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1.
J Cell Biol ; 220(8)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34160559

RESUMO

Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here, we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homologue of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT-driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologues in licensing ESCRT-III-dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1-Vps4-ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition.


Assuntos
Adenosina Trifosfatases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Corpos Multivesiculares/enzimologia , Biogênese de Organelas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Adenosina Trifosfatases/genética , Sítios de Ligação , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Ativação Enzimática , Microscopia de Fluorescência , Modelos Moleculares , Corpos Multivesiculares/genética , Mutação , Domínios Proteicos , Transporte Proteico , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/metabolismo , Ubiquitinação
2.
Proc Natl Acad Sci U S A ; 117(18): 9884-9895, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32321832

RESUMO

The factors and mechanisms involved in vacuolar transport in plants, and in particular those directing vesicles to their target endomembrane compartment, remain largely unknown. To identify components of the vacuolar trafficking machinery, we searched for Arabidopsis modified transport to the vacuole (mtv) mutants that abnormally secrete the synthetic vacuolar cargo VAC2. We report here on the identification of 17 mtv mutations, corresponding to mutant alleles of MTV2/VSR4, MTV3/PTEN2A MTV7/EREL1, MTV8/ARFC1, MTV9/PUF2, MTV10/VPS3, MTV11/VPS15, MTV12/GRV2, MTV14/GFS10, MTV15/BET11, MTV16/VPS51, MTV17/VPS54, and MTV18/VSR1 Eight of the MTV proteins localize at the interface between the trans-Golgi network (TGN) and the multivesicular bodies (MVBs), supporting that the trafficking step between these compartments is essential for segregating vacuolar proteins from those destined for secretion. Importantly, the GARP tethering complex subunits MTV16/VPS51 and MTV17/VPS54 were found at endoplasmic reticulum (ER)- and microtubule-associated compartments (EMACs). Moreover, MTV16/VPS51 interacts with the motor domain of kinesins, suggesting that, in addition to tethering vesicles, the GARP complex may regulate the motors that transport them. Our findings unveil a previously uncharacterized compartment of the plant vacuolar trafficking pathway and support a role for microtubules and kinesins in GARP-dependent transport of soluble vacuolar cargo in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Transporte Proteico/genética , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/genética , Alelos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Vesículas Citoplasmáticas/genética , Vesículas Citoplasmáticas/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Cinesinas/genética , Cinesinas/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Corpos Multivesiculares/genética , Corpos Multivesiculares/metabolismo , Mutação , Vacúolos/genética , Proteínas de Transporte Vesicular/metabolismo
3.
Cell Mol Life Sci ; 76(24): 4829-4848, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31363817

RESUMO

Exosomes, a type of small extracellular vesicles (sEVs), are secreted membrane vesicles that are derived from various cell types, including cancer cells, mesenchymal stem cells, and immune cells via multivesicular bodies (MVBs). These sEVs contain RNAs (mRNA, miRNA, lncRNA, and rRNA), lipids, DNA, proteins, and metabolites, all of which mediate cell-to-cell communication. This communication is known to be implicated in a diverse set of diseases such as cancers and their metastases and degenerative diseases. The molecular mechanisms, by which proteins are modified and sorted to sEVs, are not fully understood. Various cellular processes, including degradation, transcription, DNA repair, cell cycle, signal transduction, and autophagy, are known to be associated with ubiquitin and ubiquitin-like proteins (UBLs). Recent studies have revealed that ubiquitin and UBLs also regulate MVBs and protein sorting to sEVs. Ubiquitin-like 3 (UBL3)/membrane-anchored Ub-fold protein (MUB) acts as a post-translational modification (PTM) factor to regulate efficient protein sorting to sEVs. In this review, we focus on the mechanism of PTM by ubiquitin and UBLs and the pathway of protein sorting into sEVs and discuss the potential biological significance of these processes.


