RESUMEN
Phosphorylation of proteins on tyrosine (Tyr) residues evolved in metazoan organisms as a mechanism of coordinating tissue growth1. Multicellular eukaryotes typically have more than 50 distinct protein Tyr kinases that catalyse the phosphorylation of thousands of Tyr residues throughout the proteome1-3. How a given Tyr kinase can phosphorylate a specific subset of proteins at unique Tyr sites is only partially understood4-7. Here we used combinatorial peptide arrays to profile the substrate sequence specificity of all human Tyr kinases. Globally, the Tyr kinases demonstrate considerable diversity in optimal patterns of residues surrounding the site of phosphorylation, revealing the functional organization of the human Tyr kinome by substrate motif preference. Using this information, Tyr kinases that are most compatible with phosphorylating any Tyr site can be identified. Analysis of mass spectrometry phosphoproteomic datasets using this compendium of kinase specificities accurately identifies specific Tyr kinases that are dysregulated in cells after stimulation with growth factors, treatment with anti-cancer drugs or expression of oncogenic variants. Furthermore, the topology of known Tyr signalling networks naturally emerged from a comparison of the sequence specificities of the Tyr kinases and the SH2 phosphotyrosine (pTyr)-binding domains. Finally we show that the intrinsic substrate specificity of Tyr kinases has remained fundamentally unchanged from worms to humans, suggesting that the fidelity between Tyr kinases and their protein substrate sequences has been maintained across hundreds of millions of years of evolution.
Asunto(s)
Fosfotirosina , Proteínas Tirosina Quinasas , Especificidad por Sustrato , Tirosina , Animales , Humanos , Secuencias de Aminoácidos , Evolución Molecular , Espectrometría de Masas , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilación , Fosfotirosina/metabolismo , Proteínas Tirosina Quinasas/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Proteoma/química , Proteoma/metabolismo , Proteómica , Transducción de Señal , Dominios Homologos src , Tirosina/metabolismo , Tirosina/químicaRESUMEN
Methylation of Lys and Arg residues on non-histone proteins has emerged as a prevalent post-translational modification and as an important regulator of cellular signal transduction mediated by the MAPK, WNT, BMP, Hippo and JAK-STAT signalling pathways. Crosstalk between methylation and other types of post-translational modifications, and between histone and non-histone protein methylation frequently occurs and affects cellular functions such as chromatin remodelling, gene transcription, protein synthesis, signal transduction and DNA repair. With recent advances in proteomic techniques, in particular mass spectrometry, the stage is now set to decode the methylproteome and define its functions in health and disease.
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Ensamble y Desensamble de Cromatina/fisiología , Reparación del ADN/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Biosíntesis de Proteínas/fisiología , Transcripción Genética/fisiología , Vía de Señalización Wnt/fisiología , Animales , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Vía de Señalización Hippo , Humanos , Metilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción STAT/genética , Factores de Transcripción STAT/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMEN
Lysine methylation is a prevalent post-translational modification (PTM) used by the cell to reversibly regulate protein function. Although it has been extensively studied in the context of histones and the associated chromatin, the remaining methyllysine proteome remains largely unexplored. This SnapShot provides an overview of the current state of lysine methylation research and its emergence as a dynamic PTM occurring on histone and non-histone proteins.
