RESUMEN

γ-Hydroxybutyric acid (GHB) analogs are small molecules that bind competitively to a specific cavity in the oligomeric CaMKIIα hub domain. Binding affects conformation and stability of the hub domain, which may explain the neuroprotective action of some of these compounds. Here, we describe molecular details of interaction of the larger-type GHB analog 2-(6-(4-chlorophenyl)imidazo[1,2-b]pyridazine-2-yl)acetic acid (PIPA). Like smaller-type analogs, PIPA binding to the CaMKIIα hub domain promoted thermal stability. PIPA additionally modulated CaMKIIα activity under sub-maximal CaM concentrations and ultimately led to reduced substrate phosphorylation. A high-resolution X-ray crystal structure of a stabilized CaMKIIα (6x mutant) hub construct revealed details of the binding mode of PIPA, which involved outward placement of tryptophan 403 (Trp403), a central residue in a flexible loop close to the upper hub cavity. Small-angle X-ray scattering (SAXS) solution structures and mass photometry of the CaMKIIα wild-type hub domain in the presence of PIPA revealed a high degree of ordered self-association (stacks of CaMKIIα hub domains). This stacking neither occurred with the smaller compound 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA), nor when Trp403 was replaced with leucine (W403L). Additionally, CaMKIIα W403L hub was stabilized to a larger extent by PIPA compared to CaMKIIα hub wild type, indicating that loop flexibility is important for holoenzyme stability. Thus, we propose that ligand-induced outward placement of Trp403 by PIPA, which promotes an unforeseen mechanism of hub domain stacking, may be involved in the observed reduction in CaMKIIα kinase activity. Altogether, this sheds new light on allosteric regulation of CaMKIIα activity via the hub domain.

Asunto(s)

Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Dominios Proteicos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/química , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Cristalografía por Rayos X , Humanos , Ligandos , Modelos Moleculares , Dispersión del Ángulo Pequeño , Triptófano/química , Triptófano/metabolismo , Piridazinas/química , Piridazinas/metabolismo , Fosforilación

RESUMEN

BCR-ABL1 compound mutations can lead to resistance to ABL1 inhibitors in chronic myeloid leukemia (CML), which could be targeted by combining the ATP-site inhibitor ponatinib and the allosteric inhibitor asciminib. Here, we report the clinical validation of this approach in a CML patient, providing a basis for combination therapy to overcome such resistance.

Asunto(s)

Resistencia a Antineoplásicos , Proteínas de Fusión bcr-abl , Imidazoles , Leucemia Mielógena Crónica BCR-ABL Positiva , Mutación , Inhibidores de Proteínas Quinasas , Piridazinas , Humanos , Piridazinas/uso terapéutico , Piridazinas/farmacología , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Imidazoles/farmacología , Imidazoles/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Masculino , Femenino , Niacinamida/análogos & derivados , Pirazoles

RESUMEN

Oncogenic mutations can destabilize signaling proteins, resulting in increased or unregulated activity. Thus, there is considerable interest in mapping the relationship between mutations and the stability of signaling proteins, to better understand the consequences of oncogenic mutations and potentially inform the development of new therapeutics. Here, we develop a tool to study protein-kinase stability in live mammalian cells and the effects of the HSP90 chaperone system on the stability of these kinases. We determine the expression levels of protein kinases by monitoring the fluorescence of fluorescent proteins fused to those kinases, normalized to that of co-expressed reference fluorescent proteins. We used this tool to study the dependence of Src- and Raf-family kinases on the HSP90 system. We demonstrate that this sensor reports on destabilization induced by oncogenic mutations in these kinases. We also show that Src-homology 2 and Src-homology 3 domains, which are required for autoinhibition of Src-family kinases, stabilize these kinase domains in the cell. Our expression-calibrated sensor enables the facile characterization of the effects of mutations and small-molecule drugs on protein-kinase stability.

