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Molecular glues are proximity-inducing small molecules that have emerged as an attractive therapeutic approach. However, developing molecular glues remains challenging, requiring innovative mechanistic strategies to stabilize neoprotein interfaces and expedite discovery. Here we unveil a trans-labeling covalent molecular glue mechanism, termed 'template-assisted covalent modification'. We identified a new series of BRD4 molecular glue degraders that recruit CUL4DCAF16 ligase to the second bromodomain of BRD4 (BRD4BD2). Through comprehensive biochemical, structural and mutagenesis analyses, we elucidated how pre-existing structural complementarity between DCAF16 and BRD4BD2 serves as a template to optimally orient the degrader for covalent modification of DCAF16Cys58. This process stabilizes the formation of BRD4-degrader-DCAF16 ternary complex and facilitates BRD4 degradation. Supporting generalizability, we found that a subset of degraders also induces GAK-BRD4BD2 interaction through trans-labeling of GAK. Together, our work establishes 'template-assisted covalent modification' as a mechanism for covalent molecular glues, which opens a new path to proximity-driven pharmacology.
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A target identification platform derived from the bioorthogonal activation of reactive species is described. We explore the reactivity of halogenated enamine N-oxides and report that the previously undisclosed α,γ-halogenated enamine N-oxides can be reduced biooorthogonally by diboron reagents to produce highly electrophilic α,ß-unsaturated haloiminium ions suitable for labeling a range of amino acid residues on proteins in a 1,2- or 1,4-fashion. Affinity labeling reagents bearing this motif enable ligand-directed protein modification and afford highly sensitive and selective target identification in unbiased chemoproteomics experiments. Target identification is supported in both cell lysate and live cells.
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Whereas oncogenes can potentially be inhibited with small molecules, the loss of tumour suppressors is more common and is problematic because the tumour-suppressor proteins are no longer present to be targeted. Notable examples include SMARCB1-mutant cancers, which are highly lethal malignancies driven by the inactivation of a subunit of SWI/SNF (also known as BAF) chromatin-remodelling complexes. Here, to generate mechanistic insights into the consequences of SMARCB1 mutation and to identify vulnerabilities, we contributed 14 SMARCB1-mutant cell lines to a near genome-wide CRISPR screen as part of the Cancer Dependency Map Project1-3. We report that the little-studied gene DDB1-CUL4-associated factor 5 (DCAF5) is required for the survival of SMARCB1-mutant cancers. We show that DCAF5 has a quality-control function for SWI/SNF complexes and promotes the degradation of incompletely assembled SWI/SNF complexes in the absence of SMARCB1. After depletion of DCAF5, SMARCB1-deficient SWI/SNF complexes reaccumulate, bind to target loci and restore SWI/SNF-mediated gene expression to levels that are sufficient to reverse the cancer state, including in vivo. Consequently, cancer results not from the loss of SMARCB1 function per se, but rather from DCAF5-mediated degradation of SWI/SNF complexes. These data indicate that therapeutic targeting of ubiquitin-mediated quality-control factors may effectively reverse the malignant state of some cancers driven by disruption of tumour suppressor complexes.