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Histonas/metabolismo , Procesamiento Proteico-Postraduccional , Transducción de Señal , Animales , Humanos , Lisina , MetilaciónRESUMEN
Cell migration is an essential physiological process, and aberrant migration of epithelial cells underlies many pathological conditions. However, the molecular mechanisms governing cell migration are not fully understood. We report here that growth factor-induced epithelial cell migration is critically dependent on the crosstalk of two molecular switches, namely phosphorylation switch (P-switch) and transcriptional switch (T-switch). P-switch refers to dynamic interactions of deleted in liver cancer 1 (DLC1) and PI3K with tensin-3 (TNS3), phosphatase and tensin homolog (PTEN), C-terminal tension, and vav guanine nucleotide exchange factor 2 (VAV2) that are dictated by mitogen-activated protein kinase kinase 1/2-extracellular signal-regulated protein kinase 1/2-dependent phosphorylation of TNS3, PTEN, and VAV2. Phosphorylation of TNS3 and PTEN on specific Thr residues led to the switch of DLC1-TNS3 and PI3K-PTEN complexes to DLC1-PTEN and PI3K-TNS3 complexes, whereas Ser phosphorylation of VAV2 promotes the transition of the PI3K-TNS3/PTEN complexes to PI3K-VAV2 complex. T-switch denotes an increase in C-terminal tension transcription/expression regulated by both extracellular signal-regulated protein kinase 1/2 and signal transducer and activator of transcription 3 (STAT3) via interleukin-6-Janus kinase-STAT3 signaling pathway. We have found that, the P-switch is indispensable for both the initiation and continuation of cell migration induced by growth factors, whereas the T-switch is only required to sustain cell migration. The interplay of the two switches facilitated by the interleukin-6-Janus kinase-STAT3 pathway governs a sequence of dynamic protein-protein interactions for sustained cell migration. That a similar mechanism is employed by both normal and tumorigenic epithelial cells to drive their respective migration suggests that the P-switch and T-switch are general regulators of epithelial cell migration and potential therapeutic targets.
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Movimiento Celular/efectos de los fármacos , Células Epiteliales/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Línea Celular Tumoral , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/genética , MAP Quinasa Quinasa 2/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismoRESUMEN
Src homology 2 (SH2) domains play an essential role in cellular signal transduction by binding to proteins phosphorylated on Tyr residue. Although Tyr phosphorylation (pY) is a prerequisite for binding for essentially all SH2 domains characterized to date, different SH2 domains prefer specific sequence motifs C-terminal to the pY residue. Because all SH2 domains adopt the same structural fold, it is not well understood how different SH2 domains have acquired the ability to recognize distinct sequence motifs. We have shown previously that the EF and BG loops that connect the secondary structure elements on an SH2 domain dictate its specificity. In this study, we investigated if these surface loops could be engineered to encode diverse specificities. By characterizing a group of SH2 variants selected by different pY peptides from phage-displayed libraries, we show that the EF and BG loops of the Fyn SH2 domain can encode a wide spectrum of specificities, including all three major specificity classes (p + 2, p + 3 and p + 4) of the SH2 domain family. Furthermore, we found that the specificity of a given variant correlates with the sequence feature of the bait peptide used for its isolation, suggesting that an SH2 domain may acquire specificity by co-evolving with its ligand. Intriguingly, we found that the SH2 variants can employ a variety of different mechanisms to confer the same specificity, suggesting the EF and BG loops are highly flexible and adaptable. Our work provides a plausible mechanism for the SH2 domain to acquire the wide spectrum of specificity observed in nature through loop variation with minimal disturbance to the SH2 fold. It is likely that similar mechanisms may have been employed by other modular interaction domains to generate diversity in specificity.
Asunto(s)
Proteínas Proto-Oncogénicas c-fyn/química , Animales , Cristalografía por Rayos X , Variación Genética , Humanos , Ligandos , Modelos Moleculares , Biblioteca de Péptidos , Estructura Secundaria de Proteína , Proteínas Proto-Oncogénicas c-fyn/genética , Dominios Homologos srcRESUMEN
A large number of post-translational modifications (PTMs) in proteins are buried in the unassigned mass spectrometric (MS) spectra in shot-gun proteomics datasets. Because the modified peptide fragments are low in abundance relative to the corresponding non-modified versions, it is critical to develop tools that allow facile evaluation of assignment of PTMs based on the MS/MS spectra. Such tools will preferably have the ability to allow comparison of fragment ion spectra and retention time between the modified and unmodified peptide pairs or group. Herein, MMS2plot, an R package for visualizing peptide-spectrum matches (PSMs) for multiple peptides, is described. MMS2plot features a batch mode and generates the output images in vector graphics file format that facilitate evaluation and publication of the PSM assignment. MMS2plot is expected to play an important role in PTM discovery from large-scale proteomics datasets generated by liquid chromatography-MS/MS. The MMS2plot package is freely available at https://github.com/lileir/MMS2plot under the GPL-3 license.