Asunto(s)

Proteínas HSP90 de Choque Térmico , Humanos , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/química , Familia-src Quinasas/metabolismo , Familia-src Quinasas/química , Familia-src Quinasas/genética , Células HEK293 , Estabilidad Proteica , Mutación , Estabilidad de Enzimas , Fluorescencia

RESUMEN

The ability of mutations to facilitate adaptation is central to evolution. To understand how mutations can lead to functional adaptation in a complex molecular machine, we created a defective version of the T4 clamp-loader complex, which is essential for DNA replication. This variant, which is ∼5,000-fold less active than the wild type, was made by replacing the catalytic domains with those from another phage. A directed-evolution experiment revealed that multiple substitutions to a single negatively charged residue in the chimeric clamp loader-Asp 86-restore fitness to within ∼20-fold of wild type. These mutations remove an adventitious electrostatic repulsive interaction between Asp 86 and the sliding clamp. Thus, the fitness decrease of the chimeric clamp loader is caused by a reduction in affinity between the clamp loader and the clamp. Deep mutagenesis shows that the reduced fitness of the chimeric clamp loader is also compensated for by lysine and arginine substitutions of several DNA-proximal residues in the clamp loader or the sliding clamp. Our results demonstrate that there is a latent capacity for increasing the affinity of the clamp loader for DNA and the sliding clamp, such that even single-point mutations can readily compensate for the loss of function due to suboptimal interactions elsewhere.

Asunto(s)

Adenosina Trifosfatasas , Adenosina Trifosfato , Adenosina Trifosfatasas/genética , Adenosina Trifosfato/química , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Replicación del ADN , ADN

RESUMEN

Clamp loaders are AAA+ ATPases that facilitate high-speed DNA replication. In eukaryotic and bacteriophage clamp loaders, ATP hydrolysis requires interactions between aspartate residues in one protomer, present in conserved 'DEAD-box' motifs, and arginine residues in adjacent protomers. We show that functional defects resulting from a DEAD-box mutation in the T4 bacteriophage clamp loader can be compensated by widely distributed single mutations in the ATPase domain. Using cryo-EM, we discovered an unsuspected inactive conformation of the clamp loader, in which DNA binding is blocked and the catalytic sites are disassembled. Mutations that restore function map to regions of conformational change upon activation, suggesting that these mutations may increase DNA affinity by altering the energetic balance between inactive and active states. Our results show that there are extensive opportunities for evolution to improve catalytic efficiency when an inactive intermediate is involved.

Asunto(s)

Adenosina Trifosfatasas , Replicación del ADN , Adenosina Trifosfatasas/metabolismo , Microscopía por Crioelectrón , ADN , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Mutagénesis , Adenosina Trifosfato/metabolismo

RESUMEN

T cell responses are inhibited by acidic environments. T cell receptor (TCR)-induced protein phosphorylation is negatively regulated by dephosphorylation and/or ubiquitination, but the mechanisms underlying sensitivity to acidic environments are not fully understood. Here, we found that TCR stimulation induced a molecular complex of Cbl-b, an E3-ubiquitin ligase, with STS1, a pH-sensitive unconventional phosphatase. The induced interaction depended upon a proline motif in Cbl-b interacting with the STS1 SH3 domain. STS1 dephosphorylated Cbl-b interacting phosphoproteins. The deficiency of STS1 or Cbl-b diminished the sensitivity of T cell responses to the inhibitory effects of acid in an autocrine or paracrine manner in vitro or in vivo. Moreover, the deficiency of STS1 or Cbl-b promoted T cell proliferative and differentiation activities in vivo and inhibited tumor growth, prolonged survival, and improved T cell fitness in tumor models. Thus, a TCR-induced STS1-Cbl-b complex senses intra- or extra-cellular acidity and regulates T cell responses, presenting a potential therapeutic target for improving anti-tumor immunity.

