RESUMO
Mutations in the tyrosine phosphatase Src homology-2 domain-containing protein tyrosine phosphatase-2 (SHP2) are associated with a variety of human diseases. Most mutations in SHP2 increase its basal catalytic activity by disrupting autoinhibitory interactions between its phosphatase domain and N-terminal SH2 (phosphotyrosine recognition) domain. By contrast, some disease-associated mutations located in the ligand-binding pockets of the N- or C-terminal SH2 domains do not increase basal activity and likely exert their pathogenicity through alternative mechanisms. We lack a molecular understanding of how these SH2 mutations impact SHP2 structure, activity, and signaling. Here, we characterize five SHP2 SH2 domain ligand-binding pocket mutants through a combination of high-throughput biochemical screens, biophysical and biochemical measurements, and molecular dynamics simulations. We show that while some of these mutations alter binding affinity to phosphorylation sites, the T42A mutation in the N-SH2 domain is unique in that it also substantially alters ligand-binding specificity, despite being 8 to 10 Å from the specificity-determining region of the SH2 domain. This mutation exerts its effect on sequence specificity by remodeling the phosphotyrosine-binding pocket, altering the mode of engagement of both the phosphotyrosine and surrounding residues on the ligand. The functional consequence of this altered specificity is that the T42A mutant has biased sensitivity toward a subset of activating ligands and enhances downstream signaling. Our study highlights an example of a nuanced mechanism of action for a disease-associated mutation, characterized by a change in protein-protein interaction specificity that alters enzyme activation.
Assuntos
Simulação de Dinâmica Molecular , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Domínios de Homologia de src , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/química , Humanos , Domínios de Homologia de src/genética , Ligação Proteica , Mutação , Fosforilação , Sítios de Ligação/genética , Fosfotirosina/metabolismo , LigantesRESUMO
Cytoplasmic protein tyrosine phosphatase nonreceptor type 11 (PTPN11) and Drosophila homolog Corkscrew (Csw) regulate the mitogen-activated protein kinase (MAPK) pathway via a conserved autoinhibitory mechanism. Disease-causing loss-of-function (LoF) and gain-of-function (GoF) mutations both disrupt this autoinhibition to potentiate MAPK signaling. At the Drosophila neuromuscular junction glutamatergic synapse, LoF/GoF mutations elevate transmission strength and reduce activity-dependent synaptic depression. In both sexes of LoF/GoF mutations, the synaptic vesicles (SV)-colocalized synapsin phosphoprotein tether is highly elevated at rest, but quickly reduced with stimulation, suggesting a larger SV reserve pool with greatly heightened activity-dependent recruitment. Transmission electron microscopy of mutants reveals an elevated number of SVs clustered at the presynaptic active zones, suggesting that the increased vesicle availability is causative for the elevated neurotransmission. Direct neuron-targeted extracellular signal-regulated kinase (ERK) GoF phenocopies both increased local presynaptic MAPK/ERK signaling and synaptic transmission strength in mutants, confirming the presynaptic regulatory mechanism. Synapsin loss blocks this elevation in both presynaptic PTPN11 and ERK mutants. However, csw null mutants cannot be rescued by wild-type Csw in neurons: neurotransmission is only rescued by expressing Csw in both neurons and glia simultaneously. Nevertheless, targeted LoF/GoF mutations in either neurons or glia alone recapitulate the elevated neurotransmission. Thus, PTPN11/Csw mutations in either cell type are sufficient to upregulate presynaptic function, but a dual requirement in neurons and glia is necessary for neurotransmission. Taken together, we conclude that PTPN11/Csw acts in both neurons and glia, with LoF and GoF similarly upregulating MAPK/ERK signaling to enhance presynaptic Synapsin-mediated SV trafficking.
