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1.
Artigo em Inglês | MEDLINE | ID: mdl-32227412

RESUMO

KRAS homo-dimerization has been implicated in the activation of RAF kinases, however, the mechanism and structural basis remain elusive. We developed a system to study KRAS dimerization on nanodiscs using paramagnetic relaxation enhancement (PRE) NMR, and determined distinct structures of membrane-anchored KRAS dimers in the active GTP- and inactive GDP-loaded states. Both dimerize through an α4-α5 interface, but the relative orientation of the protomers and their contacts differ substantially. Dimerization of KRAS-GTP, stabilized by electrostatic interactions between R135 and E168, favours an orientation on the membrane that promotes accessibility of the effector-binding site. Remarkably, 'cross'-dimerization between GTP- and GDP-bound KRAS molecules is unfavorable. These models provide a vital platform to elucidate the structural basis of RAF activation by RAS and to develop newer inhibitors that can disrupt the KRAS dimerization. The methodology developed to specifically probe the intermolecular interactions within KRAS dimer is applicable to many other farnesylated small GTPases.

2.
J Mol Biol ; 432(2): 367-383, 2020 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-31626806

RESUMO

Store operated calcium (Ca2+) entry (SOCE) is the process whereby endoplasmic reticulum (ER) Ca2+ store depletion causes Orai1-composed Ca2+ channels on the plasma membrane (PM) to open, mediating a rise in cytosolic Ca2+ levels. Stromal interaction molecules (STIMs) are the proteins that directly sense ER Ca2+ content and gate Orai1 channels due to store depletion. The trigger for STIM activation is Ca2+ unbinding from the ER lumen-oriented domains, which consist of a nonconserved amino (N) terminal region and EF-hand and sterile α motif (SAM) domains (EF-SAM), highly conserved from humans to Caenorhabditis elegans. Solution NMR structures of the human EF-SAM domains have been determined at high Ca2+ concentrations; however, no direct structural view of the Ca2+ binding mode has been elucidated. Further, no atomic resolution data currently exists on EF-SAM at low Ca2+ levels. Here, we determined the X-ray crystal structure of the C. elegans STIM luminal domain, revealing that EF-SAM binds a single Ca2+ ion with pentagonal bipyramidal geometry and an ancillary α-helix formed by the N-terminal region acts as a brace to stabilize EF-SAM. Using solution NMR, we observed EF-hand domain unfolding and a conformational exchange between folded and unfolded states involving the ancillary α-helix and the canonical EF-hand in low Ca2+. Remarkably, we also detected an α-helix (+Ca2+) to ß-strand (-Ca2+) transition at the terminal SAM domain α-helix. Collectively, our analyses indicate that one canonically bound Ca2+ ion is sufficient to stabilize the quiescent luminal domain structure, precluding unfolding, conformational exchange, and secondary structure transformation.

3.
Methods Mol Biol ; 1929: 207-221, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30710275

RESUMO

Calmodulin (CaM) is a ubiquitous calcium-sensing protein that has one of the most highly conserved sequences among eukaryotes. CaM has been a useful tool for biologists studying calcium signaling for decades. In recent years, CaM has also been implicated in numerous cancer-associated pathways, and rare CaM mutations have been identified as a cause of human cardiac arrhythmias. Here, we present a collection of our most recent and effective protocols for the expression and purification of recombinant CaM from Escherichia coli, including various isotopic labeling schemes, primarily for nuclear magnetic resonance (NMR) spectroscopy and other biophysical applications.