Asunto(s)

Transducción de Señal , Linfocitos T , Ubiquitina-Proteína Ligasas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Concentración de Iones de Hidrógeno

RESUMEN

Oncogenic mutations can destabilize signaling proteins, resulting in increased or unregulated activity. Thus, there is considerable interest in mapping the relationship between mutations and the stability of proteins, to better understand the consequences of oncogenic mutations and potentially inform the development of new therapeutics. Here, we develop a tool to study protein-kinase stability in live mammalian cells and the effects of the HSP90 chaperone system on the stability of these kinases. We monitor the fluorescence of kinases fused to a fluorescent protein relative to that of a co-expressed reference fluorescent protein. We used this tool to study the dependence of Src- and Raf-family kinases on the HSP90 system. We demonstrate that this sensor reports on destabilization induced by oncogenic mutations in these kinases. We also show that Src-homology 2 (SH2) and Src-homology 3 (SH3) domains, which are required for autoinhibition of Src-family kinases, stabilize these kinase domains in the cell. Our expression-calibrated sensor enables the facile characterization of the effects of mutations and small-molecule drugs on protein-kinase stability.

RESUMEN

Genomic analysis of the unicellular choanoflagellate, Monosiga brevicollis (MB), revealed the remarkable presence of cell signaling and adhesion protein domains that are characteristically associated with metazoans. Strikingly, receptor tyrosine kinases, one of the most critical elements of signal transduction and communication in metazoans, are present in choanoflagellates. We determined the crystal structure at 1.95 Å resolution of the kinase domain of the M. brevicollis receptor tyrosine kinase C8 (RTKC8, a member of the choanoflagellate receptor tyrosine kinase C family) bound to the kinase inhibitor staurospaurine. The chonanoflagellate kinase domain is closely related in sequence to mammalian tyrosine kinases (~ 40% sequence identity to the human Ephrin kinase domain EphA3) and, as expected, has the canonical protein kinase fold. The kinase is structurally most similar to human Ephrin (EphA5), even though the extracellular sensor domain is completely different from that of Ephrin. The RTKC8 kinase domain is in an active conformation, with two staurosporine molecules bound to the kinase, one at the active site and another at the peptide-substrate binding site. To our knowledge this is the first example of staurospaurine binding in the Aurora A activation segment (AAS). We also show that the RTKC8 kinase domain can phosphorylate tyrosine residues in peptides from its C-terminal tail segment which is presumably the mechanism by which it transmits the extracellular stimuli to alter cellular function.

Asunto(s)

Coanoflagelados , Humanos , Animales , Proteínas Tirosina Quinasas Receptoras , Transducción de Señal , Proteínas Tirosina Quinasas , Efrinas , Mamíferos

RESUMEN

The Tec-family kinase Btk contains a lipid-binding Pleckstrin homology and Tec homology (PH-TH) module connected by a proline-rich linker to a 'Src module', an SH3-SH2-kinase unit also found in Src-family kinases and Abl. We showed previously that Btk is activated by PH-TH dimerization, which is triggered on membranes by the phosphatidyl inositol phosphate PIP3, or in solution by inositol hexakisphosphate (IP6) (Wang et al., 2015, https://doi.org/10.7554/eLife.06074). We now report that the ubiquitous adaptor protein growth-factor-receptor-bound protein 2 (Grb2) binds to and substantially increases the activity of PIP3-bound Btk on membranes. Using reconstitution on supported-lipid bilayers, we find that Grb2 can be recruited to membrane-bound Btk through interaction with the proline-rich linker in Btk. This interaction requires intact Grb2, containing both SH3 domains and the SH2 domain, but does not require that the SH2 domain be able to bind phosphorylated tyrosine residues - thus Grb2 bound to Btk is free to interact with scaffold proteins via the SH2 domain. We show that the Grb2-Btk interaction recruits Btk to scaffold-mediated signaling clusters in reconstituted membranes. Our findings indicate that PIP3-mediated dimerization of Btk does not fully activate Btk, and that Btk adopts an autoinhibited state at the membrane that is released by Grb2.