Assuntos
Proteínas de Drosophila , Sistema de Sinalização das MAP Quinases , Neuroglia , Neurônios , Terminações Pré-Sinápticas , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Sinapsinas , Transmissão Sináptica , Vesículas Sinápticas , Animais , Feminino , Masculino , Animais Geneticamente Modificados , Drosophila , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Mutação , Neuroglia/metabolismo , Neuroglia/fisiologia , Junção Neuromuscular/metabolismo , Junção Neuromuscular/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Sinapsinas/metabolismo , Sinapsinas/genética , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismoRESUMO
The Src Family kinase Lck sets a critical threshold for T cell activation because it phosphorylates the TCR complex and the Zap70 kinase. How a T cell controls the abundance of active Lck molecules remains poorly understood. We have identified an unappreciated role for a phosphosite, Y192, within the Lck SH2 domain that profoundly affects the amount of active Lck in cells. Notably, mutation of Y192 blocks critical TCR-proximal signaling events and impairs thymocyte development in retrogenic mice. We determined that these defects are caused by hyperphosphorylation of the inhibitory C-terminal tail of Lck. Our findings reveal that modification of Y192 inhibits the ability of CD45 to associate with Lck in cells and dephosphorylate the C-terminal tail of Lck, which prevents its adoption of an active open conformation. These results suggest a negative feedback loop that responds to signaling events that tune active Lck amounts and TCR sensitivity.
Assuntos
Antígenos Comuns de Leucócito/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Timócitos/enzimologia , Domínios de Homologia de src , Animais , Ativação Enzimática , Genótipo , Células HEK293 , Humanos , Células Jurkat , Antígenos Comuns de Leucócito/química , Antígenos Comuns de Leucócito/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/deficiência , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Mutação , Fenótipo , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Timócitos/imunologia , Fatores de Tempo , TransfecçãoRESUMO
SH2 domain containing inositol polyphosphate5-phosphatase-2 (SHIP2) is a member of the 5-phosphatase family, acting as a vital negative regulator of immune response in vertebrates. In the present study, a SHIP2 homologue (designed as CgSHIP2) was identified from Pacific oyster, Crassostrea gigas. There was a SH2 domain, an IPPc domain and a SAM domain in CgSHIP2. The mRNA transcripts of CgSHIP2 were widely expressed in all the tested tissues with the highest expression in haemolymph. The mRNA expressions of CgSHIP2 in haemocytes increased significantly at 6, 12, 48 and 72 h after Vibrio splendidus stimulation. The positive green signals of CgSHIP2 protein were mainly located in cytoplasm of haemocytes. After the expression of CgSHIP2 was inhibited by RNA interference, the mRNA transcripts of interleukin 17s (CgIL-17-1, CgIL-17-2, CgIL-17-3 and CgIL-17-6) in the haemocytes increased significantly at 24 h after V. splendidus stimulation, which were 8.15-fold (p < 0.001), 3.44-fold (p < 0.05), 2.15-fold (p < 0.01) and 4.63-fold (p < 0.05) compared with that in NC-RNAi group, respectively. Obvious branchial swelling and cilium shedding in gills were observed in CgSHIP2-RNAi group at 24 h after V. splendidus stimulation. The results suggested that CgSHIP2 played an important role in controlling inflammatory response induced by bacteria in oysters.
