RESUMO
The development of alcohol-associated liver disease (ALD) is associated with disorganized Golgi apparatus and accelerated phagophore formation. While Golgi membranes may contribute to phagophores, association between Golgi alterations and macroautophagy/autophagy remains unclear. GOLGA4/p230 (golgin A4), a dimeric Golgi matrix protein, participates in phagophore formation, but the underlying mechanism is elusive. Our prior research identified ethanol (EtOH)-induced Golgi scattering, disrupting intra-Golgi trafficking and depleting RAB3D GTPase from the trans-Golgi. Employing various techniques, we analyzed diverse cellular and animal models representing chronic and chronic/binge alcohol consumption. In trans-Golgi of non-treated hepatocytes, we found a triple complex formed between RAB3D, GOLGA4, and MYH10/NMIIB (myosin, heavy polypeptide 10, non-muscle). However, EtOH-induced RAB3D downregulation led to MYH10 segregation from the Golgi, accompanied by Golgi fragmentation and tethering of the MYH10 isoform, MYH9/NMIIA, to dispersed Golgi membranes. EtOH-activated autophagic flux is evident through increased WIPI2 recruitment to the Golgi, phagophore formation, enhanced LC3B lipidation, and reduced SQSTM1/p62. Although GOLGA4 dimerization and intra-Golgi localization are unaffected, loss of RAB3D leads to an extension of the cytoplasmic N terminal domain of GOLGA4, forming GOLGA4-positive phagophores. Autophagy inhibition by hydroxychloroquine (HCQ) prevents alcohol-mediated Golgi disorganization, restores distribution of ASGR (asialoglycoprotein receptor), and mitigates COL (collagen) deposition and steatosis. In contrast to short-term exposure to HCQ, extended co-treatment with both EtOH and HCQ results in the depletion of LC3B protein via proteasomal degradation. Thus, (a) RAB3D deficiency and GOLGA4 conformational changes are pivotal in MYH9-driven, EtOH-mediated Golgiphagy, and (b) HCQ treatment holds promise as a therapeutic approach for alcohol-induced liver injury.Abbreviation: ACTB: actin, beta; ALD: alcohol-associated liver disease; ASGR: asialoglycoprotein receptor; AV: autophagic vacuoles; EM: electron microscopy; ER: endoplasmic reticulum; EtOH: ethanol; HCQ: hydroxychloroquine; IP: immunoprecipitation; KD: knockdown; KO: knockout; MYH10/NMIIB: myosin, heavy polypeptide 10, non-muscle; MYH9/NMIIA: myosin, heavy polypeptide 9, non-muscle; PLA: proximity ligation assay; ORO: Oil Red O staining; PM: plasma membrane; TGN: trans-Golgi network; SIM: structured illumination super-resolution microscopy.
Assuntos
Autofagia , Regulação para Baixo , Etanol , Complexo de Golgi , Complexo de Golgi/metabolismo , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Humanos , Animais , Regulação para Baixo/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Etanol/farmacologia , Proteínas rab3 de Ligação ao GTP/metabolismo , Camundongos , Hepatócitos/metabolismo , Hepatócitos/efeitos dos fármacos , Camundongos Endogâmicos C57BLRESUMO
Prostate cancer progression to the lethal metastatic castration-resistant phenotype (mCRPC) is driven by αv integrins and is associated with Golgi disorganization and activation of the ATF6 branch of unfolded protein response (UPR). Overexpression of integrins requires N-acetylglucosaminyltransferase-V (MGAT5)-mediated glycosylation and subsequent cluster formation with Galectin-3 (Gal-3). However, the mechanism underlying this altered glycosylation is missing. For the first time, using HALO analysis of IHC, we found a strong association of integrin αv and Gal-3 at the plasma membrane (PM) in primary prostate cancer and mCRPC samples. We discovered that MGAT5 activation is caused by Golgi fragmentation and mislocalization of its competitor, N-acetylglucosaminyltransferase-III, MGAT3, from Golgi to the endoplasmic reticulum (ER). This was validated in an ethanol-induced model of ER stress, where alcohol treatment in androgen-refractory PC-3 and DU145 cells or alcohol consumption in patient with prostate cancer samples aggravates Golgi scattering, activates MGAT5, and enhances integrin expression at PM. This explains known link between alcohol consumption and prostate cancer mortality. ATF6 depletion significantly blocks UPR and reduces the number of Golgi fragments in both PC-3 and DU145 cells. Inhibition of autophagy by hydroxychloroquine (HCQ) restores compact Golgi, rescues MGAT3 intra-Golgi localization, blocks glycan modification via MGAT5, and abrogates delivery of Gal-3 to the cell surface. Importantly, the loss of Gal-3 leads to reduced integrins at PM and their accelerated internalization. ATF6 depletion and HCQ treatment synergistically decrease integrin αv and Gal-3 expression and temper orthotopic tumor growth and metastasis. IMPLICATIONS: Combined ablation of ATF6 and autophagy can serve as new mCRPC therapeutic.
