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1.
Nat Cell Biol ; 22(8): 973-985, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32753672

RESUMO

Autophagy is a homeostatic process with multiple functions in mammalian cells. Here, we show that mammalian Atg8 proteins (mAtg8s) and the autophagy regulator IRGM control TFEB, a transcriptional activator of the lysosomal system. IRGM directly interacted with TFEB and promoted the nuclear translocation of TFEB. An mAtg8 partner of IRGM, GABARAP, interacted with TFEB. Deletion of all mAtg8s or GABARAPs affected the global transcriptional response to starvation and downregulated subsets of TFEB targets. IRGM and GABARAPs countered the action of mTOR as a negative regulator of TFEB. This was suppressed by constitutively active RagB, an activator of mTOR. Infection of macrophages with the membrane-permeabilizing microbe Mycobacterium tuberculosis or infection of target cells by HIV elicited TFEB activation in an IRGM-dependent manner. Thus, IRGM and its interactors mAtg8s close a loop between the autophagosomal pathway and the control of lysosomal biogenesis by TFEB, thus ensuring coordinated activation of the two systems that eventually merge during autophagy.


Assuntos
Família da Proteína 8 Relacionada à Autofagia/fisiologia , Autofagia/fisiologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Calcineurina/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisossomos/fisiologia , Transporte Proteico , Proteínas Qa-SNARE/metabolismo
2.
Proc Natl Acad Sci U S A ; 117(34): 20615-20624, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32778589

RESUMO

Trafficking of photoreceptor membrane proteins from their site of synthesis in the inner segment (IS) to the outer segment (OS) is critical for photoreceptor function and vision. Here we evaluate the role of syntaxin 3 (STX3), in trafficking of OS membrane proteins such as peripherin 2 (PRPH2) and rhodopsin. Photoreceptor-specific Stx3 knockouts [Stx3 f/f(iCre75) and Stx3 f/f(CRX-Cre) ] exhibited rapid, early-onset photoreceptor degeneration and functional decline characterized by structural defects in IS, OS, and synaptic terminals. Critically, in the absence of STX3, OS proteins such as PRPH2, the PRPH2 binding partner, rod outer segment membrane protein 1 (ROM1), and rhodopsin were mislocalized along the microtubules to the IS, cell body, and synaptic region. We find that the PRPH2 C-terminal domain interacts with STX3 as well as other photoreceptor SNAREs, and our findings indicate that STX3 is an essential part of the trafficking pathway for both disc (rhodopsin) and rim (PRPH2/ROM1) components of the OS.


Assuntos
Periferinas/metabolismo , Proteínas Qa-SNARE/metabolismo , Segmento Interno das Células Fotorreceptoras da Retina/metabolismo , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Rodopsina/metabolismo , Animais , Técnicas de Silenciamento de Genes , Camundongos , Células Fotorreceptoras de Vertebrados/fisiologia , Transporte Proteico , Proteínas Qa-SNARE/genética , Segmento Interno das Células Fotorreceptoras da Retina/ultraestrutura , Segmento Externo das Células Fotorreceptoras da Retina/ultraestrutura , Proteínas SNARE/metabolismo
3.
Proc Natl Acad Sci U S A ; 117(35): 21391-21402, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817423

RESUMO

Syntaxin17, a key autophagosomal N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, can associate with ATG8 family proteins SNAP29 and VAMP8 to facilitate the membrane fusion process between the double-membraned autophagosome and single-membraned lysosome in mammalian macroautophagy. However, the inherent properties of Syntaxin17 and the mechanistic basis underlying the interactions of Syntaxin17 with its binding proteins remain largely unknown. Here, using biochemical, NMR, and structural approaches, we systemically characterized Syntaxin17 as well as its interactions with ATG8 family proteins, SNAP29 and VAMP8. We discovered that Syntaxin17 alone adopts an autoinhibited conformation mediated by a direct interaction between its Habc domain and the Qa-SNARE motif. In addition, we revealed that the Qa-SNARE region of Syntaxin17 contains one LC3-interacting region (LIR) motif, which preferentially binds to GABARAP subfamily members. Importantly, the GABARAP binding of Syntaxin17 can release its autoinhibited state. The determined crystal structure of the Syntaxin17 LIR-GABARAP complex not only provides mechanistic insights into the interaction between Syntaxin17 and GABARAP but also reveals an unconventional LIR motif with a C-terminally extended 310 helix for selectively binding to ATG8 family proteins. Finally, we also elucidated structural arrangements of the autophagic Syntaxin17-SNAP29-VAMP8 SNARE core complex, and uncovered its conserved biochemical and structural characteristics common to all other SNAREs. In all, our findings reveal three distinct states of Syntaxin17, and provide mechanistic insights into the Syntaxin17-mediated autophagosome-lysosome fusion process.