Assuntos
Complexos Multiproteicos , Mutação , Neoplasias , Proteína SMARCB1 , Animais , Feminino , Humanos , Masculino , Camundongos , Linhagem Celular Tumoral , Sistemas CRISPR-Cas , Edição de Genes , Neoplasias/genética , Neoplasias/metabolismo , Proteína SMARCB1/deficiência , Proteína SMARCB1/genética , Proteína SMARCB1/metabolismo , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteólise , Ubiquitina/metabolismoRESUMO
Small molecules that can induce protein degradation by inducing proximity between a desired target and an E3 ligase have the potential to greatly expand the number of proteins that can be manipulated pharmacologically. Current strategies for targeted protein degradation are mostly limited in their target scope to proteins with preexisting ligands. Alternate modalities such as molecular glues, as exemplified by the glutarimide class of ligands for the CUL4CRBN ligase, have been mostly discovered serendipitously. We recently reported a trans-labelling covalent glue mechanism which we named 'Template-assisted covalent modification', where an electrophile decorated small molecule binder of BRD4 was effectively delivered to a cysteine residue on an E3 ligase DCAF16 as a consequence of a BRD4-DCAF16 protein-protein interaction. Herein, we report our medicinal chemistry efforts to evaluate how various electrophilic modifications to the BRD4 binder, JQ1, affect DCAF16 trans-labeling and subsequent BRD4 degradation efficiency. We discovered a decent correlation between the ability of the electrophilic small molecule to induce ternary complex formation between BRD4 and DCAF16 with its ability to induce BRD4 degradation. Moreover, we show that a more solvent-exposed warhead presentation is optimal for DCAF16 recruitment and subsequent BRD4 degradation. Unlike the sensitivity of CUL4CRBN glue degraders to chemical modifications, the diversity of covalent attachments in this class of BRD4 glue degraders suggests a high tolerance and tunability for the BRD4-DCAF16 interaction. This offers a potential new avenue for a rational design of covalent glue degraders by introducing covalent warheads to known binders.
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Lung cancer is frequently caused by activating mutations in the epidermal growth factor receptor (EGFR). Allosteric EGFR inhibitors offer promise as the next generation of therapeutics, as they are unaffected by common ATP-site resistance mutations and synergize with the drug osimertinib. Here, we examine combinations of ATP-competitive and allosteric inhibitors to better understand the molecular basis for synergy. We identify a subset of irreversible EGFR inhibitors that display positive binding cooperativity and synergy with the allosteric inhibitor JBJ-04-125-02 in several EGFR variants. Structural analysis of these complexes reveals conformational changes occur mainly in the phosphate-binding loop (P-loop). Mutation of F723 in the P-loop reduces cooperative binding and synergy, supporting a mechanism in which F723-mediated contacts between the P-loop and the allosteric inhibitor are critical for synergy. These structural and mechanistic insights will aid in the identification and development of additional inhibitor combinations with potential clinical value.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares , Trifosfato de Adenosina , Compostos de Anilina , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/metabolismo , Humanos , Mutação , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Chagas disease is mainly transmitted by triatomine insect vectors that feed on vertebrate blood. The disease has complex domiciliary infestation patterns and parasite transmission dynamics, influenced by biological, ecological, and socioeconomic factors. In this context, feeding patterns have been used to understand vector movement and transmission risk. Recently, a new technique using Liquid chromatography tandem mass spectrometry (LC-MS/MS) targeting hemoglobin peptides has showed excellent results for understanding triatomines' feeding patterns. The aim of this study was to further develop the automated computational analysis pipeline for peptide sequence taxonomic identification, enhancing the ability to analyze large datasets data. We then used the enhanced pipeline to evaluate the feeding patterns of Triatoma dimidiata, along with domiciliary infestation risk variables, such as unkempt piles of firewood or construction material, cracks in bajareque and adobe walls and intradomiciliary animals. Our new python scripts were able to detect blood meal sources in 100% of the bugs analyzed and identified nine different species of blood meal sources. Human, chicken, and dog were the main blood sources found in 78.7%, 50.4% and 44.8% of the bugs, respectively. In addition, 14% of the bugs feeding on chicken and 15% of those feeding on dogs were captured in houses with no evidence of those animals being present. This suggests a high mobility among ecotopes and houses. Two of the three main blood sources, dog and chicken, were significantly (p < 0.05) affected by domiciliary infestation risk variables, including cracks in walls, construction material and birds sleeping in the intradomicile. This suggests that these variables are important for maintaining reproducing Triatoma dimidiata populations and that it is critical to mitigate these variables in all the houses of a village for effective control of these mobile vectors.
