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1.
Nat Struct Mol Biol ; 27(4): 392-399, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32251413

RESUMO

The endosomal sorting complexes required for transport (ESCRTs) mediate diverse membrane remodeling events. These typically require ESCRT-III proteins to stabilize negatively curved membranes; however, recent work has indicated that certain ESCRT-IIIs also participate in positive-curvature membrane-shaping reactions. ESCRT-IIIs polymerize into membrane-binding filaments, but the structural basis for negative versus positive membrane remodeling by these proteins remains poorly understood. To learn how certain ESCRT-IIIs shape positively curved membranes, we determined structures of human membrane-bound CHMP1B-only, membrane-bound CHMP1B + IST1, and IST1-only filaments by cryo-EM. Our structures show how CHMP1B first polymerizes into a single-stranded helical filament, shaping membranes into moderate-curvature tubules. Subsequently, IST1 assembles a second strand on CHMP1B, further constricting the membrane tube and reducing its diameter nearly to the fission point. Each step of constriction thins the underlying bilayer, lowering the barrier to membrane fission. Our structures reveal how a two-component, sequential polymerization mechanism drives membrane tubulation, constriction and bilayer thinning.


Assuntos
Membrana Celular/ultraestrutura , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Proteínas Oncogênicas/ultraestrutura , Membrana Celular/química , Membrana Celular/genética , Citocinese/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/química , Endossomos/genética , Endossomos/ultraestrutura , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/ultraestrutura , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Polimerização , Conformação Proteica
2.
Nat Commun ; 11(1): 1941, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32321914

RESUMO

Cytokinesis requires the constriction of ESCRT-III filaments on the side of the midbody, where abscission occurs. After ESCRT recruitment at the midbody, it is not known how the ESCRT-III machinery localizes to the abscission site. To reveal actors involved in abscission, we obtained the proteome of intact, post-abscission midbodies (Flemmingsome) and identified 489 proteins enriched in this organelle. Among these proteins, we further characterized a plasma membrane-to-ESCRT module composed of the transmembrane proteoglycan syndecan-4, ALIX and syntenin, a protein that bridges ESCRT-III/ALIX to syndecans. The three proteins are highly recruited first at the midbody then at the abscission site, and their depletion delays abscission. Mechanistically, direct interactions between ALIX, syntenin and syndecan-4 are essential for proper enrichment of the ESCRT-III machinery at the abscission site, but not at the midbody. We propose that the ESCRT-III machinery must be physically coupled to a membrane protein at the cytokinetic abscission site for efficient scission, uncovering common requirements in cytokinesis, exosome formation and HIV budding.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Citocinese , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Organelas/metabolismo , Sindecana-4/metabolismo , Sinteninas/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/genética , Membrana Celular/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/genética , Endossomos/metabolismo , Células HeLa , Humanos , Organelas/genética , Ligação Proteica , Sindecana-4/genética , Sinteninas/genética
3.
FASEB J ; 34(1): 1331-1344, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914703

RESUMO

Proteins specialized in the detection, generation, or stabilization of membrane curvature play important roles in establishing various morphologies of cells and cellular organelles. Primary cilia are cellular organelles that protrude from the cell surface using a microtubule-based cytoskeleton called the axoneme as a structural support. It is unclear whether the integrity of the high curvature of the ciliary membrane depends on membrane curvature-related proteins. Charged Multivesicular Body Protein 4B (CHMP4B), a subunit of the endosomal sorting complexes required for transport (ESCRT), can stabilize membrane curvature. Here we show that CHMP4B is involved in the assembly and maintenance of primary cilia. CHMP4B was localized to primary cilia in mammalian cells. Knockdown of CHMP4B interfered with cilium assembly and also caused fragmentation of preexisting cilia. By contrast, cilium formation was unaffected by the interruption of the ESCRT-dependent endocytic degradation pathway. Morpholino (MO)-mediated CHMP4B depletion in zebrafish embryos induced characteristic phenotypes of ciliary defects such as curved body axis, hydrocephalus, otolith malformation, and kidney cyst. Our study reveals a new role for the multifunctional protein CHMP4B as a key factor in maintaining the structural integrity of primary cilia.


