RESUMO
The transfer of endocytosed cargoes to lysosomes (LYSs) requires HOPS, a multiprotein complex that tethers late endosomes (LEs) to LYSs before fusion. Many proteins interact with HOPS on LEs/LYSs. However, it is not clear whether these HOPS interactors localize to LEs or LYSs or how they participate in tethering. Here, we biochemically characterized endosomes purified from untreated or experimentally manipulated cells to put HOPS and interacting proteins in order and to establish their functional interdependence. Our results assign Rab2a and Rab7 to LEs and Arl8 and BORC to LYSs and show that HOPS drives LE-LYS fusion by bridging late endosomal Rab2a with lysosomal BORC-anchored Arl8. We further show that Rab7 is absent from sites of HOPS-dependent tethering but promotes fusion by moving LEs toward LYSs via dynein. Thus, our study identifies the topology of the machinery for LE-LYS tethering and elucidates the role of different small GTPases in the process.
Assuntos
Endocitose , Endossomos , Endossomos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Lisossomos/metabolismo , Fusão de MembranaRESUMO
Early detection of tumor cells by identifying universal Tumor Associated Antigens (TAA) can drastically change our diagnostic, theranostic and therapeutic possibilities to cure cancer. Human Telomerase Reverse Transcriptase (hTERT), a hallmark of cancer, could act as an optimal TAA candidate. Here we report about the development of a monoclonal antibody against hTERT peptide (α-hTERT mAb) presented on the surface of cancer cells and its possible applications as a pan-cancer marker. Liquid biopsies, an innovative tool in precision oncology, comprising the noninvasive analysis of circulating tumor-derived material to counteract limitations associated with tissue biopsies. Within the tumor circulome, the US Food and Drug Administration already approved the use of circulating tumor cells (CTCs) as valid liquid biopsies. However, currently CTCs are being trapped using antibodies against specific cancer types, with anti EpCAM as the most common antibody, directed mainly against solid tumors. Moreover, the precision medicine approach is based on specific cancer type directed antibodies. Our novel mAb against the hTERT 16-mer peptide, corresponding to amino acids 611-626, is capable of detecting various types of cancer cells both in vitro and ex vivo from tumors of patients with either hematological or solid tumors. This antibody does not bind to normal lymphocytes cells. Cleavage of our antibody to F(ab')2 fragments increased its binding specificity to the tested cancer cells. Future studies may point to the use of this antibody in the procedure of capturing CTCs.
Assuntos
Células Neoplásicas Circulantes , Telomerase , Humanos , Telomerase/metabolismo , Corpo Humano , Medicina de Precisão , Anticorpos Monoclonais , Peptídeos/metabolismo , Antígenos de NeoplasiasRESUMO
Intracellular trafficking is mediated by transport carriers that originate by membrane remodeling from donor organelles. Tubular carriers contribute to the flux of membrane lipids and proteins to acceptor organelles, but how lipids and proteins impose a tubular geometry on the carriers is incompletely understood. Using imaging approaches on cells and in vitro membrane systems, we show that phosphatidylinositol-4-phosphate (PI4P) and biogenesis of lysosome-related organelles complex 1 (BLOC-1) govern the formation, stability, and functions of recycling endosomal tubules. In vitro, BLOC-1 binds and tubulates negatively charged membranes, including those containing PI4P. In cells, endosomal PI4P production by type II PI4-kinases is needed to form and stabilize BLOC-1-dependent recycling endosomal tubules. Decreased PI4KIIs expression impairs the recycling of endosomal cargoes and the life cycles of intracellular pathogens such as Chlamydia bacteria and influenza virus that exploit the membrane dynamics of recycling endosomes. This study demonstrates how a phospholipid and a protein complex coordinate the remodeling of cellular membranes into functional tubules.
