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 beta-protein (Abeta). 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 Abeta. 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 Abeta-(1-42), Abeta-(1-40), and Abeta 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 A 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 Abeta by hPreP may potentially be of importance in the pathology of Alzheimer disease.
"By virtue of their distinctive subcellular localizations and pH optima, AβDPs constitute powerful tools for manipulating different pools of Aβ and, thereby, gaining fresh insight into their potential involvement in the pathogenesis of AD (Leissring and Turner, 2013). AβDPs are present in a diverse range of subcellular compartments: insulin-degrading enzyme (IDE) in cytosol (Roth, 2004), IDE and presequence peptidase in mitochondria (Leissring et al., 2004; Falkevall et al., 2006), BACE-2 and endothelin-converting enzymes 1-and -2 in endosomes (Eckman et al., 2001; Abdul- Hay et al., 2012), and in cathepsins B and D in lysosomes (Gan et al., 2004; Leissring et al., 2009) (see Saido and Leissring, 2012; Leissring and Turner, 2013 for comprehensive reviews). Collectively, these AβDPs represent a diverse set of experimental tools for selectively manipulating different pools of Aβ. "
"Thus, all these data support the significance of ABAD to Aβ induced neuronal stress. Several enzymes, such as presequence protease (PreP), a metalloprotease containing an inverted zinc binding motif, were identified to degrade Aβ [83, 84]. Intramitochondrial localization studies demonstrated that PreP is localized within the mitochondrial matrix. "
[Show abstract][Hide abstract] ABSTRACT: Mitochondria are dynamic ATP-generating organelle which contribute to many cellular functions including bioenergetics processes, intracellular calcium regulation, alteration of reduction-oxidation potential of cells, free radical scavenging, and activation of caspase mediated cell death. Mitochondrial functions can be negatively affected by amyloid β peptide (Aβ), an important component in Alzheimer's disease (AD) pathogenesis, and Aβ can interact with mitochondria and cause mitochondrial dysfunction. One of the most accepted hypotheses for AD onset implicates that mitochondrial dysfunction and oxidative stress are one of the primary events in the insurgence of the pathology. Here, we examine structural and functional mitochondrial changes in presence of Aβ. In particular we review data concerning Aβ import into mitochondrion and its involvement in mitochondrial oxidative stress, bioenergetics, biogenesis, trafficking, mitochondrial permeability transition pore (mPTP) formation, and mitochondrial protein interaction. Moreover, the development of AD therapy targeting mitochondria is also discussed.
Oxidative medicine and cellular longevity 08/2014; 2014:780179. DOI:10.1155/2014/780179 · 3.36 Impact Factor
"Considering that hPreP can degrade mitochondria-localized Aβ peptides , this protease is clearly an important regulator of Aβ concentration within the mitochondria and therefore perturbation of hPreP activity can potentially influence Aβ accumulation . In collaboration with Yan laboratory, we have analyzed the activity of hPreP in mitochondrial matrix fractions isolated from the brain temporal lobe (an area of brain highly susceptible to Aβ accumulation) of AD patients and age-matched controls using three different substrates (Aβ1–40, Aβ1–42 and the F 1 β presequence). "
[Show abstract][Hide abstract] ABSTRACT: Mitochondrial dysfunctions associated with amyloid-β peptide (Aβ) accumulation in mitochondria have been observed in Alzheimer´s disease (AD) patients’ brains and in AD mice models. Aβ is produced by sequential action of β- and γ-secretases cleaving the amyloid precursor protein (APP). The γ-secretase complex was found in mitochondria-associated endoplasmic reticulum membranes (MAM) suggesting that this could be a potential site of Aβ production, from which Aβ is further transported into mitochondria. In vitro, Aβ was shown to be imported into mitochondria through the translocase of the outer membrane (TOM) complex. The mitochondrial presequence protease (PreP) is responsible for Aβ degradation reducing toxic effects of Aβ on mitochondrial functions. The proteolytic activity of PreP is, however, lower in AD brain temporal lobe mitochondria and in AD transgenic mice models, possibly due to an increased reactive oxygen species (ROS) production. Here, we review the intracellular mechanisms of Aβ production, its mitochondrial import and the intra-mitochondrial degradation. We also discuss the implications of a reduced efficiency of mitochondrial Aβ clearance for AD. Understanding the underlying mechanisms may provide new insights into mitochondria related pathogenesis of AD and development of drug therapy against AD. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.
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