RESUMEN
Cellular functions are frequently regulated by protein-protein interactions involving the binding of a modular domain in one protein to a specific peptide sequence in another. This mechanism may be explored to identify binding partners for proteins harboring a peptide-recognition domain. Here we report a proteomic strategy combining peptide and protein microarray screening with biochemical and cellular assays to identify modular domain-mediated protein-protein interactions in a systematic manner. We applied this strategy to Numb, a multi-functional protein containing a phosphotyrosine-binding (PTB) domain. Through the screening of a protein microarray, we identified >100 protein kinases, including both Tyr and Ser/Thr kinases, that could potentially interact with the Numb PTB domain, suggesting a general role for Numb in regulating kinase function. The putative interactions between Numb and several tyrosine kinases were subsequently validated by GST pull-down and/or co-immunoprecipitation assays. Furthermore, using the Oriented Peptide Array Library approach, we defined the specificity of the Numb PTB domain which, in turn, allowed us to predict binding partners for Numb at the genome level. The combination of the protein microarray screening with computer-aided prediction produced the most expansive interactome for Numb to date, implicating Numb in regulating phosphorylation signaling through protein kinases and phosphatases. Not only does the data generated from this study provide an important resource for hypothesis-driven research to further define the function of Numb, the proteomic strategy described herein may be employed to uncover the interactome for other peptide-recognition domains whose consensus motifs are known or can be determined.
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Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Mapeo de Interacción de Proteínas , Proteínas Quinasas/metabolismo , Secuencias de Aminoácidos , Línea Celular Tumoral , Genoma Humano , Humanos , Proteínas de la Membrana/química , Proteínas del Tejido Nervioso/química , Péptidos/metabolismo , Fosforilación , Unión Proteica , Dominios Proteicos , Reproducibilidad de los Resultados , Transducción de SeñalRESUMEN
The proto-oncogene Akt/protein kinase B (PKB) is a pivotal signal transducer for growth and survival. Growth factor stimulation leads to Akt phosphorylation at two regulatory sites (Thr-308 and Ser-473), acutely activating Akt signaling. Delineating the exact role of each regulatory site is, however, technically challenging and has remained elusive. Here, we used genetic code expansion to produce site-specifically phosphorylated Akt1 to dissect the contribution of each regulatory site to Akt1 activity. We achieved recombinant production of full-length Akt1 containing site-specific pThr and pSer residues for the first time. Our analysis of Akt1 site-specifically phosphorylated at either or both sites revealed that phosphorylation at both sites increases the apparent catalytic rate 1500-fold relative to unphosphorylated Akt1, an increase attributable primarily to phosphorylation at Thr-308. Live imaging of COS-7 cells confirmed that phosphorylation of Thr-308, but not Ser-473, is required for cellular activation of Akt. We found in vitro and in the cell that pThr-308 function cannot be mimicked with acidic residues, nor could unphosphorylated Thr-308 be mimicked by an Ala mutation. An Akt1 variant with pSer-308 achieved only partial enzymatic and cellular signaling activity, revealing a critical interaction between the γ-methyl group of pThr-308 and Cys-310 in the Akt1 active site. Thus, pThr-308 is necessary and sufficient to stimulate Akt signaling in cells, and the common use of phosphomimetics is not appropriate for studying the biology of Akt signaling. Our data also indicate that pThr-308 should be regarded as the primary diagnostic marker of Akt activity.