Asunto(s)

Proteínas Tirosina Quinasas , Dominios Homologos src , Familia-src Quinasas , Transducción de Señal , Dimerización

RESUMEN

Many tyrosine kinases cannot be expressed readily in Escherichia coli, limiting facile production of these proteins for biochemical experiments. We used ancestral sequence reconstruction to generate a spleen tyrosine kinase (Syk) variant that can be expressed in bacteria and purified in soluble form, unlike the human members of this family (Syk and zeta-chain-associated protein kinase of 70 kDa [ZAP-70]). The catalytic activity, substrate specificity, and regulation by phosphorylation of this Syk variant are similar to the corresponding properties of human Syk and ZAP-70. Taking advantage of the ability to express this novel Syk-family kinase in bacteria, we developed a two-hybrid assay that couples the growth of E. coli in the presence of an antibiotic to successful phosphorylation of a bait peptide by the kinase. Using this assay, we screened a site-saturation mutagenesis library of the kinase domain of this reconstructed Syk-family kinase. Sites of loss-of-function mutations identified in the screen correlate well with residues established previously as critical to function and/or structure in protein kinases. We also identified activating mutations in the regulatory hydrophobic spine and activation loop, which are within key motifs involved in kinase regulation. Strikingly, one mutation in an ancestral Syk-family variant increases the soluble expression of the protein by 75-fold. Thus, through ancestral sequence reconstruction followed by deep mutational scanning, we have generated Syk-family kinase variants that can be expressed in bacteria with very high yield.

Asunto(s)

Escherichia coli , Péptidos y Proteínas de Señalización Intracelular , Antibacterianos , Precursores Enzimáticos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mutagénesis , Péptidos/química , Fosforilación , Quinasa Syk/genética , Quinasa Syk/metabolismo , Tirosina/genética

RESUMEN

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available coronavirus disease 2019 vaccines and monoclonal antibody therapies through epitope change on the receptor binding domain of the viral spike glycoprotein. Hence, there is a specific urgent need for alternative antivirals that target processes less likely to be affected by mutation, such as the membrane fusion step of viral entry into the host cell. One such antiviral class includes peptide inhibitors, which block formation of the so-called heptad repeat 1 and 2 (HR1HR2) six-helix bundle of the SARS-CoV-2 spike (S) protein and thus interfere with viral membrane fusion. We performed structural studies of the HR1HR2 bundle, revealing an extended, well-folded N-terminal region of HR2 that interacts with the HR1 triple helix. Based on this structure, we designed an extended HR2 peptide that achieves single-digit nanomolar inhibition of SARS-CoV-2 in cell-based and virus-based assays without the need for modifications such as lipidation or chemical stapling. The peptide also strongly inhibits all major SARS-CoV-2 variants to date. This extended peptide is ∼100-fold more potent than all previously published short, unmodified HR2 peptides, and it has a very long inhibition lifetime after washout in virus infection assays, suggesting that it targets a prehairpin intermediate of the SARS-CoV-2 S protein. Together, these results suggest that regions outside the HR2 helical region may offer new opportunities for potent peptide-derived therapeutics for SARS-CoV-2 and its variants, and even more distantly related viruses, and provide further support for the prehairpin intermediate of the S protein.

Asunto(s)

Tratamiento Farmacológico de COVID-19 , Glicoproteína de la Espiga del Coronavirus , Antivirales/química , Antivirales/farmacología , Humanos , Péptidos/química , Péptidos/farmacología , SARS-CoV-2/efectos de los fármacos