Assuntos
Análise Química do Sangue/métodos , Doença de Chagas/transmissão , Cromatografia Gasosa-Espectrometria de Massas/métodos , Hemoglobinas/análise , Insetos Vetores/parasitologia , Triatoma/parasitologia , Animais , Galinhas/parasitologia , Cães/parasitologia , Comportamento Alimentar , Guatemala , Humanos , Modelos Logísticos , Medição de Risco , Fatores de RiscoRESUMO
Inhibitors targeting the epidermal growth factor receptor (EGFR) are an effective therapy for patients with non-small cell lung cancer harboring drug-sensitive activating mutations in the EGFR kinase domain. Drug resistance due to treatment-acquired mutations has motivated the development of successive generations of inhibitors that bind in the ATP site. The third-generation agent osimertinib is now a first-line treatment for this disease. Recently, allosteric inhibitors have been developed to overcome drug-resistant mutations that confer a resistance to osimertinib. Here, we present the structure-guided design and synthesis of a mutant-selective lead compound, which consists of a pyridinyl imidazole-fused benzylisoindolinedione scaffold that simultaneously occupies the orthosteric and allosteric sites. The compound potently inhibits enzymatic activity in L858R/T790M/C797S mutant EGFR (4.9 nM), with a significantly lower activity for wild-type EGFR (47 nM). Additionally, this compound achieves modest cetuximab-independent and mutant-selective cellular efficacies on the L858R (1.2 µM) and L858R/T790M (4.4 µM) variants.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Imidazóis/química , Mutação , Inibidores de Proteínas Quinases/farmacologia , Acrilamidas/farmacologia , Sítio Alostérico , Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologiaRESUMO
The RAF/MEK/ERK pathway is central to the control of cell physiology, and its dysregulation is associated with many cancers. Accordingly, the proteins constituting this pathway, including MEK1/2 (MEK), have been subject to intense drug discovery and development efforts. Allosteric MEK inhibitors (MEKi) exert complex effects on RAF/MEK/ERK pathway signaling and are employed clinically in combination with BRAF inhibitors in malignant melanoma. Although mechanisms and structures of MEKi bound to MEK have been described for many of these compounds, recent studies suggest that RAF/MEK complexes, rather than free MEK, should be evaluated as the target of MEKi. Here, we describe structural and biochemical studies of eight structurally diverse, clinical-stage MEKi to better understand their mechanism of action on BRAF/MEK complexes. We find that all of these agents bind in the MEK allosteric site in BRAF/MEK complexes, in which they stabilize the MEK activation loop in a conformation that is resistant to BRAF-mediated dual phosphorylation required for full activation of MEK. We also show that allosteric MEK inhibitors act most potently on BRAF/MEK complexes rather than on free active MEK, further supporting the notion that a BRAF/MEK complex is the physiologically relevant pharmacologic target for this class of compounds. Our findings provide a conceptual and structural framework for rational development of RAF-selective MEK inhibitors as an avenue to more effective and better-tolerated agents targeting this pathway.
Assuntos
MAP Quinase Quinase Quinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/metabolismo , Regulação Alostérica , Ativação Enzimática , Estabilidade Enzimática , Humanos , MAP Quinase Quinase Quinases/química , MAP Quinase Quinase Quinases/metabolismo , Fosforilação , Conformação Proteica , Transdução de SinaisRESUMO
The Discoidin, CUB, and LCCL domain-containing protein (DCBLD) family consists of two type-I transmembrane scaffolding receptors, DCBLD1 and DCBLD2, which play important roles in development and cancer. The nonreceptor tyrosine kinases FYN and ABL are known to drive phosphorylation of tyrosine residues in YXXP motifs within the intracellular domains of DCBLD family members, which leads to the recruitment of the Src homology 2 (SH2) domain of the adaptors CT10 regulator of kinase (CRK) and CRK-like (CRKL). We previously characterized the FYN- and ABL-driven phosphorylation of DCBLD family YXXP motifs. However, we have identified additional FYN- and ABL-dependent phosphorylation sites on DCBLD1 and DCBLD2. This suggests that beyond CRK and CRKL, additional DCBLD interactors may be regulated by FYN and ABL activity. Here, we report a quantitative proteomics approach in which we map the FYN- and ABL-regulated interactomes of DCBLD family members. We found FYN and ABL regulated the binding of several signaling molecules to DCBLD1 and DCBLD2, including members of the 14-3-3 family of adaptors. Biochemical investigation of the DCBLD2/14-3-3 interaction revealed ABL-induced binding of 14-3-3 family members directly to DCBLD2.