Asunto(s)
Código Genético , Imagen Molecular/métodos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina/metabolismo , Treonina/metabolismo , Células Cultivadas , Cristalografía por Rayos X , Humanos , Mutación , Fosforilación , Conformación Proteica , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-akt/química , Proteínas Proto-Oncogénicas c-akt/genética , Serina/química , Serina/genética , Treonina/química , Treonina/genéticaRESUMEN
Insulin-like growth factor binding protein (IGFBP)-1 influences fetal growth by modifying insulin-like growth factor-I (IGF-I) bioavailability. IGFBP-1 phosphorylation, which markedly increases its affinity for IGF-I, is regulated by mechanistic target of rapamycin (mTOR) and casein kinase (CSNK)-2. However, the underlying molecular mechanisms remain unknown. We examined the cellular localization and potential interactions of IGFBP-1, CSNK-2ß, and mTOR as a prerequisite for protein-protein interaction. Analysis of dual immunofluorescence images indicated a potential perinuclear co-localization between IGFBP-1 and CSNK-2ß and a nuclear co-localization between CSNK-2ß and mTOR. Proximity ligation assay (PLA) indicated proximity between IGFBP-1 and CSNK-2ß as well as mTOR and CSNK-2ß but not between mTOR and IGFBP-1. Three-dimensional rendering of the PLA images validated that IGFBP-1 and CSNK-2ß interactions were in the perinuclear region and mTOR and CSNK-2ß interactions were also predominantly perinuclear rather than nuclear as indicated by mTOR and CSNK-2ß co-localization. Compared with control, hypoxia and rapamycin treatment showed markedly amplified PLA signals for IGFBP-1 and CSNK-2ß (approximately 18-fold, P = 0.0002). Stable isotope labeling with multiple reaction monitoring-mass spectrometry demonstrated that hypoxia and rapamycin treatment increased IGFBP-1 phosphorylation at Ser98/Ser101/Ser119/Ser174 but most considerably (106-fold) at Ser169. We report interactions between CSNK-2ß and IGFBP-1 as well as mTOR and CSNK-2ß, providing strong evidence of a mechanistic link between mTOR and IGF-I signaling, two critical regulators of cell growth via CSNK-2.
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Carcinoma Hepatocelular/metabolismo , Quinasa de la Caseína II/metabolismo , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Neoplasias Hepáticas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Carcinoma Hepatocelular/patología , Técnica del Anticuerpo Fluorescente , Células Hep G2 , Humanos , Neoplasias Hepáticas/patología , Fosforilación , Transducción de Señal/fisiologíaRESUMEN
We present a new strategy for systematic identification of phosphotyrosine (pTyr) by affinity purification mass spectrometry (AP-MS) using a Src homology 2 (SH2)-domain-derived pTyr superbinder as the affinity reagent. The superbinder allows for markedly deeper coverage of the Tyr phosphoproteome than anti-pTyr antibodies when an optimal amount is used. We identified â¼20,000 distinct phosphotyrosyl peptides and >10,000 pTyr sites, of which 36% were 'novel', from nine human cell lines using the superbinder approach. Tyrosine kinases, SH2 domains and phosphotyrosine phosphatases were preferably phosphorylated, suggesting that the toolkit of kinase signaling is subject to intensive regulation by phosphorylation. Cell-type-specific global kinase activation patterns inferred from label-free quantitation of Tyr phosphorylation guided the design of experiments to inhibit cancer cell proliferation by blocking the highly activated tyrosine kinases. Therefore, the superbinder is a highly efficient and cost-effective alternative to conventional antibodies for systematic and quantitative characterization of the tyrosine phosphoproteome under normal or pathological conditions.
Asunto(s)
Fosfotirosina/metabolismo , Proteómica , Línea Celular Tumoral , Humanos , Espectrometría de Masas , Fosforilación , Fosfotirosina/química , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Dominios Homologos srcRESUMEN
Tyrosine phosphorylation (pTyr) is important for normal physiology and implicated in many human diseases, particularly cancer. Identification of pTyr sites is critical to dissecting signaling pathways and understanding disease pathologies. However, compared with serine/threonine phosphorylation (pSer/pThr), the analysis of pTyr at the proteome level is more challenging due to its low abundance. Here, we developed a biphasic affinity chromatographic approach where Src SH2 superbinder was coupled with NeutrAvidin affinity chromatography, for tyrosine phosphoproteome analysis. With the use of competitive elution agent biotin-pYEEI, this strategy can distinguish high-affinity phosphotyrosyl peptides from low-affinity ones, while the excess competitive agent is readily removed by using NeutrAvidin agarose resin in an integrated tip system. The excellent performance of this system was demonstrated by analyzing tyrosine phosphoproteome of Jurkat cells from which 3,480 unique pTyr sites were identified. The biphasic affinity chromatography method for deep Tyr phosphoproteome analysis is rapid, sensitive, robust, and cost-effective. It is widely applicable to the global analysis of the tyrosine phosphoproteome associated with tyrosine kinase signal transduction.