Assuntos
Endossomos , Membranas Intracelulares , Peptídeos e Proteínas de Sinalização Intracelular , Fosfatos de Fosfatidilinositol , Membrana Celular/metabolismo , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Transporte ProteicoRESUMO
The small GTPase ARL8 associates with endolysosomes, leading to the recruitment of several effectors that couple endolysosomes to kinesins for anterograde transport along microtubules, and to tethering factors for eventual fusion with other organelles. Herein we report the identification of the RUN- and FYVE-domain-containing proteins RUFY3 and RUFY4 as ARL8 effectors that promote coupling of endolysosomes to dynein-dynactin for retrograde transport along microtubules. Using various methodologies, we find that RUFY3 and RUFY4 interact with both GTP-bound ARL8 and dynein-dynactin. In addition, we show that RUFY3 and RUFY4 promote concentration of endolysosomes in the juxtanuclear area of non-neuronal cells, and drive redistribution of endolysosomes from the axon to the soma in hippocampal neurons. The function of RUFY3 in retrograde transport contributes to the juxtanuclear redistribution of endolysosomes upon cytosol alkalinization. These studies thus identify RUFY3 and RUFY4 as ARL8-dependent, dynein-dynactin adaptors or regulators, and highlight the role of ARL8 in the control of both anterograde and retrograde endolysosome transport.
Assuntos
Dineínas , Microtúbulos , Complexo Dinactina , Dineínas/metabolismo , Endossomos/metabolismo , Cinesinas , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismoRESUMO
Long-range movement of organelles within the cytoplasm relies on coupling to microtubule motors, a process that is often mediated by adaptor proteins. In many cases, this coupling involves organelle- or adaptor-induced activation of the microtubule motors by conformational reversal of an autoinhibited state. Herein, we show that a similar regulatory mechanism operates for an adaptor protein named SKIP (also known as PLEKHM2). SKIP binds to the small guanosine triphosphatase (GTPase) ARL8 on the lysosomal membrane to couple lysosomes to the anterograde microtubule motor kinesin-1. Structure-function analyses of SKIP reveal that the C-terminal region comprising three pleckstrin homology (PH) domains interacts with the N-terminal region comprising ARL8- and kinesin-1-binding sites. This interaction inhibits coupling of lysosomes to kinesin-1 and, consequently, lysosome movement toward the cell periphery. We also find that ARL8 does not just recruit SKIP to the lysosomal membrane but also relieves SKIP autoinhibition, promoting kinesin-1-driven, anterograde lysosome transport. Finally, our analyses show that the largely disordered middle region of SKIP mediates self-association and that this self-association enhances the interaction of SKIP with kinesin-1. These findings indicate that SKIP is not just a passive connector of lysosome-bound ARL8 to kinesin-1 but is itself subject to intra- and inter-molecular interactions that regulate its function. We anticipate that similar organelle- or GTPase-induced conformational changes could regulate the activity of other kinesin adaptors.
Assuntos
Lisossomos , Fatores de Ribosilação do ADP , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , GTP Fosfo-Hidrolases , Células HeLa , Humanos , Cinesinas , Lisossomos/metabolismo , Microtúbulos/metabolismoRESUMO
Phosphoinositides have a pivotal role in the maturation of nascent phagosomes into microbicidal phagolysosomes. Following degradation of their contents, mature phagolysosomes undergo resolution, a process that remains largely uninvestigated. Here we studied the role of phosphoinositides in phagolysosome resolution. Phosphatidylinositol-4-phosphate (PtdIns(4)P), which is abundant in maturing phagolysosomes, was depleted as they tubulated and resorbed. Depletion was caused, in part, by transfer of phagolysosomal PtdIns(4)P to the endoplasmic reticulum, a process mediated by oxysterol-binding protein-related protein 1L (ORP1L), a RAB7 effector. ORP1L formed discrete tethers between the phagolysosome and the endoplasmic reticulum, resulting in distinct regions with alternating PtdIns(4)P depletion and enrichment. Tubules emerged from PtdIns(4)P-rich regions, where ADP-ribosylation factor-like protein 8B (ARL8B) and SifA- and kinesin-interacting protein/pleckstrin homology domain-containing family M member 2 (SKIP/PLEKHM2) accumulated. SKIP binds preferentially to monophosphorylated phosphoinositides, of which PtdIns(4)P is most abundant in phagolysosomes, contributing to their tubulation. Accordingly, premature hydrolysis of PtdIns(4)P impaired SKIP recruitment and phagosome resolution. Thus, resolution involves phosphoinositides and tethering of phagolysosomes to the endoplasmic reticulum.
