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1.
J Mol Biol ; 433(13): 166983, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-33839165

RESUMO

Recombinant antibodies (Abs) against the SARS-CoV-2 virus hold promise for treatment of COVID-19 and high sensitivity and specific diagnostic assays. Here, we report engineering principles and realization of a Protein-fragment Complementation Assay (PCA) detector of SARS-CoV-2 antigen by coupling two Abs to complementary N- and C-terminal fragments of the reporter enzyme Gaussia luciferase (Gluc). Both Abs display comparably high affinities for distinct epitopes of viral Spike (S)-protein trimers. Gluc activity is reconstituted when the Abs are simultaneously bound to S-protein bringing the Ab-fused N- and C-terminal fragments close enough together (8 nm) to fold. We thus achieve high specificity both by requirement of simultaneous binding of the two Abs to the S-protein and also, in a steric configuration in which the two Gluc complementary fragments can fold and thus reconstitute catalytic activity. Gluc activity can also be reconstituted with virus-like particles that express surface S-protein with detectable signal over background within 5 min of incubation. Design principles presented here can be readily applied to develop reporters to virtually any protein with sufficient available structural details. Thus, our results present a general framework to develop reporter assays for COVID-19, and the strategy can be readily deployed in response to existing and future pathogenic threats and other diseases.


Assuntos
Anticorpos Antivirais/química , Antígenos Virais/imunologia , COVID-19/diagnóstico , COVID-19/virologia , SARS-CoV-2/isolamento & purificação , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Antígenos Virais/química , Antígenos Virais/isolamento & purificação , Epitopos/imunologia , Humanos , Imunoglobulina G/química , Imunoglobulina G/imunologia , Luciferases , Medições Luminescentes/métodos , Engenharia de Proteínas , SARS-CoV-2/química , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia
2.
Bioessays ; 42(2): e1900169, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31854021

RESUMO

How do common and rare genetic polymorphisms contribute to quantitative traits or disease risk and progression? Multiple human traits have been extensively characterized at the genomic level, revealing their complex genetic architecture. However, it is difficult to resolve the mechanisms by which specific variants contribute to a phenotype. Recently, analyses of variant effects on molecular traits have uncovered intermediate mechanisms that link sequence variation to phenotypic changes. Yet, these methods only capture a fraction of genetic contributions to phenotype. Here, in reviewing the field, it is proposed that complex traits can be understood by characterizing the dynamics of biochemical networks within living cells, and that the effects of genetic variation can be captured on these networks by using protein-protein interaction (PPI) methodologies. This synergy between PPI methodologies and the genetics of complex traits opens new avenues to investigate the molecular etiology of human diseases and to facilitate their prevention or treatment.


Assuntos
Polimorfismo de Nucleotídeo Único/genética , Mapas de Interação de Proteínas/genética , Proteoma/genética , Animais , Estudo de Associação Genômica Ampla/métodos , Genômica/métodos , Humanos , Modelos Genéticos , Fenótipo , Locos de Características Quantitativas/genética
3.
Cytokine ; 121: 154738, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31158699

RESUMO

Crohn's disease (CD) and ulcerative colitis (UC) are the two major forms of inflammatory bowel disease (IBD). These idiopathic and chronic diseases result from inflammation of the gastrointestinal tract and are mainly mediated by the immune system. Genome wide association studies link genes of the IL-12 and IL-23 biology to both CD and UC susceptibility. IL-12 and IL-23 cytokines share a functional subunit, p40, and their respective receptors also share a functional subunit, IL-12Rß1. However, clinical trials targeting p40, and thus inhibiting both IL-12 and IL-23 pathways, provided mitigated effects on IBD, suggesting context dependent effects for each cytokine. In addition to IL-12 and IL-23, genetic deficiencies in IL-10 also result in severe IBD pathology. We generated various mouse models to determine how IL-12 or IL-23 interacts with IL-10 in IBD pathology. Whereas defects in both IL-10 and IL-12R do not impact the severity of the Dextran Sulfate Sodium (DSS)-induced colitis, combined deficiencies in both IL-10 and IL-23R aggravate the disease. In contrast to DSS-induced colitis, defects in IL-12R and IL-23R both protect from the spontaneous colitis observed in IL10-/- mice. Together, these studies exemplify the complexity of genetic and environmental interactions for identifying biological pathways predictive of pathological inflammatory processes.


