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HER2 breast cancer (BC) remains a significant problem in patients with locally advanced or metastatic BC. We investigated the relationship between T helper 1 (Th1) immune response and the proteasomal degradation pathway (PDP), in HER2-sensitive and -resistant cells. HER2 overexpression is partially maintained because E3 ubiquitin ligase Cullin5 (CUL5), which degrades HER2, is frequently mutated or underexpressed, while the client-protective co-chaperones cell division cycle 37 (Cdc37) and heat shock protein 90 (Hsp90) are increased translating to diminished survival. The Th1 cytokine interferon (IFN)-γ caused increased CUL5 expression and marked dissociation of both Cdc37 and Hsp90 from HER2, causing significant surface loss of HER2, diminished growth, and induction of tumor senescence. In HER2-resistant mammary carcinoma, either IFN-γ or Th1-polarizing anti-HER2 vaccination, when administered with anti-HER2 antibodies, demonstrated increased intratumor CUL5 expression, decreased surface HER2, and tumor senescence with significant therapeutic activity. IFN-γ synergized with multiple HER2-targeted agents to decrease surface HER2 expression, resulting in decreased tumor growth. These data suggest a novel function of IFN-γ that regulates HER2 through the PDP pathway and provides an opportunity to impact HER2 responses through anti-tumor immunity.

Purpose of review FOXOs are transcription factors that regulate downstream target genes to counteract to cell stress. Here we review the function and regulation of FOXO transcription factors, the mechanism of FOXO3 activation in the kidney, and the role of FOXO3 in delaying the development of chronic kidney disease (CKD). Recent findings Progressive renal hypoxia from vascular dropout and metabolic perturbation is a pathogenic factor for the initiation and development of CKD. Hypoxia and low levels of α-ketoglutarate generated from the TCA cycle inhibit prolyl hydroxylase domain (PHD)-mediated prolyl hydroxylation of FoxO3, thus reducing FoxO3 protein degradation via the ubiquitin proteasomal pathway, similar to HIF stabilization under hypoxic conditions. FoxO3 accumulation and nuclear translocation activate two key cellular defense mechanisms, autophagy and antioxidative response in renal tubular cells, to reduce cell injury and promote cell survival. FoxO3 directly activates the expression of Atg proteins, which replenishes core components of the autophagic machinery to allow sustained autophagy in the chronically hypoxic kidney. FoxO3 protects mitochondria by stimulating the expression of superoxide dismutase 2 (SOD2), as tubular deletion of FoxO3 in mice results in reduced SOD2 levels and profound mitochondrial damage. Summary Knowledge gained from animal studies may help understand the function of stress responsive transcription factors that could be targeted to prevent or treat CKD.

Age-related macular degeneration (AMD) is a complex degenerative disease of the retina with multiple risk-modifying factors, including aging, genetics, and lifestyle choices. The combination of these factors leads to oxidative stress, inflammation, and metabolic failure in the retinal pigment epithelium (RPE) with subsequent degeneration of photoreceptors in the retina. The alternative complement pathway is tightly linked to AMD. In particular, the genetic variant in the complement factor H gene (CFH), which leads to the Y402H polymorphism in the factor H protein (FH), confers the second highest risk for the development and progression of AMD. Although the association between the FH Y402H variant and increased complement system activation is known, recent studies have uncovered novel FH functions not tied to this activity and highlighted functional relevance for intracellular FH. In our previous studies, we show that loss of CFH expression in RPE cells causes profound disturbances in cellular metabolism, increases the vulnerability towards oxidative stress, and modulates the activation of pro-inflammatory signaling pathways, most importantly the NF-kB pathway. Here, we silenced CFH in hTERT-RPE1 cells to investigate the mechanism by which intracellular FH regulates RPE cell homeostasis. We found that silencing of CFH results in hyperactivation of mTOR signaling along with decreased mitochondrial respiration and that mTOR inhibition via rapamycin can partially rescue these metabolic defects. To obtain mechanistic insight into the function of intracellular FH in hTERT-RPE1 cells, we analyzed the interactome of FH via immunoprecipitation followed by mass spectrometry-based analysis. We found that FH interacts with essential components of the ubiquitin-proteasomal pathway (UPS) as well as with factors associated with RB1/E2F signalling in a complement-pathway independent manner. Moreover, we found that FH silencing affects mRNA levels of the E3 Ubiquitin-Protein Ligase Parkin and PTEN induced putative kinase (Pink1), both of which are associated with UPS. As inhibition of mTORC1 was previously shown to result in increased overall protein degradation via UPS and as FH mRNA and protein levels were shown to be affected by inhibition of UPS, our data stress a potential regulatory link between endogenous FH activity and the UPS.

Age-related macular degeneration (AMD) is a complex degenerative disease of the retina with multiple risk-modifying factors, including aging, genetics, and lifestyle choices. The combination of these factors leads to oxidative stress, inflammation, and metabolic failure in the retinal pigment epithelium (RPE) with subsequent degeneration of photoreceptors in the retina. The alternative complement pathway is tightly linked to AMD. In particular, the genetic variant in the complement factor H gene (CFH), which leads to the Y402H polymorphism in the factor H protein (FH), confers the second highest risk for the development and progression of AMD. Although the association between the FH Y402H variant and increased complement system activation is known, recent studies have uncovered novel FH functions not tied to this activity and highlighted functional relevance for intracellular FH. In our previous studies, we show that loss of CFH expression in RPE cells causes profound disturbances in cellular metabolism, increases the vulnerability towards oxidative stress, and modulates the activation of pro-inflammatory signaling pathways, most importantly the NF-kB pathway. Here, we silenced CFH in hTERT-RPE1 cells to investigate the mechanism by which intracellular FH regulates RPE cell homeostasis. We found that silencing of CFH results in hyperactivation of mTOR signaling along with decreased mitochondrial respiration and that mTOR inhibition via rapamycin can partially rescue these metabolic defects. To obtain mechanistic insight into the function of intracellular FH in hTERT-RPE1 cells, we analyzed the interactome of FH via immunoprecipitation followed by mass spectrometry-based analysis. We found that FH interacts with essential components of the ubiquitin-proteasomal pathway (UPS) as well as with factors associated with RB1/E2F signalling in a complement-pathway independent manner. Moreover, we found that FH silencing affects mRNA levels of the E3 Ubiquitin-Protein Ligase Parkin and PTEN induced putative kinase (Pink1), both of which are associated with UPS. As inhibition of mTORC1 was previously shown to result in increased overall protein degradation via UPS and as FH mRNA and protein levels were shown to be affected by inhibition of UPS, our data stress a potential regulatory link between endogenous FH activity and the UPS.

