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1.
J Cell Sci ; 135(20)2022 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-36239192

RESUMO

Focal adhesion kinase (FAK; also known as PTK2) was discovered three decades ago and is now recognised as a key player in the regulation of cell-matrix adhesion and mesenchymal cell migration. Although it is essential during development, FAK also drives invasive cancer progression and metastasis. On a structural level, the basic building blocks of FAK have been described for some time. However, a picture of how FAK integrates into larger assemblies in various cellular environments, including one of its main cellular locations, the focal adhesion (FA) complex, is only beginning to emerge. Nano-resolution data from cellular studies, as well as atomic structures from reconstituted systems, have provided first insights, but also point to challenges that remain for obtaining a full structural understanding of how FAK is integrated in the FA complex and the structural changes occurring at different stages of FA maturation. In this Review, we discuss the known structural features of FAK, the interactions with its partners within the FA environment on the cell membrane and propose how its initial assembly in nascent FAs might change during FA maturation under force.


Assuntos
Adesões Focais , Adesão Celular , Movimento Celular , Quinase 1 de Adesão Focal/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/metabolismo , Fosforilação
2.
FASEB J ; 35(8): e21815, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34314064

RESUMO

The Src homology 2 containing inositol 5-phosphatase 2 (SHIP2) is a large multidomain enzyme that catalyzes the dephosphorylation of the phospholipid phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3 ) to form PI(3,4)P2 . PI(3,4,5)P3 is a key lipid second messenger controlling the recruitment of signaling proteins to the plasma membrane, thereby regulating a plethora of cellular events, including proliferation, growth, apoptosis, and cytoskeletal rearrangements. SHIP2, alongside PI3K and PTEN, regulates PI(3,4,5)P3 levels at the plasma membrane and has been heavily implicated in serious diseases such as cancer and type 2 diabetes; however, many aspects of its regulation mechanism remain elusive. We recently reported an activating effect of the SHIP2 C2 domain and here we describe an additional layer of regulation via the pleckstrin homology-related (PHR) domain. We show a phosphoinositide-induced transition to a high activity state of the enzyme that increases phosphatase activity up to 10-15 fold. We further show that PI(3,4)P2 directly interacts with the PHR domain to trigger this allosteric activation. Modeling of the PHR-phosphatase-C2 region of SHIP2 on the membrane suggests no major inter-domain interactions with the PHR domain, but close contacts between the two linkers offer a possible path of allosteric communication. Together, our data show that the PHR domain acts as an allosteric module regulating the catalytic activity of SHIP2 in response to specific phosphoinositide levels in the cell membrane.


Assuntos
Membrana Celular/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Domínios de Homologia à Plecstrina , Regulação Alostérica , Biocatálise , Humanos , Modelos Moleculares , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/química , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo
3.
Elife ; 62017 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-28792888

RESUMO

SH2-containing-inositol-5-phosphatases (SHIPs) dephosphorylate the 5-phosphate of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) and play important roles in regulating the PI3K/Akt pathway in physiology and disease. Aiming to uncover interdomain regulatory mechanisms in SHIP2, we determined crystal structures containing the 5-phosphatase and a proximal region adopting a C2 fold. This reveals an extensive interface between the two domains, which results in significant structural changes in the phosphatase domain. Both the phosphatase and C2 domains bind phosphatidylserine lipids, which likely helps to position the active site towards its substrate. Although located distant to the active site, the C2 domain greatly enhances catalytic turnover. Employing molecular dynamics, mutagenesis and cell biology, we identify two distinct allosteric signaling pathways, emanating from hydrophobic or polar interdomain interactions, differentially affecting lipid chain or headgroup moieties of PI(3,4,5)P3. Together, this study reveals details of multilayered C2-mediated effects important for SHIP2 activity and points towards interesting new possibilities for therapeutic interventions.


Assuntos
Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/química , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Análise Mutacional de DNA , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Fosfatidilserinas/metabolismo , Ligação Proteica , Conformação Proteica , Domínios Proteicos
4.
Protein J ; 35(3): 225-30, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27170292