Assuntos
N-Acetilglucosaminiltransferases , Neoplasias de Próstata Resistentes à Castração , Humanos , Masculino , Integrinas , Integrina alfaV , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Estresse do Retículo Endoplasmático , Autofagia , Fator 6 Ativador da Transcrição/genética , Fator 6 Ativador da Transcrição/metabolismoRESUMO
It has been a long-standing debate in the research and medical societies whether alcohol consumption is linked to the risk of prostate cancer (PCa). Many comprehensive studies from different geographical areas and nationalities have shown that moderate and heavy drinking is positively correlated with the development of PCa. Nevertheless, some observations could not confirm that such a correlation exists; some even suggest that wine consumption could prevent or slow prostate tumor growth. Here, we have rigorously analyzed the evidence both for and against the role of alcohol in PCa development. We found that many of the epidemiological studies did not consider other, potentially critical, factors, including diet (especially, low intake of fish, vegetables and linoleic acid, and excessive use of red meat), smoking, family history of PCa, low physical activity, history of high sexual activities especially with early age of first intercourse, and sexually transmitted infections. In addition, discrepancies between observations come from selectivity criteria for control groups, questionnaires about the type and dosage of alcohol, and misreported alcohol consumption. The lifetime history of alcohol consumption is critical given that a prostate tumor is typically slow-growing; however, many epidemiological observations that show no association monitored only current or relatively recent drinking status. Nevertheless, the overall conclusion is that high alcohol intake, especially binge drinking, is associated with increased risk for PCa, and this effect is not limited to any type of beverage. Alcohol consumption is also directly linked to PCa lethality as it may accelerate the growth of prostate tumors and significantly shorten the time for the progression to metastatic PCa. Thus, we recommend immediately quitting alcohol for patients diagnosed with PCa. We discuss the features of alcohol metabolism in the prostate tissue and the damaging effect of ethanol metabolites on intracellular organization and trafficking. In addition, we review the impact of alcohol consumption on prostate-specific antigen level and the risk for benign prostatic hyperplasia. Lastly, we highlight the known mechanisms of alcohol interference in prostate carcinogenesis and the possible side effects of alcohol during androgen deprivation therapy.
Assuntos
Neoplasias da Próstata , Consumo de Bebidas Alcoólicas/efeitos adversos , Consumo de Bebidas Alcoólicas/epidemiologia , Antagonistas de Androgênios , Etanol , Humanos , Masculino , Próstata/patologia , Neoplasias da Próstata/epidemiologia , Neoplasias da Próstata/etiologia , Neoplasias da Próstata/patologia , Fatores de RiscoRESUMO
BACKGROUND: The development of persistent endoplasmic reticulum (ER) stress is one of the cornerstones of prostate carcinogenesis; however, the mechanism is missing. Also, alcohol is a physiological ER stress inducer, and the link between alcoholism and progression of prostate cancer (PCa) is well documented but not well characterized. According to the canonical model, the mediator of ER stress, ATF6, is cleaved sequentially in the Golgi by S1P and S2P proteases; thereafter, the genes responsible for unfolded protein response (UPR) undergo transactivation. METHODS: Cell lines used were non-malignant prostate epithelial RWPE-1 cells, androgen-responsive LNCaP, and 22RV1 cells, as well as androgen-refractory PC-3 cells. We also utilized PCa tissue sections from patients with different Gleason scores and alcohol consumption backgrounds. Several sophisticated approaches were employed, including Structured illumination superresolution microscopy, Proximity ligation assay, Atomic force microscopy, and Nuclear magnetic resonance spectroscopy. RESULTS: Herein, we identified the trans-Golgi matrix dimeric protein GCC185 as a Golgi retention partner for both S1P and S2P, and in cells lacking GCC185, these enzymes lose intra-Golgi situation. Progression of prostate cancer (PCa) is associated with overproduction of S1P and S2P but monomerization of GCC185 and its downregulation. Utilizing different ER stress models, including ethanol administration, we found that PCa cells employ an elegant mechanism that auto-activates ER stress by fragmentation of Golgi, translocation of S1P and S2P from Golgi to ER, followed by intra-ER cleavage of ATF6, accelerated UPR, and cell proliferation. The segregation of S1P and S2P from Golgi and activation of ATF6 are positively correlated with androgen receptor signaling, different disease stages, and alcohol consumption. Finally, depletion of ATF6 significantly retarded the growth of xenograft prostate tumors and blocks production of pro-metastatic metabolites. CONCLUSIONS: We found that progression of PCa associates with translocation of S1P and S2P proteases to the ER and subsequent ATF6 cleavage. This obviates the need for ATF6 transport to the Golgi and enhances UPR and cell proliferation. Thus, we provide the novel mechanistic model of ATF6 activation and ER stress implication in the progression of PCa, suggesting ATF6 is a novel promising target for prostate cancer therapy.
