RESUMEN
The role of ADAM-8 in cancer and inflammatory diseases such as allergy, arthritis and asthma makes it an attractive target for drug development. Therefore, the catalytic domain of human ADAM-8 was expressed, purified and crystallized in complex with a hydroxamic acid inhibitor, batimastat. The crystal structure of the enzyme-inhibitor complex was refined to 2.1 Å resolution. ADAM-8 has an overall fold similar to those of other ADAM members, including a central five-stranded ß-sheet and a catalytic Zn(2+) ion. However, unique differences within the S1' binding loop of ADAM-8 are observed which might be exploited to confer specificity and selectivity to ADAM-8 competitive inhibitors for the treatment of diseases involving this enzyme.
Asunto(s)
Proteínas ADAM/química , Dominio Catalítico , Proteínas de la Membrana/química , Fenilalanina/análogos & derivados , Inhibidores de Proteasas/química , Tiofenos/química , Proteínas ADAM/metabolismo , Humanos , Ligandos , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Fenilalanina/química , Fenilalanina/metabolismo , Inhibidores de Proteasas/metabolismo , Unión Proteica , Desplegamiento Proteico , Tiofenos/metabolismoRESUMEN
The Janus kinase (JAK) family consists of four members: JAK-1, -2, -3 and tyrosine kinase 2 (TYK-2). Recent work suggests that cytokine signaling through TYK-2 may play a critical role in a number of inflammatory processes. We recently described the purification and characterization of phosphorylated isoforms of the TYK-2 kinase domain (TYK-2 KD) and its high resolution 3D structure in the presence of inhibitors. We now report the expression and a two-step purification procedure for the doubly tagged full-length construct, H6-FL-TYK-2-FLAG, and examine its properties compared to those of TYK-2 KD. In the presence of ATP and a peptide substrate, H6-FL-TYK-2-FLAG showed a marked lag in phosphopeptide product formation, while TYK-2 KD showed no such lag. This lag could be eliminated by ATP pretreatment, suggesting that the H6-FL-TYK-2-FLAG enzyme was activated by phosphorylation. The potencies of several nanomolar inhibitors were similar for TYK-2 KD and H6-FL-TYK-2-FLAG. However, these same inhibitors were about 1000 times less potent inhibiting the autophosphorylation of H6-FL-TYK-2-FLAG than they were inhibiting the phosphorylation of a peptide substrate modeled after the activation loop sequence of TYK-2. This intriguing result suggests that autophosphorylation and, thus, activation of H6-FL-TYK-2-FLAG is relatively insensitive to inhibition and that present inhibitors act to inhibit TYK-2 subsequent to activation. Inhibition of TYK-2 autophosphorylation may represent a new area of investigation for the JAK family.
Asunto(s)
TYK2 Quinasa/antagonistas & inhibidores , TYK2 Quinasa/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Dominio Catalítico , Electroforesis en Gel de Poliacrilamida , Histidina/química , Humanos , Cinética , Oligopéptidos/química , Fosforilación , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , TYK2 Quinasa/química , TYK2 Quinasa/aislamiento & purificaciónRESUMEN
Autotaxin (ATX, also known as ectonucleotide pyrophosphatase/phosphodiesterase-2, ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX-LPA signaling is involved in various pathologies including tumor progression and inflammation. However, the molecular basis of substrate recognition and catalysis by ATX and the mechanism by which it interacts with target cells are unclear. Here, we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We have identified a hydrophobic lipid-binding pocket and mapped key residues for catalysis and selection between nucleotide and phospholipid substrates. We have shown that ATX interacts with cell-surface integrins through its N-terminal somatomedin B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling and suggest new approaches for targeting ATX with small-molecule therapeutic agents.
Asunto(s)
Integrinas/metabolismo , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/metabolismo , Pirofosfatasas/química , Pirofosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Dominio Catalítico , Línea Celular , Cristalografía por Rayos X , Humanos , Lisofosfolípidos/metabolismo , Datos de Secuencia Molecular , Mutación , Hidrolasas Diéster Fosfóricas/genética , Unión Proteica , Estructura Terciaria de Proteína , Pirofosfatasas/genética , Ratas , Especificidad por SustratoRESUMEN
Rat autotaxin has been cloned, expressed, purified to homogeneity and crystallized via hanging-drop vapour diffusion using PEG 3350 as precipitant and ammonium iodide and sodium thiocyanate as salts. The crystals diffracted to a maximum resolution of 2.05 A and belonged to space group P1, with unit-cell parameters a=53.8, b=63.3, c=70.5 A, alpha=98.8, beta=106.2, gamma=99.8 degrees. Preliminary X-ray diffraction analysis indicated the presence of one molecule per asymmetric unit, with a solvent content of 47%.
