels in these genes. Hong et al. (2006) observed that elevated expression levels of the genes (over 2-fold) were induced by BPA (8.26%) in the uterus of immature rats. Examples of differentially expressed representative genes include calbindin-D9k (vitamin D-dependent calcium-binding protein), oxytocin, adipocyte complement related protein (30 kDa), lactate dehydrogenase A, and calcium-binding protein A6 (calcyclin). The mRNA levels of these genes were also increased in various phases of the menstrual cycle. This study in rats (Hong et al. 2006) supports the possibility of distinct effects of endogenous E2 and environmental endocrine-disrupting chemicals in the uterus of women. Involvement of these gene transcripts, which are present in breast, uterine, and ovarian tissues, in the environment–endocrine inter action suggests the possibility of a utero-ovarian feedback control of breast cancer chemo sensitivity effected by npcRNAs. Sladek and Somponpun (2008) studied the effect of vasopressin (VP) on the reproductive cycles of humans; their results suggest the involvement of multiple types of ERs in the VP-mediated G-protein coupled response (Hong et al. 2006). VP, acting through fluid-electrolyte mechanisms, may have a role in the mechanism of breast cancer initiation and progression, which may be prone to regulatory impacts through npcRNAs involved in the replication of dysregulating pathways of the mammary epithelium. These observations suggest that there may be a utero-ovarian feedback control mediated by ERs on the uterus that cross-talk with vasoneural pathways; the feedback control may mediate estrogen involved in chemoresponsive pathways of breast cancer. Furthermore, these pathways may be regulated by noncoding npcRNAs whose functions may be physiochemically modified by environmental toxicants such as BPA and other related chemials. Since the forum titled “Bisphenol A: An Expert Panel Examination of the Relevance of Ecological, in Vitro and Laboratory Animal Studies for Assessing Risks to Human Health” in Chapel Hill, North Carolina, on 28–30 November 2006 (Keri et al. 2007), there has been no meeting convened to discuss environmental endocrine disruptors, particularly as it relates to BPA and related chemicals. I hope that a future review panel will assess the literature on both animals and humans and evaluate the role of BPA in carcinogenesis. Such an assessment should also include recommendations for future areas of research.

Transcriptional profiles of adult male and female Schistosoma japonicum in response to insulin reveal increased expression of genes involved in growth and development

Microarray analysis was used to investigate differential gene regulation in adult male and female Schistosoma japonicum cultured in the presence or absence of insulin in vitro. A total of 1,101 genes were up- or down-regulated in response to insulin, the majority of differential expression occurring 24 h after the addition of insulin to the cultures. Genes differentially expressed in male or female worms were predominantly involved in growth and development, with significant sex-specific differences in transcriptional profiles evident. Insulin appeared to promote protein synthesis and control protein degradation more prominently in male parasites. The study also indicated that insulin plays a more pronounced role in the uptake of glucose in unpaired female parasites, as reflected in the increased stimulation of gene expression of the phosphatidylinositol 3-kinase sub-pathway of insulin signalling. Insulin may also impact on the sexual differentiation and fecundity of female schistosomes by activation of the mitogenic-activated protein kinase sub-pathway.

Down-Regulation of Akt/Mammalian Target of Rapamycin Signaling Pathway in Response to Myostatin Overexpression in Skeletal Muscle

Myostatin, a member of the TGF-beta family, has been identified as a master regulator of embryonic myogenesis and early postnatal skeletal muscle growth. However, cumulative evidence also suggests that alterations in skeletal muscle mass are associated with dysregulation in myostatin expression and that myostatin may contribute to muscle mass loss in adulthood. Two major branches of the Akt pathway are relevant for the regulation of skeletal muscle mass, the Akt/mammalian target of rapamycin (mTOR) pathway, which controls protein synthesis, and the Akt/forkhead box O (FOXO) pathway, which controls protein degradation. Here, we provide further insights into the mechanisms by which myostatin regulates skeletal muscle mass by showing that myostatin negatively regulates Akt/mTOR signaling pathway. Electrotransfer of a myostatin expression vector into the tibialis anterior muscle of Sprague Dawley male rats increased myostatin protein level and decreased skeletal muscle mass 7 d after gene electrotransfer. Using RT-PCR and immunoblot analyses, we showed that myostatin overexpression was ineffective to alter the ubiquitin-proteasome pathway. By contrast, myostatin acted as a negative regulator of Akt/mTOR pathway. This was supported by data showing that the phosphorylation of Akt on Thr308, tuberous sclerosis complex 2 on Thr1462, ribosomal protein S6 on Ser235/236, and 4E-BP1 on Thr37/46 was attenuated 7 d after myostatin gene electrotransfer. The data support the conclusion that Akt/mTOR signaling is a key target that accounts for myostatin function during muscle atrophy, uncovering a novel role for myostatin in protein metabolism and more specifically in the regulation of translation in skeletal muscle.