Assuntos
Autofagossomos/fisiologia , Lisossomos/fisiologia , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Motivos de Aminoácidos , Proteínas Reguladoras de Apoptose/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Escherichia coli , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo
4.
Nat Commun ; 11(1): 2779, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32487999

RESUMO

T cell receptor (TCR) activation is modulated by mechanisms such as TCR endocytosis, which is thought to terminate TCR signalling. Here we show that, upon internalization, TCR continues to signal from a set of specialized endosomes that are crucial for T cell functions. Mechanistically, TCR ligation leads to clathrin-mediated internalization of the TCR-CD3ζ complex, while maintaining CD3ζ signalling, in endosomal vesicles that contain the insulin responsive aminopeptidase (IRAP) and the SNARE protein Syntaxin 6. Destabilization of this compartment through IRAP deletion enhances plasma membrane expression of the TCR-CD3ζ complex, yet compromises overall CD3ζ signalling; moreover, the integrity of this compartment is also crucial for T cell activation and survival after suboptimal TCR activation, as mice engineered with a T cell-specific deletion of IRAP fail to develop efficient polyclonal anti-tumour responses. Our results thus reveal a previously unappreciated function of IRAP-dependent endosomal TCR signalling in T cell activation.


Assuntos
Cistinil Aminopeptidase/metabolismo , Endossomos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/fisiologia , Linfócitos T/metabolismo , Animais , Membrana Celular/metabolismo , Proliferação de Células , Clatrina/metabolismo , Cistinil Aminopeptidase/genética , Modelos Animais de Doenças , Endocitose/fisiologia , Células HEK293 , Humanos , Interleucina-2/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Qa-SNARE/metabolismo , Transcriptoma
5.
Hum Cell ; 33(3): 810-818, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32274658

RESUMO

Circular RNAs (circRNAs) exert pivotal effects on regulating the progression of osteosarcoma (OS). It was found through microarray analysis that circ-0002052 is abnormally expressed in OS, but the role of circ-0002052 in OS remains obscure. The results of this research manifested that relative to that in non-tumor controls, circ-0002052 level was raised in OS tissues. Up-regulated circ-0002052 was associated with advanced stage, tumor size, and metastasis. Additionally, circ-0002052 elevation indicated a low survival rate in OS patients and silencing of circ-0002052 suppressed proliferation, migration, and invasion of OS cells. It was proved that circ-0002052 sponged miR-382 and stimulated STX6 expression, thus activating Wnt/ß-catenin. The function of circ-0002052 reduction in OS cells was effectively reversed by miR-382 suppression. To sum up, it can be concluded that circ-0002052, functioning as a sponge for miR-382, enhances the activation of Wnt/ß-catenin mediated by STX6 to stimulate the progression of OS, and circ-0002052 may be an underlying treatment target and a biomarker for prognosis of osteosarcoma.


Assuntos
Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Osteossarcoma/genética , Osteossarcoma/metabolismo , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , RNA Circular/fisiologia , Linhagem Celular Tumoral , Humanos , Terapia de Alvo Molecular , Osteossarcoma/diagnóstico , Osteossarcoma/terapia , RNA Circular/genética , RNA Circular/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
6.
J Virol ; 94(12)2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32269127

