RESUMO
Dysregulated activity of A Disintegrin And Metalloproteinase 17 (ADAM17)/TNFα Converting Enzyme (TACE) is associated with inflammatory disorders and cancer progression by releasing regulatory membrane-tethered proteins like TNFα, IL6R and EGFR ligands. Although specific inhibition of TACE is thought to be a viable strategy for inflammatory disorders and for malignancies treatment, the generation of effective inhibitors in vivo has been proven to be challenging. Here we report on the development of a protein inhibitor that leverages the endogenous modulator of TACE. We have generated a stable form of the auto-inhibitory TACE prodomain (TPD), which specifically inhibits in vitro and cell-surface TACE, but not the related ADAM10, and effectively modulated TNFα secretion in cells. TPD significantly attenuated TACE-mediated disease models of sepsis, rheumatoid arthritis (RA) and inflammatory bowel disease (IBD), and reduced TNFα in synovial fluids from RA patients. Our results demonstrate that intervening with endogenous ADAM sheddase modulatory mechanisms holds potential as a general strategy for the design of ADAM inhibitors.
Assuntos
Proteína ADAM17/química , Artrite/tratamento farmacológico , Colite/tratamento farmacológico , Inibidores Enzimáticos/administração & dosagem , Choque Séptico/tratamento farmacológico , Proteína ADAM10/metabolismo , Proteína ADAM17/antagonistas & inibidores , Animais , Artrite/induzido quimicamente , Artrite/metabolismo , Células Cultivadas , Colite/induzido quimicamente , Colite/metabolismo , Colágeno/efeitos adversos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Lipopolissacarídeos/efeitos adversos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Domínios Proteicos , Choque Séptico/induzido quimicamente , Choque Séptico/metabolismo , Ácido Trinitrobenzenossulfônico/efeitos adversosRESUMO
Phospholemman (FXYD1) is a single-transmembrane protein regulator of Na,K-ATPase, expressed strongly in heart, skeletal muscle, and brain and phosphorylated by protein kinases A and C at Ser-68 and Ser-63, respectively. Binding of FXYD1 reduces Na,K-ATPase activity, and phosphorylation at Ser-68 or Ser-63 relieves the inhibition. Despite the accumulated information on physiological effects, whole cell studies provide only limited information on molecular mechanisms. As a complementary approach, we utilized purified human Na,K-ATPase (α1ß1 and α2ß1) reconstituted with FXYD1 or mutants S63E, S68E, and S63E,S68E that mimic phosphorylation at Ser-63 and Ser-68. Compared with control α1ß1, FXYD1 reduces Vmax and turnover rate and raises K0.5Na. The phosphomimetic mutants reverse these effects and reduce K0.5Na below control K0.5Na. Effects on α2ß1 are similar but smaller. Experiments in proteoliposomes reconstituted with α1ß1 show analogous effects of FXYD1 on K0.5Na, which are abolished by phosphomimetic mutants and also by increasing mole fractions of DOPS in the proteoliposomes. Stopped-flow experiments using the dye RH421 show that FXYD1 slows the conformational transition E2(2K)ATP â E1(3Na)ATP but does not affect 3NaE1P â E2P3Na. This regulatory effect is explained simply by molecular modeling, which indicates that a cytoplasmic helix (residues 60-70) docks between the αN and αP domains in the E2 conformation, but docking is weaker in E1 (also for phosphomimetic mutants). Taken together with previous work showing that FXYD1 also raises binding affinity for the Na(+)-selective site III, these results provide a rather comprehensive picture of the regulatory mechanism of FXYD1 that complements the physiological studies.