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1.
Nat Commun ; 11(1): 5559, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33144569

RESUMO

Cholesterol import in mammalian cells is mediated by the LDL receptor pathway. Here, we perform a genome-wide CRISPR screen using an endogenous cholesterol reporter and identify >100 genes involved in LDL-cholesterol import. We characterise C18orf8 as a core subunit of the mammalian Mon1-Ccz1 guanidine exchange factor (GEF) for Rab7, required for complex stability and function. C18orf8-deficient cells lack Rab7 activation and show severe defects in late endosome morphology and endosomal LDL trafficking, resulting in cellular cholesterol deficiency. Unexpectedly, free cholesterol accumulates within swollen lysosomes, suggesting a critical defect in lysosomal cholesterol export. We find that active Rab7 interacts with the NPC1 cholesterol transporter and licenses lysosomal cholesterol export. This process is abolished in C18orf8-, Ccz1- and Mon1A/B-deficient cells and restored by a constitutively active Rab7. The trimeric Mon1-Ccz1-C18orf8 (MCC) GEF therefore plays a central role in cellular cholesterol homeostasis coordinating Rab7 activation, endosomal LDL trafficking and NPC1-dependent lysosomal cholesterol export.


Assuntos
Colesterol/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/metabolismo , Multimerização Proteica , Proteínas rab de Ligação ao GTP/metabolismo , Transporte Biológico , Sistemas CRISPR-Cas/genética , LDL-Colesterol/metabolismo , Endossomos/metabolismo , Endossomos/ultraestrutura , Corantes Fluorescentes/metabolismo , Genoma Humano , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Células HeLa , Homeostase , Humanos , Hidroximetilglutaril-CoA Sintase/metabolismo , Lisossomos/ultraestrutura , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Ligação Proteica
2.
Cell ; 183(6): 1520-1535.e14, 2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-33157038

RESUMO

ß-Coronaviruses are a family of positive-strand enveloped RNA viruses that includes the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much is known regarding their cellular entry and replication pathways, but their mode of egress remains uncertain. Using imaging methodologies and virus-specific reporters, we demonstrate that ß-coronaviruses utilize lysosomal trafficking for egress rather than the biosynthetic secretory pathway more commonly used by other enveloped viruses. This unconventional egress is regulated by the Arf-like small GTPase Arl8b and can be blocked by the Rab7 GTPase competitive inhibitor CID1067700. Such non-lytic release of ß-coronaviruses results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation pathways. ß-Coronavirus-induced exploitation of lysosomal organelles for egress provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities.


Assuntos
/metabolismo , Via Secretória , Liberação de Vírus , Fatores de Ribosilação do ADP/metabolismo , Animais , /patologia , Feminino , Células HeLa , Compostos Heterocíclicos com 2 Anéis/farmacologia , Humanos , Lisossomos , Camundongos , Tioureia/análogos & derivados , Tioureia/farmacologia , Proteínas rab de Ligação ao GTP/antagonistas & inibidores , Proteínas rab de Ligação ao GTP/metabolismo
3.
Sci Rep ; 10(1): 16803, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033331

RESUMO

Tunneling nanotubes (TNTs) are F-actin rich structures that connect distant cells, allowing the transport of many cellular components, including vesicles, organelles and molecules. Rab GTPases are the major regulators of vesicle trafficking and also participate in actin cytoskeleton remodelling, therefore, we examined their role in TNTs. Rab35 functions with several proteins that are involved in vesicle trafficking such as ACAP2, MICAL-L1, ARF6 and EHD1, which are known to be involved in neurite outgrowth. Here we show that Rab35 promotes TNT formation and TNT-mediated vesicle transfer in a neuronal cell line. Furthermore, our data indicates that Rab35-GTP, ACAP2, ARF6-GDP and EHD1 act in a cascade mechanism to promote TNT formation. Interestingly, MICAL-L1 overexpression, shown to be necessary for the action of Rab35 on neurite outgrowth, showed no effect on TNTs, indicating that TNT formation and neurite outgrowth may be processed through similar but not identical pathways, further supporting the unique identity of these cellular protrusions.


