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1.
Nat Commun ; 11(1): 5031, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024112

RESUMO

VARP and TBC1D5 are accessory/regulatory proteins of retromer-mediated retrograde trafficking from endosomes. Using an NMR/X-ray approach, we determined the structure of the complex between retromer subunit VPS29 and a 12 residue, four-cysteine/Zn++ microdomain, which we term a Zn-fingernail, two of which are present in VARP. Mutations that abolish VPS29:VARP binding inhibit trafficking from endosomes to the cell surface. We show that VARP and TBC1D5 bind the same site on VPS29 and can compete for binding VPS29 in vivo. The relative disposition of VPS29s in hetero-hexameric, membrane-attached, retromer arches indicates that VARP will prefer binding to assembled retromer coats through simultaneous binding of two VPS29s. The TBC1D5:VPS29 interaction is over one billion years old but the Zn-fingernail appears only in VARP homologues in the lineage directly giving rise to animals at which point the retromer/VARP/TBC1D5 regulatory network became fully established.


Assuntos
Evolução Molecular , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Zinco/metabolismo , Microscopia Crioeletrônica , Cisteína/química , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Células HeLa , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Conformação Proteica , Proteínas de Transporte Vesicular/genética , Dedos de Zinco
2.
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
3.
PLoS One ; 15(9): e0239657, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32966336

RESUMO

Upon internalization, receptors are trafficked to sorting endosomes (SE) where they undergo sorting and are then packaged into budding vesicles that undergo fission and transport within the cell. Eps15 Homology Domain Protein 1 (EHD1), the best-characterized member of the Eps15 Homology Domain Protein (EHD) family, has been implicated in catalyzing the fission process that releases endosome-derived vesicles for recycling to the plasma membrane. Indeed, recent studies suggest that upon receptor-mediated internalization, EHD1 is recruited from the cytoplasm to endosomal membranes where it catalyzes vesicular fission. However, the mechanism by which this recruitment occurs remains unknown. Herein, we demonstrate that the EHD1 paralog, EHD4, is required for the recruitment of EHD1 to SE. We show that EHD4 preferentially dimerizes with EHD1, and knock-down of EHD4 expression by siRNA, shRNA or by CRISPR/Cas9 gene-editing leads to impaired EHD1 SE-recruitment and enlarged SE. Moreover, we demonstrate that at least 3 different asparagine-proline-phenylalanine (NPF) motif-containing EHD binding partners, Rabenosyn-5, Syndapin2 and MICAL-L1, are required for the recruitment of EHD1 to SE. Indeed, knock-down of any of these SE-localized EHD interaction partners leads to enlarged SE, presumably due to impaired endosomal fission. Overall, we identify a novel mechanistic role for EHD4 in recruitment of EHD1 to SE, thus positioning EHD4 as an essential component of the EHD1-fission machinery at SE.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Endossomos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Sítios de Ligação , Proteínas de Ligação a DNA/química , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Camundongos , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Células NIH 3T3 , Proteínas Nucleares/química , Ligação Proteica , Proteínas de Transporte Vesicular/química
4.
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
5.
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
6.
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
7.
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
8.
Am J Physiol Renal Physiol ; 319(2): F178-F191, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32567349

RESUMO

Kidney injury in hypertension and diabetes entails, among in other structures, damage in a key cell of the glomerular filtration barrier, the podocyte. Podocytes are polarized and highly differentiated cells in which vesicular transport, partly driven by Rab GTPases, is a relevant process. The aim of the present study was to analyze Rab GTPases of the Rab-Rabphilin system in human immortalized podocytes and the impact of high glucose and angiotensin II. Furthermore, alterations of the system in urine cell pellets from patients with hypertension and diabetes were studied. Apoptosis was analyzed in podocytes, and mRNA level quantification, Western blot analysis, and immunofluorescence were developed to quantify podocyte-specific molecules and Rab-Rabphilin components (Rab3A, Rab27A, and Rabphilin3A). Quantitative RT-PCR was performed on urinary cell pellet from patients. The results showed that differentiated cells had reduced protein levels of the Rab-rabphillin system compared with undifferentiated cells. After glucose overload and angiotensin II treatment, apoptosis was increased and podocyte-specific proteins were reduced. Rab3A and Rab27A protein levels were increased under glucose overload, and Rabphilin3A decreased. Furthermore, this system exhibited higher levels under stress conditions in a manner of angiotensin II dose and time treatment. Immunofluorescence imaging indicated different expression patterns of podocyte markers and Rab27A under treatments. Finally, Rab3A and Rab27A were increased in patient urine pellets and showed a direct relationship with albuminuria. Collectively, these results suggest that the Rab-Rabphilin system could be involved in the alterations observed in injured podocytes and that a mechanism may be activated to reduce damage through the vesicular transport enhancement directed by this system.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Angiotensina II/farmacologia , Glucose/efeitos adversos , Podócitos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Albuminúria/metabolismo , Apoptose/efeitos dos fármacos , Humanos , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Transporte Vesicular/metabolismo
9.
PLoS One ; 15(5): e0232532, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32357161

