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1.
Int J Mol Sci ; 22(6)2021 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-33809321

RESUMO

The revolutionary evolution in science and technology over the last few decades has made it possible to face more adequately three main challenges of modern medicine: changes in old diseases, the appearance of new diseases, and diseases that are unknown (mostly genetic), despite research efforts. In this paper we review the road travelled by pathologists in search of a method based upon the use of routine instruments and techniques which once were available for research only. The application to tissue studies of techniques from immunology, molecular biology, and genetics has allowed dynamic interpretations of biological phenomena with special regard to gene regulation and expression. That implies stepwise investigations, including light microscopy, immunohistochemistry, in situ hybridization, electron microscopy, molecular histopathology, protein crystallography, and gene sequencing, in order to progress from suggestive features detectable in routinely stained preparations to more characteristic, specific, and finally, pathognomonic features. Hematoxylin and Eosin (H&E)-stained preparations and appropriate immunohistochemical stains have enabled the recognition of phenotypic changes which may reflect genotypic alterations. That has been the case with hepatocytic inclusions detected in H&E-stained preparations, which appeared to correspond to secretory proteins that, due to genetic mutations, were retained within the rough endoplasmic reticulum (RER) and were deficient in plasma. The identification of this phenomenon affecting the molecules alpha-1-antitrypsin and fibrinogen has led to the discovery of a new field of cell organelle pathology, endoplasmic reticulum storage disease(s) (ERSD). Over fifty years, pathologists have wandered through a dark forest of complicated molecules with strange conformations, and by detailed observations in simple histopathological sections, accompanied by a growing background of molecular techniques and revelations, have been able to recognize and identify arrays of grotesque polypeptide arrangements.


Assuntos
Retículo Endoplasmático/genética , Imuno-Histoquímica , Doenças Metabólicas/patologia , alfa 1-Antitripsina/genética , Retículo Endoplasmático/patologia , Regulação da Expressão Gênica/genética , Genótipo , Humanos , Doenças Metabólicas/classificação , Doenças Metabólicas/diagnóstico , Doenças Metabólicas/genética , Mutação/genética
2.
Molecules ; 26(4)2021 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-33562298

RESUMO

Osteoarthritis (OA) is considered one of the most common arthritic diseases characterized by progressive degradation and abnormal remodeling of articular cartilage. Potential therapeutics for OA aim at restoring proper chondrocyte functioning and inhibiting apoptosis. Previous studies have demonstrated that tauroursodeoxycholic acid (TUDCA) showed anti-inflammatory and anti-apoptotic activity in many models of various diseases, acting mainly via alleviation of endoplasmic reticulum (ER) stress. However, little is known about cytoprotective effects of TUDCA on chondrocyte cells. The present study was designed to evaluate potential effects of TUDCA on interleukin-1ß (IL-1ß) and tunicamycin (TNC)-stimulated NHAC-kn chondrocytes cultured in normoxic and hypoxic conditions. Our results showed that TUDCA alleviated ER stress in TNC-treated chondrocytes, as demonstrated by reduced CHOP expression; however, it was not effective enough to prevent apoptosis of NHAC-kn cells in either normoxia nor hypoxia. However, co-treatment with TUDCA alleviated inflammatory response induced by IL-1ß, as shown by down regulation of Il-1ß, Il-6, Il-8 and Cox2, and increased the expression of antioxidant enzyme Sod2. Additionally, TUDCA enhanced Col IIα expression in IL-1ß- and TNC-stimulated cells, but only in normoxic conditions. Altogether, these results suggest that although TUDCA may display chondoprotective potential in ER-stressed cells, further analyses are still necessary to fully confirm its possible recommendation as potential candidate in OA therapy.


