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1.
J Environ Pathol Toxicol Oncol ; 39(3): 261-279, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32865917

RESUMO

Among the neurodegenerative diseases, Alzheimer's disease (AD) is a predominant public health issue, affecting 16 million people around the world. It is clinically manifested by the presence of amyloid plaques (Aß) and neurofibrillary tangles (NFT) within the brain. Due to intraneuronal processing, Aß interacts with cellular targets such as mitochondria, ER, and Golgi apparatus and hampers their normal functions. Alteration in the mitochondrial function, closely related to the production of reactive oxygen species (ROS), Ca+2 overload, and apoptosis in the brain, is one of the key pathological events studied in AD pathogenesis. It is also an important pivot for the intracellular interaction with ER and Golgi through signal transduction and membrane contact to regulate cell survival and death mechanism. Alteration in mitochondrial function is intimately connected with abnormal ER or Golgi function. Stimuli that enhance perturbation in the normal ER or Golgi organelles function can involve mitochondria mediated apoptotic cell death. In this review, we address the importance of the mitochondria and their cross talk with ER and Golgi in AD pathogenesis and animal models with a therapeutic strategy to improve the mitochondrial functions.


Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Mitocôndrias/metabolismo , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Apoptose , Encéfalo/patologia , Retículo Endoplasmático/patologia , Complexo de Golgi/patologia , Humanos , Mitocôndrias/patologia , Transdução de Sinais
2.
Nat Commun ; 11(1): 4471, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32901010

RESUMO

A human cell contains hundreds to thousands of mitochondrial DNA (mtDNA) packaged into nucleoids. Currently, the segregation and allocation of nucleoids are thought to be passively determined by mitochondrial fusion and division. Here we provide evidence, using live-cell super-resolution imaging, that nucleoids can be actively transported via KIF5B-driven mitochondrial dynamic tubulation (MDT) activities that predominantly occur at the ER-mitochondria contact sites (EMCS). We further demonstrate that a mitochondrial inner membrane protein complex MICOS links nucleoids to Miro1, a KIF5B receptor on mitochondria, at the EMCS. We show that such active transportation is a mechanism essential for the proper distribution of nucleoids in the peripheral zone of the cell. Together, our work identifies an active transportation mechanism of nucleoids, with EMCS serving as a key platform for the interplay of nucleoids, MICOS, Miro1, and KIF5B to coordinate nucleoids segregation and transportation.


Assuntos
DNA Mitocondrial/metabolismo , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/fisiologia , Animais , Transporte Biológico Ativo , Células COS , Células Cultivadas , Chlorocebus aethiops , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Cinesina/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Ratos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Proteínas rho de Ligação ao GTP/metabolismo
3.
Nat Commun ; 11(1): 4576, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917905

RESUMO

Endosome maturation depends on membrane contact sites (MCSs) formed between endoplasmic reticulum (ER) and endolysosomes (LyLEs). The mechanism underlying lipid supply for this process and its pathophysiological relevance remains unclear, however. Here, we identify PDZD8-the mammalian ortholog of a yeast ERMES subunit-as a protein that interacts with protrudin, which is located at ER-LyLE MCSs. Protrudin and PDZD8 promote the formation of ER-LyLE MCSs, and PDZD8 shows the ability to extract various lipids from the ER. Overexpression of both protrudin and PDZD8 in HeLa cells, as well as their depletion in mouse primary neurons, impairs endosomal homeostasis by inducing the formation of abnormal large vacuoles reminiscent of those apparent in spastin- or REEP1-deficient neurons. The protrudin-PDZD8 system is also essential for the establishment of neuronal polarity. Our results suggest that protrudin and PDZD8 cooperatively promote endosome maturation by mediating ER-LyLE tethering and lipid extraction at MCSs, thereby maintaining neuronal polarity and integrity.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Endossomos/fisiologia , Metabolismo dos Lipídeos , Neurônios/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Feminino , Células HEK293 , Células HeLa , Humanos , Lipídeos , Lipossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Mitocôndrias , Domínios Proteicos , Proteômica , Proteínas Recombinantes , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/genética
4.
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
5.
Nat Commun ; 11(1): 3944, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769983