RESUMO

Early endosomes, regarded as the main sorting station on endocytic pathway, are characterized by high frequency of homotypic fusions mediated by tethering protein EEA1. Despite intensive investigations, biogenesis of endosomes, boundaries between early and late endosomes, and process of cargo transition though them remain obscure. Here, using EGF/EGFR endocytosis as a model and confocal microscopy of fixed and live cells, we provide evidence favoring EEA1-vesicles being pre-existed vesicular compartment, that maintains its resident proteins' level and is sensitive to biosynthetic, but not endocytic pathway disturbance. EEA1-vesicles directly fuse with incoming EGF/EGFR-vesicles into hybrid endosomes with separated EEA1- and EGFR-domains, thus providing a platform for rapid achievement of an excess of surface-derived membrane that is used to form intraluminal vesicles (ILVs). Thus, multivesicular structures colocalized with EEA1 are still early endosomes. "EEA1-cycle" ends by exclusion of EGFR-containing domains with ILVs inside that turns into MVE and restoration of initial EEA1-vesicles population.


Assuntos
Endossomos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Vias Biossintéticas , Meios de Cultura Livres de Soro , Vesículas Citoplasmáticas/metabolismo , Endocitose , Receptores ErbB/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Microscopia Confocal , Modelos Biológicos , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo
10.
PLoS One ; 15(5): e0233502, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32433667

RESUMO

The environment within the Endoplasmic Reticulum (ER) influences Insulin biogenesis. In particular, ER stress may contribute to the development of Type 2 Diabetes (T2D) and Cystic Fibrosis Related Diabetes (CFRD), where evidence of impaired Insulin processing, including elevated secreted Proinsulin/Insulin ratios, are observed. Our group has established the role of a novel ER chaperone ERp29 (ER protein of 29 kDa) in the biogenesis of the Epithelial Sodium Channel, ENaC. The biogenesis of Insulin and ENaC share may key features, including their potential association with COP II machinery, their cleavage into a more active form in the Golgi or later compartments, and their ability to bypass such cleavage and remain in a less active form. Given these similarities we hypothesized that ERp29 is a critical factor in promoting the efficient conversion of Proinsulin to Insulin. Here, we confirmed that Proinsulin associates with the COP II vesicle cargo recognition component, Sec24D. When Sec24D expression was decreased, we observed a corresponding decrease in whole cell Proinsulin levels. In addition, we found that Sec24D associates with ERp29 in co-precipitation experiments and that ERp29 associates with Proinsulin in co-precipitation experiments. When ERp29 was overexpressed, a corresponding increase in whole cell Proinsulin levels was observed, while depletion of ERp29 decreased whole cell Proinsulin levels. Together, these data suggest a potential role for ERp29 in regulating Insulin biosynthesis, perhaps in promoting the exit of Proinsulin from the ER via Sec24D/COPII vesicles.


Assuntos
Proteínas de Choque Térmico/fisiologia , Insulina/biossíntese , Proteínas de Transporte Vesicular/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Fibrose Cística/complicações , Diabetes Mellitus Tipo 2 , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Humanos , Proinsulina/metabolismo , Transporte Proteico
11.
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
12.
Nat Commun ; 11(1): 2170, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358503

RESUMO

Plants as non-mobile organisms constantly integrate varying environmental signals to flexibly adapt their growth and development. Local fluctuations in water and nutrient availability, sudden changes in temperature or other abiotic and biotic stresses can trigger changes in the growth of plant organs. Multiple mutually interconnected hormonal signaling cascades act as essential endogenous translators of these exogenous signals in the adaptive responses of plants. Although the molecular backbones of hormone transduction pathways have been identified, the mechanisms underlying their interactions are largely unknown. Here, using genome wide transcriptome profiling we identify an auxin and cytokinin cross-talk component; SYNERGISTIC ON AUXIN AND CYTOKININ 1 (SYAC1), whose expression in roots is strictly dependent on both of these hormonal pathways. We show that SYAC1 is a regulator of secretory pathway, whose enhanced activity interferes with deposition of cell wall components and can fine-tune organ growth and sensitivity to soil pathogens.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Citocininas/metabolismo , Resistência à Doença/genética , Ácidos Indolacéticos/metabolismo , Proteínas de Membrana/metabolismo , Reguladores de Crescimento de Planta/metabolismo , Raízes de Plantas/metabolismo , Transcriptoma/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Parede Celular/química , Parede Celular/metabolismo , Endossomos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/genética , Complexo de Golgi/metabolismo , Proteínas de Membrana/genética , Raízes de Plantas/microbiologia , Plantas Geneticamente Modificadas/metabolismo , Plasmodioforídeos/patogenicidade , Via Secretória/genética , Solo , Proteínas de Transporte Vesicular/metabolismo
13.
J Biotechnol ; 318: 20-30, 2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32387451