Assuntos
Inflamação/tratamento farmacológico , Interleucina-1beta/genética , Osteoartrite/tratamento farmacológico , Ácido Tauroquenodesoxicólico/farmacologia , Fator de Transcrição CHOP/genética , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/crescimento & desenvolvimento , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/genética , Células Cultivadas , Condrócitos/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Inflamação/genética , Inflamação/patologia , Osteoartrite/genética , Osteoartrite/patologia , Ácido Tauroquenodesoxicólico/química , Tunicamicina/farmacologia
3.
Dev Cell ; 56(1): 52-66.e7, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33434526

RESUMO

ER tubules form and maintain membrane contact sites (MCSs) with endosomes. How and why these ER-endosome MCSs persist as endosomes traffic and mature is poorly understood. Here we find that a member of the reticulon protein family, Reticulon-3L (Rtn3L), enriches at ER-endosome MCSs as endosomes mature. We show that this localization is due to the long divergent N-terminal cytoplasmic domain of Rtn3L. We found that Rtn3L is recruited to ER-endosome MCSs by endosomal protein Rab9a, which marks a transition stage between early and late endosomes. Rab9a utilizes an FSV region to recruit Rtn3L via its six LC3-interacting region motifs. Consistent with our localization results, depletion or deletion of RTN3 from cells results in endosome maturation and cargo sorting defects, similar to RAB9A depletion. Together our data identify a tubular ER protein that promotes endosome maturation at ER MCSs.


Assuntos
Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transporte Proteico/genética , Motivos de Aminoácidos , Autofagossomos/genética , Autofagossomos/metabolismo , Sistemas CRISPR-Cas , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Retículo Endoplasmático/genética , Endossomos/genética , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Proteínas de Membrana/genética , Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas Nogo/genética , Proteínas Nogo/metabolismo , RNA Interferente Pequeno , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
4.
Mol Cell ; 81(4): 724-738.e9, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33476576

RESUMO

Micronuclei are aberrant nuclear compartments that can form as a result of chromosome mis-segregation. Frequent loss of micronuclear envelope integrity exposes DNA to the cytoplasm, leading to chromosome fragmentation and immune activation. Here, we use micronuclei purification to show that the endoplasmic reticulum (ER)-associated nuclease TREX1 inhibits cGAS activation at micronuclei by degrading micronuclear DNA upon micronuclear envelope rupture. We demonstrate that the ER accesses ruptured micronuclei and plays a critical role in enabling TREX1 nucleolytic attack. TREX1 mutations, previously implicated in immune disease, untether TREX1 from the ER, disrupt TREX1 localization to micronuclei, diminish micronuclear DNA damage, and enhance cGAS activation. These results establish ER-directed resection of micronuclear DNA by TREX1 as a critical regulator of cytosolic DNA sensing in chromosomally unstable cells and provide a mechanistic basis for the importance of TREX1 ER tethering in preventing autoimmunity.


Assuntos
Dano ao DNA , Retículo Endoplasmático/metabolismo , Exodesoxirribonucleases/metabolismo , Micronúcleos com Defeito Cromossômico , Mutação , Nucleotidiltransferases/metabolismo , Fosfoproteínas/metabolismo , Retículo Endoplasmático/genética , Ativação Enzimática/genética , Exodesoxirribonucleases/genética , Células HEK293 , Humanos , Nucleotidiltransferases/genética , Fosfoproteínas/genética , Transporte Proteico/genética
5.
Int J Mol Sci ; 22(2)2021 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-33430308

RESUMO

Cell survival and normal cell function require a highly coordinated and precise regulation of basal cytosolic Ca2+ concentrations. The primary source of Ca2+ entry into the cell is mediated by the Ca2+ release-activated Ca2+ (CRAC) channel. Its action is stimulated in response to internal Ca2+ store depletion. The fundamental constituents of CRAC channels are the Ca2+ sensor, stromal interaction molecule 1 (STIM1) anchored in the endoplasmic reticulum, and a highly Ca2+-selective pore-forming subunit Orai1 in the plasma membrane. The precise nature of the Orai1 pore opening is currently a topic of intensive research. This review describes how Orai1 gating checkpoints in the middle and cytosolic extended transmembrane regions act together in a concerted manner to ensure an opening-permissive Orai1 channel conformation. In this context, we highlight the effects of the currently known multitude of Orai1 mutations, which led to the identification of a series of gating checkpoints and the determination of their role in diverse steps of the Orai1 activation cascade. The synergistic action of these gating checkpoints maintains an intact pore geometry, settles STIM1 coupling, and governs pore opening. We describe the current knowledge on Orai1 channel gating mechanisms and summarize still open questions of the STIM1-Orai1 machinery.