Assuntos
Nanotubos , Neurônios/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Fatores de Ribosilação do ADP/metabolismo , Animais , Western Blotting , Linhagem Celular , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/ultraestrutura , Citometria de Fluxo , Proteínas Ativadoras de GTPase/metabolismo , Camundongos , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Nanotubos/ultraestrutura , Neurônios/ultraestrutura , Proteínas de Transporte Vesicular/metabolismo
4.
Breast Cancer Res ; 22(1): 105, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33023655

RESUMO

BACKGROUND: ErbB2/HER2 oncoprotein often drives breast cancers (BCs) which are treated with the anti-ErbB2 antibody trastuzumab. The efficacy of trastuzumab-based metastatic BC therapies is routinely assessed by imaging studies. Trastuzumab typically becomes ineffective in the case of this disease and is then replaced by other drugs. Biomarkers of BC trastuzumab response could allow imaging studies and the switch to other drugs to occur earlier than is now possible. Moreover, bone-only BC metastases can be hard to measure, and biomarkers of their trastuzumab response could facilitate further treatment decisions. Such biomarkers are presently unavailable. In this study, we searched for proteins whose levels in BC cell-emitted extracellular vesicles (EVs) potentially correlate with BC trastuzumab sensitivity. METHODS: We isolated EVs from cultured trastuzumab-sensitive and trastuzumab-resistant human BC cells before and after trastuzumab treatment and characterized these EVs by nanoparticle tracking analysis and electron microscopy. We found previously that ErbB2 drives BC by downregulating a pro-apoptotic protein PERP. We now tested whether trastuzumab-induced PERP upregulation in EVs emitted by cultured human BC cells correlates with their trastuzumab sensitivity. We also used mass spectrometry to search for additional proteins whose levels in such EVs reflect BC cell trastuzumab sensitivity. Once we identified proteins whose EV levels correlate with this sensitivity in culture, we explored the feasibility of testing whether their levels in the blood EVs of trastuzumab-treated metastatic BC patients correlate with patients' response to trastuzumab-based treatments. RESULTS: We found that neither trastuzumab nor acquisition of trastuzumab resistance by BC cells affects the size or morphology of EVs emitted by cultured BC cells. We established that EV levels of proteins PERP, GNAS2, GNA13, ITB1, and RAB10 correlate with BC cell trastuzumab response. Moreover, these proteins were upregulated during trastuzumab-based therapies in the blood EVs of a pilot cohort of metastatic BC patients that benefited from these therapies but not in those derived from patients that failed such treatments. CONCLUSIONS: Upregulation of a protein set in EVs derived from cultured breast tumor cells correlates with tumor cell trastuzumab sensitivity. It is feasible to further evaluate these proteins as biomarkers of metastatic BC trastuzumab response.


Assuntos
Biomarcadores Farmacológicos/metabolismo , Neoplasias da Mama/tratamento farmacológico , Vesículas Extracelulares/metabolismo , Proteínas de Membrana/metabolismo , Receptor ErbB-2/metabolismo , Trastuzumab/uso terapêutico , Adulto , Antineoplásicos Imunológicos/uso terapêutico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Cromograninas/metabolismo , Estudos de Coortes , Feminino , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Genes Supressores de Tumor , Humanos , Pessoa de Meia-Idade , Metástase Neoplásica , Proteômica/métodos , Receptor ErbB-2/antagonistas & inibidores , Regulação para Cima , Proteínas rab de Ligação ao GTP/metabolismo
5.
Proc Natl Acad Sci U S A ; 117(43): 26784-26794, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33055216

RESUMO

The obligate intracellular bacteria Chlamydia trachomatis, the causative agent of trachoma and sexually transmitted diseases, multiply in a vacuolar compartment, the inclusion. From this niche, they secrete "effector" proteins, that modify cellular activities to enable bacterial survival and proliferation. Here, we show that the host autophagy-related protein 16-1 (ATG16L1) restricts inclusion growth and that this effect is counteracted by the secretion of the bacterial effector CT622/TaiP (translocated ATG16L1 interacting protein). ATG16L1 is mostly known for its role in the lipidation of the human homologs of ATG8 (i.e., LC3 and homologs) on double membranes during autophagy as well as on single membranes during LC3-associated phagocytosis and other LC3-lipidation events. Unexpectedly, the LC3-lipidation-related functions of ATG16L1 are not required for restricting inclusion development. We show that the carboxyl-terminal domain of TaiP exposes a mimic of an eukaryotic ATG16L1-binding motif that binds to ATG16L1's WD40 domain. By doing so, TaiP prevents ATG16L1 interaction with the integral membrane protein TMEM59 and allows the rerouting of Rab6-positive compartments toward the inclusion. The discovery that one bacterial effector evolved to target ATG16L1's engagement in intracellular traffic rather than in LC3 lipidation brings this "secondary" activity of ATG16L1 in full light and emphasizes its importance for maintaining host cell homeostasis.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Chlamydia trachomatis/fisiologia , Interações Hospedeiro-Patógeno , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Bactérias/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas rab de Ligação ao GTP/metabolismo
6.
Nat Commun ; 11(1): 5189, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33060596