Assuntos
Calmodulina/isolamento & purificação , Calmodulina/metabolismo , Escherichia coli/crescimento & desenvolvimento , Sinalização do Cálcio , Calmodulina/genética , Cromatografia de Afinidade , Escherichia coli/genética , Expressão Gênica , Humanos , Marcação por Isótopo , Espectroscopia de Ressonância Magnética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
4.
Nat Commun ; 10(1): 224, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30644389

RESUMO

Deregulation of the RAS GTPase cycle due to mutations in the three RAS genes is commonly associated with cancer development. Protein tyrosine phosphatase SHP2 promotes RAF-to-MAPK signaling pathway and is an essential factor in RAS-driven oncogenesis. Despite the emergence of SHP2 inhibitors for the treatment of cancers harbouring mutant KRAS, the mechanism underlying SHP2 activation of KRAS signaling remains unclear. Here we report tyrosyl-phosphorylation of endogenous RAS and demonstrate that KRAS phosphorylation via Src on Tyr32 and Tyr64 alters the conformation of switch I and II regions, which stalls multiple steps of the GTPase cycle and impairs binding to effectors. In contrast, SHP2 dephosphorylates KRAS, a process that is required to maintain dynamic canonical KRAS GTPase cycle. Notably, Src- and SHP2-mediated regulation of KRAS activity extends to oncogenic KRAS and the inhibition of SHP2 disrupts the phosphorylation cycle, shifting the equilibrium of the GTPase cycle towards the stalled 'dark state'.


Assuntos
Antineoplásicos/uso terapêutico , GTP Fosfo-Hidrolases/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Células HEK293 , Humanos , Masculino , Camundongos SCID , Neoplasias Pancreáticas/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases raf/metabolismo
5.
Cell Calcium ; 77: 79-80, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30528613

RESUMO

A single calcium (Ca2+) binding site within the canonical EF-hand loop was thought to govern the stromal interaction molecule-1 (STIM1) structural changes that lead to activation of Orai1 Ca2+ channels. Recent work by Gudlur et al., published in Nat Commun [9(1):4536], suggests that the STIM1 endoplasmic reticulum (ER) luminal domain has ∼5 additional Ca2+ binding sites, which underlie a surprising new proposal for Ca2+ sensing.


Assuntos
Cálcio , Retículo Endoplasmático , Sítios de Ligação , Canais de Cálcio , Domínios Proteicos , Molécula 1 de Interação Estromal
6.
Nat Commun ; 9(1): 5121, 2018 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-30504777

RESUMO

α-catenin is a key mechanosensor that forms force-dependent interactions with F-actin, thereby coupling the cadherin-catenin complex to the actin cytoskeleton at adherens junctions (AJs). However, the molecular mechanisms by which α-catenin engages F-actin under tension remained elusive. Here we show that the α1-helix of the α-catenin actin-binding domain (αcat-ABD) is a mechanosensing motif that regulates tension-dependent F-actin binding and bundling. αcat-ABD containing an α1-helix-unfolding mutation (H1) shows enhanced binding to F-actin in vitro. Although full-length α-catenin-H1 can generate epithelial monolayers that resist mechanical disruption, it fails to support normal AJ regulation in vivo. Structural and simulation analyses suggest that α1-helix allosterically controls the actin-binding residue V796 dynamics. Crystal structures of αcat-ABD-H1 homodimer suggest that α-catenin can facilitate actin bundling while it remains bound to E-cadherin. We propose that force-dependent allosteric regulation of αcat-ABD promotes dynamic interactions with F-actin involved in actin bundling, cadherin clustering, and AJ remodeling during tissue morphogenesis.


Assuntos
Junções Aderentes/metabolismo , alfa Catenina/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Actinas/metabolismo , Animais , Caderinas/química , Caderinas/metabolismo , Humanos , Estrutura Secundária de Proteína , alfa Catenina/química
7.
Nat Commun ; 9(1): 5116, 2018 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-30504805

RESUMO

Neutrophil extracellular traps (NETs) promote cancer metastasis in preclinical models following massive exogenous inflammatory stimuli. It remains unknown whether cancer hosts under physiologic conditions experience NETosis and consequent metastasis. Here we show that plasma redox imbalance caused by albumin oxidation promotes inflammation-independent NETosis. Albumin is the major source of free thiol that maintains redox balance. Oxidation of albumin-derived free thiol is sufficient to trigger NETosis via accumulation of reactive oxygen species within neutrophils. The resultant NETs are found predominantly within lungs where they contribute to the colonization of circulating tumor cells leading to pulmonary metastases. These effects are abrogated by pharmacologic inhibition of NET formation. Moreover, albumin oxidation is associated with pulmonary metastasis in a cohort of head and neck cancer patients. These results implicate plasma redox balance as an endogenous and physiologic regulator of NETosis and pulmonary cancer metastasis, providing new therapeutic and diagnostic opportunities for combatting cancer progression.