Assuntos
Sinalização do Cálcio/genética , Cálcio/metabolismo , Proteína ORAI1/genética , Molécula 1 de Interação Estromal/genética , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Sobrevivência Celular/genética , Citosol/metabolismo , Retículo Endoplasmático/genética , Humanos
6.
Methods Mol Biol ; 2233: 253-264, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33222140

RESUMO

Proteins destined to be exposed to the extracellular space enter the secretory pathway at the level of the endoplasmic reticulum. Proteins are then transported to the Golgi apparatus and addressed to their destination compartment, such as the plasma membrane for exocytic cargos. Exocytosis constitutes the last step of the anterograde transport of secretory cargos. Exocytic vesicles fuse with the plasma membrane, releasing soluble proteins to the extracellular milieu and transmembrane proteins to the plasma membrane. In order to monitor local exocytosis of cargos, we describe in this chapter how to perform synchronization of the anterograde transport of an exocytic cargo of interest using the retention using selective hooks (RUSH) assay in combination with selective protein immobilization (SPI). SPI is based on the coating of coverslips with anti-green fluorescent protein (GFP) antibodies, which capture the GFP-tagged RUSH cargos once exposed to the cell surface after its release by the addition of biotin.


Assuntos
Exocitose/genética , Complexo de Golgi/genética , Biologia Molecular/métodos , Via Secretória/genética , Animais , Membrana Celular/genética , Retículo Endoplasmático/genética , Proteínas de Fluorescência Verde/genética , Humanos , Proteínas de Membrana/genética , Transporte Proteico/genética , Vesículas Secretórias/genética
7.
EMBO J ; 40(2): e107407, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33346928

RESUMO

The endoplasmic reticulum (ER) membrane protein complex (EMC) was identified over a decade ago in a genetic screen for ER protein homeostasis. The EMC inserts transmembrane domains (TMDs) with limited hydrophobicity. Two recent cryo-EM structures, and a third model based on partial high- and low-resolution structures, suggest how this is accomplished.


Assuntos
Retículo Endoplasmático , Proteínas de Membrana , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Biossíntese de Proteínas , Domínios Proteicos
8.
Biochim Biophys Acta Gen Subj ; 1865(3): 129834, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33358864

RESUMO

BACKGROUND: Mitochondria is a key organelle for energy production and cellular adaptive response to intracellular and extracellular stresses. Mitochondrial stress can be evoked by various stimuli such as metabolic stressors or pathogen infection, which may lead to expression of 'mitokines' such as growth differentiation factor 15 (GDF15). SCOPE OF REVIEW: This review summarizes the mechanism of GDF15 expression in response to organelle stress such as mitochondrial stress, and covers pathophysiological conditions or diseases that are associated with elevated GDF15 level. This review also illustrates the in vivo role of GDF15 expression in those stress conditions or diseases, and a potential of GDF15 as a therapeutic agent against metabolic disorders such as NASH. MAJOR CONCLUSIONS: Mitochondrial unfolded protein response (UPRmt) is a critical process to recover from mitochondrial stress. UPRmt can induce expression of secretory proteins that can exert systemic effects (mitokines) as well as mitochondrial chaperons. GDF15 can have either protective or detrimental systemic effects in response to mitochondrial stresses, suggesting its role as a mitokine. Mounting evidence shows that GDF15 is also induced by stresses of organelles other than mitochondria such as endoplasmic reticulum (ER). GDF15 level is increased in serum or tissue of mice and human subjects with metabolic diseases such as obesity or NASH. GDF15 can modulate metabolic features of those diseases. GENERAL SIGNIFICANCE: GDF15 play a role as an integrated stress response (ISR) beyond mitochondrial stress response. GDF15 is involved in the pathogenesis of metabolic diseases such as NASH, and also could be a candidate for therapeutic agent against those diseases.


Assuntos
Envelhecimento/genética , Fator 15 de Diferenciação de Crescimento/genética , Miopatias Mitocondriais/genética , Atrofia Muscular/genética , Hepatopatia Gordurosa não Alcoólica/genética , Obesidade/genética , Envelhecimento/metabolismo , Animais , Modelos Animais de Doenças , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/genética , Regulação da Expressão Gênica , Fator 15 de Diferenciação de Crescimento/metabolismo , Fator 15 de Diferenciação de Crescimento/uso terapêutico , Humanos , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Miopatias Mitocondriais/metabolismo , Miopatias Mitocondriais/patologia , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Obesidade/metabolismo , Obesidade/patologia , Resposta a Proteínas não Dobradas
9.
Biochim Biophys Acta Gen Subj ; 1865(3): 129812, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33316349

