RESUMO
A novel peptidasome PreP is responsible for degradation of targeting peptides and other unstructured peptides in mitochondria and chloroplasts. Arabidopsis thaliana contains two PreP isoforms, AtPreP1, and AtPreP2. Here we have characterized single and double prep knockout mutants. Immunoblot analysis of atprep1 and atprep2 mutants showed that both isoforms are expressed in all tissues with the highest expression in flowers and siliques; additionally, AtPreP1 accumulated to a much higher level in comparison to AtPreP2. The atprep2 mutant behaved like wild type, whereas deletion of AtPreP1 resulted in slightly pale-green seedlings. Analysis of the atprep1 atprep2 double mutant revealed a chlorotic phenotype in true leaves with diminished chlorophyll a and b content, but unchanged Chl a/b ratio indicating a proportional decrease of both PSI and PSII complexes. Mitochondrial respiratory rates (state 3) were lower and the mitochondria were partially uncoupled. EM pictures on cross sections of the first true leaves showed aberrant chloroplasts, including less grana stacking and less starch granules. Mitochondria with extremely variable size could also be observed. At later developmental stages the plants appeared almost normal. However, all through the development there was a statistically significant decrease of approximately 40% in the accumulated biomass in the double mutant plants in comparison to wild type. In mitochondria, deletion of AtPreP was not compensated by activation of any peptidolytic activity, whereas chloroplast membranes contained a minor peptidolytic activity not related to AtPreP. In summary, the AtPreP peptidasome is required for efficient plant growth and organelle function particularly during early development.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/genética , Arabidopsis/ultraestrutura , Clorofila/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Cloroplastos/ultraestrutura , Deleção de Genes , Microscopia Eletrônica de Transmissão , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/ultraestruturaRESUMO
Several studies suggest mitochondrial dysfunction as a possible mechanism underlying the development of Alzheimer disease (AD). There is data showing that amyloid-beta (A beta) peptide is present in AD brain mitochondria. The human presequence protease (hPreP) was recently shown to be the major mitochondrial A beta-degrading enzyme. We investigated if there is an increased susceptibility to AD, which can be attributed to genetic variation in the hPreP gene PITRM1 and if the proteolytic efficiency of recombinant hPreP variants is affected. When a total of 673 AD cases and 649 controls were genotyped for 18 single nucleotide polymorphisms (SNPs), no genetic association between any of the SNPs and the risk for AD was found. In contrast, functional analysis of four non-synonymous SNPs in hPreP revealed a decreased activity compared to wild type hPreP. Using A beta, the presequence of ATP synthase F(1)beta subunit and a fluorescent peptide as substrates, the lowest activity was observed for the hPreP(A525D) variant, corresponding to rs1224893, which displayed only 20-30% of wild type activity. Furthermore, the activity of all variants was restored by the addition of Mg(2+), suggesting an important role for this metal during proteolysis. In conclusion, our data suggest that genetic variation in the hPreP gene PITRM1 may potentially contribute to mitochondrial dysfunctions.
Assuntos
Doença de Alzheimer/genética , Predisposição Genética para Doença , Metaloendopeptidases/genética , Polimorfismo de Nucleotídeo Único , Idoso , Alelos , Peptídeos beta-Amiloides/metabolismo , Estudos de Casos e Controles , Feminino , Frequência do Gene , Estudos de Associação Genética , Humanos , Desequilíbrio de Ligação , Magnésio/metabolismo , Masculino , Metaloendopeptidases/metabolismo , Modelos Moleculares , Fragmentos de Peptídeos/metabolismoRESUMO
The dual-targeted mitochondrial and chloroplastic zinc metallooligopeptidase from Arabidopsis, AtPreP, functions as a peptidasome that degrades targeting peptides and other small unstructured peptides. In addition to Zn located in the catalytic site, AtPreP also contains two Mg-binding sites. We have investigated the role of Mg-binding using AtPreP variants, in which one or both sites were rendered unable to bind Mg(2+). Our results show that metal binding besides that of the active site is crucial for AtPreP proteolysis, particularly the inner site appears essential for normal proteolytic function. This is also supported by its evolutionary conservation among all plant species of PreP.