RESUMO
Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique to solve the structure of biomolecular complexes at atomic resolution in solution. Small proteins such as Src-homology 2 (SH2) domains have fast tumbling rates and long-lived NMR signals, making them particularly suited to be studied by standard NMR methods. SH2 domains are modular proteins whose function is the recognition of sequences containing phosphotyrosines. In this chapter, we describe the application of NMR to assess the interaction between SH2 domains and phosphopeptides and determine the structure of the resulting complexes.
Assuntos
Fosfopeptídeos , Domínios de Homologia de src , Imageamento por Ressonância Magnética , Fosfotirosina , Espectroscopia de Ressonância MagnéticaRESUMO
Nuclear magnetic resonance (NMR) spectroscopy is the method of choice for studying the dynamics of biological macromolecules in solution. By exploiting the intricate interplay between the effects of protein motion (both overall rotational diffusion and internal mobility) and nuclear spin relaxation, NMR allows molecular motion to be probed at atomic resolution over a wide range of timescales, including picosecond (bond vibrations and methyl-group rotations), nanosecond (loop motions and rotational diffusion), and microsecond-millisecond (ligand binding, allostery). In this chapter, we describe different NMR pulse schemes (R1, R1ρ, heteronuclear NOE, and CPMG relaxation dispersion) to characterize the dynamics of SH2 domains. As an example, we use the N-SH2 domain of protein tyrosine phosphatase SHP2 in complex with two phosphopeptides derived from immune checkpoint receptor PD-1 (ITIM and ITSM).
Assuntos
Fosfopeptídeos , Domínios de Homologia de src , Imageamento por Ressonância Magnética , Difusão , Espectroscopia de Ressonância MagnéticaRESUMO
Src Homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1) interacts specifically with GluN2A subunit of N-methyl-D-aspartate (NMDA) subtype of glutamate receptors in spinal cord dorsal horn. This molecular interaction is involved in the development of GluN2A-dependent spinal sensitization of nociceptive behaviors. Intrathecal application of a GluN2A-derived polypeptide (short for pep-GluN2A) has been shown to disturb spinal GluN2A/SHP1 interaction and inhibit inflammatory pain. Here we found that SHP1 was also located at dorsal root ganglion (DRG) neurons and formed complexes with GluN2A subunit. Peripheral inflammation activated SHP1 in DRG neurons, which promoted GluN2A tyrosine phosphorylation. The SHP1 binding to GluN2A facilitated the glutamate release from primary afferent fibers and exaggerated nociceptive synaptic transmission onto postsynaptic spinal cord neurons. Our data showed that intradermal application of pep-GluN2A disrupted GluN2A/SHP1 interaction in DRG neurons, attenuated the ability of GluN2A subunit-containing NMDA receptors to regulate the presynaptic glutamate release and more importantly, alleviated the pain hypersensitivity caused by carrageenan, complete Freund's adjuvant and formalin. The neuropathic pain induced by spared nerve injury was also ameliorated by intradermal pep-GluN2A application. These data suggested that disruption of GluN2A/SHP1 interaction in DRG neurons generated an effective analgesic action against pathological pain.