RESUMO

BACKGROUND: The quality of proteins destined for the secretory pathway is ensured by two distinct mechanisms in the endoplasmic reticulum (ER): productive folding of newly synthesized proteins, which is assisted by ER-localized molecular chaperones and in most cases also by disulfide bond formation and transfer of an oligosaccharide unit; and ER-associated degradation (ERAD), in which proteins unfolded or misfolded in the ER are recognized and processed for delivery to the ER membrane complex, retrotranslocated through the complex with simultaneous ubiquitination, extracted by AAA-ATPase to the cytosol, and finally degraded by the proteasome. SCOPE OF REVIEW: We describe the mechanisms of productive folding and ERAD, with particular attention to glycoproteins versus non-glycoproteins, and to yeast versus mammalian systems. MAJOR CONCLUSION: Molecular mechanisms of the productive folding of glycoproteins and non-glycoproteins mediated by molecular chaperones and protein disulfide isomerases are well conserved from yeast to mammals. Additionally, mammals have gained an oligosaccharide structure-dependent folding cycle for glycoproteins. The molecular mechanisms of ERAD are also well conserved from yeast to mammals, but redundant expression of yeast orthologues in mammals has been encountered, particularly for components involved in recognition and processing of glycoproteins and components of the ER membrane complex involved in retrotranslocation and simultaneous ubiquitination of glycoproteins and non-glycoproteins. This may reflect an evolutionary consequence of increasing quantity or quality needs toward mammals. GENERAL SIGNIFICANCE: The introduction of innovative genome editing technology into analysis of the mechanisms of mammalian ERAD, as exemplified here, will provide new insights into the pathogenesis of various diseases.


Assuntos
Degradação Associada com o Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Glicoproteínas/metabolismo , Chaperonas Moleculares/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Retículo Endoplasmático/genética , Edição de Genes/métodos , Glicoproteínas/genética , Glicosilação , Chaperonas Moleculares/genética , Oligossacarídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Biossíntese de Proteínas , Isomerases de Dissulfetos de Proteínas/genética , Dobramento de Proteína , Transporte Proteico , Proteólise , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ubiquitinação
10.
Elife ; 92020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33317697

RESUMO

Mitophagy plays an important role in mitochondrial homeostasis. In yeast, the phosphorylation of the mitophagy receptor Atg32 by casein kinase 2 is essential for mitophagy. This phosphorylation is counteracted by the yeast equivalent of the STRIPAK complex consisting of the PP2A-like protein phosphatase Ppg1 and Far3-7-8-9-10-11 (Far complex), but the underlying mechanism remains elusive. Here we show that two subpopulations of the Far complex reside in the mitochondria and endoplasmic reticulum, respectively, and play distinct roles; the former inhibits mitophagy via Atg32 dephosphorylation, and the latter regulates TORC2 signaling. Ppg1 and Far11 form a subcomplex, and Ppg1 activity is required for the assembling integrity of Ppg1-Far11-Far8. The Far complex preferentially interacts with phosphorylated Atg32, and this interaction is weakened by mitophagy induction. Furthermore, the artificial tethering of Far8 to Atg32 prevents mitophagy. Taken together, the Ppg1-mediated Far complex formation and its dissociation from Atg32 are crucial for mitophagy regulation.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Mitocôndrias/enzimologia , Mitofagia , Fosfoproteínas Fosfatases/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas Relacionadas à Autofagia/genética , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/genética , Regulação Fúngica da Expressão Gênica , Mitocôndrias/genética , Complexos Multiproteicos , Fosfoproteínas Fosfatases/genética , Fosforilação , Receptores Citoplasmáticos e Nucleares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais
11.
Sci Rep ; 10(1): 22315, 2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33339897

