A Functional Ubiquitin-Proteasome System is Required for Efficient Replication of New World Mayaro and Una Alphaviruses.

Mayaro (MAYV) and Una (UNAV) are emerging arboviruses belonging to the Alphavirus genus of the Togaviridae family. These viruses can produce febrile disease with symptoms such as fever, headache, myalgia, skin rash and incapacitating poly-arthralgia. Serological studies indicate that both viruses are circulating in different countries in Latin America. Viruses need the host cell machinery and resources to replicate effectively. One strategy to find new antivirals consists of identifying key cellular pathways or factors that are essential for virus replication. In this study, we analyzed the role of the ubiquitin-proteasome system (UPS) in MAYV and UNAV replication. Vero-E6 or HeLa cells were treated with the proteasome inhibitors MG132 or Lactacystin, and viral progeny production was quantified using a plaque assay method. In addition, the synthesis of viral proteins was analyzed by Western blot and confocal microscopy. Our results indicate that treatment with proteasome inhibitors decreases MAYV and UNAV protein synthesis, and also causes a significant dose-dependent decrease in MAYV and UNAV replication. Proteasome activity seems to be important at the early stages of MAYV replication. These findings suggest that the ubiquitin-proteasome system is a possible pharmacological target to inhibit these neglected alphaviruses.

Proteasome Dependent Actin Remodeling Facilitates Antigen Extraction at the Immune Synapse of B Cells.

Engagement of the B cell receptor (BCR) with surface-tethered antigens leads to the formation of an immune synapse (IS), where cell signaling and antigen uptake are tightly coordinated. Centrosome re-orientation to the immune synapse has emerged as a critical regulatory step to guide the local recruitment and secretion of lysosomes, which can facilitate the extraction of immobilized antigens. This process is coupled to actin remodeling at the centrosome and at the immune synapse, which is crucial to promote cell polarity. How B cells balance both pools of actin cytoskeleton to achieve a polarized phenotype during the formation of an immune synapse is not fully understood. Here, we reveal that B cells rely on proteasome activity to achieve this task. The proteasome is a multi-catalytic protease that degrades cytosolic and nuclear proteins and its dysfunction is associated with diseases, such as cancer and autoimmunity. Our results show that resting B cells contain an active proteasome pool at the centrosome, which is required for efficient actin clearance at this level. As a result of proteasome inhibition, activated B cells do not deplete actin at the centrosome and are unable to separate the centrosome from the nucleus and thus display impaired polarity. Consequently, lysosome recruitment to the immune synapse, antigen extraction and presentation are severely compromised in B cells with diminished proteasome activity. Additionally, we found that proteasome inhibition leads to impaired actin remodeling at the immune synapse, where B cells display defective spreading responses and distribution of key signaling molecules at the synaptic membrane. Overall, our results reveal a new role for the proteasome in regulating the immune synapse of B cells, where the intracellular compartmentalization of proteasome activity controls cytoskeleton remodeling between the centrosome and synapse, with functional repercussions in antigen extraction and presentation.

Acute intermittent hypoxia in rats activates muscle proteolytic pathways through a gluccorticoid-dependent mechanism.