RESUMO

Among the various host cellular processes that are hijacked by flaviviruses, few mechanisms have been described with regard to viral egress. Here we investigate how flaviviruses exploit Src family kinases (SFKs) for exit from infected cells. We identify Lyn as a critical component for secretion of Dengue and Zika infectious particles and their corresponding virus like particles (VLPs). Pharmacological inhibition or genetic depletion of the SFKs, Lyn in particular, block virus secretion. Lyn-/- cells are impaired in virus release and are rescued when reconstituted with wild-type Lyn, but not a kinase- or palmitoylation-deficient Lyn mutant. We establish that virus particles are secreted in two distinct populations - one as free virions and the other enclosed within membranes. Lyn is critical for the latter, which consists of proteolytically processed, infectious virus progenies within autophagosome-derived vesicles. This process depends on Ulk1, Rab GTPases and SNARE complexes implicated in secretory but not degradative autophagy and occur with significantly faster kinetics than the conventional secretory pathway. Our study reveals a previously undiscovered Lyn-dependent exit route of flaviviruses in LC3+ secretory organelles that enables them to evade circulating antibodies and might affect tissue tropism.


Assuntos
Autofagossomos/metabolismo , Autofagossomos/virologia , Flavivirus/metabolismo , Quinases da Família src/metabolismo , Animais , Autofagia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Linhagem Celular , Chlorocebus aethiops , Dengue , Vírus da Dengue/metabolismo , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas SNARE/metabolismo , Via Secretória , Células Vero , Vírion/metabolismo , Liberação de Vírus , Zika virus/metabolismo , Infecção por Zika virus , Proteínas rab de Ligação ao GTP/metabolismo , Quinases da Família src/genética
7.
Sci Rep ; 10(1): 15741, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978479

RESUMO

Regenerative medicine approaches to enhancing beta cell growth and survival represent potential treatments for diabetes. It is known that growth factors such as insulin, IGF-1 and HGF support beta cell growth and survival, but in people with type 2 diabetes the destructive effects of metabolic stress predominate and beta cell death or dysfunction occurs. In this study we explore the novel hypothesis that regulation of growth factor receptor trafficking can be used to promote islet beta cell survival. Growth factor signalling is dependent on the presence of cell surface receptors. Endosomal trafficking and subsequent recycling or degradation of these receptors is controlled by the Rab GTPase family of proteins. We show that Rab7a siRNA inhibition enhances IGF-1 and HGF signalling in beta cells and increases expression of the growth factor receptors IGF-1R and c-Met. Furthermore, Rab7a inhibition promotes beta cell growth and islet survival, and protects against activation of apoptosis and autophagy pathways under conditions of metabolic stress. This study therefore demonstrates that Rab7a-mediated trafficking of growth factor receptors controls beta cell survival. Pharmaceutical Rab7a inhibition may provide a means to promote beta cell survival in the context of metabolic stress and prevent the onset of type 2 diabetes.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Ilhotas Pancreáticas/citologia , Proteínas Proto-Oncogênicas c-met/metabolismo , RNA Interferente Pequeno/farmacologia , Receptor IGF Tipo 1/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Apoptose , Autofagia , Proliferação de Células , Células Cultivadas , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Camundongos , Transporte Proteico/efeitos dos fármacos , Estresse Fisiológico , Regulação para Cima , Proteínas rab de Ligação ao GTP/antagonistas & inibidores
8.
Sci Rep ; 10(1): 15804, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978434