RESUMO

In experimental studies, pancreatic islet microvasculature is essential for islet endocrine function and mass, and islet vascular morphology is altered in diabetic subjects. Even so, almost no information is available concerning human islet microvascular endothelial cell (MVEC) physiology and gene expression. In this study, islets and exocrine pancreatic tissue were acquired from organ donors with normoglycemia or impaired glucose metabolism (IGM) immediately after islet isolation. Following single-cell dissociation, primary islet- and exocrine MVECs were obtained through fluorescence-activated cell sorting (FACS) and transcriptional profiles were generated using AmpliSeq. Multiple gene sets involved in general vascular development and extracellular matrix remodeling were enriched in islet MVEC. In exocrine MVEC samples, multiple enriched gene sets that relate to biosynthesis and biomolecule catabolism were found. No statistically significant enrichment was found in gene sets related to autophagy or endoplasmic reticulum (ER) stress. Although ample differences were found between islet- and exocrine tissue endothelial cells, no differences could be observed between normoglycemic donors and donors with IGM at gene or gene set level. Our data is consistent with active angiogenesis and vascular remodeling in human islets and support the notion of ongoing endocrine pancreas tissue repair and regeneration even in the adult human.


Assuntos
Diabetes Mellitus/genética , Glucose/metabolismo , Ilhotas Pancreáticas/metabolismo , Pâncreas Exócrino/metabolismo , Adulto , Idoso , Autofagia/genética , Metabolismo dos Carboidratos/genética , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Regulação da Expressão Gênica/genética , Humanos , Ilhotas Pancreáticas/patologia , Masculino , Microvasos/metabolismo , Pessoa de Meia-Idade , Pâncreas Exócrino/patologia , Análise de Célula Única , Transcriptoma/genética
12.
Nat Commun ; 11(1): 5614, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33154382

RESUMO

Adult mammalian central nervous system axons have intrinsically poor regenerative capacity, so axonal injury has permanent consequences. One approach to enhancing regeneration is to increase the axonal supply of growth molecules and organelles. We achieved this by expressing the adaptor molecule Protrudin which is normally found at low levels in non-regenerative neurons. Elevated Protrudin expression enabled robust central nervous system regeneration both in vitro in primary cortical neurons and in vivo in the injured adult optic nerve. Protrudin overexpression facilitated the accumulation of endoplasmic reticulum, integrins and Rab11 endosomes in the distal axon, whilst removing Protrudin's endoplasmic reticulum localization, kinesin-binding or phosphoinositide-binding properties abrogated the regenerative effects. These results demonstrate that Protrudin promotes regeneration by functioning as a scaffold to link axonal organelles, motors and membranes, establishing important roles for these cellular components in mediating regeneration in the adult central nervous system.


Assuntos
Axônios/fisiologia , Sistema Nervoso Central/fisiologia , Retículo Endoplasmático/metabolismo , Regeneração Nervosa , Proteínas de Transporte Vesicular/metabolismo , Animais , Axônios/metabolismo , Células Cultivadas , Retículo Endoplasmático/genética , Endossomos/metabolismo , Feminino , Humanos , Integrinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Regeneração Nervosa/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/fisiologia , Fármacos Neuroprotetores/administração & dosagem , Traumatismos do Nervo Óptico/tratamento farmacológico , Traumatismos do Nervo Óptico/metabolismo , Traumatismos do Nervo Óptico/patologia , Fosforilação , Domínios Proteicos , Ratos , Ratos Sprague-Dawley , Retina/efeitos dos fármacos , Retina/fisiologia , Proteínas de Transporte Vesicular/administração & dosagem , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética
13.
Proc Natl Acad Sci U S A ; 117(40): 24974-24985, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32958637

RESUMO

The antigen-presenting molecule MR1 (MHC class I-related protein 1) presents metabolite antigens derived from microbial vitamin B2 synthesis to activate mucosal-associated invariant T (MAIT) cells. Key aspects of this evolutionarily conserved pathway remain uncharacterized, including where MR1 acquires ligands and what accessory proteins assist ligand binding. We answer these questions by using a fluorophore-labeled stable MR1 antigen analog, a conformation-specific MR1 mAb, proteomic analysis, and a genome-wide CRISPR/Cas9 library screen. We show that the endoplasmic reticulum (ER) contains a pool of two unliganded MR1 conformers stabilized via interactions with chaperones tapasin and tapasin-related protein. This pool is the primary source of MR1 molecules for the presentation of exogenous metabolite antigens to MAIT cells. Deletion of these chaperones reduces the ER-resident MR1 pool and hampers antigen presentation and MAIT cell activation. The MR1 antigen-presentation pathway thus co-opts ER chaperones to fulfill its unique ability to present exogenous metabolite antigens captured within the ER.


