RESUMO
The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular and in vitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS. A previously unrecognized apparatus defined here controls HyPAS biogenesis and mammalian autophagosomal precursor membranes. HyPAS can be modulated by pharmacological agents whereas its formation is inhibited upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or by expression of SARS-CoV-2 nsp6. These findings reveal the origin of mammalian autophagosomal membranes, which emerge via convergence of secretory and endosomal pathways, and show that this process is targeted by microbial factors such as coronaviral membrane-modulating proteins.
Assuntos
Autofagossomos/virologia , COVID-19/virologia , Autofagia , COVID-19/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Endossomos/fisiologia , Endossomos/virologia , Complexo de Golgi/fisiologia , Células HEK293 , Células HeLa , Humanos , Fusão de Membrana , Microscopia Confocal , Fagossomos/metabolismo , Fagossomos/virologia , Proteínas Qa-SNARE/biossíntese , Receptores sigma/biossíntese , SARS-CoV-2 , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/biossíntese , Sinaptotagminas/biossíntese , Receptor Sigma-1RESUMO
The Coatomer protein complex subunit beta 2 (COPB2) is involved in the formation of the COPI coatomer protein complex and is responsible for the transport of vesicles between the Golgi apparatus and the endoplasmic reticulum. It plays an important role in maintaining the integrity of these cellular organelles, as well as in maintaining cell homeostasis. More importantly, COPB2 plays key roles in embryonic development and tumor progression. COPB2 is regarded as a vital oncogene in several cancer types and has been implicated in tumor cell proliferation, survival, invasion, and metastasis. Here, we summarize the current knowledge on the roles of COPB2 in cancer development and progression in the context of the hallmarks of cancer.
Assuntos
Proteína Coatomer/fisiologia , Neoplasias/etiologia , Animais , Apoptose/genética , Apoptose/fisiologia , Morte Celular Autofágica/fisiologia , Ciclo Celular/fisiologia , Proliferação de Células/genética , Sobrevivência Celular/genética , Proteína Coatomer/genética , Modelos Animais de Doenças , Progressão da Doença , Desenvolvimento Embrionário , Retículo Endoplasmático/fisiologia , Complexo de Golgi/fisiologia , Homeostase , Humanos , Camundongos , Invasividade Neoplásica/genética , Invasividade Neoplásica/fisiopatologia , Metástase Neoplásica/genética , Metástase Neoplásica/fisiopatologia , Neoplasias/patologia , Vesículas Transportadoras/fisiologiaRESUMO
Proper Golgi complex function depends on the activity of Arf1, a GTPase whose effectors assemble and transport outgoing vesicles. Phosphatidylinositol 4-phosphate (PI4P) generated at the Golgi by the conserved PI 4-kinase Pik1 (PI4KIIIß) is also essential for Golgi function, although its precise roles in vesicle formation are less clear. Arf1 has been reported to regulate PI4P production, but whether Pik1 is a direct Arf1 effector is not established. Using a combination of live-cell time-lapse imaging analyses, acute PI4P depletion experiments, and in vitro protein-protein interaction assays on Golgi-mimetic membranes, we present evidence for a model in which Arf1 initiates the final stages of Golgi maturation by tightly controlling PI4P production through direct recruitment of the Pik1-Frq1 PI4-kinase complex. This PI4P serves as a critical signal for AP-1 and secretory vesicle formation, the final events at maturing Golgi compartments. This work therefore establishes the regulatory and temporal context surrounding Golgi PI4P production and its precise roles in Golgi maturation.
Assuntos
Complexo de Golgi/fisiologia , Fosfatos de Fosfatidilinositol/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , 1-Fosfatidilinositol 4-Quinase/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Ligação ao Cálcio/fisiologia , Diglicerídeos/metabolismo , Ligação Proteica , Saccharomycetales , Vesículas Secretórias/fisiologia , Imagem com Lapso de TempoRESUMO
Eukaryotic cells are complex systems compartmentalized in membrane-bound organelles. Visualization of organellar electrical activity in living cells requires both a suitable reporter and non-invasive imaging at high spatiotemporal resolution. Here we present hVoSorg, an optical method to monitor changes in the membrane potential of subcellular membranes. This method takes advantage of a FRET pair consisting of a membrane-bound voltage-insensitive fluorescent donor and a non-fluorescent voltage-dependent acceptor that rapidly moves across the membrane in response to changes in polarity. Compared to the currently available techniques, hVoSorg has advantages including simple and precise subcellular targeting, the ability to record from individual organelles, and the potential for optical multiplexing of organellar activity.