Assuntos
Retículo Endoplasmático/metabolismo , Monócitos/metabolismo , Fagossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Receptores de Esteroides/genética , Transdução de Sinais , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Animais , Sistemas CRISPR-Cas , Retículo Endoplasmático/ultraestrutura , Edição de Genes , Regulação da Expressão Gênica , Humanos , Camundongos , Monócitos/ultraestrutura , Fagocitose , Fagossomos/ultraestrutura , Cultura Primária de Células , Proteólise , Células RAW 264.7 , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Esteroides/antagonistas & inibidores , Receptores de Esteroides/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Ubiquitylation is an eukaryotic signal that regulates most cellular pathways. However, four major hurdles pose challenges to study ubiquitylation: (1) high redundancy between ubiquitin (Ub) cascades, (2) ubiquitylation is tightly regulated in the cell, (3) the transient nature of the Ub signal, and (4) difficulties to purify functional ubiquitylation apparatus for in vitro assay. Here, we present systems that express functional Ub cascades in E. coli, which lacks deubiquitylases, Ub-dependent degradations, and control mechanisms for ubiquitylation. Therefore, expression of an ubiquitylation cascade results in the accumulation of stable ubiquitylated protein that can be genetically selected or purified, thus circumventing the above challenges. Co-expression of split antibiotic resistance protein fragments tethered to Ub and ubiquitylation targets along with ubiquitylation enzymes (E1, E2, and E3) gives rise to bacterial growth on selective media. We show that ubiquitylation rate is highly correlated with growth efficiency. Hence, genetic libraries and simple manipulations in the selection system facilitate the identification and characterization of components and interfaces along Ub cascades. The bacterial expression system also facilitates the detection of ubiquitylated proteins. Furthermore, the expression system allows affinity chromatography-based purification of milligram quantities of ubiquitylated proteins for downstream biochemical, biophysical, and structural studies.
Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas/genética , Proteínas/isolamento & purificação , Proteínas/metabolismo , Ordem dos Genes , Vetores Genéticos/genética , Modelos Moleculares , Conformação Proteica , Proteínas/química , Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Clinicians continue to rely on invasive tissue biopsies as a mean to assess a patient's disease and prescribe appropriate treatment regimens. Biopsies not only are risky and expensive but also limit the understanding of disease. Circulating tumor cells (CTCs) can be isolated from a simple blood draw and offer a promising potential to both diagnose and monitor cancer progression. The VTX-1 Liquid Biopsy System automates the isolation of clinically relevant CTC populations, while simplifying their collection for easy analysis, ultimately expanding the clinical possibilities for CTCs. We present here the key features and performance of this automated system for isolating CTCs directly from whole blood, both with cell spiking experiments and patient samples. As a first step toward the characterization of CTCs for research applications and transfer to clinical practice, we present workflows for both molecular analyses and automated cell enumeration and biomarker quantification with the BioView imaging platform. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
Assuntos
Biópsia Líquida/métodos , Neoplasias/patologia , Células Neoplásicas Circulantes/patologia , Automação Laboratorial/métodos , Contagem de Células/métodos , Linhagem Celular Tumoral , Separação Celular/métodos , Humanos , Coloração e Rotulagem/métodosRESUMO
BACKGROUND: Early detection decreases lung cancer mortality. The Target-FISH Lung Cancer Detection (LCD) Test is a non-invasive test designed to detect chromosomal changes (deletion or amplification) via Fluorescence in situ Hybridization (FISH) in sputum specimens from persons suspected of having lung cancer. We evaluated the performance of the LCD test in patients with highly suspicious pulmonary nodules who were scheduled for a biopsy procedure. METHODS: Induced sputum was collected from patients who were scheduled for biopsy of a solitary pulmonary nodule (0.8-3â¯cm) in one of 6 tertiary medical centers in the US and Israel. The lung cancer detection (LCD) Test combined sputum cytology and Target-FISH analysis on the same target cells and the results were compared to the pathology. Participants with non-surgical negative biopsy results were followed for 2 years to determine their final diagnosis. RESULTS: Of the 173 participants who were evaluated, 112 were available for analysis. Overall, the LCD test had a sensitivity of 85.5% (95% CI, 76.1-92.3), specificity of 69% (95% CI, 49.2-84.7) and an accuracy of 81.3% (95% CI, 72.8-88). The positive and negative predictive values (PPV, NPV) were 88.8% and 62.5%, respectively. The LCD test was positive in 9 of 11 lung cancer patients who had an initial negative biopsy. CONCLUSIONS: In a cohort of patients with highly suspicious lung nodules, the LCD test is a non-invasive option with good sensitivity and a high positive predictive value. A positive LCD test reinforces the need to aggressively pursue a definitive diagnosis of suspicious nodules.
