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1.
Nat Commun ; 11(1): 495, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980630

RESUMO

Maize rough dwarf disease (MRDD), caused by various species of the genus Fijivirus, threatens maize production worldwide. We previously identified a quantitative locus qMrdd1 conferring recessive resistance to one causal species, rice black-streaked dwarf virus (RBSDV). Here, we show that Rab GDP dissociation inhibitor alpha (RabGDIα) is the host susceptibility factor for RBSDV. The viral P7-1 protein binds tightly to the exon-10 and C-terminal regions of RabGDIα to recruit it for viral infection. Insertion of a helitron transposon into RabGDIα intron 10 creates alternative splicing to replace the wild-type exon 10 with a helitron-derived exon 10. The resultant splicing variant RabGDIα-hel has difficulty being recruited by P7-1, thus leading to quantitative recessive resistance to MRDD. All naturally occurring resistance alleles may have arisen from a recent single helitron insertion event. These resistance alleles are valuable to improve maize resistance to MRDD and potentially to engineer RBSDV resistance in other crops.


Assuntos
Resistência à Doença , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Doenças das Plantas/virologia , Proteínas de Plantas/metabolismo , Vírus de Plantas/fisiologia , Zea mays/virologia , Alelos , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Modelos Biológicos , Mapeamento Físico do Cromossomo , Doenças das Plantas/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Ligação Proteica , Locos de Características Quantitativas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Proteínas Virais/metabolismo , Zea mays/genética , Zea mays/ultraestrutura
2.
Small GTPases ; 10(3): 227-242, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-29065764

RESUMO

The small GTPase Rab5 is the key regulator of early endosomal fusion. It is post-translationally modified by covalent attachment of two geranylgeranyl (GG) chains to adjacent cysteine residues of the C-terminal hypervariable region (HVR). The GDP dissociation inhibitor (GDI) recognizes membrane-associated Rab5(GDP) and serves to release it into the cytoplasm where it is kept in a soluble state. A detailed new structural and dynamic model for human Rab5(GDP) recognition and binding with human GDI at the early endosome membrane and in its dissociated state is presented. In the cytoplasm, the GDI protein accommodates the GG chains in a transient hydrophobic binding pocket. In solution, two different binding modes of the isoprenoid chains inserted into the hydrophobic pocket of the Rab5(GDP):GDI complex can be identified. This equilibrium between the two states helps to stabilize the protein-protein complex in solution. Interprotein contacts between the Rab5 switch regions and characteristic patches of GDI residues from the Rab binding platform (RBP) and the C-terminus coordinating region (CCR) reveal insight on the formation of such a stable complex. GDI binding to membrane-anchored Rab5(GDP) is initially mediated by the solvent accessible switch regions of the Rab-specific RBP. Formation of the membrane-associated Rab5(GDP):GDI complex induces a GDI reorientation to establish additional interactions with the Rab5 HVR. These results allow to devise a detailed structural model for the process of extraction of GG-Rab5(GDP) by GDI from the membrane and the dissociation from targeting factors and effector proteins prior to GDI binding.


Assuntos
Diterpenos/química , Inibidores de Dissociação do Nucleotídeo Guanina/química , Simulação de Dinâmica Molecular , Complexos Multiproteicos , Prenilação de Proteína , Proteínas rab5 de Ligação ao GTP/química , Animais , Bovinos , Diterpenos/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Humanos , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo
3.
Nat Commun ; 9(1): 1025, 2018 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-29523789

RESUMO

Asymmetric cell divisions balance stem cell proliferation and differentiation to sustain tissue morphogenesis and homeostasis. During asymmetric divisions, fate determinants and niche contacts segregate unequally between daughters, but little is known on how this is achieved mechanistically. In Drosophila neuroblasts and murine mammary stem cells, the association of the spindle orientation protein LGN with the stem cell adaptor Inscuteable has been connected to asymmetry. Here we report the crystal structure of Drosophila LGN in complex with the asymmetric domain of Inscuteable, which reveals a tetrameric arrangement of intertwined molecules. We show that Insc:LGN tetramers constitute stable cores of Par3-Insc-LGN-GαiGDP complexes, which cannot be dissociated by NuMA. In mammary stem cells, the asymmetric domain of Insc bound to LGN:GαiGDP suffices to drive asymmetric fate, and reverts aberrant symmetric divisions induced by p53 loss. We suggest a novel role for the Insc-bound pool of LGN acting independently of microtubule motors to promote asymmetric fate specification.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Divisão Celular Assimétrica , Proteínas do Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/citologia , Drosophila/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células-Tronco/citologia , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Drosophila/química , Drosophila/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Inibidores de Dissociação do Nucleotídeo Guanina/química , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Modelos Moleculares , Ligação Proteica , Células-Tronco/química , Células-Tronco/metabolismo
4.
Cancer Immunol Immunother ; 67(1): 67-77, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28916862

