SLURP1 is a secreted member of the LY6/PLAUR protein family. Mutations in the SLURP1 gene are the cause of Mal de Meleda (MDM), a rare autosomal recessive genetic disease, characterized by inflammatory palmoplantar keratoderma. In this study, we have analyzed the expression of SLURP1 in normal and MDM skin. SLURP1 was found to be a marker of late differentiation, predominantly expressed in the granular layer of skin, notably the acrosyringium. Moreover, SLURP1 was also identified in several biological fluids such as sweat, saliva, tears, and urine from normal volunteers. In palmoplantar sections from MDM patients, as well as in their sweat, mutant SLURP1, including the new variant R71H-SLURP1, was either absent or barely detectable. Transfected human embryonic kidney 293T cells expressed the MDM mutant SLURP1 containing the single amino-acid substitution G86R but did not tolerate the MDM mutation W15R located in the signal peptide. Thus, most MDM mutations in SLURP1 affect either the expression, integrity, or stability of the protein, suggesting that a simple immunologic test could be used as a rapid screening procedure.

CAG/CTG repeat expansions cause over 13 neurological diseases that remain without a cure. Because longer tracts cause more severe phenotypes, contracting them may provide a therapeutic avenue. No currently known agent can specifically generate contractions. Using a GFP-based chromosomal reporter that monitors expansions and contractions in the same cell population, here we find that inducing double-strand breaks within the repeat tract causes instability in both directions. In contrast, the CRISPR-Cas9 D10A nickase induces mainly contractions independently of single-strand break repair. Nickase-induced contractions depend on the DNA damage response kinase ATM, whereas ATR inhibition increases both expansions and contractions in a MSH2- and XPA-dependent manner. We propose that DNA gaps lead to contractions and that the type of DNA damage present within the repeat tract dictates the levels and the direction of CAG repeat instability. Our study paves the way towards deliberate induction of CAG/CTG repeat contractions in vivo.

γ-Secretase, an integral membrane protein complex, catalyzes the intramembrane cleavage of the β-amyloid precursor protein (APP) during the neuronal production of the amyloid β-peptide (Aβ). As such, the protease has emerged as a key target for developing agents to treat and prevent Alzheimer's disease. Existing biochemical studies conflict on the oligomeric assembly state of the protease complex, and its detailed structure is not known. Here, we report that purified active human γ-secretase in digitonin has a total molecular mass of ~230 kDa when measured by scanning transmission electron microscopy. This result supports a complex that is monomeric for each of the four component proteins. We further report the 3-dimensional structure of the γ-secretase complex at 12 Å resolution, as obtained by cryo-EM and single particle image reconstruction. The structure reveals several domains on the extracellular side, three solvent-accessible low-density cavities and a potential substrate-binding surface groove in the transmembrane region of the complex.

γ‐Secretase is an unconventional aspartyl protease that processes many type 1 membrane proteins within the lipid bilayer. Because its cleavage of amyloid‐β precursor protein generates the amyloid‐β protein (Aβ) of Alzheimer’s disease, partially inhibiting γ‐secretase is an attractive therapeutic strategy, but the structure of the protease remains poorly understood. We recently used electron microscopy and single particle image analysis on the purified enzyme to generate the first 3D reconstruction of γ‐secretase, but at low resolution (15 Å). The limited amount of purified γ‐secretase that can be produced using currently available cell lines and procedures has prevented the achievement of a high resolution crystal structure by X‐ray crystallography or 2D crystallization. We report here the generation and characterization of a new mammalian cell line (S‐20) that overexpresses strikingly high levels of all four γ‐secretase components (presenilin, nicastrin, Aph‐1 and Pen‐2). We then used these cells to develop a rapid protocol for the high‐grade purification of proteolytically active γ‐secretase. The cells and purification methods detailed here provide a key step towards crystallographic studies of this ubiquitous enzyme.

BackgroundMutations linked to early onset, familial forms of Alzheimer's disease (FAD) are found most frequently in PSEN1, the gene encoding presenilin-1 (PS1). Together with nicastrin (NCT), anterior pharynx-defective protein 1 (APH1), and presenilin enhancer 2 (PEN2), the catalytic subunit PS1 constitutes the core of the γ-secretase complex and contributes to the proteolysis of the amyloid precursor protein (APP) into amyloid-beta (Aβ) peptides. Although there is a growing consensus that FAD-linked PS1 mutations affect Aβ production by enhancing the Aβ1–42/Aβ1–40 ratio, it remains unclear whether and how they affect the generation of APP intracellular domain (AICD). Moreover, controversy exists as to how PS1 mutations exert their effects in different experimental systems, by either increasing Aβ1–42 production, decreasing Aβ1–40 production, or both. Because it could be explained by the heterogeneity in the composition of γ-secretase, we purified to homogeneity complexes made of human NCT, APH1aL, PEN2, and the pathogenic PS1 mutants L166P, ΔE9, or P436Q.Methodology/Principal FindingsWe took advantage of a mouse embryonic fibroblast cell line lacking PS1 and PS2 to generate different stable cell lines overexpressing human γ-secretase complexes with different FAD-linked PS1 mutations. A multi-step affinity purification procedure was used to isolate semi-purified or highly purified γ-secretase complexes. The functional characterization of these complexes revealed that all PS1 FAD-linked mutations caused a loss of γ-secretase activity phenotype, in terms of Aβ1–40, Aβ1–42 and APP intracellular domain productions in vitro.Conclusion/SignificanceOur data support the view that PS1 mutations lead to a strong γ-secretase loss-of-function phenotype and an increased Aβ1–42/Aβ1–40 ratio, two mechanisms that are potentially involved in the pathogenesis of Alzheimer's disease.