Assuntos
Retículo Endoplasmático/genética , Antígenos de Histocompatibilidade Classe I/genética , Metaboloma/genética , Antígenos de Histocompatibilidade Menor/genética , Proteômica , Apresentação do Antígeno/genética , Antígenos/genética , Antígenos/imunologia , Sistemas CRISPR-Cas/genética , Humanos , Ligantes , Ativação Linfocitária/genética , Proteínas de Membrana Transportadoras/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/imunologia , Células T Invariáveis Associadas à Mucosa/imunologia , Riboflavina/genética
14.
Nat Commun ; 11(1): 4056, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792483

RESUMO

Autophagy has been associated with oncogenesis with one of its emerging key functions being its contribution to the metabolism of tumors. Therefore, deciphering the mechanisms of how autophagy supports tumor cell metabolism is essential. Here, we demonstrate that the inhibition of autophagy induces an accumulation of lipid droplets (LD) due to a decrease in fatty acid ß-oxidation, that leads to a reduction of oxidative phosphorylation (OxPHOS) in acute myeloid leukemia (AML), but not in normal cells. Thus, the autophagic process participates in lipid catabolism that supports OxPHOS in AML cells. Interestingly, the inhibition of OxPHOS leads to LD accumulation with the concomitant inhibition of autophagy. Mechanistically, we show that the disruption of mitochondria-endoplasmic reticulum (ER) contact sites (MERCs) phenocopies OxPHOS inhibition. Altogether, our data establish that mitochondria, through the regulation of MERCs, controls autophagy that, in turn finely tunes lipid degradation to fuel OxPHOS supporting proliferation and growth in leukemia.


Assuntos
Autofagia/fisiologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia/metabolismo , Mitocôndrias/metabolismo , Animais , Autofagia/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/fisiologia , Citometria de Fluxo , Humanos , Leucemia/genética , Leucemia Mieloide Aguda/patologia , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Lipogênese/genética , Lipogênese/fisiologia , Camundongos , Mitocôndrias/genética , Oxirredução , Fosforilação Oxidativa
15.
Mol Cell ; 79(6): 963-977.e3, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32735772

RESUMO

Autophagic degradation of the endoplasmic reticulum (ER-phagy) is triggered by ER stress in diverse organisms. However, molecular mechanisms governing ER stress-induced ER-phagy remain insufficiently understood. Here we report that ER stress-induced ER-phagy in the fission yeast Schizosaccharomyces pombe requires Epr1, a soluble Atg8-interacting ER-phagy receptor. Epr1 localizes to the ER through interacting with integral ER membrane proteins VAPs. Bridging an Atg8-VAP association is the main ER-phagy role of Epr1, as it can be bypassed by an artificial Atg8-VAP tether. VAPs contribute to ER-phagy not only by tethering Atg8 to the ER membrane, but also by maintaining the ER-plasma membrane contact. Epr1 is upregulated during ER stress by the unfolded protein response (UPR) regulator Ire1. Loss of Epr1 reduces survival against ER stress. Conversely, increasing Epr1 expression suppresses the ER-phagy defect and ER stress sensitivity of cells lacking Ire1. Our findings expand and deepen the molecular understanding of ER-phagy.


Assuntos
Estresse do Retículo Endoplasmático/genética , Endorribonucleases/genética , Proteínas R-SNARE/genética , Autofagossomos/metabolismo , Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/genética , Retículo Endoplasmático/genética , Regulação Fúngica da Expressão Gênica/genética , Proteólise , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Resposta a Proteínas não Dobradas/genética
16.
Proc Natl Acad Sci U S A ; 117(28): 16638-16648, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601188