Botulinum neurotoxins (BoNTs) are known as the most potent bacterial toxins, which can cause potentially deadly disease botulism. BoNT Serotype A (BoNT/A) is the most studied serotype as it is responsible for most human botulism cases, and its formulations are extensively utilized in clinics for therapeutic and cosmetic applications. BoNT/A has the longest-lasting effect in neurons compared to other serotypes, and there has been high interest in understanding how BoNT/A manages to escape protein degradation machinery in neurons for months. Recent work demonstrated that an E3 ligase, HECTD2, leads to efficient ubiquitination of the BoNT/A Light Chain (A/LC); however, the dominant activity of a deubiquitinase (DUB), VCIP135, inhibits the degradation of the enzymatic component. Another DUB, USP9X, was also identified as a potential indirect contributor to A/LC degradation. In this study, we screened a focused ubiquitin-proteasome pathway inhibitor library, including VCIP135 and USP9X inhibitors, and identified ten potential lead compounds affecting BoNT/A mediated SNAP-25 cleavage in neurons in pre-intoxication conditions. We then tested the dose-dependent effects of the compounds and their potential toxic effects in cells. A subset of the lead compounds demonstrated efficacy on the stability and ubiquitination of A/LC in cells. Three of the compounds, WP1130 (degrasyn), PR-619, and Celastrol, further demonstrated efficacy against BoNT/A holotoxin in an in vitro post-intoxication model. Excitingly, PR-619 and WP1130 are known inhibitors of VCIP135 and USP9X, respectively. Modulation of BoNT turnover in cells by small molecules can potentially lead to the development of effective countermeasures against botulism.

Simple Summary The tumor suppressor TAp73 is a member of the p53 family, which is inhibited in many human solid and hematological tumors. In contrast to those in the p53 gene, mutations in the p73 gene are very rare in tumors, suggesting that the decrease in TAp73 activity and expression detected in those tumors are caused mainly by coordinated post-translational modifications of TAp73. Thus, understanding and investigating these post-translational modifications will allow for the identification of new targets to overcome the downregulation of TAp73 and, ultimately, the development of novel cancer therapeutics. This review highlights the multiple post-translational modifications underlying TAp73 regulation in cancer cells and the growing importance of targeting their trigger enzymes as a promising antitumor strategy. Abstract The tumor suppressor p73 is a member of the p53 family and is expressed as different isoforms with opposing properties. The TAp73 isoforms act as tumor suppressors and have pro-apoptotic effects, whereas the ΔNp73 isoforms lack the N-terminus transactivation domain and behave as oncogenes. The TAp73 protein has a high degree of similarity with both p53 function and structure, and it induces the regulation of various genes involved in the cell cycle and apoptosis. Unlike those of the p53 gene, the mutations in the p73 gene are very rare in tumors. Cancer cells have developed several mechanisms to inhibit the activity and/or expression of p73, from the hypermethylation of its promoter to the modulation of the ratio between its pro- and anti-apoptotic isoforms. The p73 protein is also decorated by a panel of post-translational modifications, including phosphorylation, acetylation, ubiquitin proteasomal pathway modifications, and small ubiquitin-related modifier (SUMO)ylation, that regulate its transcriptional activity, subcellular localization, and stability. These modifications orchestrate the multiple anti-proliferative and pro-apoptotic functions of TAp73, thereby offering multiple promising candidates for targeted anti-cancer therapies. In this review, we summarize the current knowledge of the different pathways implicated in the regulation of TAp73 at the post-translational level. This review also highlights the growing importance of targeting the post-translational modifications of TAp73 as a promising antitumor strategy, regardless of p53 status.

Hematopoietic stem cells (HSCs) are responsible for the production of blood cells throughout the human life span. Single HSCs can give rise to at least eight distinct blood cell lineages. Together, hematopoiesis, erythropoiesis and angiogenesis coordinate several biological processes, such as cellular interactions in development and proliferation, guided migration, lineage programming and reprogramming by transcription factors. Any dysregulation of these processes may result in hematological disorders and/or malignancies. Several studies of the molecular mechanisms governing HSC maintenance have demonstrated that protein regulation by the ubiquitin proteasomal pathway is crucial for normal HSC function. Recent studies have shown that the reversal of ubiquitination by deubiquitinating enzymes (DUBs) plays an equally important role in hematopoiesis; however, there is only limited additional information regarding the biological function of DUBs. In this review, we focus on recent discoveries that have led to a better understanding of the physiological roles of DUBs in hematopoiesis, erythropoiesis and angiogenesis. In addition, we discuss the DUBs associated with common hematological disorders and malignancies, which may potentially be therapeutic drug targets.