Assuntos
Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Neoplasias da Próstata/metabolismo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Progressão da Doença , Complexo de Golgi/metabolismo , Xenoenxertos , Humanos , Masculino , Metaloendopeptidases/metabolismo , Camundongos , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Pró-Proteína Convertases/metabolismo , Neoplasias da Próstata/etiologia , Neoplasias da Próstata/patologia , Ligação Proteica , Transporte Proteico , Serina Endopeptidases/metabolismoRESUMO
Alcohol consumption worsens hepatitis B virus (HBV) infection pathogenesis. We have recently reported that acetaldehyde suppressed HBV peptide-major histocompatibility complex I (MHC class I) complex display on hepatocytes, limiting recognition and subsequent removal of the infected hepatocytes by HBV-specific cytotoxic T lymphocytes (CTLs). This suppression was attributed to impaired processing of antigenic peptides by the proteasome. However, in addition to proteasome dysfunction, alcohol may induce endoplasmic reticulum (ER) stress and Golgi fragmentation in HBV-infected liver cells to reduce uploading of viral peptides to MHC class I and/or trafficking of this complex to the hepatocyte surface. Hence, the aim of this study was to elucidate whether alcohol-induced ER stress and Golgi fragmentation affect HBV peptide-MHC class I complex presentation on HBV+ hepatocytes. Here, we demonstrate that, while both acetaldehyde and HBV independently cause ER stress and Golgi fragmentation, the combined exposure provided an additive effect. Thus we observed an activation of the inositol-requiring enzyme 1α-X-box binding protein 1 and activation transcription factor (ATF)6α, but not the phospho PKR-like ER kinase-phospho eukaryotic initiation factor 2α-ATF4-C/EBP homologous protein arms of ER stress in HBV-transfected cells treated with acetaldehyde-generating system (AGS). In addition, Golgi proteins trans-Golgi network 46, GM130, and Giantin revealed punctate distribution, indicating Golgi fragmentation upon AGS exposure. Furthermore, the effects of acetaldehyde were reproduced by treatment with ER stress inducers, thapsigargin and tunicamycin, which also decreased the display of this complex and MHC class I turnover in HepG2.2.15 cells and HBV-infected primary human hepatocytes. Taken together, alcohol-induced ER stress and Golgi fragmentation contribute to the suppression of HBV peptide-MHC class I complex presentation on HBV+ hepatocytes, which may diminish their recognition by CTLs and promote persistence of HBV infection in hepatocytes.NEW & NOTEWORTHY Our current findings show that acetaldehyde accelerates endoplasmic reticulum (ER) stress by activating the unfolded protein response arms inositol-requiring enzyme 1α-X-box binding protein 1 and activation transcription factor (ATF)6α but not phospho PKR-like ER kinase-p eukaryotic initiation factor 2α-ATF4-C/EBP homologous protein in hepatitis B virus (HBV)-transfected HepG2.2.15 cells. It also potentiates Golgi fragmentation, as evident by punctate distribution of Golgi proteins, GM130, trans-Golgi network 46, and Giantin. While concomitantly increasing HBV DNA and HBV surface antigen titers, acetaldehyde-induced ER stress suppresses the presentation of HBV peptide-major histocompatibility complex I complexes on hepatocyte surfaces, thereby promoting the persistence of HBV infection in the liver.
Assuntos
Apresentação de Antígeno/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Vírus da Hepatite B/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Fígado/virologia , Acetaldeído , Estresse do Retículo Endoplasmático/genética , Expressão Gênica/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Antígeno HLA-A2/análise , Células Hep G2 , Vírus da Hepatite B/genética , Antígenos de Histocompatibilidade Classe I/efeitos dos fármacos , Humanos , Fígado/imunologia , RNA Mensageiro/análise , Transfecção , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Resposta a Proteínas não Dobradas/genéticaRESUMO
BACKGROUND: The Golgi apparatus undergoes disorganization in response to stress, but it is able to restore compact and perinuclear structure under recovery. This self-organization mechanism is significant for cellular homeostasis, but remains mostly elusive, as does the role of giantin, the largest Golgi matrix dimeric protein. METHODS: In HeLa and different prostate cancer cells, we used the model of cellular stress induced by Brefeldin A (BFA). The conformational structure of giantin was assessed by proximity ligation assay and atomic force microscopy. The post-BFA distribution of Golgi resident enzymes was examined by 3D SIM high-resolution microscopy. RESULTS: We detected that giantin is rather flexible than an extended coiled-coil dimer and BFA-induced Golgi disassembly was associated with giantin monomerization. A fusion of the nascent Golgi membranes after BFA washout is forced by giantin re-dimerization via disulfide bond in its luminal domain and assisted by Rab6a GTPase. GM130-GRASP65-dependent enzymes are able to reach the nascent Golgi membranes, while giantin-sensitive enzymes appeared at the Golgi after its complete recovery via direct interaction of their cytoplasmic tail with N-terminus of giantin. CONCLUSION: Post-stress recovery of Golgi is conducted by giantin dimer and Golgi proteins refill membranes according to their docking affiliation rather than their intra-Golgi location.