RESUMEN

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available COVID-19 vaccines and monoclonal antibody therapies through epitope change on the receptor binding domain of the viral spike glycoprotein. Hence, there is a specific urgent need for alternative antivirals that target processes less likely to be affected by mutation, such as the membrane fusion step of viral entry into the host cell. One such antiviral class includes peptide inhibitors which block formation of the so-called HR1HR2 six-helix bundle of the SARS-CoV-2 spike (S) protein and thus interfere with viral membrane fusion. Here we performed structural studies of the HR1HR2 bundle, revealing an extended, well-folded N-terminal region of HR2 that interacts with the HR1 triple helix. Based on this structure, we designed an extended HR2 peptide that achieves single-digit nanomolar inhibition of SARS-CoV-2 in cell-based fusion, VSV-SARS-CoV-2 chimera, and authentic SARS-CoV-2 infection assays without the need for modifications such as lipidation or chemical stapling. The peptide also strongly inhibits all major SARS-CoV-2 variants to date. This extended peptide is ~100-fold more potent than all previously published short, unmodified HR2 peptides, and it has a very long inhibition lifetime after washout in virus infection assays, suggesting that it targets a pre-hairpin intermediate of the SARS-CoV-2 S protein. Together, these results suggest that regions outside the HR2 helical region may offer new opportunities for potent peptide-derived therapeutics for SARS-CoV-2 and its variants, and even more distantly related viruses, and provide further support for the pre-hairpin intermediate of the S protein. Significance Statement: SARS-CoV-2 infection requires fusion of viral and host membranes, mediated by the viral spike glycoprotein (S). Due to the importance of viral membrane fusion, S has been a popular target for developing vaccines and therapeutics. We discovered a simple peptide that inhibits infection by all major variants of SARS-CoV-2 with nanomolar efficacies. In marked contrast, widely used shorter peptides that lack a key N-terminal extension are about 100 x less potent than this peptide. Our results suggest that a simple peptide with a suitable sequence can be a potent and cost-effective therapeutic against COVID-19 and they provide new insights at the virus entry mechanism.

RESUMEN

We reconstitute a phosphotyrosine-mediated protein condensation phase transition of the ∼200 residue cytoplasmic tail of the epidermal growth factor receptor (EGFR) and the adaptor protein, Grb2, on a membrane surface. The phase transition depends on phosphorylation of the EGFR tail, which recruits Grb2, and crosslinking through a Grb2-Grb2 binding interface. The Grb2 Y160 residue plays a structurally critical role in the Grb2-Grb2 interaction, and phosphorylation or mutation of Y160 prevents EGFR:Grb2 condensation. By extending the reconstitution experiment to include the guanine nucleotide exchange factor, SOS, and its substrate Ras, we further find that the condensation state of the EGFR tail controls the ability of SOS, recruited via Grb2, to activate Ras. These results identify an EGFR:Grb2 protein condensation phase transition as a regulator of signal propagation from EGFR to the MAPK pathway.

Asunto(s)

Receptores ErbB , Transducción de Señal , Receptores ErbB/metabolismo , Proteína Adaptadora GRB2/metabolismo , Fosforilación , Fosfotirosina/metabolismo

RESUMEN

Cancer mutations in Ras occur predominantly at three hotspots: Gly 12, Gly 13, and Gln 61. Previously, we reported that deep mutagenesis of H-Ras using a bacterial assay identified many other activating mutations (Bandaru et al., 2017). We now show that the results of saturation mutagenesis of H-Ras in mammalian Ba/F3 cells correlate well with the results of bacterial experiments in which H-Ras or K-Ras are co-expressed with a GTPase-activating protein (GAP). The prominent cancer hotspots are not dominant in the Ba/F3 data. We used the bacterial system to mutagenize Ras constructs of different stabilities and discovered a feature that distinguishes the cancer hotspots. While mutations at the cancer hotspots activate Ras regardless of construct stability, mutations at lower-frequency sites (e.g. at Val 14 or Asp 119) can be activating or deleterious, depending on the stability of the Ras construct. We characterized the dynamics of three non-hotspot activating Ras mutants by using NMR to monitor hydrogen-deuterium exchange (HDX). These mutations result in global increases in HDX rates, consistent with destabilization of Ras. An explanation for these observations is that mutations that destabilize Ras increase nucleotide dissociation rates, enabling activation by spontaneous nucleotide exchange. A further stability decrease can lead to insufficient levels of folded Ras - and subsequent loss of function. In contrast, the cancer hotspot mutations are mechanism-based activators of Ras that interfere directly with the action of GAPs. Our results demonstrate the importance of GAP surveillance and protein stability in determining the sensitivity of Ras to mutational activation.