RESUMO

Niemann-Pick type C (NPC) is an autosomal recessive lysosomal storage disorder due to mutations in NPC1 (95 % cases) or NPC2 genes, encoding NPC1 and NPC2 proteins, respectively. Both NPC1 and NPC2 proteins are involved in transport of intracellular cholesterol and their alteration leads to the accumulation of unesterified cholesterol and other lipids within the lysosomes. The disease is characterized by visceral, neurological and psychiatric symptoms. However, the pathogenic mechanisms that lead to the fatal neurodegeneration are still unclear. To date, several mutations leading to the generation of aberrant splicing variants or mRNA degradation in NPC1 and NPC2 genes have been reported. In addition, different lines of experimental evidence have highlighted the possible role of RNA-binding proteins and RNA-metabolism, in the onset and progression of many neurodegenerative disorders, that could explain NPC neurological features and in general, the disease pathogenesis. In this review, we will provide an overview of the impact of mRNA processing and metabolism on NPC disease pathology.


Assuntos
Doença de Niemann-Pick Tipo C/patologia , Processamento Pós-Transcricional do RNA/genética , Proteínas de Ligação a RNA/metabolismo , Colesterol/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/metabolismo , Mutação , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/metabolismo , RNA/metabolismo , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Proteínas de Ligação a RNA/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
14.
J Neurosci ; 40(19): 3862-3879, 2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32291328

RESUMO

Hydrocephalus is a pathologic condition associated with various brain diseases, including Alzheimer's disease (AD). Dysfunctional ependymal cells (EpCs) are believed to contribute to the development of hydrocephalus. It is thus of interest to investigate EpCs' development and function. Here, we report that vacuolar protein sorting-associated protein 35 (VPS35) is critical for EpC differentiation, ciliogenesis, and survival, and thus preventing neonatal hydrocephalus. VPS35 is abundantly expressed in EpCs. Mice with conditional knock-out (cKO) of Vps35 in embryonic (Vps35GFAP-Cre and Vps35Emx1-Cre) or postnatal (Vps35Foxj1-CreER) EpC progenitors exhibit enlarged lateral ventricles (LVs) and hydrocephalus-like pathology. Further studies reveal marked reductions in EpCs and their cilia in both Vps35GFAP-Cre and Vps35Foxj1-CreER mutant mice. The reduced EpCs appear to be due to impairments in EpC differentiation and survival. Additionally, both Vps35GFAP-Cre and Vps35Foxj1-CreER neonatal pups exhibit increased cell proliferation and death largely in a region close to LV-EpCs. Many microglia close to the mutant LV-EpC region become activated. Depletion of the microglia by PLX3397, an antagonist of colony-stimulating factor 1 receptor (CSF1R), restores LV-EpCs and diminishes the pathology of neonatal hydrocephalus in Vps35Foxj1-CreER mice. Taken together, these observations suggest unrecognized functions of Vps35 in EpC differentiation, ciliogenesis, and survival in neonatal LV, and reveal pathologic roles of locally activated microglia in EpC homeostasis and hydrocephalus development.SIGNIFICANCE STATEMENT This study reports critical functions of vacuolar protein sorting-associated protein 35 (VPS35) not only in promoting ependymal cell (EpC) differentiation, ciliogenesis, and survival, but also in preventing local microglial activation. The dysfunctional EpCs and activated microglia are likely to induce hydrocephalus.


Assuntos
Epêndima/metabolismo , Células Ependimogliais/metabolismo , Hidrocefalia/metabolismo , Microglia/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Animais Recém-Nascidos , Diferenciação Celular/fisiologia , Sobrevivência Celular , Epêndima/citologia , Hidrocefalia/fisiopatologia , Camundongos , Camundongos Knockout
15.
Proc Natl Acad Sci U S A ; 117(19): 10565-10574, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32345721