Assuntos
Técnicas Biossensoriais , Retículo Endoplasmático/fisiologia , Complexo de Golgi/fisiologia , Potenciais da Membrana , Microscopia de Fluorescência , Imagem Óptica , Animais , Retículo Endoplasmático/metabolismo , Transferência Ressonante de Energia de Fluorescência , Genes Reporter , Complexo de Golgi/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Células MCF-7 , Optogenética , Células PC12 , RatosRESUMO
Rare genetic diseases preponderantly affect the nervous system causing neurodegeneration to neurodevelopmental disorders. This is the case for both Menkes and Wilson disease, arising from mutations in ATP7A and ATP7B, respectively. The ATP7A and ATP7B proteins localize to the Golgi and regulate copper homeostasis. We demonstrate genetic and biochemical interactions between ATP7 paralogs with the conserved oligomeric Golgi (COG) complex, a Golgi apparatus vesicular tether. Disruption of Drosophila copper homeostasis by ATP7 tissue-specific transgenic expression caused alterations in epidermis, aminergic, sensory, and motor neurons. Prominent among neuronal phenotypes was a decreased mitochondrial content at synapses, a phenotype that paralleled with alterations of synaptic morphology, transmission, and plasticity. These neuronal and synaptic phenotypes caused by transgenic expression of ATP7 were rescued by downregulation of COG complex subunits. We conclude that the integrity of Golgi-dependent copper homeostasis mechanisms, requiring ATP7 and COG, are necessary to maintain mitochondria functional integrity and localization to synapses.SIGNIFICANCE STATEMENT Menkes and Wilson disease affect copper homeostasis and characteristically afflict the nervous system. However, their molecular neuropathology mechanisms remain mostly unexplored. We demonstrate that copper homeostasis in neurons is maintained by two factors that localize to the Golgi apparatus, ATP7 and the conserved oligomeric Golgi (COG) complex. Disruption of these mechanisms affect mitochondrial function and localization to synapses as well as neurotransmission and synaptic plasticity. These findings suggest communication between the Golgi apparatus and mitochondria through homeostatically controlled cellular copper levels and copper-dependent enzymatic activities in both organelles.
Assuntos
Cobre/fisiologia , Complexo de Golgi/fisiologia , Homeostase/fisiologia , Biogênese de Organelas , Sinapses/fisiologia , Adenosina Trifosfatases/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Cobre/toxicidade , ATPases Transportadoras de Cobre/genética , Drosophila , Estimulação Elétrica , Espaço Extracelular/metabolismo , Feminino , Humanos , Masculino , RNA Interferente Pequeno , Sinapses/ultraestruturaRESUMO
Nuclear recycling is essential for cell and organismal homeostasis. Nuclear architecture perturbations, such as nuclear loss or nuclear enlargement, have been observed in several pathological conditions. Apart from proteasomal components which reside in the nucleus, specific autophagic proteins also shuttle between the nucleus and the cytoplasm. Until recently, only the microautophagic degradation of nuclear components had been described. Recent studies, dissecting nuclear material recycling in organisms ranging from yeast to mammals, provide insight relevant to other forms of nucleophagy and the mediators involved. Nucleophagy has also been implicated in pathology. Lamins are degraded in cancer through direct interaction with LC3 in the nucleus. Similarly, in neurodegeneration, Golgi-associated nucleophagy is exacerbated. The physiological role of nucleophagy and its contribution to other pathologies remain to be elucidated. Here we discus recent findings that shed light into the molecular mechanisms and pathways that mediate the autophagic recycling of nuclear material.