RESUMO

Small extracellular vesicles (sEVs), 50-150 nm in diameter, have been proposed to mediate cell-cell communication with important implications in tumor microenvironment interactions, tumor growth, and metastasis. We previously showed that mutant KRAS colorectal cancer (CRC) cells release sEVs containing Rab13 protein and mRNA. Previous work had shown that disruption of intracellular Rab13 trafficking inhibits epithelial cell proliferation and invasiveness. Here, we show that Rab13 additionally regulates the secretion of sEVs corresponding to both traditional exosomes and a novel subset of vesicles containing both ß1-integrin and Rab13. We find that exposure of recipient cells to sEVs from KRAS mutant donor cells increases proliferation and tumorigenesis and that knockdown of Rab13 blocks these effects. Thus, Rab13 serves as both a cargo protein and as a regulator of sEV secretion. Our data support a model whereby Rab13 can mediate its effects on cell proliferation and invasiveness via autocrine and paracrine signaling.


Assuntos
Neoplasias Colorretais/patologia , Exossomos/metabolismo , Vesículas Extracelulares/metabolismo , Integrina beta1/metabolismo , Mutação , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Comunicação Celular , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Humanos , Integrina beta1/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Células Tumorais Cultivadas , Microambiente Tumoral , Proteínas rab de Ligação ao GTP/genética
9.
PLoS Pathog ; 16(8): e1008822, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866204

RESUMO

Shigella flexneri invades host cells by entering within a bacteria-containing vacuole (BCV). In order to establish its niche in the host cytosol, the bacterium ruptures its BCV. Contacts between S. flexneri BCV and infection-associated macropinosomes (IAMs) formed in situ have been reported to enhance BCV disintegration. The mechanism underlying S. flexneri vacuolar escape remains however obscure. To decipher the molecular mechanism priming the communication between the IAMs and S. flexneri BCV, we performed mass spectrometry-based analysis of the magnetically purified IAMs from S. flexneri-infected cells. While proteins involved in host recycling and exocytic pathways were significantly enriched at the IAMs, we demonstrate more precisely that the S. flexneri type III effector protein IpgD mediates the recruitment of the exocyst to the IAMs through the Rab8/Rab11 pathway. This recruitment results in IAM clustering around S. flexneri BCV. More importantly, we reveal that IAM clustering subsequently facilitates an IAM-mediated unwrapping of the ruptured vacuole membranes from S. flexneri, enabling the naked bacterium to be ready for intercellular spread via actin-based motility. Taken together, our work untangles the molecular cascade of S. flexneri-driven host trafficking subversion at IAMs to develop its cytosolic lifestyle, a crucial step en route for infection progression at cellular and tissue level.


Assuntos
Disenteria Bacilar , Shigella flexneri , Transdução de Sinais , Vacúolos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Disenteria Bacilar/genética , Disenteria Bacilar/metabolismo , Células HeLa , Humanos , Shigella flexneri/genética , Shigella flexneri/metabolismo , Shigella flexneri/patogenicidade , Vacúolos/genética , Vacúolos/metabolismo , Vacúolos/microbiologia , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
10.
Nat Commun ; 11(1): 4187, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826901

RESUMO

EHBP1 is an adaptor protein that regulates vesicular trafficking by recruiting Rab8 family members and Eps15-homology domain-containing proteins 1/2 (EHD1/2). It also links endosomes to the actin cytoskeleton. However, the underlying molecular mechanism of activation of EHBP1 actin-binding activity is unclear. Here, we show that both termini of EHBP1 have membrane targeting potential. EHBP1 associates with PI(3)P, PI(5)P, and phosphatidylserine via its N-terminal C2 domain. We show that in the absence of Rab8 family members, the C-terminal bivalent Mical/EHBP Rab binding (bMERB) domain forms an intramolecular complex with its central calponin homology (CH) domain and auto-inhibits actin binding. Rab8 binding to the bMERB domain relieves this inhibition. We have analyzed the CH:bMERB auto-inhibited complex and the active bMERB:Rab8 complex biochemically and structurally. Together with structure-based mutational studies, this explains how binding of Rab8 frees the CH domain and allows it to interact with the actin cytoskeleton, leading to membrane tubulation.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/genética , Proteínas dos Microfilamentos/genética , Modelos Moleculares , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico/fisiologia , Alinhamento de Sequência , Proteínas de Transporte Vesicular , Proteínas rab de Ligação ao GTP/genética
11.
Parasitol Res ; 119(9): 2991-3003, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32748038