Assuntos
Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Brefeldina A/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células HeLa , Humanos , Imunoprecipitação , Masculino , Proteínas de Membrana/metabolismo , Microscopia de Força Atômica , Microscopia Confocal , Neoplasias da Próstata/metabolismo , Ligação ProteicaRESUMO
Proteasome inhibitors (PIs) have markedly improved response rates as well as the survival of multiple myeloma (MM) patients over the past decade and have become an important foundation in the treatment of MM patients. Unfortunately, the majority of patients either relapses or becomes refractory to proteasome inhibition. This report describes that both PI sensitive and resistant MM cells display enhanced sensitivity to PI in the presence of synthetic amphiphilic block copolymers, Pluronics (SP1017). SP1017 effectively overcomes both acquired resistance and tumor microenvironment-mediated resistance to PIs. The combination of bortezomib and SP1017 augments accumulation of ubiquitinated proteins, increases markers of proteotoxic and ER stress, and ultimately induces both the intrinsic and extrinsic drug-induced apoptotic pathways in MM cells. Notably, co-treatment of bortezomib and SP1017 intensifies SP1017-induced disorganization of the Golgi complex and significantly reduces secretion of paraproteins. Using a human MM/SCID mice model, the combination of bortezomib and SP1017 exerted enhanced antitumor efficacy as compared to bortezomib alone, delaying disease progression, but without additional toxicity. Collectively, these findings provide proof of concept for the utility of combining PI with SP1017 and present a new approach to enhance the efficacy of current treatment options for MM patients.
Assuntos
Mieloma Múltiplo/tratamento farmacológico , Poloxâmero/farmacologia , Inibidores de Proteassoma/farmacologia , Fator 6 Ativador da Transcrição/metabolismo , Animais , Bortezomib/farmacologia , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Feminino , Camundongos , Camundongos SCID , Mieloma Múltiplo/metabolismo , Oligopeptídeos/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
Multiple epidemiologic observations and meta-analysis clearly indicate the link between alcohol abuse and the incidence and progression of prostate cancer; however, the mechanism remains enigmatic. Recently, it was found that ethanol (EtOH) induces disorganization of the Golgi complex caused by impaired function of the largest Golgi matrix protein, giantin (GOLGB1), which, in turn, alters the Golgi docking of resident Golgi proteins. Here, it is determined that in normal prostate cells, histone deacetylase 6 (HDAC6), the known regulator of androgen receptor (AR) signaling, localizes in the cytoplasm and nucleus, while its kinase, glycogen synthase kinase ß (GSK3ß), primarily resides in the Golgi. Progression of prostate cancer is accompanied by Golgi scattering, translocation of GSK3ß from the Golgi to the cytoplasm, and the cytoplasmic shift in HDAC6 localization. Alcohol dehydrogenase-generated metabolites induces Golgi disorganization in androgen-responsive LNCaP and 22Rv1 cells, facilitates tumor growth in a mouse xenograft model and activates anchorage-independent proliferation, migration, and cell adhesion. EtOH-treated cells demonstrate reduced giantin and subsequent cytoplasmic GSK3ß; this phenomenon was validated in giantin-depleted cells. Redistribution of GSK3ß to the cytoplasm results in phosphorylation of HDAC6 and its retention in the cytoplasm, which, in turn, stimulates deacetylation of HSP90, AR import into the nucleus, and secretion of prostate-specific antigen (PSA). Finally, the relationship between Golgi morphology, HDAC6 cytoplasmic content, and clinicopathologic features was assessed in human prostate cancer patient specimens with and without a history of alcohol dependence. IMPLICATIONS: This study demonstrates the importance of alcohol-induced Golgi fragmentation in the activation of AR-mediated proliferation.
