RESUMO
The proteasome is responsible for removal of ubiquitinated proteins. Although several aspects of its regulation (e.g., assembly, composition, and post-translational modifications) have been unraveled, studying its adaptive compartmentalization in response to stress is just starting to emerge. We found that following amino acid starvation, the proteasome is translocated from its large nuclear pool to the cytoplasm-a response regulated by newly identified mTOR-agonistic amino acids-Tyr, Trp, and Phe (YWF). YWF relay their signal upstream of mTOR through Sestrin3 by disrupting its interaction with the GATOR2 complex. The triad activates mTOR toward its downstream substrates p62 and transcription factor EB (TFEB), affecting both proteasomal and autophagic activities. Proteasome translocation stimulates cytosolic proteolysis which replenishes amino acids, thus enabling cell survival. In contrast, nuclear sequestration of the proteasome following mTOR activation by YWF inhibits this proteolytic adaptive mechanism, leading to cell death, which establishes this newly identified pathway as a key stress-coping mechanism.
Assuntos
Aminoácidos Aromáticos , Complexo de Endopeptidases do Proteassoma , Sobrevivência Celular , Aminoácidos , Serina-Treonina Quinases TOR/genéticaRESUMO
NF-κB is a key transcriptional regulator involved in inflammation and cell proliferation, survival, and transformation. Several key steps in its activation are mediated by the ubiquitin (Ub) system. One uncharacterized step is limited proteasomal processing of the NF-κB1 precursor p105 to the p50 active subunit. Here, we identify KPC1 as the Ub ligase (E3) that binds to the ankyrin repeats domain of p105, ubiquitinates it, and mediates its processing both under basal conditions and following signaling. Overexpression of KPC1 inhibits tumor growth likely mediated via excessive generation of p50. Also, overabundance of p50 downregulates p65, suggesting that a p50-p50 homodimer may modulate transcription in place of the tumorigenic p50-p65. Transcript analysis reveals increased expression of genes associated with tumor-suppressive signals. Overall, KPC1 regulation of NF-κB1 processing appears to constitute an important balancing step among the stimulatory and inhibitory activities of the transcription factor in cell growth control.
Assuntos
Subunidade p50 de NF-kappa B/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Sistema Livre de Células , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Subunidade p50 de NF-kappa B/química , Neoplasias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Estrutura Terciária de Proteína , Alinhamento de Sequência , Transdução de Sinais , Ubiquitina-Proteína Ligases/isolamento & purificação , UbiquitinaçãoRESUMO
Missense mutations in the p53 tumor suppressor abound in human cancer. Common ("hotspot") mutations endow mutant p53 (mutp53) proteins with oncogenic gain of function (GOF), including enhanced cell migration and invasiveness, favoring cancer progression. GOF is usually attributed to transcriptional effects of mutp53. To elucidate transcription-independent effects of mutp53, we characterized the protein interactome of the p53R273H mutant in cells derived from pancreatic ductal adenocarcinoma (PDAC), where p53R273H is the most frequent p53 mutant. We now report that p53R273H, but not the p53R175H hotspot mutant, interacts with SQSTM1/p62 and promotes cancer cell migration and invasion in a p62-dependent manner. Mechanistically, the p53R273H-p62 axis drives the proteasomal degradation of several cell junctionassociated proteins, including the gap junction protein Connexin 43, facilitating scattered cell migration. Concordantly, down-regulation of Connexin 43 augments PDAC cell migration, while its forced overexpression blunts the promigratory effect of the p53R273H-p62 axis. These findings define a mechanism of mutp53 GOF.