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Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Sequência de Aminoácidos , Células HEK293 , Humanos , Proteínas de Membrana/química , Modelos Biológicos , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Fosforilação , Mapas de Interação de ProteínasRESUMO
The triggering receptor expressed on myeloid cells (TREM) protein family forms a class of type I transmembrane proteins expressed in immune cells that play important roles in innate and adaptive immune responses. The TREM family member TREM-like transcript 1 (TLT-1, also TREML1) is expressed in megakaryocytes and packaged into platelet granules. TLT-1 binds fibrinogen and plays a role in bleeding initiated by inflammatory insults. Here, we describe a proteomics screen that maps the TLT-1 interactome in resting and activated human platelets. Several identified TLT-1 interactors are involved in cell adhesion and migration, as well as platelet activation. Select interactors, including ß3-integrin, RACK1, GRB2, and Rabs 5A, 7, and 11A, were additionally characterized in co-immunoprecipitation/immunoblotting experiments. Finally, several phosphorylation sites were found on immunoprecipitated TLT-1, including Thr280, a novel, regulated site on a conserved residue near the TLT-1 ITIM regulatory sequence. SIGNIFICANCE: Platelet function relies on the secretion of active molecules from intracellular vesicles, or granules, which contain soluble and membrane-bound proteins that are essential for platelet aggregation, coagulation reactions, and pathogen defense mechanisms. TLT-1 is sequestered in α-granules and transported to the plasma membrane, where it plays a unique role in hemostasis after inflammatory insults. Despite the known importance of TLT-1 in platelet biology, our knowledge of TLT-1 mechanistic signaling is limited. This study defines the TLT-1 interactome in resting and active human platelets, identifying several novel TLT-1 interactors, as well as TLT-1 phosphorylation sites, all with likely signaling implications in platelet aggregation dynamics.
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Plaquetas , Receptores Imunológicos , Fibrinogênio , Humanos , Proteínas de Neoplasias , Ativação Plaquetária , Agregação Plaquetária , Receptores de Quinase C AtivadaRESUMO
The Vel blood group antigen is carried on the short extracellular segment of the 78-amino-acid-long, type II transmembrane protein SMIM1 of unknown function. Here, using biochemical analysis and flow cytometry of cells expressing wild-type and mutant alleles of SMIM1, we demonstrate that dimerization of SMIM1 promotes cell surface display of the Vel epitope. We show that SMIM1 dimerization is mediated both by an extracellular Cys77-dependent, homomeric disulfide linkage and via a GxxxG helix-helix interaction motif in the transmembrane domain. These results provide important context for the observed variability in reactivity patterns of clinically important anti-Vel identified in patient sera.
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Antígenos de Grupos Sanguíneos/imunologia , Epitopos/metabolismo , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Cisteína/química , Dissulfetos/química , Dissulfetos/metabolismo , Eritrócitos/efeitos dos fármacos , Eritrócitos/imunologia , Células HEK293 , Humanos , Células K562 , Proteínas de Membrana/genética , Mutação , Multimerização ProteicaRESUMO
Chagas disease, a neglected tropical disease endemic in Latin America, is caused by the protozoan parasite Trypanosoma cruzi and is responsible for significant health impacts, especially in rural communities. The parasite is transmitted by insect vectors in the Triatominae subfamily and due to lack of vaccines and limited treatment options, vector control is the main way of controlling the disease. Knowing what vectors are feeding on directly enhances our understanding of the ecology and biology of the different vector species and can potentially aid in engaging communities in active disease control, a concept known as Ecohealth management. We evaluated bloodmeals in rural community, house-caught insect vectors previously evaluated for bloodmeals via DNA analysis as part of a larger collaborative project from three countries in Central America, including Guatemala. In addition to identifying bloodmeals in 100% of all samples using liquid chromatography tandem mass spectrometry (LC-MS/MS) (nâ¯=â¯50), strikingly for 53% of these samples there was no evidence of a recent bloodmeal by DNA-PCR. As individual vectors often feed on multiple sources, we developed an enhanced detection pipeline, and showed the ability to quantify a bloodmeal using stable-isotope-containing synthetic references peptides, a first step in further exploration of species-specific bloodmeal composition. Furthermore, we show that a lower resolution mass spectrometer is sufficient to correctly identify taxa from bloodmeals, an important and strong attribute of our LC-MS/MS-based method, opening the door to using proteomics in countries where Chagas disease is endemic.