Assuntos
Etanol/toxicidade , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Neoplasias da Próstata/induzido quimicamente , Receptores Androgênicos/metabolismo , Álcool Desidrogenase/metabolismo , Animais , Linhagem Celular Tumoral , Etanol/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Complexo de Golgi/patologia , Xenoenxertos , Desacetilase 6 de Histona/metabolismo , Humanos , Masculino , Camundongos , Camundongos Nus , Fosforilação , Próstata/efeitos dos fármacos , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
In hepatocytes and alcohol-metabolizing cultured cells, Golgi undergoes ethanol (EtOH)-induced disorganization. Perinuclear and organized Golgi is important in liver homeostasis, but how the Golgi remains intact is unknown. Work from our laboratories showed that EtOH-altered cellular function could be reversed after alcohol removal; we wanted to determine whether this recovery would apply to Golgi. We used alcohol-metabolizing HepG2 (VA-13) cells (cultured with or without EtOH for 72 h) and rat hepatocytes (control and EtOH-fed (Lieberâ»DeCarli diet)). For recovery, EtOH was removed and replenished with control medium (48 h for VA-13 cells) or control diet (10 days for rats). Results: EtOH-induced Golgi disassembly was associated with de-dimerization of the largest Golgi matrix protein giantin, along with impaired transport of selected hepatic proteins. After recovery from EtOH, Golgi regained their compact structure, and alterations in giantin and protein transport were restored. In VA-13 cells, when we knocked down giantin, Rab6a GTPase or non-muscle myosin IIB, minimal changes were observed in control conditions, but post-EtOH recovery was impaired. Conclusions: These data provide a link between Golgi organization and plasma membrane protein expression and identify several proteins whose expression is important to maintain Golgi structure during the recovery phase after EtOH administration.
Assuntos
Etanol/efeitos adversos , Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Fígado/metabolismo , Fígado/patologia , Animais , Modelos Animais de Doenças , Células Hep G2 , Humanos , Cirrose Hepática Alcoólica/metabolismo , Masculino , Camundongos , Miosinas/metabolismo , Ratos Wistar , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Abnormalities in the Golgi apparatus function are important to the development of alcoholic liver injury. We recently reported that Golgi disorganization in ethanol (EtOH)-treated hepatocytes is caused by impaired dimerization of the largest Golgi matrix protein, giantin. However, little is known about the mechanism which forces fragmentation. Here, in both HepG2 cells overexpressing alcohol dehydrogenase and in rat hepatocytes, we found that EtOH administration reduces the complex between giantin and Rab6a GTPase and results in the S1943 phosphorylation of non-muscle Myosin IIA (NMIIA) heavy chain, thus facilitating NMIIA association with Golgi enzymes, as detected by biochemical approaches and 3D Structured Illumination Microscopy. We revealed that NMIIA-P-S1943 competes with giantin for the Rab6a dimer, which was converted to monomer after Golgi fragmentation. Therefore, Rab6a plays a dual role in the Golgi, serving as master regulator of Golgi organization and disorganization, and that NMIIA and giantin engage in a "tug-of-war". However, the inhibition of F-actin and downregulation of NMIIA or overexpression of NMHC-IIAΔtailpiece, as well the overexpression of dominant negative Rab6a(T27N), preserved a compact Golgi phenotype. Thus, the actomyosin complex forces EtOH-induced Golgi disorganization, and the targeting of NMIIA-P-S1943 may be important for preventing the damaging effects of alcohol metabolism on the cell.
Assuntos
Etanol/farmacologia , Complexo de Golgi/efeitos dos fármacos , Miosina não Muscular Tipo IIA/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Álcool Desidrogenase/metabolismo , Animais , Técnicas de Cultura de Células , Complexo de Golgi/enzimologia , Proteínas da Matriz do Complexo de Golgi/metabolismo , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Masculino , Fosforilação , Ratos WistarRESUMO
The hepatic asialoglycoprotein receptor (ASGP-R) is posttranslationally modified in the Golgi en route to the plasma membrane, where it mediates clearance of desialylated serum glycoproteins. It is known that content of plasma membrane-associated ASGP-R is decreased after ethanol exposure, although the mechanisms remain elusive. Previously, we found that formation of compact Golgi requires dimerization of the largest Golgi matrix protein giantin. We hypothesize that ethanol-impaired giantin function may be related to altered trafficking of ASGP-R. Here we report that in HepG2 cells expressing alcohol dehydrogenase and hepatocytes of ethanol-fed rats, ethanol metabolism results in Golgi disorganization. This process is initiated by dysfunction of SAR1A GTPase followed by altered COPII vesicle formation and impaired Golgi delivery of the protein disulfide isomerase A3 (PDIA3), an enzyme that catalyzes giantin dimerization. Additionally, we show that SAR1A gene silencing in hepatocytes mimics the effect of ethanol: dedimerization of giantin, arresting PDIA3 in the endoplasmic reticulum (ER) and large-scale alterations in Golgi architecture. Ethanol-induced Golgi fission has no effect on ER-to-Golgi transportation of ASGP-R, however, it results in its deposition in cis-medial-, but not trans-Golgi. Thus, alcohol-induced deficiency in COPII vesicle formation predetermines Golgi fragmentation which, in turn, compromises the Golgi-to-plasma membrane transportation of ASGP-R.