RESUMO

Intravesical Bacillus Calmette-Guerin (BCG) is the best treatment modality for progression of non-muscle invasive bladder cancer. We aimed to monitor changes at the proteome level to identify putative protein biomarkers associated with the response of urothelial precancerous lesions to intravesical BCG treatment. The rats were divided into three groups (n = 10/group): control, non-treated, and BCG-treated groups. The non-treated and BCG-treated groups received N-methyl-N-nitrosourea intravesically. BCG Tice-strain was instilled into bladder in BCG-treated group. At the endpoint of experiment, all surviving rat bladders were collected and equally divided into two portions vertically from dome to neck. Half of each bladder was assessed immunohistopathologically and the other half was used for 2D-based comparative proteomic analysis. Differentially expressed proteins were validated by Western blot analysis. Precancerous lesions of bladder cancer were more common in non-treated group (77.8%) than in BCG-treated group (50%) and the control group (0%). Greater than twofold changes occurred in the expression of a number of proteins. Among them, Rab-GDIß, aldehyde dehydrogenase 2 (ALDH2) and 14-3-3 zeta/delta were important since they were previously reported to be associated with cancer and their expression levels were found to be lower in BCG-treated group in comparison to the non-treated group. ALDH2 and 14-3-3 zeta/delta were also found to be highly expressed in the non-treated group compared to the control group. The down-regulation of these proteins and Rab-GDIß was achieved with BCG; this result indicates that they may be used as putative biomarkers for monitoring changes in bladder carcinogenesis in response to BCG immunotherapy.


Assuntos
Vacina BCG/imunologia , Vacinas Anticâncer/imunologia , Imunoterapia/métodos , Neoplasias Urológicas/terapia , Urotélio/metabolismo , Administração Intravesical , Animais , Modelos Animais de Doenças , Feminino , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Humanos , Lesões Pré-Cancerosas , Proteoma , Ratos , Ratos Wistar , Neoplasias Urológicas/imunologia , Urotélio/patologia
5.
Proc Natl Acad Sci U S A ; 114(48): E10319-E10328, 2017 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-29133411

RESUMO

Activation of heterotrimeric G proteins by cytoplasmic nonreceptor proteins is an alternative to the classical mechanism via G protein-coupled receptors (GPCRs). A subset of nonreceptor G protein activators is characterized by a conserved sequence named the Gα-binding and activating (GBA) motif, which confers guanine nucleotide exchange factor (GEF) activity in vitro and promotes G protein-dependent signaling in cells. GBA proteins have important roles in physiology and disease but remain greatly understudied. This is due, in part, to the lack of efficient tools that specifically disrupt GBA motif function in the context of the large multifunctional proteins in which they are embedded. This hindrance to the study of alternative mechanisms of G protein activation contrasts with the wealth of convenient chemical and genetic tools to manipulate GPCR-dependent activation. Here, we describe the rational design and implementation of a genetically encoded protein that specifically inhibits GBA motifs: GBA inhibitor (GBAi). GBAi was engineered by introducing modifications in Gαi that preclude coupling to every known major binding partner [GPCRs, Gßγ, effectors, guanine nucleotide dissociation inhibitors (GDIs), GTPase-activating proteins (GAPs), or the chaperone/GEF Ric-8A], while favoring high-affinity binding to all known GBA motifs. We demonstrate that GBAi does not interfere with canonical GPCR-G protein signaling but blocks GBA-dependent signaling in cancer cells. Furthermore, by implementing GBAi in vivo, we show that GBA-dependent signaling modulates phenotypes during Xenopus laevis embryonic development. In summary, GBAi is a selective, efficient, and convenient tool to dissect the biological processes controlled by a GPCR-independent mechanism of G protein activation mediated by cytoplasmic factors.


