Full-length cellular β-secretase has a trimeric subunit stoichiometry, and its sulfur-rich transmembrane interaction site modulates cytosolic copper compartmentalization

Abstract

The β-secretase (BACE1) initiates processing of the amyloid precursor protein (APP) into Aβ peptides, which have been implicated as central players in the pathology of Alzheimer disease. BACE1 has been described as a copper-binding protein and its oligomeric state as being monomeric, dimeric, and/or multimeric, but the native cellular stoichiometry has remained elusive. Here, by using single-molecule fluorescence and cross-linking experiments with photo-activatable unnatural amino acids, we show that full-length BACE1, independently of its subcellular localization, exists as trimers in human cells. We found that trimerization requires the BACE1 transmembrane sequences (TMSs) and cytoplasmic domains, with residues Ala and Cys buried within the trimer interface of the sulfur-rich core of the TMSs. Our 3D model predicts that the sulfur-rich core of the trimeric BACE1 TMS is accessible to metal ions, but copper ions did not trigger trimerization. The results of functional assays of endogenous BACE1 suggest that it has a role in intracellular copper compartmentalization by transferring cytosolic copper to intracellular compartments, while leaving the overall cellular copper concentration unaltered. Adding to existing physiological models, our results provide novel insight into the atypical interactions between copper and BACE1 and into its non-enzymatic activities. In conclusion, therapeutic Alzheimer disease prevention strategies aimed at decreasing BACE1 protein levels should be regarded with caution, because adverse effects in copper homeostasis may occur.

Optimal fluorescent protein tags for quantifying protein oligomerization in living cells

“…Coli GlnA 37 , and transmembrane proteins such as the Influenza A virus HA. An equivalent correction approach was used before in single molecule subunit counting studies, albeit mostly restricted to (m)EGFP 10 , 11 , 24 . Our results clearly show that a precise correction of the non-fluorescent FP fraction and knowledge of the p f for all involved FPs are absolutely necessary for a correct quantification of protein oligomerization in FFS techniques.…”

“…All together, these observations challenge the suitability of FPs for quantitative brightness analysis 5 . In this context, partially contradicting results are reported: studies performing subunit counting typically indicate apparent fluorescence probability (p f ) values of 50–80% 10 , 11 , 23 , 24 for GFPs. Very few investigations utilizing FFS approaches report similar values 25 , 26 , while very often it is simply assumed that all FPs are fluorescent.…”

Quantifying protein oligomerization in living cells: A systematic comparison of fluorescent proteins

“…It is worth emphasizing that this procedure works well not only for mEGFP-homo-oligomers, but also for large self-assembling protein complexes such as the 12-meric E.Coli GlnA 32 , and transmembrane proteins such as the Influenza A virus HA. An equivalent correction approach was used before in single molecule subunit counting studies, albeit mostly restricted to (m)EGFP 10,11,21 . Our results clearly show that a precise correction of the non-fluorescent FP fraction and knowledge of the pf for all involved FPs are absolutely necessary for a correct quantification of protein oligomerization in FFS techniques.…”

“…All together, these observations challenge the suitability of FPs for quantitative brightness analysis 5 . In this context, partially contradicting results are reported: studies performing subunit counting typically indicate apparent fluorescence probability (pf) values of 50-80% 10,11,20,21 for GFPs. Very few investigations utilizing FFS approaches report similar values 22,23 , while very often it is simply assumed that all FPs are fluorescent.…”

Dysregulation of Neuronal Iron Homeostasis as an Alternative Unifying Effect of Mutations Causing Familial Alzheimer’s Disease

“…Indeed, the functional existence of such complexes appears inconsistent with other data on the subcellular localization of these enzyme activities (e.g., see Pera et al, 2017 ). Note that the BACE1 protein that provides β-secretase activity was recently shown to be involved in intracellular Cu 2+ homeostasis ( Liebsch et al, 2017 ).…”