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Cromatografía de Afinidad/métodos , Fosfopéptidos/análisis , Proteoma/análisis , Avidina/química , Avidina/metabolismo , Humanos , Células Jurkat , Fosfopéptidos/aislamiento & purificación , Fosforilación , Proteoma/metabolismo , Tirosina/metabolismo , Dominios Homologos srcRESUMEN
The non-receptor isoform of protein-tyrosine phosphatase ϵ (cyt-PTPe) supports adhesion of bone-resorbing osteoclasts by activating Src downstream of integrins. Loss of cyt-PTPe reduces Src activity in osteoclasts, reduces resorption of mineralized matrix both in vivo and in cell culture, and induces mild osteopetrosis in young female PTPe KO mice. Activation of Src by cyt-PTPe is dependent upon this phosphatase undergoing phosphorylation at its C-terminal Tyr-638 by partially active Src. To understand how cyt-PTPe activates Src, we screened 73 Src homology 2 (SH2) domains for binding to Tyr(P)-638 of cyt-PTPe. The SH2 domain of GRB2 bound Tyr(P)-638 of cyt-PTPe most prominently, whereas the Src SH2 domain did not bind at all, suggesting that GRB2 may link PTPe with downstream molecules. Further studies indicated that GRB2 is required for activation of Src by cyt-PTPe in osteoclast-like cells (OCLs) in culture. Overexpression of GRB2 in OCLs increased activating phosphorylation of Src at Tyr-416 and of cyt-PTPe at Tyr-638; opposite results were obtained when GRB2 expression was reduced by shRNA or by gene inactivation. Phosphorylation of cyt-PTPe at Tyr-683 and its association with GRB2 are integrin-driven processes in OCLs, and cyt-PTPe undergoes autodephosphorylation at Tyr-683, thus limiting Src activation by integrins. Reduced GRB2 expression also reduced the ability of bone marrow precursors to differentiate into OCLs and reduced the fraction of OCLs in which podosomal adhesion structures assume organization typical of active, resorbing cells. We conclude that GRB2 physically links cyt-PTPe with Src and enables cyt-PTPe to activate Src downstream of activated integrins in OCLs.
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Proteína Adaptadora GRB2/fisiología , Osteoclastos/enzimología , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores/fisiología , Familia-src Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Adhesión Celular , Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular , Células HEK293 , Humanos , Ratones de la Cepa 129 , Ratones Noqueados , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Procesamiento Proteico-PostraduccionalRESUMEN
Aberrant or elevated levels of reactive oxygen species (ROS) can mediate deleterious cellular effects, including neuronal toxicity and degeneration observed in the etiology of a number of pathological conditions, including Alzheimer's and Parkinson's diseases. Nevertheless, ROS can be generated in a controlled manner and can regulate redox sensitive transcription factors such as NFκB, AP-1 and NFAT. Moreover, ROS can modulate the redox state of tyrosine phosphorylated proteins, thereby having an impact on many transcriptional networks and signaling cascades important for neurogenesis. A large body of literature links the controlled generation of ROS at low-to-moderate levels with the stimulation of differentiation in certain developmental programs such as neurogenesis. In this regard, ROS are involved in governing the acquisition of the neural fate-from neural induction to the elaboration of axons. Here, we summarize and discuss the growing body of literature that describe a role for ROS signaling in neuronal development.
