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1.
Nat Commun ; 11(1): 4576, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917905

RESUMO

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


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Endossomos/fisiologia , Metabolismo dos Lipídeos , Neurônios/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Feminino , Células HEK293 , Células HeLa , Humanos , Lipídeos , Lipossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Mitocôndrias , Domínios Proteicos , Proteômica , Proteínas Recombinantes , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/genética
2.
Nat Commun ; 11(1): 3848, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737286

RESUMO

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease characterized by the degeneration of upper and lower motor neurons (MNs). We find a significant reduction of the retromer complex subunit VPS35 in iPSCs-derived MNs from ALS patients, in MNs from ALS post mortem explants and in MNs from SOD1G93A mice. Being the retromer involved in trafficking of hydrolases, a pathological hallmark in ALS, we design, synthesize and characterize an array of retromer stabilizers based on bis-guanylhydrazones connected by a 1,3-phenyl ring linker. We select compound 2a as a potent and bioavailable interactor of VPS35-VPS29. Indeed, while increasing retromer stability in ALS mice, compound 2a attenuates locomotion impairment and increases MNs survival. Moreover, compound 2a increases VPS35 in iPSCs-derived MNs and shows brain bioavailability. Our results clearly suggest the retromer as a valuable druggable target in ALS.


Assuntos
Esclerose Amiotrófica Lateral/tratamento farmacológico , Hidrazonas/farmacologia , Neurônios Motores/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Proteínas de Transporte Vesicular/genética , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Diferenciação Celular , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Hidrazonas/síntese química , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/síntese química , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica , Relação Estrutura-Atividade , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Proteínas de Transporte Vesicular/metabolismo
3.
PLoS One ; 15(7): e0235864, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645101

RESUMO

In eukaryotic cells, KDEL receptors (KDELRs) facilitate the retrieval of endoplasmic reticulum (ER) luminal proteins from the Golgi compartment back to the ER. Apart from the well-documented retention function, recent findings reveal that the cellular KDELRs have more complex roles, e.g. in cell signalling, protein secretion, cell adhesion and tumorigenesis. Furthermore, several studies suggest that a sub-population of KDELRs is located at the cell surface, where they could form and internalize KDELR/cargo clusters after K/HDEL-ligand binding. However, so far it has been unclear whether there are species- or cell-type-specific differences in KDELR clustering. By comparing ligand-induced KDELR clustering in different mouse and human cell lines via live cell imaging, we show that macrophage cell lines from both species do not develop any clusters. Using RT-qPCR experiments and numerical analysis, we address the role of KDELR expression as well as endocytosis and exocytosis rates on the receptor clustering at the plasma membrane and discuss how the efficiency of directed transport to preferred docking sites on the membrane influences the exponent of the power-law distribution of the cluster size.


Assuntos
Receptores de Peptídeos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Endocitose , Exocitose , Expressão Gênica , Humanos , Macrófagos/metabolismo , Camundongos , Transporte Proteico , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores de Peptídeos/análise , Receptores de Peptídeos/genética , Proteínas de Transporte Vesicular/análise , Proteínas de Transporte Vesicular/genética
4.
Proc Natl Acad Sci U S A ; 117(25): 14158-14167, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513738

RESUMO

Eukaryotic N-degron pathways are proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal (Nt) degradation signals called N-degrons, and to target these proteins for degradation by the 26S proteasome or autophagy. GID4, a subunit of the GID ubiquitin ligase, is the main recognition component of the proline (Pro)/N-degron pathway. GID4 targets proteins through their Nt-Pro residue or a Pro at position 2, in the presence of specific downstream sequence motifs. Here we show that human GID4 can also recognize hydrophobic Nt-residues other than Pro. One example is the sequence Nt-IGLW, bearing Nt-Ile. Nt-IGLW binds to wild-type human GID4 with a K d of 16 µM, whereas the otherwise identical Nt-Pro-bearing sequence PGLW binds to GID4 more tightly, with a K d of 1.9 µM. Despite this difference in affinities of GID4 for Nt-IGLW vs. Nt-PGLW, we found that the GID4-mediated Pro/N-degron pathway of the yeast Saccharomyces cerevisiae can target an Nt-IGLW-bearing protein for rapid degradation. We solved crystal structures of human GID4 bound to a peptide bearing Nt-Ile or Nt-Val. We also altered specific residues of human GID4 and measured the affinities of resulting mutant GID4s for Nt-IGLW and Nt-PGLW, thereby determining relative contributions of specific GID4 residues to the GID4-mediated recognition of Nt-Pro vs. Nt-residues other than Pro. These and related results advance the understanding of targeting by the Pro/N-degron pathway and greatly expand the substrate recognition range of the GID ubiquitin ligase in both human and yeast cells.