RESUMO

Triacylglycerols (TG) are synthesized at the endoplasmic reticulum (ER) bilayer and packaged into organelles called lipid droplets (LDs). LDs are covered by a single phospholipid monolayer contiguous with the ER bilayer. This connection is used by several monotopic integral membrane proteins, with hydrophobic membrane association domains (HDs), to diffuse between the organelles. However, how proteins partition between ER and LDs is not understood. Here, we employed synthetic model systems and found that HD-containing proteins strongly prefer monolayers and returning to the bilayer is unfavorable. This preference for monolayers is due to a higher affinity of HDs for TG over membrane phospholipids. Protein distribution is regulated by PC/PE ratio via alterations in monolayer packing and HD-TG interaction. Thus, HD-containing proteins appear to non-specifically accumulate to the LD surface. In cells, protein editing mechanisms at the ER membrane would be necessary to prevent unspecific relocation of HD-containing proteins to LDs.


Assuntos
Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismo , Gotículas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Triglicerídeos/metabolismo , Dicroísmo Circular , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/química , Simulação de Dinâmica Molecular , Domínios Proteicos , Transporte Proteico , Triglicerídeos/química
6.
Nat Commun ; 11(1): 4012, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32782388

RESUMO

Transmembrane B cell lymphoma 2-associated X protein inhibitor motif-containing (TMBIM) 6, a Ca2+ channel-like protein, is highly up-regulated in several cancer types. Here, we show that TMBIM6 is closely associated with survival in patients with cervical, breast, lung, and prostate cancer. TMBIM6 deletion or knockdown suppresses primary tumor growth. Further, mTORC2 activation is up-regulated by TMBIM6 and stimulates glycolysis, protein synthesis, and the expression of lipid synthesis genes and glycosylated proteins. Moreover, ER-leaky Ca2+ from TMBIM6, a unique characteristic, is shown to affect mTORC2 assembly and its association with ribosomes. In addition, we identify that the BIA compound, a potentialTMBIM6 antagonist, prevents TMBIM6 binding to mTORC2, decreases mTORC2 activity, and also regulates TMBIM6-leaky Ca2+, further suppressing tumor formation and progression in cancer xenograft models. This previously unknown signaling cascade in which mTORC2 activity is enhanced via the interaction with TMBIM6 provides potential therapeutic targets for various malignancies.


Assuntos
Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Indenos/farmacologia , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Cálcio/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Neoplasias/genética , Neoplasias/patologia , Ligação Proteica , Ribossomos/metabolismo , Transdução de Sinais , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra
7.
Chemosphere ; 258: 127408, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32782161

RESUMO

This study investigates the impacts of exposure to an environment Ca2+ challenge and the mechanism of action of dibutyl phthalate (DBP) on Ca2+ influx in the gills of Danio rerio. In vitro profile of 45Ca2+ influx in gills was verified through the basal time-course. Fish were exposed to low, normal and high Ca2+ concentrations (0.02, 0.7 and 2 mM) for 12 h. So, gills were morphologically analysed and ex vivo45Ca2+ influx at 30 and 60 min was determined. For the in vitro studies, gills were treated for 60 min with DBP (1 pM, 1 nM and 1 µM) with/without blockers/activators of ionic channels, Ca2+ chelator, inhibitors of ATPases, ionic exchangers and protein kinase C to study the mechanism of DBP-induced 45Ca2+ influx. Exposure to high environmental Ca2+ augmented 45Ca2+ influx when compared to fish exposed to normal and low Ca2+ concentrations. Additionally, histopathological changes were observed in the gills of fish maintained for 12 h in low and high Ca2+. In vitro exposure of gills to DBP (1 pM) disturbed Ca2+ homeostasis. DBP stimulated 45Ca2+ influx in gills through the transitory receptor potential vanilloid 1 (TRPV1), and reverse-mode Na+/Ca2+ exchanger (NCX) activation, protein kinase C and K+ channels and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). These data suggest that in vivo short-term exposure of gills to low and high Ca2+ leads to 45Ca2+ influx and histopathological changes. Additionally, the DBP-induced rapid 45Ca2+ influx is mediated by TRPV1, NCX activation with the involvement of PKC, K+-channels and SERCA, thereby altering Ca2+ homeostasis.