RESUMO

Positive-strand RNA [(+)RNA] viruses assemble numerous membrane-bound viral replicase complexes (VRCs) with the help of viral replication proteins and co-opted host proteins within large viral replication compartments in the cytosol of infected cells. In this study, we found that deletion or depletion of Sac1 phosphatidylinositol 4-phosphate [PI(4)P] phosphatase reduced tomato bushy stunt virus (TBSV) replication in yeast (Saccharomyces cerevisiae) and plants. We demonstrate a critical role for Sac1 in TBSV replicase assembly in a cell-free replicase reconstitution assay. The effect of Sac1 seems to be direct, based on its interaction with the TBSV p33 replication protein, its copurification with the tombusvirus replicase, and its presence in the virus-induced membrane contact sites and within the TBSV replication compartment. The proviral functions of Sac1 include manipulation of lipid composition, sterol enrichment within the VRCs, and recruitment of additional host factors into VRCs. Depletion of Sac1 inhibited the recruitment of Rab5 GTPase-positive endosomes and enrichment of phosphatidylethanolamine in the viral replication compartment. We propose that Sac1 might be a component of the assembly hub for VRCs, likely in collaboration with the co-opted the syntaxin18-like Ufe1 SNARE protein within the TBSV replication compartments. This work also led to demonstration of the enrichment of PI(4)P phosphoinositide within the replication compartment. Reduction in the PI(4)P level due to chemical inhibition in plant protoplasts; depletion of two PI(4)P kinases, Stt4p and Pik1p; or sequestration of free PI(4)P via expression of a PI(4)P-binding protein in yeast strongly inhibited TBSV replication. Altogether, Sac1 and PI(4)P play important proviral roles during TBSV replication.IMPORTANCE Replication of positive-strand RNA viruses depends on recruitment of host components into viral replication compartments or organelles. Using TBSV, we uncovered the critical roles of Sac1 PI(4)P phosphatase and its substrate, PI(4)P phosphoinositide, in promoting viral replication. Both Sac1 and PI(4)P are recruited to the site of viral replication to facilitate the assembly of the viral replicase complexes, which perform viral RNA replication. We found that Sac1 affects the recruitment of other host factors and enrichment of phosphatidylethanolamine and sterol lipids within the subverted host membranes to promote optimal viral replication. In summary, this work demonstrates the novel functions of Sac1 and PI(4)P in TBSV replication in the model host yeast and in plants.


Assuntos
Interações Hospedeiro-Patógeno/genética , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Tombusvirus/genética , Replicação Viral/genética , 1-Fosfatidilinositol 4-Quinase/genética , 1-Fosfatidilinositol 4-Quinase/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/virologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Endossomos/metabolismo , Regulação da Expressão Gênica , Fosfatidiletanolaminas/genética , Fosfatidiletanolaminas/metabolismo , Monoéster Fosfórico Hidrolases/deficiência , Monoéster Fosfórico Hidrolases/metabolismo , Células Vegetais/metabolismo , Células Vegetais/virologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/virologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Protoplastos/metabolismo , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , /metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/virologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Esteróis/metabolismo , Tabaco/genética , Tabaco/metabolismo , Tabaco/virologia , Tombusvirus/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo
7.
Nat Commun ; 11(1): 1127, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32111841

RESUMO

Although viruses must navigate the complex host endomembrane system to infect cells, the strategies used to achieve this is unclear. During entry, polyomavirus SV40 is sorted from the late endosome (LE) to the endoplasmic reticulum (ER) to cause infection, yet how this is accomplished remains enigmatic. Here we find that EMC4 and EMC7, two ER membrane protein complex (EMC) subunits, support SV40 infection by promoting LE-to-ER targeting of the virus. They do this by engaging LE-associated Rab7, presumably to stabilize contact between the LE and ER. These EMC subunits also bind to the ER-resident fusion machinery component syntaxin18, which is required for SV40-arrival to the ER. Our data suggest that EMC4 and EMC7 act as molecular tethers, inter-connecting two intracellular compartments to enable efficient transport of a virus between these compartments. As LE-to-ER transport of cellular cargos is unclear, our results have broad implications for illuminating inter-organelle cargo transport.


Assuntos
Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Internalização do Vírus , Animais , Sítios de Ligação , Células COS , Linhagem Celular , Chlorocebus aethiops , Retículo Endoplasmático/virologia , Endossomos/metabolismo , Endossomos/virologia , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Membranas Intracelulares/virologia , Proteínas de Membrana/genética , Ligação Proteica , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Vírus 40 dos Símios/fisiologia , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
8.
Sci Rep ; 10(1): 2907, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32076023

RESUMO

Reconstitution assays with proteoliposomes provide a powerful tool to elucidate the mechanism of neurotransmitter release, but it is important to understand how these assays report on membrane fusion, and recent studies with yeast vacuolar SNAREs uncovered asymmetry in the results of lipid mixing assays. We have investigated whether such asymmetry also occurs in reconstitution assays with the neuronal SNAREs, using syntaxin-1-SNAP-25-containing liposomes and liposomes containing synaptobrevin (T and V liposomes, respectively), and fluorescent probes to monitor lipid and content mixing simultaneously. Switching the fluorescent probes placed on the T and V liposomes, we observed a striking asymmetry in both lipid and content mixing stimulated by a fragment spanning the two C2 domains of synaptotagmin-1, or by a peptide that spans the C-terminal half of the synaptobrevin SNARE motif. However, no such asymmetry was observed in assays performed in the presence of Munc18-1, Munc13-1, NSF and αSNAP, which coordinate the assembly-disassembly cycle of neuronal SNARE complexes. Our results show that switching fluorescent probes between the two types of liposomes provides a useful approach to better understand the reactions that occur between liposomes and detect heterogenous behavior in these reactions.