Assuntos
Prolina/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/química , Proteínas de Transporte Vesicular/química , Humanos , Modelos Moleculares , Prolina/metabolismo , Complexo de Endopeptidases do Proteassoma , Conformação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
5.
DNA Cell Biol ; 39(6): 1041-1050, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32401536

RESUMO

Ovarian cancer (OC) is one of gynecological malignancies that seriously affects women's health. Mounting evidence demonstrated that long noncoding RNAs (lncRNAs), microRNAs (miRNAs), and messenger RNAs (mRNAs) play important roles in various biological processes related to the pathogenesis of OC. This research aimed to investigate the regulatory mechanism of lncRNA SCAMP1/miR-137/CXCL12 (C-X-C motif chemokine ligand 12) axis on OC progression. In this study, we found that SCAMP1 was highly expressed in OC cells, which promoted OC cell invasion and angiogenesis. In addition, our research confirmed that SCAMP1 could bind with miR-137, and SCAMP1 sponged miR-137 to accelerate the progression of OC. We also observed that CXCL12 was a downstream target gene for miR-137, and miR-137 targeted CXCL12 to participate in the regulation of OC. Finally, through TCGA database, we found that SCAMP1 (or CXCL12) was upregulated as well as miR-137 was downregulated in OC tissues, and high (or low) level of them was associated with poor prognosis. miR-137 expression was negatively correlated with SCAMP1 (or CXCL12) expression, and SCAMP1 expression was positively correlated with CXCL12 expression in OC. In summary, our study clarified the role of SCAMP1/miR-137/CXCL12 axis in OC, and this finding may provide a potential therapeutic target of OC.


Assuntos
Quimiocina CXCL12/genética , MicroRNAs/genética , Neovascularização Patológica/genética , Neoplasias Ovarianas/patologia , RNA Longo não Codificante/genética , Proteínas de Transporte Vesicular/genética , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica/genética , Neoplasias Ovarianas/irrigação sanguínea , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/genética , Prognóstico
6.
Proc Natl Acad Sci U S A ; 117(24): 13468-13479, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32467162

RESUMO

The functions of nervous and neuroendocrine systems rely on fast and tightly regulated release of neurotransmitters stored in secretory vesicles through SNARE-mediated exocytosis. Few proteins, including tomosyn (STXBP5) and amisyn (STXBP6), were proposed to negatively regulate exocytosis. Little is known about amisyn, a 24-kDa brain-enriched protein with a SNARE motif. We report here that full-length amisyn forms a stable SNARE complex with syntaxin-1 and SNAP-25 through its C-terminal SNARE motif and competes with synaptobrevin-2/VAMP2 for the SNARE-complex assembly. Furthermore, amisyn contains an N-terminal pleckstrin homology domain that mediates its transient association with the plasma membrane of neurosecretory cells by binding to phospholipid PI(4,5)P2 However, unlike synaptrobrevin-2, the SNARE motif of amisyn is not sufficient to account for the role of amisyn in exocytosis: Both the pleckstrin homology domain and the SNARE motif are needed for its inhibitory function. Mechanistically, amisyn interferes with the priming of secretory vesicles and the sizes of releasable vesicle pools, but not vesicle fusion properties. Our biochemical and functional analyses of this vertebrate-specific protein unveil key aspects of negative regulation of exocytosis.