Asunto(s)

Proteínas Activadoras de GTPasa , Neoplasias , Animales , Mamíferos , Mutagénesis , Mutación , Nucleótidos , Proteínas Activadoras de ras GTPasa

RESUMEN

Son of Sevenless (SOS) is a Ras guanine nucleotide exchange factor (GEF) that plays a central role in numerous cellular signaling pathways. Like many other signaling molecules, SOS is autoinhibited in the cytosol and activates only after recruitment to the membrane. The mean activation time of individual SOS molecules has recently been measured to be ∼60 s, which is unexpectedly long and seemingly contradictory with cellular signaling timescales, which have been measured to be as fast as several seconds. Here, we rectify this discrepancy using a first-passage time analysis to reconstruct the effective signaling timescale of multiple SOS molecules from their single-molecule activation kinetics. Along with corresponding experimental measurements, this analysis reveals how the functional response time, comprised of many slowly activating molecules, can become substantially faster than the average molecular kinetics. This consequence stems from the enzymatic processivity of SOS in a highly out-of-equilibrium reaction cycle during receptor triggering. Ultimately, rare, early activation events dominate the macroscopic reaction dynamics.

Asunto(s)

Modelos Químicos , Proteínas Son Of Sevenless/metabolismo , Proteínas ras/metabolismo , Activación Enzimática , Cinética , Imagen Individual de Molécula

RESUMEN

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that couples the binding of extracellular ligands, such as EGF and transforming growth factor-α (TGF-α), to the initiation of intracellular signaling pathways. EGFR binds to EGF and TGF-α with similar affinity, but generates different signals from these ligands. To address the mechanistic basis of this phenomenon, we have carried out cryo-EM analyses of human EGFR bound to EGF and TGF-α. We show that the extracellular module adopts an ensemble of dimeric conformations when bound to either EGF or TGF-α. The two extreme states of this ensemble represent distinct ligand-bound quaternary structures in which the membrane-proximal tips of the extracellular module are either juxtaposed or separated. EGF and TGF-α differ in their ability to maintain the conformation with the membrane-proximal tips of the extracellular module separated, and this conformation is stabilized preferentially by an oncogenic EGFR mutation. Close proximity of the transmembrane helices at the junction with the extracellular module has been associated previously with increased EGFR activity. Our results show how EGFR can couple the binding of different ligands to differential modulation of this proximity, thereby suggesting a molecular mechanism for the generation of ligand-sensitive differential outputs in this receptor family.

Asunto(s)

Células Cultivadas/fisiología , Receptores ErbB/química , Ligandos , Transducción de Señal/efectos de los fármacos , Spodoptera/fisiología , Factores de Crecimiento Transformadores/química , Animales , Humanos , Modelos Moleculares

RESUMEN

The catalytic activity of Syk-family tyrosine kinases is regulated by a tandem Src homology 2 module (tSH2 module). In the autoinhibited state, this module adopts a conformation that stabilizes an inactive conformation of the kinase domain. The binding of the tSH2 module to phosphorylated immunoreceptor tyrosine-based activation motifs necessitates a conformational change, thereby relieving kinase inhibition and promoting activation. We determined the crystal structure of the isolated tSH2 module of Syk and find, in contrast to ZAP-70, that its conformation more closely resembles that of the peptide-bound state, rather than the autoinhibited state. Hydrogen-deuterium exchange by mass spectrometry, as well as molecular dynamics simulations, reveal that the dynamics of the tSH2 modules of Syk and ZAP-70 differ, with most of these differences occurring in the C-terminal SH2 domain. Our data suggest that the conformational landscapes of the tSH2 modules in Syk and ZAP-70 have been tuned differently, such that the autoinhibited conformation of the Syk tSH2 module is less stable. This feature of Syk likely contributes to its ability to more readily escape autoinhibition when compared to ZAP-70, consistent with tighter control of downstream signaling pathways in T cells.