Assuntos
Radioisótopos de Cálcio/metabolismo , Cálcio/metabolismo , Dibutilftalato/toxicidade , Brânquias/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/metabolismo , Animais , Cálcio/toxicidade , Dibutilftalato/metabolismo , Retículo Endoplasmático/metabolismo , Brânquias/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Canais de Cátion TRPV/metabolismo , Poluentes Químicos da Água/metabolismo
8.
PLoS One ; 15(8): e0237982, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32817700

RESUMO

Some organisms, like Trichomonas vaginalis, contain mitochondria-related hydrogen-producing organelles, called hydrogenosomes. The protein targeting into these organelles is proposed to be similar to the well-studied mitochondria import. Indeed, S. cerevisiae mitochondria and T. vaginalis hydrogenosomes share some components of protein import complexes. However, it is still unknown whether targeting signals directing substrate proteins to hydrogenosomes can support in other eukaryotes specific mitochondrial localization. To address this issue, we investigated the intracellular localization of three hydrogenosomal tail-anchored proteins expressed in yeast cells. We observed that these proteins were targeted to both mitochondria and ER with a variable dependency on the mitochondrial MIM complex. Our results suggest that the targeting signal of TA proteins are only partially conserved between hydrogenosomes and yeast mitochondria.


Assuntos
Retículo Endoplasmático/metabolismo , Regulação Fúngica da Expressão Gênica , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Trichomonas vaginalis/citologia , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Trichomonas vaginalis/genética , Trichomonas vaginalis/metabolismo
9.
Mol Cell ; 79(5): 768-781.e7, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32738194

RESUMO

Misfolded proteins in the endoplasmic reticulum (ER) are degraded by ER-associated degradation (ERAD). Although ERAD components involved in degradation of luminal substrates are well characterized, much less is known about quality control of membrane proteins. Here, we analyzed the degradation pathways of two short-lived ER membrane model proteins in mammalian cells. Using a CRISPR-Cas9 genome-wide library screen, we identified an ERAD branch required for quality control of a subset of membrane proteins. Using biochemical and mass spectrometry approaches, we showed that this ERAD branch is defined by an ER membrane complex consisting of the ubiquitin ligase RNF185, the ubiquitin-like domain containing proteins TMUB1/2 and TMEM259/Membralin, a poorly characterized protein. This complex cooperates with cytosolic ubiquitin ligase UBE3C and p97 ATPase in degrading their membrane substrates. Our data reveal that ERAD branches have remarkable specificity for their membrane substrates, suggesting that multiple, perhaps combinatorial, determinants are involved in substrate selection.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Sistema Enzimático do Citocromo P-450/metabolismo , Células HEK293 , Células HeLa , Humanos , Domínios Proteicos , Dobramento de Proteína , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Esterol 14-Desmetilase/metabolismo
10.
DNA Cell Biol ; 39(10): 1760-1766, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32808810

RESUMO

Congenital cataract refers to a lens opacity caused by multiple etiological factors, including genetic mutation, abnormal metabolism of the lens, and infection. Currently, there are >100 known disease-causing genes as well as 60 known mutations in the Cx46 gene (Gap junction alpha-3, GJA3) associated with congenital cataracts. Dysfunction of gap junctions impairs homeostasis in lens cells, thereby inducing cataract pathogenesis. This study aims to identify the disease-causing mutation in a family with congenital cataract, and to further explore the possible pathogenic mechanism resulting from this mutation. We identified that a recurrent heterozygous missense mutation c.T148C (p.S50P) in GJA3 was the pathogenic mutation in this family. Previously, this mutation was found in a British family causing bilateral congenital cataract. We further demonstrated that CX46 wild type (WT) was coupled through functional gap junctions in HeLa cells, while mutant Cx46 S50P lost this ability. Moreover, the half-life of Cx46 S50P was longer than that of Cx46 WT, Cx46 S50P protein was also localized to the endoplasmic reticulum and induced more reactive oxygen species compared to Cx46 WT, which may lead to dysregulation of Cx46-formed gap junction. Collectively, our study defines the genetics basis of a congenital cataract family as well as the cellular mechanisms of mutant Cx46 S50P, and provides useful information for further studies of the pathogenesis and therapeutic strategy for treating congenital cataract.