Assuntos
Colite/metabolismo , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Interleucina-23/metabolismo , Transdução de Sinais , Animais , Sulfato de Dextrana , Modelos Animais de Doenças , Inflamação/patologia , Doenças Inflamatórias Intestinais/patologia , Interleucina-10/deficiência , Camundongos Endogâmicos C57BL , Receptores de Interleucina/deficiência , Receptores de Interleucina/metabolismo
4.
Curr Opin Struct Biol ; 54: 171-178, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30978654

RESUMO

There are emerging interests in understanding higher order assemblies of biopolymers within and between cells, such as protein-protein and protein-RNA biomolecular condensates. These biomolecular condensates are thought to assemble/disassemble via multivalent interactions, including those mediated particularly by unique repeated amino acid motifs (URM). We asked how common are proteins with such URMs, their incidence and abundance, by exhaustively enumerating repeating motifs of length 3-10 in the human proteome. We found that URMs are very common and widely distributed across the human proteome. Moreover, the number of repetitions and intervals between them do not correlate with their lengths, which suggests that the number of repeats among proteins in the proteome is independent of length, contrary to the notion that short motifs are more abundant then long motifs. Finally, we describe two examples of URMs in proteins known to form higher order biopolymer assemblies: multi-PDZ domain-containing proteins and the FUS family of RNA binding proteins. For the FUS family, we predicted a known sequence 'grammar', specific motifs and interval sequence compositions that are essential to phase separation and material properties of condensates formed by this family of proteins. In PDZ domain-containing proteins we found a novel repeated motif that was surprisingly both within and between individual PDZ domains. We speculate that these motifs could be binding sites for multivalent interactions, a residual result of the mechanism by which PDZ-domain duplications occurred or that the linker sequences between PDZ domains may encode cryptic PDZ domains.


Assuntos
Proteoma/química , Proteoma/genética , Sequências Repetitivas de Aminoácidos , Motivos de Aminoácidos , Animais , Humanos , Dobramento de Proteína , Proteoma/metabolismo
5.
J Am Chem Soc ; 141(19): 7751-7757, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-31017394

RESUMO

Biomolecular condensates formed by liquid-liquid phase separation of proteins and nucleic acids have been recently discovered to be prevalent in biology. These dynamic condensates behave like biochemical reaction vessels, but little is known about their structural organization and biophysical properties, which are likely related to condensate size. Thus, it is critical that we study them on scales found in vivo. However, previous in vitro studies of condensate assembly and physical properties have involved condensates up to 1000 times larger than those found in vivo. Here, we apply confinement microscopy to visualize condensates and control their sizes by creating appropriate confinement length scales relevant to the cell environment. We observe anomalous diffusion of probe particles embedded within confined condensates, as well as heterogeneous dynamics in condensates formed from PEG/dextran and in ribonucleoprotein complexes of RNA and the RNA-binding protein Dhh1. We propose that the observed non-Gaussian dynamics indicate a hopping diffusion mechanism inside condensates. We also observe that, for dextran-rich condensates, but not for ribonucleo condensates, probe particle diffusion depends on condensate size.


Assuntos
Microambiente Celular , Dextranos/química , Polietilenoglicóis/química , Difusão , Microscopia de Fluorescência
6.
Nat Methods ; 16(2): 205, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30602782

RESUMO

The version of Supplementary Table 1 originally published online with this article contained incorrect localization annotations for one plate. This error has been corrected in the online Supplementary Information.

7.
Sci Immunol ; 3(30)2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30578352

RESUMO

Inherited IL-12Rß1 and TYK2 deficiencies impair both IL-12- and IL-23-dependent IFN-γ immunity and are rare monogenic causes of tuberculosis, each found in less than 1/600,000 individuals. We show that homozygosity for the common TYK2 P1104A allele, which is found in about 1/600 Europeans and between 1/1000 and 1/10,000 individuals in regions other than East Asia, is more frequent in a cohort of patients with tuberculosis from endemic areas than in ethnicity-adjusted controls (P = 8.37 × 10-8; odds ratio, 89.31; 95% CI, 14.7 to 1725). Moreover, the frequency of P1104A in Europeans has decreased, from about 9% to 4.2%, over the past 4000 years, consistent with purging of this variant by endemic tuberculosis. Surprisingly, we also show that TYK2 P1104A impairs cellular responses to IL-23, but not to IFN-α, IL-10, or even IL-12, which, like IL-23, induces IFN-γ via activation of TYK2 and JAK2. Moreover, TYK2 P1104A is properly docked on cytokine receptors and can be phosphorylated by the proximal JAK, but lacks catalytic activity. Last, we show that the catalytic activity of TYK2 is essential for IL-23, but not IL-12, responses in cells expressing wild-type JAK2. In contrast, the catalytic activity of JAK2 is redundant for both IL-12 and IL-23 responses, because the catalytically inactive P1057A JAK2, which is also docked and phosphorylated, rescues signaling in cells expressing wild-type TYK2. In conclusion, homozygosity for the catalytically inactive P1104A missense variant of TYK2 selectively disrupts the induction of IFN-γ by IL-23 and is a common monogenic etiology of tuberculosis.


