The amyloid ␤-peptide (A␤) has been suggested to exert its toxicity intracellularly. Mitochondrial functions can be negatively affected by A␤ and accumulation of A␤ has been detected in mitochondria. Because A␤ is not likely to be produced locally in mitochondria, we decided to investigate the mechanisms for mitochondrial A␤ uptake. Our results from rat mitochondria show that A␤ is transported into mitochondria via the translocase of the outer membrane (TOM) machinery. The import was insensitive to valinomycin, indicating that it is independent of the mitochondrial membrane potential. Subfractionation studies following the import experiments revealed A␤ association with the inner membrane fraction, and immunoelectron microscopy after import showed localization of A␤ to mitochondrial cristae. A similar distribution pattern of A␤ in mitochondria was shown by immunoelectron microscopy in human cortical brain biopsies obtained from living subjects with normal pressure hydrocephalus. Thus, we present a unique import mechanism for A␤ in mitochondria and demonstrate both in vitro and in vivo that A␤ is located to the mitochondrial cristae. Importantly, we also show that extracellulary applied A␤ can be internalized by human neuroblastoma cells and can colocalize with mitochondrial markers. Together, these results provide further insight into the mitochondrial uptake of A␤, a peptide considered to be of major significance in Alzheimer's disease.Alzheimer disease ͉ protein import ͉ human brain biopsies T he amyloid-␤ peptide (A␤) is produced by regulated intramembrane proteolysis of the A␤ precursor protein (APP) by the sequential cleavage by ␤-and ␥-secretases (1-2). Plaques consisting mainly of aggregated A␤ are detected in the neuropil in aged subjects and in particular in subjects with Alzheimer's disease (AD) (3-5). Recently, it has been argued that it is A␤ oligomers and fibrils that cause toxicity, loss of synapses, and ultimately neuronal death (6-9). The exact mechanisms of how A␤ damages the neurons are still unknown; however, several lines of evidence implicate that A␤ exerts its toxicity intracellularly (10, 11) and point toward a role of mitochondria in this process (12). It has been reported that mitochondrial A␤ accumulation impairs neuronal function and, thus, contributes to cellular dysfunction in a transgenic APP mouse model (13). It is noteworthy that in AD at an early stage there is already a reduction in the number of mitochondria (14), the brain glucose metabolism is decreased (15), and the activities of both tricarboxylic acid cycle enzymes (16) and cytochrome c oxidase (COX) are reduced (17)(18)(19)(20). In vitro studies with isolated mitochondria suggest that A␤ 1-42 inhibits COX activity in a copper-dependent manner (21). Furthermore, mitochondrial A␤-binding alcohol dehydrogenase (ABAD) has been found to be up-regulated in neurons from AD patients (22), and A␤ has been shown to interact with ABAD, resulting in free radical production and neuronal apoptosis. Recently, we have shown that preseque...

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 ␤-protein (A␤). PreP belongs to the pitrilysin oligopeptidase family M16C containing an inverted zincbinding 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 A␤. We have cloned, overexpressed, and purified recombinant hPreP and its mutant with catalytic base Glu 78 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 A␤-(1-42), A␤-(1-40), and A␤ 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 Cys 90 and Cys 527 that form disulfide bridges under oxidized conditions and might be involved in redox regulation of the enzyme. Degradation of the mitochondrial A␤ by hPreP may potentially be of importance in the pathology of Alzheimer disease.Several human disorders are associated with the deposition of aggregated peptides. One of them is Alzheimer disease (AD) 4 in which the polymerization of amyloid ␤-protein (A␤) into insoluble fibrils in brain seems to be a pathological event. An extracellular accumulation of A␤ has been the main focus of molecular studies associated with AD (1). However, increasing attention is directed toward intracellular events including the mitochondrial role in AD (2). There are many links between mitochondrial dysfunctions and AD (3, 4). Impairment of mitochondrial energy metabolism and altered cytochrome c oxidase activity are among the earliest detectable defects in AD (5, 6). It has been shown that Alzheimer amyloid precursor protein (APP) 695 is not only targeted to the plasma membrane but also to mitochondria (5). Accumulation of APP in the outer mitochondrial membrane caused dysfunctions and impaired energy metabolism. The active ␥-secretase complex including presenilin, nicastrin, APH-1, and PEN-2, which cleave APP to generate A␤, has been shown to be present in the mitochondrial outer membrane (7). Furthermore, the occurrence of A␤ in mitochondria of AD patients and its direct binding to ABAD (A␤-binding alcohol dehydrogenase, also called ERAB) induces apoptosis and free radical generation in neurons (8). A recent study demonstrated that A␤ is present in the mitochondrial matrix in A...

Intracellular amyloid-β peptide (Aβ) has been implicated in the pathogenesis of Alzheimer's disease (AD). Mitochondria were found to be the target both for amyloid precursor protein (APP) that accumulates in the mitochondrial import channels and for Aβ that interacts with several proteins inside mitochondria and leads to mitochondrial dysfunction. Here, we have studied the role of mitochondrial γ-secretase in processing different substrates. We found that a significant proportion of APP is associated with mitochondria in cultured cells and that γ-secretase cleaves the shedded C-terminal part of APP identified as C83 associated with the outer membrane of mitochondria (OMM). Moreover, we have established the topology of the C83 in the OMM and found the APP intracellular domain (AICD) to be located inside mitochondria. Our data show for the first time that APP is a substrate for the mitochondrial γ-secretase and that AICD is produced inside mitochondria. Thus, we provide a mechanistic view of the mitochondria-associated APP metabolism where AICD, P3 peptide and potentially Aβ are produced locally and may contribute to mitochondrial dysfunction in AD.

FK506 binding protein of 51 kDa (FKBP51) is a heat shock protein 90 (Hsp90) co-chaperone involved in the regulation of steroid hormone receptors activity. It is known for its role in various regulatory pathways implicated in mood and stress-related disorders, cancer, obesity, Alzheimer’s disease and corticosteroid resistant asthma. It consists of two FKBP12 like active peptidyl prolyl isomerase (PPIase) domains (an active FK1 and inactive FK2 domain) and one tetratricopeptide repeat (TPR) domain that mediates interaction with Hsp90 via its C-terminal MEEVD peptide. Here, we report a combined x-ray crystallography and molecular dynamics study to reveal the binding mechanism of Hsp90 MEEVD peptide to the TPR domain of FKBP51. The results demonstrated that the Hsp90 C-terminal peptide binds to the TPR domain of FKBP51 with the help of di-carboxylate clamp involving Lys272, Glu273, Lys352, Asn322, and Lys329 which are conserved throughout several di-carboxylate clamp TPR proteins. Interestingly, the results from molecular dynamics study are also in agreement to the complex structure where all the contacts between these two partners were consistent throughout the simulation period. In a nutshell, our findings provide new opportunity to engage this important protein-protein interaction target by small molecules designed by structure based drug design strategy.