RESUMO

The Orai1 channel is regulated by stromal interaction molecules STIM1 and STIM2 within endoplasmic reticulum (ER)-plasma membrane (PM) contact sites. Ca2+ signals generated by Orai1 activate Ca2+-dependent gene expression. When compared with STIM1, STIM2 is a weak activator of Orai1, but it has been suggested to have a unique role in nuclear factor of activated T cells 1 (NFAT1) activation triggered by Orai1-mediated Ca2+ entry. In this study, we examined the contribution of STIM2 in NFAT1 activation. We report that STIM2 recruitment of Orai1/STIM1 to ER-PM junctions in response to depletion of ER-Ca2+ promotes assembly of the channel with AKAP79 to form a signaling complex that couples Orai1 channel function to the activation of NFAT1. Knockdown of STIM2 expression had relatively little effect on Orai1/STIM1 clustering or local and global [Ca2+]i increases but significantly attenuated NFAT1 activation and assembly of Orai1 with AKAP79. STIM1ΔK, which lacks the PIP2-binding polybasic domain, was recruited to ER-PM junctions following ER-Ca2+ depletion by binding to Orai1 and caused local and global [Ca2+]i increases comparable to those induced by STIM1 activation of Orai1. However, in contrast to STIM1, STIM1ΔK induced less NFAT1 activation and attenuated the association of Orai1 with STIM2 and AKAP79. Orai1-AKAP79 interaction and NFAT1 activation were recovered by coexpressing STIM2 with STIM1ΔK. Replacing the PIP2-binding domain of STIM1 with that of STIM2 eliminated the requirement of STIM2 for NFAT1 activation. Together, these data demonstrate an important role for STIM2 in coupling Orai1-mediated Ca2+ influx to NFAT1 activation.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Cálcio/metabolismo , Fatores de Transcrição NFATC/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/metabolismo , Proteínas de Ancoragem à Quinase A/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Fatores de Transcrição NFATC/genética , Proteínas de Neoplasias/genética , Proteína ORAI1/genética , Ligação Proteica , Transdução de Sinais , Molécula 1 de Interação Estromal/genética , Molécula 2 de Interação Estromal/genética
17.
Proc Natl Acad Sci U S A ; 117(28): 16401-16408, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601215

RESUMO

Proteins have evolved by incorporating several structural units within a single polypeptide. As a result, multidomain proteins constitute a large fraction of all proteomes. Their domains often fold to their native structures individually and vectorially as each domain emerges from the ribosome or the protein translocation channel, leading to the decreased risk of interdomain misfolding. However, some multidomain proteins fold in the endoplasmic reticulum (ER) nonvectorially via intermediates with nonnative disulfide bonds, which were believed to be shuffled to native ones slowly after synthesis. Yet, the mechanism by which they fold nonvectorially remains unclear. Using two-dimensional (2D) gel electrophoresis and a conformation-specific antibody that recognizes a correctly folded domain, we show here that shuffling of nonnative disulfide bonds to native ones in the most N-terminal region of LDL receptor (LDLR) started at a specific timing during synthesis. Deletion analysis identified a region on LDLR that assisted with disulfide shuffling in the upstream domain, thereby promoting its cotranslational folding. Thus, a plasma membrane-bound multidomain protein has evolved a sequence that promotes the nonvectorial folding of its upstream domains. These findings demonstrate that nonvectorial folding of a multidomain protein in the ER of mammalian cells is more coordinated and elaborated than previously thought. Thus, our findings alter our current view of how a multidomain protein folds nonvectorially in the ER of living cells.


Assuntos
Retículo Endoplasmático/metabolismo , Receptores de LDL/química , Receptores de LDL/genética , Retículo Endoplasmático/química , Retículo Endoplasmático/genética , Células HeLa , Humanos , Biossíntese de Proteínas , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , Receptores de LDL/metabolismo
18.
Proc Natl Acad Sci U S A ; 117(31): 18530-18539, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690699

RESUMO

Endoplasmic reticulum (ER) macroautophagy (hereafter called ER-phagy) uses autophagy receptors to selectively degrade ER domains in response to starvation or the accumulation of aggregation-prone proteins. Autophagy receptors package the ER into autophagosomes by binding to the ubiquitin-like yeast protein Atg8 (LC3 in mammals), which is needed for autophagosome formation. In budding yeast, cortical and cytoplasmic ER-phagy requires the autophagy receptor Atg40. While different ER autophagy receptors have been identified, little is known about other components of the ER-phagy machinery. In an effort to identify these components, we screened the genome-wide library of viable yeast deletion mutants for defects in the degradation of cortical ER following treatment with rapamycin, a drug that mimics starvation. Among the mutants we identified was vps13Δ. While yeast has one gene that encodes the phospholipid transporter VPS13, humans have four vacuolar protein-sorting (VPS) protein 13 isoforms. Mutations in all four human isoforms have been linked to different neurological disorders, including Parkinson's disease. Our findings have shown that Vps13 acts after Atg40 engages the autophagy machinery. Vps13 resides at contact sites between the ER and several organelles, including late endosomes. In the absence of Vps13, the cortical ER marker Rtn1 accumulated at late endosomes, and a dramatic decrease in ER packaging into autophagosomes was observed. Together, these studies suggest a role for Vps13 in the sequestration of the ER into autophagosomes at late endosomes. These observations may have important implications for understanding Parkinson's and other neurological diseases.