Assuntos
Exocitose , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Membrana Celular/metabolismo , Células Cultivadas , Células Cromafins/metabolismo , Humanos , Lipossomos/metabolismo , Fusão de Membrana , Células PC12 , Domínios de Homologia à Plecstrina , Ligação Proteica , Ratos , Proteínas SNARE/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Sintaxina 1/metabolismo , Vertebrados , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética
7.
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 , Cinesina/genética , Cinesina/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Corpos Multivesiculares/genética , Corpos Multivesiculares/metabolismo , Mutação , Vacúolos/genética , Proteínas de Transporte Vesicular/metabolismo
8.
PLoS Biol ; 18(4): e3000704, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32251437

RESUMO

Lymph nodes (LNs) are highly organized secondary lymphoid organs that mediate adaptive immune responses to antigens delivered via afferent lymphatic vessels. Lymphatic endothelial cells (LECs) line intranodal lymphatic sinuses and organize lymph and antigen distribution. LECs also directly regulate T cells, mediating peripheral tolerance to self-antigens, and play a major role in many diseases, including cancer metastasis. However, little is known about the phenotypic and functional heterogeneity of LN LECs. Using single-cell RNA sequencing, we comprehensively defined the transcriptome of LECs in murine skin-draining LNs and identified new markers and functions of distinct LEC subpopulations. We found that LECs residing in the subcapsular sinus (SCS) have an unanticipated function in scavenging of modified low-density lipoprotein (LDL) and also identified a specific cortical LEC subtype implicated in rapid lymphocyte egress from LNs. Our data provide new, to our knowledge, insights into the diversity of LECs in murine LNs and a rich resource for future studies into the regulation of immune responses by LN LECs.


Assuntos
Linfonodos/citologia , Análise de Célula Única/métodos , Animais , Biomarcadores/metabolismo , Células Endoteliais/citologia , Endotélio Linfático/citologia , Citometria de Fluxo , Imunofluorescência , Perfilação da Expressão Gênica , Humanos , Integrina alfa2/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Receptores CCR/genética , Receptores CCR/metabolismo , Análise de Sequência de RNA , Proteínas de Transporte Vesicular/genética
9.
BMC Med Genet ; 21(1): 59, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32209057

RESUMO

BACKGROUND: Intellectual disability (ID) is both a clinically diverse and genetically heterogeneous group of disorder, with an onset of cognitive impairment before the age of 18 years. ID is characterized by significant limitations in intellectual functioning and adaptive behaviour. The identification of genetic variants causing ID and neurodevelopmental disorders using whole-exome sequencing (WES) has proven to be successful. So far more than 1222 primary and 1127 candidate genes are associated with ID. METHODS: To determine pathogenic variants causative of ID in three unrelated consanguineous Pakistani families, we used a combination of WES, homozygosity-by-descent mapping, de-deoxy sequencing and bioinformatics analysis. RESULTS: Rare pathogenic single nucleotide variants identified by WES which passed our filtering strategy were confirmed by traditional Sanger sequencing and segregation analysis. Novel and deleterious variants in VPS53, GLB1, and MLC1, genes previously associated with variable neurodevelopmental anomalies, were found to segregate with the disease in the three families. CONCLUSIONS: This study expands our knowledge on the molecular basis of ID as well as the clinical heterogeneity associated to different rare genetic causes of neurodevelopmental disorders. This genetic study could also provide additional knowledge to help genetic assessment as well as clinical and social management of ID in Pakistani families.


Assuntos
Consanguinidade , Deficiência Intelectual/genética , Proteínas de Membrana/genética , Polimorfismo Genético , Proteínas de Transporte Vesicular/genética , beta-Galactosidase/genética , Criança , Pré-Escolar , Família , Feminino , Genes Recessivos/genética , Heterogeneidade Genética , Testes Genéticos , Homozigoto , Humanos , Deficiência Intelectual/complicações , Deficiência Intelectual/patologia , Masculino , Transtornos do Neurodesenvolvimento/complicações , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/patologia , Paquistão/epidemiologia , Linhagem , Sequenciamento Completo do Exoma
10.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(4): 441-444, 2020 Apr 10.
Artigo em Chinês | MEDLINE | ID: mdl-32219832

RESUMO

OBJECTIVE: To detect pathological variant in two patients with Chediak-Higashi syndrome (CHS) from a consanguineous family and to explore its genotype-phenotype correlation. METHODS: Clinical data was collected for this pedigree. Genomic DNA was prepared from probands' peripheral leukocytes and their relatives' fingernail. Whole exome sequencing and Sanger sequencing were carried out to detect potential variant of the LYST gene. RESULTS: The proband presented with partial oculocutaneous albinism, immunodeficiency and acidophilic inclusion body in bone marrow and blood smears. A novel homozygous nonsense variant c.8782C>T (p.Gln2928*) was identified in exon 34 of the LYST gene in the sib pair. The same variant was found to be in heterozygous status in 6 unaffected individuals from the pedigree. CONCLUSION: Above result enriched the mutational spectrum of CHS and provided a basis for genetic counseling and prenatal diagnosis for this pedigree.


Assuntos
Síndrome de Chediak-Higashi/genética , Linhagem , Proteínas de Transporte Vesicular/genética , Éxons , Heterozigoto , Humanos , Mutação , Análise de Sequência de DNA , Sequenciamento Completo do Exoma
11.
Epilepsia ; 61(4): 810-821, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32112430

RESUMO

OBJECTIVE: Genetic variants in STXBP1, which encodes the conserved exocytosis protein Munc18-1, are associated with a variety of infantile epilepsy syndromes. We aimed to develop an in vivo Caenorhabditis elegans model that could be used to test the pathogenicity of such variants in a cost-effective manner. METHODS: The CRISPR/Cas9 method was used to introduce a null mutation into the unc-18 gene (the C. elegans orthologue of STXBP1), thereby creating a paralyzed worm strain. We subsequently rescued this strain with transgenes encoding the human STXBP1/Munc18-1 protein (wild-type and eight different epilepsy-associated missense variants). The resulting humanized worm strains were then analyzed via behavioral, electrophysiological, and biochemical approaches. RESULTS: Transgenic expression of wild-type human STXBP1 protein fully rescued locomotion in both solid and liquid media to the same level as the standard wild-type worm strain, Bristol N2. Six variant strains (E59K, V84D, C180Y, R292H, L341P, R551C) exhibited impaired locomotion, whereas two (P335L, R406H) were no different from worms expressing wild-type STXBP1. Electrophysiological recordings revealed that all eight variant strains displayed less frequent and more irregular pharyngeal pumping in comparison to wild-type STXBP1-expressing strains. Four strains (V84D, C180Y, R292H, P335L) exhibited pentylenetetrazol-induced convulsions in an acute assay of seizure-like activity, in contrast to worms expressing wild-type STXBP1. No differences were seen between wild-type and variant STXBP1 strains in terms of mRNA abundance. However, STXBP1 protein levels were reduced to 20%-30% of wild-type in all variants, suggesting that the mutations result in STXBP1 protein instability. SIGNIFICANCE: The approach described here is a cost-effective in vivo method for establishing the pathogenicity of genetic variants in STXBP1 and potentially other conserved neuronal proteins. Furthermore, the humanized strains we created could potentially be used in the future for high-throughput drug screens to identify novel therapeutics.


Assuntos
Modelos Animais de Doenças , Epilepsia/genética , Proteínas Munc18/genética , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Humanos , Mutação , Proteínas de Transporte Vesicular/genética
12.
Genes Cells ; 25(6): 391-401, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32167217

RESUMO

Vesicular transport serves as an important mechanism for cell shape regulation during development. Although the semaphorin signaling molecule, a well-known regulator of axon guidance, induces endocytosis in the growth cone and the axonal transport of vertebrate neurons, the underlying molecular mechanisms remain largely unclear. Here, we show that the Caenorhabditis elegans SNT-1/synaptotagmin-UNC-41/stonin2 system, whose role in synaptic vesicle recycling in neurons has been studied extensively, is involved in semaphorin-regulated vesicular transport in larval epidermal cells. Mutations in the snt-1/unc-41 genes strongly suppressed the cell shape defects of semaphorin mutants. The null mutation in the semaphorin receptor gene, plx-1, altered the expression and localization pattern of endocytic and exocytic markers in the epidermal cells while repressing the transport of SNT-1-containing vesicles toward late endosome/lysosome pathways. Our findings suggest that the nematode semaphorins regulate the vesicular transport in epidermal cells in a manner distinct from that of vertebrate semaphorins in neurons.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Células Epidérmicas/metabolismo , Semaforinas/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptotagminas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Animais Geneticamente Modificados , Transporte Biológico Ativo/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Endocitose/genética , Endossomos/genética , Endossomos/metabolismo , Exocitose/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Lisossomos/genética , Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Interferência de RNA , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Semaforinas/genética , Transdução de Sinais/genética , Sinaptotagminas/genética , Proteínas de Transporte Vesicular/genética
13.
Arterioscler Thromb Vasc Biol ; 40(5): 1311-1324, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32188273

RESUMO

OBJECTIVE: TMEM55B (transmembrane protein 55B) is a phosphatidylinositol-(4,5)-bisphosphate (PI[4,5]P2) phosphatase that regulates cellular cholesterol, modulates LDLR (low-density lipoprotein receptor) decay, and lysosome function. We tested the effects of Tmem55b knockdown on plasma lipids in mice and assessed the roles of LDLR lysosomal degradation and change in (PI[4,5]P2) in mediating these effects. Approach and Results: Western diet-fed C57BL/6J mice were treated with antisense oligonucleotides against Tmem55b or a nontargeting control for 3 to 4 weeks. Hepatic Tmem55b transcript and protein levels were reduced by ≈70%, and plasma non-HDL (high-density lipoprotein) cholesterol was increased ≈1.8-fold (P<0.0001). Immunoblot analysis of fast protein liquid chromatography (FPLC) fractions revealed enrichment of ApoE-containing particles in the LDL size range. In contrast, Tmem55b knockdown had no effect on plasma cholesterol in Ldlr-/- mice. In primary hepatocytes and liver tissues from Tmem55b knockdown mice, there was decreased LDLR protein. In the hepatocytes, there was increased lysosome staining and increased LDLR-lysosome colocalization. Impairment of lysosome function (incubation with NH4Cl or knockdown of the lysosomal proteins LAMP1 or RAB7) abolished the effect of TMEM55B knockdown on LDLR in HepG2 (human hepatoma) cells. Colocalization of the recycling endosome marker RAB11 (Ras-related protein 11) with LDLR in HepG2 cells was reduced by 50% upon TMEM55B knockdown. Finally, knockdown increased hepatic PI(4,5)P2 levels in vivo and in HepG2 cells, while TMEM55B overexpression in vitro decreased PI(4,5)P2. TMEM55B knockdown decreased, whereas overexpression increased, LDL uptake in HepG2 cells. Notably, the TMEM55B overexpression effect was reversed by incubation with PI(4,5)P2. Conclusions: These findings indicate a role for TMEM55B in regulating plasma cholesterol levels by affecting PI(4,5)P2-mediated LDLR lysosomal degradation.


Assuntos
Colesterol/sangue , Hepatócitos/metabolismo , Fígado/metabolismo , Lisossomos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatases de Fosfoinositídeos/metabolismo , Receptores de LDL/metabolismo , Animais , Dieta Hiperlipídica , Regulação para Baixo , Feminino , Células Hep G2 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatases de Fosfoinositídeos/genética , Transporte Proteico , Proteólise , Receptores de LDL/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
14.
PLoS Genet ; 16(3): e1008674, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32196494

RESUMO

Epithelial cell polarity defects support cancer progression. It is thus crucial to decipher the functional interactions within the polarity protein network. Here we show that Drosophila Girdin and its human ortholog (GIRDIN) sustain the function of crucial lateral polarity proteins by inhibiting the apical kinase aPKC. Loss of GIRDIN expression is also associated with overgrowth of disorganized cell cysts. Moreover, we observed cell dissemination from GIRDIN knockdown cysts and tumorspheres, thereby showing that GIRDIN supports the cohesion of multicellular epithelial structures. Consistent with these observations, alteration of GIRDIN expression is associated with poor overall survival in subtypes of breast and lung cancers. Overall, we discovered a core mechanism contributing to epithelial cell polarization from flies to humans. Our data also indicate that GIRDIN has the potential to impair the progression of epithelial cancers by preserving cell polarity and restricting cell dissemination.


Assuntos
Proteínas de Drosophila/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Células CACO-2 , Diferenciação Celular/fisiologia , Polaridade Celular/fisiologia , Proliferação de Células/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/genética , Morfogênese/fisiologia , Mapas de Interação de Proteínas , Proteína Quinase C/metabolismo , Proteínas de Transporte Vesicular/genética
15.
Proc Natl Acad Sci U S A ; 117(14): 7776-7781, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32193351

