Participation of transmembrane domain 1 of presenilin 1 in the catalytic pore structure of the γ-secretase.

γ-Secretase is an intramembrane-cleaving protease that is responsible for the generation of amyloid-ß peptides linked to the pathogenesis of Alzheimer's disease. Using a substituted cysteine accessibility method, we have previously shown that the hydrophilic "catalytic pore" structure of γ-secretase is formed by the transmembrane domains (TMDs) 6, 7, and 9 of presenilin 1 (PS1), the catalytic subunit of γ-secretase, within the membrane. Here, we analyzed the structure in and around the first hydrophobic region, the putative TMD1, of PS1, of which the precise function as well as three-dimensional location within γ-secretase remained unknown. We found that TMD1 is located in proximity to the catalytic GxGD and PAL motifs within the C-terminal fragment of PS1, facing directly the catalytic pore. Competition experiments using known γ-secretase inhibitors suggested that the N-terminal region of TMD1 functions as a subsite during proteolytic action of the γ-secretase. