RESUMO
The Menkes (ATP7A) P(1B)-type ATPase is a transmembrane copper-translocating protein. It contains six similar high-affinity metal-binding domains (MBDs) in the N-terminal cytoplasmic tail that are important for sensing intracellular copper and regulating ATPase function through the transfer of copper between domains. Molecular characterization of copper-binding and transfer is predominantly dependent on NMR structures derived from E. coli expression systems. A limitation of these models is the exclusion of post-translational modifications. We have previously shown that the third copper-binding domain, MBD3, uniquely contains two phosphorylated residues: Thr-327, which is phosphorylated only in the presence of elevated copper; and Ser-339, which is constitutively phosphorylated independent of copper levels. Here, using molecular dynamic simulations, we have incorporated these phosphorylated residues into a model based on the NMR structures of MBD3. Our data suggests that constitutively phosphorylated Ser-339, which is in a loop facing the copper-binding site, may facilitate the copper transfer process by exposing the CxxC copper-binding region of MBD3. Copper-induced phosphorylation of Thr327 is predicted to stabilize this change in conformation. This offers new molecular insights into how cell signaling (phosphorylation) can affect MBD structure and dynamics and how this may in turn affect copper-binding and thus copper-translocation functions of ATP7A.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Conformação Proteica , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte de Cátions/genética , Cobre/química , ATPases Transportadoras de Cobre , Estabilidade Enzimática , Síndrome dos Cabelos Torcidos/enzimologia , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Alinhamento de Sequência , Eletricidade EstáticaRESUMO
BACKGROUND: Proteases play a major role in inflammatory diseases of the gastrointestinal tract. Activatable probes are a major technological advance, enabling sensitive detection of active proteases in tissue samples. Our aim was to synthesize an activatable probe for cathepsin S and validate its use in a mouse model of colitis. METHODS: We designed and synthesized a new fluorescent activatable probe, NB200, for the detection of active cathepsin S. Colitis was induced in C57BL/6 mice by the administration of 3% dextran sulfate sodium (DSS). Homogenized mouse colons, with or without the addition of the specific cathepsin S inhibitor MV026031, were incubated with NB200 in a fluorescent plate reader. KEY RESULTS: NB200 selectively detected purified cathepsin S and not other common inflammatory proteases. Homogenates of colon from mice with DSS colitis induced a significant fluorescent increase when compared to control animals (control vs DSS: p < 0.05 at 200 min and p < 0.01 at 220-240 min), indicating cathepsin S activation. The cathepsin S inhibitor abolished this increase in fluorescence (DSS vs DSS + MV026031: p < 0.05 at 140 min, p < 0.01 at 180 min, p < 0.001 at 200-240 min), which confirms cathepsin S activation. Cathepsin S activity correlated with the disease activity index (Spearman r = 0.77, p = 0.017). CONCLUSIONS & INFERENCES: Our investigation has demonstrated the utility of activatable probes for detecting protease activity in intestinal inflammation. Panels of such probes may allow 'signature' protease profiles to be established for a range of inflammatory diseases and disorders.