Assuntos
Movimento Celular , Neoplasias Pancreáticas , Proteína Supressora de Tumor p53 , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Genes p53 , Humanos , Mutação , Neoplasias Pancreáticas/genética , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
INTRODUCTION: Arrhythmogenic cardiomyopathy (AC) is an inherited cardiomyopathy characterized by fibro-fatty replacement of cardiomyocytes, leading to life-threatening ventricular arrhythmia and heart failure. Pathogenic variants of desmoglein2 gene (DSG2) have been reported as genetic etiologies of AC. In contrast, many reported DSG2 variants are benign or variants of uncertain significance. Correct genetic variant classification is crucial for determining the best medical therapy for the patient and family members. METHODS: Pathogenicity of the DSG2 Ser194Leu variant that was identified by whole exome sequencing in a patient, who presented with ventricular tachycardia and was diagnosed with AC, was investigated by electron microscopy and immunohistochemical staining of endomyocardial biopsy sample. RESULTS: Electron microscopy demonstrated a widened gap in the adhering junction and a less well-organized intercalated disk region in the mutated cardiomyocytes compared to the control. Immunohistochemical staining in the proband diagnosed with AC showed reduced expression of desmoglein 2 and connexin 43 and intercalated disc distortion. Reduced expression of DSG2 and Connexin 43 were observed in cellular cytoplasm and gap junctions. Additionally, we detected perinuclear accumulation of DSG2 and Connexin 43 in the proband sample. CONCLUSION: Ser194Leu is a missense pathogenic mutation of DSG2 gene associated with arrhythmogenic left ventricular cardiomyopathy.
Assuntos
Displasia Arritmogênica Ventricular Direita , Cardiomiopatias , Taquicardia Ventricular , Humanos , Conexina 43/genética , Conexina 43/metabolismo , Displasia Arritmogênica Ventricular Direita/genética , Cardiomiopatias/complicações , Mutação/genética , Arritmias Cardíacas/complicações , Taquicardia Ventricular/genética , Taquicardia Ventricular/complicações , Miócitos Cardíacos/metabolismo , Desmogleína 2/genética , Desmogleína 2/metabolismoRESUMO
Degradation of a protein by the ubiquitin-proteasome system (UPS) is a multistep process catalyzed by sequential reactions. Initially, ubiquitin is conjugated to the substrate in a process mediated by concerted activity of three enzymes; the last of them-a ubiquitin ligase (E3)-belongs to a family of several hundred members, each recognizing a few specific substrates. This is followed by repeated addition of ubiquitin moieties to the previously conjugated one to generate a ubiquitin chain that serves as a recognition element for the proteasome, which then degrades the substrate. Ubiquitin is recycled via the activity of deubiquitinating enzymes (DUBs). It stands to reason that efficiency of such a complex process would depend on colocalization of the different components in an assembly that allows the reactions to be carried out sequentially and processively. Here we describe nuclear condensates that are dynamic in their composition. They contain p62 as an essential component. These assemblies are generated by liquid-liquid phase separation (LLPS) and also contain ubiquitinated targets, 26S proteasome, the three conjugating enzymes, and DUBs. Under basal conditions, they serve as efficient centers for proteolysis of nuclear proteins (e.g., c-Myc) and unassembled subunits of the proteasome, suggesting they are involved in cellular protein quality control. Supporting this notion is the finding that such foci are also involved in degradation of misfolded proteins induced by heat and oxidative stresses, following recruitment of heat shock proteins and their associated ubiquitin ligase CHIP.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ubiquitina/metabolismo , Deleção de Genes , Regulação da Expressão Gênica/fisiologia , Células HeLa , Temperatura Alta , Humanos , Pressão Osmótica , Estresse Oxidativo , Complexo de Endopeptidases do Proteassoma/genética , Proteínas de Ligação a RNA/genética , Estresse Fisiológico , Ubiquitina/genéticaRESUMO
The ubiquitin-proteasome system and autophagy are the two main proteolytic systems involved in, among other functions, the maintenance of cell integrity by eliminating misfolded and damaged proteins and organelles. Both systems remove their targets after their conjugation with ubiquitin. An interesting, yet incompletely understood problem relates to the fate of the components of the two systems. Here we provide evidence that amino acid starvation enhances polyubiquitination on specific sites of the proteasome, a modification essential for its targeting to the autophagic machinery. The uptake of the ubiquitinated proteasome is mediated by its interaction with the ubiquitin-associated domain of p62/SQSTM1, a process that also requires interaction with LC3. Importantly, deletion of the PB1 domain of p62, which is important for the targeting of ubiquitinated substrates to the proteasome, has no effect on stress-induced autophagy of this proteolytic machinery, suggesting that the domain of p62 that binds to the proteasome determines the function of p62 in either targeting substrates to the proteasome or targeting the proteasome to autophagy.
Assuntos
Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Sequestossoma-1/química , Proteína Sequestossoma-1/metabolismo , Ubiquitina/metabolismo , Aminoácidos/metabolismo , Autofagia , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Domínios Proteicos , Proteólise , Deleção de Sequência , Estresse FisiológicoRESUMO
OBJECTIVE: Factors and mechanisms that activate macrophages in atherosclerotic plaques are incompletely understood. We examined the capacity of heparanase to activate macrophages. METHODS AND RESULTS: Highly purified heparanase was added to mouse peritoneal macrophages and macrophage-like J774 cells, and the levels of tumor necrosis factor-α, matrix metalloproteinase-9, interlukin-1, and monocyte chemotactic protein-1 were evaluated by ELISA. Gene expression was determined by RT-PCR. Cells collected from Toll-like receptor-2 and Toll-like receptor-4 knockout mice were evaluated similarly. Heparanase levels in the plasma of patients with acute myocardial infarction, stable angina, and healthy subjects were determined by ELISA. Immunohistochemistry was applied to detect the expression of heparanase in control specimens and specimens of patients with stable angina or acute myocardial infarction. Addition or overexpression of heparanase variants resulted in marked increase in tumor necrosis factor-α, matrix metalloproteinase-9, interlukin-1, and monocyte chemotactic protein-1 levels. Mouse peritoneal macrophages harvested from Toll-like receptor-2 or Toll-like receptor-4 knockout mice were not activated by heparanase. Plasma heparanase level was higher in patients with acute myocardial infarction, compared with patients with stable angina and healthy subjects. Pathologic coronary specimens obtained from vulnerable plaques showed increased heparanase staining compared with specimens of stable plaque and controls. CONCLUSIONS: Heparanase activates macrophages, resulting in marked induction of cytokine expression associated with plaque progression toward vulnerability.
Assuntos
Aterosclerose/enzimologia , Glucuronidase/metabolismo , Ativação de Macrófagos , Macrófagos Peritoneais/enzimologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Angina Estável/sangue , Angina Estável/enzimologia , Animais , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/patologia , Linhagem Celular , Quimiocina CCL2/metabolismo , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/enzimologia , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica , Glucuronidase/sangue , Glucuronidase/genética , Humanos , Imuno-Histoquímica , Interleucina-1/metabolismo , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/patologia , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Infarto do Miocárdio/sangue , Infarto do Miocárdio/enzimologia , Placa Aterosclerótica , Reação em Cadeia da Polimerase , Ruptura Espontânea , Transdução de Sinais , Fatores de Tempo , Receptor 2 Toll-Like/deficiência , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genética , Transfecção , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Activity of heparanase is implicated strongly in dissemination of metastatic tumor cells and cells of the immune system. In addition, heparanase enhances the phosphorylation of selected signaling molecules, including SRC and EGFR, in a manner that requires secretion but not enzymatic activity of heparanase and is mediated by its C-terminal domain. Clinically, heparanase staining is associated with larger tumors and increased EGFR phosphorylation in head and neck carcinoma. We hypothesized that signal transducer and activator of transcription (STAT) proteins mediate the protumorigenic function of heparanase downstream of the EGFR. We provide evidence that heparanase enhances the phosphorylation of STAT3 and STAT5b but not STAT5a. Moreover, enhanced proliferation of heparanase transfected cells was attenuated by STAT3 and STAT5b siRNA, but not STAT5a or STAT1 siRNA. Clinically, STAT3 phosphorylation was associated with head and neck cancer progression, EGFR phosphorylation, and heparanase expression and cellular localization. Notably, cytoplasmic rather than nuclear phospho-STAT3 correlated with increased tumor size (T-stage; p = 0.007), number of metastatic neck lymph nodes (p = 0.05), and reduced survival of patients (p = 0.04).