Assuntos
Membrana Celular/metabolismo , Cílios/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Membrana Celular/genética , Cílios/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Deleção de Genes , Humanos , Camundongos , Células NIH 3T3 , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
4.
Biochem Soc Trans ; 47(6): 1867-1879, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31845722

RESUMO

Protein modification by ubiquitin is one of the most versatile posttranslational regulations and counteracted by almost 100 deubiquitinating enzymes (DUBs). USP8 was originally identified as a growth regulated ubiquitin-specific protease and is like many other DUBs characterized by its multidomain architecture. Besides the catalytic domain, specific protein-protein interaction modules were characterized which contribute to USP8 substrate recruitment, regulation and targeting to distinct protein complexes. Studies in mice and humans impressively showed the physiological relevance and non-redundant function of USP8 within the context of the whole organism. USP8 knockout (KO) mice exhibit early embryonic lethality while induced deletion in adult animals rapidly causes lethal liver failure. Furthermore, T-cell specific ablation disturbs T-cell development and function resulting in fatal autoimmune inflammatory bowel disease. In human patients, somatic mutations in USP8 were identified as the underlying cause of adrenocorticotropic hormone (ACTH) releasing pituitary adenomas causing Cushing's disease (CD). Here we provide an overview of the versatile molecular, cellular and pathology associated function and regulation of USP8 which appears to depend on specific protein binding partners, substrates and the cellular context.


Assuntos
Enzimas Desubiquitinantes/metabolismo , Endopeptidases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Apoptose/fisiologia , Autofagia/fisiologia , Cílios/metabolismo , Endopeptidases/genética , Endopeptidases/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Endossomos/metabolismo , Humanos , Camundongos , Camundongos Knockout , Mitofagia/fisiologia , Mutação , Hipersecreção Hipofisária de ACTH/genética , Ligação Proteica , Transdução de Sinais , Linfócitos T/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/fisiologia
5.
PLoS Genet ; 15(11): e1008387, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31738769

RESUMO

The ubiquitin-proteasome system regulates numerous cellular processes and is central to protein homeostasis. In proliferating yeast and many mammalian cells, proteasomes are highly enriched in the nucleus. In carbon-starved yeast, proteasomes migrate to the cytoplasm and collect in proteasome storage granules (PSGs). PSGs dissolve and proteasomes return to the nucleus within minutes of glucose refeeding. The mechanisms by which cells regulate proteasome homeostasis under these conditions remain largely unknown. Here we show that AMP-activated protein kinase (AMPK) together with endosomal sorting complexes required for transport (ESCRTs) drive a glucose starvation-dependent microautophagy pathway that preferentially sorts aberrant proteasomes into the vacuole, thereby biasing accumulation of functional proteasomes in PSGs. The proteasome core particle (CP) and regulatory particle (RP) are regulated differently. Without AMPK, the insoluble protein deposit (IPOD) serves as an alternative site that specifically sequesters CP aggregates. Our findings reveal a novel AMPK-controlled ESCRT-mediated microautophagy mechanism in the regulation of proteasome trafficking and homeostasis under carbon starvation.


Assuntos
Proteínas Quinases Ativadas por AMP/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Microautofagia/genética , Complexo de Endopeptidases do Proteassoma/genética , Citoplasma/genética , Citoplasma/metabolismo , Glucose/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Proteico/genética , Saccharomyces cerevisiae/genética , Inanição/genética , Inanição/metabolismo , Ubiquitina/genética , Ubiquitinação/genética , Vacúolos/genética , Vacúolos/metabolismo
6.
Eur J Endocrinol ; 181(6): 615-627, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31581124

RESUMO

Objective: Pituitary corticotroph adenomas commonly cause Cushing's disease (CD) but part of these tumours are hormonally inactive (silent corticotroph adenomas, SCA). USP8 mutations are well-known driver mutations in corticotrophinomas. Differences in transcriptomic profiles between functioning and silent tumours or tumours with different USP8 status have not been investigated. Design and methods: Forty-eight patients (28 CD, 20 SCA) were screened for USP8 mutations with Sanger sequencing. Twenty-four patients were included in transcriptomic profiling with Ampliseq Transcriptome Human Gene Expression Core Panel. The entire patients group was included in qRT-PCR analysis of selected genes expression. Immunohistochemistry was used for visualization of selected protein. Results: We found USP8 mutation in 15 patients with CD and 4 SCAs. USP8 mutations determine molecular profile of the tumours as showed by hierarchical clustering and identification of 1648 genes differentially expressed in USP8-mutated and USP8-wild-type tumours. Mutations affect many molecular pathways as observed in Gene Set Enrichment analysis. USP8-mutated adenomas showed higher level of POMC, CDC25A, MAPK4 but lower level of CCND2, CDK6, CDKN1B than USP8-wt tumours. Eighty-seven genes differentially expressed between CD-related adenomas and SCAs were found, including those involved in cell signalling (GLI2, DLC1, TBX2, RASSF6), cell adhesion (GJA1, CDH6), ion transport (KCNN4, KCNJ5) and GABA signalling (GABBR2, GABRD). Conclusion: USP8 mutations occur in functioning and silent corticotrophinomas. They have pleiotropic effect, not limited to EGFR signalling, and affect expression levels of many genes involved in different pathways. Expression of GABA-related genes GABBR2, GNAL, GABARD and KCNJ5 correspond to functional status of the tumours.


