RESUMO
Presenilin (PS) mutations are associated with early-onset Alzheimer's disease and PS proteins are involved with gamma-secretase cleavage of the amyloid precursor protein, APP. We have shown previously that alpha-, beta- and gamma-secretase cleavages of APP are conserved in Pichia pastoris. Here, we report co-expression of APP and PSI in P. pastoris and show by immunoelectron microscopy colocalization of these two proteins in expanded endoplasmic reticulum. Western blot analysis indicates a drastic reduction of both alpha- and beta-secretase products. A relative increase in beta-secretase product derived from immature APP is also observed, pointing to a beta-secretase activity of P. pastoris associated with the early secretory pathway.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Pichia/enzimologia , Pichia/genética , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/biossíntese , Precursor de Proteína beta-Amiloide/genética , Ácido Aspártico Endopeptidases/metabolismo , Western Blotting , Células Clonais/citologia , Células Clonais/enzimologia , Células Clonais/metabolismo , Endopeptidases , Retículo Endoplasmático/metabolismo , Glicosilação , Imuno-Histoquímica , Microscopia Eletrônica , Modelos Biológicos , Pichia/citologia , Pichia/ultraestrutura , Presenilina-1 , Processamento de Proteína Pós-Traducional , TransfecçãoRESUMO
betaA4 (Abeta) amyloid peptide, a major component of Alzheimer's disease (AD) plaques, is a proteolytic product of the amyloid precursor protein (APP). Endoproteases, termed beta- and gamma-secretase, release respectively the N- and C-termini of the peptide. APP default secretion involves cleavage within the betaA4 domain by alpha-secretase. To study the conservation of APP processing in lower eukaryotes, the yeast Pichia pastoris was transfected with human APP695 cDNA. In addition to the full-length integral transmembrane protein found in the cell lysate, soluble/secreted APP (sAPP) was detected in the culture medium. Most sAPP comprised the N-terminal moiety of betaA4 and corresponds to sAPPalpha, the product of alpha-secretase. The culture medium also contained minor secreted forms detected by a monoclonal antibody specific for sAPPbeta (the ectodomain released by beta-secretase cleavage). Analysis of the cell lysates with specific antibodies also detected membrane-associated C-terminal fragments corresponding to the products of alpha and beta cleavages. Moreover, immunoprecipitation of the culture medium with three antibodies directed at distinct epitopes of the betaA4 domain yielded a 4 kDa product with the same electrophoretic mobility as betaA4 synthetic peptide. These results suggest that the alpha-, beta-, and gamma-secretase cleavages are conserved in yeast and that P. pastoris may offer an alternative to mammalian cells to identify the proteases involved in the generation of AD betaA4 amyloid.
Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Endopeptidases/metabolismo , Pichia/genética , Pichia/metabolismo , Processamento de Proteína Pós-Traducional , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/imunologia , Ácido Aspártico Endopeptidases , Western Blotting , Clonagem Molecular , Endopeptidases/imunologia , Humanos , Hidrólise , Neuroblastoma , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Testes de Precipitina , Processamento de Proteína Pós-Traducional/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Células Tumorais CultivadasRESUMO
The action of serine (and cysteine) proteases on peptide esters proceeds, as a generalization, orders of magnitude faster than the corresponding enzymatic hydrolysis of peptide bonds or peptide amides. Esterolysis liberates an alcohol while generating a free carboxyl group on the peptide; the proton produced can be detected by the use of an appropriate indicator. The action of trypsin on benzyloxycarbonylalanylarginine methyl ester was used as a model for the development of a simple microtiter plate assay procedure that takes advantage of the speed of these reactions and the ease of detection afforded by the color change of the indicator. A family of ester substrates of the form benzyloxycarbonylalanyl-X-methyl ester, in which X is one of the 20 common amino acids, was synthesized to allow the determination of the primary specificity profiles of serine proteases. Using a 96-well microtiter plate the specificity profiles of four enzymes with all 20 substrates can be carried out in approximately 4 h per enzyme, including setting up and data processing. The primary substrate preferences of trypsin, chymotrypsin, thrombin, pancreatic elastase, alpha-lytic protease, subtilisin, and proteinase K were determined to demonstrate the method and were found to be in good general agreement with reported specificities established by more conventional means.
Assuntos
Esterases/metabolismo , Serina Endopeptidases/análise , Serina Endopeptidases/metabolismo , Quimotripsina/metabolismo , Endopeptidase K , Concentração de Íons de Hidrogênio , Indicadores e Reagentes , Cinética , Microquímica/métodos , Elastase Pancreática/metabolismo , Especificidade por Substrato , Subtilisinas/metabolismo , Trombina/metabolismo , Tripsina/metabolismoRESUMO
In the past, several point mutations have been introduced individually into the substrate-binding site of alpha-lytic protease (EC 3.4.21.12) and shown to affect its specificity in a predictable manner [Bone, R., Silen, J. L., & Agard, D. A. (1989) Nature 339, 191-195]. One of the resulting mutant enzymes (Met190Ala in the numbering system of Fujinaga et al.) [Fujinaga, M., Delbaere, L. T. J., Brayer, G. D., & James, M. N. G. (1985) J. Mol. Biol. 183, 479-502] cleaves at large hydrophobic residues. We chose this enzyme as the parent for a library of mutant proteases. The library was constructed by effecting combinatorial random substitution of up to four other residues (Gly191, Arg192, Met213, and Val218) thought likely to influence the primary specificity of the protease. Active enzymes in the library were screened with a range of synthetic substrates (encompassing 19 different amino acids in the P1 position) in order to evaluate their primary cleavage preferences. The amino acid sequences of active mutants revealed a strong preference for the replacement of Met213 with a His residue. This substitution also had the greatest observed effect on specificity, conferring a greatly increased and, in some cases, dominant ability to cleave at His residues in synthetic amide substrates. Mutant enzymes with greatly increased proteolytic activity were also found in the library.