Assuntos
Armadilhas Extracelulares/metabolismo , Neoplasias Pulmonares/sangue , Espécies Reativas de Oxigênio/sangue , Albuminas/metabolismo , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Neutrófilos/metabolismo , Oxirredução
8.
Cell Chem Biol ; 25(11): 1327-1336.e4, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30122370

RESUMO

KRAS is frequently mutated in several of the most lethal types of cancer; however, the KRAS protein has proven a challenging drug target. K-RAS4B must be localized to the plasma membrane by prenylation to activate oncogenic signaling, thus we endeavored to target the protein-membrane interface with small-molecule compounds. While all reported lead compounds have low affinity for KRAS in solution, the potency of Cmpd2 was strongly enhanced when prenylated K-RAS4B is associated with a lipid bilayer. We have elucidated a unique mechanism of action of Cmpd2, which simultaneously engages a shallow pocket on KRAS and associates with the lipid bilayer, thereby stabilizing KRAS in an orientation in which the membrane occludes its effector-binding site, reducing RAF binding and impairing activation of RAF. Furthermore, enrichment of Cmpd2 on the bilayer enhances potency by promoting interaction with KRAS. This insight reveals a novel approach to developing inhibitors of membrane-associated proteins.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Piperidinas/química , Piperidinas/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Animais , Descoberta de Drogas , Humanos , Indóis/química , Indóis/farmacologia , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo
9.
Cell Calcium ; 73: 88-94, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29698850

RESUMO

Stromal interaction molecule (STIM)-1 and -2 are multi-domain, single-pass transmembrane proteins involved in sensing changes in compartmentalized calcium (Ca2+) levels and transducing this cellular signal to Orai1 channel proteins. Our understanding of the molecular mechanisms underlying STIM signaling has been dramatically improved through available X-ray crystal and solution NMR structures. This high-resolution structural data has revealed that intricate intramolecular and intermolecular protein-protein interactions are involved in converting STIMs from the quiescent to activation-competent states. This review article summarizes the current high resolution structural data on specific EF-hand, sterile α motif and coiled-coil interactions which drive STIM function in the activation of Orai1 channels. Further, the work discusses the effects of post-translational modifications on the structure and function of STIMs. Future structural studies on larger STIM:Orai complexes will be critical to fully defining the molecular bases for STIM function and how post-translational modifications influence these mechanisms.


Assuntos
Proteínas de Neoplasias/química , Proteínas de Neoplasias/fisiologia , Molécula 1 de Interação Estromal/química , Molécula 1 de Interação Estromal/fisiologia , Molécula 2 de Interação Estromal/química , Molécula 2 de Interação Estromal/fisiologia , Animais , Citosol/fisiologia , Humanos , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
10.
J Am Chem Soc ; 140(13): 4473-4476, 2018 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-29543440

RESUMO

Small GTPases (sGTPases) are critical switch-like regulators that mediate several important cellular functions and are often mutated in human cancers. They are activated by guanine nucleotide exchange factors (GEFs), which specifically catalyze the exchange of GTP for GDP. GEFs coordinate signaling networks in normal cells, and are frequently deregulated in cancers. sGTPase signaling pathways are complex and interconnected; however, most GEF assays do not reveal such complexity. In this Communication, we describe the development of a unique real-time NMR-based multiplexed GEF assay that employs distinct isotopic labeling schemes for each sGTPase protein to enable simultaneous observation of six proteins of interest. We monitor nucleotide exchange of KRas, Rheb, RalB, RhoA, Cdc42 and Rac1 in a single system, and assayed the activities of GEFs in lysates of cultured human cells and 3D organoids derived from pancreatic cancer patients. We observed potent activation of RhoA by lysates of HEK293a cells transfected with GEF-H1, along with weak stimulation of Rac1, which we showed is indirect. Our functional analyses of pancreatic cancer-derived organoids revealed higher GEF activity for RhoA than other sGTPases, in line with RNA-seq data indicating high expression of RhoA-specific GEFs.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Bioensaio , Fatores de Troca do Nucleotídeo Guanina/classificação , Células HEK293 , Humanos , Espectroscopia de Ressonância Magnética , Neoplasias Pancreáticas/patologia , Proteína rhoA de Ligação ao GTP/química
11.
Sci Signal ; 10(503)2017 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-29089450