Assuntos
Regulação para Baixo , Etanol/farmacologia , Complexo de Golgi/metabolismo , Hepatócitos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Álcool Desidrogenase/metabolismo , Animais , Receptor de Asialoglicoproteína/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Regulação para Baixo/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Inativação Gênica/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/ultraestrutura , Proteínas da Matriz do Complexo de Golgi , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Humanos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Proteínas de Membrana/metabolismo , Metaboloma/efeitos dos fármacos , Camundongos , Modelos Biológicos , Fenótipo , Isomerases de Dissulfetos de Proteínas/metabolismo , Multimerização Proteica/efeitos dos fármacos , RatosRESUMO
Core 2 N-acetylglucosaminyltransferase 2/M (C2GnT-M) synthesizes all three ß6GlcNAc branch structures found in secreted mucins. Loss of C2GnT-M leads to development of colitis and colon cancer. Recently we have shown that C2GnT-M targets the Golgi at the Giantin site and is recycled by binding to non-muscle myosin IIA, a motor protein, via the cytoplasmic tail (CT). But how this enzyme is retained in the Golgi is not known. Proteomics analysis identifies keratin type II cytoskeletal 1 (KRT1) as a protein pulled down with anti-c-Myc antibody or C2GnT-M CT from the lysate of Panc1 cells expressing bC2GnT-M tagged with c-Myc. Yeast two-hybrid analysis shows that the rod domain of KRT1 interacts directly with the WKR(6) motif in the C2GnT-M CT. Knockdown of KRT1 does not affect Golgi morphology but increases the interaction of C2GnT-M with non-muscle myosin IIA and its transportation to the endoplasmic reticulum, ubiquitination, and degradation. During Golgi recovery after brefeldin A treatment, C2GnT-M forms a complex with Giantin before KRT1, demonstrating CT-mediated sequential events of Golgi targeting and retention of C2GnT-M. In HeLa cells transiently expressing C2GnT-M-GFP, knockdown of KRT1 does not affect Golgi morphology but leaves C2GnT-M outside of the Golgi, resulting in the formation of sialyl-T antigen. Interaction of C2GnT-M and KRT1 was also detected in the goblet cells of human colon epithelial tissue and primary culture of colonic epithelial cells. The results indicate that glycosylation and thus the function of glycoconjugates can be regulated by a protein that helps retain a glycosyltransferase in the Golgi.
Assuntos
Complexo de Golgi/metabolismo , Queratina-1/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Transporte Proteico , Animais , Brefeldina A/farmacologia , Citoplasma/genética , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/genética , Complexo de Golgi/ultraestrutura , Proteínas da Matriz do Complexo de Golgi , Células HeLa , Humanos , Queratina-1/química , Queratina-1/genética , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , N-Acetilglucosaminiltransferases/químicaRESUMO
The Golgi apparatus-complex is a highly dynamic organelle which is considered the "heart" of intracellular transportation. Since its discovery by Camillo Golgi in 1873, who described it as the "black reaction," and despite the enormous volume of publications about Golgi, this apparatus remains one of the most enigmatic of the cytoplasmic organelles. A typical mammalian Golgi consists of a parallel series of flattened, disk-shaped cisternae which align into stacks. The tremendous volume of Golgi-related incoming and outgoing traffic is mediated by different motor proteins, including members of the dynein, kinesin, and myosin families. Yet in spite of the strenuous work it performs, Golgi contrives to maintain its monolithic morphology and orchestration of matrix and residential proteins. However, in response to stress, alcohol, and treatment with many pharmacological drugs over time, Golgi undergoes a kind of disorganization which ranges from mild enlargement to critical scattering. While fragmentation of the Golgi was confirmed in cancer by electron microscopy almost fifty years ago, it is only in recent years that we have begun to understand the significance of Golgi fragmentation in the biology of tumors. Below author would like to focus on how Golgi fragmentation opens the doors for cascades of fatal pathways which may facilitate cancer progression and metastasis. Among the issues addressed will be the most important cancer-specific hallmarks of Golgi fragmentation, including aberrant glycosylation, abnormal expression of the Ras GTPases, dysregulation of kinases, and hyperactivity of myosin motor proteins.