Assuntos
Processamento de Proteína Pós-Traducional/genética , Proteínas/genética , Ubiquitina/genética , Ubiquitinas/genética , Autofagia/genética , Exossomos/genética , Vesículas Extracelulares/genética , Humanos , Células-Tronco Mesenquimais/metabolismo , Corpos Multivesiculares/genética , Corpos Multivesiculares/metabolismo , Proteínas/metabolismo , Transdução de Sinais
4.
Proc Natl Acad Sci U S A ; 116(14): 6858-6867, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30894482

RESUMO

The formation of multivesicular endosomes (MVEs) mediates the turnover of numerous integral membrane proteins and has been implicated in the down-regulation of growth factor signaling, thereby exhibiting properties of a tumor suppressor. The endosomal sorting complex required for transport (ESCRT) machinery plays a key role in MVE biogenesis, enabling cargo selection and intralumenal vesicle (ILV) budding. However, the spatiotemporal pattern of endogenous ESCRT complex assembly and disassembly in mammalian cells remains poorly defined. By combining CRISPR/Cas9-mediated genome editing and live cell imaging using lattice light sheet microscopy (LLSM), we determined the native dynamics of both early- and late-acting ESCRT components at MVEs under multiple growth conditions. Specifically, our data indicate that ESCRT-0 accumulates quickly on endosomes, typically in less than 30 seconds, and its levels oscillate in a manner dependent on the downstream recruitment of ESCRT-I. Similarly, levels of the ESCRT-I complex also fluctuate on endosomes, but its average residency time is more than fivefold shorter compared with ESCRT-0. Vps4 accumulation is the most transient, however, suggesting that the completion of ILV formation occurs rapidly. Upon addition of epidermal growth factor (EGF), both ESCRT-I and Vps4 are retained at endosomes for dramatically extended periods of time, while ESCRT-0 dynamics are only modestly affected. Our findings are consistent with a model in which growth factor stimulation stabilizes late-acting components of the ESCRT machinery at endosomes to accelerate the rate of ILV biogenesis and attenuate signal transduction initiated by receptor activation.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Corpos Multivesiculares/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Transformada , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Edição de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Corpos Multivesiculares/genética , Transporte Proteico/fisiologia
5.
Autophagy ; 15(3): 527-542, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30335568

RESUMO

50% of Caucasians carry a Thr300Ala variant (T300A) in the protein encoded by the macroautophagy/autophagy gene ATG16L1. Here, we show that the T300A variant confers protection against urinary tract infections (UTIs), the most common infectious disease in women. Using knockin mice carrying the human T300A variant, we show that the variant limits the UTI-causing bacteria, uropathogenic Escherichia coli (UPEC), from establishing persistent intracellular reservoirs, which can seed UTI recurrence. This phenotype is recapitulated in mice lacking Atg16l1 or Atg7 exclusively in the urothelium. We further show that mice with the T300A variant exhibit urothelial cellular abnormalities, including vesicular congestion and aberrant accumulation of UPK (uroplakin) proteins. Importantly, presence of the T300A variant in humans is associated with similar urothelial architectural abnormalities, indicating an evolutionarily conserved impact. Mechanistically, we show that the reduced bacterial persistence is independent of basal autophagic flux or proinflammatory cytokine responses and does not involve Atg14 or Epg5. However, the T300A variant is associated with increased expression of the small GTPase Rab33b; RAB33B interacts with ATG16L1, as well as other secretory RABs, RAB27B and RAB11A, important for UPEC exocytosis from the urothelium. Finally, inhibition of secretory RABs in bladder epithelial cells increases intracellular UPEC load. Together, our results reveal that UPEC selectively utilize genes important for autophagosome formation to persist in the urothelium, and that the presence of the T300A variant in ATG16L1 is associated with changes in urothelial vesicle trafficking, which disrupts the ability of UPEC to persist, thereby limiting the risk of recurrent UTIs. Abbreviations: 3-PEHPC: 3-pyridinyl ethylidene hydroxyl phosphonocarboxylate; ATG: autophagy; ATG16L1: autophagy related 16 like 1; BECs: bladder epithelial cells; dpi: days post infection; hpi: hours post infection; IF: immunofluorescence; IL1B: interleukin 1 beta; IL6: interleukin 6; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MVB: multivesicular bodies; T300A: Thr300Ala; TNF: tumor necrosis factor; QIR(s): quiescent intracellular reservoir(s); siRNA: short interfering RNA; UPEC: uropathogenic Escherichia coli; UTI(s): urinary tract infection(s); TEM: transmission electron microscopy; WT: wild type.