Asunto(s)
Hidrolasas Diéster Fosfóricas/química , Pirofosfatasas/química , Animales , Cristalización , Cristalografía por Rayos X , RatasRESUMEN
A disintegrin and metalloprotease-8 (ADAM8) is thought to play a role in cancer and inflammatory diseases such as allergy, arthritis, and asthma. Despite the implication of ADAM8 in these diseases, the functional role of ADAM8 catalytic activity remains unclear. In this report, we demonstrate that an early critical autolytic event, we have termed pre-processing, is accelerated at acidic pH (pH 5.5) while autolytic activation is abrogated under the same conditions. Likewise, we found that pre-processing is hindered and autolytic activation is facilitated in neutral pH conditions, and thus demonstrates a pH-dependent shift in substrate selectivity. This finding is further supported by two peptide substrates corresponding to the pre-processing and C-terminal scissile bonds that were preferentially cleaved at acidic and neutral pH, respectively. Lastly, we found fibronectin cleavage to be attenuated at pH 5.5, while two novel substrates, brevican, and vitronectin, were readily cleaved in neutral or acidic conditions.
Asunto(s)
Proteínas ADAM/metabolismo , Proteínas de la Membrana/metabolismo , Proteoglicanos/metabolismo , Proteínas ADAM/química , Proteínas ADAM/genética , Proteínas ADAM/aislamiento & purificación , Biocatálisis , Activación Enzimática , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Cinética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Péptidos/metabolismo , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
Members of the ADAM (a disintegrin and metalloproteinase) family of proteins possess a multidomain architecture which permits functionalities as adhesion molecules, signalling intermediates and proteolytic enzymes. ADAM8 is found on immune cells and is induced by multiple pro-inflammatory stimuli suggesting a role in inflammation. Here we describe an activation mechanism for recombinant human ADAM8 that is independent from classical PC (pro-protein convertase)-mediated activation. N-terminal sequencing revealed that, unlike other ADAMs, ADAM8 undergoes pre-processing at Glu(158), which fractures the Pro (pro-segment)-domain before terminal activation takes place to remove the putative cysteine switch (Cys(167)). ADAM8 lacking the DIS (disintegrin) and/or CR (cysteine-rich) and EGF (epidermal growth factor) domains displayed impaired ability to complete this event. Thus pre-processing of the Pro-domain is co-ordinated by DIS and CR/EGF domains. Furthermore, by placing an EK (enterokinase) recognition motif between the Pro- and catalytic domains of multiple constructs, we were able to artificially remove the pro-segment prior to pre-processing. In the absence of pre-processing of the Pro-domain a marked decrease in specific activity was observed with the autoactivated enzyme, suggesting that the Pro-domain continued to associate and inhibit active enzyme. Thus, pre-processing of the Pro-domain of human ADAM8 is important for enzyme maturation by preventing re-association of the pro-segment with the catalytic domain. Given the observed necessity of DIS and CR/EGF for pre-processing, we conclude that these domains are crucial for the proper activation and maturation of human ADAM8.
Asunto(s)
Proteínas ADAM/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas ADAM/química , Proteínas ADAM/genética , Proteínas ADAM/aislamiento & purificación , Catálisis , Dominio Catalítico , Activación Enzimática , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ProteínaRESUMEN
One member of the progenipoietin (ProGP) family of engineered proteins, ProGP-2, is a chimaeric dual cytokine receptor agonist, expressed in mammalian cells, that stimulates both human fetal liver tyrosine kinase-3 (Flt3) and the granulocyte-colony-stimulating-factor (G-CSF) receptor. The production of ProGP-2 on a small and large scale using either anti-(Flt3 ligand) antibody-affinity chromatography, or a combination of (NH4)2SO4 fractionation, anion-exchange chromatography, hydrophobic-interaction chromatography and preparative reverse-phase chromatography is described. ProGP-2 was produced in hollow-fibre reactors containing stably transfected NS0 cells. The productivity of ProGP-2 was initially high, but was found to decrease 3-4-fold over time. When the yield of ProGP-2 decreased, the combination of three conventional chromatography steps was required to meet protein purity similar to that achieved by the anti-(Flt3 ligand) chromatography method. In addition, a protease activity was observed in conditioned media from the hollow-fibre reactors that resulted in increased degradation of ProGP-2 that was removed by hydrophobic-interaction chromatography at higher pH. Together the results demonstrated a method for production and purification of ProGP-2 for additional studies on its haematopoietic activity.