Assuntos
Crassostrea , Regulação da Expressão Gênica , RNA Mensageiro , Vibrio , Animais , Crassostrea/imunologia , Crassostrea/genética , Vibrio/fisiologia , Regulação da Expressão Gênica/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Imunidade Inata/genética , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-17/metabolismo , Filogenia , Sequência de Aminoácidos , Perfilação da Expressão Gênica/veterinária , Alinhamento de Sequência/veterinária , Hemócitos/imunologiaRESUMO
There are many genes that produce proteins related to diseases and these proteins can be targeted with drugs as a potential therapeutic approach. Recent advancement in drug discovery techniques have created new opportunities for treating variety of diseases by targeting disease-related proteins. Structure-based drug discovery is a faster and more cost-effective approach than traditional methods. SHP2 phosphatase, encoded by the PTPN11 gene, has been the focus of much attention due to its involvement in many types of diseases. The biological function of SHP2 is enabled mostly by protein-protein interaction through its SH2 domains. In this study, we report the identification of a potential small molecule inhibitor for the N-SH2 domain of SHP2 by structure-based drug discovery approach. We utilized molecular docking studies, followed by molecular dynamics simulations and MM/PBSA calculations, to analyze compounds retrieved from the Broad's Drug Repurposing Hub and ZINC15 databases. We selected 10 hit compounds with the best docking scores from the libraries and examined their binding properties in the N-SH2 domain. We found that compound CID 60838 (Irinotecan) was the most suitable compound with a binding free energy value of - 64.45 kcal/mol and significant interactions with the target residues in the domain.
RESUMO
The non-catalytic region of tyrosine kinase (Nck) family of adaptors, consisting of Nck1 and Nck2, contributes to selectivity and specificity in the flow of cellular information by recruiting components of signaling networks. Known to play key roles in cytoskeletal remodeling, Nck adaptors modulate host cell-pathogen interactions, immune cell receptor activation, cell adhesion and motility, and intercellular junctions in kidney podocytes. Genetic inactivation of both members of the Nck family results in embryonic lethality; however, viability of mice lacking either one of these adaptors suggests partial functional redundancy. In this Cell Science at a Glance and the accompanying poster, we highlight the molecular organization and functions of the Nck family, focusing on key interactions and pathways, regulation of cellular processes, development, homeostasis and pathogenesis, as well as emerging and non-redundant functions of Nck1 compared to those of Nck2. This article thus aims to provide a timely perspective on the biology of Nck adaptors and their potential as therapeutic targets.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Oncogênicas , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Transporte , Citoesqueleto/metabolismo , Camundongos , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Transdução de Sinais , Domínios de Homologia de srcRESUMO
Suppressor Of Cytokine Signaling (SOCS) 1 is a critical negative regulator of cytokine signaling and required to protect against an excessive inflammatory response. Genetic deletion of Socs1 results in unrestrained cytokine signaling and neonatal lethality, characterised by an inflammatory immune infiltrate in multiple organs. Overexpression and structural studies have suggested that the SOCS1 kinase inhibitory region (KIR) and Src homology 2 (SH2) domain are important for interaction with and inhibition of the receptor-associated JAK1, JAK2 and TYK2 tyrosine kinases, which initiate downstream signaling. To investigate the role of the KIR and SH2 domain in SOCS1 function, we independently mutated key conserved residues in each domain and analysed the impact on cytokine signaling, and the in vivo impact on SOCS1 function. Mutation of the SOCS1-KIR or SH2 domain had no impact on the integrity of the SOCS box complex, however, mutation within the phosphotyrosine binding pocket of the SOCS1-SH2 domain specifically disrupted SOCS1 interaction with phosphorylated JAK1. In contrast, mutation of the KIR did not affect the interaction with JAK1, but did prevent SOCS1 inhibition of JAK1 autophosphorylation. In human and mouse cell lines, both mutants impacted the ability of SOCS1 to restrain cytokine signaling, and crucially, Socs1-R105A and Socs1-F59A mice displayed a neonatal lethality and excessive inflammatory phenotype similar to Socs1-null mice. This study defines a critical and non-redundant role for both the KIR and SH2 domain in endogenous SOCS1 function.