Assuntos
Autofagia/genética , Infecções por Escherichia coli/metabolismo , Infecções Urinárias/metabolismo , Escherichia coli Uropatogênica , Urotélio/microbiologia , Animais , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Linhagem Celular , Células Epiteliais/metabolismo , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Feminino , Variação Genética , Humanos , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Corpos Multivesiculares/genética , Corpos Multivesiculares/microbiologia , Corpos Multivesiculares/patologia , Bexiga Urinária/microbiologia , Infecções Urinárias/genética , Infecções Urinárias/microbiologia , Infecções Urinárias/patologia , Uroplaquinas/metabolismo , Urotélio/citologia , Urotélio/metabolismo , Urotélio/ultraestrutura , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
6.
Prog Mol Subcell Biol ; 57: 107-135, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30097773

RESUMO

The ubiquitin-dependent degradation of membrane proteins via the multivesicular body (MVB) pathway requires the Endosomal Sorting Complexes Required for Transport (ESCRT). This molecular machinery is composed of five distinct multi-subunit complexes. On the surface of endosomes, ESCRT-0, -I and -II bind to ubiquitinated membrane proteins, while ESCRT-III and Vps4 bud intraluminal vesicles (ILVs) into the lumen of the endosomes. By working together, ESCRTs package membrane proteins into ILVs and thereby generate MVBs. The fusion of mature MVBs with lysosomes delivers ILVs into the lysosomal lumen where the membrane proteins are degraded. Besides generating ILVs, the ESCRT machinery mediates for topologically related membrane budding processes at the plasma membrane and the nuclear envelop. In this chapter, we briefly discuss membrane protein ubiquitination, endocytosis, and summarize current knowledge on the ESCRT machinery in the MVB pathway.


Assuntos
Endocitose/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/genética , Ubiquitinação/genética , ATPases Associadas a Diversas Atividades Celulares/genética , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Proteínas de Membrana/genética , Corpos Multivesiculares/genética , Corpos Multivesiculares/metabolismo , Transporte Proteico/genética , ATPases Vacuolares Próton-Translocadoras/genética
7.
Int J Biol Sci ; 14(6): 633-643, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29904278

RESUMO

Saliva, which contains biological information, is considered a valuable diagnostic tool for local and systemic diseases and conditions because, similar to blood, it contains important molecules like DNA, RNA, and proteins. Exosomes are cell-derived vesicles 30-100 nm in diameter with substantial biological functions, including intracellular communication and signalling. These vesicles, which are present in bodily fluids, including saliva, are released upon fusion of multivesicular bodies (MVBs) with the cellular plasma membrane. Salivary diagnosis has notable advantages, which include noninvasiveness, ease of collection, absence of coagulation, and a similar content as plasma, as well as increased patient compliance compared to other diagnostic approaches. However, investigation of the roles of salivary exosomes is still in its early years. In this review, we first describe the characteristics of endocytosis and secretion of salivary exosomes, as well as database and bioinformatics analysis of exosomes. Then, we describe strategies for the isolation of exosomes from human saliva and the emerging role of salivary exosomes as potential biomarkers of oral and other systemic diseases. Given the ever-growing role of salivary exosomes, defining their functions and understanding their specific mechanisms will provide novel insights into possible applications of salivary exosomes in the diagnosis and treatment of systemic diseases.