A new generation of heterobifunctional small molecules, termed proteolysis targeting chimeras (PROTACs), targets proteins for degradation through recruitment to E3 ligases and holds significant therapeutic potential. Despite numerous successful examples, PROTAC small molecule development remains laborious and unpredictable, involving testing compounds for end-point degradation activity at fixed times and concentrations without resolving or optimizing for the important biological steps required for the process. Given the complexity of the ubiquitin proteasomal pathway, technologies that enable real-time characterization of PROTAC efficacy and mechanism of action are critical for accelerating compound development, profiling, and improving guidance of chemical structure-activity relationship. Here, we present an innovative, modular live-cell platform utilizing endogenous tagging technologies and apply it to monitoring PROTAC-mediated degradation of the bromodomain and extra-terminal family members. We show comprehensive real-time degradation and recovery profiles for each target, precisely quantifying degradation rates, maximal levels of degradation ( D

The tumor suppressor p53 and its homologues, p63 and p73, play a pivotal role in the regulation of the DNA damage response, cellular homeostasis, development, aging, and metabolism. A number of mouse studies have shown that a genetic defect in the p53 family could lead to spontaneous tumor development, embryonic lethality, or severe tissue abnormality, indicating that the activity of the p53 family must be tightly regulated to maintain normal cellular functions. While the p53 family members are regulated at the level of gene expression as well as post-translational modification, they are also controlled at the level of protein stability through the ubiquitin proteasomal pathway. Over the last 20 years, many ubiquitin E3 ligases have been discovered that directly promote protein degradation of p53, p63, and p73 in vitro and in vivo. Here, we provide an overview of such E3 ligases and discuss their roles and functions.

Rheumatoid arthritis is a chronic, inflammatory joint disease leading to inflammation of synovial membrane that lines the joints. This inflammation further progresses and results in destruction of joints and surrounding cartilages. The underlying factors can be oxidative stress, pro-inflammatory mediators, imbalance and attenuation between various enzymes and proteins (like nuclear factor erythroid 2 related factor 2/Nrf2 and ubiquitin). Protein degradation pathways comprises of lysosomal, proteasomal pathway, and autophagosome (that are carried out in mammalian cells) are regulated through ubiquitin. Ubiquitin proteasomal system is dominating pathway for carrying out non-lysosomal proteolysis of intracellularly proteins. Fundamental processes including cell cycle progression, process of division, apoptosis, modulation of immune responses and cell trafficking are regulated by process of ubiquitination. Ubiquitin proteasomal pathway (UPP) includes ubiquitin moieties which are covalently attached to proteins and guides them proteasome for degradation. Misfolded, oxidized and damaged proteins which are responsible for critical processes, are major targets of degradation process. Any alteration in this system leads to dysregulated cellular homeostasis; progressively leading to numerous diseases including rheumatoid arthritis. Factors including TAK1, TRAF6 undergo are required for the progression of disease and thus contributes towards pathology of inflammatory disorders such as rheumatoid arthritis. This review will include all linked aspects which contribute its major role in rheumatoid arthritis.

Aims Dysferlinopathies are autosomal recessive neuromuscular disorders arising from mutations of the protein dysferlin that preferentially affect the limbs which waste and weaken. The pathomechanisms of the diseases are not known and effective treatment is not available. Although free radicals and upstream signaling by the redox sensitive transcription factor, NF-κB, in activation of the ubiquitin pathway are shown to occur in several muscle wasting disorders, their involvement in dysferlinopathy is not known. This study analyzed the role of oxidative stress, NF-κB and the ubiquitin pathway in dysferlinopathic muscle and in dysferlin knockdown human myoblasts and myotubes. Main methods Fourteen dysferlinopathic muscle biopsies and 8 healthy control muscle biopsies were analyzed for oxidative stress, NF-κB activation and protein ubiquitinylation and human primary myoblasts and myotubes knocked down for dysferlin were studied for their state of oxidative stress. Key findings Dysferlinopathic muscle biopsies showed NF-κB p65 signaling induced protein ubiquitinylation in response to oxidative stress. Dysferlin knock down primary muscle cell cultures confirmed that oxidative stress is induced in the absence of dysferlin in muscle. Significance Anti-oxidants that also inhibit nitrosative stress and NF-κB activation, might prove to be of therapeutic benefit in slowing the progression of muscle wasting that occurs with dysferlinopathy.

The neuromuscular disorder, calpainopathy (LGMD 2A), is a major muscular dystrophy classified under limb girdle muscular dystrophies. Genetic mutations of the enzyme calpain 3 cause LGMD 2A. Calpainopathy is phenotypically observed as progressive muscle wasting and weakness. Pathomechanisms of muscle wasting of calpainopathy remain poorly understood. Oxidative stress, NF-κB and the ubiquitin proteasomal pathway underlie the pathology of several muscle wasting conditions but their role in calpainopathic dystrophy is not known. Oxidative and nitrosative stress, the source of reactive oxygen species, NF-κB signaling and protein ubiquitinylation were studied in 15 calpainopathic and 8 healthy control human muscle biopsies. Oxidative stress and NF-κB/IKK β signaling were increased in calpainopathic muscle and may contribute to increased protein ubiquitinylation and muscle protein loss. Preventing oxidative stress or inhibition of NF-κB signaling could be considered for treatment of LGMD 2A.

Protein S-nitrosylation is a covalent post-translational modification through coupling of a nitric oxide (NO) moiety with the reactive thiol group of a protein cysteine residue to form an S-nitrosothiol (SNO). S-nitrosylation is a key mechanism in the transmission of NO-based cellular signals in the vital cellular processes, including transcription regulation, DNA repair, and apoptosis. Contemporary research has implicated dysregulation of S-nitrosylation in severe pathological events, including cancer onset, progression, and treatment resistance. The S-nitrosylation status may be directly linked to many cancer therapy outcomes as well as therapeutic-resistance, emphasizing the need to develop S-nitrosylation-related anti-cancer therapeutics. The role of S-nitrosylated proteins in the development and progression of cancer are varied, generating a critical need for a thorough review of the current dynamic research in this area.