Assuntos
Citocinas , Proteína 1 Supressora da Sinalização de Citocina , Domínios de Homologia de src , Animais , Humanos , Camundongos , Citocinas/metabolismo , Fosforilação , Transdução de Sinais/fisiologia , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , TYK2 Quinase/metabolismoRESUMO
BACKGROUND: SH2 domain containing 1A (SH2D1A) expression has been linked to cancer progression. However, the functions of SH2D1A in hepatocellular carcinoma (HCC) have not been reported. METHODS: The effects of SH2D1A on the proliferation, migration, and invasion of HCC cells and the related pathways were re-explored in cell models with SH2D1A overexpression using the CCK-8, migration and invasion assays and western blotting. The functions and mechanisms of genes co-expressed with SH2D1A were analyzed using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The relationship between SH2D1A expression and immune microenvironment features in HCC was explored. RESULTS: Elevated SH2D1A expression promoted cell proliferation, migration, and invasion, which was related to the overexpression of p-Nf-κB and BCL2A1 protein levels in HCC. SH2D1A expression was related to the immune, stromal, and ESTIMATE scores, and the abundance of immune cells, such as B cells, CD8+ T cells, and T cells. SH2D1A expression was significantly related to the expression of immune cell markers, such as PDCD1, CD8A, and CTLA4 in HCC. CONCLUSION: SH2D1A overexpression was found to promote cell growth and metastasis via the Nf-κB signaling pathway and may be related to the immune microenvironment in HCC. The findings indicate that SH2D1A can function as a biomarker in HCC.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária , Humanos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Linfócitos T CD8-Positivos , Biologia Computacional , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Processos Neoplásicos , NF-kappa B , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária/genética , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária/imunologia , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
Abnormal activation of receptor tyrosine kinases (RTKs) contributes to tumorigenesis, while protein tyrosine phosphatases (PTPs) contribute to tumor control. One of the most representative PTPs is Src homology region 2 (SH2) domain-containing phosphatase 1 (SHP-1), which is associated with either an increased or decreased survival rate depending on the cancer type. Hypermethylation in the promoter region of PTPN6, the gene for the SHP-1 protein, is a representative epigenetic regulation mechanism that suppresses the expression of SHP-1 in tumor cells. SHP-1 comprises two SH2 domains (N-SH2 and C-SH2) and a catalytic PTP domain. Intramolecular interactions between the N-SH2 and PTP domains inhibit SHP-1 activity. Opening of the PTP domain by a conformational change in SHP-1 increases enzymatic activity and contributes to a tumor control phenotype by inhibiting the activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT3) pathway. Although various compounds that increase SHP-1 activation or expression have been proposed as tumor therapeutics, except sorafenib and its derivatives, few candidates have demonstrated clinical significance. In some cancers, SHP-1 expression and activation contribute to a tumorigenic phenotype by inducing a tumor-friendly microenvironment. Therefore, developing anticancer drugs targeting SHP-1 must consider the effect of SHP-1 on both cell biological mechanisms of SHP-1 in tumor cells and the tumor microenvironment according to the target cancer type. Furthermore, the use of combination therapies should be considered.
Assuntos
Carcinogênese , Epigênese Genética , Humanos , Terapia Combinada , Domínio Catalítico , Janus Quinases , Microambiente TumoralRESUMO
Deletion of c-Src, a ubiquitously expressed tyrosine kinase, results in osteoclast dysfunction and osteopetrosis, in which bones harden into "stone." In contrast, deletion of the genes encoding other members of the Src family kinase (SFK) fails to produce an osteopetrotic phenotype. This suggests that c-Src performs a unique function in the osteoclast that cannot be compensated for by other SFKs. We aimed to identify the molecular basis of this unique role in osteoclasts and bone resorption. We found that c-Src, Lyn, and Fyn were the most highly expressed SFKs in WT osteoclasts, whereas Hck, Lck, Blk, and Fgr displayed low levels of expression. Formation of the podosome belt, clusters of unique actin assemblies, was disrupted in src-/- osteoclasts; introduction of constitutively activated SFKs revealed that only c-Src and Fyn could restore this process. To identify the key structural domains responsible, we constructed chimeric Src-Hck and Src-Lyn constructs in which the unique, SH3, SH2, or catalytic domains had been swapped. We found that the Src unique, SH3, and kinase domains were each crucial to establish Src functionality. The SH2 domain could however be substituted with Lyn or Hck SH2 domains. Furthermore, we demonstrate that c-Src's functionality is, in part, derived from an SH3-proximal proline-rich domain interaction with c-Cbl, leading to phosphorylation of c-Cbl Tyr700. These data help clarify Src's unique functionality in the organization of the cytoskeleton in osteoclasts, required for efficient bone resorption and explain why c-Src cannot be replaced, in osteoclasts, by other SFKs.