RESUMO

The Saccharomyces cerevisiae protein Ddi1 and its homologs in higher eukaryotes have been proposed to serve as shuttling factors that deliver ubiquitinated substrates to the proteasome. Although Ddi1 contains both ubiquitin-interacting UBA and proteasome-interacting UBL domains, the UBL domain is atypical, as it binds ubiquitin. Furthermore, unlike other shuttling factors, Ddi1 and its homologs contain a conserved helical domain (helical domain of Ddi1, HDD) and a retroviral-like protease (RVP) domain. The RVP domain is probably responsible for cleavage of the precursor of the transcription factor Nrf1 in higher eukaryotes, which results in the up-regulation of proteasomal subunit genes. However, enzymatic activity of the RVP domain has not yet been demonstrated, and the function of Ddi1 remains poorly understood. Here, we show that Ddi1 is a ubiquitin-dependent protease, which cleaves substrate proteins only when they are tagged with long ubiquitin chains (longer than about eight ubiquitins). The RVP domain is inactive in isolation, in contrast to its retroviral counterpart. Proteolytic activity of Ddi1 requires the HDD domain and is stimulated by the UBL domain, which mediates high-affinity interaction with the polyubiquitin chain. Compromising the activity of Ddi1 in yeast cells results in the accumulation of polyubiquitinated proteins. Aside from the proteasome, Ddi1 is the only known endoprotease that acts on polyubiquitinated substrates. Ddi1 and its homologs likely cleave polyubiquitinated substrates under conditions where proteasome function is compromised.


Assuntos
Chaperonas Moleculares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Ubiquitina/genética , Proteínas de Transporte Vesicular/genética , Poliubiquitina/genética , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica/genética , Domínios Proteicos/genética , Homologia de Sequência
16.
BMC Med Genet ; 21(1): 47, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-32131761

RESUMO

BACKGROUND: Choreoacanthocytosis (ChAc), is a rare neurodegenerative disease, characterized by movement disorders and acanthocytosis in the peripheral blood smears, and various neurological, neuropsychiatric and neuromuscular signs. It is caused by mutations in VPS13A gene with autosomal recessive pattern of inheritance. CASE PRESENTATION: Here we report two patients belonging to a consanguineous Moroccan family who present with movement disorder pathology. They were suspected to have choreoacanthocytosis according to biological, clinical and radiological finding. Thus, whole-exome sequencing was performed for precise diagnosis and identified a homozygous novel nonsense mutation c.337C > T (p.Gln113*) in exon 5 of VPS13A in the two affected siblings. CONCLUSION: Here, we report a novel nonsense p.Gln113* mutation in VPS13A identified by whole-exome sequencing, which caused ChAc in a Moroccan family. This is the first description of ChAc in Morocco with genetic confirmation, that expands the mutation diversity of VPS13A and provide clinical, neuroimaging and deep brain stimulation findings.


Assuntos
Neuroacantocitose/genética , Polimorfismo de Nucleotídeo Único , Proteínas de Transporte Vesicular/genética , Adulto , Códon sem Sentido , Consanguinidade , Feminino , Humanos , Marrocos , Neuroacantocitose/patologia , Linhagem , Convulsões/complicações , Convulsões/genética , Irmãos , Espasmo/complicações , Espasmo/genética
17.
Nat Commun ; 11(1): 649, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-32005855

RESUMO

Synapse formation is induced by transsynaptic interaction of neuronal cell-adhesion molecules termed synaptic organizers. Type IIa receptor protein tyrosine phosphatases (IIa RPTPs) function as presynaptic organizers. The cytoplasmic domain of IIa RPTPs consists of two phosphatase domains, and the membrane-distal one (D2) is essential for synapse formation. Liprin-α, which is an active zone protein critical for synapse formation, interacts with D2 via its C-terminal domain composed of three tandem sterile alpha motifs (tSAM). Structural mechanisms of this critical interaction for synapse formation remain elusive. Here, we report the crystal structure of the complex between mouse PTPδ D2 and Liprin-α3 tSAM at 1.91 Å resolution. PTPδ D2 interacts with the N-terminal helix and the first and second SAMs (SAM1 and SAM2, respectively) of Liprin-α3. Structure-based mutational analyses in vitro and in cellulo demonstrate that the interactions with Liprin-α SAM1 and SAM2 are essential for the binding and synaptogenic activity.