Assuntos
Proteínas Ativadoras de GTPase/genética , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas Nucleares/genética , Engenharia de Proteínas/métodos , Receptores Acoplados a Proteínas-G/genética , Proteínas de Transporte Vesicular/genética , Motivos de Aminoácidos , Animais , Clonagem Molecular , Embrião não Mamífero , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HEK293 , Humanos , Células MCF-7 , Proteínas Nucleares/metabolismo , Ratos , Receptores Acoplados a Proteínas-G/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Proteínas de Transporte Vesicular/metabolismo , Xenopus laevis/genética , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismo
6.
Nat Commun ; 8(1): 1383, 2017 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-29123099

RESUMO

Asymmetric cell division, creating sibling cells with distinct developmental potentials, can be manifested in sibling cell size asymmetry. This form of physical asymmetry occurs in several metazoan cells, but the underlying mechanisms and function are incompletely understood. Here we use Drosophila neural stem cells to elucidate the mechanisms involved in physical asymmetry establishment. We show that Myosin relocalizes to the cleavage furrow via two distinct cortical Myosin flows: at anaphase onset, a polarity induced, basally directed Myosin flow clears Myosin from the apical cortex. Subsequently, mitotic spindle cues establish a Myosin gradient at the lateral neuroblast cortex, necessary to trigger an apically directed flow, removing Actomyosin from the basal cortex. On the basis of the data presented here, we propose that spatiotemporally controlled Myosin flows in conjunction with spindle positioning and spindle asymmetry are key determinants for correct cleavage furrow placement and cortical expansion, thereby establishing physical asymmetry.


Assuntos
Miosinas/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Fuso Acromático/metabolismo , Actomiosina/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/citologia , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular , Tamanho Celular , Cromatina/genética , Cromatina/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Larva , Miosinas/genética , Fuso Acromático/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
7.
J Cell Biol ; 216(12): 4165-4182, 2017 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-29051265

RESUMO

Isoprenylcysteine carboxyl methyltransferase (ICMT) methylesterifies C-terminal prenylcysteine residues of CaaX proteins and some RAB GTPases. Deficiency of either ICMT or NOTCH1 accelerates pancreatic neoplasia in Pdx1-Cre;LSL-KrasG12D mice, suggesting that ICMT is required for NOTCH signaling. We used Drosophila melanogaster wing vein and scutellar bristle development to screen Rab proteins predicted to be substrates for ICMT (ste14 in flies). We identified Rab7 and Rab8 as ICMT substrates that when silenced phenocopy ste14 deficiency. ICMT, RAB7, and RAB8 were all required for efficient NOTCH1 signaling in mammalian cells. Overexpression of RAB8 rescued NOTCH activation after ICMT knockdown both in U2OS cells expressing NOTCH1 and in fly wing vein development. ICMT deficiency induced mislocalization of GFP-RAB7 and GFP-RAB8 from endomembrane to cytosol, enhanced binding to RABGDI, and decreased GTP loading of RAB7 and RAB8. Deficiency of ICMT, RAB7, or RAB8 led to mislocalization and diminished processing of NOTCH1-GFP. Thus, NOTCH signaling requires ICMT in part because it requires methylated RAB7 and RAB8.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , GTP Fosfo-Hidrolases/genética , Regulação Neoplásica da Expressão Gênica , Metiltransferases de Proteína/genética , Receptor Notch1/genética , Proteínas rab de Ligação ao GTP/genética , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Animais , Linhagem Celular Tumoral , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Metilação , Camundongos , Osteoblastos/metabolismo , Osteoblastos/patologia , Metiltransferases de Proteína/deficiência , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Transativadores/genética , Transativadores/metabolismo , Asas de Animais , Proteínas rab de Ligação ao GTP/metabolismo
8.
Plant Sci ; 263: 1-11, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28818364

RESUMO

Physiological responses of plants to salinity stress requires the coordinated activation of many genes. A salt-induced gene was isolated from roots of the wild tomato species Solanum chilense and named SchRabGDI1 because it encodes a protein with high identity to GDP dissociation inhibitors of plants. These proteins are regulators of the RabGTPase cycle that play key roles in intracellular vesicular trafficking. The expression pattern of SchRabGDI1 showed an early up-regulation in roots and leaves under salt stress. Functional activity of SchRabGDI1 was shown by restoring the defective phenotype of the yeast sec19-1 mutant and the capacity of SchRabGDI1 to interact with RabGTPase was demonstrated through BiFC assays. Expression of SchRabGDI1 in Arabidopsis thaliana plants resulted in increased salt tolerance. Also, the root cells of transgenic plants showed higher rate of endocytosis under normal growth conditions and higher accumulation of sodium in vacuoles and small vesicular structures under salt stress than wild type. Our results suggest that in salt tolerant species such as S. chilense, bulk endocytosis is one of the early mechanisms to avoid salt stress, which requires the concerted expression of regulatory genes involved in vesicular trafficking of the endocytic pathway.