Assuntos
Interferon gama/imunologia , Interleucina-23/imunologia , Mutação de Sentido Incorreto/genética , TYK2 Quinase/genética , Tuberculose/imunologia , Células Cultivadas , Homozigoto , Humanos , Interleucina-23/deficiência , TYK2 Quinase/imunologia
8.
Cell ; 175(5): 1418-1429.e9, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30454649

RESUMO

We report here a simple and global strategy to map out gene functions and target pathways of drugs, toxins, or other small molecules based on "homomer dynamics" protein-fragment complementation assays (hdPCA). hdPCA measures changes in self-association (homomerization) of over 3,500 yeast proteins in yeast grown under different conditions. hdPCA complements genetic interaction measurements while eliminating the confounding effects of gene ablation. We demonstrate that hdPCA accurately predicts the effects of two longevity and health span-affecting drugs, the immunosuppressant rapamycin and the type 2 diabetes drug metformin, on cellular pathways. We also discovered an unsuspected global cellular response to metformin that resembles iron deficiency and includes a change in protein-bound iron levels. This discovery opens a new avenue to investigate molecular mechanisms for the prevention or treatment of diabetes, cancers, and other chronic diseases of aging.


Assuntos
Ferro/metabolismo , Metaloproteínas/metabolismo , Metformina/farmacologia , Saccharomyces cerevisiae/metabolismo , Sirolimo/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Teste de Complementação Genética , Humanos , Metaloproteínas/genética , Saccharomyces cerevisiae/genética
9.
Nat Methods ; 15(8): 617-622, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29988094

RESUMO

Yeast libraries revolutionized the systematic study of cell biology. To extensively increase the number of such libraries, we used our previously devised SWAp-Tag (SWAT) approach to construct a genome-wide library of ~5,500 strains carrying the SWAT NOP1promoter-GFP module at the N terminus of proteins. In addition, we created six diverse libraries that restored the native regulation, created an overexpression library with a Cherry tag, or enabled protein complementation assays from two fragments of an enzyme or fluorophore. We developed methods utilizing these SWAT collections to systematically characterize the yeast proteome for protein abundance, localization, topology, and interactions.


Assuntos
Genoma Fúngico , Biblioteca Genômica , Proteoma/genética , Saccharomyces cerevisiae/genética , Teste de Complementação Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Mapeamento de Interação de Proteínas , Proteoma/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sitios de Sequências Rotuladas
10.
J Mol Biol ; 430(23): 4754-4761, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-29913159

RESUMO

The spontaneous nature of biopolymer phase separation in cells entails that the resulting condensates can be thermodynamic machines, which, in the process of condensing, can take on distinct forms themselves and deform neighboring cellular structures. We introduce here general notions of material and mechanical properties of protein condensates with an emphasis on how molecular arrangements and intermolecular interaction within condensates determine their ability to do work on their surroundings. We further propose functional implications of these concepts to cellular and subcellular morphology and biogenesis.


Assuntos
Biopolímeros/química , Biopolímeros/metabolismo , Proteínas/química , Animais , Fenômenos Bioquímicos , Humanos , Proteínas/metabolismo , Termodinâmica
12.
Cell Chem Biol ; 24(10): 1196-1197, 2017 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-29053950

RESUMO

In this issue of Cell Chemical Biology, Diaz et al. (2017) report a strategy to achieve temporal, spatial, and stoichiometric control over the protein kinase cAbl in living cells. They achieve this by splitting cAbl into two inactive fragments that form an active kinase upon small molecule addition, potentially providing a general way to probe the wiring of signal transduction networks.