Assuntos
Carcinoma de Células Escamosas/metabolismo , Glucuronidase/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma de Células Escamosas/patologia , Células Cultivadas , Progressão da Doença , Receptores ErbB/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/enzimologia , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Neoplasias Laríngeas/metabolismo , Neoplasias Laríngeas/patologia , Masculino , Camundongos , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Neoplasias Nasais/metabolismo , Neoplasias Nasais/patologia , Fosforilação/fisiologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT5/genética , Neoplasias da Língua/metabolismo , Neoplasias da Língua/patologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
BACKGROUND: Prolonged neutrophil survival is evident in various cardiovascular and respiratory morbidities, in hypoxic conditions in-vitro and in patients with obstructive sleep apnea (OSA) characterized by nightly intermittent hypoxia (IH). This may lead to persistent inflammation, tissue injury and dysfunction. We therefore investigated by a translational approach the potential contribution of the intrinsic stress-induced mitochondrial pathway in extending neutrophil survival under IH conditions. Thus, neutrophils of healthy individuals treated with IH in-vitro and neutrophils of OSA patients undergoing nightly IH episodes in-vivo were investigated. Specifically, the balance between pro-apoptotic Bax and anti-apoptotic Mcl-1 protein expression, and the potential involvement of p38MAPK and ERK1/2 signaling pathways in the control of Mcl-1 expression were investigated. METHODS: Purified neutrophils were exposed to IH and compared to normoxia and to sustained hypoxia (SH) using a BioSpherix-OxyCycler C42 system. Bax and Mcl-1 levels, and p38MAPK and ERK1/2 phosphorylation were determined by western blotting. Also, Bax/Mcl-1 expression and Bax translocation to the mitochondria were assessed by confocal microscopy in pre-apoptotic neutrophils, before the appearance of apoptotic morphology. Co-localization of Bax and mitochondria was quantified by LSM 510 CarlZeiss MicroImaging using Manders Overlap Coefficient. A paired two-tailed t test, with Bonferroni correction for multiple comparisons, was used for statistical analysis. RESULTS: Compared to normoxia, IH and SH up-regulated the anti-apoptotic Mcl-1 by about 2-fold, down-regulated the pro-apoptotic Bax by 41% and 27%, respectively, and inhibited Bax co-localization with mitochondria before visible morphological signs of apoptosis were noted. IH induced ERK1/2 and p38MAPKs phosphorylation, whereas SH induced only p38MAPK phosphorylation. Accordingly, both ERK and p38MAPK inhibitors attenuated the IH-induced Mcl-1 increase. In SH, only p38MAPK inhibition decreased Mcl-1 expression. Similar to neutrophils of healthy subjects exposed to IH (0.97± 0.2), in OSA neutrophils, Bax/Mcl-1 ratio was significantly lower compared to normoxic controls (1.0±0.5 vs.1.99±0.3, p=0.015), and Bax did not co-localize with mitochondria. CONCLUSIONS: These findings suggest that decreased Bax/Mcl-1 balance promotes neutrophil survival in IH in-vitro as well as in OSA patients. Moreover, Bax/Mcl-1 protein function in IH and SH might be regulated by different signal transduction pathways, highlighting a novel regulatory function through ERK1/2 signaling in IH.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hipóxia/patologia , Neutrófilos/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Apneia Obstrutiva do Sono/patologia , Proteína X Associada a bcl-2/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Adulto , Estudos de Casos e Controles , Sobrevivência Celular/efeitos dos fármacos , Demografia , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Humanos , Hipóxia/complicações , Hipóxia/enzimologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides , Neutrófilos/efeitos dos fármacos , Oxigênio/farmacologia , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Apneia Obstrutiva do Sono/complicações , Apneia Obstrutiva do Sono/enzimologia , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Heparanase activity is highly