Assuntos
Regulação da Expressão Gênica de Plantas , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Solanum/genética , Arabidopsis/genética , Arabidopsis/fisiologia , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Modelos Estruturais , Folhas de Planta/genética , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Brotos de Planta/genética , Brotos de Planta/fisiologia , Transporte Proteico , Salinidade , Tolerância ao Sal , Cloreto de Sódio/metabolismo , Solanum/fisiologia , Estresse Fisiológico , Vesículas Transportadoras/metabolismo , Regulação para Cima
9.
EMBO Rep ; 18(9): 1509-1520, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28684399

RESUMO

In many cell types, mitotic spindle orientation relies on the canonical "LGN complex" composed of Pins/LGN, Mud/NuMA, and Gαi subunits. Membrane localization of this complex recruits motor force generators that pull on astral microtubules to orient the spindle. Drosophila Pins shares highly conserved functional domains with its two vertebrate homologs LGN and AGS3. Whereas the role of Pins and LGN in oriented divisions is extensively documented, involvement of AGS3 remains controversial. Here, we show that AGS3 is not required for planar divisions of neural progenitors in the mouse neocortex. AGS3 is not recruited to the cell cortex and does not rescue LGN loss of function. Despite conserved interactions with NuMA and Gαiin vitro, comparison of LGN and AGS3 functional domains in vivo reveals unexpected differences in the ability of these interactions to mediate spindle orientation functions. Finally, we find that Drosophila Pins is unable to substitute for LGN loss of function in vertebrates, highlighting that species-specific modulations of the interactions between components of the Pins/LGN complex are crucial in vivo for spindle orientation.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Drosophila/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Fuso Acromático/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Ciclo Celular , Divisão Celular , Polaridade Celular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Inibidores de Dissociação do Nucleotídeo Guanina/química , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Camundongos , Microtúbulos/metabolismo , Neocórtex/fisiologia , Proteínas Nucleares/metabolismo , Ligação Proteica , Domínios Proteicos , Fuso Acromático/genética
10.
Neuroscience ; 344: 346-359, 2017 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-28057534

RESUMO

RAB-GDP dissociation inhibitor 1 (GDI1) loss-of-function mutations are responsible for a form of non-specific X-linked Intellectual Disability (XLID) where the only clinical feature is cognitive impairment. GDI1 patients are impaired in specific aspects of executive functions and conditioned response, which are controlled by fronto-striatal circuitries. Previous molecular and behavioral characterization of the Gdi1-null mouse revealed alterations in the total number/distribution of hippocampal and cortical synaptic vesicles as well as hippocampal short-term synaptic plasticity, and memory deficits. In this study, we employed cognitive protocols with high translational validity to human condition that target the functionality of cortico-striatal circuitry such as attention and stimulus selection ability with progressive degree of complexity. We previously showed that Gdi1-null mice are impaired in some hippocampus-dependent forms of associative learning assessed by aversive procedures. Here, using appetitive-conditioning procedures we further investigated associative learning deficits sustained by the fronto-striatal system. We report that Gdi1-null mice are impaired in attention and associative learning processes, which are a key part of the cognitive impairment observed in XLID patients.


Assuntos
Lobo Frontal/fisiopatologia , Inibidores de Dissociação do Nucleotídeo Guanina/deficiência , Deficiência Intelectual/fisiopatologia , Neostriado/fisiopatologia , Tonsila do Cerebelo/diagnóstico por imagem , Tonsila do Cerebelo/fisiopatologia , Animais , Aprendizagem por Associação/fisiologia , Atenção/fisiologia , Condicionamento Psicológico/fisiologia , Discriminação Psicológica/fisiologia , Modelos Animais de Doenças , Dopamina/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Lobo Frontal/diagnóstico por imagem , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Inibição Psicológica , Deficiência Intelectual/diagnóstico por imagem , Deficiência Intelectual/psicologia , Masculino , Camundongos Knockout , Neostriado/diagnóstico por imagem , Vias Neurais/diagnóstico por imagem , Vias Neurais/fisiopatologia , Distribuição Aleatória , Vesículas Sinápticas/metabolismo , Percepção do Tempo/fisiologia , Técnicas de Cultura de Tecidos
11.
Mol Neurobiol ; 54(4): 2458-2468, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-26971292