RESUMO

The PAR-1-MARK pathway controls cell polarity through the phosphorylation of microtubule-associated proteins. Rho-Rac guanine nucleotide exchange factor 2 (ARHGEF2), which activates Ras homolog family member A (RHOA), is anchored to the microtubule network and sequestered in an inhibited state through binding to dynein light chain Tctex-1 type 1 (DYNLT1). We showed in mammalian cells that liver kinase B1 (LKB1) activated the microtubule affinity-regulating kinase 3 (MARK3), which in turn phosphorylated ARHGEF2 at Ser151 This modification disrupted the interaction between ARHGEF2 and DYNLT1 by generating a 14-3-3 binding site in ARHGEF2, thus causing ARHGEF2 to dissociate from microtubules. Phosphorylation of ARHGEF2 by MARK3 stimulated RHOA activation and the formation of stress fibers and focal adhesions, and was required for organized cellular architecture in three-dimensional culture. Protein phosphatase 2A (PP2A) dephosphorylated Ser151 in ARHGEF2 to restore the inhibited state. Thus, we have identified a regulatory switch controlled by MARK3 that couples microtubules to the actin cytoskeleton to establish epithelial cell polarity through ARHGEF2.


Assuntos
Citoesqueleto de Actina/metabolismo , Polaridade Celular/fisiologia , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Animais , Células COS , Dineínas/genética , Dineínas/metabolismo , Adesões Focais/metabolismo , Células HEK293 , Humanos , Fosforilação , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Serina/metabolismo , Fibras de Estresse/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
12.
Nat Commun ; 8(1): 1099, 2017 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-29062045

RESUMO

Elucidation of activation mechanisms governing protein fusions is essential for therapeutic development. MLL undergoes rearrangement with numerous partners, including a recurrent translocation fusing the epigenetic regulator to a cytoplasmic RAS effector, AF6/afadin. We show here that AF6 employs a non-canonical, evolutionarily conserved α-helix to bind RAS, unique to AF6 and the classical RASSF effectors. Further, all patients with MLL-AF6 translocations express fusion proteins missing only this helix from AF6, resulting in exposure of hydrophobic residues that induce dimerization. We provide evidence that oligomerization is the dominant mechanism driving oncogenesis from rare MLL translocation partners and employ our mechanistic understanding of MLL-AF6 to examine how dimers induce leukemia. Proteomic data resolve association of dimerized MLL with gene expression modulators, and inhibiting dimerization disrupts formation of these complexes while completely abrogating leukemogenesis in mice. Oncogenic gene translocations are thus selected under pressure from protein structure/function, underscoring the complex nature of chromosomal rearrangements.


Assuntos
Evolução Molecular , Cinesina/metabolismo , Leucemia/genética , Miosinas/metabolismo , Proteína Oncogênica p21(ras)/metabolismo , Sequência de Aminoácidos , Dimerização , Humanos , Cinesina/química , Cinesina/genética , Leucemia/enzimologia , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Modelos Moleculares , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Miosinas/química , Miosinas/genética , Proteína Oncogênica p21(ras)/química , Proteína Oncogênica p21(ras)/genética , Ligação Proteica , Domínios Proteicos , Translocação Genética
13.
Biomol NMR Assign ; 11(1): 21-24, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27804064

RESUMO

α-Catenin is a filamentous actin (F-actin) binding protein that links the classical cadherin-catenin complex to the actin cytoskeleton at adherens junctions (AJs). Its C-terminal F-actin binding domain is required for regulating the dynamic interaction between AJs and the actin cytoskeleton during tissue development. Thus, obtaining the molecular details of this interaction is a crucial step towards understanding how α-catenin plays critical roles in biological processes, such as morphogenesis, cell polarity, wound healing and tissue maintenance. Here we report the backbone atom (1HN, 15N, 13Cα, 13Cß and 13C') resonance assignments of the C-terminal F-actin binding domain of αN-catenin.