RESUMO

The Src homology 2 containing inositol 5-phosphatase 2 (SHIP2) catalyses the dephosphorylation of the phospholipid phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3) to form PI(3,4)P2. PI(3,4,5)P3 is a key lipid second messenger, which can recruit signalling proteins to the plasma membrane and subsequently initiate numerous downstream signalling pathways responsible for the regulation of a plethora of cellular events such as proliferation, growth, apoptosis and cytoskeletal rearrangements. SHIP2 has been heavily implicated with several serious diseases such as cancer and type 2 diabetes but its regulation remains poorly understood. In order to gain insight into the mechanisms of SHIP2 regulation, a fragment of human SHIP2 containing the phosphatase domain and a region proposed to resemble a C2 domain was crystallized. Currently, no structural information is available on the putative C2-related domain or its relative position with respect to the phosphatase domain. Initial crystals were polycrystalline, but were optimized to obtain diffraction data to a resolution of 2.1 Å. Diffraction data analysis revealed a P212121 space group with unit cell parameters a = 136.04 Å, b = 175.84 Å, c = 176.89 Å. The Matthews coefficient is 2.54 Å(3) Da(-1) corresponding to 8 molecules in the asymmetric unit with a solvent content of 51.7 %.


Assuntos
Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/química , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/isolamento & purificação , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Domínios Proteicos , Transdução de Sinais
5.
Biophys J ; 108(3): 698-705, 2015 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-25650936

RESUMO

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that regulates cell signaling, proliferation, migration, and development. A major mechanism of regulation of FAK activity is an intramolecular autoinhibitory interaction between two of its domains--the catalytic and FERM domains. Upon cell adhesion to the extracellular matrix, FAK is being translocated toward focal adhesion sites and activated. Interactions of FAK with phosphoinositide phosphatidylinsositol-4,5-bis-phosphate (PIP2) are required to activate FAK. However, the molecular mechanism of the activation remains poorly understood. Recent fluorescence resonance energy transfer experiments revealed a closure of the FERM-kinase interface upon ATP binding, which is reversed upon additional binding of PIP2. Here, we addressed the allosteric regulation of FAK by performing all-atom molecular-dynamics simulations of a FAK fragment containing the catalytic and FERM domains, and comparing the dynamics in the absence or presence of ATP and PIP2. As a major conformational change, we observe a closing and opening motion upon ATP and additional PIP2 binding, respectively, in good agreement with the fluorescence resonance energy transfer experiments. To reveal how the binding of the regulatory PIP2 to the FERM F2 lobe is transduced to the very distant F1/N-lobe interface, we employed force distribution analysis. We identified a network of mainly charged residue-residue interactions spanning from the PIP2 binding site to the distant interface between the kinase and FERM domains, comprising candidate residues for mutagenesis to validate the predicted mechanism of FAK activation.


Assuntos
Trifosfato de Adenosina/farmacologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Fosfatos de Inositol/farmacologia , Regulação Alostérica/efeitos dos fármacos , Animais , Proteína-Tirosina Quinases de Adesão Focal/química , Simulação de Dinâmica Molecular , Análise de Componente Principal , Conformação Proteica , Termodinâmica
6.
Cancer Lett ; 348(1-2): 88-99, 2014 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-24657306

RESUMO

FAK and FGFR2 signaling pathways play important roles in cancer development, progression and tumor angiogenesis. PHM16 is a novel ATP competitive inhibitor of FAK and FGFR2. To evaluate the therapeutic efficacy of this agent, we examined its anti-angiogenic effect in HUVEC and its anti-tumor effect in different cancer cell lines. We showed PHM16 inhibited endothelial cell viability, adherence and tube formation along with the added ability to induce endothelial cell apoptosis. This compound significantly delayed tumor cell growth. Together, these data showed that inhibition of both FAK and FGFR2 signaling pathways can enhance anti-tumor and anti-angiogenic activities.


Assuntos
Inibidores da Angiogênese/farmacologia , Quinase 1 de Adesão Focal/antagonistas & inibidores , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Neoplasias/enzimologia , Neovascularização Fisiológica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Inibidores da Angiogênese/metabolismo , Apoptose/efeitos dos fármacos , Sítios de Ligação , Adesão Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática , Quinase 1 de Adesão Focal/metabolismo , Células HCT116 , Células Endoteliais da Veia Umbilical Humana/enzimologia , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Modelos Moleculares , Neoplasias/patologia , Fosforilação , Inibidores de Proteínas Quinases/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo
7.
Bioorg Med Chem Lett ; 23(16): 4552-6, 2013 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-23845217

RESUMO

We report herein the synthesis of novel diarylamino-1,3,5-triazine derivatives as FAK (focal adhesion kinase) inhibitors and the evaluation of their anti-angiogenic activity on HUVEC cells. Generally, the effects of these compounds on endothelial cells could be correlated with their kinase inhibitory activity. The most efficient compounds displayed inhibition of viability against HUVEC cells in the micromolar range, as observed with TAE-226, which was designed by Novartis Pharma AG. X-ray crystallographic analysis of the co-crystal structure for compound 34 revealed that the mode of interaction with the FAK kinase domain is highly similar to that observed in the complex of TAE-226.