RESUMO

X-linked non-syndromic intellectual disability (XLID) is a common mental disorder recognized by cognitive and behavioral deficits. Mutations in the brain-specific αGDI, shown to alter a subset of RAB GTPases redistribution in cells, are linked to XLID, likely via changes in vesicle traffic in neurons. Here, we show directly that isolated XLID mice astrocytes, devoid of pathologic tissue environment, exhibit vesicle mobility deficits. Contrary to previous studies, we show that astrocytes express two GDI proteins. The siRNA-mediated suppression of expression of αGDI especially affected vesicle dynamics. A similar defect was recorded in astrocytes from the Gdi1 -/Y mouse model of XLID and in astrocytes with recombinant mutated human XLID αGDI. Endolysosomal vesicles studied here are involved in the release of gliosignaling molecules as well as in regulating membrane receptor density; thus, the observed changes in astrocytic vesicle mobility may, over the long time-course, profoundly affect signaling capacity of these cells, which optimize neural activity.


Assuntos
Astrócitos/metabolismo , Vesículas Citoplasmáticas/metabolismo , Genes Ligados ao Cromossomo X , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Deficiência Intelectual/genética , Animais , Astrócitos/patologia , Modelos Animais de Doenças , Endossomos/metabolismo , Inativação Gênica , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Lisossomos/metabolismo , Masculino , Camundongos , Mutação/genética , Ratos , Transfecção
12.
Gene ; 599: 78-86, 2017 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-27836664

RESUMO

Vesicle shuttling is critical for many cellular and organismal processes, including embryonic development. GDI proteins contribute to vesicle shuttling by regulating the activity of Rab GTPases, controlling their cycling between the inactive cytosol and active membrane bound states. While identifying genes controlled by A-form DNA sequences we discovered a previously unknown member of the GDI family, GDI3. The GDI3 gene is found only in amphibians and fish and is developmentally expressed in Xenopus from neurula stages onwards in the neural plate, and subsequently in both dorsal and anterior structures. Depletion or over-expression of the GDI3 protein in Xenopus embryos gives rise to very similar phenotypes, suggesting that strict control of GDI3 protein levels is required for correct embryonic development. Our analysis suggests the evolutionary origins of GDI3 and that it is functionally distinct from GDI1. Predicted structural analysis of GDI3 suggests that the key difference between GDI1 and GDI3 lies in their lipid binding pockets.


Assuntos
Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Neurogênese/fisiologia , Proteínas de Xenopus/metabolismo , Xenopus/embriologia , Xenopus/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Sequência de Bases , Clonagem Molecular , DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Inibidores de Dissociação do Nucleotídeo Guanina/química , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Modelos Moleculares , Neurogênese/genética , Filogenia , Xenopus/genética , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/genética
13.
J Clin Invest ; 126(12): 4537-4553, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27797340

RESUMO

Certain secretory proteins are known to be critical for maintaining the stemness of stem cells through autocrine signaling. However, the processes underlying the biogenesis, maturation, and secretion of these proteins remain largely unknown. Here we demonstrate that many secretory proteins produced by hematopoietic stem cells (HSCs) undergo exosomal maturation and release that is controlled by vacuolar protein sorting protein 33b (VPS33B). Deletion of VPS33B in either mouse or human HSCs resulted in impaired exosome maturation and secretion as well as loss of stemness. Additionally, VPS33B deficiency led to a dramatic delay in leukemogenesis. Exosomes purified from either conditioned medium or human plasma could partially rescue the defects of HSCs and leukemia-initiating cells (LICs). VPS33B co-existed in exosomes with GDI2, VPS16B, FLOT1, and other known exosome markers. Mechanistically, VPS33B interacted with the GDI2/RAB11A/RAB27A pathway to regulate the trafficking of secretory proteins as exosomes. These findings reveal an essential role for VPS33B in exosome pathways in HSCs and LICs. Moreover, they shed light on the understanding of vesicle trafficking in other stem cells and on the development of improved strategies for cancer treatment.


