RESUMO
Pokeweed antiviral protein (PAP) isolated from Phytolacca americana is a ribosome-inactivating protein (RIP) that has RNA N-glycosidase (RNG) activity towards both eukaryotic and prokaryotic ribosomes. In contrast, karasurin-A (KRN), a RIP from Trichosanthes kirilowii var. japonica, is active only on eukaryotic ribosomes. Stepwise selection of chimera proteins between PAP and KRN indicated that the C-terminal region of PAP (residues 209-225) was critical for RNG activity toward prokaryotic ribosomes. When the region of PAP (residues 209-225) was replaced with the corresponding region of KRN the PAP chimera protein, like KRN, was active only on eukaryotic ribosomes. Furthermore, insertion of the region of PAP (residues 209-225) into the KRN chimera protein resulted not only in the detectable RNG activity toward prokaryotic ribosome, but also activity toward the eukaryotic ribosomes as well that was seven-fold higher than for the original KRN. In this study, the possibility of genetic manipulation of the activity and substrate specificity of RIPs is demonstrated.
Assuntos
Células Procarióticas/fisiologia , RNA , Proteínas Inativadoras de Ribossomos Tipo 1/genética , Proteínas Inativadoras de Ribossomos/fisiologia , Ribossomos/fisiologia , Sequência de Aminoácidos , Cristalografia por Raios X , Ativação Enzimática , Modelos Moleculares , Dados de Sequência Molecular , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/fisiologia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Conformação Proteica , RNA/química , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Inativadoras de Ribossomos/química , Proteínas Inativadoras de Ribossomos/genética , Proteínas Inativadoras de Ribossomos Tipo 1/química , Proteínas Inativadoras de Ribossomos Tipo 1/fisiologia , Alinhamento de Sequência , Relação Estrutura-Atividade , Especificidade por SubstratoAssuntos
Regulação da Expressão Gênica/fisiologia , Células do Corno Posterior/patologia , Receptores da Neurocinina-1/biossíntese , Proteínas Inativadoras de Ribossomos Tipo 1/toxicidade , Substância P/análogos & derivados , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunotoxinas/metabolismo , Imunotoxinas/toxicidade , Dor/induzido quimicamente , Dor/genética , Dor/metabolismo , Células do Corno Posterior/efeitos dos fármacos , Receptores da Neurocinina-1/genética , Receptores da Neurocinina-1/fisiologia , Proteínas Inativadoras de Ribossomos Tipo 1/fisiologia , Saporinas , Substância P/fisiologia , Substância P/toxicidadeRESUMO
Ribosome-inactivating proteins (RIPs, EC 3.2.2.22) are plant enzymes that can inhibit the translation process by removing single adenine residues of the large rRNA. These enzymes are known to function in defense against pathogens, but their biological role is unknown, partly due to the absence of work on RIPs in a model plant. In this study, we purified a protein showing RIP activity from Arabidopsis thaliana by employing chromatography separations coupled with an enzymatic activity. Based on N-terminal and internal amino acid sequencing, the RIP purified was identified as a mature form of pectin methylesterase (PME, At1g11580). The purified native protein showed both PME and RIP activity. PME catalyzes pectin deesterification, releasing acid pectin and methanol, which cause cell wall changes. We expressed the full-length and mature form of cDNA clones into an expression vector and transformed it in Escherichia coli for protein expression. The recombinant PME proteins (full-length and mature) expressed in E. coli did not show either PME or RIP activity, suggesting that post-translational modifications are important for these enzymatic activities. This study demonstrates a new function for an old enzyme identified in a model plant and discusses the possible role of a protein's conformational changes corresponding to its dual enzymatic activity.