Assuntos
Deficiência de Prolidase/tratamento farmacológico , Prolina/uso terapêutico , Úlcera Cutânea/tratamento farmacológico , Administração Tópica , Adulto , Albuminúria/diagnóstico , Feminino , Humanos , Deficiência de Prolidase/patologia , Prolina/administração & dosagem , Úlcera Cutânea/patologia , Resultado do TratamentoRESUMO
Prolidase is a metallopeptidase that cleaves iminodipeptides containing a proline (Pro) or hydroxyproline (Hyp) residue at their C-terminal end. The disease prolidase deficiency (PD) is a rare recessive human disorder characterized by reduced prolidase activity. PD manifests itself by a wide range of severe clinical symptoms, most commonly as skin ulceration, recurrent infections of the respiratory tract, and mental retardation. Several mutations in the PEPD gene have been identified that are responsible for the loss or the reduction of prolidase activity. In contrast, the structural basis of enzyme inactivation has so far remained elusive. In this study, we present high resolution crystal structures of a number of human prolidase (HsProl) variants, in which single amino acids are either substituted by others or deleted. The observed implications of the mutations on the three-dimensional structure of HsProl are reported and discussed and related to their enzymatic activity. The resulting structures may be divided into four groups depending on the presumed effect of the corresponding mutations on the reaction mechanism. The four possible inactivation mechanisms, which could be elucidated, are disruption of the catalytic Mn2 (OH- )-center, introduction of chain disorder along with the displacement of important active site residues, rigidification of the active site, and flexibilization of the active site. DATABASE: All refined structure coordinates as well as the corresponding structure factor amplitudes have been deposited in the PDB under the accession numbers 5MBY, 5MBZ, 5MC0, 5MC1, 5MC2, 5MC3, 5MC4, 5MC5, 6H2P, 6H2Q.
Assuntos
Dipeptidases/química , Proteínas Mutantes/química , Mutação , Deficiência de Prolidase/patologia , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Dipeptidases/genética , Dipeptidases/metabolismo , Humanos , Hidroxiprolina/metabolismo , Manganês/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Deficiência de Prolidase/enzimologia , Prolina/metabolismo , Ligação Proteica , Conformação Proteica , Especificidade por SubstratoAssuntos
Aberrações Cromossômicas , Análise Mutacional de DNA , Dipeptidases/genética , Dermatoses da Perna/genética , Deficiência de Prolidase/genética , Úlcera Cutânea/genética , Adolescente , Adulto , Criança , Cromossomos Humanos Par 19/genética , Terapia Combinada , Dipeptídeos/urina , Fácies , Feminino , Seguimentos , Humanos , Dermatoses da Perna/diagnóstico , Dermatoses da Perna/patologia , Dermatoses da Perna/terapia , Cooperação do Paciente , Deficiência de Prolidase/diagnóstico , Deficiência de Prolidase/patologia , Deficiência de Prolidase/terapia , Recidiva , Análise de Sequência de DNA , Pele/patologia , Úlcera Cutânea/diagnóstico , Úlcera Cutânea/patologia , Úlcera Cutânea/terapia , Adulto JovemRESUMO
Prolidase is the only human enzyme responsible for the digestion of iminodipeptides containing proline or hydroxyproline at their C-terminal end, being a key player in extracellular matrix remodeling. Prolidase deficiency (PD) is an intractable loss of function disease, characterized by mutations in the prolidase gene. The exact causes of activity impairment in mutant prolidase are still unknown. We generated three recombinant prolidase forms, hRecProl-231delY, hRecProl-E412K and hRecProl-G448R, reproducing three mutations identified in homozygous PD patients. The enzymes showed very low catalytic efficiency, thermal instability and changes in protein conformation. No variation of Mn(II) cofactor affinity was detected for hRecProl-E412K; a compromised ability to bind the cofactor was found in hRecProl-231delY and Mn(II) was totally absent in hRecProl-G448R. Furthermore, local structure perturbations for all three mutants were predicted by in silico analysis. Our biochemical investigation of the three causative alleles identified in perturbed folding/instability, and in consequent partial prolidase degradation, the main reasons for enzyme inactivity. Based on the above considerations we were able to rescue part of the prolidase activity in patients' fibroblasts through the induction of Heath Shock Proteins expression, hinting at new promising avenues for PD treatment.