Assuntos
Catarata/genética , Conexinas/genética , Mutação , Adulto , Catarata/metabolismo , Catarata/patologia , Criança , Conexinas/metabolismo , Retículo Endoplasmático/metabolismo , Feminino , Junções Comunicantes/metabolismo , Junções Comunicantes/patologia , Células HeLa , Humanos , Masculino , Linhagem , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo
11.
PLoS One ; 15(8): e0237930, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841274

RESUMO

Chinese hamster ovary cells have been the workhorse for the production of recombinant proteins in mammalian cells. Since biochemical, cellular and omics studies are usually affected by the lack of suitable fractionation procedures to isolate compartments from these cells, differential and isopycnic centrifugation based techniques were characterized and developed specially for them. Enriched fractions in intact nuclei, mitochondria, peroxisomes, cis-Golgi, trans-Golgi and endoplasmic reticulum (ER) were obtained in differential centrifugation steps and subsequently separated in discontinuous sucrose gradients. Nuclei, mitochondria, cis-Golgi, peroxisomes and smooth ER fractions were obtained as defined bands in 30-60% gradients. Despite the low percentage represented by the microsomes of the total cell homogenate (1.7%), their separation in a novel sucrose gradient (10-60%) showed enough resolution and efficiency to quantitatively separate their components into enriched fractions in trans-Golgi, cis-Golgi and ER. The identity of these organelles belonging to the classical secretion pathway that came from 10-60% gradients was confirmed by proteomics. Data are available via ProteomeXchange with identifier PXD019778. Components from ER and plasma membrane were the most frequent contaminants in almost all obtained fractions. The improved sucrose gradient for microsomal samples proved being successful in obtaining enriched fractions of low abundance organelles, such as Golgi apparatus and ER components, for biochemical and molecular studies, and suitable for proteomic research, which makes it a useful tool for future studies of this and other mammalian cell lines.


Assuntos
Microssomos/metabolismo , Proteômica , Animais , Células CHO , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Centrifugação , Cricetinae , Cricetulus , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Ontologia Genética , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Microssomos/ultraestrutura , Mitocôndrias/ultraestrutura , Proteoma/metabolismo , Software , Frações Subcelulares/metabolismo
12.
Nat Commun ; 11(1): 4285, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855390

RESUMO

Plant hormone cytokinins are perceived by a subfamily of sensor histidine kinases (HKs), which via a two-component phosphorelay cascade activate transcriptional responses in the nucleus. Subcellular localization of the receptors proposed the endoplasmic reticulum (ER) membrane as a principal cytokinin perception site, while study of cytokinin transport pointed to the plasma membrane (PM)-mediated cytokinin signalling. Here, by detailed monitoring of subcellular localizations of the fluorescently labelled natural cytokinin probe and the receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) fused to GFP reporter, we show that pools of the ER-located cytokinin receptors can enter the secretory pathway and reach the PM in cells of the root apical meristem, and the cell plate of dividing meristematic cells. Brefeldin A (BFA) experiments revealed vesicular recycling of the receptor and its accumulation in BFA compartments. We provide a revised view on cytokinin signalling and the possibility of multiple sites of perception at PM and ER.


Assuntos
Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Citocininas/metabolismo , Retículo Endoplasmático/metabolismo , Corantes Fluorescentes/química , Proteínas Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Brefeldina A/farmacologia , Citocininas/química , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Meristema/citologia , Meristema/metabolismo , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos
13.
Phys Rev Lett ; 125(5): 058101, 2020 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-32794890

RESUMO

Diffusion of tracer particles in the cytoplasm of mammalian cells is often anomalous with a marked heterogeneity even within individual particle trajectories. Despite considerable efforts, the mechanisms behind these observations have remained largely elusive. To tackle this problem, we performed extensive single-particle tracking experiments on quantum dots in the cytoplasm of living mammalian cells at varying conditions. Analyses of the trajectories reveal a strong, microtubule-dependent subdiffusion with antipersistent increments and a substantial heterogeneity. Furthermore, particles stochastically switch between different mobility states, most likely due to transient associations with the cytoskeleton-shaken endoplasmic reticulum network. Comparison to simulations highlight that all experimental observations can be fully described by an intermittent fractional Brownian motion, alternating between two states of different mobility.


