The amyloid precursor protein (APP) is an ubiquitously expressed cell surface protein and a key molecule in the etiology of Alzheimer disease. Amyloidogenic processing of APP through secretases leads to the generation of toxic amyloid β (Aβ) peptides, which are regarded as the molecular cause of the disease. We report here an alternative processing pathway of APP through the mammalian intramembrane rhomboid protease RHBDL4. RHBDL4 efficiently cleaves APP inside the cell, thus bypassing APP from amyloidogenic processing, leading to reduced Aβ levels. RHBDL4 cleaves APP multiple times in the ectodomain, resulting in several N- and C-terminal fragments that are not further degraded by classical APP secretases. Knockdown of endogenous RHBDL4 results in decreased levels of C-terminal fragments derived from endogenous APP. Similarly, we found the APP family members APLP1 and APLP2 to be substrates of RHBDL4. We conclude that RHBDL4-mediated APP processing provides insight into APP and rhomboid physiology and qualifies for further investigations to elaborate its impact on Alzheimer disease pathology.

Amyloid-β (Aβ) peptides are likely the molecular cause of neurodegeneration observed in Alzheimer's disease. In the brain, Aβ42 and Aβ40 are toxic and the most important proteolytic fragments generated through sequential processing of the amyloid precursor protein (APP) by β- and γ-secretases. Impeding the generation of Aβ42 and Aβ40 is thus considered as a promising strategy to prevent Alzheimer's disease. We therefore wanted to determine key parameters of the APP transmembrane sequence enabling production of these Aβ species. Here we show that the hydrophilicity of amino acid residues G33, T43, and T48 critically determines the generation of Aβ42 and Aβ40 peptides (amino acid numbering according to Aβ nomenclature starting with aspartic acid 1). First, we performed a comprehensive mutational analysis of glycine residue G33 positioned within the N-terminal half of the APP transmembrane sequence by exchanging it against the 19 other amino acids. We found that hydrophilicity of the residue at position 33 positively correlated with Aβ42 and Aβ40 generation. Second, we analyzed two threonine residues at positions T43 and T48 in the C-terminal half of the APP-transmembrane sequence. Replacement of single threonine residues by hydrophobic valines inversely affected Aβ42 and Aβ40 generation. We observed that threonine mutants affected the initial γ-secretase cut, which is associated with levels of Aβ42 or Aβ40. Overall, hydrophilic residues of the APP transmembrane sequence decide on the exact initial γ-cut and the amounts of Aβ42 and Aβ40.

Proteases, sharp yet unforgivable tools of every cell, require tight regulation to ensure specific non-aberrant cleavages. The relatively recent discovered class of intramembrane proteases has gained increasing interest due to their involvement in important signaling pathways linking them to diseases including Alzheimer's disease and cancer. Despite tremendous efforts, their regulatory mechanisms have only started to unravel. There is evidence that the membrane composition itself can regulate intramembrane protease activity and specificity. In this review, we highlight the work on γ-secretase and rhomboid proteases and summarize several studies as to how different lipids impact on enzymatic activity.

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