Inhibition of proteasome activity blocks Trypanosoma cruzi growth and metacyclogenesis

Proteasomes are intracellular complexes that control protein degradation in organisms ranging from Archaebacteria to mammals. In some parasitic protozoa, the proteasome is involved in cell differentiation and replication. In this study, we have used proteasome inhibitors to determine the biological role of proteasomes during the replication and in vitro metacyclogenesis of Trypanosoma cruzi. We used light and transmission electron microscopy to analyze morphological data and flow cytometry to analyze changes in the cell cycle. The growth of T. cruzi epimastigote culture forms in liver infusion tryptose medium was inhibited by the presence of up to 10 microM lactacystin. Inhibition was dose-dependent, with IC50 (50% inhibitory concentration) of 4.35 microM after 24 or 72 h. The metacyclogenesis process in vitro was strongly (95%) inhibited by 5 microM lactacystin treatment. The adhesion phase was not affected, but the epimastigotes did not differentiate into metacyclic trypomastigotes. Most treated epimastigotes had replicated DNA, with swelling of the mitochondrion and an altered distribution of nuclear and kinetoplast DNA. Our findings suggest that inhibition of the ubiquitin-proteasome pathway in T. cruzi epimastigotes does not block adhesion, but disrupts cell division and affects factors triggering differentiation.

Polyglutamine gene function and dysfunction in the ageing brain

The coordinated regulation of gene expression and protein interactions determines how mammalian nervous systems develop and retain function and plasticity over extended periods of time such as a human life span. By studying mutations that occur in a group of genes associated with chronic neurodegeneration, the polyglutamine (polyQ) disorders, it has emerged that CAG/glutamine stretches play important roles in transcriptional regulation and protein-protein interactions. However, it is still unclear what the many structural and functional roles of CAG and other low-complexity sequences in eukaryotic genomes are, despite being the most commonly shared peptide fragments in such proteomes. In this review we examine the function of genes responsible for at least 10 polyglutamine disorders in relation to the nervous system and how expansion mutations lead to neuronal dysfunction, by particularly focusing on Huntington's disease (HD). We argue that the molecular and cellular pathways that turn out to be dysfunctional during such diseases, as a consequence of a CAG expansion, are also involved in the ageing of the central nervous system. These are pathways that control protein degradation systems (including molecular chaperones), axonal transport, redox-homeostasis and bioenergetics. CAG expansion mutations confer novel properties on proteins that lead to a slow-progressing neuronal pathology and cell death similar to that found in other age-related conditions such as Alzheimer's and Parkinson's diseases.

New targets for lymphoma treatment

The rapid development in understanding the biology oflymphoid cells has led to the identification of multiplepathways and targets for therapy in recent years. These rangefrom cell surface interactions involving the B-cell receptor(BCR), through signalling pathways such as that mediated viathe mammalian target of rapamycin (mTOR) and NF-jB,modulation of gene expression by histone acetylation, controlof protein degradation by the proteosome or heat shockproteins, and regulation of apoptosis by both surface deathreceptors and intracellular proteins. In addition, a variety ofstrategies for driving immune responses to lymphoma havebeen investigated, including the use of small moleculeimmunomodulators, and agonistic antibodies which targetimmune cells rather than the malignant cells themselves.Finally, the idiotype remains an attractive target for B-celllymphoma, being wholly patient specific, and vaccinationstrategies aimed at raising humoral and cellular responses are atan advanced stage of clinical study. The development of allthese agents remains at an experimental stage, with none havingas yet delivered clear advantages in terms of overall survival inphase III trials, although some are getting close. In many casesnovel combinations are in clinical testing, with eitherconventional cytotoxics, monoclonal antibodies or several newagents together. A deeper understanding of their mechanismsof action should allow the rational development of suchcombinations in the near future.