Assuntos
Biomarcadores/metabolismo , Exossomos/metabolismo , Saliva/metabolismo , Animais , Exossomos/genética , Genômica , Humanos , Corpos Multivesiculares/genética , Corpos Multivesiculares/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
8.
Cancer Lett ; 414: 107-115, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29107112

RESUMO

Exosomes are a new means of intercellular information exchange that have aroused great research interest. Long neglected in research, exosomes were deemed nonfunctional cellular components to be discarded. However, it has been gradually revealed that exosomes are an important tool for the exchange of intercellular information and material. Exosomes contain specific repertoires of non-coding RNAs (ncRNAs, including microRNA and lncRNA), indicating that a specific RNA sorting mechanism may exist. Correspondingly, intracellular multivesicular bodies (MVBs) are produced after fusion with the cell membrane to release exosomes rather than inducing autophagy, which reveals that there may be a specific regulatory mechanism for MVB secretion. Cells can trigger cancer-related disorders after the recognition and uptake of circulating exosomal ncRNAs, providing indications for early tumor biopsy and treatment. The use of exosomes as a biological carrier in targeted therapy has been demonstrated. However, there may be a specific, unknown switch for loading drugs. This review focuses on the mechanisms of exosome biogenesis, release, and uptake. We also review the promotion of tumor development by exosomal ncRNAs including chemotherapy resistance, metastasis and the prospective use of exosomes in cancer diagnosis and treatment.


Assuntos
Exossomos/genética , MicroRNAs/genética , Neoplasias/genética , RNA Longo não Codificante/genética , Animais , Transporte Biológico , Comunicação Celular , Membrana Celular/genética , Membrana Celular/metabolismo , Humanos , MicroRNAs/metabolismo , Corpos Multivesiculares/genética , Corpos Multivesiculares/metabolismo , Neoplasias/metabolismo , Neoplasias/terapia , RNA Longo não Codificante/metabolismo
9.
PLoS Genet ; 12(9): e1006311, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27618555

RESUMO

Previous studies have shown that multivesicular bodies (MVBs)/endosomes-mediated vesicular trafficking may play key roles in plant immunity and cell death. However, the molecular regulation is poorly understood in rice. Here we report the identification and characterization of a MVBs-localized AAA ATPase LRD6-6 in rice. Disruption of LRD6-6 leads to enhanced immunity and cell death in rice. The ATPase activity and homo-dimerization of LRD6-6 is essential for its regulation on plant immunity and cell death. An ATPase inactive mutation (LRD6-6E315Q) leads to dominant-negative inhibition in plants. The LRD6-6 protein co-localizes with the MVBs marker protein RabF1/ARA6 and interacts with ESCRT-III components OsSNF7 and OsVPS2. Further analysis reveals that LRD6-6 is required for MVBs-mediated vesicular trafficking and inhibits the biosynthesis of antimicrobial compounds. Collectively, our study shows that the AAA ATPase LRD6-6 inhibits plant immunity and cell death most likely through modulating MVBs-mediated vesicular trafficking in rice.


Assuntos
Adenosina Trifosfatases/biossíntese , Imunidade Celular/genética , Corpos Multivesiculares/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Adenosina Trifosfatases/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Morte Celular/genética , Resistência à Doença/genética , Resistência à Doença/imunologia , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/genética , Endossomos/metabolismo , Regulação da Expressão Gênica de Plantas , Corpos Multivesiculares/imunologia , Mutação , Oryza/genética , Oryza/crescimento & desenvolvimento , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Folhas de Planta/genética , Folhas de Planta/imunologia , Plantas Geneticamente Modificadas/imunologia , Transporte Proteico/genética , Proteínas rab de Ligação ao GTP/genética
10.
Mod Pathol ; 29(8): 928-38, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27150162