Orientador: Maria Alice da Cruz Höfling Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: O veneno botrópico apresenta efeitos miotóxico, hemorrágico e proteolítico que são amplamente conhecidos. Acidentes com a serpente Bothrops jararacussu mostram que as alterações locais no músculo, geralmente de membros inferiores, são rápidas. Dados experimentais revelam que a miotoxicidade deve-se à ação direta das miotoxinas Bothropstoxina I e II, e indiretamente pela anóxia do tecido, devido à falência da microcirculação causada por toxinas hemorrágicas, ambas presentes no veneno. Nesse trabalho foi caracterizada a expressão dos fatores de transcrição miogênicos MyoD e Myog, via ubiquitina-proteassomo (Fbx-32 e MuRF-1) e gene GDF-8. Camundongos Swiss (Mus musculus) machos, adultos jovens (6 - 8 semanas/22 ¿ 25 g) foram divididos em 4 grupos Bjussu, BthTx1, BthTx2 e Sham. Os animais do grupo Bjussu receberam injeção intramuscular (mm. gastrocnêmio direito) (20 µl) do veneno liofilizado (diluído em NaCl 0,9%) de serpente do gênero Bothrops jararacussu (Bjssu) na dose de 1 mg/Kg. BthTx-I e BthTx-II receberam injeção i.m. de bothropstoxina I e II respectivamente, isoladas do veneno de B. jararacussu, na dose de 2,5 mg/Kg e o grupo Sham recebeu injeção i.m. de NaCl 0,9%. Animais de todos os grupos foram sacrificados e tiveram o músculo gastrocnêmio direito coletado nos períodos de 24, 48, 72 e 96 horas pós-tratamento para análise de expressão qualitativa e localização proteica (imunohistoquímica) e quantitativa (Western Blotting). Além disso, foi realizada análise da função motora dinâmica por meio do sistema CatWalk para os grupos Sham e Bjussu, nos períodos pré-lesão, 3, 24, 48, 72 e 96 horas. Os animais do grupo Bjussu não realizaram apoio do membro posterior direito (mantiveram postura flexora reflexa) no período de 3 horas pós-injúria (p=0.001) diferentemente do grupo Sham. Após 24 horas a função motora foi reestabelecida. Na análise de imunohistoquímica foi observada expressão de MyoD, Myog, GDF-8, Fbx-32 e MuRF-1 em todos os períodos analisados para o grupo Bjussu; no grupo Sham não foi possível observar expressão de nenhuma das proteínas. Na análise por Western Blotting e comparando-se o grupo Bjussu com Sham foi observado aumento de expressão de MyoD e GDF-8 no período de 96 horas (p=0.01), Myog em 72 horas (p=0.01) e queda de expressão de MuRF-1 em 96 horas (p=0.05), sendo que a expressão de Fbx-32 permaneceu inalterada. Para BthTx-I observam-se aumentos significativos para Myog (p=0.001), GDF-8 (p=0.01), Fbx-32 (p=0.01) e MuRF-1 (p=0.01) no período de 48 horas e Fbx-32 no período de 96 horas (p=0.05). Na análise de BthTx-II observa-se queda na expressão de MyoD (p=0.01), GDF-8 (p=0.01), Fbx-32 (p=0.01) e MuRF-1 (p=0.01) no período de 24 horas , e elevação da expressão de Myog (p=0.01) e GDF-8 (p=0.01) no período de 48 horas. De maneira geral, podemos sugerir que o veneno bruto de Bothrops jararacussu e a BthTx-II são capazes de ativar o processo molecular de regeneração, ao contrário do que ocorre com a BthTx-I. Considerando ainda que a via ubiquitina-proteassomo não sofreu alterações em nenhum dos modelos de injúria reproduzidos Abstract: Bothropic venom presents myotoxic, hemorrhagic and hemolytic effects that are widely known. Bothrops jararacussu snakebite accidents show that local changes in skeletal muscle occurs in a fast pace, usually affecting lower limbs. Experimental data show that local changes in skeletal muscle usually affect the lower limbs and evolve fast due to direct myotoxin action (mainly Bothropstoxin I and II) and also indirectly by tissue anoxia, due to failure in microcirculation caused by hemorrhagic toxins, both present in the venom. In this work, we characterized the expression of Myogenic Regulatory Factors MyoD and Myogenin, the GDF-8 gene and the ubiquitin-proteasome pathway (Fbx-32 and MuRF-1). Young adult male Swiss mice (Mus musculus) (6-8 weeks/22-25g) were divided in four groups Bjussu, BthTx1, BthTx2 and Sham. Animals from Bjussu received intramuscular injection (right gastrocnemius) (20µl) of lyophilized Bothrops jararacussu venom (eluted in NaCl 0,9%) at a concentration of 1mg/Kg. BthTx1, BthTx2 received i.m. injection of lyophilized bothropstoxin 1 and 2 respectively, isolated from Bothrops jararacussu crude venom at a concentration of 2,5mg/Kg, similarly Sham group was administered a 0,9% NaCl i.m. injection. Animals from all groups were sacrificed and had their muscles collected 24, 48, 72 and 96 hours post treatment for qualitative protein expression (immunohistochemistry) and quantitative (Western Blotting). In addition, the groups Sham and Bjussu went through dynamic function motor analysis (CatWalk system) in the periods of pre-injury, 3, 24, 48, 72 and 96 hours post injury. The animals from Bjussu did not perform support of the right hindlimb (kept a flexed in a reflex posture) 3 hours post injury (p=0.001), unlikely Sham group. Motor function was reestablished 24 hours post injury. For the immunohistochemistry analysis staining for MyoD, Myog, GDF-8, Fbx-32 and MuRF-1 was observed in all periods studied in Bjussu group, no staining was observed in Sham. In Western Blotting analysis, comparing Bjussu with Sham, it was possible to observe increased expression of MyoD and GDF-8 in 96 h (p=0.01), Myog in 72 h(p=0.01), and decreased expression of MuRF-1 in 96 h (p=0.05), taking into account that Fbx-32 expression remained inaltered. In BthTx1 expression occurs to increase for Myog (p=0.001), GDF-8 (p=0.01), Fbx-32 (p=0.01) and MuRF-1 (p=0.01) in 48 h and for Fbx-32 in the 96 h post injury (p=0.05). Analysis for BthTx2 revealed decrease for MyoD (p=0.01), GDF-8 (p=0.01), Fbx-32 (p=0.01) and MuRF-1 (p=0.01) in 24 h and increase for Myog (p=0.01) and GDF-8 (p=0.01) in 48 h. We suggest that crude Bothrops jararacussu venom and BthTx-2 are capable of activating the molecular regeneration process, in contrary to BthTx-1 inability. Considering that, the ubiquitin-proteasome pathway has remained inaltered in all present injury models Mestrado Biologia Celular Mestre em Biologia Celular e Estrutural CAPES FAPESP CNPQ FAEPEX