RESUMO

Visceral leishmaniasis (VL, also known as kala-azar) is a vector borne disease caused by obligate intracellular protozoan parasite Leishmania donovani. To overcome the limitations of currently available drugs for VL, molecular target-based study is a promising tool to develop new drugs to treat this neglected tropical disease. One such target we recently identified from L. donovani (Ld) genome (WGS, clinical Indian isolate; BHU 1220, AVPQ01000001) is a small GTP-binding protein, Rab6 protein. We now report a specific inhibitor of the GTPase activity of Rab6 protein of L. donovani (LdRab6) without restricting host enzyme activity. First, to understand the nature of LdRab6 protein, we generated recombinant LdRab6 mutant proteins (rLdRab6) by systematically introducing deletion (two cysteine residues at C-terminal) and mutations [single amino acid substitutions in the conserved region of GTP (Q84L)/GDP(T38N) coding sequence]. The GTPase activity of rLdRab6:GTP and rLdRab6:GDP locked mutant proteins showed ~ 8-fold and ~ 1.5-fold decreases in enzyme activity, respectively, compared to the wild type enzyme activity. The mutant protein rLdRab6:ΔC inhibited the GTPase activity. Sequence alignment analysis of Rab6 protein of L. donovani with Homo sapiens showed identical amino acids in the G conserved region (GTP/GDP-binding sites) but it differed in the C-terminal region. We then evaluated the inhibitory activity of trans-dibenzalacetone (DBA, a synthetic analog of curcumin with strong antileishmanial activity reported earlier by us) in the GTPase activity of LdRab6 protein. Comparative molecular docking analysis of DBA and specific inhibitors of Rab proteins (Lovastatin, BFA, Zoledronate, and NE10790) indicated that DBA had optimum binding affinity with LdRab6 protein. This was further confirmed by the GTPase activity of DBA-treated LdRab6 which showed a basal GTP level significantly lower than that of the wild-type rLdRab6. The results confirm that DBA inhibits the GTPase activity of LdRab6 protein from L. donovani (LdRab6), a potential target for its antileishmanial effect.


Assuntos
Antiprotozoários/farmacologia , Inibidores Enzimáticos/farmacologia , Leishmania donovani/efeitos dos fármacos , Leishmaniose Visceral/parasitologia , Pentanonas/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Proteínas rab de Ligação ao GTP/antagonistas & inibidores , Sequência de Aminoácidos , Sítios de Ligação , Curcumina/farmacologia , Humanos , Leishmania donovani/química , Leishmania donovani/enzimologia , Leishmania donovani/genética , Leishmaniose Visceral/tratamento farmacológico , Simulação de Acoplamento Molecular , Pentanonas/química , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
12.
PLoS One ; 15(8): e0235551, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833964

RESUMO

VPS34 is a key regulator of endomembrane dynamics and cargo trafficking, and is essential in cultured cell lines and in mice. To better characterize the role of VPS34 in cell growth, we performed unbiased cell line profiling studies with the selective VPS34 inhibitor PIK-III and identified RKO as a VPS34-dependent cellular model. Pooled CRISPR screen in the presence of PIK-III revealed endolysosomal genes as genetic suppressors. Dissecting VPS34-dependent alterations with transcriptional profiling, we found the induction of hypoxia response and cholesterol biosynthesis as key signatures. Mechanistically, acute VPS34 inhibition enhanced lysosomal degradation of transferrin and low-density lipoprotein receptors leading to impaired iron and cholesterol uptake. Excess soluble iron, but not cholesterol, was sufficient to partially rescue the effects of VPS34 inhibition on mitochondrial respiration and cell growth, indicating that iron limitation is the primary driver of VPS34-dependency in RKO cells. Loss of RAB7A, an endolysosomal marker and top suppressor in our genetic screen, blocked transferrin receptor degradation, restored iron homeostasis and reversed the growth defect as well as metabolic alterations due to VPS34 inhibition. Altogether, our findings suggest that impaired iron mobilization via the VPS34-RAB7A axis drive VPS34-dependence in certain cancer cells.