Assuntos
Proteínas Relacionadas à Autofagia/fisiologia , Autofagia , Núcleo Celular , Animais , Autofagia/fisiologia , Caenorhabditis elegans/citologia , Caenorhabditis elegans/fisiologia , Nucléolo Celular/ultraestrutura , Complexo de Golgi/fisiologia , Humanos , Corpos de Inclusão Intranuclear/patologia , Laminopatias/patologia , Laminas/genética , Mamíferos/fisiologia , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Neoplasias/patologia , Doenças Neurodegenerativas/patologia , Membrana Nuclear/metabolismo , Proteólise , Estabilidade de RNA , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/fisiologiaRESUMO
Reactive oxygen species (ROS)-induced oxidative stress has been associated with diseases such as amyotrophic lateral sclerosis, stroke, and cancer. While the effect of ROS on mitochondria and endoplasmic reticulum (ER) has been well documented, its consequence on the Golgi apparatus is less well understood. In this study, we characterized the Golgi structure and function in HeLa cells after exposure to hydrogen peroxide (H2O2), a reagent commonly used to introduce ROS to cells. Treatment of cells with 1 mM H2O2 for 10 min resulted in the degradation of Arl1 and dissociation of GRIP domain-containing proteins Golgin-97 and Golgin-245 from the trans-Golgi. This effect could be rescued by treatment of cells with a ROS scavenger N-acetyl cysteine or protease inhibitors. Structurally, H2O2 treatment reduced the number of cisternal membranes per Golgi stack, suggesting a loss of trans-Golgi cisternae. Functionally, H2O2 treatment inhibited both anterograde and retrograde protein transport, consistent with the loss of membrane tethers on the trans-Golgi cisternae. This study revealed membrane tethers at the trans-Golgi as novel specific targets of ROS in cells.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Estresse Oxidativo/fisiologia , Fatores de Ribosilação do ADP/efeitos dos fármacos , Autoantígenos/metabolismo , Transporte Biológico/fisiologia , Complexo de Golgi/fisiologia , Proteínas da Matriz do Complexo de Golgi/metabolismo , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Membranas Intracelulares/metabolismo , Proteínas de Membrana/efeitos dos fármacos , Mitocôndrias/metabolismo , Transporte Proteico/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Rede trans-Golgi/metabolismoRESUMO
BACKGROUND: As the major storage protein in rice seeds, glutelins are synthesized at the endoplasmic reticulum (ER) as proglutelins and transported to protein storage vacuoles (PSVs) called PBIIs (Protein body IIs), where they are cleaved into mature forms by the vacuolar processing enzymes. However, the molecular mechanisms underlying glutelin trafficking are largely unknown. RESULTS: In this study, we report a rice mutant, named glutelin precursor accumulation6 (gpa6), which abnormally accumulates massive proglutelins. Cytological analyses revealed that in gpa6 endosperm cells, proglutelins were mis-sorted, leading to the presence of dense vesicles (DVs) and the formation paramural bodies (PMBs) at the apoplast, consequently, smaller PBII were observed. Mutated gene in gpa6 was found to encode a Na+/H+ antiporter, OsNHX5. OsNHX5 is expressed in all tissues analyzed, and its expression level is much higher than its closest paralog OsNHX6. The OsNHX5 protein colocalizes to the Golgi, the trans-Golgi network (TGN) and the pre-vacuolar compartment (PVC) in tobacco leaf epidermal cells. In vivo pH measurements indicated that the lumens of Golgi, TGN and PVC became more acidic in gpa6. CONCLUSIONS: Our results demonstrated an important role of OsNHX5 in regulating endomembrane luminal pH, which is essential for seed storage protein trafficking in rice.