Assuntos
Adenoma Hipofisário Secretor de ACT/genética , Adenoma Hipofisário Secretor de ACT/metabolismo , Endopeptidases/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Mutação/genética , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/metabolismo , Ubiquitina Tiolesterase/genética , Adulto , Idoso , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Conexina 43/genética , Conexina 43/metabolismo , Feminino , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteína Gli2 com Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/metabolismo
7.
PLoS Biol ; 17(10): e3000512, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31658248

RESUMO

Endocytosis of membrane proteins in yeast requires α-arrestin-mediated ubiquitylation by the ubiquitin ligase Rsp5. Yet, the diversity of α-arrestin targets studied is restricted to a small subset of plasma membrane (PM) proteins. Here, we performed quantitative proteomics to identify new targets of 12 α-arrestins and gained insight into the diversity of pathways affected by α-arrestins, including the cell wall integrity pathway and PM-endoplasmic reticulum contact sites. We found that Art2 is the main regulator of substrate- and stress-induced ubiquitylation and endocytosis of the thiamine (vitamin B1) transporters: Thi7, nicotinamide riboside transporter 1 (Nrt1), and Thi72. Genetic screening allowed for the isolation of transport-defective Thi7 mutants, which impaired thiamine-induced endocytosis. Coexpression of inactive mutants with wild-type Thi7 revealed that both transporter conformation and transport activity are important to induce endocytosis. Finally, we provide evidence that Art2 mediated Thi7 endocytosis is regulated by the target of rapamycin complex 1 (TORC1) and requires the Sit4 phosphatase but is not inhibited by the Npr1 kinase.


Assuntos
Arrestinas/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Transporte de Nucleosídeos/genética , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Tiamina/metabolismo , Arrestinas/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Parede Celular/metabolismo , Endocitose/genética , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Mutação , Proteínas de Transporte de Nucleosídeos/metabolismo , Ligação Proteica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Estrutura Secundária de Proteína , Proteômica/métodos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Tiamina/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Complexos Ubiquitina-Proteína Ligase/genética , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitinação
8.
Mol Med Rep ; 20(6): 5145-5151, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31638258

RESUMO

To the best of our knowledge, the present study reported the case of the first Chinese patient with microcephaly­capillary malformation (MIC­CAP) syndrome caused by a novel compound heterozygous mutation in the STAMBP gene, which encodes STAM binding protein. The present study also provides a review of relevant previously published studies. A boy with MIC­CAP syndrome with developmental delay, intractable epilepsy and prominent dyskinesia was examined. A pathogenic mutation was identified by whole­exome sequencing, and the protein structure and function affected by this mutation were predicted using bioinformatics analysis. Finally, the clinical features of 16 other cases reported in previous studies were reviewed and compared. A novel compound heterozygous mutation of the STAMBP (c.1119­1G>T, c.968A>G) was identified in the present study and epilepsy was refractory, consistent with previously reported cases. The present study also highlighted the fact that STAMBP mutation­associated MIC­CAP often presents as intractable early­life epilepsy, which may lead to mortality.