implicated in cell dissemination associated with tumor metastasis, angiogenesis, and inflammation. Heparanase expression is induced in many hematological and solid tumors, associated with poor prognosis. Heparanase homolog, termed heparanase 2 (Hpa2), was cloned based on sequence homology. Detailed characterization of Hpa2 at the biochemical, cellular, and clinical levels has not been so far reported, and its role in normal physiology and pathological disorders is obscure. We provide evidence that unlike heparanase, Hpa2 is not subjected to proteolytic processing and exhibits no enzymatic activity typical of heparanase. Notably, the full-length Hpa2c protein inhibits heparanase enzymatic activity, likely due to its high affinity to heparin and heparan sulfate and its ability to associate physically with heparanase. Hpa2 expression was markedly elevated in head and neck carcinoma patients, correlating with prolonged time to disease recurrence (follow-up to failure; p = 0.006) and inversely correlating with tumor cell dissemination to regional lymph nodes (N-stage; p = 0.03). Hpa2 appears to restrain tumor metastasis, likely by attenuating heparanase enzymatic activity, conferring a favorable outcome of head and neck cancer patients.
Assuntos
Glucuronidase/antagonistas & inibidores , Glucuronidase/metabolismo , Heparitina Sulfato/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Glucuronidase/química , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Dados de Sequência Molecular , Metástase Neoplásica , Ligação Proteica , Transporte ProteicoRESUMO
Heparanase is a mammalian endo-beta-d-glucuronidase that can cleave heparan sulfate side chains, an activity strongly implicated in tumor cell dissemination. The current study aimed to identify and characterize heparanase splice variants. LEADS, Compugen's alternative splicing modeling platform (Compugen, Tel Aviv, Israel), was used to search for splice variants in silico; tumor-derived cell lines (i.e., CAG myeloma) and tumor biopsies were utilized to validate T5 expression in vivo; signaling (i.e., Src phosphorylation) was evaluated following T5 gene silencing or overexpression and correlated with cell proliferation, colony formation, and tumor xenograft development. A novel spliced form of human heparanase, termed T5, was identified. In this splice variant, 144 bp of intron 5 are joined with exon 4, which results in a truncated, enzymatically inactive protein. T5 overexpression resulted in increased cell proliferation and larger colonies in soft agar, mediated by Src activation. Furthermore, T5 overexpression markedly enhanced tumor xenograft development. T5 expression is up-regulated in 75% of human renal cell carcinoma biopsies examined, which suggests that this splice variant is clinically relevant. Controls included cells overexpressing wild-type heparanase or an empty plasmid and normal-looking tissue adjacent the carcinoma lesion. T5 is a novel functional splice variant of human heparanase endowed with protumorigenic characteristics.-Barash, U., Cohen-Kaplan, V., Arvatz, G., Gingis-Velitski, S., Levy-Adam, F., Nativ, O., Shemesh, R., Ayalon-Sofer, M., Ilan, N., Vlodavsky, I. A novel human heparanase splice variant, T5, endowed with protumorigenic characteristics.
Assuntos
Processamento Alternativo , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glucuronidase/biossíntese , Proteínas de Neoplasias/biossíntese , Animais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Inativação Gênica , Glucuronidase/genética , Heparitina Sulfato/metabolismo , Humanos , Masculino , Camundongos , Camundongos Nus , Modelos Genéticos , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Transplante HeterólogoRESUMO
Membraneless condensates have recently caught the attention of biologists as hubs for cellular components required for catalysis of basic processes. Whether they are real has become the center of heated discussion where the main issues are their mechanism of assembly and function. A recent study describing these condensates as hubs for protein degradation by the ubiquitin system may shed a new light on this recent development in cell biology.
