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1.
J Biol Chem ; 300(2): 105643, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38199574

RESUMO

Intestinal epithelia express two long myosin light-chain kinase (MLCK) splice variants, MLCK1 and MLCK2, which differ by the absence of a complete immunoglobulin (Ig)-like domain 3 within MLCK2. MLCK1 is preferentially associated with the perijunctional actomyosin ring at steady state, and this localization is enhanced by inflammatory stimuli including tumor necrosis factor (TNF). Here, we sought to identify MLCK1 domains that direct perijunctional MLCK1 localization and their relevance to disease. Ileal biopsies from Crohn's disease patients demonstrated preferential increases in MLCK1 expression and perijunctional localization relative to healthy controls. In contrast to MLCK1, MLCK2 expressed in intestinal epithelia is predominantly associated with basal stress fibers, and the two isoforms have distinct effects on epithelial migration and barrier regulation. MLCK1(Ig1-4) and MLCK1(Ig1-3), but not MLCK2(Ig1-4) or MLCK1(Ig3), directly bind to F-actin in vitro and direct perijunctional recruitment in intestinal epithelial cells. Further study showed that Ig1 is unnecessary, but that, like Ig3, the unstructured linker between Ig1 and Ig2 (Ig1/2us) is essential for recruitment. Despite being unable to bind F-actin or direct recruitment independently, Ig3 does have dominant negative functions that allow it to displace perijunctional MLCK1, increase steady-state barrier function, prevent TNF-induced MLCK1 recruitment, and attenuate TNF-induced barrier loss. These data define the minimal domain required for MLCK1 localization and provide mechanistic insight into the MLCK1 recruitment process. Overall, the results create a foundation for development of molecularly targeted therapies that target key domains to prevent MLCK1 recruitment, restore barrier function, and limit inflammatory bowel disease progression.


Assuntos
Actinas , Actomiosina , Humanos , Actinas/metabolismo , Actomiosina/metabolismo , Citocinese , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Miosinas/metabolismo , Junções Íntimas/metabolismo , Células CACO-2 , Fator de Necrose Tumoral alfa/metabolismo
2.
Genome Res ; 17(4): 536-43, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17322287

RESUMO

The availability of an annotated genome sequence for the yeast Saccharomyces cerevisiae has made possible the proteome-scale study of protein function and protein-protein interactions. These studies rely on availability of cloned open reading frame (ORF) collections that can be used for cell-free or cell-based protein expression. Several yeast ORF collections are available, but their use and data interpretation can be hindered by reliance on now out-of-date annotations, the inflexible presence of N- or C-terminal tags, and/or the unknown presence of mutations introduced during the cloning process. High-throughput biochemical and genetic analyses would benefit from a "gold standard" (fully sequence-verified, high-quality) ORF collection, which allows for high confidence in and reproducibility of experimental results. Here, we describe Yeast FLEXGene, a S. cerevisiae protein-coding clone collection that covers over 5000 predicted protein-coding sequences. The clone set covers 87% of the current S. cerevisiae genome annotation and includes full sequencing of each ORF insert. Availability of this collection makes possible a wide variety of studies from purified proteins to mutation suppression analysis, which should contribute to a global understanding of yeast protein function.


Assuntos
Genômica/métodos , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Composição de Bases , Sequência de Bases , Western Blotting , Clonagem Molecular , DNA Fúngico/química , DNA Fúngico/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Fases de Leitura Aberta/genética , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Sequência de DNA
3.
Science ; 313(5785): 324-8, 2006 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-16794039

RESUMO

Alpha-synuclein (alphaSyn) misfolding is associated with several devastating neurodegenerative disorders, including Parkinson's disease (PD). In yeast cells and in neurons alphaSyn accumulation is cytotoxic, but little is known about its normal function or pathobiology. The earliest defect following alphaSyn expression in yeast was a block in endoplasmic reticulum (ER)-to-Golgi vesicular trafficking. In a genomewide screen, the largest class of toxicity modifiers were proteins functioning at this same step, including the Rab guanosine triphosphatase Ypt1p, which associated with cytoplasmic alphaSyn inclusions. Elevated expression of Rab1, the mammalian YPT1 homolog, protected against alphaSyn-induced dopaminergic neuron loss in animal models of PD. Thus, synucleinopathies may result from disruptions in basic cellular functions that interface with the unique biology of particular neurons to make them especially vulnerable.


Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Neurônios/fisiologia , Transtornos Parkinsonianos/fisiopatologia , Transporte Proteico , alfa-Sinucleína/metabolismo , Proteínas rab1 de Ligação ao GTP/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Dopamina/fisiologia , Drosophila , Expressão Gênica , Biblioteca Gênica , Humanos , Camundongos , Degeneração Neural , Neurônios/citologia , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Proteínas/química , Proteínas/metabolismo , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab1 de Ligação ao GTP/genética
4.
Nat Chem Biol ; 2(2): 103-9, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16415861

RESUMO

Identification of the cellular targets of small-molecule hits in phenotypic screens is a central challenge in the development of small molecules as biological tools and potential therapeutics. To facilitate the process of small-molecule target identification, we developed a global, microarray-based method for monitoring the growth of pools of yeast strains, each overexpressing a different protein, in the presence of small molecules. Specifically, the growth of Saccharomyces cerevisiae strains harboring approximately 3,900 different overexpression plasmids was monitored in the presence of rapamycin, which inhibits the target of rapamycin (TOR) proteins. TOR was successfully identified as a candidate rapamycin target, and many additional gene products were implicated in the TOR signaling pathway. We also characterized the mechanism of LY-83583, a small-molecule suppressor of rapamycin-induced growth inhibition. These data enabled functional links to be drawn between groups of genes implicated in the TOR pathway, identified several candidate targets for LY-83583, and suggested a role for mitochondrial respiration in mediating rapamycin sensitivity.


Assuntos
Aminoquinolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sirolimo/farmacologia , Antifúngicos/farmacologia , Desenho de Fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Plasmídeos/genética , Análise Serial de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
5.
Genome Res ; 14(10B): 2020-8, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15489321

RESUMO

The creation of genome-scale clone resources is a difficult and costly process, making it essential to maximize the efficiency of each step of clone creation. In this review, we compare the available commercial and open-source recombinational cloning methods with regard to their use in creating comprehensive open reading frame (ORF) clone collections with an emphasis on the properties requisite to use in a high-throughput setting. The most efficient strategy to the creation of ORF clone resources is to build a master clone collection that serves as a quality validated source for producing collections of expression clones. We examine the methods for recombinational cloning available for both the creation of master clones and their conversion into expression clones. Alternative approaches to creating clones involving mixing of cloning methods, including gap-repair cloning, are also explored.


Assuntos
Clonagem Molecular/métodos , Técnicas de Transferência de Genes , Engenharia Genética , Vetores Genéticos/genética , Fases de Leitura Aberta/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Animais , Expressão Gênica , Humanos , Proteínas Recombinantes/isolamento & purificação
6.
Nat Biotechnol ; 21(8): 921-6, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12872131

RESUMO

There is a growing need for techniques that can identify and characterize protein modifications on a large or global scale. We report here a proteomics approach to enrich, recover, and identify ubiquitin conjugates from Saccharomyces cerevisiae lysate. Ubiquitin conjugates from a strain expressing 6xHis-tagged ubiquitin were isolated, proteolyzed with trypsin and analyzed by multidimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for amino acid sequence determination. We identified 1,075 proteins from the sample. In addition, we detected 110 precise ubiquitination sites present in 72 ubiquitin-protein conjugates. Finally, ubiquitin itself was found to be modified at seven lysine residues providing evidence for unexpected diversity in polyubiquitin chain topology in vivo. The methodology described here provides a general tool for the large-scale analysis and characterization of protein ubiquitination.


Assuntos
Mapeamento de Interação de Proteínas/métodos , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência/métodos , Análise de Sequência de Proteína/métodos , Ubiquitina/química , Ubiquitina/metabolismo , Sequência de Aminoácidos , Regulação Fúngica da Expressão Gênica/fisiologia , Dados de Sequência Molecular , Proteoma/química , Proteoma/metabolismo , Homologia de Sequência de Aminoácidos
7.
Arch Med Res ; 33(4): 318-24, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12234520

RESUMO

Thanks to the results of the multiple completed and ongoing genome sequencing projects and to the newly available recombination-based cloning techniques, it is now possible to build gene repositories with no precedent in their composition, formatting, and potential. This new type of gene repository is necessary to address the challenges imposed by the post-genomic era, i.e., experimentation on a genome-wide scale. We are building the FLEXGene (Full Length EXpression-ready) repository. This unique resource will contain clones representing the complete ORFeome of different organisms, including Homo sapiens as well as several pathogens and model organisms. It will consist of a comprehensive, characterized (sequence-verified), and arrayed gene repository. This resource will allow full exploitation of the genomic information by enabling genome-wide scale experimentation at the level of functional/phenotypic assays as well as at the level of protein expression, purification, and analysis. Here we describe the rationale and construction of this resource and focus on the data obtained from the Saccharomyces cerevisiae project.


Assuntos
Clonagem Molecular , Genoma , Fases de Leitura Aberta , Proteômica , Animais , Biologia Computacional , Bases de Dados Genéticas , Genoma Humano , Genômica , Humanos
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