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Ração Animal/análise , Doença de Chagas/transmissão , DNA/análise , Proteômica/métodos , Triatoma/patogenicidade , Trypanosoma cruzi/patogenicidade , Animais , América Central , Cromatografia Líquida , Feminino , Humanos , Proteínas de Insetos/metabolismo , Insetos Vetores/metabolismo , Insetos Vetores/parasitologia , Masculino , População Rural , Especificidade da Espécie , Espectrometria de Massas em Tandem , Triatoma/genética , Triatoma/metabolismo , Triatoma/parasitologiaRESUMO
The discoidin, CUB, and LCCL domain-containing (DCBLD) receptor family are composed of the type-I transmembrane proteins DCBLD1 and DCBLD2 (also ESDN and CLCP1). These proteins are highly conserved across vertebrates and possess similar domain structure to that of neuropilins, which act as critical co-receptors in developmental processes. Although DCBLD1 remains largely uncharacterized, the functional and mechanistic roles of DCBLD2 are emerging. This review provides a comprehensive discussion of this presumed receptor family, ranging from structural and signaling aspects to their associations with cancer, physiology, and development.
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Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Transdução de Sinais , Animais , Humanos , Neoplasias/patologiaRESUMO
The cAMP-dependent protein kinase A (PKA) is a serine/threonine kinase involved in many fundamental cellular processes, including migration and proliferation. Recently, we found that the Src family kinase Fyn phosphorylates the catalytic subunit of PKA (PKA-C) at Y69, thereby increasing PKA kinase activity. We also showed that Fyn induced the phosphorylation of cellular proteins within the PKA preferred target motif. This led to the hypothesis that Fyn could affect proteins in complex with PKA. To test this, we employed a quantitative mass spectrometry approach to identify Fyn-dependent binding partners in complex with PKA-C. We found Fyn enhanced the binding of PKA-C to several cytoskeletal regulators that localize to the centrosome and Golgi apparatus. Three of these Fyn-induced PKA interactors, AKAP9, PDE4DIP, and CDK5RAP2, were validated biochemically and were shown to exist in complex with Fyn and PKA in a glioblastoma cell line. Intriguingly, the complexes formed between PKA-C and these known AKAPs were dependent upon Fyn catalytic activity and expression levels. In addition, we identified Fyn-regulated phosphorylation sites on proteins in complex with PKA-C. We also identified and biochemically validated a novel PKA-C interactor, LARP4, which complexed with PKA in the absence of Fyn. These results demonstrate the ability of Fyn to influence the docking of PKA to specific cellular scaffolds and suggest that Fyn may affect the downstream substrates targeted by PKA.