Assuntos
Citoplasma/metabolismo , Modelos Biológicos , Citoesqueleto de Actina/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Simulação por Computador , Citocalasina D/farmacologia , Citoplasma/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Difusão , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Nocodazol/farmacologia , Pontos Quânticos , Processos Estocásticos , Tiazolidinas/farmacologia
14.
Proc Natl Acad Sci U S A ; 117(29): 17369-17380, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32641503

RESUMO

Voltage-gated L-type Ca2+ channel (Cav1.2) blockers (LCCBs) are major drugs for treating hypertension, the preeminent risk factor for heart failure. Vascular smooth muscle cell (VSMC) remodeling is a pathological hallmark of chronic hypertension. VSMC remodeling is characterized by molecular rewiring of the cellular Ca2+ signaling machinery, including down-regulation of Cav1.2 channels and up-regulation of the endoplasmic reticulum (ER) stromal-interacting molecule (STIM) Ca2+ sensor proteins and the plasma membrane ORAI Ca2+ channels. STIM/ORAI proteins mediate store-operated Ca2+ entry (SOCE) and drive fibro-proliferative gene programs during cardiovascular remodeling. SOCE is activated by agonists that induce depletion of ER Ca2+, causing STIM to activate ORAI. Here, we show that the three major classes of LCCBs activate STIM/ORAI-mediated Ca2+ entry in VSMCs. LCCBs act on the STIM N terminus to cause STIM relocalization to junctions and subsequent ORAI activation in a Cav1.2-independent and store depletion-independent manner. LCCB-induced promotion of VSMC remodeling requires STIM1, which is up-regulated in VSMCs from hypertensive rats. Epidemiology showed that LCCBs are more associated with heart failure than other antihypertensive drugs in patients. Our findings unravel a mechanism of LCCBs action on Ca2+ signaling and demonstrate that LCCBs promote vascular remodeling through STIM-mediated activation of ORAI. Our data indicate caution against the use of LCCBs in elderly patients or patients with advanced hypertension and/or onset of cardiovascular remodeling, where levels of STIM and ORAI are elevated.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Hipertensão/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/metabolismo , Moléculas de Interação Estromal/metabolismo , Remodelação Vascular/fisiologia , Animais , Anti-Hipertensivos/farmacologia , Cálcio/metabolismo , Canais de Cálcio Tipo L/efeitos dos fármacos , Membrana Celular/metabolismo , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Insuficiência Cardíaca , Humanos , Proteínas de Membrana/genética , Miócitos de Músculo Liso , Proteínas de Neoplasias , Proteína ORAI1/genética , Ratos , Molécula 1 de Interação Estromal/genética , Molécula 2 de Interação Estromal/genética
15.
Proc Natl Acad Sci U S A ; 117(32): 19399-19407, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719124

RESUMO

The source proteins from which CD8+ T cell-activating peptides are derived remain enigmatic. Glycoproteins are particularly challenging in this regard owing to several potential trafficking routes within the cell. By engineering a glycoprotein-derived epitope to contain an N-linked glycosylation site, we determined that optimal CD8+ T cell expansion and function were induced by the peptides that are rapidly produced from the exceedingly minor fraction of protein mislocalized to the cytosol. In contrast, peptides derived from the much larger fraction that undergoes translocation and quality control are produced with delayed kinetics and induce suboptimal CD8+ T cell responses. This dual system of peptide generation enhances CD8+ T cell participation in diversifying both antigenicity and the kinetics of peptide display.


