Therese Eneqvist

Degradation of the amyloid beta-protein by the novel mitochondrial peptidasome, PreP.

Recently we have identified the novel mitochondrial peptidase responsible for degrading presequences and other short unstructured peptides in mitochondria, the presequence peptidase, which we named PreP peptidasome. In the present study we have identified and characterized the human PreP homologue, hPreP, in brain mitochondria, and we show its capacity to degrade the amyloid β-protein (Aβ). PreP belongs to the pitrilysin oligopeptidase family M16C containing an inverted zinc-binding motif. We show that hPreP is localized to the mitochondrial matrix. In situ immuno-inactivation studies in human brain mitochondria using anti-hPreP antibodies showed complete inhibition of proteolytic activity against Aβ. We have cloned, overexpressed, and purified recombinant hPreP and its mutant with catalytic base Glu78 in the inverted zinc-binding motif replaced by Gln. In vitro studies using recombinant hPreP and liquid chromatography nanospray tandem mass spectrometry revealed novel cleavage specificities against Aβ-(1-42), Aβ-(1-40), and Aβ Arctic, a protein that causes increased protofibril formation an early onset familial variant of Alzheimer disease. In contrast to insulin degrading enzyme, which is a functional analogue of hPreP, hPreP does not degrade insulin but does degrade insulin B-chain. Molecular modeling of hPreP based on the crystal structure at 2.1 Å resolution of AtPreP allowed us to identify Cys90 and Cys527 that form disulfide bridges under oxidized conditions and might be involved in redox regulation of the enzyme. Degradation of the mitochondrial Aβ by hPreP may potentially be of importance in the pathology of Alzheimer disease.

High resolution crystal structures of piscine transthyretin reveal different binding modes for triiodothyronine and thyroxine.

Transthyretin (TTR) is an extracellular transport protein involved in the distribution of thyroid hormones and vitamin A. So far, TTR has only been found in vertebrates, of which piscine TTR displays the lowest sequence identity with human TTR (47%). Human and piscine TTR bind both thyroid hormones 3,5,3′-triiodo-l-thyronine (T3) and 3,5,3′,5′-tetraiodo-l-thyronine (thyroxine, T4). Human TTR has higher affinity for T4 than T3, whereas the reverse holds for piscine TTR. X-ray structures of Sparus aurata (sea bream) TTR have been determined as the apo-protein at 1.75 Å resolution and bound to ligands T3 and T4, both at 1.9 Å resolution. The apo structure is similar to human TTR with structural changes only at β-strand D. This strand forms an extended loop conformation similar to the one in chicken TTR. The piscine TTR·T4 complex shows the T4-binding site to be similar but not identical to human TTR, whereas the TTR·T3 complex shows the I3′ halogen situated at the site normally occupied by the hydroxyl group of T4. The significantly wider entrance of the hormone-binding channel in sea bream TTR, in combination with its narrower cavity, provides a structural explanation for the different binding affinities of human and piscine TTR to T3 and T4.

The beta-slip: a novel concept in transthyretin amyloidosis.

Transthyretin is a tetrameric plasma protein associated with two forms of amyloid disease. The structure of the highly amyloidogenic transthyretin triple mutant TTRG53S/E54D/L55S determined at 2.3 A resolution reveals a novel conformation: the beta-slip. A three-residue shift in beta strand D places Leu-58 at the position normally occupied by Leu-55 now mutated to serine. The beta-slip is best defined in two of the four monomers, where it makes new protein-protein interactions to an area normally involved in complex formation with retinol-binding protein. This interaction creates unique packing arrangements, where two protein helices combine to form a double helix in agreement with fiber diffraction and electron microscopy data. Based on these findings, a novel model for transthyretin amyloid formation is presented.

Structural distribution of mutations associated with familial amyloidotic polyneuropathy in human transthyretin.

The human plasma protein transthyretin (TTR) is a highly stable soluble homotetrameric protein. Still, confor-mational changes in the wild type protein can lead to self-assembly into insoluble amyloid fibrils. In addition, 74 point mutations are known to enhance amyloid formation causing familial amyloidotic polyneuropathy (FAP). Alignment of TTR sequences from twenty different species shows that only six of these mutations occur as natural amino acids in other organisms. In this paper we analyse the distribution of FAP mutations within the three-dimensional structure of TTR. Contradictoty to what might be expected from protein stability studies, the mutations are not restricted to structurally rigid parts of the molecule, nor are they concentrated at the monomer interaction sites.

Proteolytic mechanism of a novel mitochondrial and chloroplastic PreP peptidasome.

Abstract The 2.1-Å-resolution crystal structure of the novel mitochondrial and chloroplastic metalloendopeptidase, AtPreP1, revealed a unique peptidasome structure, in which the two halves of the enzyme completely enfold a huge proteolytic cavity. Based on the structure, we proposed a novel mechanism for proteolysis involving hinge-bending motions, which cause the protease to open and close in response to substrate binding. We generated four double-mutants of AtPreP1 by introducing cysteines at positions where disulfide bonds can be formed in order to lock and unlock the protease and tested the activity under oxidizing and reducing conditions. The overall results support the proposed mechanism.