RESUMO

Exosomes are small membrane vesicles that have important roles in transporting a great variety of bioactive molecules between epithelial compartment and their microenvironment during tumor formation including colorectal adenoma-carcinoma sequence. We tested the mRNA expression of the top 25 exosome-related markers based on ExoCharta database in healthy (n=49), adenoma (n=49) and colorectal carcinoma (n=49) patients using Affymetrix HGU133 Plus2.0 microarrays. Most related genes showed significantly elevated expression including PGK1, PKM, ANXA5, ENO1, HSP90AB1 and MSN during adenoma-carcinoma sequence. Surprisingly, the expression of ALIX (ALG 2-interacting protein X), involved in multivesicular body (MVB) and exosome formation, was significantly reduced in normal vs adenoma (P=5.02 × 10(-13)) and in normal vs colorectal carcinoma comparisons (P=1.51 × 10(-10)). ALIX also showed significant reduction (P<0.05) at the in situ protein level in the epithelial compartment of adenoma (n=35) and colorectal carcinoma (n=37) patients compared with 27 healthy individuals. Furthermore, significantly reduced ALIX protein levels were accompanied by their gradual transition from diffuse cytoplasmic expression to granular signals, which fell into the 0.6-2 µm diameter size range of MVBs. These ALIX-positive particles were seen in the tumor nests, including tumor-stroma border, which suggest their exosome function. MVB-like structures were also detected in tumor microenvironment including α-smooth muscle actin-positive stromal cells, budding off cancer cells in the tumor front as well as in cancer cells entrapped within lymphoid vessels. In conclusion, we determined the top aberrantly expressed exosome-associated markers and revealed the transition of diffuse ALIX protein signals into a MVB-like pattern during adenoma-carcinoma sequence. These tumor-associated particles seen both in the carcinoma and the surrounding microenvironment can potentially mediate epithelial-stromal interactions involved in the regulation of tumor growth, metastatic invasion and therapy response.


Assuntos
Adenoma/química , Biomarcadores Tumorais/análise , Proteínas de Ligação ao Cálcio/análise , Carcinoma/química , Proteínas de Ciclo Celular/análise , Neoplasias Colorretais/química , Complexos Endossomais de Distribuição Requeridos para Transporte/análise , Exossomos/química , Corpos Multivesiculares/química , Adenoma/genética , Adenoma/patologia , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Proteínas de Ligação ao Cálcio/genética , Carcinoma/genética , Carcinoma/patologia , Estudos de Casos e Controles , Proteínas de Ciclo Celular/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Exossomos/genética , Exossomos/patologia , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Corpos Multivesiculares/genética , Corpos Multivesiculares/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Microambiente Tumoral
11.
Elife ; 52016 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-27074665

RESUMO

The endosomal sorting complexes required for transport (ESCRT) pathway facilitates multiple fundamental membrane remodeling events. Previously, we determined X-ray crystal structures of ESCRT-III subunit Snf7, the yeast CHMP4 ortholog, in its active and polymeric state (Tang et al., 2015). However, how ESCRT-III activation is coordinated by the upstream ESCRT components at endosomes remains unclear. Here, we provide a molecular explanation for the functional divergence of structurally similar ESCRT-III subunits. We characterize novel mutations in ESCRT-III Snf7 that trigger activation, and identify a novel role of Bro1, the yeast ALIX ortholog, in Snf7 assembly. We show that upstream ESCRTs regulate Snf7 activation at both its N-terminal core domain and the C-terminus α6 helix through two parallel ubiquitin-dependent pathways: the ESCRT-I-ESCRT-II-Vps20 pathway and the ESCRT-0-Bro1 pathway. We therefore provide an enhanced understanding for the activation of the spatially unique ESCRT-III-mediated membrane remodeling.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Regulação Fúngica da Expressão Gênica , Corpos Multivesiculares/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Corpos Multivesiculares/genética , Corpos Multivesiculares/ultraestrutura , Mutação , Domínios Proteicos , Estrutura Secundária de Proteína , Transporte Proteico , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Proteínas Ubiquitinadas/genética , Proteínas Ubiquitinadas/metabolismo , Ubiquitinação
13.
Cell Death Differ ; 23(1): 99-109, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26045048