Assuntos
Biocatálise , Engenharia de Proteínas , Proteínas Proto-Oncogênicas c-abl/metabolismo , Ativação Enzimática/efeitos dos fármacos , Células HeLa , Humanos , Proteínas Proto-Oncogênicas c-abl/genética , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/genética
13.
PLoS Comput Biol ; 13(4): e1005499, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28426660

RESUMO

High-throughput in vitro methods have been extensively applied to identify linear information that encodes peptide recognition. However, these methods are limited in number of peptides, sequence variation, and length of peptides that can be explored, and often produce solutions that are not found in the cell. Despite the large number of methods developed to attempt addressing these issues, the exhaustive search of linear information encoding protein-peptide recognition has been so far physically unfeasible. Here, we describe a strategy, called DALEL, for the exhaustive search of linear sequence information encoded in proteins that bind to a common partner. We applied DALEL to explore binding specificity of SH3 domains in the budding yeast Saccharomyces cerevisiae. Using only the polypeptide sequences of SH3 domain binding proteins, we succeeded in identifying the majority of known SH3 binding sites previously discovered either in vitro or in vivo. Moreover, we discovered a number of sites with both non-canonical sequences and distinct properties that may serve ancillary roles in peptide recognition. We compared DALEL to a variety of state-of-the-art algorithms in the blind identification of known binding sites of the human Grb2 SH3 domain. We also benchmarked DALEL on curated biological motifs derived from the ELM database to evaluate the effect of increasing/decreasing the enrichment of the motifs. Our strategy can be applied in conjunction with experimental data of proteins interacting with a common partner to identify binding sites among them. Yet, our strategy can also be applied to any group of proteins of interest to identify enriched linear motifs or to exhaustively explore the space of linear information encoded in a polypeptide sequence. Finally, we have developed a webserver located at http://michnick.bcm.umontreal.ca/dalel, offering user-friendly interface and providing different scenarios utilizing DALEL.


Assuntos
Sítios de Ligação , Biologia Computacional/métodos , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Algoritmos , Proteína Adaptadora GRB2 , Humanos , Proteínas de Saccharomyces cerevisiae , Análise de Sequência de Proteína , Domínios de Homologia de src
14.
BMC Syst Biol ; 11(1): 38, 2017 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-28298210

RESUMO

BACKGROUND: Kinases and phosphatases (KP) form complex self-regulating networks essential for cellular signal processing. In spite of having a wealth of data about interactions among KPs and their substrates, we have very limited models of the structures of the directed networks they form and consequently our ability to formulate hypotheses about how their structure determines the flow of information in these networks is restricted. RESULTS: We assembled and studied the largest bona fide kinase-phosphatase network (KP-Net) known to date for the yeast Saccharomyces cerevisiae. Application of the vertex sort (VS) algorithm on the KP-Net allowed us to elucidate its hierarchical structure in which nodes are sorted into top, core and bottom layers, forming a bow tie structure with a strongly connected core layer. Surprisingly, phosphatases tend to sort into the top layer, implying they are less regulated by phosphorylation than kinases. Superposition of the widest range of KP biological properties over the KP-Net hierarchy shows that core layer KPs: (i), receive the largest number of inputs; (ii), form bottlenecks implicated in multiple pathways and in decision-making; (iii), and are among the most regulated KPs both temporally and spatially. Moreover, top layer KPs are more abundant and less noisy than those in the bottom layer. Finally, we showed that the VS algorithm depends on node degrees without biasing the biological results of the sorted network. The VS algorithm is available as an R package ( https://cran.r-project.org/web/packages/VertexSort/index.html ). CONCLUSIONS: The KP-Net model we propose possesses a bow tie hierarchical structure in which the top layer appears to ensure highest fidelity and the core layer appears to mediate signal integration and cell state-dependent signal interpretation. Our model of the yeast KP-Net provides both functional insight into its organization as we understand today and a framework for future investigation of information processing in yeast and eukaryotes in general.