Assuntos
Osteoclastos/metabolismo , Podossomos/metabolismo , Domínios de Homologia de src , Quinases da Família src/metabolismo , Animais , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Diferenciação Celular , Células HEK293 , Humanos , Camundongos , Osteoclastos/citologia , Quinases da Família src/genéticaRESUMO
Prostate cancer ranks among the most commonly diagnosed malignancies for men and has become a non-negligible threat for public health. Interplay between inflammatory factors and cancer cells renders inflammatory tissue environment as a predisposing condition for cancer development. The Hippo pathway is a conserved signaling pathway across multiple species during evolution that regulates tissue homeostasis and organ development. Nevertheless, whether Hippo pathway regulates cancer-related inflammatory factors remains elusive. Here, we show that high cell density-mediated activation of the Hippo pathway blunts STAT3 activity in prostate cancer cells. Hippo pathway component MST2 kinase phosphorylates STAT3 at T622, which is located in the SH2 domain of STAT3. This phosphorylation blocks the SH2 domain in one STAT3 molecule to bind with the phosphorylated Y705 site in another STAT3 molecule, which further counteracts IL6-induced STAT3 dimerization and activation. Expression of a nonphosphorylatable STAT3 T622A mutant enhances STAT3 activity and IL6 expression at high cell density and promotes tumor growth in a mice xenograft model. Our findings demonstrate that STAT3 is a novel phosphorylation substrate for MST2 and thereby highlight a regulatory cascade underlying the crosstalk between inflammation and the Hippo pathway in prostate cancer cells.
Assuntos
Via de Sinalização Hippo , Neoplasias da Próstata , Animais , Humanos , Interleucina-6/metabolismo , Masculino , Camundongos , Fosforilação/fisiologia , Neoplasias da Próstata/patologia , Fator de Transcrição STAT3/metabolismo , Transdução de SinaisRESUMO
The nonreceptor tyrosine kinase ITK is a key component of the T cell receptor (TCR) signaling pathway and is required for cytokine production by CD4+ T cells that have differentiated into Th2 cells. Structural and biochemical studies suggest that contacts between the SH2 and SH3 domains of ITK mediate intermolecular self-association, forming a structure that restrains ITK activity by interfering with interactions between ITK and other components of the TCR signaling pathway. Wild-type (WT) ITK and a panel of ITK mutants containing amino acid substitutions in the SH2 and SH3 domains were tested for self-association and for binding to the adaptor protein SLP76, a key ligand for the ITK SH2 domain. WT and ITK mutants were also expressed in Itk-deficient CD4+ T cells via retroviral-mediated gene delivery to analyze their ability to support TCR signaling and cytokine production by Th2 cells. Specific amino acid substitutions in the ITK SH2 or SH3 domains impaired self-association, with the greatest effects being seen when both intermolecular SH2-SH3 domain contacts were disrupted. Two of the SH2 domain substitutions tested reduced ITK self-association but had no effect on binding to SLP-76. When their function was analyzed in Th2 cells, ITK proteins with diminished self-association activity supported greater IL-4 production and calcium flux in response to TCR stimulation compared to WT ITK. Our findings indicate that intermolecular contacts between ITK molecules can restrain the amplitude of TCR signaling, suggesting ITK is a limiting factor for responses by CD4+ T cells.