Assuntos
Bioensaio/métodos , Lipídeos/química , Neurônios/metabolismo , Proteolipídeos/metabolismo , Proteínas SNARE/metabolismo , Animais , Bovinos , Cricetulus , Transferência Ressonante de Energia de Fluorescência , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Ratos
9.
Nat Chem Biol ; 16(3): 327-336, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32080624

RESUMO

The retrograde transport inhibitor Retro-2 has a protective effect on cells and in mice against Shiga-like toxins and ricin. Retro-2 causes toxin accumulation in early endosomes and relocalization of the Golgi SNARE protein syntaxin-5 to the endoplasmic reticulum. The molecular mechanisms by which this is achieved remain unknown. Here, we show that Retro-2 targets the endoplasmic reticulum exit site component Sec16A, affecting anterograde transport of syntaxin-5 from the endoplasmic reticulum to the Golgi. The formation of canonical SNARE complexes involving syntaxin-5 is not affected in Retro-2-treated cells. By contrast, the interaction of syntaxin-5 with a newly discovered binding partner, the retrograde trafficking chaperone GPP130, is abolished, and we show that GPP130 must indeed bind to syntaxin-5 to drive Shiga toxin transport from the endosomes to the Golgi. We therefore identify Sec16A as a druggable target and provide evidence for a non-SNARE function for syntaxin-5 in interaction with GPP130.


Assuntos
Benzamidas/metabolismo , Proteínas Qa-SNARE/metabolismo , Tiofenos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Benzamidas/farmacologia , Transporte Biológico , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Transporte Proteico , Ricina/metabolismo , Toxina Shiga/metabolismo , Toxinas Shiga/metabolismo , Tiofenos/farmacologia , Proteínas de Transporte Vesicular/fisiologia
10.
Sci Rep ; 10(1): 709, 2020 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-31959797

RESUMO

Recent evidence suggests that SNARE fusion machinery play critical roles in postsynaptic neurotransmitter receptor trafficking, which is essential for synaptic plasticity. However, the key SNAREs involved remain highly controversial; syntaxin-3 and syntaxin-4 are leading candidates for the syntaxin isoform underlying postsynaptic plasticity. In a previous study, we showed that pyramidal-neuron specific conditional knockout (cKO) of syntaxin-4 significantly reduces basal transmission, synaptic plasticity and impairs postsynaptic receptor trafficking. However, this does not exclude a role for syntaxin-3 in such processes. Here, we generated and analyzed syntaxin-3 cKO mice. Extracellular field recordings in hippocampal slices showed that syntaxin-3 cKO did not exhibit significant changes in CA1 basal neurotransmission or in paired-pulse ratios. Importantly, there were no observed differences during LTP in comparison to control mice. Syntaxin-3 cKO mice performed similarly as the controls in spatial and contextual learning tasks. Consistent with the minimal effects of syntaxin-3 cKO, syntaxin-3 mRNA level was very low in hippocampal and cortex pyramidal neurons, but strongly expressed in the corpus callosum and caudate axon fibers. Together, our data suggest that syntaxin-3 is dispensable for hippocampal basal neurotransmission and synaptic plasticity, and further supports the notion that syntaxin-4 is the major isoform mediating these processes.


Assuntos
Região CA1 Hipocampal/fisiologia , Plasticidade Neuronal/genética , Plasticidade Neuronal/fisiologia , Proteínas Qa-SNARE/fisiologia , Transmissão Sináptica/genética , Transmissão Sináptica/fisiologia , Animais , Região CA1 Hipocampal/metabolismo , Corpo Caloso/metabolismo , Expressão Gênica , Técnicas In Vitro , Potenciação de Longa Duração/fisiologia , Camundongos Knockout , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , RNA Mensageiro/metabolismo
11.
Proc Natl Acad Sci U S A ; 117(2): 1036-1041, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31888993