Although it is well known that chronic hypoxia induces muscle wasting, the effects of intermittent hypoxia on skeletal muscle protein metabolism remain unclear. We hypothesized that acute intermittent hypoxia (AIH), a challenge that activates the hypothalamic-pituitary-adrenal axis, would alter muscle protein homeostasis through a glucocorticoid-dependent mechanism. Three-week-old rats were submitted to adrenalectomy (ADX) and exposed to 8 h of AIH (6% O2 for 40 s at 9-min intervals). Animals were euthanized, and the soleus and extensor digitorum longus (EDL) muscles were harvested and incubated in vitro for measurements of protein turnover. AIH increased plasma levels of corticosterone and induced insulin resistance as estimated by the insulin tolerance test and lower rates of muscle glucose oxidation and the HOMA index. In both soleus and EDL muscles, rates of overall proteolysis increased after AIH. This rise was accompanied by an increased proteolytic activities of the ubiquitin(Ub)-proteasome system (UPS) and lysosomal and Ca2+-dependent pathways. Furthermore, AIH increased Ub-protein conjugates and gene expression of atrogin-1 and MuRF-1, two key Ub-protein ligases involved in muscle atrophy. In parallel, AIH increased the mRNA expression of the autophagy-related genes LC3b and GABARAPl1. In vitro rates of protein synthesis in skeletal muscles did not differ between AIH and control rats. ADX completely blocked the insulin resistance in hypoxic rats and the AIH-induced activation of proteolytic pathways and atrogene expression in both soleus and EDL muscles. These results demonstrate that AIH induces insulin resistance in association with activation of the UPS, the autophagic-lysosomal process, and Ca2+-dependent proteolysis through a glucocorticoid-dependent mechanism.NEW & NOTEWORTHY Since hypoxia is a condition in which the body is deprived of adequate oxygen supply and muscle wasting is induced, the present work provides evidence linking hypoxia to proteolysis through a glucocorticoid-dependent mechanism. We show that the activation of proteolytic pathways, atrophy-related genes, and insulin resistance in rats exposed to acute intermittent hypoxia was abolished by surgical removal of adrenal gland. This finding will be helpful for understanding of the muscle wasting in hypoxemic conditions.

Forgetting of what was once learned: Exploring the role of postsynaptic ionotropic glutamate receptors on memory formation, maintenance, and decay.

Over the past years, extensive research in experimental cognitive neuroscience has provided a comprehensive understanding about the role of ionotropic glutamate receptor (IGluR)-dependent signaling underpinning postsynaptic plasticity induced by long-term potentiation (LTP), the leading cellular basis of long-term memory (LTM). However, despite the fact that iGluR-mediated postsynaptic plasticity regulates the formation and persistence of LTP and LTM, here we discuss the state-of-the-art regarding the mechanisms underpinning both LTP and LTM decay. First, we review the crucial roles that iGluRs play on memory encoding and stabilization. Second, we discuss the latest findings in forgetting considering hippocampal GluA2-AMPAR trafficking at postsynaptic sites as well as dendritic spine remodeling possibly involved in LTP decay. Third, on the role of retrieving consolidated LTMs, we discuss the mechanisms involved in memory destabilization that occurs followed reactivation that share striking similarities with the neurobiological basis of forgetting. Fourth, since different AMPAR subunits as well as postsynaptic scaffolding proteins undergo ubiquitination, the ubiquitin-proteasome system (UPS) is discussed in light of memory decay. In conclusion, we provide an integrated overview revealing some of the mechanisms determining memory forgetting that are mediated by iGluRs. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'.

Two waves of proteasome-dependent protein degradation in the hippocampus are required for recognition memory consolidation.

Healthy neuronal function and synaptic modification require a concert of synthesis and degradation of proteins. Increasing evidence indicates that protein turnover mediated by proteasome activity is involved in long-term synaptic plasticity and memory. However, its role in different phases of memory remains debated, and previous studies have not examined the possible requirement of protein degradation in recognition memory. Here, we show that the proteasome inhibitor, lactacystin (LAC), infused into the CA1 area of the hippocampus at two specific time points during consolidation, impairs 24-retention of memory for object recognition in rats. Administration of LAC after retrieval did not affect retention. These findings provide the first evidence for a requirement of proteasome activity in recognition memory, indicate that protein degradation in the hippocampus is necessary during selective time windows of memory consolidation, and further our understanding of the role of protein turnover in memory formation.

Aerobic exercise training improves oxidative stress and ubiquitin proteasome system activity in heart of spontaneously hypertensive rats.