Assuntos
Capilares/anormalidades , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Epilepsia/genética , Predisposição Genética para Doença , Microcefalia/genética , Mutação , Ubiquitina Tiolesterase/genética , Malformações Vasculares/genética , Idade de Início , Criança , Análise Mutacional de DNA , Epilepsia/diagnóstico , Estudos de Associação Genética , Humanos , Masculino , Microcefalia/diagnóstico , Linhagem , Análise de Sequência de DNA , Síndrome , Malformações Vasculares/diagnóstico
9.
Int J Mol Sci ; 20(21)2019 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-31661793

RESUMO

Vasopressin-dependent trafficking of AQP2 in the renal collecting duct is crucial for the regulation of water homeostasis. This process involves the targeting of AQP2 to the apical membrane during dehydration as well as its removal when hydration levels have been restored. The latter involves AQP2 endocytosis and sorting into multivesicular bodies (MVB), from where it may be recycled, degraded in lysosomes, or released into urine via exosomes. The lysosomal trafficking regulator-interacting protein 5 (LIP5) plays a crucial role in this by coordinating the actions of the endosomal sorting complex required for transport III (ESCRT-III) and vacuolar protein sorting 4 (Vps4) ATPase, resulting in the insertion of AQP2 into MVB inner vesicles. While the interaction between LIP5 and the ESCRT-III complex and Vps4 is well characterized, very little is known about how LIP5 interacts with AQP2 or any other membrane protein cargo. Here, we use a combination of fluorescence spectroscopy and computer modeling to provide a structural model of how LIP5 interacts with human AQP2. We demonstrate that, the AQP2 tetramer binds up to two LIP5 molecules and that the interaction is similar to that seen in the complex between LIP5 and the ESCRT-III component, charged multivesicular body protein 1B (CHMP1B). These studies give the very first structural insights into how LIP5 enables membrane protein insertion into MVB inner vesicles and significantly increase our understanding of the AQP2 trafficking mechanism.


Assuntos
Aquaporina 2/química , Aquaporina 2/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Corpos Multivesiculares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Adenosina Trifosfatases/metabolismo , Aquaporina 2/genética , Endocitose/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Humanos , Simulação de Acoplamento Molecular , Multimerização Proteica/genética , Transporte Proteico/fisiologia , Espectrometria de Fluorescência , ATPases Vacuolares Próton-Translocadoras/metabolismo
10.
Mol Microbiol ; 112(5): 1499-1518, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31442344

RESUMO

Assimilation of heme is mediated by the cell surface protein Shu1 in Schizosaccharomyces pombe. Shu1 undergoes internalization from the cell surface to the vacuole in response to high concentrations of hemin. Here, we have identified cellular components that are involved in mediating vacuolar targeting of Shu1. Cells deficient in heme biosynthesis and lacking the polyubiquitin gene ubi4+ exhibit poor growth in the presence of exogenous hemin as a sole source of heme. Microscopic analyses of hem1Δ shu1Δ ubi4Δ cells expressing a functional HA4 -tagged Shu1 show that Shu1 localizes to the cell surface. Ubiquitinated Nbr1 functions as a receptor for the endosomal sorting complexes required for transport (ESCRT) that delivers cargos to the vacuole. Inactivation of nbr1+ , ESCRT-0 hse1+ or ESCRT-I sst6+ results in hem1Δ cells being unable to use exogenous hemin for the growth. Using lysate preparations from hemin-treated cells, Shu1-Nbr1 and Shu1-Hse1 complexes are detected by coimmunoprecipitation experiments. Further analysis by immunofluorescence microscopy shows that Shu1 is unable to reach vacuoles of hemin-treated cells harboring a deletion for one of the following genes: ubi4+ , nbr1+ , hse1+ and sst6+ . Together, these results reveal that hemin-mediated vacuolar targeting of Shu1 requires Ubi4-dependent ubiquitination, the receptor Nbr1 and the ESCRT proteins Hse1 and Sst6.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Heme/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/crescimento & desenvolvimento , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Proteínas de Membrana/genética , Transporte Proteico/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Ubiquitina C/genética , Ubiquitina C/metabolismo , Ubiquitinação
11.
Differentiation ; 109: 16-27, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31404815