Autosomal recessive juvenile parkinsonism (AR-JP), a common familial form of Parkinson's disease, is caused by mutations of human Parkin. To deepen the understanding of Parkin biology in an in vivo model of Drosophila, we attempted to characterize the function of Drosophila melanogaster Parkin and found that D. melanogaster Parkin exhibited UbcH8-dependent E3 ubiquitin-protein ligase activity. Using E2 binding and in vitro ubiquitination assays, UbcH8 preferentially was found to bind to Parkin mutants harboring functional RING1 domains, but failed to bind to mutants harboring point mutants with complete loss of function. This inability of UbcH8 binding to such mutants was accompanied by abrogation of an E3 ligase activity, indicating that D. melanogaster Parkin as an E3 ligase interacts with UbcH8 through its RING1 domain. An in vivo ubiquitination assay revealed that D. melanogaster Parkin existed in ubiquitinated form in vivo. Moreover, peanut and septin1, D. melanogaster septin proteins, were also ubiquitinated by D. melanogaster Parkin. Co-immunoprecipitation with membrane protein Syntaxin indicated direct binding of septin proteins to syntaxin, implicating their relevance in the exocytosis of dopamine in cells. Western blot analysis and DNA fragmentation indicated that the rate and efficiency of p53-dependent apoptosis were significantly higher in the presence of dopamine than without the septin proteins. Therefore, our findings in the present study demonstrate that Parkin possibly influences septin protein effects on p53-mediated apoptosis, helping to extend the utility of Drosophila as a model system for the study of neurodegeneration.

Complex I impairment with rotenone produces damage though a mechanism thought to be distinct from effects on mitochondrial respiration. The outcome of chronic rotenone on energy status in relation to toxicity, however, is unknown. To examine this, mesencephalic cultures were exposed to chronic, low-dose rotenone (5-100 nM, 8 days in vitro) or acute, high-dose rotenone (500 nM, 1-24 h), and ATP/ADP levels and toxicity were measured. Chronic exposure to 5-50 nM rotenone produced selective dopamine cell loss. High-dose rotenone produced nonselective damage at all exposure times. Chronic, low-dose rotenone (37.5 nM) decreased ATP/ADP gradually over several days to 40% of controls, whereas high-dose rotenone (500 nM, 1-6 h), collapsed ATP/ADP by 1 h of exposure. The ubiquitin proteasomal pathway, an ATP-dependent pathway, is implicated in Parkinson's disease and, thus, various rotenone exposures were examined for effects on ubiquitin proteasomal function. Chronic, low-dose rotenone (25-50 nM, 8 days), but not acute, high-dose rotenone (500 nM, 1-6 h), caused accumulation of ubiquitinated proteins, E1-ubiquitin activation, and increased proteasomal activities prior to toxicity even though both exposures increased free radical production. Findings show that selective dopamine cell loss and alterations in ubiquitin proteasomal function only occur with rotenone exposures that partially maintain ATP/ADP. High concentrations of rotenone that collapse energy status kill neurons in a nonselective manner independent of the ubiquitin proteasomal pathway.

Proteostasis, the precisely orchestrated balance between protein synthesis and degradation, plays a critical role in the maintenance of cell function. This is particularly the case for postmitotic cells such as cardiomyocytes and neurons, which rarely re-enter the cell cycle to divide and hence survive for decades. Disease-related stress can promote dysregulation of proteostasis, leading to accumulation of damaged or misfolded proteins as toxic aggregates capable of triggering cell death.1,2 Indeed, in both dilated cardiomyopathy and ischemic heart disease, perturbations in proteostasis are evidenced by accumulation of polyubiquitinated proteins.3 Additionally, genetic disorders that lead to aggregate formation can trigger cardiomyopathy.4 For example, specific mutations in the coding region of the molecular chaperone αB-crystallin (CryAB) disrupt its chaperone function, leading to a distinctive myofibrillar myopathy characterized by accumulation of toxic aggregates in cardiac myocytes reminiscent of Alzheimer disease or polyglutamine expansion disorders. Article see p 365 Cells can be protected from accumulation of abnormal protein aggregates by either a decrease in their formation or an increase in their clearance. Protein aggregate clearance is accomplished by 2 major catabolic processes: the ubiquitin proteasome pathway (UPP) or the autophagy-lysosomal pathway (Figure 1). Inhibition of either pathway provokes increases in toxic, intracellular aggregates that can promote disease pathogenesis. Figure 1. Damaged or misfolded proteins are targeted primarily for degradation by the ubiquitin proteasomal pathway (UPP). When an excess of these proteins accumulates, they aggregate and are degraded by autophagic pathways. If misfolded proteins continue to accumulate, they can be ultimately sequestered by the cell into ubiquitin-rich cytoplasmic inclusions called aggresomes. Autophagy is a highly conserved catabolic process underlying bulk removal of cytoplasmic proteins and organelles. Autophagy acts as an adaptive response both under conditions of starvation, where recycling of intracellular contents is required to replenish life-sustaining nutrients, and in the setting of …

A variety of mutations in parkin are linked to autosomal recessive Parkinson's disease (PD). Mutations in parkin may be one of the most common causes of autosomal recessive PD. Parkin is an E3 ubiquitin-protein ligase and familial associated mutations disrupt the E3 ligase activity, thus implicating derangements in the ubiquitin proteasomal pathway in the pathogenesis of PD. We review the clinical and neuropathological features of Parkinson's disease caused by parkin mutations. In addition, we discuss the emerging evidence that parkin is an E3 ubiquitin-protein ligase and how derangements in its E3 ligase activity might cause autosomal recessive PD.