RESUMO
UNLABELLED: Prostate cancer progression is associated with upregulation of sialyl-T antigen produced by ß-galactoside α-2,3-sialyltransferase-1 (ST3Gal1) but not with core 2-associated polylactosamine despite expression of core 2 N-acetylglucosaminyltransferase-L (C2GnT-L/GCNT1). This property allows androgen-refractory prostate cancer cells to evade galectin-1 (LGALS1)-induced apoptosis, but the mechanism is not known. We have recently reported that Golgi targeting of glycosyltransferases is mediated by golgins: giantin (GOLGB1) for C2GnT-M (GCNT3) and GM130 (GOLGA2)-GRASP65 (GORASP1) or GM130-giantin for core 1 synthase. Here, we show that for Golgi targeting, C2GnT-L also uses giantin exclusively whereas ST3Gal1 uses either giantin or GM130-GRASP65. In addition, the compact Golgi morphology is detected in both androgen-sensitive prostate cancer and normal prostate cells, but fragmented Golgi and mislocalization of C2GnT-L are found in androgen-refractory cells as well as primary prostate tumors (Gleason grade 2-4). Furthermore, failure of giantin monomers to be phosphorylated and dimerized prevents Golgi from forming compact morphology and C2GnT-L from targeting the Golgi. On the other hand, ST3Gal1 reaches the Golgi by an alternate site, GM130-GRASP65. Interestingly, inhibition or knockdown of non-muscle myosin IIA (MYH9) motor protein frees up Rab6a GTPase to promote phosphorylation of giantin by polo-like kinase 3 (PLK3), which is followed by dimerization of giantin assisted by protein disulfide isomerase A3 (PDIA3), and restoration of compact Golgi morphology and targeting of C2GnT-L. Finally, the Golgi relocation of C2GnT-L in androgen-refractory cells results in their increased susceptibility to galectin-1-induced apoptosis by replacing sialyl-T antigen with polylactosamine. IMPLICATIONS: This study demonstrates the importance of Golgi morphology and regulation of glycosylation and provides insight into how the Golgi influences cancer progression and metastasis.
Assuntos
Galectina 1/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Mucinas/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Neoplasias da Próstata/patologia , Sialiltransferases/metabolismo , Apoptose , Autoantígenos/metabolismo , Linhagem Celular Tumoral , Dimerização , Glicosilação , Proteínas da Matriz do Complexo de Golgi , Humanos , Masculino , Proteínas de Membrana/química , Fosforilação , Neoplasias da Próstata/metabolismo , Especificidade por Substrato , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
The Golgi apparatus is a highly dynamic organelle which frequently undergoes morphological changes in certain normal physiological processes or in response to stress. The mechanisms are largely not known. We have found that heat shock of Panc1 cells expressing core 2 N-acetylglucosaminyltransferase-M (Panc1-C2GnT-M) induces Golgi disorganization by increasing non-muscle myosin IIA (NMIIA)-C2GnT-M complexes and polyubiquitination and proteasomal degradation of C2GnT-M. These effects are prevented by inhibition or knockdown of NMIIA. Also, the speed of Golgi fragmentation induced by heat shock is found to be positively correlated with the levels of C2GnT-M in the Golgi. The results are reproduced in LNCaP cells expressing high levels of two endogenous glycosyltransferases-core 2 N-acetylglucosaminyltransferase-L:1 and ß-galactoside:α2-3 sialyltransferase 1. Further, during recovery after heat shock, Golgi reassembly as monitored by a Golgi matrix protein giantin precedes the return of C2GnT-M to the Golgi. The results are consistent with the roles of giantin as a building block of the Golgi architecture and a docking site for transport vesicles carrying glycosyltransferases. In addition, inhibition/depletion of HSP70 or HSP90 in Panc1-C2GnT-M cells also causes an increase of NMIIA-C2GnT-M complexes and NMIIA-mediated Golgi fragmentation but results in accumulation or degradation of C2GnT-M, respectively. These results can be explained by the known functions of these two HSP: participation of HSP90 in protein folding and HSP70 in protein folding and degradation. We conclude that NMIIA is the master regulator of Golgi fragmentation induced by heat shock or inhibition/depletion of HSP70/90.
Assuntos
Complexo de Golgi/metabolismo , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Resposta ao Choque Térmico , N-Acetilglucosaminiltransferases/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Sialiltransferases/metabolismo , Linhagem Celular Tumoral , Forma Celular , Citoplasma/metabolismo , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Miosina não Muscular Tipo IIA/antagonistas & inibidores , Ligação Proteica , RNA Interferente Pequeno/metabolismo , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
The Golgi apparatus undergoes morphological changes under stress or malignant transformation, but the precise mechanisms are not known. We recently showed that non-muscle myosin IIA (NMIIA) binds to the cytoplasmic tail of Core 2 N-acetylglucosaminyltransferase mucus-type (C2GnT-M) and transports it to the endoplasmic reticulum for recycling. Here, we report that Golgi fragmentation induced by brefeldin A (BFA) or coatomer protein (ß-COP) knockdown (KD) in Panc1-bC2GnT-M (c-Myc) cells is accompanied by the increased association of NMIIA with C2GnT-M and its degradation by proteasomes. Golgi fragmentation is prevented by inhibition or KD of NMIIA. Using multiple approaches, we have shown that the speed of BFA-induced Golgi fragmentation is positively correlated with the levels of this enzyme in the Golgi. The observation is reproduced in LNCaP cells which express high levels of two endogenous glycosyltransferases--C2GnT-L and ß-galactoside α2,3 sialyltransferase 1. NMIIA is found to form complexes with these two enzymes but not Golgi matrix proteins. The KD of both enzymes or the prevention of Golgi glycosyltransferases from exiting endoplasmic reticulum reduced Golgi-associated NMIIA and decreased the BFA-induced fragmentation. Interestingly, the fragmented Golgi detected in colon cancer HT-29 cells can be restored to a compact morphology after inhibition or KD of NMIIA. The Golgi disorganization induced by the microtubule or actin destructive agent is NMIIA-independent and does not affect the levels of glycosyltransferases. We conclude that NMIIA interacts with Golgi residential but not matrix proteins, and this interaction is responsible for Golgi fragmentation induced by ß-COP KD or BFA treatment. This is a novel non-enzymatic function of Golgi glycosyltransferases.