RESUMO
Activity of heparanase, responsible for cleavage of heparan sulfate (HS), is strongly implicated in tumor metastasis. This is due primarily to remodeling of the extracellular matrix (ECM) that becomes more prone to invasion by metastatic tumor cells. In addition, heparanase promotes the development of blood and lymph vessels that mobilize disseminated cells to distant organs. Here, we provide evidence for an additional mechanism by which heparanase affects cell motility, namely the destruction of E-cadherin based adherent junctions (AJ). We found that overexpression of heparanase or its exogenous addition results in reduced E-cadherin levels in the cell membrane. This was associated with a substantial increase in the phosphorylation levels of E-cadherin, ß-catenin, and p120-catenin, the latter recognized as a substrate of Src. Indeed, we found that Src phosphorylation is increased in heparanase overexpressing cells, associating with a marked decrease in the interaction of E-cadherin with ß-catenin, which is instrumental for AJ integrity and cell-cell adhesion. Notably, the association of E-cadherin with ß-catenin in heparanase overexpressing cells was restored by Src inhibitor, along with reduced cell migration. These results imply that heparanase promotes tumor metastasis by virtue of its enzymatic activity responsible for remodeling of the ECM, and by signaling aspects that result in Src-mediated phosphorylation of E-cadherin/catenins and loosening of cell-cell contacts that are required for maintaining the integrity of epithelial sheets.
RESUMO
Almost 70 years after the discovery of the lysosome, and about four decades following the unraveling of ubiquitin as a specific "mark of death," the field of protein turnover-the numerous processes it regulates, the pathologies resulting from its dysregulation, and the drugs that have been developed to target them-is still growing exponentially. Accordingly, the need for new technologies and methods is ever growing. One interesting question in the field is the mechanism(s) by which the "predators become prey". We have reported recently that the 26S proteasome, the catalytic arm of the ubiquitin system, is degraded by the autophagy-lysosome machinery, in a process requiring specific ubiquitination of the proteasome, and subsequent recognition by the shuttle protein p62/SQSTM1. Studying the modification(s), recognition sites, engulfment, and breakdown of the 26S proteasome via such "proteaphagy" has required the use of microscopy, subcellular fractionation, 'classical biochemistry', and proteomics. In this chapter, we present the essentials of these protocols, with emphasis on the refinements we have introduced in order for them to better suit the particular study of proteaphagy.
Assuntos
Autofagossomos/metabolismo , Microscopia de Fluorescência/métodos , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Autofagossomos/ultraestrutura , Autofagia , Imunofluorescência/métodos , Células HeLa , Humanos , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Complexo de Endopeptidases do Proteassoma/análise , Proteólise , UbiquitinaçãoRESUMO
Heparanase is an endoglycosidase that specifically cleaves heparan sulfate side chains, a class of glycosaminoglycans abundantly present in the extracellular matrix and on the cell surface. Heparanase activity is strongly implicated in tumor angiogenesis and metastasis attributed to remodeling of the subepithelial and subendothelial basement membranes. We hypothesized that similar to its proangiogenic capacity, heparanase is also engaged in lymphangiogenesis and utilized the D2-40 monoclonal antibody to study lymphangiogenesis in tumor specimens obtained from 65 head and neck carcinoma patients. Lymphatic density was analyzed for association with clinical parameters and heparanase staining. We provide evidence that lymphatic vessel density (LVD) correlates with head and neck lymph node metastasis (N-stage, p = 0.007) and inversely correlates with tumor cell differentiation (p = 0.007). Notably, heparanase staining correlated with LVD (p = 0.04) and, moreover, with VEGF C levels (p = 0.01). We further demonstrate that heparanase overexpression by epidermoid, breast, melanoma and prostate carcinoma cells induces a 3- to 5-fold elevation in VEGF C expression in vitro and facilitates tumor xenograft lymphangiogenesis in vivo, whereas heparanase gene silencing was associated with decreased VEGF C levels. These findings suggest that heparanase plays a unique dual role in tumor metastasis, facilitating tumor cell invasiveness and inducing VEGF C expression, thereby increasing the density of lymphatic vessels that mobilize metastatic cells.