RESUMO

Multivesicular bodies (MVBs) are endocytic compartments that enclose intraluminal vesicles (ILVs) formed by inward budding from the limiting membrane of endosomes. In T lymphocytes, these ILV contain Fas ligand (FasL) and are secreted as 'lethal exosomes' following activation-induced fusion of the MVB with the plasma membrane. Diacylglycerol (DAG) and diacylglycerol kinase α (DGKα) regulate MVB maturation and polarized traffic, as well as subsequent secretion of pro-apoptotic exosomes, but the molecular basis underlying these phenomena remains unclear. Here we identify protein kinase D (PKD) family members as DAG effectors involved in MVB genesis and secretion. We show that the inducible secretion of exosomes is enhanced when a constitutively active PKD1 mutant is expressed in T lymphocytes, whereas exosome secretion is impaired in PKD2-deficient mouse T lymphoblasts and in PKD1/3-null B cells. Analysis of PKD2-deficient T lymphoblasts showed the presence of large, immature MVB-like vesicles and demonstrated defects in cytotoxic activity and in activation-induced cell death. Using pharmacological and genetic tools, we show that DGKα regulates PKD1/2 subcellular localization and activation. Our studies demonstrate that PKD1/2 is a key regulator of MVB maturation and exosome secretion, and constitutes a mediator of the DGKα effect on MVB secretory traffic.


Assuntos
Morte Celular/genética , Diacilglicerol Quinase/genética , Proteína Quinase C/genética , Proteínas Quinases/genética , Animais , Linfócitos B/metabolismo , Diacilglicerol Quinase/metabolismo , Exossomos/genética , Exossomos/metabolismo , Proteína Ligante Fas , Camundongos , Corpos Multivesiculares/genética , Corpos Multivesiculares/metabolismo , Proteína Quinase C/metabolismo , Proteína Quinase D2 , Proteínas Quinases/metabolismo , Linfócitos T/metabolismo
14.
Mol Biol Cell ; 26(25): 4700-17, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26490120

RESUMO

Hedgehog (Hh) is a secreted morphogen involved in both short- and long-range signaling necessary for tissue patterning during development. It is unclear how this dually lipidated protein is transported over a long range in the aqueous milieu of interstitial spaces. We previously showed that the long-range signaling of Hh requires its oligomerization. Here we show that Hh is secreted in the form of exovesicles. These are derived by the endocytic delivery of cell surface Hh to multivesicular bodies (MVBs) via an endosomal sorting complex required for transport (ECSRT)-dependent process. Perturbations of ESCRT proteins have a selective effect on long-range Hh signaling in Drosophila wing imaginal discs. Of importance, oligomerization-defective Hh is inefficiently incorporated into exovesicles due to its poor endocytic delivery to MVBs. These results provide evidence that nanoscale organization of Hh regulates the secretion of Hh on ESCRT-derived exovesicles, which in turn act as a vehicle for long-range signaling.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Endocitose/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Proteínas Hedgehog/genética , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas Hedgehog/metabolismo , Discos Imaginais/crescimento & desenvolvimento , Discos Imaginais/metabolismo , Redes e Vias Metabólicas/genética , Corpos Multivesiculares/genética , Corpos Multivesiculares/metabolismo , Transporte Proteico/genética , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismo
15.
J Huazhong Univ Sci Technolog Med Sci ; 34(5): 640-645, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25318871

RESUMO

Microvesicles (MVs) are the heterogeneous mixtures of vesicles. MVs released by leukemia cells constitute an important part of the leukemia microenvironment. MVs might act as important reservoirs of microRNAs (miRNAs). It is worth evaluating whether MVs possess some unique miRNA contents that are valuable in understanding the pathogenesis. In this study, we investigated the miRNA expression patterns of Nalm-6-derived MVs, Jurkat-derived MVs and normal cell-derived MVs using miRNA microarrays. The potential target genes regulated by differentially expressed miRNAs were also predicted and analyzed. Results demonstrated that 182 miRNAs and 166 miRNAs were differentially expressed in Nalm-6-MVs and Jurkat-MVs, respectively. Many oncogenes, tumor suppressors and signal pathway genes were targeted by these aberrantly expressed miRNAs, which might contribute to the development of B-ALL or T-ALL. Our findings expanded the potential diagnostic markers of ALL and provided useful information for ALL pathogenesis.