Assuntos
Complexo de Golgi/enzimologia , Proteínas Motores Moleculares/metabolismo , Cadeias Pesadas de Miosina/metabolismo , N-Acetilglucosaminiltransferases/fisiologia , Compostos Benzidrílicos/farmacologia , Brefeldina A/farmacologia , Proteína Coatomer/genética , Proteína Coatomer/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/enzimologia , Complexo de Golgi/efeitos dos fármacos , Células HEK293 , Células HT29 , Proteínas de Choque Térmico/antagonistas & inibidores , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise , Pirrolidinonas/farmacologia , Sialiltransferases/metabolismo , Tunicamicina/farmacologia , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
Core 2 N-acetylglucosaminyltransferase 1 (C2GnT1) is a key enzyme participating in the synthesis of core 2-associated sialyl Lewis x (C2-O-sLe(x)), a ligand involved in selectin-mediated leukocyte trafficking and cancer metastasis. To accomplish that, C2GnT1 needs to be localized to the Golgi and this step requires interaction of its cytoplasmic tail (CT) with a protein that has not been identified. Employing C2GnT1 CT as the bait to perform a yeast two-hybrid screen, we have identified Golgi phosphoprotein 3 (GOLPH3) as a principal candidate protein that interacts with C2GnT1 and demonstrated that C2GnT1 binds to GOLPH3 via the LLRRR(9) sequence in the CT. Confocal fluorescence microscopic analysis shows substantial Golgi co-localization of C2GnT1 and GOLPH3. Upon GOLPH3 knockdown, C2GnT1 is found mainly in the endoplasmic reticulum and decorated with complex-type N-glycans, indicating that the enzyme has been transported to the Golgi but is not retained. Also, we have found that a recombinant protein consisting of C2GnT1 CT(1-16)-Leu(17-32)-Gly(33-42)-GFP is localized to the Golgi although the same construct with mutated CT (AAAAA(9)) is not. The data demonstrate that the C2GnT1 CT is necessary and sufficient for Golgi localization of C2GnT1. Furthermore, GOLPH3 knockdown results in reduced synthesis of C2-O-sLe(x) associated with P-selectin glycoprotein ligand-1, reduced cell tethering to and rolling on immobilized P- or E-selectin, and compromised E-selectin-induced activation of spleen tyrosine kinase and cell adhesion to intercellular adhesion molecule-1 under dynamic flow. Our results reveal that GOLPH3 can regulate cell-cell interaction by controlling Golgi retention of C2GnT1.
Assuntos
Comunicação Celular/fisiologia , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Motivos de Aminoácidos , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Técnicas de Silenciamento de Genes , Complexo de Golgi/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células K562 , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , N-Acetilglucosaminiltransferases/genética , Ligação Proteica , Transporte Proteico/fisiologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Quinase SykRESUMO
Glycosylation of secreted and membrane-bound mucins is carried out by glycosyltransferases localized to specific Golgi compartments according to the step in which each enzyme participates. However, the Golgi-targeting mechanisms of these enzymes are not clear. Herein, we investigate the Golgi-targeting mechanisms of core 1 ß3 galactosyltransferase (C1GalT1) and core 2 ß1,6-N-acetylglucosaminyltransferase-2 or mucus type (C2GnT-M), which participate in the early O-glycosylation steps. siRNAs, co-immunoprecipitation, and confocal fluorescence microscopy were employed to identify the golgins involved in the Golgi docking of vesicular complexes (VCs) that carry these two enzymes. We have found that these VCs use different golgins for docking: C2GnT-M-carrying VC (C2GnT-M-VC) utilizes Giantin, whereas C1GalT1-VC employs GM130-GRASP65 complex. However, in the absence of GRASP65, C1GalT1-VC utilizes GM130-Giantin complex. Also, we have found that these VCs are 1.1-1.2 µm in diameter, specific for each enzyme, and independent of coat protein complex II and I (COPII and COPI). These two fluorescently tagged enzymes exhibit different fluorescence recovery times in the Golgi after photobleaching. Thus, novel enzyme-specific Golgi-targeting mechanisms are employed by glycosyltransferases, and multiple Golgi docking strategies are utilized by C1GalT1.