RESUMO

Charged multivesicular body protein 4B (CHMP4B) functions as a core component of the endosome sorting complex required for transport-III (ESCRT-III) machinery that facilitates diverse membrane remodeling and scission processes in eukaryotes. Mutations in the human CHMP4B gene underlie rare, inherited forms of early-onset lens opacities or cataract. Here we have characterized the lens phenotypes of mutant (knock-in) mice harboring a human cataract-associated mutation (p.D129V) in CHMP4B (Chmp4b-mutant) and conditional knockdown mice deficient in lens CHMP4B (Chmp4b-CKD). In situ hybridization localized Chmp4b transcripts to lens epithelial cells and elongating fiber cells at the lens equator. Heterozygous Chmp4b-mutant (D/V) mice were viable and fertile with lenses grossly similar to those of wild-type. However, homozygous Chmp4b-mutant (V/V) mice died by embryonic day 15.5 (E15.5) with grossly abnormal eye and brain histology. Chmp4b-CKD mice displayed variable degrees of lens dysmorphology including lens ablation. Immuno-localization of aquaporin-0 (AQP0) revealed lens fiber cell degeneration in homozygous Chmp4b-mutant (V/V) mouse embryos and in embryonic and postnatal Chmp4b-CKD mice. DNA fragmentation (TUNEL) analysis revealed global cell death in homozygous Chmp4b-mutant (V/V) embryos, whereas, cell death was confined to the lens of Chmp4b-CKD mice. Immuno-localization of the monocyte/macrophage marker macrosialin (CD68) suggested that severe lens degeneration in Chmp4b-CKD mice resulted in an ocular immune cell response. Collectively, these mouse data suggest that (1) heterozygous, germ-line mutations in Chmp4b may not manifest as cataract, (2) homozygous, germ-line mutations in Chmp4b are embryonic lethal, and (3) conditional loss of Chmp4b results in arrest of lens growth and differentiation.


Assuntos
Diferenciação Celular , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/fisiologia , Cristalino/citologia , Mutação , Animais , Feminino , Técnicas de Introdução de Genes , Humanos , Cristalino/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfogênese , Fenótipo
12.
ACS Chem Biol ; 14(9): 1874-1878, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31411851

RESUMO

The budding of HIV from infected cells is driven by the protein-protein interaction between the p6 domain of the HIV Gag protein and the UEV domain of the human TSG101 protein. We report the development of a cyclic peptide inhibitor of the p6/UEV interaction, from a non cell-permeable parent that was identified in a SICLOPPS screen. Amino acids critical for the activity of the parent cyclic peptide were uncovered using alanine-scanning, and a series of non-natural analogues synthesized and assessed. The most potent molecule disrupts the p6/UEV interaction with an IC50 of 6.17 ± 0.24 µM by binding to UEV with a Kd of 11.9 ± 2.8 µM. This compound is cell permeable and active in a cellular virus-like particle budding assay with an IC50 of ∼2 µM. This work further demonstrates the relative simplicity with which the potency and activity of cyclic peptides identified from SICLOPPS libraries can be optimized.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Peptídeos Cíclicos/farmacologia , Ligação Proteica/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Desenvolvimento de Medicamentos , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Escherichia coli/genética , Células HEK293 , HIV/química , HIV/efeitos dos fármacos , Células HeLa , Humanos , Peptídeos Cíclicos/toxicidade , Domínios Proteicos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Liberação de Vírus/efeitos dos fármacos
13.
Biochem J ; 476(14): 2031-2046, 2019 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-31273031

RESUMO

The endosomal sorting complex required for transport (ESCRT)-III is associated with a multitude of cellular processes involving membrane remodeling and abscission. The exact composition of ESCRT-III and the contribution of individual ESCRT-III family members to these diverse functions is unclear. Most of the currently available information about ESCRT-III was obtained with tagged, largely non-functional proteins, which may not correctly reflect the in vivo situation. Here, we performed a comprehensive biochemical analysis of ESCRT-III localization and composition in yeast under purely native conditions. Most of our findings are in line with the current concepts about ESCRT-III, but some findings are unexpected and call for adjustments to the model. In particular, our data suggest that the distinction between bona fide ESCRT-III components and ESCRT-III associated proteins is not justified. We detected a single complex containing all ESCRT-III members (except of Chm7) with Did2 as its main component. The classical core components were present in equimolar amounts. Our analysis of the impact of single deletions on the composition of ESCRT-III confirmed the central role of Snf7 for ESCRT-III assembly. For the other ESCRT-III family members predictions could be made about their role in ESCRT-III assembly. Furthermore, our cell fractionation points to a role of Vps20 at the endoplasmic reticulum.


