Proteases that degrade the amyloid-␤ peptide (A␤) are important in protecting against Alzheimer's disease (AD), and understanding these proteases is critical to understanding AD pathology. Endopeptidases sensitive to inhibition by thiorphan and phosphoramidon are especially important, because these inhibitors induce dramatic A␤ accumulation (ϳ30-to 50-fold) and pathological deposition in rodents. The A␤-degrading enzyme neprilysin (NEP) is the best known target of these inhibitors. However, genetic ablation of NEP results in only modest increases (ϳ1.5-to 2-fold) in A␤, indicating that other thiorphan/phosphoramidon-sensitive endopeptidases are at work. Of particular interest is the NEP homolog neprilysin 2 (NEP2), which is thiorphan/ phosphoramidon-sensitive and degrades A␤. We investigated the role of NEP2 in A␤ degradation in vivo through the use of gene knockout and transgenic mice. Mice deficient for the NEP2 gene showed significant elevations in total A␤ species in the hippocampus and brainstem/diencephalon (ϳ1.5-fold). Increases in A␤ accumulation were more dramatic in NEP2 knockout mice crossbred with APP transgenic mice. In NEP/NEP2 double-knockout mice, A␤ levels were marginally increased (ϳ1.5-to 2-fold), compared with NEP Alzheimer's disease (AD) is a neurodegenerative disorder currently affecting more than 26 million people worldwide and, as advances in modern medicine prolong lifespan, this number is expected to quadruple by 2050. 1 A major factor believed to be involved in the progression of AD pathology is the accumulation of amyloid-␤ peptide (A␤). Studying the mechanisms of A␤ clearance is, therefore, very important to understanding AD.Currently, enzymatic degradation is thought to play an integral role in the removal of A␤. Of the A␤-degrading enzymes, neprilysin (NEP) has been shown to be highly critical for cerebral A␤ control.2 NEP expression has also been inversely correlated with amyloid pathology in humans and mice, and NEP gene transfer has been reported to reduce amyloid pathology in transgenic mice (reviewed by Marr and Spencer 3 ). Despite the importance of NEP-mediated A␤ degradation, NEP knockout (KO) mice show only moderately elevated A␤ levels (1.5-to 2-fold), insufficient to cause plaque deposition. 4 However, when treated with thiorphan, an NEP endopeptidase inhibitor, mice and rats demonstrate pathological accumulations of A␤ after only 1 month. 2,5 This was also found in mice treated with phosphoramidon, another NEP inhibitor. 6 These results indicate that there may exist additional NEPlike endopeptidases in the metalloprotease 13 (M13) family that are central to the A␤ clearance pathway.The NEP homolog neprilysin 2 (NEP2) is one such endopeptidase. NEP2 (also known as MMEL1/2, SEP, NL1, NE-PLP) possesses a 55% sequence identity to NEP and has been shown to degrade vasoactive peptides. 7,8 In addition, the membrane-bound ␣-splice form of murine NEP2 has demonstrated A␤-degrading properties in membrane fractions. 9 In transduced HEK293T cells, our research group previously sho...

In Parkinson's disease (PD), misfolded and aggregated α-synuclein protein accumulates in degenerating midbrain dopaminergic neurons. The amino acid alanine-76 in α-synuclein and phosphorylation at serine-87 and serine-129 are thought to regulate its aggregation and toxicity. However, their exact contributions to α-synuclein membrane association are less clear. We found that α-synuclein is indeed phosphorylated in fission yeast and budding yeast, the two models that we employed for assessing α-synuclein aggregation and membrane association properties, respectively. Surprisingly, blocking serine phosphorylation (S87A, S129A, and S87A/S129A) or mimicking it (S87D, S129D) altered α-synuclein aggregation in fission yeast. Either blocking or mimicking this phosphorylation increased endomembrane association in fission yeast, but only mimicking it decreased plasma membrane association in budding yeast. Polar substitution mutations of alanine-76 (A76E and A76R) decreased α-synuclein membrane association in budding yeast and decreased aggregation in fission yeast. These yeast studies extend our understanding of serine phosphorylation and alanine-76 contributions to α-synuclein aggregation and are the first to detail their impact on α-synuclein's plasma membrane and endomembrane association.

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In Parkinson's disease (PD), midbrain dopaminergic neuronal death is linked to the accumulation of aggregated α-synuclein. The familial PD mutant form of α-synuclein, E46K, has not been thoroughly evaluated yet in an organismal model system. Here, we report that E46K resembled wild-type (WT) α-synuclein in Saccharomyces cerevisiae in that it predominantly localized to the plasma membrane, and it did not induce significant toxicity or accumulation. In contrast, in Schizosaccharomyces pombe, E46K did not associate with the plasma membrane. Instead, in one strain, it extensively aggregated in the cytoplasm and was as toxic as WT. Remarkably, in another strain, E46K extensively associated with the endomembrane system and was more toxic than WT. Our studies recapitulate and extend aggregation and phospholipid membrane association properties of E46K previously observed in vitro and cell culture. Furthermore, it supports the notion that E46K generates toxicity partly due to increased association with endomembrane systems within cells.

contains several prion elements, which are epigenetically transmitted as self-perpetuating protein conformations. One such prion is [ ], whose protein determinant is Swi1, a subunit of the SWI/SNF chromatin-remodeling complex. We previously reported that [ ] formation results in a partial loss-of-function phenotype of poor growth in nonglucose medium and abolishment of multicellular features. We also showed that the first 38 amino acids of Swi1 propagated []. We show here that a region as small as the first 32 amino acids of Swi1 (Swi1) can decorate [] aggregation and stably maintain and transmit [] independently of full-length Swi1. Regions smaller than Swi1 are either incapable of aggregation or unstably propagate []. When fused to Sup35MC, the [ ] determinant lacking its PrD, Swi1 and Swi1 can act as transferable ionomains (PrDs). The resulting fusions give rise to a novel chimeric prion, [], exhibiting []-like nonsense suppression. Thus, an NH-terminal region of ∼30 amino acids of Swi1 contains all the necessary information for prion formation, maintenance, and transmission. This PrD is unique in size and composition: glutamine free, asparagine rich, and the smallest defined to date. Our findings broaden our understanding of what features allow a protein region to serve as a PrD.