Assuntos
Glucuronidase/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Linfangiogênese , Fator C de Crescimento do Endotélio Vascular/metabolismo , Idoso , Animais , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Glucuronidase/genética , Neoplasias de Cabeça e Pescoço/genética , Humanos , Imuno-Histoquímica , Metástase Linfática/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Estadiamento de Neoplasias , Taxa de Sobrevida , Fator C de Crescimento do Endotélio Vascular/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ubiquitin (Ub) is a small protein (8 kDa) found in all eukaryotic cells, which is conjugated covalently to numerous proteins, tagging them for recognition by a downstream effector. One of the best characterized functions of Ub is targeting proteins for either selective degradation by the proteasome, or for bulk degradation by the autophagy-lysosome system. The executing arm of the UPS is the 26S proteasome, a large multicatalytic complex. While much is known about the synthesis and assembly of the proteasome's subunits, the mechanism(s) underlying its removal has remained obscure, similar to that of many other components of the ubiquitin-proteasome system. Our recent study identified autophagy as the degrading mechanism for the mammalian proteasome, mostly under stress conditions. Amino acid starvation induces specific ubiquitination of certain 19S proteasomal subunits that is essential for its binding to SQSTM1/p62, the protein that shuttles the ubiquitinated proteasome to the autophagic machinery. SQSTM1 delivers ubiquitinated substrates for proteasomal degradation via interaction of its PB1 domain with the 19S proteasomal subunit PSMD4/Rpn10, in situations where the proteasome serves as a "predator." In contrast, we found that the UBA domain of SQSTM1 is essential for its interaction with the ubiquitinated proteasome and its delivery to the autophagosome, rendering the proteasome a "prey."
Assuntos
Autofagia , Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Receptores de Superfície Celular/metabolismo , Estresse Fisiológico , Ubiquitinação , Aminoácidos/deficiência , Arabidopsis/citologia , Arabidopsis/metabolismo , Modelos BiológicosRESUMO
The living cell is an ever changing, responsive, and adaptive environment where proteins play key roles in all processes and functions. While the scientific community focused for a long time on the decoding of the information required for protein synthesis, little attention was paid to the mechanisms by which proteins are removed from the cell. We now realize that the timely and proper activity of proteins is regulated to a large extent by their degradation; that cellular coping with different physiological cues and stress conditions depends on different catabolic pathways; and that many pathological states result from improper protein breakdown. There are two major protein degradation systems in all eukaryotic cells-the ubiquitin- proteasome and the autophagy-lysosome. The two systems are highly regulated, and-via degradation of a broad array of proteins-are responsible for maintenance of protein homeostasis and adaptation to environmental changes. Each is comprised of numerous components responsible for its coordinated function, and together they encompass a considerable fraction of the entire genome. In this review, we shall discuss the common and diverse characteristics of the ubiquitin-proteasome system (UPS) and autophagy-their substructure, mechanisms of action, function and concerted regulation under varying pathophysiological conditions.
Assuntos
Autofagia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Animais , HumanosRESUMO
The 26S proteasome is a large, â¼2.5 MDa, multi-catalytic ATP-dependent protease complex that serves as the degrading arm of the ubiquitin system, which is the major pathway for regulated degradation of cytosolic, nuclear and membrane proteins in all eukaryotic organisms.