Assuntos
Modelos Biológicos , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Quinases/metabolismo , Saccharomyces cerevisiae/metabolismo , Espaço Intracelular/metabolismo , Fosforilação , Transporte Proteico , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia
15.
Cold Spring Harb Protoc ; 2016(11)2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27803252

RESUMO

Protein-fragment complementation assays (PCAs) can be used to study protein-protein interactions (PPIs) in any living cell, in vivo or in vitro, in any subcellular compartment or membranes. Here, we present a detailed protocol for performing and analyzing a high-throughput PCA screening to study PPIs in yeast, using dihydrofolate reductase (DHFR) as the reporter protein. The DHFR PCA is a simple survival-selection assay in which Saccharomyces cerevisiae DHFR (scDHFR) is inhibited by methotrexate, thus preventing nucleotide synthesis and causing arrest of cell division. Complementation of cells with a methotrexate-insensitive murine DHFR restores nucleotide synthesis, allowing cell proliferation. The methotrexate-resistant DHFR has two mutations (L22F and F31S) and is 10,000 times less sensitive to methotrexate than wild-type scDHFR, but retains full catalytic activity. The DHFR PCA is sensitive enough for PPIs to be detected for open reading frame (ORF)-PCA fragments expressed off of their endogenous promoters.


Assuntos
Testes Genéticos/métodos , Viabilidade Microbiana , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Seleção Genética , Tetra-Hidrofolato Desidrogenase/metabolismo , Animais , Genes Reporter , Camundongos , Tetra-Hidrofolato Desidrogenase/genética
16.
Cold Spring Harb Protoc ; 2016(11)2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27803254

RESUMO

Here, we present a detailed protocol for studying in yeast cells the contingent interaction between a substrate and its multisubunit enzyme complex by using a death selection technique known as the optimized yeast cytosine deaminase protein-fragment complementation assay (OyCD PCA). In yeast, the enzyme cytosine deaminase (encoded by FCY1) is involved in pyrimidine metabolism. The PCA is based on an engineered form of yeast cytosine deaminase optimized by directed evolution for maximum activity (OyCD), which acts as a reporter converting the pro-drug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), a toxic compound that kills the cell. Cells that have OyCD PCA activity convert 5-FC to 5-FU and die. Using this assay, it is possible to assess how regulatory subunits of an enzyme contribute to the overall interaction between the catalytic subunit and the potential substrates. Furthermore, OyCD PCA can be used to dissect different functions of mutant forms of a protein as a mutant can disrupt interaction with one partner, while retaining interaction with others. As it is scalable to a medium- or high-throughput format, OyCD PCA can be used to study hundreds to thousands of pairwise protein-protein interactions in different deletion strains. In addition, OyCD PCA vectors (pAG413GAL1-ccdB-OyCD-F[1] and pAG415GAL1-ccdB-OyCD-F[2]) have been designed to be compatible with the proprietary Gateway technology. It is therefore easy to generate fusion genes with the OyCD reporter fragments. As an example, we will focus on the yeast cyclin-dependent protein kinase 1 (Cdk1, encoded by CDC28), its regulatory cyclin subunits, and its substrates or binding partners.


Assuntos
Citosina Desaminase/metabolismo , Teste de Complementação Genética , Viabilidade Microbiana , Complexos Multiproteicos/genética , Mapeamento de Interação de Proteínas/métodos , Saccharomyces cerevisiae/genética , Seleção Genética , Citosina Desaminase/genética
17.
Cold Spring Harb Protoc ; 2016(11)2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27803255

RESUMO

Here, we present detailed protocols for direct, real-time protein-fragment complementation assays (PCAs) for studying the spatiotemporal dynamics of protein-protein interactions (PPIs). The assays require the use of two fluorescent reporter proteins-the "Venus" version of yellow fluorescent protein (vYFP), and the monomeric infrared fluorescent protein 1.4 (IFP 1.4)-or two luciferase reporter proteins-Renilla (Rluc) and Gaussia (Gluc). The luciferase PCAs can be used to study the temporal dynamics of PPIs in any cellular compartment and on membranes. The full reversibility of these PCAs assures accurate measurements of the kinetics of PPI assembly/disassembly for processes that occur anywhere in a living cell and over time frames of seconds to hours. vYFP PCA, and all PCAs based on green fluorescent protein and its variants, are irreversible and can be used to trap and visualize rare and transient complexes and follow dynamic relocalization of constitutive complexes. vYFP PCA is limited in that accurate measurements of temporal changes in PPIs are not possible owing to the slow maturation time of vYFP (minutes to hours) and the irreversibility of its PCA that traps the complexes, thereby preventing the dissociation of PPIs that, in some instances, might cause spurious mislocalization of protein complexes. The limitations of vYFP PCA are overcome with IFP PCA, which is fully reversible and thus can be used to study spatiotemporal dynamics of PPIs on the timescale of seconds. All of these PCAs are sensitive enough to detect interactions among proteins expressed at endogenous levels in vivo.