Assuntos
Glutens/metabolismo , Homeostase , Oryza/metabolismo , Endosperma/metabolismo , Complexo de Golgi/fisiologia , Concentração de Íons de Hidrogênio , Transporte Proteico , Vacúolos/metabolismoRESUMO
BACKGROUND: Pseudorabies virus (PRV) protein UL56 (pUL56) has been implicated in viral dissemination and virulence in vivo. However, the properties of PRV pUL56 remain largely unknown. In the present study, we aim to investigate the subcellular localization of pUL56 and the underlying molecular basis in transfected cells. METHODS: Constructs of N-terminal green fluorescent protein (GFP) fused pUL56 and its truncations were employed for investigating subcellular localization and further identifying amino acids crucial for pUL56 localization in transfected Vero cells. Finally, the identified amino acids were replaced with alanine for confirming if these mutations could impair the specific localization of pUL56. RESULTS: The pUL56 predominantly localized at the Golgi and trans-Golgi network (TGN) through its predicted C-terminal transmembrane helix in transfected Vero cells. A Golgi-associated protein Rab6a, independent of interaction with pUL56, was significantly downregulated by pUL56. Further, we found three truncated pUL56 C-terminal fragments (174-184, 175-185 and 191-195) could restrict GFP in the perinuclear region, respectively. Within these truncations, the 174proline (P), 181leucine (L), 185L and 191L were essential for maintaining perinuclear accumulation, thus suggesting an important role of leucine. Alanine (A) mutagenesis assays were employed to generate a series of pUL56 C-terminal mutants on the basis of leucine. Finally, a pUL56 mutant M10 (174P/A-177L/A-181L/A-185L/A-191L/A-194L/A-195I/A-196-197L/A-200L/A) lost Golgi-TGN localization. Thus, our data revealed that the leucine-rich transmembrane helix was responsible for pUL56 Golgi-TGN localization and retention, probably through specific intracellular membrane insertion. CONCLUSION: Our data indicated that the C-terminal transmembrane helix was responsible for the Golgi-TGN localization of pUL56. In addition, the leucines within C-terminal transmembrane helix were essential for maintaining pUL56 Golgi-TGN retention in cells. Further, the pUL56 can induce downregulation of Golgi-associated protein Rab6a.
Assuntos
Complexo de Golgi/fisiologia , Leucina/química , Pseudorraiva , Proteínas Estruturais Virais/metabolismo , Rede trans-Golgi/fisiologia , Animais , Chlorocebus aethiops , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Transporte Proteico , Transfecção , Células Vero , Proteínas Estruturais Virais/genéticaRESUMO
Coronaviruses (CoVs) assemble by budding into the lumen of the early Golgi complex prior to exocytosis. The small CoV envelope (E) protein plays roles in assembly, virion release, and pathogenesis. CoV E has a single hydrophobic domain (HD), is targeted to Golgi membranes, and has cation channel activity in vitro The E protein from avian infectious bronchitis virus (IBV) has dramatic effects on the secretory system, which require residues in the HD. Mutation of the HD of IBV E in a recombinant virus background results in impaired growth kinetics, impaired release of infectious virions, accumulation of IBV spike (S) protein on the plasma membrane compared to wild-type (WT) IBV-infected cells, and aberrant cleavage of IBV S on virions. We previously reported the formation of two distinct oligomeric pools of IBV E in transfected and infected cells. Disruption of the secretory pathway by IBV E correlates with a form that is likely monomeric, suggesting that the effects on the secretory pathway are independent of E ion channel activity. Here, we present evidence suggesting that the monomeric form of IBV E correlates with an increased Golgi luminal pH. Infection with IBV or expression of IBV E induces neutralization of Golgi pH, promoting a model in which IBV E alters the secretory pathway through interaction with host cell factors, protecting IBV S from premature cleavage and leading to the efficient release of infectious virus from the cells. This is the first demonstration of a coronavirus-induced alteration in the microenvironment of the secretory pathway.IMPORTANCE Coronaviruses are important human pathogens with significant zoonotic potential. Progress has been made toward identifying potential vaccine candidates for highly pathogenic human CoVs, including the use of attenuated viruses that lack the CoV E protein or express E mutants. However, no approved vaccines or antiviral therapeutics exist. Understanding the role of the CoV E protein in virus assembly and release is thus an important prerequisite for potential vaccines as well as in identifying novel antiviral therapeutics.
