Binding of divalent cations is essential for the activity of the organellar peptidasome in Arabidopsis thaliana, AtPreP

AbstractThe 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 Mg2+. 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.Structured summaryMINT-7231937, MINT-7232017, MINT-7232035, MINT-7232051, MINT-7232070, MINT-7232090:AtPreP1 (uniprotkb:Q9LJL3) enzymaticly reacts (MI:0414) pF1 beta (uniprotkb:P17614) by protease assay (MI:0435)MINT-7232132:AtPreP1 (uniprotkb:Q9LJL3) enzymaticly reacts (MI:0414) galanin (uniprotkb:P22466) by protease assay (MI:0435)MINT-7232175:AtPreP1 (uniprotkb:Q9LJL3) enzymaticly reacts (MI:0414) Cecropin A (uniprotkb:P14954) by protease assay (MI:0435)MINT-7232163:AtPreP1 (uniprotkb:Q9LJL3) enzymaticly reacts (MI:0414) hPrPss (uniprotkb:P04156) by protease assay (MI:0435

The transthyretin-related protein family

A number of proteins related to the homotetrameric transport protein transthyretin (TTR) forms a highly conserved protein family, which we present in an integrated analysis of data from different sources combined with an initial biochemical characterization. Homologues of the transthyretinrelated protein (TRP) can be found in a wide range of species including bacteria, plants and animals, whereas transthyretins have so far only been identified in vertebrates. A multiple sequence alignment of 49 TRP sequences from 47 species to TTR suggests that the tertiary and quaternary features of the three-dimensional structure are most likely preserved. Interestingly, while some of the TRP orthologues show as little as 30% identity, the residues at the putative ligandbinding site are almost entirely conserved. RT/PCR analysis in Caenorhabditis elegans confirms that one TRP gene is transcribed, spliced and predominantly expressed in the worm, which suggests that at least one of the two C. elegans TRP genes encodes a functional protein. We used doublestranded RNA-mediated interference techniques in order to determine the loss-of-function phenotype for the two TRP genes in C. elegans but detected no apparent phenotype. The cloning and initial characterization of purified TRP from Escherichia coli reveals that, while still forming a homotetramer, this protein does not recognize thyroid hormones that are the natural ligands of TTR. The ligand for TRP is not known; however, genomic data support a functional role involving purine catabolism especially linked to urate oxidase (uricase) activity

The closed structure of presequence protease PreP forms a unique 10 000 Å(3) chamber for proteolysis

Presequence protease PreP is a novel protease that degrades targeting peptides as well as other unstructured peptides in both mitochondria and chloroplasts. The first structure of PreP from Arabidopsis thaliana refined at 2.1 Å resolution shows how the 995-residue polypeptide forms a unique proteolytic chamber of more than 10 000 Å(3) in which the active site resides. Although there is no visible opening to the chamber, a peptide is bound to the active site. The closed conformation places previously unidentified residues from the C-terminal domain at the active site, separated by almost 800 residues in sequence to active site residues located in the N-terminal domain. Based on the structure, a novel mechanism for proteolysis is proposed involving hinge-bending motions that cause the protease to open and close in response to substrate binding. In support of this model, cysteine double mutants designed to keep the chamber covalently locked show no activity under oxidizing conditions. The manner in which substrates are processed inside the chamber is reminiscent of the proteasome; therefore, we refer to this protein as a peptidasome