Signal Peptide Peptidase-Like 2c (SPPL2c) impairs vesicular transport and cleavage of SNARE proteins

Members of the GxGD-type intramembrane aspartyl proteases have emerged as key players not only in fundamental cellular processes such as B-cell development or protein glycosylation, but also in development of pathologies, such as Alzheimer's disease or hepatitis virus infections. However, one member of this protease family, signal peptide peptidase-like 2c (SPPL2c), remains orphan and its capability of proteolysis as well as its physiological function is still enigmatic. Here, we demonstrate that SPPL2c is catalytically active and identify a variety of SPPL2c candidate substrates using proteomics. The majority of the SPPL2c candidate substrates cluster to the biological process of vesicular trafficking. Analysis of selected SNARE proteins reveals proteolytic processing by SPPL2c that impairs vesicular transport and causes retention of cargo proteins in the endoplasmic reticulum. As a consequence, the integrity of subcellular compartments, in particular the Golgi, is disturbed. Together with a strikingly high physiological SPPL2c expression in testis, our data suggest involvement of SPPL2c in acrosome formation during spermatogenesis

Mouse brain proteomics establishes MDGA1 and CACHD1 as in vivo substrates of the Alzheimer protease BACE1

The protease beta-site APP cleaving enzyme 1 (BACE1) has fundamental functions in the nervous system. Its inhibition is a major therapeutic approach in Alzheimer's disease, because BACE1 cleaves the amyloid precursor protein (APP), thereby catalyzing the first step in the generation of the pathogenic amyloid beta (A beta) peptide. Yet, BACE1 cleaves numerous additional membrane proteins besides APP. Most of these substrates have been identified in vitro, but only few were further validated or characterized in vivo. To identify BACE1 substrates with in vivo relevance, we used isotope label-based quantitative proteomics of wild type and BACE1-deficient (BACE1 KO) mouse brains. This approach identified known BACE1 substrates, including Close homolog of L1 and contactin-2, which were found to be enriched in the membrane fraction of BACE1 KO brains. VWFA and cache domain-containing protein 1 (CACHD)1 and MAM domain-containing glycosylphosphatidylinositol anchor protein 1 (MDGA1), which have functions in synaptic transmission, were identified and validated as new BACE1 substrates in vivo by immunoblots using primary neurons and mouse brains. Inhibition or deletion of BACE1 from primary neurons resulted in a pronounced inhibition of substrate cleavage and a concomitant increase in full-length protein levels of CACHD1 and MDGA1. The BACE1 cleavage site in both proteins was determined to be located within the juxtamembrane domain. In summary, this study identifies and validates CACHD1 and MDGA1 as novel in vivo substrates for BACE1, suggesting that cleavage of both proteins may contribute to the numerous functions of BACE1 in the nervous system

Data from: Computational and experimental characterization of dVHL establish a Drosophila model of VHL syndrome

The von Hippel-Lindau (VHL) cancer syndrome is associated with mutations in the VHL gene. The pVHL protein is involved in response to changes in oxygen availability as part of an E3-ligase that targets the Hypoxia-Inducible Factor for degradation. pVHL has a molten globule configuration with marginal thermodynamic stability. The cancer-associated mutations further destabilize it. The Drosophila homolog, dVHL, has relatively low sequence similarity to pVHL, and is also involved in regulating HIF1-α. Using in silico, in vitro and in vivo approaches we demonstrate high similarity between the structure and function of dVHL and pVHL. These proteins have a similar fold, secondary and tertiary structures, as well as thermodynamic stability. Key functional residues in dVHL are evolutionary conserved. This structural homology underlies functional similarity of both proteins, evident by their ability to bind their reciprocal partner proteins, and by the observation that transgenic pVHL can fully maintain normal dVHL-HIF1-α downstream pathways in flies. This novel transgenic Drosophila model is thus useful for studying the VHL syndrome, and for testing drug candidates to treat it

Total proteome turbidity assay for tracking global protein aggregation in the natural cellular environment