Assuntos
Gânglios Espinais/efeitos dos fármacos , Neuralgia/tratamento farmacológico , Peptídeos/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sequência de Aminoácidos , Animais , Comportamento Animal/efeitos dos fármacos , Gânglios Espinais/patologia , Masculino , Neuralgia/metabolismo , Neuralgia/patologia , Neuralgia/fisiopatologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Nociceptividade/efeitos dos fármacos , Peptídeos/química , Peptídeos/uso terapêutico , Ligação Proteica/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Commercial antibodies raised against phosphotyrosine have been widely used as reagents to detect or isolate tyrosine-phosphorylated proteins from cellular samples. However, these antibodies are costly and are not amenable to in-house production in an academic lab setting. In this chapter, we describe a method to generate super-high affinity SH2 domains, dubbed the phosphotyrosine superbinders, by evolving a natural SH2 domain using the phage display technology. The superbinders are stable and can be easily produced in Escherichia coli in large quantities. The strategy presented here may also be applied to other protein domains to generate domain variants with markedly enhanced affinities for a specific post-translational modification.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Modelos Moleculares , Fosfotirosina/metabolismo , Domínios e Motivos de Interação entre Proteínas , Domínios de Homologia de src , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/genética , Técnicas de Visualização da Superfície Celular , Biologia Computacional/métodos , Sequência Conservada , Ensaio de Imunoadsorção Enzimática , Evolução Molecular , Vetores Genéticos/genética , Cinese , Mutagênese Sítio-Dirigida , Biblioteca de Peptídeos , Fosforilação , Fosfotirosina/química , Matrizes de Pontuação de Posição Específica , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Análise de Sequência de DNA , Software , Fluxo de Trabalho , Domínios de Homologia de src/genéticaRESUMO
Fluorescent proteins (FPs) displaying distinct spectra have shed their light on a wide range of biological functions. Moreover, sophisticated biosensors engineered to contain single or multiple FPs, including Förster resonance energy transfer (FRET)-based biosensors, spatiotemporally reveal the molecular mechanisms underlying a variety of pathophysiological processes. However, their usefulness for applied life sciences has yet to be fully explored. Recently, our research group has begun to expand the potential of FPs from basic biological research to the clinic. Here, we describe a method to evaluate the responsiveness of leukemia cells from patients to tyrosine kinase inhibitors using a biosensor based on FP technology and the principle of FRET. Upon phosphorylation of the tyrosine residue of the biosensor, binding of the SH2 domain to phosphotyrosine induces conformational change of the biosensor and brings the donor and acceptor FPs into close proximity. Therefore, kinase activity and response to kinase inhibitors can be monitored by an increase and a decrease in FRET efficiency, respectively. As in basic research, this biosensor resolves hitherto arduous tasks and may provide innovative technological advances in clinical laboratory examinations. State-of-the-art detection devices that enable such innovation are also introduced.
Assuntos
Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fusão bcr-abl/química , Proteínas de Fusão bcr-abl/metabolismo , Domínios de Homologia de src , Técnicas Biossensoriais/instrumentação , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Citometria de Fluxo , Transferência Ressonante de Energia de Fluorescência/instrumentação , Proteínas de Fusão bcr-abl/genética , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Processamento de Imagem Assistida por Computador , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Microscopia de Fluorescência , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão , Software , Transfecção , Células Tumorais CultivadasRESUMO
Targeted therapy with inhibitors of epidermal growth factor receptor (EGFR) has produced a noticeable benefit to non-small cell lung cancer (NSCLC) patients whose tumors carry activating mutations (e.g. L858R) in EGFR. Unfortunately, these patients develop drug resistance after treatment, due to acquired secondary gatekeeper mutations in EGFR (e.g. T790M). Given the critical role of SHP2 in growth factor receptor signaling, we sought to determine whether targeting SHP2 could have therapeutic value for EGFR inhibitor resistant NSCLC. We show that SHP2 is required for EGF-stimulated ERK1/2 phosphorylation and proliferation in EGFR inhibitor resistant NSCLC cell line H1975, which harbors the EGFR T790M/L858R double-mutant. We demonstrate that treatment of H1975 cells with II-B08, a specific SHP2 inhibitor, phenocopies the observed growth inhibition and reduced ERK1/2 activation seen in cells treated with SHP2 siRNA. Importantly, we also find that II-B08 exhibits marked anti-tumor activity in H1975 xenograft mice. Finally, we observe that combined inhibition of SHP2 and PI3K impairs both the ERK1/2 and PI3K/AKT signaling axes and produces significantly greater effects on repressing H1975 cell growth than inhibition of either protein individually. Collectively, these results suggest that targeting SHP2 may represent an effective strategy for treatment of EGFR inhibitor resistant NSCLCs.