Assuntos
Citodiagnóstico/métodos , Detecção Precoce de Câncer/métodos , Hibridização in Situ Fluorescente/métodos , Neoplasias Pulmonares/diagnóstico , Escarro/citologia , Idoso , Biópsia , Diagnóstico Diferencial , Feminino , Humanos , Pulmão/patologia , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Nódulo Pulmonar Solitário/diagnóstico , Nódulo Pulmonar Solitário/patologiaRESUMO
Lysosomes play key roles in the cellular response to amino acid availability. Depletion of amino acids from the medium turns off a signaling pathway involving the Ragulator complex and the Rag guanosine triphosphatases (GTPases), causing release of the inactive mammalian target of rapamycin complex 1 (mTORC1) serine/threonine kinase from the lysosomal membrane. Decreased phosphorylation of mTORC1 substrates inhibits protein synthesis while activating autophagy. Amino acid depletion also causes clustering of lysosomes in the juxtanuclear area of the cell, but the mechanisms responsible for this phenomenon are poorly understood. Herein we show that Ragulator directly interacts with BLOC-1-related complex (BORC), a multi-subunit complex previously found to promote lysosome dispersal through coupling to the small GTPase Arl8 and the kinesins KIF1B and KIF5B. Interaction with Ragulator exerts a negative regulatory effect on BORC that is independent of mTORC1 activity. Amino acid depletion strengthens this interaction, explaining the redistribution of lysosomes to the juxtanuclear area. These findings thus demonstrate that amino acid availability controls lysosome positioning through Ragulator-dependent, but mTORC1-independent, modulation of BORC.
Assuntos
Aminoácidos/metabolismo , Proteínas de Transporte/metabolismo , Lisossomos/metabolismo , Complexos Multiproteicos/metabolismo , Neurônios/metabolismo , Proteínas/metabolismo , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Aminoácidos/farmacologia , Autofagia , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Cinesinas/genética , Cinesinas/metabolismo , Lectinas/genética , Lectinas/metabolismo , Lisossomos/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Complexos Multiproteicos/genética , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fosforilação , Proteínas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
Ubiquitylation controls protein function and degradation. Therefore, ubiquitin ligases need to be tightly controlled. We discovered an evolutionarily conserved allosteric restraint mechanism for Nedd4 ligases and demonstrated its function with diverse substrates: the yeast soluble proteins Rpn10 and Rvs167, and the human receptor tyrosine kinase FGFR1 and cardiac IKS potassium channel. We found that a potential trimerization interface is structurally blocked by the HECT domain α1-helix, which further undergoes ubiquitylation on a conserved lysine residue. Genetic, bioinformatics, biochemical and biophysical data show that attraction between this α1-conjugated ubiquitin and the HECT ubiquitin-binding patch pulls the α1-helix out of the interface, thereby promoting trimerization. Strikingly, trimerization renders the ligase inactive. Arginine substitution of the ubiquitylated lysine impairs this inactivation mechanism and results in unrestrained FGFR1 ubiquitylation in cells. Similarly, electrophysiological data and TIRF microscopy show that NEDD4 unrestrained mutant constitutively downregulates the IKS channel, thus confirming the functional importance of E3-ligase autoinhibition.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas dos Microfilamentos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Multimerização Proteica , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Humanos , Proteínas dos Microfilamentos/química , Ubiquitina-Proteína Ligases Nedd4 , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Complexo de Endopeptidases do Proteassoma/química , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/química , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Lysosomes have been classically considered terminal degradative organelles, but in recent years they have been found to participate in many other cellular processes, including killing of intracellular pathogens, antigen presentation, plasma membrane repair, cell adhesion and migration, tumor invasion and metastasis, apoptotic cell death, metabolic signaling and gene regulation. In addition, lysosome dysfunction has been shown to underlie not only rare lysosome storage disorders but also more common diseases, such as cancer and neurodegeneration. The involvement of lysosomes in most of these processes is now known to depend on the ability of lysosomes to move throughout the cytoplasm. Here, we review recent findings on the mechanisms that mediate the motility and positioning of lysosomes, and the importance of lysosome dynamics for cell physiology and pathology.
