👤 Michael J Emanuele

High cholesterol levels are a risk factor for the development of Alzheimer's disease. Experiments investigating the influence of cholesterol on the proteolytic processing of the amyloid precursor protein (APP) by the β-secretase Bace1 and on their proximity in cells have led to conflicting results. By using a fluorescence bioassay coupled with flow cytometry we found a direct correlation between the increase in membrane cholesterol amount and the degree of APP shedding in living human neuroblastoma cells. Analogue results were obtained for cells overexpressing an APP mutant that cannot be processed by α-secretase, highlighting the major influence of cholesterol enrichment on the cleavage of APP carried out by Bace1. By contrast, the cholesterol content was not correlated with changes in membrane dynamics of APP and Bace1 analyzed with single molecule tracking, indicating that the effect of cholesterol enrichment on APP processing by Bace1 is uncoupled from changes in their lateral diffusion. Show less

The interplay between E2 and E3 enzymes regulates the polyubiquitination of substrates in eukaryotes. Among the several RING-domain E3 ligases in humans, many utilize two distinct E2s for polyubiquitination. For example, the cell cycle regulatory E3, human anaphase-promoting complex/cyclosome (APC/C), relies on UBE2C to prime substrates with ubiquitin (Ub) and on UBE2S to extend polyubiquitin chains. However, the potential coordination between these steps in ubiquitin chain formation remains undefined. While numerous studies have unveiled how RING E3s stimulate individual E2s for Ub transfer, here we change perspective to describe a case where the chain-elongating E2 UBE2S feeds back and directly stimulates the E3 APC/C to promote substrate priming and subsequent multiubiquitination by UBE2C. Our work reveals an unexpected model for the mechanisms of RING E3-dependent ubiquitination and for the diverse and complex interrelationship between components of the ubiquitination cascade. Show less