The activity of the ubiquitin proteasome system (UPS) and the level of oxidative stress contribute to the transition from compensated cardiac hypertrophy to heart failure in hypertension. Moreover, aerobic exercise training (AET) is an important therapy for the treatment of hypertension, but its effects on the UPS are not completely known. The aim of this study was to evaluate the effect of AET on UPS's activity and oxidative stress level in heart of spontaneously hypertensive rats (SHR). A total of 53 Wistar and SHR rats were randomly divided into sedentary and trained groups. The AET protocol was 5×/week in treadmill for 13 weeks. Exercise tolerance test, non-invasive blood pressure measurement, echocardiographic analyses, and left ventricle hemodynamics were performed during experimental period. The expression of ubiquitinated proteins, 4-hydroxynonenal (4-HNE), Akt, phospho-Akt(ser473), GSK3ß, and phospho-GSK3ß(ser9) were analyzed by western blotting. The evaluation of lipid hydroperoxide concentration was performed using the xylenol orange method, and the proteasomal chymotrypsin-like activity was measured by fluorimetric assay. Sedentary hypertensive group presented cardiac hypertrophy, unaltered expression of total Akt, phospho-Akt, total GSK3ß and phospho-GSK3ß, UPS hyperactivity, increased lipid hydroperoxidation as well as elevated expression of 4-HNE but normal cardiac function. In contrast, AET significantly increased exercise tolerance, decreased resting systolic blood pressure and heart rate in hypertensive animals. In addition, the AET increased phospho-Akt expression, decreased phospho-GSK3ß, and did not alter the expression of total Akt, total GSK3ß, and ubiquitinated proteins, however, significantly attenuated 4-HNE levels, lipid hydroperoxidation, and UPS's activity toward normotensive group levels. Our results provide evidence for the main effect of AET on attenuating cardiac ubiquitin proteasome hyperactivity and oxidative stress in SHR rats.

Protein degradation by ubiquitin-proteasome system in formation and labilization of contextual conditioning memory.

The ubiquitin-proteasome system (UPS) of protein degradation has been evaluated in different forms of neural plasticity and memory. The role of UPS in such processes is controversial. Several results support the idea that the activation of this system in memory consolidation is necessary to overcome negative constrains for plasticity. In this case, the inhibition of the UPS during consolidation impairs memory. Similar results were reported for memory reconsolidation. However, in other cases, the inhibition of UPS had no effect on memory consolidation and reconsolidation but impedes the amnesic action of protein synthesis inhibition after retrieval. The last finding suggests a specific action of the UPS inhibitor on memory labilization. However, another interpretation is possible in terms of the synthesis/degradation balance of positive and negative elements in neural plasticity, as was found in the case of long-term potentiation. To evaluate these alternative interpretations, other reconsolidation-interfering drugs than translation inhibitors should be tested. Here we analyzed initially the UPS inhibitor effect in contextual conditioning in crabs. We found that UPS inhibition during consolidation impaired long-term memory. In contrast, UPS inhibition did not affect memory reconsolidation after contextual retrieval but, in fact, impeded memory labilization, blocking the action of drugs that does not affect directly the protein synthesis. To extend these finding to vertebrates, we performed similar experiments in contextual fear memory in mice. We found that the UPS inhibitor in hippocampus affected memory consolidation and blocked memory labilization after retrieval. These findings exclude alternative interpretations to the requirement of UPS in memory labilization and give evidence of this mechanism in both vertebrates and invertebrates.

Cardiomyocyte ryanodine receptor degradation by chaperone-mediated autophagy.

AIMS: Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins bearing the sequence KFERQ. These proteins are targeted by chaperones and delivered to lysosomes where they are translocated into the lysosomal lumen and degraded via the lysosome-associated membrane protein type 2A (LAMP-2A). Mutations in LAMP2 that inhibit autophagy result in Danon disease characterized by hypertrophic cardiomyopathy. The ryanodine receptor type 2 (RyR2) plays a key role in cardiomyocyte excitation-contraction and its dysfunction can lead to cardiac failure. Whether RyR2 is degraded by CMA is unknown. METHODS AND RESULTS: To induce CMA, cultured neonatal rat cardiomyocytes were treated with geldanamycin (GA) to promote protein degradation through this pathway. GA increased LAMP-2A levels together with its redistribution and colocalization with Hsc70 in the perinuclear region, changes indicative of CMA activation. The inhibition of lysosomes but not proteasomes prevented the loss of RyR2. The recovery of RyR2 content after incubation with GA by siRNA targeting LAMP-2A suggests that RyR2 is degraded via CMA. In silico analysis also revealed that the RyR2 sequence harbours six KFERQ motifs which are required for the recognition Hsc70 and its degradation via CMA. Our data suggest that presenilins are involved in RyR2 degradation by CMA. CONCLUSION: These findings are consistent with a model in which oxidative damage of the RyR2 targets it for turnover by presenilins and CMA, which could lead to removal of damaged or leaky RyR2 channels.