The ubiquitin-proteasome system is thought to play a major role in normal muscle protein turnover and to contribute to diabetes-induced protein wasting in skeletal muscle. However, its importance in cardiac muscle is not clear. We measured heart muscle mRNA for ubiquitin and for the C2 and C8 proteasomal subunits, the amount of free ubiquitin and the proteasome chymotrypsin-like proteolytic activity in control and diabetic rats. Results were compared to those in skeletal muscle (rectus). Heart ubiquitin, C2 and C8 subunit mRNA and proteolytic activity were significantly greater than in skeletal muscle (P

Parkinson’s disease (PD) is the second most common neurodegenerative disease among the elderly. While sporadic PD constitutes 99% of the cases, the remaining 1% is of genetic origin. The neuropathological hallmarks of PD are progressive degeneration of dopaminergic (DA) neurons and presence of Lewy neurites and Lewy bodies (LBs) intracytoplasmic proteinaceous inclusions that contain -synuclein (SYN), synphilin-1, components of the ubiquitin proteasomal pathway and parkin (Dawson, 2006). The loss of DA neurons in substantia nigra pars compacta (SNpc) results in decreased signalling in the striatum thereby giving rise to motor defects like resting tremor, bradykinesia, rigidity and posture instability. Besides DA neuronal loss, microglial activation and increased astroglial and lymphocyte infiltration also occur in PD. A role for inflammation in PD has been inferred from the identification of human leukocyte antigen (HLA)-DR positive reactive microglia in the brains of PD patients (McGeer et al., 1988). Additionally, levels of proinflammatory cytokines like IL-6, IL-1, TNF have been found to be elevated in the blood and cerebrospinal fluid (CSF) of PD patients (Nagatsu & Sawada, 2005; Dawson, 2006) Although these inflammatory components might serve as useful biomarkers, the aetiology of striatal DA degeneration still remains enigmatic. In the last decade, identification of mutations in several distinct genes (LRRK2, parkin, PINK1, DJ-1, -synuclein, MAPT, UCHL1 etc) linked to different forms of familial Parkinsonism has imparted a new direction to understanding PD pathogenesis (Tong & Shen, 2009). The question as to how seemingly divergent genes cause PD still remains unanswered, as there is no common molecular pathway involving these gene products. While parkin, -synuclein (SYN) and ubiquitin C-terminal hydrolase L1 (UCHL1) are functionally associated with the cellular ubiquitin proteasomal system (UPS), DJ-1 and PINK1 protect against oxidative stress and mitochondrial dysfunction. More recently, microarray analysis of SNpc from parkinsonian brain (Mandel et al., 2005) has shown that 68 genes related to protein degradation, signal transduction, dopaminergic transmission, iron transport and glycolysis are downregulated. Prominent among these are the protein chaperone HSC-70, subunits of the UPS and SKP1A, a member of the E3 ubiquitin ligase complex. Therefore, it is most likely that impairment in energy metabolism and/or alterations in UPS are the underlying mechanisms for PD pathogenesis (Eriksen et al., 2005; Mandel et al., 2005). Current PD treatment regimes can be divided into three categories: symptomatic, protective and restorative. Only symptomatic treatment via the administration of L-dopa and other

Ubiquitin proteasomal pathway (UPP) is the principle mechanism for protein catabolism and affects cellular processes critical for survival and proliferation. Levels of tumor suppressor protein p53 are very low in cells due to its rapid turnover by UPP-mediated degradation. While p53 is mutated in human cancers, most human melanomas maintain wild-type conformation. In this study, to investigate the effects of UPP inhibitor in vitro and in vivo, we used a genetically-engineered mouse model (GEMM) that has the same genetic alterations as those of human melanomas. Melanoma cells were established from mouse tumors and named 8B20 cells. Treatment of 8B20 cells with the UPP inhibitors, MG132 and clasto-lactacystin-β-lactone, led to an increase in levels of p53 while treatment with non-proteasomal inhibitors did not alter p53 levels. UPP inhibitors induced formation of heavy molecular weight ubiquitinated proteins, a hallmark of UPP inhibition, and p53-specific poly-ubiquitinated products in 8B20 cells. To further decipher the mechanism of p53 stabilization, we investigated half-life of p53 in cells treated with cycloheximide to block de novo protein synthesis. Treatment of 8B20 cells with MG132 led to an increase in the half-life of p53. Further analysis revealed that p53 stabilization was not mediated by phosphorylation of Ser-15 and Ser-20 residues. In vivo studies showed that MG132 induced p53 overexpression and reduced tumor growth, suggesting an important role of p53 stabilization in controlling melanoma. Taken together, our studies provide a proof of principle for using a GEMM to address the mechanisms of action and efficacy of melanoma treatment.

Akt plays a role in protecting macrophages from apoptosis induced by some oxysterols. Previously we observed enhanced degradation of Akt in P388D1 moncocyte/macrophages following treatment with 25-hydroxycholesterol (25-OH) or 7-ketocholesterol (7-KC). In the present report we examine the role of the ubiquitin proteasomal pathway in this process. We show that treatment with 25-OH or 7-KC results in the accumulation of poly-ubiquitinated Akt, an effect that is enhanced by co-treatment with the proteasome inhibitor MG-132. Modification of Akt by the addition of a Gly-Ala repeat (GAr), a domain known to block ubiquitin-dependent targeting of proteins to the proteasome, resulted in a chimeric protein that is resistant to turn-over induced by 25-OH or 7-KC and provides protection from apoptosis induced by these oxysterols. These results uncover a new aspect of oxysterol regulation of Akt in macrophages; oxysterol-stimulated poly-ubiquitination of Akt and degradation by the proteasomal pathway.