Assuntos
Transdução de Sinais , Domínios de Homologia de src , Linfócitos T CD4-Positivos/metabolismo , Citocinas/metabolismo , Ligação Proteica , Receptores de Antígenos de Linfócitos T/genética , Células Th2RESUMO
Isocryptotanshinone (ICTS), a natural product with potential signal transducer and activator of transcription-3 (STAT3) signaling pathway inhibitory activity, shows significant inhibitory activity against several tumors. In this study, a series of ICTS derivatives and simplified analogs containing a 1, 4-naphthoquinone core was designed, synthesized, and evaluated. The results demonstrated that most target compounds were potent STAT3 signaling pathway inhibitors based on their mechanism of inhibition of STAT3 phosphorylation. Moreover, based on the obtained data, the structure-activity relationship (SAR) was rationally deduced. Simultaneously, molecular docking of the compound 16r suggested its possible interaction mode with STAT3. To further verify anticancer activity, all target compounds were tested using HCT116, HepG2, MCF-7, A549, and U251 cell lines. Interestingly, compared with different tumor cell lines, the HCT-116 cell line was determined to be the most sensitive. Furthermore, compounds 21e, 16r, 28a, and 16e showed a dose-dependent inhibition of the growth of HCT116 cells. Thus, the SAR of ICTS derivatives and its simplified analogs was determined, and some of them were discovered to be potential anticancer candidates owing to their ability to inhibit the STAT3 signaling pathway.
Assuntos
Antineoplásicos , Abietanos , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Quinonas/farmacologia , Fator de Transcrição STAT3 , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
SHIP2 is a multi-domain inositol 5-phosphatase binding to a variety of phosphotyrosine (pY)-containing proteins through its SH2 domain, so as to regulate various cell signaling pathways by modulating the phosphatidylinositol level in the plasma membrane. Unfavorably, Helicobacter pylori can hijack SHIP2 through the CagA protein to induce gastric cell carcinogenesis. To date, the interaction between SHIP2 and CagA was not analyzed from a structural point of view. Here, the binding of SHIP2-SH2 with Tyr-phosphorylated peptides from four EPIYA motifs (A/B/C/D) in CagA was studied using NMR spectroscopy. The results showed that EPIYA-C and -D bind to a similar interface of SHIP2-SH2, including a pY-binding pocket and a hydrophobic pocket, to achieve high affinity, while EPIYA-A and -B bind to a smaller interface of SHIP2-SH2 with weak affinity. By summarizing the interface and affinity of SHIP2-SH2 for CagA EPIYA-A/B/C/D, c-MET and FcgR2B ITIM, it was proposed that, potentially, SHIP2-SH2 has a selective preference for L > I > V for the aliphatic residues at the pY+3 position in its ligand. This study reveals the rule of the ligand sequence bound by SHIP2-SH2 and the mechanism by which CagA protein hijacks SHIP2, which will help design a peptide inhibitor against SHIP2-SH2.
Assuntos
Helicobacter pylori , Motivos de Aminoácidos , Sequência de Aminoácidos , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Carcinogênese , Helicobacter pylori/metabolismo , Humanos , Inositol Polifosfato 5-Fosfatases/metabolismo , Ligantes , Peptídeos/química , Fosfatidilinositóis/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Isoformas de Proteínas/metabolismoRESUMO
The localization and activity of the SHP2 tyrosine phosphatase across different cellular compartments to the target substrates are steered by the binding of phosphotyrosine (pY) peptides to the tandem SH2 domains. The most N-terminal domain (N-SH2) can also keep the enzyme inactive by intramolecular occlusion of the catalytic site. Enzyme activity can be recovered by an allosteric disruption of this self-inhibitory docking upon the binding of pY peptides to the N-SH2 domain. Prior to this, the N-SH2 domain must abandon the closed conformation because it impedes the access of pY peptides to the binding cleft. Although it cooperates with the self-inhibitory docking in the negative regulation of the phosphatase activity, the structural determinants of the stability of the closed conformation in the self-inhibited phosphatase are still elusive. To address this issue, a molecular dynamics simulation study is carried out. It is shown that the closed conformation is stabilized by the interaction of the N-SH2 domain with a conserved peptide portion in the region encoded by PTPN11 exon 6 (E6).