Assuntos
Retículo Endoplasmático , Complexos Endossomais de Distribuição Requeridos para Transporte , Deleção de Genes , Saccharomyces cerevisiae , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Transporte Proteico/genética , Coelhos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
14.
Pituitary ; 22(4): 435-442, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31273566

RESUMO

PURPOSE: Cushing's disease (CD) is a severe illness generally caused by microcorticotropinomas (MICs) and in approximately 7-20% of patients by macrocorticotropinomas (MACs). USP8-mutations have been identified as a major genetic cause of CD (~ 50%). Few studies have reported the distribution between MICs-MACs related to USP8-mutations and their genotype-phenotype correlations. Therefore, we aimed to evaluate USP8-mutations in a cohort of MICs-MACs from a unique center and to perform a systematic review and meta-analysis. METHODS: DNA-tumor-tissues from 47 corticotropinomas (16 MICs and 31 MACs) were sequenced. Clinical-biochemical data, radiological imaging data and remission/recurrence rates were evaluated. In addition, we performed a meta-analysis of nine published series (n = 630). RESULTS: We identified four different USP8-mutations previously described, in 11 out of 47 (23.4%) corticotropinomas; 8 out of 11 were MACs. The urinary cortisol levels of our patients with corticotrophin USP8-mutated-alleles were lower than those of patients with wild-type (WT) alleles (p ≤ 0.017). The frequency of USP8-mutated-alleles among the series was approximately 30% with a higher prevalence in female-patients (p < 0.1 × 10-4). Among the 5 series, the remission rates were higher in patients with USP8-mutated-alleles than in those with the USP8-WT-alleles (p < 0.1 × 10-4). CONCLUSION: Our data, as well as the retrospective review of CD series associated with USP8-mutated alleles, show heterogeneous findings among the series. Several drawbacks included the lack of a systematic protocol to evaluate these patients before surgery and follow-up. Further prospective studies using a systematic protocol will provide more consistent information about the influence of the corticotropinomas with USP8-mutated alleles on the phenotype, responses to treatment and outcome of patients with CD.


Assuntos
Endopeptidases/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Mutação/genética , Hipersecreção Hipofisária de ACTH/etiologia , Hipersecreção Hipofisária de ACTH/genética , Ubiquitina Tiolesterase/genética , Alelos , Estudos de Associação Genética , Humanos , Hipersecreção Hipofisária de ACTH/epidemiologia
15.
Elife ; 82019 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-31246173

RESUMO

Self-assembly of ESCRT-III complex is a critical step in all ESCRT-dependent events. ESCRT-III hetero-polymers adopt variable architectures, but the mechanisms of inter-subunit recognition in these hetero-polymers to create flexible architectures remain unclear. We demonstrate in vivo and in vitro that the Saccharomyces cerevisiae ESCRT-III subunit Snf7 uses a conserved acidic helix to recruit its partner Vps24. Charge-inversion mutations in this helix inhibit Snf7-Vps24 lateral interactions in the polymer, while rebalancing the charges rescues the functional defects. These data suggest that Snf7-Vps24 assembly occurs through electrostatic interactions on one surface, rather than through residue-to-residue specificity. We propose a model in which these cooperative electrostatic interactions in the polymer propagate to allow for specific inter-subunit recognition, while sliding of laterally interacting polymers enable changes in architecture at distinct stages of vesicle biogenesis. Our data suggest a mechanism by which interaction specificity and polymer flexibility can be coupled in membrane-remodeling heteropolymeric assemblies.


Assuntos
Biopolímeros/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Eletricidade Estática , Sequência de Aminoácidos , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Mutação/genética , Ligação Proteica , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Supressão Genética
16.
Methods Mol Biol ; 1998: 13-29, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31250291

RESUMO

Mosaic analysis in Drosophila represents a convenient entry point for studying the role of ESCRT (Endosomal Sorting Complex Required for Transport) genes in multiple cell processes crucial for organ development and homeostasis. Here, we describe the procedure to generate populations of ESCRT-mutant cells within Drosophila larval epithelial organs and to study them in whole-mount preparations using confocal microscopy. The use of antibodies directed to endocytic cargoes, vesicular trafficking, cell proliferation, death, and polarity markers allows one to investigate the consequences of loss of ESCRT activity at the subcellular and tissue level. The protocols described here can be used in fixed tissue as well as in unfixed tissue using endocytic uptake assays.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Técnicas de Silenciamento de Genes/métodos , Discos Imaginais/diagnóstico por imagem , Discos Imaginais/metabolismo , Imuno-Histoquímica/métodos , Larva , Microscopia Confocal/métodos , Mutação , Coloração e Rotulagem/métodos , Fixação de Tecidos/métodos
17.
Methods Mol Biol ; 1998: 31-47, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31250292

