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1.
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.

2.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
Biochem Biophys Res Commun ; 460(1): 5-21, 2015 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-25998729

RESUMO

The calcium (Ca(2+)) ion is a universal signalling messenger which plays vital physiological roles in all eukaryotes. To decode highly regulated intracellular Ca(2+) signals, cells have evolved a number of sensor proteins that are ideally adapted to respond to a specific range of Ca(2+) levels. Among many such proteins, calmodulin (CaM) is a multi-functional cytoplasmic Ca(2+) sensor with a remarkable ability to interact with and regulate a plethora of structurally diverse target proteins. CaM achieves this 'multi-talented' functionality through two EF-hand domains, each with an independent capacity to bind targets, and an adaptable flexible linker. By contrast, stromal interaction molecule-1 and -2 (STIMs) have evolved for a specific role in endoplasmic reticulum (ER) Ca(2+) sensing using EF-hand machinery analogous to CaM; however, whereas CaM structurally adjusts to dissimilar binding partners, STIMs use the EF-hand machinery to self-regulate the stability of the Ca(2+) sensing domain. The molecular mechanisms underlying the Ca(2+)-dependent signal transduction by CaM and STIMs have revealed a remarkable repertoire of actions and underscore the flexibility of nature in molecular evolution and adaption to discrete Ca(2+) levels. Recent genomic sequencing efforts have uncovered a number of disease-associated mutations in both CaM and STIM1. This article aims to highlight the most recent key structural and functional findings in the CaM and STIM fields, and discusses how these two Ca(2+) sensor proteins execute their biological functions.


Assuntos
Arritmias Cardíacas/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Moléculas de Adesão Celular/metabolismo , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Arritmias Cardíacas/genética , Sinalização do Cálcio/fisiologia , Calmodulina/genética , Moléculas de Adesão Celular/genética , Retículo Endoplasmático/genética , Humanos , Proteínas de Membrana/genética , Modelos Biológicos , Proteínas de Neoplasias/genética , Molécula 1 de Interação Estromal , Molécula 2 de Interação Estromal
9.
Org Lett ; 15(2): 322-5, 2013 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-23268733

RESUMO

Siladenoserinols A-L were isolated from a tunicate as inhibitors of p53-Hdm2 interaction, a promising target for cancer chemotherapy. Their structures including the absolute configurations were elucidated to be new sulfonated serinol derivatives, each of which contains a 6,8-dioxabicyclo[3.2.1]octane unit and either glycerophosphocholine or glycerophosphoethanolamine moiety. They inhibited p53-Hdm2 interaction with IC(50) values of 2.0-55 µM. Among them, siladenoserinol A and B exhibited the strongest inhibition with an IC(50) value of 2.0 µM.


Assuntos
Propilenoglicóis/isolamento & purificação , Propilenoglicóis/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/efeitos dos fármacos , Proteína Supressora de Tumor p53/efeitos dos fármacos , Urocordados/química , Animais , Humanos , Estrutura Molecular , Propanolaminas , Propilenoglicóis/química , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo
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