Asunto(s)
Sistema Nervioso/metabolismo , Neurogénesis , Especies Reactivas de Oxígeno/metabolismo , Animales , Femenino , Humanos , Masculino , Ratones , NADPH Oxidasas/metabolismo , Sistema Nervioso/crecimiento & desarrollo , Neuronas/enzimología , Transducción de SeñalRESUMEN
A distinct feature of hepatocellular carcinoma (HCC) is the tendency of tumor cells to disperse throughout the liver. Nck family adaptor proteins function to couple tyrosine phosphorylation signals to regulate actin cytoskeletal reorganization that leads to cell motility. In order to explore the role of Nck in HCC development, we performed GST pull-down assay using the SH2 domain of Nck1 as bait. The resulting precipitates were separated by 2-DE. Mass spectrometry analysis revealed a group of Nck1 SH2 domain-binding proteins that were differentially expressed in HCC. One of these proteins, dermcidin (DCD), and its interaction with Nck1, was further validated in vitro. GST pull-down assay revealed that Nck1 SH2 domain binds to the phosphotyrosine residue at position 20 (Y20) of the DCD. Pervandate treatment significantly enhanced the interaction between DCD and Nck1. Moreover, we demonstrated that forced expression of DCD could activate Rac1 and Cdc42 and promoted cell migration. Taken together, these data suggest a role of DCD in tumor metastasis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Carcinoma Hepatocelular/patología , Movimiento Celular , Neoplasias Hepáticas/patología , Proteínas Oncogénicas/metabolismo , Péptidos/fisiología , Adulto , Anciano , Carcinoma Hepatocelular/química , Línea Celular Tumoral , Femenino , Humanos , Hígado/química , Neoplasias Hepáticas/química , Masculino , Persona de Mediana Edad , Péptidos/análisis , Fosforilación , Unión Proteica , Proteómica , Tirosina/metabolismo , Proteínas de Unión al GTP rho/fisiología , Dominios Homologos srcRESUMEN
SH2 domains are long known prominent players in the field of phosphotyrosine recognition within signaling protein networks. However, over the years they have been joined by an increasing number of other protein domain families that can, at least with some of their members, also recognise pTyr residues in a sequence-specific context. This superfamily of pTyr recognition modules, which includes substantial fractions of the PTB domains, as well as much smaller, or even single member fractions like the HYB domain, the PKCδ and PKCθ C2 domains and RKIP, represents a fascinating, medically relevant and hence intensely studied part of the cellular signaling architecture of metazoans. Protein tyrosine phosphorylation clearly serves a plethora of functions and pTyr recognition domains are used in a similarly wide range of interaction modes, which encompass, for example, partner protein switching, tandem recognition functionalities and the interaction with catalytically active protein domains. If looked upon closely enough, virtually no pTyr recognition and regulation event is an exact mirror image of another one in the same cell. Thus, the more we learn about the biology and ultrastructural details of pTyr recognition domains, the more does it become apparent that nature cleverly combines and varies a few basic principles to generate a sheer endless number of sophisticated and highly effective recognition/regulation events that are, under normal conditions, elegantly orchestrated in time and space. This knowledge is also valuable when exploring pTyr reader domains as diagnostic tools, drug targets or therapeutic reagents to combat human diseases.
RESUMEN
The glomerular filtration barrier in the kidney is formed in part by a specialized intercellular junction known as the slit diaphragm, which connects adjacent actin-based foot processes of kidney epithelial cells (podocytes). Mutations affecting a number of slit diaphragm proteins, including nephrin (encoded by NPHS1), lead to renal disease owing to disruption of the filtration barrier and rearrangement of the actin cytoskeleton, although the molecular basis for this is unclear. Here we show that nephrin selectively binds the Src homology 2 (SH2)/SH3 domain-containing Nck adaptor proteins, which in turn control the podocyte cytoskeleton in vivo. The cytoplasmic tail of nephrin has multiple YDxV sites that form preferred binding motifs for the Nck SH2 domain once phosphorylated by Src-family kinases. We show that this Nck-nephrin interaction is required for nephrin-dependent actin reorganization. Selective deletion of Nck from podocytes of transgenic mice results in defects in the formation of foot processes and in congenital nephrotic syndrome. Together, these findings identify a physiological signalling pathway in which nephrin is linked through phosphotyrosine-based interactions to Nck adaptors, and thus to the underlying actin cytoskeleton in podocytes. Simple and widely expressed SH2/SH3 adaptor proteins can therefore direct the formation of a specialized cellular morphology in vivo.