RESUMO

Munc13-1 is a large multifunctional protein essential for synaptic vesicle fusion and neurotransmitter release. Its dysfunction has been linked to many neurological disorders. Evidence suggests that the MUN domain of Munc13-1 collaborates with Munc18-1 to initiate SNARE assembly, thereby priming vesicles for fast calcium-triggered vesicle fusion. The underlying molecular mechanism, however, is poorly understood. Recently, it was found that Munc18-1 catalyzes neuronal SNARE assembly through an obligate template complex intermediate containing Munc18-1 and 2 SNARE proteins-syntaxin 1 and VAMP2. Here, using single-molecule force spectroscopy, we discovered that the MUN domain of Munc13-1 stabilizes the template complex by ∼2.1 kBT. The MUN-bound template complex enhances SNAP-25 binding to the templated SNAREs and subsequent full SNARE assembly. Mutational studies suggest that the MUN-bound template complex is functionally important for SNARE assembly and neurotransmitter release. Taken together, our observations provide a potential molecular mechanism by which Munc13-1 and Munc18-1 cooperatively chaperone SNARE folding and assembly, thereby regulating synaptic vesicle fusion.


Assuntos
Chaperonas Moleculares/metabolismo , Proteínas Munc18/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas SNARE/metabolismo , Exocitose/fisiologia , Cinética , Fusão de Membrana/fisiologia , Chaperonas Moleculares/química , Proteínas Munc18/química , Proteínas do Tecido Nervoso/química , Neurônios/metabolismo , Pinças Ópticas , Ligação Proteica , Domínios Proteicos , Proteínas Qa-SNARE/metabolismo , Proteínas SNARE/química , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Proteína 25 Associada a Sinaptossoma/química , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo
12.
Mol Carcinog ; 59(1): 62-72, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31674708

RESUMO

Prostate cancer (PCa) deaths are typically the result of metastatic castration-resistant PCa (mCRPC). Recently, enzalutamide (Enz), an oral androgen receptor inhibitor, was approved for treating patients with mCRPC. Invariably, all PCa patients eventually develop resistance against Enz. Therefore, novel strategies aimed at overcoming Enz resistance are needed to improve the survival of PCa patients. The role of exosomes in drug resistance has not been fully elucidated in PCa. Therefore, we set out to better understand the exosome's role in the mechanism underlying Enz-resistant PCa. Results showed that Enz-resistant PCa cells (C4-2B, CWR-R1, and LNCaP) secreted significantly higher amounts of exosomes (2-4 folds) compared to Enz-sensitive counterparts. Inhibition of exosome biogenesis in resistant cells by GW4869 and dimethyl amiloride strongly decreased their cell viability. Mechanistic studies revealed upregulation of syntaxin 6 as well as its increased colocalization with CD63 in Enz-resistant PCa cells compared to Enz-sensitive cells. Syntaxin 6 knockdown by specific small interfering RNAs in Enz-resistant PCa cells (C4-2B and CWR-R1) resulted in reduced cell number and increased cell death in the presence of Enz. Furthermore, syntaxin 6 knockdown significantly reduced the exosome secretion in both Enz-resistant C4-2B and CWR-R1 cells. The Cancer Genome Atlas analysis showed increased syntaxin 6 expressions associated with higher Gleason score and decreased progression-free survival in PCa patients. Importantly, IHC analysis showed higher syntaxin 6 expression in cancer tissues from Enz-treated patients compared to Enz naïve patients. Overall, syntaxin 6 plays an important role in the secretion of exosomes and increased survival of Enz-resistant PCa cells.


Assuntos
Antineoplásicos/farmacologia , Exossomos/metabolismo , Feniltioidantoína/análogos & derivados , Neoplasias da Próstata/tratamento farmacológico , Proteínas Qa-SNARE/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Exossomos/efeitos dos fármacos , Humanos , Masculino , Feniltioidantoína/farmacologia , Neoplasias da Próstata/metabolismo
13.
J Cell Biol ; 219(1)2020 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-31825461

RESUMO

The surfacing of the glucose transporter GLUT4 driven by insulin receptor activation provides the prototypic example of a homeostasis response dependent on mobilization of an intracellular storage compartment. Here, we generalize this concept to a G protein-coupled receptor, somatostatin receptor subtype 2 (SSTR2), in pituitary cells. Following internalization in corticotropes, SSTR2 moves to a juxtanuclear syntaxin-6-positive compartment, where it remains until the corticotropes are stimulated with corticotropin releasing factor (CRF), whereupon SSTR2 exits the compartment on syntaxin-6-positive vesicular/tubular carriers that depend on Rab10 for their fusion with the plasma membrane. As SSTR2 activation antagonizes CRF-mediated hormone release, this storage/resurfacing mechanism may allow for a physiological homeostatic feedback system. In fact, we find that SSTR2 moves from an intracellular compartment to the cell surface in pituitary gland somatotropes, concomitant with increasing levels of serum growth hormone (GH) during natural GH cycles. Our data thus provide a mechanism by which signaling-mediated plasma membrane resurfacing of SSTR2 can fine-tune pituitary hormone release.