Proteome homeostasis is crucial for optimal cellular function and survival in the face of various stressful impacts. This entails preservation of a balance between protein synthesis, folding, degradation, and trafficking collectively termed proteostasis. A hallmark of proteostasis failure, which underlies various diseases, is enhanced misfolding and aggregation of proteins. Here we adapted the measurement of protein turbidity, which is commonly used to evaluate aggregation of single purified proteins, for monitoring propensity for aggregation of the entire soluble cellular proteome incubated in vitro for several hours. We show that over-expression of an aggregation-prone protein or applying endoplasmic-reticulum (ER) stress to either cells in culture or to the intact organism, Drosophila, enhances the rise in turbidity of the global soluble proteome compared to untreated cells. Additionally, given that Alzheimer’s disease (AD) is known to involve ER stress and aggregation of proteins, we demonstrate that the soluble fraction of brain extracts from AD patients displays markedly higher rise of global proteome turbidity than in healthy counterparts. This assay could be valuable for various biological, medical and biotechnological applications

Data from: Computational and experimental characterization of dVHL establish a Drosophila model of VHL syndrome

The von Hippel-Lindau (VHL) cancer syndrome is associated with mutations in the VHL gene. The pVHL protein is involved in response to changes in oxygen availability as part of an E3-ligase that targets the Hypoxia-Inducible Factor for degradation. pVHL has a molten globule configuration with marginal thermodynamic stability. The cancer-associated mutations further destabilize it. The Drosophila homolog, dVHL, has relatively low sequence similarity to pVHL, and is also involved in regulating HIF1-α. Using in silico, in vitro and in vivo approaches we demonstrate high similarity between the structure and function of dVHL and pVHL. These proteins have a similar fold, secondary and tertiary structures, as well as thermodynamic stability. Key functional residues in dVHL are evolutionary conserved. This structural homology underlies functional similarity of both proteins, evident by their ability to bind their reciprocal partner proteins, and by the observation that transgenic pVHL can fully maintain normal dVHL-HIF1-α downstream pathways in flies. This novel transgenic Drosophila model is thus useful for studying the VHL syndrome, and for testing drug candidates to treat it

dVHLModel

Coordinate file (PDB format) of the model structure of the Drosophila VHL protein

Analysis from k2d2 program of pVHL and dVHL suggest similar secondary structure content of α-helix, β-sheet and random structure.

<p>Analysis from k2d2 program of pVHL and dVHL suggest similar secondary structure content of α-helix, β-sheet and random structure.</p

The GFP fluorescence signal caused by over-expression of ODD-GFP is lowered by co-expression of either dVHL, pVHL19, or pVHL30.

<p>(<b>A</b>) <b>GFP levels</b> were analyzed by confocal microscopy. Genotypes: GMR-Gal4; UAS-ODD-GFP/UAS-RFP (left), GMR-Gal4; UAS-ODD-GFP/UAS-dVHL (second from left) GMR-Gal4; UAS-ODD-GFP/UAS-pVHL19 (third from the left) and GMR-Gal4; UAS-ODD-GFP; UAS-pVHL30 (right). Scale bar, 20 µm (<b>B) Western blot</b> analysis showing the levels of ODD-GFP protein extract from flies heads. β-actin was used as loading control. (<b>1</b>) GMR-Gal4; UAS-ODD-GFP/UAS-RFP. (<b>2</b>) GMR-Gal4. (<b>3</b>) GMR-Gal4; UAS-ODD-GFP/UAS-dVHL. (<b>4</b>) GMR-Gal4; UAS-ODD-GFP/UAS-pVHL19. (<b>5</b>) GMR-Gal4; UAS-ODD-GFP/UAS-pVHL30.</p

Subcellular localization of pVHL in transgenic Drosophila.

<p>Immunofluorescence staining of the <i>Drosophila</i> eye imaginal discs over expressing pVHL30 by GMR Gal4. (<b>A</b>) Bright field (<b>B</b>) Nuclei stained using DAPI (blue) (<b>C</b>) Actin stained using phalloidin 568 (red) (<b>D</b>) Anti-pVHL antibody was used for identification of pVHL protein (purple). (<b>E</b>) Merge. Upper panel scale bar: 20 µm; lower panel scale bar: 10 µm (zoom in).</p