Assuntos
Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Ferro/metabolismo , Neoplasias/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Proliferação de Células , Colesterol/biossíntese , Colesterol/genética , Classe III de Fosfatidilinositol 3-Quinases/genética , Endossomos/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Receptores de LDL/metabolismo , Transferrina/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
13.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1865(12): 158805, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32829064

RESUMO

Charcot-Marie Tooth type 2B (CMT2B) is a rare inherited peripheral neuropathy caused by five missense mutations in the RAB7A gene, which encodes a small GTPase of the RAB family. Currently, no cure is available for this disease. In this study, we approached the disease by comparing the lipid metabolism of CMT2B-derived fibroblasts to that of healthy controls. We found that CMT2B cells showed increased monounsaturated fatty acid level and increased expression of key enzymes of monounsaturated and polyunsaturated fatty acid synthesis. Moreover, in CMT2B cells a higher expression of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), key enzymes of de novo fatty acid synthesis, with a concomitantly increased [1-14C]acetate incorporation into fatty acids, was observed. The expression of diacylglycerol acyltransferase 2, a rate-limiting enzyme in triacylglycerol synthesis, as well as triacylglycerol levels were increased in CMT2B compared to control cells. In addition, as RAB7A controls lipid droplet breakdown and lipid droplet dynamics have been linked to diseases, we analyzed these organelles and showed that in CMT2B cells there is a strong accumulation of lipid droplets compared to control cells, thus reinforcing our data on abnormal lipid metabolism in CMT2B. Furthermore, we demonstrated that ACC and FAS expression levels changed upon RAB7 silencing or overexpression in HeLa cells, thus suggesting that metabolic modifications observed in CMT2B-derived fibroblasts can be, at least in part, related to RAB7 mutations.


Assuntos
Doença de Charcot-Marie-Tooth/metabolismo , Metabolismo dos Lipídeos , Células Cultivadas , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , /patologia , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/patologia , Mutação de Sentido Incorreto , Triglicerídeos/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
14.
Sci Rep ; 10(1): 14136, 2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32839520

RESUMO

Rab27 is an essential molecule of vesicle fusion and trafficking in exosome secretion process, which plays important roles in cancer progression and metastasis. Recent studies reported that Rab27 expression is also associated with cancer prognosis. Therefore, we performed a meta-analysis to reveal the prognostic significance of Rab27 expression in solid cancer. Data were extracted by searching on PubMed, Embase and Cochrane library until February 15 2020. Pooled hazard ratio (HR) with confidence interval (CI) was calculated to evaluate the association between Rab27 expression and survival in solid cancer. Ten studies with 1434 cancer patients were including for this meta-analysis. High expression of Rab27 was associated with poor survival (HR 2.67, 95% CI 1.52-4.69, p = 0.001). High expression of Rab27A was significantly associated with lymph node metastasis (HR 1.53, 95% CI 1.00-2.34, p = 0.048). High expression of Rab27B was significantly correlated with lymph node and distant metastasis (HR 2.15, 95% CI 1.56-2.95, p < 0.001; HR 6.80, 95% CI 3.12-14.85, p < 0.001), and higher TNM stage (HR 2.55, 95% CI 1.78-3.65, p < 0.001). This meta-analysis revealed that Rab27 expression could be a potential prognostic marker in solid cancer.


Assuntos
Metástase Linfática/patologia , Neoplasias/patologia , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab27 de Ligação ao GTP/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Humanos , Metástase Linfática/genética , Estadiamento de Neoplasias , Neoplasias/mortalidade , Prognóstico , Proteínas rab de Ligação ao GTP/genética , Proteínas rab27 de Ligação ao GTP/genética
15.
Mol Cancer Ther ; 19(9): 1930-1942, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32737155