Assuntos
Coronavirus/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas do Envelope Viral/metabolismo , Animais , Bronquite/imunologia , Bronquite/virologia , Membrana Celular/metabolismo , Chlorocebus aethiops , Coronavirus/patogenicidade , Proteínas do Envelope de Coronavírus , Infecções por Coronavirus/virologia , Complexo de Golgi/fisiologia , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Vírus da Bronquite Infecciosa/imunologia , Via Secretória , Células Vero , Proteínas do Envelope Viral/fisiologia , Vírion/metabolismo , Montagem de Vírus , Viroses/metabolismoRESUMO
Recent research has demonstrated that small heat shock protein (sHsp) phosphorylation plays a variety of roles in neural cells. While the phosphorylation of serine 16 (Ser16) is blocked, Hsp20 no longer has neuroprotective effects. To further investigate the mechanism underlying this process, oxygen-glucose deprivation and reperfusion (OGD/R) was used with human SH-SY5Y cells and mouse N2a neuroblastoma cells. When SH-SY5Y and N2a cells were transfected with pEGFP-Hsp20(WT), pEGFP-Hsp20(S16A), and pEGFP-Hsp20(S16D) plasmids, the Golgi apparatus (GA) became more swollen and scattered, and many small fragments formed in the MOCK and S16A groups after OGD/R (P < 0.05). Meanwhile, the endoplasmic reticulum (ER) network was reduced, and the lamellar structure increased. However, these changes were not as obvious in the WT and S16D groups. Additionally, after OGD/R, Golgi Stress related protein contents were increased in the WT and S16D groups compared with the MOCK and S16A groups (P < 0.05). However, ER Stress related protein contents were decreased in the WT and S16D groups compared with the MOCK and S16A groups (P < 0.05). Our study demonstrates that Hsp20 phosphorylation on Ser16 protects against not only OGD/R-induced GA fragmentation in SH-SY5Y cells and N2a cells via Golgi stress but also OGD/R-induced ER structural changes in SH-SY5Y cells via ER stress. These findings suggest that Hsp20 is a potential drug target for ischemia stroke treatment.
Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Glucose/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Choque Térmico HSP20/metabolismo , Oxigênio/metabolismo , Fosforilação/fisiologia , Serina/metabolismo , Animais , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/fisiologia , Complexo de Golgi/fisiologia , Humanos , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/fisiopatologiaRESUMO
SREBPs are master regulators of lipid homeostasis and undergo sterol-regulated export from ER to Golgi apparatus for processing and activation via COPII-coated vesicles. While COPII recognizes SREBP through its escort protein SCAP, factor(s) specifically promoting SREBP/SCAP loading to the COPII machinery remains unknown. Here, we show that the ER/lipid droplet-associated protein Cideb selectively promotes the loading of SREBP/SCAP into COPII vesicles. Sterol deprivation releases SCAP from Insig and enhances ER export of SREBP/SCAP by inducing SCAP-Cideb interaction, thereby modulating sterol sensitivity. Moreover, Cideb binds to the guanine nucleotide exchange factor Sec12 to enrich SCAP/SREBP at ER exit sites, where assembling of COPII complex initiates. Loss of Cideb inhibits the cargo loading of SREBP/SCAP, reduces SREBP activation, and alleviates diet-induced hepatic steatosis. Our data point to a linchpin role of Cideb in regulated ER export of SREBP and lipid homeostasis.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/fisiologia , Retículo Endoplasmático/fisiologia , Complexo de Golgi/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Esteróis/farmacologia , Animais , Proteínas Reguladoras de Apoptose/genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/efeitos dos fármacos , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/fisiologia , Retículo Endoplasmático/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Células HEK293 , Células Hep G2 , Homeostase , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Transporte Proteico , Proteína de Ligação a Elemento Regulador de Esterol 1/genéticaRESUMO
Biallelic loss-of-function mutations in TRIP11, encoding the golgin GMAP-210, cause the lethal human chondrodysplasia achondrogenesis 1A (ACG1A). We now find that a homozygous splice-site mutation of the lamin B receptor (LBR) gene results in the same phenotype. Intrigued by the genetic heterogeneity, we compared GMAP-210- and LBR-deficient primary cells to unravel how particular mutations in LBR cause a phenocopy of ACG1A. We could exclude a regulatory interaction between LBR and GMAP-210 in patients' cells. However, we discovered a common disruption of Golgi apparatus architecture that was accompanied by decreased secretory trafficking in both cases. Deficiency of Golgi-dependent glycan processing indicated a similar downstream effect of the disease-causing mutations upon Golgi function. Unexpectedly, our results thus point to a common pathogenic mechanism in GMAP-210- and LBR-related diseases attributable to defective secretory trafficking at the Golgi apparatus.