Assuntos
Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , MicroRNAs/genética , Corpos Multivesiculares/genética , Humanos , Células Jurkat , Análise de Sequência com Séries de Oligonucleotídeos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
Nat Commun ; 5: 3477, 2014 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-24637612

RESUMO

Exosomes are small vesicles that are secreted by cells and act as mediators of cell to cell communication. Because of their potential therapeutic significance, important efforts are being made towards characterizing exosomal contents. However, little is known about the mechanisms that govern exosome biogenesis. We have recently shown that the exosomal protein syntenin supports exosome production. Here we identify the small GTPase ADP ribosylation factor 6 (ARF6) and its effector phospholipase D2 (PLD2) as regulators of syntenin exosomes. ARF6 and PLD2 affect exosomes by controlling the budding of intraluminal vesicles (ILVs) into multivesicular bodies (MVBs). ARF6 also controls epidermal growth factor receptor degradation, suggesting a role in degradative MVBs. Yet ARF6 does not affect HIV-1 budding, excluding general effects on Endosomal Sorting Complexes Required for Transport. Our study highlights a novel pathway controlling ILV budding and exosome biogenesis and identifies an unexpected role for ARF6 in late endosomal trafficking.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Exossomos/metabolismo , Corpos Multivesiculares/metabolismo , Fosfolipase D/metabolismo , Sinteninas/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Receptores ErbB/metabolismo , Exossomos/enzimologia , Exossomos/genética , Infecções por HIV/genética , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos , Corpos Multivesiculares/enzimologia , Corpos Multivesiculares/genética , Fosfolipase D/genética , Transporte Proteico , Sinteninas/genética
17.
J Cell Sci ; 126(Pt 22): 5143-52, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-24006264

RESUMO

Multivesicular endosomes/bodies (MVBs) deliver proteins, such as activated EGF receptor (EGFR), to the lysosome for degradation, and, in pigmented cells, MVBs containing PMEL are an initial stage in melanosome biogenesis. The mechanisms regulating numbers and fate of different populations of MVB are unclear. Here, we focus on the role of the G-protein-coupled receptor OA1 (also known as GPR143), which is expressed exclusively in pigmented cells and mutations in which cause the most common type of ocular albinism. When exogenously expressing PMEL, HeLa cells have been shown to form MVBs resembling early stage melanosomes. To focus on the role of OA1 in the initial stages of melanosome biogenesis we take advantage of the absence of the later stages of melanosome maturation in HeLa cells to determine whether OA1 activity can regulate MVB number and fate. Expression of wild-type but not OA1 mutants carrying inactivating mutations or deletions causes MVB numbers to increase. Whereas OA1 expression has no effect on delivery of EGFR-containing MVBs to the lysosome, it inhibits the lysosomal delivery of PMEL and PMEL-containing MVBs accumulate. We propose that OA1 activity delays delivery of PMEL-containing MVBs to the lysosome to allow time for melanin synthesis and commitment to melanosome biogenesis.