RESUMO

Although intravesical bacillus Calmette-Guérin (BCG) immunotherapy has been the gold standard for nonsurgical management of non-muscle-invasive bladder cancer, a considerable number of patients exhibit resistance to the adjuvant treatment with unexplained mechanisms. This study aimed to investigate whether and how androgen receptor (AR) signals modulate BCG cytotoxicity in bladder cancer. AR knockdown or overexpression in bladder cancer lines resulted in induction or reduction, respectively, in intracellular BCG quantity and its cytotoxic activity. Microarray screening identified Rab27b, a small GTPase known to mediate bacterial exocytosis, which was upregulated in BCG-resistant cells and downregulated in AR-shRNA cells. Knockdown of Rab27b, or its effector SYTL3, or overexpression of Rab27b also induced or reduced, respectively, BCG quantity and cytotoxicity. In addition, treatment with GW4869, which was previously shown to inhibit Rab27b-dependent secretion, induced them and reduced Rab27b expression in bladder cancer cells. Meanwhile, AR expression was upregulated in BCG-resistant lines, compared with respective controls. In a mouse orthotopic xenograft model, Rab27b/SYTL3 knockdown or GW4869 treatment enhanced the amount of BCG within tumors and its suppressive effect on tumor growth. Moreover, in non-muscle-invasive bladder cancer specimens from patients subsequently undergoing BCG therapy, positivity of AR/Rab27b expression was associated with significantly higher risks of tumor recurrence. AR activation thus correlates with resistance to BCG treatment, presumably via upregulating Rab27b expression. Mechanistically, it is suggested that BCG elimination from urothelial cells is induced by Rab27b/SYTL3-mediated exocytosis. Accordingly, Rab27b inactivation, potentially via antiandrogenic drugs and/or exocytosis inhibition are anticipated to sensitize the efficacy of BCG therapy, especially in patients with BCG-refractory AR/Rab27b-positive bladder cancer.


Assuntos
Vacina BCG/uso terapêutico , Exocitose/efeitos dos fármacos , Imunoterapia/métodos , Receptores Androgênicos/metabolismo , Neoplasias da Bexiga Urinária/tratamento farmacológico , Proteínas rab de Ligação ao GTP/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Vacina BCG/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Transdução de Sinais
16.
Science ; 369(6502): 450-455, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32703879

RESUMO

The guanosine triphosphatase (GTPase) Rab32 coordinates a cell-intrinsic host defense mechanism that restricts the replication of intravacuolar pathogens such as Salmonella Here, we show that this mechanism requires aconitate decarboxylase 1 (IRG1), which synthesizes itaconate, a metabolite with antimicrobial activity. We find that Rab32 interacts with IRG1 on Salmonella infection and facilitates the delivery of itaconate to the Salmonella-containing vacuole. Mice defective in IRG1 rescued the virulence defect of a S. enterica serovar Typhimurium mutant specifically defective in its ability to counter the Rab32 defense mechanism. These studies provide a link between a metabolite produced in the mitochondria after stimulation of innate immune receptors and a cell-autonomous defense mechanism that restricts the replication of an intracellular bacterial pathogen.


Assuntos
Hidroliases/imunologia , Infecções por Salmonella/imunologia , Salmonella enterica , Salmonella typhimurium , Proteínas rab de Ligação ao GTP/imunologia , Animais , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Hidroliases/metabolismo , Camundongos , Infecções por Salmonella/metabolismo , Infecções por Salmonella/microbiologia , Succinatos , Virulência , Proteínas rab de Ligação ao GTP/metabolismo
17.
PLoS Biol ; 18(7): e3000778, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32678845

RESUMO

The evolution of transformed cancer cells into metastatic tumors is, in part, driven by altered intracellular signaling downstream of receptor tyrosine kinases (RTKs). The surface levels and activity of RTKs are governed mainly through clathrin-mediated endocytosis (CME), endosomal recycling, or degradation. In turn, oncogenic signaling downstream of RTKs can reciprocally regulate endocytic trafficking by creating feedback loops in cells to enhance tumor progression. We previously showed that FCH/F-BAR and Double SH3 Domain-Containing Protein (FCHSD2) has a cancer-cell specific function in regulating CME in non-small-cell lung cancer (NSCLC) cells. Here, we report that FCHSD2 loss impacts recycling of the RTKs, epidermal growth factor receptor (EGFR) and proto-oncogene c-Met (MET), and shunts their trafficking into late endosomes and lysosomal degradation. Notably, FCHSD2 depletion results in the nuclear translocation of active extracellular signal-regulated kinase 1 and 2 (ERK1/2), leading to enhanced transcription and up-regulation of EGFR and MET. The small GTPase, Ras-related protein Rab-7A (Rab7), is essential for the FCHSD2 depletion-induced effects. Correspondingly, FCHSD2 loss correlates to higher tumor grades of NSCLC. Clinically, NSCLC patients expressing high FCHSD2 exhibit elevated survival, whereas patients with high Rab7 expression display decreased survival rates. Our study provides new insight into the molecular nexus for crosstalk between oncogenic signaling and RTK trafficking that controls cancer progression.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Transporte/metabolismo , Endocitose , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/metabolismo , Oncogenes , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Linhagem Celular Tumoral , Progressão da Doença , Endossomos/metabolismo , Receptores ErbB/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/enzimologia , Transporte Proteico , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores da Transferrina/metabolismo , Regulação para Cima , Proteínas rab de Ligação ao GTP/metabolismo
18.
Arterioscler Thromb Vasc Biol ; 40(9): 2054-2069, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32640907