RESUMO

Numerous mutations that impair retrograde membrane trafficking between endosomes and the Golgi apparatus lead to neurodegenerative diseases. For example, mutations in the endosomal retromer complex are implicated in Alzheimer's and Parkinson's diseases, and mutations of the Golgi-associated retrograde protein (GARP) complex cause progressive cerebello-cerebral atrophy type 2 (PCCA2). However, how these mutations cause neurodegeneration is unknown. GARP mutations in yeast, including one causing PCCA2, result in sphingolipid abnormalities and impaired cell growth that are corrected by treatment with myriocin, a sphingolipid synthesis inhibitor, suggesting that alterations in sphingolipid metabolism contribute to cell dysfunction and death. Here we tested this hypothesis in wobbler mice, a murine model with a homozygous partial loss-of-function mutation in Vps54 (GARP protein) that causes motor neuron disease. Cytotoxic sphingoid long-chain bases accumulated in embryonic fibroblasts and spinal cords from wobbler mice. Remarkably, chronic treatment of wobbler mice with myriocin markedly improved their wellness scores, grip strength, neuropathology, and survival. Proteomic analyses of wobbler fibroblasts revealed extensive missorting of lysosomal proteins, including sphingolipid catabolism enzymes, to the Golgi compartment, which may contribute to the sphingolipid abnormalities. Our findings establish that altered sphingolipid metabolism due to GARP mutations contributes to neurodegeneration and suggest that inhibiting sphingolipid synthesis might provide a useful strategy for treating these disorders.


Assuntos
Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Esfingolipídeos/metabolismo , Animais , Modelos Animais de Doenças , Endossomos/metabolismo , Ácidos Graxos Monoinsaturados/farmacologia , Feminino , Fibroblastos/metabolismo , Complexo de Golgi/metabolismo , Masculino , Camundongos , Camundongos Mutantes Neurológicos , Doença dos Neurônios Motores/genética , Doença dos Neurônios Motores/metabolismo , Doença dos Neurônios Motores/patologia , Neurônios Motores/metabolismo , Células-Tronco Embrionárias Murinas , Mutação , Malformações do Sistema Nervoso/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Transporte Proteico , Proteômica , Proteínas de Transporte Vesicular/metabolismo
16.
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
17.
Parasitol Res ; 119(5): 1629-1640, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32219551

RESUMO

Rab proteins constitute the largest group of small GTPases and act as molecular switches in a wide variety of cellular processes, including proliferation, cytoskeleton assembly, and membrane trafficking in all eukaryotic cells. Rab21 has been reported in several eukaryotic cells, and our results suggest that in Entamoeba histolytica, Rab21 is involved in the vesicular traffic associated with the Golgi apparatus, where its function appears to be important to maintain the structure of this organelle. In addition, proteins such as Rab1A and Sec24, identified in this work associated with EhRab21, participate in the traffic of COPII vesicles from the endoplasmic reticulum to the Golgi apparatus and are necessary to maintain the latter's structure in human cells. In addition, EhRab21 probably affects the lysosome biogenesis, as indicated by an increase in the number of lysosomes as a result of the increase in EhRab21 activity. The participation of EhRab21 in the pathogenesis of amebiasis was verified on the amoebic liver abscess formation model using hamsters (Mesocricetus auratus), in which the overexpression of EhRab21Q64L (positive dominant mutant protein) decreased the number of liver abscesses formed.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Entamoeba histolytica/metabolismo , Complexo de Golgi/metabolismo , Transporte Proteico/fisiologia , Proteínas rab de Ligação ao GTP/metabolismo , Amebíase/patologia , Animais , Cricetinae , Retículo Endoplasmático/metabolismo , Humanos , Abscesso Hepático Amebiano/patologia , Lisossomos/metabolismo , Proteínas de Transporte Vesicular/metabolismo
18.
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
19.
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
20.
Nat Commun ; 11(1): 1368, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170195

RESUMO

MCFD2 and ERGIC-53, which are the products of causative genes of combined factor V and factor VIII deficiency, form a cargo receptor complex responsible for intracellular transport of these coagulation factors in the early secretory pathway. In this study, using an NMR technique, we successfully identified an MCFD2-binding segment from factor VIII composed of a 10 amino acid sequence that enhances its secretion. This prompted us to examine possible effects of attaching this sequence to recombinant glycoproteins on their secretion. We found that the secretion level of recombinant erythropoietin was significantly increased simply by tagging it with the passport sequence. Our findings not only provide molecular basis for the intracellular trafficking of coagulation factors and their genetic deficiency but also offer a potentially useful tool for increasing the production yields of recombinant glycoproteins of biopharmaceutical interest.


Assuntos
Proteínas de Transporte/metabolismo , Glicoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Polissacarídeos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Retículo Endoplasmático/fisiologia , Eritropoetina/metabolismo , Fator V , Fator VIII/metabolismo , Glicoproteínas/genética , Complexo de Golgi/fisiologia , Humanos , Lectinas de Ligação a Manose/metabolismo , Transporte Proteico , Via Secretória
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