Assuntos
Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/química , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Animais , Cristalização , Camundongos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Sinapses/genética , Sinapses/metabolismo , Proteínas de Transporte Vesicular/genética
18.
Proc Natl Acad Sci U S A ; 117(7): 3789-3796, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32015134

RESUMO

The facultative intracellular pathogen Listeria monocytogenes uses an actin-based motility process to spread within human tissues. Filamentous actin from the human cell forms a tail behind bacteria, propelling microbes through the cytoplasm. Motile bacteria remodel the host plasma membrane into protrusions that are internalized by neighboring cells. A critical unresolved question is whether generation of protrusions by Listeria involves stimulation of host processes apart from actin polymerization. Here we demonstrate that efficient protrusion formation in polarized epithelial cells involves bacterial subversion of host exocytosis. Confocal microscopy imaging indicated that exocytosis is up-regulated in protrusions of Listeria in a manner that depends on the host exocyst complex. Depletion of components of the exocyst complex by RNA interference inhibited the formation of Listeria protrusions and subsequent cell-to-cell spread of bacteria. Additional genetic studies indicated important roles for the exocyst regulators Rab8 and Rab11 in bacterial protrusion formation and spread. The secreted Listeria virulence factor InlC associated with the exocyst component Exo70 and mediated the recruitment of Exo70 to bacterial protrusions. Depletion of exocyst proteins reduced the length of Listeria protrusions, suggesting that the exocyst complex promotes protrusion elongation. Collectively, these results demonstrate that Listeria exploits host exocytosis to stimulate intercellular spread of bacteria.


Assuntos
Exocitose , Listeria monocytogenes/fisiologia , Listeriose/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células CACO-2 , Quinases do Centro Germinativo/genética , Quinases do Centro Germinativo/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Listeria monocytogenes/genética , Listeriose/genética , Listeriose/metabolismo , Listeriose/fisiopatologia , Ligação Proteica , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
19.
Nat Commun ; 11(1): 1044, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32098966

RESUMO

The inclusion of familial myeloid malignancies as a separate disease entity in the revised WHO classification has renewed efforts to improve the recognition and management of this group of at risk individuals. Here we report a cohort of 86 acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) families with 49 harboring germline variants in 16 previously defined loci (57%). Whole exome sequencing in a further 37 uncharacterized families (43%) allowed us to rationalize 65 new candidate loci, including genes mutated in rare hematological syndromes (ADA, GP6, IL17RA, PRF1 and SEC23B), reported in prior MDS/AML or inherited bone marrow failure series (DNAH9, NAPRT1 and SH2B3) or variants at novel loci (DHX34) that appear specific to inherited forms of myeloid malignancies. Altogether, our series of MDS/AML families offer novel insights into the etiology of myeloid malignancies and provide a framework to prioritize variants for inclusion into routine diagnostics and patient management.


Assuntos
Mutação em Linhagem Germinativa , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenosina Desaminase/genética , Dineínas do Axonema/genética , Estudos de Coortes , Humanos , Degradação do RNAm Mediada por Códon sem Sentido , Linhagem , Perforina/genética , Glicoproteínas da Membrana de Plaquetas/genética , RNA Helicases/genética , Receptores de Interleucina-17/genética , Proteínas de Transporte Vesicular/genética , Sequenciamento Completo do Exoma
20.
J Biol Chem ; 295(10): 3257-3268, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32005660

RESUMO

Eukaryotic cells are compartmentalized to form organelles, whose functions rely on proper phospholipid and protein transport. Here we determined the crystal structure of human VAT-1, a cytosolic soluble protein that was suggested to transfer phosphatidylserine, at 2.2 Å resolution. We found that VAT-1 transferred not only phosphatidylserine but also other acidic phospholipids between membranes in vitro Structure-based mutational analyses showed the presence of a possible lipid-binding cavity at the interface between the two subdomains, and two tyrosine residues in the flexible loops facilitated phospholipid transfer, likely by functioning as a gate to this lipid-binding cavity. We also found that a basic and hydrophobic loop with two tryptophan residues protruded from the molecule and facilitated binding to the acidic-lipid membranes, thereby achieving efficient phospholipid transfer.


Assuntos
Fosfolipídeos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Sítios de Ligação , Transporte Biológico , Cristalografia por Raios X , Humanos , Lipossomos/química , Lipossomos/metabolismo , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Fosfatidilserinas/metabolismo , Domínios Proteicos , Estrutura Terciária de Proteína , Triptofano/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética
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