Introduction: The mechanism by which cigarette smoking leads to the development of chronic obstructive pulmonary disease is still unclear. Cigarette smoke contributes to the oxidative damage of proteins inside the lung. There are two main systems to protect eucariotic cells from unfolded and damaged proteins: heat shock proteins and the ubiquitin-proteasome pathway. Any stressful condition that causes damage to cell proteins can induce heat shock protein synthesis. Degradation of proteins via the ubiquitin-proteasome pathway involves conjugation of multiple ubiquitin moieties and degradation of the tagged protein by the proteasome into small peptides. We have tested if the proteasome is involved in the increase of amino acid content inside the gas phase cigarette smoke-treated airway epithelial cells. Furthermore, we hypothesize that cigarette smoke causes protein damage, induces HSP70 expression and increases amount of ubiquitin-protein conjugates in epithelial cells. We have also examined weather cigarette smoke disturbs proteasomal degradation of damaged proteins. Materials and methods: A549 cells were exposed to gas phase cigarette smoke (GPCS) or cigarette smoke extract (CSE) for 4 or 24 hours. Free amino acids from GPCS-treated A549 cell lysates were quantified by Liquid Chromatography - Mass Spectrometry using stable isotope labeling. HSP70 and ubiquitin - protein conjugates were analyzed by Western blot. Proteasomal subunits were labeled by using the fluorescent activity-based probe MV151 and visualised by sensitive in-gel detection method. Proteasomal activities were determined by using fluorogenic substrates. Results: GPCS and CSE cause an increase of ubiquitin - protein conjugates in A549 cells. The proteasome is involved in the increase of free amino acids inside GPCS-treated A549 cells (p < 0.05). CSE causes an increase of HSP70 expression (biological duplicate). Proteasomal active sites were not disturbed by GPCS or CSE, except after 24-hour incubation of A549 cells stimulated with GPCS. GPCS changes all three kinds of activities of the proteasome. Conclusions: Our data suggest that cigarette smoke induces damage to proteins which are subsequently ubiquitinated and tagged for proteasomal degradation. Both proteasome and HSP70 are involved in the elimination of proteins damaged by cigarette smoke. Although proteasomal active sites remain intact, cigarette smoke significantly disturbs proteasomal activity which may contribute to the aggregation of damaged proteins and enhanced cell death.

Parkinson disease (PD) is the most frequent neurodegenerative disorder, affecting about 1% of people over 50 years old. It is caused by the progressive loss of dopaminergic (DA) neurons, accompanied by the accumulation of Lewy bodies, which are abnormal structures inside nerve cells that contain proteins such as a-synuclein, Parkin, and components of the ubiquitin proteasomal pathway (a cellular-degradation pathway). Patients are usually treated with levodopa, and although the drug initially improves motor symptoms, many patients later develop a range of abnormal or uncontrolled muscle movements, called dyskinesias. More than 90% of PD cases are sporadic, but rare genetic forms may yield invaluable information on the pathogenesis of both familial and spontaneous PD. After mutations affecting Parkin, mutations in PINK1 (PTEN-induced putative kinase 1) are the second-most common cause of autosomal recessive PD (where both parents must contribute a defective gene for PD to arise in the offspring). Point or truncation mutations in PINK1 produce PD with a broad phenotypic spectrum, from early-onset with atypical features to typical late-onset PD. Pathogenic PINK1 mutations—of which about 20 have been identified—annihilate or reduce the kinase activity of the protein. A study by Julia W. Pridgeon (Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia, United States) and colleages published in this issue of PLoS Biology shows that the failure of PINK1 to phosphorylate one particular substrate, TRAP1, can sensitize cells to the lethal effects of reactive oxygen species.

The interplay between influenza virus and host factors to support the viral life cycle is well documented. Influenza A virus (IAV) proteins interact with an array of cellular proteins and hijack host pathways which are at the helm of cellular responses to facilitate virus invasion. The multifaceted nature of the ubiquitination pathway for protein regulation makes it a vulnerable target of many viruses including IAV. To this end we conducted a yeast two-hybrid screen to search for cellular ubiquitin ligases important for influenza virus replication. We identified host protein, RING finger protein 43 (RNF43), a RING-type E3 ubiquitin ligase, as a novel interactor of nucleoprotein (NP) of IAV and an essential partner to induce NP-driven p53-mediated apoptosis in IAV-infected cells. In this study, we demonstrate that IAV leads to attenuation of RNF43 transcripts and hence its respective protein levels in the cellular milieu whereas in RNF43 depleted cells, viral replication was escalated several folds. Moreover, RNF43 polyubiquitinates p53 which further leads to its destabilization resulting in a decrease in induction of the p53 apoptotic pathway, a hitherto unknown process targeted by NP for p53 stabilization and accumulation. Collectively, these results conclude that NP targets RNF43 to modulate p53 ubiquitination levels and hence causes p53 stabilization which is conducive to an enhanced apoptosis level in the host cells. In conclusion, our study unravels a novel strategy adopted by IAV for utilizing the much conserved ubiquitin proteasomal pathway.

Excessive accumulation of alpha synuclein (a-syn) in the brain has been implicated in several degenerative neurological disorders, most notably Parkinson's disease. The aggregation of a-syn is the major component of intraneuronal inclusions, Lewy bodies, which are neuropathological features, observed in Parkinson's disease, Lewy body dementia, and other synucleopathies. Diverse cellular events can contribute to a-syn accumulation, aggregation, and to subsequent Lewy body formation. These factors include genetic mutations of synuclein, parkin, or the deubiquitinating enzyme, ubiquitin C-terminal hydrolase (UCH-L1), leading to reduced clearance of a-syn by the ubiquitin proteasomal pathway (UPP). Furthermore, intracellular insults include environmental factors and an age-related decrement in antioxidant defense systems that increase oxidative stress and can affect either the accumulation or clearance of a-syn. We have dynamically modeled a-syn processing in normal and in several disease states; focusing upon alterations in the aggregation and clearance of a-syn as influenced by the UPP and the oxidative stress pathways. Simulation of increased oxidative stress generates a free radical profile analogous to that reported in vivo following exposure to the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Varying model parameters of oxidative stress, UPP dysfunction, or both pathways, simulate kinetics of a-syn that corresponds with the neuropathology described for the sporadic and genetic forms of Parkinson's disease. This in silico model provides a mathematical framework that enables kinetic appraisal of pathway components to better identify and validate important pharmacological targets.