Assuntos
Simulação de Dinâmica Molecular , Domínios de Homologia de src , Domínio Catalítico , Peptídeos/química , Fosfotirosina/metabolismo , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismoRESUMO
Antiretroviral therapy has revolutionized the treatment of AIDS, turning a deadly disease into a manageable chronic condition. Life-long treatment is required because existing drugs do not eradicate HIV-infected cells. The emergence of drug-resistant viral strains and uncertain vaccine prospects highlight the pressing need for new therapeutic approaches with the potential to clear the virus. The HIV-1 accessory protein Nef is essential for viral pathogenesis, making it a promising target for antiretroviral drug discovery. Nef enhances viral replication and promotes immune escape of HIV-infected cells but lacks intrinsic enzymatic activity. Instead, Nef works through diverse interactions with host cell proteins primarily related to kinase signaling pathways and endosomal trafficking. This review emphasizes the structure, function, and biological relevance of Nef interactions with host cell protein-tyrosine kinases in the broader context of Nef functions related to enhancement of the viral life cycle and immune escape. Drug discovery targeting Nef-mediated kinase activation has allowed identification of promising inhibitors of multiple Nef functions. Pharmacological inhibitors of Nef-induced MHC-I down-regulation restore the adaptive immune response to HIV-infected cells in vitro and have the potential to enhance immune recognition of latent viral reservoirs as part of a strategy for HIV clearance.
Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Antígenos CD4/metabolismo , Cristalografia por Raios X , Regulação para Baixo , HIV-1/patogenicidade , Evasão da Resposta Imune , Complexo Principal de Histocompatibilidade , Proteínas de Membrana/metabolismo , Proteínas Quinases/efeitos dos fármacos , Transporte Proteico , Relação Estrutura-Atividade , Produtos do Gene nef do Vírus da Imunodeficiência Humana/químicaRESUMO
Protein domain interactions with short linear peptides, such as those of the Src homology 2 (SH2) domain with phosphotyrosine-containing peptide motifs (pTyr), are ubiquitous and important to many biochemical processes of the cell. The desire to map and quantify these interactions has resulted in the development of high-throughput (HTP) quantitative measurement techniques, such as microarray or fluorescence polarization assays. For example, in the last 15 years, experiments have progressed from measuring single interactions to covering 500,000 of the 5.5 million possible SH2-pTyr interactions in the human proteome. However, high variability in affinity measurements and disagreements about positive interactions between published data sets led us here to reevaluate the analysis methods and raw data of published SH2-pTyr HTP experiments. We identified several opportunities for improving the identification of positive and negative interactions and the accuracy of affinity measurements. We implemented model-fitting techniques that are more statistically appropriate for the nonlinear SH2-pTyr interaction data. We also developed a method to account for protein concentration errors due to impurities and degradation or protein inactivity and aggregation. Our revised analysis increases the reported affinity accuracy, reduces the false-negative rate, and increases the amount of useful data by adding reliable true-negative results. We demonstrate improvement in classification of binding versus nonbinding when using machine-learning techniques, suggesting improved coherence in the reanalyzed data sets. We present revised SH2-pTyr affinity results and propose a new analysis pipeline for future HTP measurements of domain-peptide interactions.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Peptídeos/química , Domínios de Homologia de src , Humanos , Ligação Proteica , Reprodutibilidade dos TestesRESUMO
The Src homology 2 (SH2) domain has a highly conserved architecture that recognizes linear phosphotyrosine motifs and is present in a wide range of signaling pathways across different evolutionary taxa. A hallmark of SH2 domains is the arginine residue in the conserved FLVR motif that forms a direct salt bridge with bound phosphotyrosine. Here, we solve the X-ray crystal structures of the C-terminal SH2 domain of p120RasGAP (RASA1) in its apo and peptide-bound form. We find that the arginine residue in the FLVR motif does not directly contact pTyr1087 of a bound phosphopeptide derived from p190RhoGAP; rather, it makes an intramolecular salt bridge to an aspartic acid. Unexpectedly, coordination of phosphotyrosine is achieved by a modified binding pocket that appears early in evolution. Using isothermal titration calorimetry, we find that substitution of the FLVR arginine R377A does not cause a significant loss of phosphopeptide binding, but rather a tandem substitution of R398A (SH2 position ßD4) and K400A (SH2 position ßD6) is required to disrupt the binding. These results indicate a hitherto unrecognized diversity in SH2 domain interactions with phosphotyrosine and classify the C-terminal SH2 domain of p120RasGAP as "FLVR-unique."