Assuntos
Acondroplasia/genética , Proteínas Nucleares/genética , Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição/genética , Acondroplasia/patologia , Transporte Biológico Ativo/genética , Proliferação de Células , Sobrevivência Celular , Colesterol/análise , Proteínas do Citoesqueleto , Retículo Endoplasmático/ultraestrutura , Feminino , Feto , Fibroblastos/patologia , Doenças Genéticas Inatas/genética , Complexo de Golgi/fisiologia , Complexo de Golgi/ultraestrutura , Humanos , Mutação , Linhagem , Fenótipo , Análise de Sequência de Proteína , Esteróis/análise , Receptor de Lamina BRESUMO
Glycosylation is a fundamental modification of proteins and membrane lipids. Toxins that utilize glycans as their receptors have served as powerful tools to identify key players in glycosylation processes. Here, we carried out Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9-mediated genome-wide loss-of-function screens using two related bacterial toxins, Shiga-like toxins (Stxs) 1 and 2, which use a specific glycolipid, globotriaosylceramide (Gb3), as receptors, and the plant toxin ricin, which recognizes a broad range of glycans. The Stxs screens identified major glycosyltransferases (GTs) and transporters involved in Gb3 biosynthesis, while the ricin screen identified GTs and transporters involved in N-linked protein glycosylation and fucosylation. The screens also identified lysosomal-associated protein transmembrane 4 alpha (LAPTM4A), a poorly characterized four-pass membrane protein, as a factor specifically required for Stxs. Mass spectrometry analysis of glycolipids and their precursors demonstrates that LAPTM4A knockout (KO) cells lack Gb3 biosynthesis. This requirement of LAPTM4A for Gb3 synthesis is not shared by its homolog lysosomal-associated protein transmembrane 4 beta (LAPTM4B), and switching the domains between them determined that the second luminal domain of LAPTM4A is required, potentially acting as a specific "activator" for the GT that synthesizes Gb3. These screens also revealed two Golgi proteins, Transmembrane protein 165 (TMEM165) and Transmembrane 9 superfamily member 2 (TM9SF2), as shared factors required for both Stxs and ricin. TMEM165 KO and TM9SF2 KO cells both showed a reduction in not only Gb3 but also other glycosphingolipids, suggesting that they are required for maintaining proper levels of glycosylation in general in the Golgi. In addition, TM9SF2 KO cells also showed defective endosomal trafficking. These studies reveal key Golgi proteins critical for regulating glycosylation and glycolipid synthesis and provide novel therapeutic targets for blocking Stxs and ricin toxicity.
Assuntos
Ricina/genética , Toxinas Shiga/genética , Toxinas Bacterianas/metabolismo , Sistemas CRISPR-Cas , Endossomos/metabolismo , Estudo de Associação Genômica Ampla/métodos , Glicolipídeos/metabolismo , Glicoesfingolipídeos , Glicosilação , Complexo de Golgi/metabolismo , Complexo de Golgi/fisiologia , Células HEK293 , Células HeLa , Humanos , Mutação com Perda de Função/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/fisiologia , Proteínas Oncogênicas/metabolismo , Transporte Proteico , Ricina/metabolismo , Toxinas Shiga/metabolismo , Triexosilceramidas/metabolismo , Triexosilceramidas/fisiologiaRESUMO
Preclinical evidence depicts the capacity of redaporfin (Redp) to act as potent photosensitizer, causing direct antineoplastic effects as well as indirect immune-dependent destruction of malignant lesions. Here, we investigated the mechanisms through which photodynamic therapy (PDT) with redaporfin kills cancer cells. Subcellular localization and fractionation studies based on the physicochemical properties of redaporfin revealed its selective tropism for the endoplasmic reticulum (ER) and the Golgi apparatus (GA). When activated, redaporfin caused rapid reactive oxygen species-dependent perturbation of ER/GA compartments, coupled to ER stress and an inhibition of the GA-dependent secretory pathway. This led to a general inhibition of protein secretion by PDT-treated cancer cells. The ER/GA play a role upstream of mitochondria in the lethal signaling pathway triggered by redaporfin-based PDT Pharmacological perturbation of GA function or homeostasis reduces mitochondrial permeabilization. In contrast, removal of the pro-apoptotic multidomain proteins BAX and BAK or pretreatment with protease inhibitors reduced cell killing, yet left the GA perturbation unaffected. Altogether, these results point to the capacity of redaporfin to kill tumor cells via destroying ER/GA function.