RESUMO

OBJECTIVE: Increased CTSS (cathepsin S) has been reported to play a critical role in atherosclerosis progression. Both CTSS synthesis and secretion are essential for exerting its functions. However, the underlying mechanisms contributing to CTSS synthesis and secretion in atherosclerosis remain unclear. Approach and Results: In this study, we showed that nicotine activated autophagy and upregulated CTSS expression in vascular smooth muscle cells and in atherosclerotic plaques. Western blotting and immunofluorescent staining showed that nicotine inhibited the mTORC1 (mammalian target of rapamycin complex 1) activity, promoted the nuclear translocation of TFEB (transcription factor EB), and upregulated the expression of CTSS. Chromatin immunoprecipitation-qualificative polymerase chain reaction, electrophoretic mobility shift assay, and luciferase reporter assay further demonstrated that TFEB directly bound to the CTSS promoter. mTORC1 inhibition by nicotine or rapamycin promoted lysosomal exocytosis and CTSS secretion. Live cell assays and IP-MS (immunoprecipitation-mass spectrometry) identified that the interactions involving Rab10 (Rab10, member RAS oncogene family) and mTORC1 control CTSS secretion. Nicotine promoted vascular smooth muscle cell migration by upregulating CTSS, and CTSS inhibition suppressed nicotine-induced atherosclerosis in vivo. CONCLUSIONS: We concluded that nicotine mediates CTSS synthesis and secretion through regulating the autophagy-lysosomal machinery, which offers a potential therapeutic target for atherosclerosis treatment.


Assuntos
Aterosclerose/tratamento farmacológico , Autofagia/efeitos dos fármacos , Catepsinas/biossíntese , Lisossomos/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Nicotina/farmacologia , Animais , Aterosclerose/enzimologia , Aterosclerose/genética , Aterosclerose/patologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Catepsinas/genética , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Exocitose , Lisossomos/enzimologia , Lisossomos/ultraestrutura , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos Knockout para ApoE , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/ultraestrutura , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/ultraestrutura , Via Secretória , Transdução de Sinais , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
19.
Life Sci ; 257: 118126, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32707053

RESUMO

AIMS: Rab31, a Rab5 subfamily member, has emerged as a modulator of membrane trafficking. Our study serves to clarify the role and mechanism of Rab31 in colorectal carcinoma (CRC) pathogenesis. MATERIALS AND METHODS: The differential expression of Rab31 was examined in paired normal and cancerous colonic tissues by quantitative PCR, western blot and immunochemistry. The prognostic significance of Rab31 was analysed by univariate and multivariate survival analyses. We also investigated the effects of Rab31 on tumour growth in vitro. KEY FINDINGS: We observed that Rab31, which is related to histological differentiation in CRC, was markedly overexpressed in CRC cells. Moreover, patients who showed higher Rab31 levels had a shortened survival period relative to those with low Rab31 levels. Rab31 knockdown significantly downregulated cyclin D1, p-mTOR, and p-p70S6K expression. Moreover, the expression of Rab31-induced p-p70S6K was almost inhibited by rapamycin, a well-established inhibitor of mTOR. Similarly, rapamycin also significantly decreased the stimulatory effect of Rab31 on the expression of cyclin D1. SIGNIFICANCE: These findings suggested that Rab31 enhanced proliferation, promoted cell cycle progression, and inhibited apoptosis of colorectal carcinoma cells through the mTOR pathway.


Assuntos
Neoplasias Colorretais/metabolismo , Ciclina D1/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Idoso , Apoptose , Western Blotting , Células CACO-2 , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Feminino , Células HCT116 , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sobrevida , Regulação para Cima
20.
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
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