Asunto(s)

Simulación de Dinámica Molecular , Quinasa Syk/química , Proteína Tirosina Quinasa ZAP-70/química , Inmunidad Adaptativa , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Sitios de Unión , Evolución Biológica , Clonación Molecular , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes , Transducción de Señal , Quinasa Syk/genética , Quinasa Syk/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Proteína Tirosina Quinasa ZAP-70/genética , Proteína Tirosina Quinasa ZAP-70/inmunología

RESUMEN

Although oxidative phosphorylation is best known for producing ATP, it also yields reactive oxygen species (ROS) as invariant byproducts. Depletion of ROS below their physiological levels, a phenomenon known as reductive stress, impedes cellular signaling and has been linked to cancer, diabetes, and cardiomyopathy. Cells alleviate reductive stress by ubiquitylating and degrading the mitochondrial gatekeeper FNIP1, yet it is unknown how the responsible E3 ligase CUL2FEM1B can bind its target based on redox state and how this is adjusted to changing cellular environments. Here, we show that CUL2FEM1B relies on zinc as a molecular glue to selectively recruit reduced FNIP1 during reductive stress. FNIP1 ubiquitylation is gated by pseudosubstrate inhibitors of the BEX family, which prevent premature FNIP1 degradation to protect cells from unwarranted ROS accumulation. FEM1B gain-of-function mutation and BEX deletion elicit similar developmental syndromes, showing that the zinc-dependent reductive stress response must be tightly regulated to maintain cellular and organismal homeostasis.

Asunto(s)

Estrés Fisiológico , Aminoácidos/química , Animales , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Femenino , Humanos , Iones , Ratones , Proteínas Mutantes/metabolismo , Mutación/genética , Unión Proteica/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Estrés Fisiológico/efectos de los fármacos , Relación Estructura-Actividad , Especificidad por Sustrato/efectos de los fármacos , Complejos de Ubiquitina-Proteína Ligasa/química , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitinación/efectos de los fármacos , Zinc/farmacología

RESUMEN

BRAF is a highly regulated protein kinase that controls cell fate in animal cells. Recent structural analyses have revealed how active and inactive forms of BRAF bind to dimers of the scaffold protein 14-3-3. Inactive BRAF binds to 14-3-3 as a monomer and is held in an inactive conformation by interactions with ATP and the substrate kinase MEK, a striking example of enzyme inhibition by substrate binding. A change in the phosphorylation state of BRAF shifts the stoichiometry of the BRAF:14-3-3 complex from 1:2 to 2:2, resulting in stabilization of the active dimeric form of the kinase. These new findings uncover unexpected features of the regulatory mechanisms underlying Raf biology and help explain the paradoxical activation of Raf by small-molecule inhibitors.

Asunto(s)

Proteínas Quinasas , Proteínas Proto-Oncogénicas B-raf , Animales , Mutación , Fosforilación , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo

RESUMEN

Ca2+/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) is a key neuronal signaling protein and an emerging drug target. The central hub domain regulates the activity of CaMKIIα by organizing the holoenzyme complex into functional oligomers, yet pharmacological modulation of the hub domain has never been demonstrated. Here, using a combination of photoaffinity labeling and chemical proteomics, we show that compounds related to the natural substance γ-hydroxybutyrate (GHB) bind selectively to CaMKIIα. By means of a 2.2-Å x-ray crystal structure of ligand-bound CaMKIIα hub, we reveal the molecular details of the binding site deep within the hub. Furthermore, we show that binding of GHB and related analogs to this site promotes concentration-dependent increases in hub thermal stability believed to alter holoenzyme functionality. Selectively under states of pathological CaMKIIα activation, hub ligands provide a significant and sustained neuroprotection, which is both time and dose dependent. This is demonstrated in neurons exposed to excitotoxicity and in a mouse model of cerebral ischemia with the selective GHB analog, HOCPCA (3-hydroxycyclopent-1-enecarboxylic acid). Together, our results indicate a hitherto unknown mechanism for neuroprotection by a highly specific and unforeseen interaction between the CaMKIIα hub domain and small molecule brain-penetrant GHB analogs. This establishes GHB analogs as powerful tools for investigating CaMKII neuropharmacology in general and as potential therapeutic compounds for cerebral ischemia in particular.

Asunto(s)

Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Oxibato de Sodio/metabolismo , Sitios de Unión , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Ácidos Carboxílicos/farmacología , Cristalografía por Rayos X , Ciclopentanos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Neuroprotección , Unión Proteica , Dominios Proteicos , Transducción de Señal