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BACKGROUND: Chagas disease is highly prevalent in Latin America, and vector control is the most effective control strategy to date. We have previously shown that liquid chromatography tandem mass spectrometry (LC-MS/MS) is a valuable tool for identifying triatomine vector blood meals. OBJECTIVES: The purpose of this study was to determine blood meal detection ability as a function of method [polymerase chain reaction (PCR) vs. LC-MS/MS], time since feeding, and the effect of molting in mouse-fed triatomine insect vectors targeting hemoglobin and albumin proteins with LC-MS/MS and short interspersed nuclear elements (SINE)-based PCR. METHODS: We experimentally fed Triatoma protracta on mice and used LC-MS/MS to detect hemoglobin and albumin peptides over time post-feeding and post-molting (≤ 12 weeks). We compared LC-MS/MS results with those of a standard PCR method based on SINEs. FINDINGS: Hemoglobin-based LC-MS/MS detected blood meals most robustly at all time points post-feeding. Post-molting, no blood meals were detected with PCR, whereas LC-MS/MS detected mouse hemoglobin and albumin up to 12 weeks. MAIN CONCLUSIONS: In our study, the hemoglobin signature in the insect abdomen lasted longer than that of albumin and DNA. LC-MS/MS using hemoglobin shows promise for identifying triatomine blood meals over long temporal scales and even post-molting. Clarifying the frequency of blood-feeding on different hosts can foster our understanding of vector behavior and may help devise sounder disease-control strategies, including Ecohealth (community based ecosystem management) approaches.
Assuntos
Albuminas/análise , Comportamento Alimentar/fisiologia , Hemoglobinas/análise , Insetos Vetores/fisiologia , Refeições , Triatoma/fisiologia , Animais , Sangue , Doença de Chagas/transmissão , Cromatografia Gasosa-Espectrometria de Massas , Camundongos , Muda , Reação em Cadeia da Polimerase , Fatores de TempoRESUMO
The type I cGMP-dependent protein kinase (PKG I) is an essential regulator of vascular tone. It has been demonstrated that the type Iα isoform can be constitutively activated by oxidizing conditions. However, the amino acid residues implicated in this phenomenon are not fully elucidated. To investigate the molecular basis for this mechanism, we studied the effects of oxidation using recombinant WT, truncated, and mutant constructs of PKG I. Using an in vitro assay, we observed that oxidation with hydrogen peroxide (H2O2) resulted in constitutive, cGMP-independent activation of PKG Iα. PKG Iα C42S and a truncation construct that does not contain Cys-42 (Δ53) were both constitutively activated by H2O2 In contrast, oxidation of PKG Iα C117S maintained its cGMP-dependent activation characteristics, although oxidized PKG Iα C195S did not. To corroborate these results, we also tested the effects of our constructs on the PKG Iα-specific substrate, the large conductance potassium channel (KCa 1.1). Application of WT PKG Iα activated by either cGMP or H2O2 increased the open probabilities of the channel. Neither cGMP nor H2O2 activation of PKG Iα C42S significantly increased channel open probabilities. Moreover, cGMP-stimulated PKG Iα C117S increased KCa 1.1 activity, but this effect was not observed under oxidizing conditions. Finally, we observed that PKG Iα C42S caused channel flickers, indicating dramatically altered KCa 1.1 channel characteristics compared with channels exposed to WT PKG Iα. Cumulatively, these results indicate that constitutive activation of PKG Iα proceeds through oxidation of Cys-117 and further suggest that the formation of a sulfur acid is necessary for this phenotype.
Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , GMP Cíclico/metabolismo , Cisteína/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Proteína Quinase Dependente de GMP Cíclico Tipo I/química , Cisteína/química , Modelos Moleculares , Óxido Nítrico/metabolismo , Oxirredução , Fosforilação , Conformação Proteica , Homologia de SequênciaRESUMO
Motivation: The development of proteomic methods for the characterization of domain/motif interactions has greatly expanded our understanding of signal transduction. However, proteomics-based binding screens have limitations including that the queried tissue or cell type may not harbor all potential interacting partners or post-translational modifications (PTMs) required for the interaction. Therefore, we sought a generalizable, complementary in silico approach to identify potentially novel motif and PTM-dependent binding partners of high priority. Results: We used as an initial example the interaction between the Src homology 2 (SH2) domains of the adaptor proteins CT10 regulator of kinase (CRK) and CRK-like (CRKL) and phosphorylated-YXXP motifs. Employing well-curated, publicly-available resources, we scored and prioritized potential CRK/CRKL-SH2 interactors possessing signature characteristics of known interacting partners. Our approach gave high priority scores to 102 of the >9000 YXXP motif-containing proteins. Within this 102 were 21 of the 25 curated CRK/CRKL-SH2-binding partners showing a more than 80-fold enrichment. Several predicted interactors were validated biochemically. To demonstrate generalized applicability, we used our workflow to predict protein-protein interactions dependent upon motif-specific arginine methylation. Our data demonstrate the applicability of our approach to, conceivably, any modular binding domain that recognizes a specific post-translationally modified motif. Supplementary information: Supplementary data are available at Bioinformatics online.