Assuntos
Lisossomos/metabolismo , Animais , Transporte Biológico , Movimento Celular , Humanos , Microtúbulos/metabolismo , Modelos BiológicosRESUMO
Ubiquitin receptors decode ubiquitin signals into many cellular responses. Ubiquitin receptors also undergo coupled monoubiquitylation, and rapid deubiquitylation has hampered the characterization of the ubiquitylated state. Using bacteria that express a ubiquitylation apparatus, we purified and determined the crystal structure of the proteasomal ubiquitin-receptor Rpn10 in its ubiquitylated state. The structure shows a novel ubiquitin-binding patch that directs K84 ubiquitylation. Superimposition of ubiquitylated-Rpn10 onto electron-microscopy models of proteasomes indicates that the Rpn10-conjugated ubiquitin clashes with Rpn9, suggesting that ubiquitylation might be involved in releasing Rpn10 from the proteasome. Indeed, ubiquitylation on immobilized proteasomes dissociates the modified Rpn10 from the complex, while unmodified Rpn10 mainly remains associated. In vivo experiments indicate that contrary to wild type, Rpn10-K84R is stably associated with the proteasomal subunit Rpn9. Similarly Rpn10, but not ubiquitylated-Rpn10, binds Rpn9 in vitro. Thus we suggest that ubiquitylation functions to dissociate modified ubiquitin receptors from their targets, a function that promotes cyclic activity of ubiquitin receptors.
Assuntos
Regulação Fúngica da Expressão Gênica , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina/química , DNA/análise , Microscopia Eletrônica , Conformação Molecular , Fases de Leitura Aberta , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , Proteínas de Saccharomyces cerevisiae/genética , Ressonância de Plasmônio de Superfície , UbiquitinaçãoRESUMO
About one-third of the eukaryotic proteome undergoes ubiquitylation, but the enzymatic cascades leading to substrate modification are largely unknown. We present a genetic selection tool that utilizes Escherichia coli, which lack deubiquitylases, to identify interactions along ubiquitylation cascades. Coexpression of split antibiotic resistance protein tethered to ubiquitin and ubiquitylation target together with a functional ubiquitylation apparatus results in a covalent assembly of the resistance protein, giving rise to bacterial growth on selective media. We applied the selection system to uncover an E3 ligase from the pathogenic bacteria EHEC and to identify the epsin ENTH domain as an ultraweak ubiquitin-binding domain. The latter was complemented with a structure-function analysis of the ENTH-ubiquitin interface. We also constructed and screened a yeast fusion library, discovering Sem1 as a novel ubiquitylation substrate of Rsp5 E3 ligase. Collectively, our selection system provides a robust high-throughput approach for genetic studies of ubiquitylation cascades and for small-molecule modulator screening.
Assuntos
Enzimas Desubiquitinantes/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Seleção Genética , Tioléster Hidrolases/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética , Farmacorresistência Bacteriana/genética , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Modelos Moleculares , Plasmídeos , Transdução de Sinais/genética , Ubiquitina/metabolismoRESUMO
The ubiquitylation signal promotes trafficking of endogenous and retroviral transmembrane proteins. The signal is decoded by a large set of ubiquitin (Ub) receptors that tether Ub-binding domains (UBDs) to the trafficking machinery. We developed a structure-based procedure to scan the protein data bank for hidden UBDs. The screen retrieved many of the known UBDs. Intriguingly, new potential UBDs were identified, including the ALIX-V domain. Pull-down, cross-linking and E3-independent ubiquitylation assays biochemically corroborated the in silico findings. Guided by the output model, we designed mutations at the postulated ALIX-V:Ub interface. Biophysical affinity measurements using microscale-thermophoresis of wild-type and mutant proteins revealed some of the interacting residues of the complex. ALIX-V binds mono-Ub with a K(d) of 119 µM. We show that ALIX-V oligomerizes with a Hill coefficient of 5.4 and IC(50) of 27.6 µM and that mono-Ub induces ALIX-V oligomerization. Moreover, we show that ALIX-V preferentially binds K63 di-Ub compared with mono-Ub and K48 di-Ub. Finally, an in vivo functionality assay demonstrates the significance of ALIX-V:Ub interaction in equine infectious anaemia virus budding. These results not only validate the new procedure, but also demonstrate that ALIX-V directly interacts with Ub in vivo and that this interaction can influence retroviral budding.