Assuntos
Proteínas do Olho/biossíntese , Lisossomos/metabolismo , Melanossomas/metabolismo , Glicoproteínas de Membrana/biossíntese , Corpos Multivesiculares/metabolismo , Endossomos/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento , Células HeLa , Humanos , Lisossomos/genética , Melanossomas/genética , Glicoproteínas de Membrana/genética , Corpos Multivesiculares/genética , Mutação , Antígeno gp100 de Melanoma/metabolismo
18.
J Exp Bot ; 64(10): 2817-29, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23682115

RESUMO

Arabidopsis thaliana ARA7 (AtRabF2b), a member of the plant Rab5 small GTPases functioning in the vacuolar transport pathway, localizes to pre-vacuolar compartments (PVCs), known as multivesicular bodies (MVBs) in plant cells. Overexpression of the constitutively active GTP-bound mutant of ARA7, ARA7(Q69L), induces the formation of large ring-like structures (1-2 µm in diameter). To better understand the biology of these ARA7(Q69L)-induced ring-like structures, transgenic Arabidopsis cell lines expressing ARA7(Q69L) tagged with green fluorescent protein (GFP) under the control of a heat shock-inducible promoter were generated. In these transgenic cells, robust ring-like structures were formed after 4 h of heat shock induction. Transient co-expression, confocal imaging, and immunogold electron microscopy (immunogold-EM) experiments demonstrated that these GFP-ARA7(Q69L)-labelled ring-like structures were distinct from the Golgi apparatus and trans-Golgi network, but were labelled with an antibody against an MVB marker protein. In addition, live cell imaging and detailed EM analysis showed that the GFP-ARA7(Q69L)-induced spherical structures originated from the homotypic fusion of MVBs. In summary, it was demonstrated that GFP-ARA7(Q69L) expression is an efficient tool for studying PVC/MVB-mediated protein trafficking and vacuolar degradation in plant cells.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Corpos Multivesiculares/enzimologia , Mutação de Sentido Incorreto , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Temperatura Alta , Corpos Multivesiculares/genética , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
19.
J Biol Chem ; 287(52): 43899-908, 2012 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-23105107

RESUMO

The AAA-ATPase Vps4 is critical for function of the multivesicular body sorting pathway, which impacts cellular phenomena ranging from receptor down-regulation to viral budding to cytokinesis. Vps4 activity is stimulated by the interaction between Vta1 and Vps60, but the structural basis for this interaction is unclear. The fragment Vps60(128-186) was reported to display the full activity of Vps60. Vta1 interacts with Vps60 using its N-terminal domain (Vta1NTD). In this work, the structure of Vps60(128-186) in complex with Vta1NTD was determined using NMR techniques, demonstrating a novel recognition mode of the microtubule-interacting and transport (MIT) domain in which Vps60(128-186) interacts with Vta1NTD through helices α4' and α5', extending over Vta1NTD MIT2 domain helices 1-3. The Vps60 binding does not result in Vta1 conformational changes, further revealing the fact that Vps4 ATPase is enhanced by the interaction between Vta1 and Vps60 in an unanticipated manner.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Multiproteicos/química , Corpos Multivesiculares/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Corpos Multivesiculares/genética , Corpos Multivesiculares/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade
20.
Traffic ; 13(1): 9-18, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21752167

RESUMO

It is becoming clear that intracellular signaling events are intimately linked with the membrane transport processes. In addition to the long known role of endocytosis in downregulating plasma membrane receptors, more recent data uncover several sophisticated modes by which endocytosis affects the type and duration of signals. Particularly striking are various roles of endocytic compartments as membrane platforms for compartmentalized assembly or sequestration of specific signaling complexes. Here we review some recent examples illustrating how endosomes may mediate ligand-stimulated apoptotic signaling and how multivesicular bodies affect Wnt signaling by regulated sequestration of signaling molecules or their secretion in exosomes. We also discuss evidence documenting the involvement of endocytic proteins in the regulation of p53 activity and stability, which suggests a possible cross-talk between endocytic processes and transcriptional responses.


Assuntos
Endocitose/fisiologia , Transdução de Sinais/fisiologia , Animais , Exossomos/genética , Exossomos/metabolismo , Exossomos/fisiologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Biológicos , Corpos Multivesiculares/genética , Corpos Multivesiculares/metabolismo , Corpos Multivesiculares/fisiologia , Estabilidade Proteica , Transporte Proteico , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo
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