Assuntos
Actinas/metabolismo , Ressonância Magnética Nuclear Biomolecular , alfa Catenina/química , alfa Catenina/metabolismo , Animais , Camundongos , Domínios Proteicos
14.
Nat Chem Biol ; 13(1): 62-68, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27820802

RESUMO

RAS GTPases are important mediators of oncogenesis in humans. However, pharmacological inhibition of RAS has proved challenging. Here we describe a functionally critical region, located outside the effector lobe of RAS, that can be targeted for inhibition. We developed NS1, a synthetic binding protein (monobody) that bound with high affinity to both GTP- and GDP-bound states of H-RAS and K-RAS but not N-RAS. NS1 potently inhibited growth factor signaling and oncogenic H-RAS- and K-RAS-mediated signaling and transformation but did not block oncogenic N-RAS, BRAF or MEK1. NS1 bound the α4-ß6-α5 region of RAS, which disrupted RAS dimerization and nanoclustering and led to blocking of CRAF-BRAF heterodimerization and activation. These results establish the importance of the α4-ß6-α5 interface in RAS-mediated signaling and define a previously unrecognized site in RAS for inhibiting RAS function.


Assuntos
Sítio Alostérico/efeitos dos fármacos , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Proteínas ras/antagonistas & inibidores , Proteínas ras/química , Animais , Anticorpos Monoclonais/química , Células COS , Células Cultivadas , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Proteínas ras/metabolismo
15.
Cell Calcium ; 63: 3-7, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-27914753

RESUMO

In 1986, J.W. Putney presented a model for capacitative calcium (Ca2+) entry conveying that depletion of endoplasmic reticulum stored Ca2+ levels leads to activation of plasma membrane Ca2+ channels which mediate influx of Ca2+ from the extracellular space into cells. Presently, the biomolecules regulating this process, more widely known as store operated Ca2+ entry (SOCE) which is vital to myriad signaling pathways in health and disease, are known and the focus of intense structural biology research aiming to illuminate the atomic mechanisms of function. This brief review highlights the known structures with respect to the mechanisms of SOCE regulation and possible future directions in this field developed out of Putney's conceptualization of this process.


Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Animais , Humanos
16.
Adv Exp Med Biol ; 981: 215-251, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29594864

RESUMO

All eukaryotic cells have adapted the use of the calcium ion (Ca2+) as a universal signaling element through the evolution of a toolkit of Ca2+ sensor, buffer and effector proteins. Among these toolkit components, integral and peripheral proteins decorate biomembranes and coordinate the movement of Ca2+ between compartments, sense these concentration changes and elicit physiological signals. These changes in compartmentalized Ca2+ levels are not mutually exclusive as signals propagate between compartments. For example, agonist induced surface receptor stimulation can lead to transient increases in cytosolic Ca2+ sourced from endoplasmic reticulum (ER) stores; the decrease in ER luminal Ca2+ can subsequently signal the opening surface channels which permit the movement of Ca2+ from the extracellular space to the cytosol. Remarkably, the minuscule compartments of mitochondria can function as significant cytosolic Ca2+ sinks by taking up Ca2+ in a coordinated manner. In non-excitable cells, inositol 1,4,5 trisphosphate receptors (IP3Rs) on the ER respond to surface receptor stimulation; stromal interaction molecules (STIMs) sense the ER luminal Ca2+ depletion and activate surface Orai1 channels; surface Orai1 channels selectively permit the movement of Ca2+ from the extracellular space to the cytosol; uptake of Ca2+ into the matrix through the mitochondrial Ca2+ uniporter (MCU) further shapes the cytosolic Ca2+ levels. Recent structural elucidations of these key Ca2+ toolkit components have improved our understanding of how they function to orchestrate precise cytosolic Ca2+ levels for specific physiological responses. This chapter reviews the atomic-resolution structures of IP3R, STIM1, Orai1 and MCU elucidated by X-ray crystallography, electron microscopy and NMR and discusses the mechanisms underlying their biological functions in their respective compartments within the cell.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Mitocôndrias Musculares/metabolismo , Animais , Cálcio/química , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Citosol/química , Retículo Endoplasmático/química , Humanos , Receptores de Inositol 1,4,5-Trifosfato/química , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Mitocôndrias Musculares/química , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/química , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/química , Molécula 1 de Interação Estromal/metabolismo
17.
Nat Cell Biol ; 18(11): 1244-1252, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27694890