REGγ is a strong candidate for the regulation of cell cycle, proliferation and the invasion by poorly differentiated thyroid carcinoma cells

REGγ is a proteasome activator that facilitates the degradation of small peptides. Abnormally high expression of REGγ has been observed in thyroid carcinomas. The purpose of the present study was to explore the role of REGγ in poorly differentiated thyroid carcinoma (PDTC). For this purpose, small interfering RNA (siRNA) was introduced to down-regulate the level of REGγ in the PDTC cell line SW579. Down-regulation of REGγ at the mRNA and protein levels was confirmed by RT-PCR and Western blot analyses. FACS analysis revealed cell cycle arrest at the G1/S transition, the MTT assay showed inhibition of cell proliferation, and the Transwell assay showed restricted cell invasion. Furthermore, the expression of the p21 protein was increased, the expression of proliferating cell nuclear antigen (PCNA) protein decreased, and the expression of the p27 protein was unchanged as shown by Western blot analyses. REGγ plays a critical role in the cell cycle, proliferation and invasion of SW579 cells. The alteration of p21 and PCNA proteins related to the down-regulation of REGγ suggests that p21 and PCNA participate in the process of REGγ regulation of cell cycle progression and cell proliferation. Thus, targeting REGγ has a therapeutic potential in the management of PDTC patients.

REGγ is a strong candidate for the regulation of cell cycle, proliferation and the invasion by poorly differentiated thyroid carcinoma cells.

REGγ is a proteasome activator that facilitates the degradation of small peptides. Abnormally high expression of REGγ has been observed in thyroid carcinomas. The purpose of the present study was to explore the role of REGγ in poorly differentiated thyroid carcinoma (PDTC). For this purpose, small interfering RNA (siRNA) was introduced to down-regulate the level of REGγ in the PDTC cell line SW579. Down-regulation of REGγ at the mRNA and protein levels was confirmed by RT-PCR and Western blot analyses. FACS analysis revealed cell cycle arrest at the G1/S transition, the MTT assay showed inhibition of cell proliferation, and the Transwell assay showed restricted cell invasion. Furthermore, the expression of the p21 protein was increased, the expression of proliferating cell nuclear antigen (PCNA) protein decreased, and the expression of the p27 protein was unchanged as shown by Western blot analyses. REGγ plays a critical role in the cell cycle, proliferation and invasion of SW579 cells. The alteration of p21 and PCNA proteins related to the down-regulation of REGγ suggests that p21 and PCNA participate in the process of REGγ regulation of cell cycle progression and cell proliferation. Thus, targeting REGγ has a therapeutic potential in the management of PDTC patients.

Regulation of cell polarity by controlled proteolytic systems.

Epithelial and neuronal cells are highly asymmetric, with discrete regions responsible for different roles that underlie the generation of specific compartments within cells that are distinct in biochemical composition, structure, and morphology that ultimately lead to distinct functions. Controlled and specific molecular targeting and sorting have been studied to understand the generation of asymmetric domains inside cells. Recently, a new and complementary explanation has emerged to account for the generation of domains that are enriched by a subset of proteins or polarization determinants: local proteolysis. In this review, we discuss the most conspicuous proteolytic systems that may contribute to the generation of cell polarity, namely the ubiquitin-proteosome and the calpain systems. Specifically, we focus this review on two cellular processes that depend on the acquisition of cell polarity; cell migration and the establishment of an axon in a neuronal cell.