Assuntos
Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Triazinas/síntese química , Inibidores da Angiogênese/química , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Células Endoteliais/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Células Endoteliais da Veia Umbilical Humana , Humanos , Estrutura Molecular , Morfolinas/química , Morfolinas/farmacologia , Triazinas/química , Triazinas/farmacologia
8.
Proc Natl Acad Sci U S A ; 108(35): 14649-53, 2011 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-21873231

RESUMO

Anticipatory ligand binding through massive protein sequence variation is rare in biological systems, having been observed only in the vertebrate adaptive immune response and in a phage diversity-generating retroelement (DGR). Earlier work has demonstrated that the prototypical DGR variable protein, major tropism determinant (Mtd), meets the demands of anticipatory ligand binding by novel means through the C-type lectin (CLec) fold. However, because of the low sequence identity among DGR variable proteins, it has remained unclear whether the CLec fold is a general solution for DGRs. We have addressed this problem by determining the structure of a second DGR variable protein, TvpA, from the pathogenic oral spirochete Treponema denticola. Despite its weak sequence identity to Mtd (∼16%), TvpA was found to also have a CLec fold, with predicted variable residues exposed in a ligand-binding site. However, this site in TvpA was markedly more variable than the one in Mtd, reflecting the unprecedented approximate 10(20) potential variability of TvpA. In addition, similarity between TvpA and Mtd with formylglycine-generating enzymes was detected. These results provide strong evidence for the conservation of the formylglycine-generating enzyme-type CLec fold among DGRs as a means of accommodating massive sequence variation.


Assuntos
Lectinas Tipo C/química , Dobramento de Proteína , Retroelementos , Treponema/genética , Imunidade Adaptativa , Proteínas de Bactérias/química
9.
PLoS Biol ; 6(6): e131, 2008 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-18532877

RESUMO

Diversity-generating retroelements (DGRs) recognize novel ligands through massive protein sequence variation, a property shared uniquely with the adaptive immune response. Little is known about how recognition is achieved by DGR variable proteins. Here, we present the structure of the Bordetella bacteriophage DGR variable protein major tropism determinant (Mtd) bound to the receptor pertactin, revealing remarkable adaptability in the static binding sites of Mtd. Despite large dissimilarities in ligand binding mode, principles underlying selective recognition were strikingly conserved between Mtd and immunoreceptors. Central to this was the differential amplification of binding strengths by avidity (i.e., multivalency), which not only relaxed the demand for optimal complementarity between Mtd and pertactin but also enhanced distinctions among binding events to provide selectivity. A quantitatively similar balance between complementarity and avidity was observed for Bordetella bacteriophage DGR as occurs in the immune system, suggesting that variable repertoires operate under a narrow set of conditions to recognize novel ligands.


Assuntos
Variação Genética , Retroelementos/genética , Proteínas Virais/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Bacteriófagos , Sítios de Ligação , Bordetella/virologia , Ligantes , Ligação Proteica , Proteínas Virais/metabolismo , Fatores de Virulência de Bordetella/metabolismo
10.
Biochemistry ; 47(11): 3484-92, 2008 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-18293939

RESUMO

Canavan disease is a fatal neurological disorder caused by the malfunctioning of a single metabolic enzyme, aspartoacylase, that catalyzes the deacetylation of N-acetyl-L-aspartate to produce L-aspartate and acetate. The structure of human brain aspartoacylase has been determined in complex with a stable tetrahedral intermediate analogue, N-phosphonomethyl-L-aspartate. This potent inhibitor forms multiple interactions between each of its heteroatoms and the substrate binding groups arrayed within the active site. The binding of the catalytic intermediate analogue induces the conformational ordering of several substrate binding groups, thereby setting up the active site for catalysis. The highly ordered binding of this inhibitor has allowed assignments to be made for substrate binding groups and provides strong support for a carboxypeptidase-type mechanism for the hydrolysis of the amide bond of the substrate, N-acetyl- l-aspartate.