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Proteômica , Proteínas Adaptadoras de Transdução de Sinal , Fosforilação , Ligação Proteica , Transdução de Sinais , Domínios de Homologia de srcRESUMO
Plexins (Plxns) are semaphorin (Sema) receptors that play important signaling roles, particularly in the developing nervous system and vasculature. Sema-Plxn signaling regulates cellular processes such as cytoskeletal dynamics, proliferation, and differentiation. However, the receptor-proximal signaling mechanisms driving Sema-Plxn signal transduction are only partially understood. Plxn tyrosine phosphorylation is thought to play an important role in these signaling events as receptor and nonreceptor tyrosine kinases have been shown to interact with Plxn receptors. The Src family kinase Fyn can induce the tyrosine phosphorylation of PlxnA1 and PlxnA2. However, the Fyn-dependent phosphorylation sites on these receptors have not been identified. Here, using mass spectrometry-based approaches, we have identified highly conserved, Fyn-induced PlexinA (PlxnA) tyrosine phosphorylation sites. Mutation of these sites to phenylalanine results in significantly decreased Fyn-dependent PlxnA tyrosine phosphorylation. Furthermore, in contrast to wild-type human PLXNA2 mRNA, mRNA harboring these point mutations cannot rescue eye developmental defects when coinjected with a plxnA2 morpholino in zebrafish embryos. Together these data suggest that Fyn-dependent phosphorylation at two critical tyrosines is a key feature of vertebrate PlxnA1 and PlxnA2 signal transduction.
Assuntos
Olho/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Receptores de Superfície Celular/metabolismo , Tirosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Olho/embriologia , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/genética , Fosforilação , Receptores de Superfície Celular/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais/genética , Tirosina/genética , Peixe-ZebraRESUMO
BACKGROUND Chagas disease is highly prevalent in Latin America, and vector control is the most effective control strategy to date. We have previously shown that liquid chromatography tandem mass spectrometry (LC-MS/MS) is a valuable tool for identifying triatomine vector blood meals. OBJECTIVES The purpose of this study was to determine blood meal detection ability as a function of method [polymerase chain reaction (PCR) vs. LC-MS/MS], time since feeding, and the effect of molting in mouse-fed triatomine insect vectors targeting hemoglobin and albumin proteins with LC-MS/MS and short interspersed nuclear elements (SINE)-based PCR. METHODS We experimentally fed Triatoma protracta on mice and used LC-MS/MS to detect hemoglobin and albumin peptides over time post-feeding and post-molting (≤ 12 weeks). We compared LC-MS/MS results with those of a standard PCR method based on SINEs. FINDINGS Hemoglobin-based LC-MS/MS detected blood meals most robustly at all time points post-feeding. Post-molting, no blood meals were detected with PCR, whereas LC-MS/MS detected mouse hemoglobin and albumin up to 12 weeks. MAIN CONCLUSIONS In our study, the hemoglobin signature in the insect abdomen lasted longer than that of albumin and DNA. LC-MS/MS using hemoglobin shows promise for identifying triatomine blood meals over long temporal scales and even post-molting. Clarifying the frequency of blood-feeding on different hosts can foster our understanding of vector behavior and may help devise sounder disease-control strategies, including Ecohealth (community based ecosystem management) approaches.