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Actinas/metabolismo , Citoesqueleto/metabolismo , Riñón/citología , Riñón/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Oncogénicas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencias de Aminoácidos , Animales , Línea Celular , Citoesqueleto/química , Humanos , Riñón/patología , Proteínas de la Membrana/genética , Ratones , Mutación/genética , Síndrome Nefrótico/congénito , Síndrome Nefrótico/genética , Síndrome Nefrótico/metabolismo , Síndrome Nefrótico/patología , Proteínas Oncogénicas/química , Proteínas Oncogénicas/deficiencia , Proteínas Oncogénicas/genética , Fosforilación , Fosfotirosina/metabolismo , Dominios Homologos srcRESUMEN
BACKGROUND: There is significant interest in treatment de-escalation for human papillomavirus-associated (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) patients given the generally favourable prognosis. However, 15-30% of patients recur after primary treatment, reflecting a need for improved risk-stratification tools. We sought to develop a molecular test to risk stratify HPV+ OPSCC patients. METHODS: We created an immune score (UWO3) associated with survival outcomes in six independent cohorts comprising 906 patients, including blinded retrospective and prospective external validations. Two aggressive radiation de-escalation cohorts were used to assess the ability of UWO3 to identify patients who recur. Multivariate Cox models were used to assess the associations between the UWO3 immune class and outcomes. FINDINGS: A three-gene immune score classified patients into three immune classes (immune rich, mixed, or immune desert) and was strongly associated with disease-free survival in six datasets, including large retrospective and prospective datasets. Pooled analysis demonstrated that the immune rich group had superior disease-free survival compared to the immune desert (HR = 9.0, 95% CI: 3.2-25.5, P = 3.6 × 10-5) and mixed (HR = 6.4, 95% CI: 2.2-18.7, P = 0.006) groups after adjusting for age, sex, smoking status, and AJCC8 clinical stage. Finally, UWO3 was able to identify patients from two small treatment de-escalation cohorts who remain disease-free after aggressive de-escalation to 30 Gy radiation. INTERPRETATION: With additional prospective validation, the UWO3 score could enable biomarker-driven clinical decision-making for patients with HPV+ OPSCC based on robust outcome prediction across six independent cohorts. Prospective de-escalation and intensification clinical trials are currently being planned. FUNDING: CIHR, European Union, and the NIH.
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Neoplasias de Cabeza y Cuello , Neoplasias Orofaríngeas , Infecciones por Papillomavirus , Humanos , Infecciones por Papillomavirus/complicaciones , Estudios Retrospectivos , Recurrencia Local de Neoplasia , Neoplasias Orofaríngeas/terapia , Carcinoma de Células Escamosas de Cabeza y Cuello , Pronóstico , Biomarcadores , Virus del Papiloma Humano , PapillomaviridaeRESUMEN
We have developed a rapid, accurate, and cost-effective serologic test for SARS-CoV-2 virus, which caused the COVID-19 pandemic, on the basis of antibody-dependent agglutination of antigen-coated latex particles. When validated using plasma samples that are positive or negative for SARS-CoV-2, the agglutination assay detected antibodies against the receptor-binding domain of the spike (S-RBD) or the nucleocapsid protein of SARS-CoV-2 with 100% specificity and â¼98% sensitivity. Furthermore, we found that the strength of the S-RBD antibody response measured by the agglutination assay correlated with the efficiency of the plasma in blocking RBD binding to the angiotensin-converting enzyme 2 in a surrogate neutralization assay, suggesting that the agglutination assay might be used to identify individuals with virus-neutralizing antibodies. Intriguingly, we found that >92% of patients had detectable antibodies on the day of a positive viral RNA test, suggesting that the agglutination antibody test might complement RNA testing for the diagnosis of SARS-CoV-2 infection.