Assuntos
Corticotrofos/metabolismo , Hormônio do Crescimento Humano/metabolismo , Hipófise/metabolismo , Proteínas Qa-SNARE/metabolismo , Receptores de Somatostatina/metabolismo , Proteínas rab de Ligação ao GTP/fisiologia , Animais , Hormônio Liberador da Corticotropina , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hipófise/citologia , Proteínas Qa-SNARE/genética , Receptores de Somatostatina/genética , Transdução de Sinais
14.
Cells ; 8(12)2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31861136

RESUMO

Syntaxin 16, a Qa-SNARE (soluble N-ethylmaleimide-sensitive factor activating protein receptor), is involved in a number of membrane-trafficking activities, particularly transport processes at the trans-Golgi network (TGN). Recent works have now implicated syntaxin 16 in the autophagy process. In fact, syntaxin 16 appears to have dual roles, firstly in facilitating the transport of ATG9a-containing vesicles to growing autophagosomes, and secondly in autolysosome formation. The former involves a putative SNARE complex between syntaxin 16, VAMP7 and SNAP-47. The latter occurs via syntaxin 16's recruitment by Atg8/LC3/GABARAP family proteins to autophagosomes and endo-lysosomes, where syntaxin 16 may act in a manner that bears functional redundancy with the canonical autophagosome Qa-SNARE syntaxin 17. Here, I discuss these recent findings and speculate on the mechanistic aspects of syntaxin 16's newly found role in autophagy.


Assuntos
Autofagia/fisiologia , Sintaxina 16/metabolismo , Animais , Autofagossomos/metabolismo , Autofagossomos/fisiologia , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Transporte/metabolismo , Humanos , Lisossomos/metabolismo , Fusão de Membrana/fisiologia , Proteínas de Membrana/metabolismo , Transporte Proteico/fisiologia , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Sintaxina 16/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Rede trans-Golgi/metabolismo , Rede trans-Golgi/fisiologia
15.
Cell Rep ; 29(12): 3958-3973.e7, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31851926

RESUMO

Salmonella is a human and animal pathogen that causes gastro-enteric diseases. The key to Salmonella infection is its entry into intestinal epithelial cells, where the bacterium resides within a Salmonella-containing vacuole (SCV). Salmonella entry also induces the formation of empty macropinosomes, distinct from the SCV, in the vicinity of the entering bacteria. A few minutes after its formation, the SCV increases in size through fusions with the surrounding macropinosomes. Salmonella also induces membrane tubules that emanate from the SCV and lead to SCV shrinkage. Here, we show that these antipodal events are utilized by Salmonella to either establish a vacuolar niche or to be released into the cytosol by SCV rupture. We identify the molecular machinery underlying dynamic SCV growth and shrinkage. In particular, the SNARE proteins SNAP25 and STX4 participate in SCV inflation by fusion with macropinosomes. Thus, host compartment size control emerges as a pathogen strategy for intracellular niche regulation.


Assuntos
Citosol/patologia , Proteínas Qa-SNARE/metabolismo , Infecções por Salmonella/patologia , Salmonella typhimurium/crescimento & desenvolvimento , Proteína 25 Associada a Sinaptossoma/metabolismo , Vacúolos/patologia , Células CACO-2 , Citosol/metabolismo , Citosol/microbiologia , Células HeLa , Humanos , Proteínas Qa-SNARE/genética , Infecções por Salmonella/metabolismo , Infecções por Salmonella/microbiologia , Salmonella typhimurium/metabolismo , Proteína 25 Associada a Sinaptossoma/genética , Vacúolos/metabolismo , Vacúolos/microbiologia
16.
J Biol Chem ; 294(50): 19119-19136, 2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31694913