RESUMO

The polarity protein Scribble (SCRIB) regulates apical-basal polarity, directional migration and tumour suppression in Drosophila and mammals. Here we report that SCRIB is an important regulator of myeloid cell functions including bacterial infection and inflammation. SCRIB interacts directly with the NADPH oxidase (NOX) complex in a PSD95/Dlg/ZO-1 (PDZ)-domain-dependent manner and is required for NOX-induced reactive oxygen species (ROS) generation in culture and in vivo. On bacterial infection, SCRIB localized to phagosomes in a leucine-rich repeat-dependent manner and promoted ROS production within phagosomes to kill bacteria. Unexpectedly, SCRIB loss promoted M1 macrophage polarization and inflammation. Thus, SCRIB uncouples ROS-dependent bacterial killing activity from M1 polarization and inflammatory functions of macrophages. Modulating the SCRIB-NOX pathway can therefore identify ways to manage infection and inflammation with implications for chronic inflammatory diseases, sepsis and cancer.


Assuntos
Membrana Celular/metabolismo , Polaridade Celular/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Humanos , Inflamação/metabolismo , Camundongos , Células Mieloides/metabolismo , Fagossomos/metabolismo , Espécies Reativas de Oxigênio/metabolismo
18.
Plant Cell ; 28(7): 1738-51, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27335451

RESUMO

Ca(2+) signaling is critical to plant immunity; however, the channels involved are poorly characterized. Cyclic nucleotide-gated channels (CNGCs) are nonspecific, Ca(2+)-permeable cation channels. Plant CNGCs are hypothesized to be negatively regulated by the Ca(2+) sensor calmodulin (CaM), and previous work has focused on a C-terminal CaM-binding domain (CaMBD) overlapping with the cyclic nucleotide binding domain of plant CNGCs. However, we show that the Arabidopsis thaliana isoform CNGC12 possesses multiple CaMBDs at cytosolic N and C termini, which is reminiscent of animal CNGCs and unlike any plant channel studied to date. Biophysical characterizations of these sites suggest that apoCaM interacts with a conserved isoleucine-glutamine (IQ) motif in the C terminus of the channel, while Ca(2+)/CaM binds additional N- and C-terminal motifs with different affinities. Expression of CNGC12 with a nonfunctional N-terminal CaMBD constitutively induced programmed cell death, providing in planta evidence of allosteric CNGC regulation by CaM. Furthermore, we determined that CaM binding to the IQ motif was required for channel function, indicating that CaM can both positively and negatively regulate CNGC12. These data indicate a complex mode of plant CNGC regulation by CaM, in contrast to the previously proposed competitive ligand model, and suggest exciting parallels between plant and animal channels.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Calmodulina/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sítios de Ligação , Cálcio/metabolismo , Calmodulina/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Imunidade Vegetal/genética , Imunidade Vegetal/fisiologia , Ligação Proteica/genética , Ligação Proteica/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
19.
J Biol Chem ; 291(30): 15641-52, 2016 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-27226556