RESUMO

A large number of studies have shown that proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) can trigger the biogenesis of different types of Extracellular Vesicles (EV). The functions that these vesicular carriers exert in vivo remain, however, poorly understood. In this chapter, we describe a series of experimental approaches that we established in the Drosophila wing imaginal disc to study the importance of ESCRT-positive EVs for the extracellular transport of signaling molecules, as exemplified by a functional analysis of the mechanism of secretion and propagation of the major developmental morphogen Hedgehog (Hh).Through the combined use of genetic, cell biological, and imaging approaches, we investigate four important aspects of exovesicle biology: (1) The genetic identification of ESCRT proteins that are specifically required for Hh secretion. (2) The imaging of ESCRT and Hh-positive EVs in the lumenal space of both living and fixed wing imaginal discs. (3) The receptor-mediated capture of Hh-containing EVs on the surface of Hh-receiving cells. (4) The effect of manipulations of ESCRT function on the extracellular pool of Hh ligands.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Vesículas Extracelulares/metabolismo , Proteínas Hedgehog/metabolismo , Microscopia Intravital/métodos , Animais , Animais Geneticamente Modificados , Drosophila melanogaster/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Discos Imaginais/diagnóstico por imagem , Discos Imaginais/metabolismo , Larva , Ligantes , Microscopia de Fluorescência , Ligação Proteica , Fixação de Tecidos/métodos , Asas de Animais/diagnóstico por imagem , Asas de Animais/metabolismo
18.
Methods Mol Biol ; 1998: 105-116, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31250297

RESUMO

Budding yeast Saccharomyces cerevisiae is an ideal model organism to study membrane trafficking pathways. The ESCRT (endosomal sorting complexes required for transport) pathway was first identified in this organism. Upon recognition of endocytosed ubiquitinated membrane proteins at endosomes, ESCRTs assemble at these organelles to catalyze the biogenesis of multivesicular bodies (MVBs). Formation of MVBs leads to the trafficking of these membrane proteins to vacuoles for degradation. Here, we describe genetic and biochemical approaches to study ESCRT function. We outline in vivo endocytosis assays using two model cargoes in Saccharomyces cerevisiae and also describe an in vitro approach to analyze ESCRT-III polymerization on lipid monolayers.


Assuntos
Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Corpos Multivesiculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/genética , Endocitose/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/isolamento & purificação , Proteínas de Fluorescência Verde/química , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Membranas Artificiais , Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Mutação , Multimerização Proteica/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Vacúolos/metabolismo
19.
Methods Mol Biol ; 1998: 117-128, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31250298

RESUMO

Live imaging of microfluidically isolated axons permits study of the dynamic behavior of fluorescently tagged proteins and vesicles in these neuronal processes. We use this technique to study the motility and transport of ESCRT proteins in axons of primary hippocampal neurons. This chapter details the preparation of microfluidic chambers, as well as the seeding, fluidic isolation, and lentiviral transduction of hippocampal neurons in these chambers, optimized for the study of ESCRT protein dynamics.


Assuntos
Axônios/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Microscopia Intravital/métodos , Técnicas Analíticas Microfluídicas/métodos , Imagem Molecular/métodos , Animais , Transporte Axonal , Células Cultivadas , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Corantes Fluorescentes/química , Vetores Genéticos , Células HEK293 , Hipocampo/citologia , Humanos , Lentivirus/genética , Técnicas Analíticas Microfluídicas/instrumentação , Sondas Moleculares/química , Sondas Moleculares/genética , Cultura Primária de Células/métodos , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção
20.
Methods Mol Biol ; 1998: 163-174, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31250301

RESUMO

Localization studies are important to understand the function of diverse proteins. The endosomal trafficking pathway is very complex, and a lot of proteins function in this pathway, primarily the endosomal sorting complexes required for transport (ESCRTs). Some of the ESCRT-related proteins or mutant variants cannot be stably expressed in planta due to the toxicity of their expression. Therefore, a transient expression system is necessary to study their function. Transient expression in protoplasts from Arabidopsis root cell-derived culture serves as a fast and reliable method for the expression and cell biological and biochemical analyses of otherwise toxic constructs.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/metabolismo , Técnicas de Cultura de Células/métodos , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Protoplastos/metabolismo , Adenosina Trifosfatases/genética , Arabidopsis/citologia , Proteínas de Arabidopsis/genética , Western Blotting/métodos , Células Cultivadas , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Vetores Genéticos/genética , Mutação , Raízes de Plantas/citologia , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção
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