RESUMO

Mutations in the centrosomal protein 290 (CEP290) gene cause various ciliopathies involving retinal degeneration. CEP290 proteins localize to the ciliary transition zone and are thought to act as a gatekeeper that controls ciliary protein trafficking. However, precise roles of CEP290 in photoreceptors and pathomechanisms of retinal degeneration in CEP290-associated ciliopathies are not sufficiently understood. Using conditional Cep290 mutant mice, in which the C-terminal myosin-tail homology domain of CEP290 is disrupted after the connecting cilium is assembled, we show that this domain is essential for protein confinement between the inner and the outer segments. Upon disruption of the myosin-tail homology domain, inner segment plasma membrane proteins, including syntaxin 3 (STX3), synaptosome-associated protein 25 (SNAP25), and interphotoreceptor matrix proteoglycan 2 (IMPG2), rapidly accumulated in the outer segment. In contrast, localization of endomembrane proteins was not altered. Trafficking and confinement of most outer segment-resident proteins appeared to be unaffected or only minimally affected in Cep290 mutant mice. One notable exception was rhodopsin (RHO), which severely mislocalized to inner segments during the initial stage of degeneration. Similar mislocalization phenotypes were observed in Cep290rd16 mice. These results suggest that a failure of protein confinement at the connecting cilium and consequent accumulation of inner segment membrane proteins in the outer segment, along with insufficient RHO delivery, is part of the disease mechanisms that cause retinal degeneration in CEP290-associated ciliopathies. Our study provides insights into the pathomechanisms of retinal degenerations associated with compromised ciliary gates.


Assuntos
Antígenos de Neoplasias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Miosinas/metabolismo , Células Fotorreceptoras/metabolismo , Proteoglicanas/metabolismo , Proteínas Qa-SNARE/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Animais , Antígenos de Neoplasias/genética , Proteínas de Ciclo Celular/genética , Células Cultivadas , Cílios/metabolismo , Cílios/patologia , Proteínas do Citoesqueleto/genética , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Mutação
17.
Cells ; 8(11)2019 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-31671609

RESUMO

Cancer cells modulate their metabolism to proliferate and survive under the metabolic stress condition, which is known as endoplasmic reticulum (ER) stress. Therefore, cancer cells should suppress ER stress-mediated cell death and induce autophagy-which recycles metabolites to provide energy and new macromolecules. In this study, we demonstrate that the ER membrane protein BAP31 acts to suppress adaptation to ER stress conditions, induce cell death, and suppress autophagy by forming a BAP31-STX17 protein complex. The loss of BAP31 stimulates tumor growth in metabolic stress conditions in vivo and enhances invasion activity. Therefore, BAP31 stimulates cell death and inhibits autophagy, and it can be considered a novel tumor suppressor factor that acts by preventing ER stress adaptation.


Assuntos
Autofagia , Neoplasias Ósseas/patologia , Estresse do Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Osteossarcoma/patologia , Proteínas Qa-SNARE/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Apoptose , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Osteossarcoma/genética , Osteossarcoma/metabolismo , Proteínas Qa-SNARE/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Biochem J ; 476(20): 3081-3107, 2019 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-31665227

RESUMO

The serum- and glucocorticoid-regulated kinase (SGK) isoforms contribute resistance to cancer therapies targeting the PI3K pathway. SGKs are homologous to Akt and these kinases display overlapping specificity and phosphorylate several substrates at the same residues, such as TSC2 to promote tumor growth by switching on the mTORC1 pathway. The SGK3 isoform is up-regulated in breast cancer cells treated with PI3K or Akt inhibitors and recruited and activated at endosomes, through its phox homology domain binding to PtdIns(3)P. We undertook genetic and pharmacological phosphoproteomic screens to uncover novel SGK3 substrates. We identified 40 potential novel SGK3 substrates, including four endosomal proteins STX7 (Ser126) and STX12 (Ser139), RFIP4 (Ser527) and WDR44 (Ser346) that were efficiently phosphorylated in vitro by SGK3 at the sites identified in vivo, but poorly by Akt. We demonstrate that these substrates are inefficiently phosphorylated by Akt as they possess an n + 1 residue from the phosphorylation site that is unfavorable for Akt phosphorylation. Phos-tag analysis revealed that stimulation of HEK293 cells with IGF1 to activate SGK3, promoted phosphorylation of a significant fraction of endogenous STX7 and STX12, in a manner that was blocked by knock-out of SGK3 or treatment with a pan SGK inhibitor (14H). SGK3 phosphorylation of STX12 enhanced interaction with the VAMP4/VTI1A/STX6 containing the SNARE complex and promoted plasma membrane localization. Our data reveal novel substrates for SGK3 and suggest a mechanism by which STX7 and STX12 SNARE complexes are regulated by SGK3. They reveal new biomarkers for monitoring SGK3 pathway activity.