Assuntos
Amidoidrolases/química , Ácido Aspártico/análogos & derivados , Encéfalo/enzimologia , Compostos Organofosforados/metabolismo , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/genética , Amidoidrolases/metabolismo , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Sítios de Ligação/genética , Carboxipeptidases/química , Carboxipeptidases/metabolismo , Catálise , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Glicosilação , Humanos , Mutagênese Sítio-Dirigida , Compostos Organofosforados/química , Ligação Proteica/genética
11.
J Biol Chem ; 281(41): 31031-40, 2006 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-16895909

RESUMO

Aspartate-beta-semialdehyde dehydrogenase (ASADH) catalyzes a critical branch point transformation in amino acid bio-synthesis. The products of the aspartate pathway are essential in microorganisms, and this entire pathway is absent in mammals, making this enzyme an attractive target for antibiotic development. The first structure of an ASADH from a Gram-positive bacterium, Streptococcus pneumoniae, has now been determined. The overall structure of the apoenzyme has a similar fold to those of the Gram-negative and archaeal ASADHs but contains some interesting structural variations that can be exploited for inhibitor design. Binding of the coenzyme NADP, as well as a truncated nucleotide analogue, into an alternative conformation from that observed in Gram-negative ASADHs causes an enzyme domain closure that precedes catalysis. The covalent acyl-enzyme intermediate was trapped by soaking the substrate into crystals of the coenzyme complex, and the structure of this elusive intermediate provides detailed insights into the catalytic mechanism.


Assuntos
Archaea/enzimologia , Aspartato-Semialdeído Desidrogenase/química , Bactérias Gram-Positivas/enzimologia , Streptococcus pneumoniae/enzimologia , Calorimetria , Catálise , Domínio Catalítico , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Termodinâmica
12.
Biochemistry ; 45(18): 5878-84, 2006 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-16669630

RESUMO

Aspartoacylase catalyzes the deacetylation of N-acetylaspartic acid (NAA) to produce acetate and L-aspartate and is the only brain enzyme that has been shown to effectively metabolize NAA. Although the exact role of this enzymatic reaction has not yet been completely elucidated, the metabolism of NAA appears to be necessary in the formation of myelin lipids, and defects in this enzyme lead to Canavan disease, a fatal neurological disorder. The low catalytic activity and inherent instability observed with the Escherichia coli-expressed form of aspartoacylase suggested the need for a suitable eukaryotic expression system that would be capable of producing a fully functional, mature enzyme. Human aspartoacylase has now been successfully expressed in Pichia pastoris. While the expression yields are lower than in E. coli, the purified enzyme is significantly more stable. This enzyme form has the same substrate specificity but is 150-fold more active than the E. coli-expressed enzyme. The molecular weight of the purified enzyme, measured by mass spectrometry, is higher than predicted, suggesting the presence of some post-translational modifications. Deglycosylation of aspartoacylase or mutation at the glycosylation site causes decreased enzyme stability and diminished catalytic activity. A carbohydrate component has been removed and characterized by mass spectrometry. In addition to this carbohydrate moiety, the enzyme has also been shown to contain one zinc atom per subunit. Chelation studies to remove the zinc result in a reversible loss of catalytic activity, thus establishing aspartoacylase as a zinc metalloenzyme.


Assuntos
Amidoidrolases/metabolismo , Doença de Canavan/enzimologia , Amidoidrolases/genética , Clonagem Molecular , Estabilidade Enzimática , Glicosilação , Humanos , Pichia/genética , Reação em Cadeia da Polimerase , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
13.
Arch Biochem Biophys ; 413(1): 1-8, 2003 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-12706335

RESUMO

Aspartoacylase catalyzes the deacetylation of N-acetylaspartic acid (NAA) in the brain to produce acetate and L-aspartate. An aspartoacylase deficiency, with concomitant accumulation of NAA, is responsible for Canavan disease, a lethal autosomal recessive disorder. To examine the mechanism of this enzyme the genes encoding murine and human aspartoacylase were cloned and expressed in Escherichia coli. A significant portion of the enzyme is expressed as soluble protein, with the remainder found as inclusion bodies. A convenient enzyme-coupled continuous spectrophotometric assay has been developed for measuring aspartoacylase activity. Kinetic parameters were determined with the human enzyme for NAA and for selected N-acyl analogs that demonstrate relaxed substrate specificity with regard to the nature of the acyl group. The clinically relevant E285A mutant reveals an altered enzyme with poor stability and barely detectable activity, while a more conservative E285D substitution leads to only fivefold lower activity than native aspartoacylase.


Assuntos
Amidoidrolases/isolamento & purificação , Ácido Aspártico/análogos & derivados , Encéfalo/enzimologia , Amidoidrolases/genética , Amidoidrolases/metabolismo , Substituição de Aminoácidos , Animais , Ácido Aspártico/metabolismo , Doença de Canavan/enzimologia , Doença de Canavan/genética , Cobalto/farmacologia , Humanos , Técnicas In Vitro , Cinética , Camundongos , Mutagênese Sítio-Dirigida , Mutação Puntual , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Zinco/farmacologia
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