Assuntos
Proteínas Luminescentes/análise , Mapeamento de Interação de Proteínas/métodos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/química , Genes Reporter , Análise Espaço-Temporal
18.
Cold Spring Harb Protoc ; 2016(11)2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27803260

RESUMO

Protein-fragment complementation assays (PCAs) comprise a family of assays that can be used to study protein-protein interactions (PPIs), conformation changes, and protein complex dimensions. We developed PCAs to provide simple and direct methods for the study of PPIs in any living cell, subcellular compartments or membranes, multicellular organisms, or in vitro. Because they are complete assays, requiring no cell-specific components other than reporter fragments, they can be applied in any context. PCAs provide a general strategy for the detection of proteins expressed at endogenous levels within appropriate subcellular compartments and with normal posttranslational modifications, in virtually any cell type or organism under any conditions. Here we introduce a number of applications of PCAs in budding yeast, Saccharomyces cerevisiae These applications represent the full range of PPI characteristics that might be studied, from simple detection on a large scale to visualization of spatiotemporal dynamics.


Assuntos
Mapeamento de Interação de Proteínas/métodos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/química , Análise Espaço-Temporal
19.
Mol Cell Biol ; 36(20): 2596-611, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27503856

RESUMO

Macropinocytosis is a normal cellular process by which cells internalize extracellular fluids and nutrients from their environment and is one strategy that Ras-transformed pancreatic cancer cells use to increase uptake of amino acids to meet the needs of rapid growth. Paradoxically, in non-Ras transformed medulloblastoma brain tumors, we have shown that expression and activation of the receptor tyrosine kinase TrkA overactivates macropinocytosis, resulting in the catastrophic disintegration of the cell membrane and in tumor cell death. The molecular basis of this uncontrolled form of macropinocytosis has not been previously understood. Here, we demonstrate that the overactivation of macropinocytosis is caused by the simultaneous activation of two TrkA-mediated pathways: (i) inhibition of RhoB via phosphorylation at Ser(185) by casein kinase 1, which relieves actin stress fibers, and (ii) FRS2-scaffolded Src and H-Ras activation of RhoA, which stimulate actin reorganization and the formation of lamellipodia. Since catastrophic macropinocytosis results in brain tumor cell death, improved understanding of the mechanisms involved will facilitate future efforts to reprogram tumors, even those resistant to apoptosis, to die.


Assuntos
Caseína Quinase I/metabolismo , Neoplasias Cerebelares/metabolismo , Meduloblastoma/metabolismo , Pinocitose , Receptor trkA/metabolismo , Proteína rhoB de Ligação ao GTP/metabolismo , Actinas/metabolismo , Morte Celular , Linhagem Celular Tumoral , Humanos , Fosforilação , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Serina/metabolismo , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/metabolismo
20.
Proc Natl Acad Sci U S A ; 113(27): E3862-71, 2016 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-27317745

RESUMO

Evolution of complexity in eukaryotic proteomes has arisen, in part, through emergence of modular independently folded domains mediating protein interactions via binding to short linear peptides in proteins. Over 30 years, structural properties and sequence preferences of these peptides have been extensively characterized. Less successful, however, were efforts to establish relationships between physicochemical properties and functions of domain-peptide interactions. To our knowledge, we have devised the first strategy to exhaustively explore the binding specificity of protein domain-peptide interactions. We applied the strategy to SH3 domains to determine the properties of their binding peptides starting from various experimental data. The strategy identified the majority (∼70%) of experimentally determined SH3 binding sites. We discovered mutual relationships among binding specificity, binding affinity, and structural properties and evolution of linear peptides. Remarkably, we found that these properties are also related to functional diversity, defined by depth of proteins within hierarchies of gene ontologies. Our results revealed that linear peptides evolved to coadapt specificity and affinity to functional diversity of domain-peptide interactions. Thus, domain-peptide interactions follow human-constructed gene ontologies, which suggest that our understanding of biological process hierarchies reflect the way chemical and thermodynamic properties of linear peptides and their interaction networks, in general, have evolved.


Assuntos
Evolução Molecular , Proteínas de Membrana/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Domínios de Homologia de src , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Ontologia Genética , Saccharomyces cerevisiae
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