Assuntos
Comunicação Autócrina , Transformação Celular Neoplásica/metabolismo , Exossomos/metabolismo , Hematopoese , Leucemia/metabolismo , Transdução de Sinais , Proteínas de Transporte Vesicular/metabolismo , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Exossomos/genética , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Células HEK293 , Humanos , Leucemia/genética , Leucemia/patologia , Camundongos , Camundongos Knockout , Transporte Proteico/genética , Proteínas de Transporte Vesicular/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab27 de Ligação ao GTP
14.
Sci Rep ; 6: 37058, 2016 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-27841328

RESUMO

The role of GDP dissociation inhibitor (GDI) protein in regulation of Rab cycle in Leishmania is not known. Here, we have cloned and characterized the functions of GDI homologue in vivo in Leishmania. Our results have shown that LdGDI:WT along with GDP removes the Rab5 from purified endosomes and inhibits the homotypic fusion between early endosomes. Whereas, LdGDI:R239A, a dominant negative mutant of GDI, under the same condition neither removes the Rab5 from endosome nor inhibits fusion. To determine the role of Ld-GDI in vivo, transgenic parasites overexpressing GFP-LdGDI:WT or GFP-LdGDI:R239A, are co-expressed with RFP-LdRab5:WT, RFP-LdRab7:WT or RFP-LdRab1:WT. Our results have shown that overexpression of GFP-LdGDI:WT extracts the RFP-LdRab5, RFP-LdRab7 or RFP-LdRab1 from their discrete endomembrane predominantly into cytosol. No change in the distribution of indicated Rabs is detected with overexpression of GFP-LdGDI:R239A. To determine the functional significance, we have used hemoglobin as an endocytic marker and gp63 as a marker for secretory pathway. We have found that overexpression of GFP-LdGDI:WT enhances the lysosomal targeting of internalized hemoglobin and the secretion of gp63 in the parasites possibly by triggering Rab cycle. This is the first demonstration of a single GDI ubiquitously regulating both endocytic and secretory pathways in Leishmania.


Assuntos
Endocitose/fisiologia , Endossomos/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Leishmania donovani/metabolismo , Metaloendopeptidases/metabolismo , Proteínas de Protozoários/metabolismo , Endossomos/genética , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Leishmania donovani/genética , Metaloendopeptidases/genética , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Proteínas de Protozoários/genética , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo
15.
Genes Immun ; 17(6): 321-7, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27307211

RESUMO

G Protein Signaling Modulator-3 (GPSM3) is a leukocyte-specific regulator of G protein-coupled receptors (GPCRs), which binds inactivated Gαi·GDP subunits and precludes their reassociation with Gßγ subunits. GPSM3 deficiency protects mice from inflammatory arthritis and, in humans, GPSM3 single-nucleotide polymorphisms (SNPs) are inversely associated with the risk of rheumatoid arthritis development; recently, these polymorphisms were linked to one particular SNP (rs204989) that decreases GPSM3 transcript abundance. However, the precise role of GPSM3 in leukocyte biology is unknown. Here, we show that GPSM3 is induced in the human promyelocytic leukemia NB4 cell line following retinoic acid treatment, which differentiates this cell line into a model of neutrophil physiology (NB4*). Reducing GPSM3 expression in NB4* cells, akin to the effect ascribed to the rs204989 C>T transition, disrupts cellular migration toward leukotriene B4 (LTB4) and (to a lesser extent) interleukin-8 (a.k.a. IL-8 or CXCL8), but not migration toward formylated peptides (fMLP). As the chemoattractants LTB4 and CXCL8 are involved in recruitment of neutrophils to the arthritic joint, our results suggest that the arthritis-protective GPSM3 SNP rs204989 may act to decrease neutrophil chemoattractant responsiveness.


Assuntos
Artrite Reumatoide/genética , Quimiotaxia de Leucócito , Inibidores de Dissociação do Nucleotídeo Guanina/fisiologia , Neutrófilos/metabolismo , Artrite Reumatoide/imunologia , Linhagem Celular Tumoral , Quimiotaxia de Leucócito/genética , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Humanos , Interleucina-8/metabolismo , Leucopoese , Leucotrieno B4/metabolismo , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Polimorfismo de Nucleotídeo Único , Tretinoína/metabolismo
16.
Sci Rep ; 6: 24948, 2016 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-27109834

RESUMO

Mammalian neuroglobin (Ngb) protects neuronal cells under conditions of oxidative stress. We previously showed that human Ngb acts as a guanine nucleotide dissociation inhibitor (GDI) for the α-subunits of heterotrimeric Gi/o proteins and inhibits the decrease in cAMP concentration, leading to protection against cell death. In the present study, we used an eukaryotic expression vector driving high-level expression of human wild-type Ngb or Ngb mutants that either exhibit or lack GDI activities in human cells. We demonstrate that the GDI activity of human Ngb is tightly correlated with its neuroprotective activity. We further demonstrate that Glu53, Glu60, and Glu118 of human Ngb are crucial for both the neuroprotective activity and interaction with Gαi1. Moreover, we show that Lys46, Lys70, Arg208, Lys209, and Lys210 residues of Gαi1 are important for binding to human Ngb. We propose a molecular docking model of the complex between human Ngb and Gαi1.


Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/antagonistas & inibidores , Globinas/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Mapas de Interação de Proteínas , Linhagem Celular , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Globinas/genética , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Humanos , Simulação de Acoplamento Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas do Tecido Nervoso/genética , Neuroglobina , Ligação Proteica , Mapeamento de Interação de Proteínas
17.
Mol Biol Evol ; 33(7): 1833-42, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27034425

RESUMO

A complex endomembrane system is one of the hallmarks of Eukaryotes. Vesicle trafficking between compartments is controlled by a diverse protein repertoire, including Rab GTPases. These small GTP-binding proteins contribute identity and specificity to the system, and by working as molecular switches, trigger multiple events in vesicle budding, transport, and fusion. A diverse collection of Rab GTPases already existed in the ancestral Eukaryote, yet, it is unclear how such elaborate repertoire emerged. A novel archaeal phylum, the Lokiarchaeota, revealed that several eukaryotic-like protein systems, including small GTPases, are present in Archaea. Here, we test the hypothesis that the Rab family of small GTPases predates the origin of Eukaryotes. Our bioinformatic pipeline detected multiple putative Rab-like proteins in several archaeal species. Our analyses revealed the presence and strict conservation of sequence features that distinguish eukaryotic Rabs from other small GTPases (Rab family motifs), mapping to the same regions in the structure as in eukaryotic Rabs. These mediate Rab-specific interactions with regulators of the REP/GDI (Rab Escort Protein/GDP dissociation Inhibitor) family. Sensitive structure-based methods further revealed the existence of REP/GDI-like genes in Archaea, involved in isoprenyl metabolism. Our analysis supports a scenario where Rabs differentiated into an independent family in Archaea, interacting with proteins involved in membrane biogenesis. These results further support the archaeal nature of the eukaryotic ancestor and provide a new insight into the intermediate stages and the evolutionary path toward the complex membrane-associated signaling circuits that characterize the Ras superfamily of small GTPases, and specifically Rab proteins.


Assuntos
Archaea/enzimologia , Proteínas rab de Ligação ao GTP/metabolismo , Archaea/genética , Archaea/metabolismo , Evolução Biológica , Células Eucarióticas/metabolismo , Evolução Molecular , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Filogenia , Ligação Proteica , Transporte Proteico , Análise de Sequência de Proteína/métodos , Proteínas rab de Ligação ao GTP/genética
18.
Biochem J ; 473(10): 1379-90, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-26987813

RESUMO

Transient receptor potential canonical 4 (TRPC4) forms non-selective cation channels implicated in the regulation of diverse physiological functions. Previously, TRPC4 was shown to be activated by the Gi/o subgroup of heterotrimeric G-proteins involving Gαi/o, rather than Gßγ, subunits. Because the lifetime and availability of Gα-GTP are regulated by regulators of G-protein signalling (RGS) and Gαi/o-Loco (GoLoco) domain-containing proteins via their GTPase-activating protein (GAP) and guanine-nucleotide-dissociation inhibitor (GDI) functions respectively, we tested how RGS and GoLoco domain proteins affect TRPC4 currents activated via Gi/o-coupled receptors. Using whole-cell patch-clamp recordings, we show that both RGS and GoLoco proteins [RGS4, RGS6, RGS12, RGS14, LGN or activator of G-protein signalling 3 (AGS3)] suppress receptor-mediated TRPC4 activation without causing detectable basal current or altering surface expression of the channel protein. The inhibitory effects are dependent on the GAP and GoLoco domains and facilitated by enhancing membrane targeting of the GoLoco protein AGS3. In addition, RGS, but not GoLoco, proteins accelerate desensitization of receptor-activation evoked TRPC4 currents. The inhibitory effects of RGS and GoLoco domains are additive and are most prominent with RGS12 and RGS14, which contain both RGS and GoLoco domains. Our data support the notion that the Gα, but not Gßγ, arm of the Gi/o signalling is involved in TRPC4 activation and unveil new roles for RGS and GoLoco domain proteins in fine-tuning TRPC4 activities. The versatile and diverse functions of RGS and GoLoco proteins in regulating G-protein signalling may underlie the complexity of receptor-operated TRPC4 activation in various cell types under different conditions.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Canais de Cátion TRPC/metabolismo , Western Blotting , Eletrofisiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/genética , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Células HEK293 , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutação Puntual , Ligação Proteica , Proteínas RGS/genética , Proteínas RGS/metabolismo , Transdução de Sinais , Canais de Cátion TRPC/genética
19.
Genes Immun ; 17(2): 139-47, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26821282