Assuntos
Retículo Endoplasmático/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/farmacologia , Sulfonamidas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Retículo Endoplasmático/fisiologia , Feminino , Complexo de Golgi/fisiologia , Humanos , Luz , Camundongos Endogâmicos C57BL , Fármacos Fotossensibilizantes/efeitos da radiação , Fármacos Fotossensibilizantes/uso terapêutico , Porfirinas/efeitos da radiação , Porfirinas/uso terapêutico , Sulfonamidas/efeitos da radiação , Sulfonamidas/uso terapêuticoRESUMO
CREB-H, an ER-anchored transcription factor, plays a key role in regulating secretion in metabolic pathways, particularly triglyceride homeostasis. It controls the production both of secretory pathway components and cargoes, including apolipoproteins ApoA-IV and ApoC-II, contributing to VLDL/HDL distribution and lipolysis. The key mechanism controlling CREB-H activity involves its ER retention and forward transport to the Golgi, where it is cleaved by Golgi-resident proteases, releasing the N-terminal product, which traffics to the nucleus to effect transcriptional responses. Here we show that a serine-rich motif termed the P-motif, located in the N-terminus between serines 73 and 90, controls release of the precursor transmembrane form from the ER and its forward transport to the Golgi. This motif is subject to GSK-3 phosphorylation, promoting ER retention, while mutation of target serines and drug inhibition of GSK-3 activity coordinately induce both forward transport of the precursor and cleavage, resulting in nuclear import. We previously showed that for the nuclear product, the P-motif is subject to multiple phosphorylations, which regulate stability by targeting the protein to the SCFFbw1a E3 ubiquitin ligase. Thus phosphorylation at the P-motif provides integrated control of CREB-H function, coupling intercompartmental transport in the cytoplasm with stabilization of the active form in the nucleus.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Apolipoproteínas/metabolismo , Apolipoproteínas A , Células COS , Técnicas de Cultura de Células , Núcleo Celular/metabolismo , Chlorocebus aethiops , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/fisiologia , Quinase 3 da Glicogênio Sintase/metabolismo , Complexo de Golgi/metabolismo , Complexo de Golgi/fisiologia , Células Hep G2 , Humanos , Lipólise , Fosforilação , Via Secretória , Fatores de Transcrição/metabolismoRESUMO
Exit of secretory cargo from the endoplasmic reticulum (ER) takes place at specialized domains called ER exit sites (ERESs). In mammals, loss of TANGO1 and other MIA/cTAGE (melanoma inhibitory activity/cutaneous T cell lymphoma-associated antigen) family proteins prevents ER exit of large cargoes such as collagen. Here, we show that Drosophila melanogaster Tango1, the only MIA/cTAGE family member in fruit flies, is a critical organizer of the ERES-Golgi interface. Tango1 rings hold COPII (coat protein II) carriers and Golgi in close proximity at their center. Loss of Tango1, present at ERESs in all tissues, reduces ERES size and causes ERES-Golgi uncoupling, which impairs secretion of not only collagen, but also all other cargoes we examined. Further supporting an organizing role of Tango1, its overexpression creates more and larger ERESs. Our results suggest that spatial coordination of ERES, carrier, and Golgi elements through Tango1's multiple interactions increases secretory capacity in Drosophila and allows secretion of large cargo.
Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/fisiologia , Animais , Transporte Biológico/fisiologia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/fisiologia , Drosophila melanogaster/metabolismo , Drosophila melanogaster/fisiologia , Complexo de Golgi/metabolismo , Complexo de Golgi/fisiologia , Ligação Proteica/fisiologia , Transporte Proteico/fisiologia , Proteínas de Transporte Vesicular/metabolismoRESUMO
The molecular mechanism of stress-induced hepatic steatosis is not well known. Human leucine zipper protein (LZIP) regulates the expression of genes involved in inflammation, cell migration, and stress response. The aim of this study was to determine the regulatory role of LZIP in stress-induced hepatic steatosis. We used a microarray analysis to identify LZIP-induced genes involved in hepatic lipid metabolism. LZIP increased the expression of apolipoprotein A-IV (APOA4) mRNA. In the presence of stress inducer, APOA4 promoter analysis was performed, and LZIP-induced lipid accumulation was monitored in mouse primary cells and human tissues. Under Golgi stress conditions, LZIP underwent proteolytic cleavage and was phosphorylated by AKT to protect against proteasome degradation. The stabilized N-terminal LZIP was translocated to the nucleus, where it directly bound to the APOA4 promoter, leading to APOA4 induction. LZIP-induced APOA4 expression resulted in increased absorption of surrounding free fatty acids. LZIP also promoted hepatic steatosis in mouse liver. Both LZIP and APOA4 were highly expressed in human steatosis samples. Our findings indicate that LZIP is a novel modulator of APOA4 expression and hepatic lipid metabolism. LZIP might be a therapeutic target for developing treatment strategies for hepatic steatosis and related metabolic diseases.-Kang, M., Kim, J., An, H.-T., Ko, J. Human leucine zipper protein promotes hepatic steatosis via induction of apolipoprotein A-IV.
Assuntos
Apolipoproteínas A/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fígado Gorduroso/metabolismo , Apolipoproteínas A/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Fígado Gorduroso/genética , Regulação da Expressão Gênica/fisiologia , Complexo de Golgi/fisiologia , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Mutação , Ácido Oleico/metabolismo , Fosforilação , Plasmídeos , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estresse FisiológicoRESUMO
The role of Golgi apparatus during phagocytic uptake by macrophages has been ruled out in the past. Notably, all such reports were limited to Fcγ receptor-mediated phagocytosis. Here, we unravel a highly devolved mechanism for recruitment of Golgi-derived secretory vesicles during phagosome biogenesis, which was important for uptake of most cargos, except the IgG-coated ones. We report recruitment of mannosidase-II-positive Golgi-derived vesicles during uptake of diverse targets, including latex beads, Escherichia coli, Salmonella typhimurium, and Mycobacterium tuberculosis in human and mouse macrophages. The recruitment of mannosidase-II vesicles was an early event mediated by focal exocytosis and coincided with the recruitment of transferrin receptor, VAMP3, and dynamin-2. Brefeldin A treatment inhibited mannosidase-II recruitment and phagocytic uptake of serum-coated or -uncoated latex beads and E. coli However, consistent with previous studies, brefeldin A treatment did not affect uptake of IgG-coated latex beads. Mechanistically, recruitment of mannosidase-II vesicles during phagocytic uptake required Ca2+ from both extra- and intracellular sources apart from PI3K, microtubules, and dynamin-2. Extracellular Ca2+ via voltage-gated Ca2+ channels established a Ca2+-dependent local phosphatidylinositol 1,4,5-trisphosphate gradient, which guides the focal movement of Golgi-derived vesicles to the site of uptake. We confirmed Golgi-derived vesicles recruited during phagocytosis were secretory vesicles as their recruitment was sensitive to depletion of VAMP2 or NCS1, whereas recruitment of the recycling endosome marker VAMP3 was unaffected. Depletion of both VAMP2 and NCS1 individually resulted in the reduced uptake by macrophages. Together, the study provides a previously unprecedented role of Golgi-derived secretory vesicles in phagocytic uptake, the key innate defense function.
Assuntos
Cálcio/farmacologia , Exocitose/fisiologia , Complexo de Golgi/fisiologia , Macrófagos/metabolismo , Fagocitose/fisiologia , Vesículas Secretórias/fisiologia , Animais , Linhagem Celular , Humanos , Imunidade Inata , Manosidases/metabolismo , Camundongos , Polifosfatos/metabolismo , Células RAW 264.7 , Vesículas Secretórias/metabolismoRESUMO
We found that a localization artifact can arise from common immunofluorescence methods. Specifically, cell fixation and permeabilization can cause mislocalization of a type II membrane-bound protein, ER mannosidase I, from its native localization in vesicles to the Golgi complex. Live cell microscopy and interestingly also mild cell fixation with paraformaldehyde without membrane permeabilization do not present this artifact.