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COVID-19 , SARS-CoV-2 , Humanos , Pandemias , COVID-19/diagnóstico , Anticuerpos Antivirales , AglutinaciónRESUMEN
BACKGROUNDThe role of humoral immunity in COVID-19 is not fully understood, owing, in large part, to the complexity of antibodies produced in response to the SARS-CoV-2 infection. There is a pressing need for serology tests to assess patient-specific antibody response and predict clinical outcome.METHODSUsing SARS-CoV-2 proteome and peptide microarrays, we screened 146 COVID-19 patients' plasma samples to identify antigens and epitopes. This enabled us to develop a master epitope array and an epitope-specific agglutination assay to gauge antibody responses systematically and with high resolution.RESULTSWe identified linear epitopes from the spike (S) and nucleocapsid (N) proteins and showed that the epitopes enabled higher resolution antibody profiling than the S or N protein antigen. Specifically, we found that antibody responses to the S-811-825, S-881-895, and N-156-170 epitopes negatively or positively correlated with clinical severity or patient survival. Moreover, we found that the P681H and S235F mutations associated with the coronavirus variant of concern B.1.1.7 altered the specificity of the corresponding epitopes.CONCLUSIONEpitope-resolved antibody testing not only affords a high-resolution alternative to conventional immunoassays to delineate the complex humoral immunity to SARS-CoV-2 and differentiate between neutralizing and non-neutralizing antibodies, but it also may potentially be used to predict clinical outcome. The epitope peptides can be readily modified to detect antibodies against variants of concern in both the peptide array and latex agglutination formats.FUNDINGOntario Research Fund (ORF) COVID-19 Rapid Research Fund, Toronto COVID-19 Action Fund, Western University, Lawson Health Research Institute, London Health Sciences Foundation, and Academic Medical Organization of Southwestern Ontario (AMOSO) Innovation Fund.
Asunto(s)
Pruebas de Aglutinación/métodos , Formación de Anticuerpos/inmunología , Prueba Serológica para COVID-19/métodos , COVID-19/inmunología , Epítopos de Linfocito B/inmunología , SARS-CoV-2/inmunología , Secuencia de Aminoácidos , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos/inmunología , COVID-19/sangre , COVID-19/mortalidad , Epítopos/inmunología , Epítopos de Linfocito B/química , Epítopos de Linfocito B/genética , Humanos , Inmunidad Humoral , Análisis por Micromatrices/métodos , Nucleocápside/química , Nucleocápside/genética , Nucleocápside/inmunología , Péptidos/inmunología , SARS-CoV-2/genética , Índice de Severidad de la Enfermedad , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/inmunologíaRESUMEN
Src homology 2 (SH2) domains are the largest family of interaction modules encoded by the human genome to recognize tyrosine-phosphorylated sequences and thereby play pivotal roles in transducing and controlling cellular signals emanating from protein-tyrosine kinases. Different SH2 domains select for distinct phosphopeptides, and the function of a given SH2 domain is often dictated by the specific motifs that it recognizes. Therefore, deciphering the phosphotyrosyl peptide motif recognized by an SH2 domain is the key to understanding its cellular function. Here we cloned all 120 SH2 domains identified in the human genome and determined the phosphotyrosyl peptide binding properties of 76 SH2 domains by screening an oriented peptide array library. Of these 76, we defined the selectivity for 43 SH2 domains and refined the binding motifs for another 33 SH2 domains. We identified a number of novel binding motifs, which are exemplified by the BRDG1 SH2 domain that selects specifically for a bulky, hydrophobic residue at P + 4 relative to the Tyr(P) residue. Based on the oriented peptide array library data, we developed scoring matrix-assisted ligand identification (or SMALI), a Web-based program for predicting binding partners for SH2-containing proteins. When applied to SH2D1A/SAP (SLAM-associated protein), a protein whose mutation or deletion underlies the X-linked lymphoproliferative syndrome, SMALI not only recapitulated known interactions but also identified a number of novel interacting proteins for this disease-associated protein. SMALI also identified a number of potential interactors for BRDG1, a protein whose function is largely unknown. Peptide in-solution binding analysis demonstrated that a SMALI score correlates well with the binding energy of a peptide to a given SH2 domain. The definition of the specificity space of the human SH2 domain provides both the necessary molecular basis and a platform for future exploration of the functions for SH2-containing proteins in cells.