Assuntos
Vírus da Anemia Infecciosa Equina/metabolismo , Complexos Multienzimáticos , Mutação , Ubiquitina-Proteína Ligases , Liberação de Vírus/fisiologia , Animais , Humanos , Vírus da Anemia Infecciosa Equina/genética , Camundongos , Modelos Biológicos , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Estrutura Terciária de Proteína , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Protein ubiquitylation controls nearly all cellular pathways in eukaryotes. A repertoire of proteins named ubiquitin (Ub) receptors harbouring ubiquitin-binding domains (UBDs) recognize ubiquitylated proteins. These Ub receptors decode the Ub signal by tethering a UBD or UBDs to a functional domain or domains, thus linking the ubiquitylated target to a specific function. The rapid dynamics of ubiquitylation/deubiquitylation has impeded the characterization of ubiquitylated proteins. To bypass this obstacle, a recently developed synthetic system that reconstructs the entire eukaryotic ubiquitylation cascade in Escherichia coli was used to purify the mono-ubiquitylated form of the regulatory proteasomal non-ATPase subunit (Ub-Rpn10) from Saccharomyces cerevisiae. Here, the first crystallization and data collection of Ub-Rpn10 is reported. Purified Ub-Rpn10 was crystallized in 12%(w/v) PEG 20,000, 0.1 M MES pH 6.5 and yielded thin rhombus-shaped crystals. X-ray analysis revealed that these crystals belonged to the monoclinic system C2, with unit-cell parameters a = 107.3, b = 49.7, c = 81.3 Å, α = γ = 90.0, ß = 130.5°. A full synchrotron data set has been collected, merged and scaled with a diffraction limit of 3.14 Å.
Assuntos
Complexo de Endopeptidases do Proteassoma/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Cristalização , Complexo de Endopeptidases do Proteassoma/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/isolamento & purificaçãoRESUMO
Covalent modification of proteins with ubiquitin (Ub) is widely implicated in the control of protein function and fate. Over 100 deubiquitylating enzymes rapidly reverse this modification, posing challenges to the biochemical and biophysical characterization of ubiquitylated proteins. We circumvented this limitation with a synthetic biology approach of reconstructing the entire eukaryotic Ub cascade in bacteria. Co-expression of affinity-tagged substrates and Ub with E1, E2 and E3 enzymes allows efficient purification of ubiquitylated proteins in milligram quantity. Contrary to in-vitro assays that lead to spurious modification of several lysine residues of Rpn10 (regulatory proteasomal non-ATPase subunit), the reconstituted system faithfully recapitulates its monoubiquitylation on lysine 84 that is observed in vivo. Mass spectrometry revealed the ubiquitylation sites on the Mind bomb E3 ligase and the Ub receptors Rpn10 and Vps9. Förster resonance energy transfer (FRET) analyses of ubiquitylated Vps9 purified from bacteria revealed that although ubiquitylation occurs on the Vps9-GEF domain, it does not affect the guanine nucleotide exchanging factor (GEF) activity in vitro. Finally, we demonstrated that ubiquitylated Vps9 assumes a closed structure, which blocks additional Ub binding. Characterization of several ubiquitylated proteins demonstrated the integrity, specificity and fidelity of the system, and revealed new biological findings.
Assuntos
Escherichia coli/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Biologia Sintética/métodos , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina/metabolismo , Ubiquitinação/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Marcadores de Afinidade , Clonagem Molecular/métodos , Transferência Ressonante de Energia de Fluorescência , Vetores Genéticos/genética , Fatores de Troca do Nucleotídeo Guanina , Guanosina Difosfato/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Complexos Ubiquitina-Proteína Ligase/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Transporte Vesicular , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
BACKGROUND: Lung cancer results from a multistep process, whereby genetic and epigenetic alterations lead to a malignant phenotype. Somatic mutations, deletions, and amplifications can be detected in the tumor itself, but they can also be found in histologically normal bronchial epithelium as a result of field cancerization. The present feasibility study describes a computer-assisted analysis of induced sputum employing morphology and fluorescence in situ hybridization (target-FISH), using 2 biomarkers located at chromosomes 3p22.1 and 10q22.3. METHODS: Induced sputum samples were collected using a standardized protocol from 12 patients with lung cancer and from 15 healthy, nonsmoking controls. We used an automated scanning system that allows consecutive scans of morphology and FISH of the same slide. Cells derived for the lower airways were analyzed for the presence of genetic alterations in the 3p22.1 and 10q22.3 loci. RESULTS: The cutoff for a positive diagnosis was defined as >4% of cells showing genetic alterations. Eleven of 12 lung cancer patients and 12 of 15 controls were identified correctly, giving an overall sensitivity and specificity of 91.66% and 80%, respectively. CONCLUSIONS: This study describes a new technology for detecting lung cancer noninvasively in induced sputum via a combination of morphology and FISH analysis (target-FISH) using computer-assisted technology. This approach may potentially be utilized for mass screening of high-risk populations.