RESUMO

G protein signaling modulator 3 (GPSM3) is a regulator of G protein-coupled receptor signaling, with expression restricted to leukocytes and lymphoid organs. Previous genome-wide association studies have highlighted single-nucleotide polymorphisms (SNPs; rs204989 and rs204991) in a region upstream of the GPSM3 transcription start site as being inversely correlated to the prevalence of rheumatoid arthritis (RA)-this association is supported by the protection afforded to Gpsm3-deficient mice in models of inflammatory arthritis. Here, we assessed the functional consequences of these polymorphisms. We collected biospecimens from 50 volunteers with RA diagnoses, 50 RA-free volunteers matched to the aforementioned group and 100 unmatched healthy young volunteers. We genotyped these individuals for GPSM3 (rs204989, rs204991), CCL21 (rs2812378) and HLA gene region (rs6457620) polymorphisms, and found no significant differences in minor allele frequencies between the RA and disease-free cohorts. However, we identified that individuals homozygous for SNPs rs204989 and rs204991 had decreased GPSM3 transcript abundance relative to individuals homozygous for the major allele. In vitro promoter activity studies suggest that SNP rs204989 is the primary cause of this decrease in transcript levels. Knockdown of GPSM3 in THP-1 cells, a human monocytic cell line, was found to disrupt ex vivo migration to the chemokine MCP-1.


Assuntos
Artrite Reumatoide/genética , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/genética , Adulto , Idoso , Alelos , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Estudos de Casos e Controles , Linhagem Celular , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiotaxia , Feminino , Expressão Gênica , Frequência do Gene , Genótipo , Inibidores de Dissociação do Nucleotídeo Guanina/antagonistas & inibidores , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Monócitos/patologia , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
20.
PLoS Genet ; 12(1): e1005786, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26765257

RESUMO

Invadopodia are specialized membrane protrusions composed of F-actin, actin regulators, signaling proteins, and a dynamically trafficked invadopodial membrane that drive cell invasion through basement membrane (BM) barriers in development and cancer. Due to the challenges of studying invasion in vivo, mechanisms controlling invadopodia formation in their native environments remain poorly understood. We performed a sensitized genome-wide RNAi screen and identified 13 potential regulators of invadopodia during anchor cell (AC) invasion into the vulval epithelium in C. elegans. Confirming the specificity of this screen, we identified the Rho GTPase cdc-42, which mediates invadopodia formation in many cancer cell lines. Using live-cell imaging, we show that CDC-42 localizes to the AC-BM interface and is activated by an unidentified vulval signal(s) that induces invasion. CDC-42 is required for the invasive membrane localization of WSP-1 (N-WASP), a CDC-42 effector that promotes polymerization of F-actin. Loss of CDC-42 or WSP-1 resulted in fewer invadopodia and delayed BM breaching. We also characterized a novel invadopodia regulator, gdi-1 (Rab GDP dissociation inhibitor), which mediates membrane trafficking. We show that GDI-1 functions in the AC to promote invadopodia formation. In the absence of GDI-1, the specialized invadopodial membrane was no longer trafficked normally to the invasive membrane, and instead was distributed to plasma membrane throughout the cell. Surprisingly, the pro-invasive signal(s) from the vulval cells also controls GDI-1 activity and invadopodial membrane trafficking. These studies represent the first in vivo screen for genes regulating invadopodia and demonstrate that invadopodia formation requires the integration of distinct cellular processes that are coordinated by an extracellular cue.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ligação ao GTP/genética , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Neoplasias/genética , Podossomos/genética , Animais , Membrana Basal/crescimento & desenvolvimento , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/biossíntese , Proteínas de Ciclo Celular/biossíntese , Modelos Animais de Doenças , Matriz Extracelular/genética , Proteínas de Ligação ao GTP/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Inibidores de Dissociação do Nucleotídeo Guanina/biossíntese , Humanos , Neoplasias/patologia , Podossomos/patologia , Transdução de Sinais
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