Splice isoform and pharmacological studies reveal that sterol depletion relocalizes α-synuclein and enhances its toxicity.

Synucleinopathies are neurodegenerative diseases associated with toxicity of the lipid-binding protein α-synuclein (α-syn). When expressed in yeast, α-syn associates with membranes at the endoplasmic reticulum and traffics with vesicles out to the plasma membrane. At higher levels it elicits a number of phenotypes, including blocking vesicle trafficking. The expression of α-syn splice isoforms varies with disease, but how these isoforms affect protein function is unknown. We investigated two of the most abundant isoforms, resulting in deletion of exon four (α-synΔ4) or exon six (α-synΔ6). α-SynΔ4, missing part of the lipid-binding domain, had reduced toxicity and membrane binding. α-SynΔ6, missing part of the protein-protein interaction domain, had reduced toxicity but no reduction in membrane binding. To compare the mechanism by which the splice isoforms exert toxicity, equally toxic strains were probed with genetic modifiers of α-syn-induced toxicity. Most modifiers equally altered the toxicity induced by the splice isoforms and full-length α-syn (α-synFL). However, the splice isoform strains responded differently to a sterol-binding protein, leading us to examine the effect of sterols on α-syn-induced toxicity. Upon inhibition of sterol synthesis, α-synFL and α-synΔ6, but not α-synΔ4, showed decreased plasma membrane association, increased vesicular association, and increased cellular toxicity. Thus, higher membrane sterol concentrations favor plasma membrane binding of α-synFL and α-synΔ6 and may be protective of synucleinopathy progression. Given the common use of cholesterol-reducing statins and these potential effects on membrane binding proteins, further investigation of how sterol concentration and α-syn splice isoforms affect vesicular trafficking in synucleinopathies is warranted.

The mammalian UPR boosts glycoprotein ERAD by suppressing the proteolytic downregulation of ER mannosidase I.

The secretory pathway provides a physical route through which only correctly folded gene products are delivered to the eukaryotic cell surface. The efficiency of endoplasmic reticulum (ER)-associated degradation (ERAD), which orchestrates the clearance of structurally aberrant proteins under basal conditions, is boosted by the unfolded protein response (UPR) as one of several means to relieve ER stress. However, the underlying mechanism that links the two systems in higher eukaryotes has remained elusive. Herein, the results of transient expression, RNAi-mediated knockdown and functional studies demonstrate that the transcriptional elevation of EDEM1 boosts the efficiency of glycoprotein ERAD through the formation of a complex that suppresses the proteolytic downregulation of ER mannosidase I (ERManI). The results of site-directed mutagenesis indicate that this capacity does not require that EDEM1 possess inherent mannosidase activity. A model is proposed in which ERManI, by functioning as a downstream effector target of EDEM1, represents a checkpoint activation paradigm by which the mammalian UPR coordinates the boosting of ERAD.

Small molecules block the polymerization of Z alpha1-antitrypsin and increase the clearance of intracellular aggregates.

The Z mutant of alpha1-antitrypsin (Glu342Lys) causes a domain swap and the formation of intrahepatic polymers that aggregate as inclusions and predispose the homozygote to cirrhosis. We have identified an allosteric cavity that is distinct from the interface involved in polymerization for rational structure-based drug design to block polymer formation. Virtual ligand screening was performed on 1.2 million small molecules and 6 compounds were identified that reduced polymer formation in vitro. Modeling the effects of ligand binding on the cavity and re-screening the library identified an additional 10 compounds that completely blocked polymerization. The best antagonists were effective at ratios of compound to Z alpha1-antitrypsin of 2.5:1 and reduced the intracellular accumulation of Z alpha1-antitrypsin by 70% in a cell model of disease. Identifying small molecules provides a novel therapy for the treatment of liver disease associated with the Z allele of alpha1-antitrypsin.

Human endoplasmic reticulum mannosidase I is subject to regulated proteolysis.

In the early secretory pathway, opportunistic cleavage of asparagine-linked oligosaccharides by endoplasmic reticulum (ER) mannosidase I targets misfolded glycoproteins for dislocation into the cytosol and destruction by 26 S proteasomes. The low basal concentration of the glycosidase is believed to coordinate the glycan cleavage with prolonged conformation-based ER retention, ensuring that terminally misfolded glycoproteins are preferentially targeted for destruction. Herein the intracellular fate of human ER mannosidase I was monitored to determine whether a post-translational process might contribute to the regulation of its intracellular concentration. The transiently expressed recombinant human glycosidase was subject to rapid intracellular turnover in mouse hepatoma cells, as was the endogenous mouse ortholog. Incubation with either chloroquine or leupeptin, but not lactacystin, led to intracellular stabilization, implicating the involvement of lysosomal acid hydrolases. Inhibition of protein synthesis with cycloheximide led to intracellular depletion of the glycosidase and concomitant ablation of asparagine-linked glycoprotein degradation, confirming the physiologic relevance of the destabilization process. Metabolic incorporation of radiolabeled phosphate, detection by anti-phosphoserine antiserum, and the stabilizing effect of general serine kinase inhibition implied that ER mannosidase I is subjected to regulated proteolysis. Stabilization in response to genetically engineered removal of the amino-terminal cytoplasmic tail, a postulated regulatory domain, and colocalization of green fluorescent protein fusion proteins with Lamp1 provided two additional lines of evidence to support the hypothesis. A model is proposed in which proteolytically driven checkpoint control of ER mannosidase I contributes to the establishment of an equitable glycoprotein quality control standard by which the efficiency of asparagine-linked glycoprotein conformational maturation is measured.