Assuntos
Apresentação do Antígeno , Linfócitos T CD8-Positivos/imunologia , Epitopos/imunologia , Epitopos/metabolismo , Animais , Linhagem Celular , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Glicosilação , Antígenos de Histocompatibilidade Classe I/metabolismo , Cinética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/genética , Peptídeos/metabolismo , Sinais Direcionadores de Proteínas/genética
16.
Nat Commun ; 11(1): 3306, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620754

RESUMO

The endoplasmic reticulum (ER) is selectively degraded by autophagy (ER-phagy) through proteins called ER-phagy receptors. In Saccharomyces cerevisiae, Atg40 acts as an ER-phagy receptor to sequester ER fragments into autophagosomes by binding Atg8 on forming autophagosomal membranes. During ER-phagy, parts of the ER are morphologically rearranged, fragmented, and loaded into autophagosomes, but the mechanism remains poorly understood. Here we find that Atg40 molecules assemble in the ER membrane concurrently with autophagosome formation via multivalent interaction with Atg8. Atg8-mediated super-assembly of Atg40 generates highly-curved ER regions, depending on its reticulon-like domain, and supports packing of these regions into autophagosomes. Moreover, tight binding of Atg40 to Atg8 is achieved by a short helix C-terminal to the Atg8-family interacting motif, and this feature is also observed for mammalian ER-phagy receptors. Thus, this study significantly advances our understanding of the mechanisms of ER-phagy and also provides insights into organelle fragmentation in selective autophagy of other organelles.


Assuntos
Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Família da Proteína 8 Relacionada à Autofagia/química , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/química , Proteínas Relacionadas à Autofagia/genética , Sítios de Ligação/genética , Estresse do Retículo Endoplasmático/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Mutação , Ligação Proteica , Domínios Proteicos , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
17.
Nat Commun ; 11(1): 3645, 2020 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-32686675

RESUMO

Endosomes are compositionally dynamic organelles that regulate signaling, nutrient status and organelle quality by specifying whether material entering the cells will be shuttled back to the cell surface or degraded by the lysosome. Recently, membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and endosomes have emerged as important players in endosomal protein sorting, dynamics and motility. Here, we show that PDZD8, a Synaptotagmin-like Mitochondrial lipid-binding Proteins (SMP) domain-containing ER transmembrane protein, utilizes distinct domains to interact with Rab7-GTP and the ER transmembrane protein Protrudin and together these components localize to an ER-late endosome MCS. At these ER-late endosome MCSs, mitochondria are also recruited to form a three-way contact. Thus, our data indicate that PDZD8 is a shared component of two distinct MCSs and suggest a role for SMP-mediated lipid transport in the regulation of endosome function.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Mitocôndrias/metabolismo , Transporte Biológico , Linhagem Celular , Humanos , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Transporte Proteico , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
18.
Nat Commun ; 11(1): 3298, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620747

RESUMO

Communication between organelles is essential for their cellular homeostasis. Neurodegeneration reflects the declining ability of neurons to maintain cellular homeostasis over a lifetime, where the endolysosomal pathway plays a prominent role by regulating protein and lipid sorting and degradation. Here we report that TMEM16K, an endoplasmic reticulum lipid scramblase causative for spinocerebellar ataxia (SCAR10), is an interorganelle regulator of the endolysosomal pathway. We identify endosomal transport as a major functional cluster of TMEM16K in proximity biotinylation proteomics analyses. TMEM16K forms contact sites with endosomes, reconstituting split-GFP with the small GTPase RAB7. Our study further implicates TMEM16K lipid scrambling activity in endosomal sorting at these sites. Loss of TMEM16K function led to impaired endosomal retrograde transport and neuromuscular function, one of the symptoms of SCAR10. Thus, TMEM16K-containing ER-endosome contact sites represent clinically relevant platforms for regulating endosomal sorting.


Assuntos
Anoctaminas/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Animais , Anoctaminas/genética , Transporte Biológico , Células COS , Linhagem Celular Tumoral , Células Cultivadas , Chlorocebus aethiops , Retículo Endoplasmático/ultraestrutura , Endossomos/ultraestrutura , Células HEK293 , Humanos , Metabolismo dos Lipídeos , Lisossomos/ultraestrutura , Camundongos Knockout , Microscopia Eletrônica , Mutação , Transporte Proteico , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo
19.
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
20.
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
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