Assuntos
Autofagossomos/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Autofagia , Linhagem Celular , Retículo Endoplasmático/genética , Endossomos/genética , Endossomos/metabolismo , Humanos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
19.
DNA Cell Biol ; 39(8): 1431-1443, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32598172

RESUMO

Mitochondria contain their own genome, mitochondrial DNA (mtDNA), essential to support their fundamental intracellular role in ATP production and other key metabolic and homeostatic pathways. Mitochondria are highly dynamic organelles that communicate with all the other cellular compartments, through sites of high physical proximity. Among all, their crosstalk with the endoplasmic reticulum (ER) appears particularly important as its derangement is tightly implicated with several human disorders. Population-specific mtDNA variants clustered in defining the haplogroups have been shown to exacerbate or mitigate these pathological conditions. The exact mechanisms of the mtDNA background-modifying effect are not completely clear and a possible explanation is the outcome of mitochondrial efficiency on retrograde signaling to the nucleus. However, the possibility that different haplogroups shape the proximity and crosstalk between mitochondria and the ER has never been proposed neither investigated. In this study, we pose and discuss this question and provide preliminary data to answer it. Besides, we also address the possibility that single, disease-causing mtDNA point mutations may act also by reshaping organelle communication. Overall, this perspective review provides a theoretical platform for future studies on the interaction between mtDNA variants and organelle contact sites.


Assuntos
DNA Mitocondrial/genética , Retículo Endoplasmático/genética , Mitocôndrias/genética , Doenças Mitocondriais/genética , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Genoma Mitocondrial/genética , Humanos , Mitocôndrias/patologia , Doenças Mitocondriais/patologia
20.
J Gen Virol ; 101(6): 622-634, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32375993

RESUMO

Zika virus (ZIKV) has recently emerged as an important human pathogen due to the strong evidence that it causes disease of the central nervous system, particularly microcephaly and Guillain-Barré syndrome. The pathogenesis of disease, including mechanisms of neuroinvasion, may include both invasion via the blood-brain barrier and via peripheral (including cranial) nerves. Cellular responses to infection are also poorly understood. This study characterizes the in vitro infection of laboratory-adapted ZIKV African MR766 and two Asian strains of (1) brain endothelial cells (hCMEC/D3 cell line) and (2) olfactory ensheathing cells (OECs) (the neuroglia populating cranial nerve I and the olfactory bulb; both human and mouse OEC lines) in comparison to kidney epithelial cells (Vero cells, in which ZIKV infection is well characterized). Readouts included infection kinetics, intracellular virus localization, viral persistence and cytokine responses. Although not as high as in Vero cells, viral titres exceeded 104 plaque-forming units (p.f.u.) ml-1 in the endothelial/neuroglial cell types, except hOECs. Despite these substantial titres, a relatively small proportion of neuroglial cells were primarily infected. Immunolabelling of infected cells revealed localization of the ZIKV envelope and NS3 proteins in the cytoplasm; NS3 staining overlapped with that of dsRNA replication intermediate and the endoplasmic reticulum (ER). Infected OECs and endothelial cells produced high levels of pro-inflammatory chemokines. Nevertheless, ZIKV was also able to establish persistent infection in hOEC and hCMEC/D3 cells. Taken together, these results provide basic insights into ZIKV infection of endothelial and neuroglial cells and will form the basis for further study of ZIKV disease mechanisms.


Assuntos
Encéfalo/virologia , Células Endoteliais/virologia , Neuroglia/virologia , Infecção por Zika virus/virologia , Zika virus/patogenicidade , Animais , Barreira Hematoencefálica/virologia , Linhagem Celular , Chlorocebus aethiops , Retículo Endoplasmático/genética , Humanos , Camundongos , Células Vero , Replicação Viral/genética
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