Assuntos
Evolução Molecular , Proteína p120 Ativadora de GTPase/química , Cristalografia por Raios X , Humanos , Proteína p120 Ativadora de GTPase/genética , Proteína p120 Ativadora de GTPase/metabolismo , Domínios de Homologia de srcRESUMO
Eph receptors are a family of receptor tyrosine kinases that control directional cell movement during various biological processes, including embryogenesis, neuronal pathfinding, and tumor formation. The biochemical pathways of Eph receptors are context-dependent in part because of the varied composition of a heterotypic, oligomeric, active Eph receptor complex. Downstream of the Eph receptors, little is known about the essential phosphorylation events that define the context and instruct cell movement. Here, we define a pathway that is required for Eph receptor B2 (EphB2)-mediated cell sorting and is conserved among multiple Eph receptors. Utilizing a HEK293 model of EphB2+/ephrinB1+ cell segregation, we found that the scaffold adaptor protein SH2 domain-containing adaptor protein B (Shb) is essential for EphB2 functionality. Further characterization revealed that Shb interacts with known modulators of cytoskeletal rearrangement and cell mobility, including Nck adaptor protein (Nck), p120-Ras GTPase-activating protein (RasGAP), and the α- and ß-Chimaerin Rac GAPs. We noted that phosphorylation of Tyr297, Tyr246, and Tyr336 of Shb is required for EphB2-ephrinB1 boundary formation, as well as binding of Nck, RasGAP, and the chimaerins, respectively. Similar complexes were formed in the context of EphA4, EphA8, EphB2, and EphB4 receptor activation. These results indicate that phosphotyrosine-mediated signaling through Shb is essential in EphB2-mediated heterotypic cell segregation and suggest a conserved function for Shb downstream of multiple Eph receptors.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Quimerinas/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptor EphB2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Separação Celular , Proteínas Quimerinas/química , Efrina-B1/genética , Efrina-B1/metabolismo , Células HEK293 , Humanos , Espectrometria de Massas , Proteínas Oncogênicas/química , Fosforilação , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/química , Receptor EphB2/química , Receptor EphB2/genética , Transdução de Sinais , Domínios de Homologia de srcRESUMO
Homeostatic scaling of the synapse, such as synaptic down-scaling, has been proposed to offset deleterious effects induced by sustained synaptic strength enhancement. Proper function and subcellular distribution of Src homology 2 domain-containing nonreceptor protein tyrosine phosphatase (SHP2) are required for synaptic plasticity. However, the role of SHP2 in synaptic down-scaling remains largely unknown. Here, using biochemical assays and cell-imaging techniques, we found that synaptic SHP2 levels are temporally regulated during synaptic down-scaling in cultured hippocampal neurons. Furthermore, we observed that a Noonan syndrome-associated mutation of SHP2, resulting in a D61G substitution, prevents synaptic down-scaling. We further show that this effect is due to an inability of the SHP2-D61G variant to properly disassociate from postsynaptic density protein 95, leading to impaired SHP2 dispersion from synaptic sites after synaptic down-scaling. Our findings reveal a molecular mechanism of the Noonan syndrome-associated genetic variant SHP2-D61G that contributes to deficient synaptic down-scaling.