RESUMO

RAS-like protein expressed in many tissues 1 (RIT1) is a disease-associated RAS subfamily small guanosine triphosphatase (GTPase). Recent studies revealed that germ-line and somatic RIT1 mutations can cause Noonan syndrome (NS), and drive proliferation of lung adenocarcinomas, respectively, akin to RAS mutations in these diseases. However, the locations of these RIT1 mutations differ significantly from those found in RAS, and do not affect the three mutational "hot spots" of RAS. Moreover, few studies have characterized the GTPase cycle of RIT1 and its disease-associated mutants. Here we developed a real-time NMR-based GTPase assay for RIT1 and investigated the effect of disease-associated mutations on GTPase cycle. RIT1 exhibits an intrinsic GTP hydrolysis rate similar to that of H-RAS, but its intrinsic nucleotide exchange rate is ∼4-fold faster, likely as a result of divergent residues near the nucleotide binding site. All of the disease-associated mutations investigated increased the GTP-loaded, activated state of RIT1 in vitro, but they could be classified into two groups with different intrinsic GTPase properties. The S35T, A57G, and Y89H mutants exhibited more rapid nucleotide exchange, whereas F82V and T83P impaired GTP hydrolysis. A RAS-binding domain pulldown assay indicated that RIT1 A57G and Y89H were highly activated in HEK293T cells, whereas T83P and F82V exhibited more modest activation. All five mutations are associated with NS, whereas two (A57G and F82V) have also been identified in urinary tract cancers and myeloid malignancies. Characterization of the effects on the GTPase cycle of RIT1 disease-associated mutations should enable better understanding of their role in disease processes.


Assuntos
Adenocarcinoma , Neoplasias Pulmonares , Mutação de Sentido Incorreto , Proteínas de Neoplasias , Síndrome de Noonan , Neoplasias Urológicas , Proteínas ras , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma de Pulmão , Substituição de Aminoácidos , Linhagem Celular , Guanosina Trifosfato/química , Humanos , Hidrólise , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Síndrome de Noonan/genética , Síndrome de Noonan/metabolismo , Domínios Proteicos , Neoplasias Urológicas/genética , Neoplasias Urológicas/metabolismo , Proteínas ras/química , Proteínas ras/genética , Proteínas ras/metabolismo
20.
Mol Biol Cell ; 27(4): 669-85, 2016 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-26700321

RESUMO

Syntaxin-1 is the central SNARE protein for neuronal exocytosis. It interacts with Munc18-1 through its cytoplasmic domains, including the N-terminal peptide (N-peptide). Here we examine the role of the N-peptide binding in two conformational states ("closed" vs. "open") of syntaxin-1 using PC12 cells and Caenorhabditis elegans. We show that expression of "closed" syntaxin-1A carrying N-terminal single point mutations (D3R, L8A) that perturb interaction with the hydrophobic pocket of Munc18-1 rescues impaired secretion in syntaxin-1-depleted PC12 cells and the lethality and lethargy of unc-64 (C. elegans orthologue of syntaxin-1)-null mutants. Conversely, expression of the "open" syntaxin-1A harboring the same mutations fails to rescue the impairments. Biochemically, the L8A mutation alone slightly weakens the binding between "closed" syntaxin-1A and Munc18-1, whereas the same mutation in the "open" syntaxin-1A disrupts it. Our results reveal a striking interplay between the syntaxin-1 N-peptide and the conformational state of the protein. We propose that the N-peptide plays a critical role in intracellular trafficking of syntaxin-1, which is dependent on the conformational state of this protein. Surprisingly, however, the N-peptide binding mode seems dispensable for SNARE-mediated exocytosis per se, as long as the protein is trafficked to the plasma membrane.


Assuntos
Proteínas de Caenorhabditis elegans/química , Exocitose , Proteínas Munc18/metabolismo , Neurônios/fisiologia , Sintaxina 1/química , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Técnicas de Silenciamento de Genes , Dados de Sequência Molecular , Neurônios/metabolismo , Células PC12 , Peptídeos/química , Peptídeos/metabolismo , Mutação Puntual , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Ratos , Sintaxina 1/genética , Sintaxina 1/metabolismo
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