Participation of the sperm proteasome during in vitro fertilisation and the acrosome reaction in cattle.

In this work, we have investigated the role of the bovine sperm proteasome during in vitro fertilisation (IVF) and the acrosome reaction (AR). Motile spermatozoa, obtained by a swim-up method in Sperm-Talp medium, were capacitated for 3.5 h and incubated in the presence or absence of the specific proteasome inhibitor epoxomicin for 30 and 60 min. Then, the spermatozoa were co-incubated with mature bovine cumulus oocytes and after 48 h the cleavage rate of inseminated oocytes was evaluated. In addition, we evaluated the participation of the sperm proteasome during the progesterone-induced AR. Capacitated spermatozoa were incubated for 30 min with or without epoxomicin, then progesterone was added and the ARs were evaluated using the dual fluorescent staining technique 'Hoechst and chlortetracycline'. The results indicate that the proteasome inhibitor decreased the cleavage rate of oocytes inseminated with treated spermatozoa. In addition, acrosomal exocytosis levels were statistically significantly higher in the samples treated with the AR inducer progesterone than in control samples in the absence of the inducer. However, the progesterone-induced AR was significantly reduced by previous treatment of the spermatozoa with epoxomicin (P < 0.001). These observations indicate that the bovine sperm proteasome participates in the IVF and AR processes.

Regulation of Pax7 protein levels by caspase-3 and proteasome activity in differentiating myoblasts.

The transcription factor Pax7 negatively regulates the activity of the muscle regulatory transcription factor MyoD, preventing muscle precursor cells from undergoing terminal differentiation. In this context, the ratio between Pax7 and MyoD protein levels is thought to be critical in allowing myogenesis to proceed or to maintain the undifferentiated muscle precursor state. We have previously shown that Pax7 is subject to rapid down regulation in differentiating myoblasts, via a proteasome-dependent pathway. Here we present evidence indicating that Pax7 is also subject to caspase-3-dependent regulation. Furthermore, simultaneous inhibition of caspase-3 and proteasome activity induced further accumulation of Pax7 protein in differentiating myoblasts. These results suggest that at early stages of muscle differentiation, Pax7 levels are regulated by at least two independent mechanisms involving caspase-3 and proteasome activity.

Regulation of Pax7 protein levels by caspase-3 and proteasome activity in differentiating myoblasts

The transcription factor Pax7 negatively regulates the activity of the muscle regulatory transcription factor MyoD, preventing muscle precursor cells from undergoing terminal differentiation. In this context, the ratio between Pax7 and MyoD protein levels is thought to be critical in allowing myogenesis to proceed or to maintain the undifferentiated muscle precursor state. We have previously shown that Pax7 is subject to rapid down regulation in differentiating myoblasts, via a proteasome-dependent pathway. Here we present evidence indicating that Pax7 is also subject to caspase-3-dependent regulation. Furthermore, simultaneous inhibition of caspase-3 and proteasome activity induced further accumulation of Pax7 protein in differentiating myoblasts. These results suggest that at early stages of muscle differentiation, Pax7 levels are regulated by at least two independent mechanisms involving caspase-3 and proteasome activity.

Regulation of cell polarity by controlled proteolytic systems

Epithelial and neuronal cells are highly asymmetric, with discrete regions responsible for different roles that underlie the generation of specific compartments within cells that are distinct in biochemical composition, structure, and morphology that ultimately lead to distinct functions. Controlled and specific molecular targeting and sorting have been studied to understand the generation of asymmetric domains inside cells. Recently, a new and complementary explanation has emerged to account for the generation of domains that are enriched by a subset of proteins or polarization determinants: local proteolysis. In this review, we discuss the most conspicuous proteolytic systems that may contribute to the generation of cell polarity, namely the ubiquitin-proteosome and the calpain systems. Specifically, we focus this review on two cellular processes that depend on the acquisition of cell polarity; cell migration and the establishment of an axon in a neuronal cell.