Parkinson’s Disease (PD) is a common neurodegenerative disorder that is characterized by the progressive loss of dopamine (DA) neurons. Accompanying the loss the of DA neurons is the accumulation of Lewy bodies and neurites, intracytoplasmic proteinaceous inclusions that contain alphasynuclein, synphilin-1, components of the ubiquitin proteasomal pathway and parkin. Recent advances indicate that PD is due in some individuals to genetic mutations in alpha-synuclein, DJ-1, PINK-1, LRRK2, and parkin. Understanding the molecular mechanisms by which mutations in familial-linked genes cause PD holds great promise for unraveling the mechanisms by which DA neurons degenerate in PD. Parkin is E3-ubiquitinprotein ligase that ubiquitinates itself and promotes its own degradation. Familial associated mutations of parkin have impaired ubiquitin ligase function suggesting that this may be the cause of familial autosomal recessive PD. Parkin might be required for formation of Lewy bodies as Lewy bodies are absent in patients with parkin mutations. Parkin interacts with and ubiquitinates the alphasynuclein interacting protein, synphilin-1. Formation of Lewy-body-like ubiquitinpositive cytosolic inclusions occurs upon coexpression of alpha-synuclein, synphilin-1 and parkin. Nitric oxide inhibits Parkin’s E-3 ligase activity and its protective function by nitric oxide through S-nitrosylation both in vitro and in vivo. Nitrosative and oxidative stress link parkin function with the more common sporadic form of Parkinson’s disease and the related α-synucleinopathy, DLBD. Development of new therapies for PD and other disorders associated with nitrosative and oxidative stress may follow the elucidation of the pathways by which NO S-nitrosylates and inhibits parkin. Moreover, parkin and alpha-synuclein are linked in common pathogenic mechanism through their interaction with synphilin-1 and parkin may be important for the formation of Lewy bodies.

International audience; The ubiquitin-proteasomal pathway of degradation of proteins is activated or repressed in response to a number of environmental stresses and thereby plays an essential role in cell function and survival. Hypoxic stress, resulting from a decrease in the concentration of oxygen in tissues, is encountered in both physiological and pathological situations, in particular in cancer. The transcriptional complex hypoxia-inducible factor (HIF) is the key player in the signalling pathway that controls the hypoxic response of mammalian cells. Under hypoxic conditions it transactivates an impressive number of genes involved in a multitude of cellular functions. Tight regulation of this response in part involves the ubiquitin-proteasomal system where oxygen-dependent prolyl-4-hydroxylation of the alpha subunit of HIF triggers a cascade of events that leads to its degradation by the 26S proteasome. Inhibition of the proteasome in conjunction with topoisomerase inhibition has shown some promise in the treatment of experimental cancer. Such treatment may impact on the hypoxic adaptation of tumour cells.

606 Background: Topoisomerase I (topo-I) degradation by ubiquitin proteasomal pathway (UPP), in response to camptothecins (CPTs) is linked to the CPT response. But the mechanism is not understood. Methods: Improvised GST pull down experiments were performed to isolate topo-I interacting proteins which were analyzed by SDS-PAGE and identified by mass spectrometry. DNA-PKcs mediated phosphorylation of GST-topo-I was analyzed by SDS-PAGE and autoradiography. Immunoblot analysis of anti-BRCA1 precipitates from HCT15 cell lines was performed. GST-topo-I phosphorylated by DNA-PKcs, was incubated with the BRCA1/BARD1 heterodimer in the presence of E1, UbCH5c and ATP in ubiquitination buffer. Four triple negative breast cancer cell lines were selected to compare the degree of topo-I degradation. Colorectal cancer (CRC) tissues were immunostained with 1C1.H5.H7 to determine topo-I-pS10 level and identify a correlation between the topo-I-pS10 level and CPT response. Results: We isolated a topo-I interacting protein complex and determined that Ku70/Ku80/DNA-PKcs complex associates with topo-I, and DNA-PKcs phosphorylates topo-I at Serine10. We showed that cells with higher topo-I-pS10 level rapidly degrade topo-I and are CPT resistant. The cells with non-detectable level of topo-I-pS10 fail to degrade topo-I and are CPT sensitive. Retrospective study with 48 CRC tissues immuno-stained with anti-topo-I-pS10 supported our cell line data. Percent DAB positive nuclei demonstrated statistically significant differential staining between CPT responders and non-responders, and ROC analysis confirmed that nearly all non-responders exhibit >35% positive nuclei, indicating that topo-I-pS10 level determines topo-I degradation and a potential predictive biomarker for CPT response. Conclusions: Colorectal cancers are treated with topo-I inhibitors, but the response rate is low. We have shown that topo-I degradation rate is linked to CPT response. We have determined that high topo-I-pS10 level is indicative of rapid topoI degradation and can serve as a biomarker for CPT resistance.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder. Recent advances in genetics and pathophysiology have led to new insights into the pathogenesis of PD. Ten loci have been linked to hereditary PD. Mutations in alpha-synuclein and ubiquitin carboxy hydrolase L1 (UchL1) cause autosomal dominant PD and mutations in parkin and DJ-1 cause autosomal recessive PD. alpha-Synuclein has emerged as an important protein in the pathogenesis of PD, as it appears to be the major structural component of Lewy bodies and its accumulation/aggregation seems to play a prominent role in sporadic PD. Mutations in parkin are the most common cause of hereditary PD, and mutations in parkin are thought to lead to a loss of parkin's ubiquitin E3 ligase activity. Derangements in parkin function as well as mutations in UCH-L1 fit with the notion that derangements in the ubiquitin proteasomal pathway (UPP) may play important roles in the demise of dopamine neurons in PD. DJ-1 is a protein of unknown function that is linked to autosomal recessive PD. Oxidative stress and impairment in mitochondrial complex I activity are important in sporadic PD, and there is emerging interest in the role of herbicides, fungicides and insecticides that inhibit mitochondrial complex I activity and their role in contributing to the development of PD. These important findings serve as the foundation for discovering new pathways that may lead to the development of new therapies for PD.