Assuntos
Biomarcadores Tumorais/genética , Hibridização in Situ Fluorescente/métodos , Neoplasias Pulmonares/genética , Escarro/metabolismo , Cromossomos Humanos Par 10/genética , Cromossomos Humanos Par 3/genética , Citodiagnóstico/métodos , Diagnóstico por Computador/métodos , Estudos de Viabilidade , Feminino , Testes Genéticos/métodos , Humanos , Neoplasias Pulmonares/diagnóstico , Masculino , Proteína A Associada a Surfactante Pulmonar/genética , Reprodutibilidade dos Testes , Proteínas Ribossômicas/genética , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Bladder cancer is among the 5 most common malignancies worldwide. Patients with bladder cancer are closely followed with periodic cystoscopies and urine cytology analyses due to the significant risk of tumor recurrence. The UroVysion fluorescence in situ hybridization (FISH) test demonstrated higher sensitivity over urine cytology in detecting bladder cancer by most comparative studies. METHODS: In the current study, the diagnostic usefulness of a combined cytology and FISH analysis approach was tested using the Duet automatic scanning system in patients with benign urine cytology who were being monitored for recurrent urothelial carcinoma or being assessed for various urologic symptoms. RESULTS: By combining the benefits of conventional cytology with molecular diagnostics, a more sensitive detection of bladder cancer was attained. All patients who had positive cystoscopy concomitantly with urine sampling were detected by combined analysis. Additional patients that developed transitional cell carcinoma during a follow-up period of 24 months had a previous positive result on combined analysis. Only 2 patients with a negative combined analysis result presented with late disease recurrence (20 months and 22 months, respectively, after the negative test). Therefore, negative combined analysis was found to be predictive of a lack of disease recurrence for at least 12 months. In this timeframe, the overall sensitivity, specificity, negative predictive value (NPV), and positive predictive values of the combined analysis test were 100%, 65%, 100%, and 44%, respectively. CONCLUSIONS: Given the absolute sensitivity and NPV of the combined analysis test, the management of patients with a negative combined analysis result might be revised and allow for more flexible assessment and management of bladder cancer patients relying more on urine bound tests.
Assuntos
Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/urina , Urina/citologia , Citodiagnóstico , Feminino , Seguimentos , Humanos , Hibridização in Situ Fluorescente , Masculino , Recidiva Local de Neoplasia/diagnóstico , Sensibilidade e EspecificidadeRESUMO
OBJECTIVES: To evaluate a combined analysis approach that involves cytologic evaluation and fluorescence in situ hybridization analysis for detecting cancer cells in voided urine samples using an automated scanning station. METHODS: Voided urine samples from 41 patients suspected of having transitional cell carcinoma were stained with May-Grünwald Giemsa stain, scanned for atypical or suspicious cells, destained, and hybridized with a mixture of fluorescent-labeled probes. Samples were tested using either the UroVysion probe or by a mix of chromosomes 3, 7, and 17 centromeric probes. A case was regarded as positive when at least one cell was abnormal in both aspects, morphology and fluorescence in situ hybridization. Patients were evaluated concomitantly by cytology, cytoscopy, and biopsy, if indicated. RESULTS: Overall, 26 samples were positive by combined analysis. Biopsy-proven transitional cell carcinoma was positive by combined analysis in all cases (100%) and in 13 cases (61.9%) by cytology (P = 0.0133). The advantage of the combined analysis was noted mostly in low-grade and superficial tumors for which the sensitivity of cytology reached 30% (P = 0.023) and 27.27% (P = 0.0133), respectively. Specificity was 100%. CONCLUSIONS: Our results have shown that combined analysis for the presence of transitional cell carcinoma cells is a powerful tool, providing high sensitivity and specificity, and may offer a new scheme for bladder cancer management.