Assuntos
Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Endossomos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Qa-SNARE/metabolismo , Biomarcadores/metabolismo , Membrana Celular/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Qa-SNARE/genética , Especificidade por Substrato , Transfecção
19.
EMBO J ; 38(22): e101994, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31625181

RESUMO

Mammalian homologs of yeast Atg8 protein (mAtg8s) are important in autophagy, but their exact mode of action remains ill-defined. Syntaxin 17 (Stx17), a SNARE with major roles in autophagy, was recently shown to bind mAtg8s. Here, we identified LC3-interacting regions (LIRs) in several SNAREs that broaden the landscape of the mAtg8-SNARE interactions. We found that Syntaxin 16 (Stx16) and its cognate SNARE partners all have LIR motifs and bind mAtg8s. Knockout of Stx16 caused defects in lysosome biogenesis, whereas a Stx16 and Stx17 double knockout completely blocked autophagic flux and decreased mitophagy, pexophagy, xenophagy, and ribophagy. Mechanistic analyses revealed that mAtg8s and Stx16 control several properties of lysosomal compartments including their function as platforms for active mTOR. These findings reveal a broad direct interaction of mAtg8s with SNAREs with impact on membrane remodeling in eukaryotic cells and expand the roles of mAtg8s to lysosome biogenesis.


Assuntos
Autofagossomos/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Autofagia , Lisossomos/metabolismo , Proteínas Qa-SNARE/metabolismo , Sintaxina 16/metabolismo , Motivos de Aminoácidos , Família da Proteína 8 Relacionada à Autofagia/genética , Células HEK293 , Células HeLa , Humanos , Redes e Vias Metabólicas , Ligação Proteica , Domínios Proteicos , Proteínas Qa-SNARE/antagonistas & inibidores , Proteínas Qa-SNARE/genética , RNA Interferente Pequeno/genética , Sintaxina 16/antagonistas & inibidores , Sintaxina 16/genética
20.
Plant Cell ; 31(12): 3015-3032, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31597687

RESUMO

Plant phospholipase Ds (PLDs), essential regulators of phospholipid signaling, function in multiple signal transduction cascades; however, the mechanisms regulating PLDs in response to pathogens remain unclear. Here, we found that Arabidopsis (Arabidopsis thaliana) PLDδ accumulated in cells at the entry sites of the barley powdery mildew fungus, Blumeria graminis f. sp hordei Using fluorescence recovery after photobleaching and single-molecule analysis, we observed higher PLDδ density in the plasma membrane after chitin treatment; PLDδ also underwent rapid exocytosis. Fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy showed that the interaction between PLDδ and the microdomain marker AtREMORIN1.3 (AtREM1.3) increased in response to chitin, indicating that exocytosis facilitates rapid, efficient sorting of PLDδ into microdomains upon pathogen stimulus. We further unveiled a trade-off between brefeldin A (BFA)-resistant and -sensitive pathways in secretion of PLDδ under diverse conditions. Upon pathogen attack, PLDδ secretion involved syntaxin-associated VAMP721/722-mediated exocytosis sensitive to BFA. Analysis of phosphatidic acid (PA), hydrogen peroxide, and jasmonic acid (JA) levels and expression of related genes indicated that the relocalization of PLDδ is crucial for its activation to produce PA and initiate reactive oxygen species and JA signaling pathways. Together, our findings revealed that the translocation of PLDδ to papillae is modulated by exocytosis, thus triggering PA-mediated signaling in plant innate immunity.plantcell;31/12/3015/FX1F1fx1.


Assuntos
Arabidopsis/imunologia , Membrana Celular/metabolismo , Imunidade Inata , Fosfolipase D/metabolismo , Doenças das Plantas/imunologia , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/metabolismo , Ascomicetos/patogenicidade , Brefeldina A/imunologia , Brefeldina A/metabolismo , Quitina/imunologia , Quitina/metabolismo , Ciclopentanos/metabolismo , Exocitose/efeitos dos fármacos , Exocitose/imunologia , Peróxido de Hidrogênio/metabolismo , Imunidade Inata/efeitos dos fármacos , Oxilipinas/metabolismo , Ácidos Fosfatídicos/metabolismo , Fosfolipase D/genética , Doenças das Plantas/microbiologia , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Transdução de Sinais/imunologia , Transdução de Sinais/fisiologia
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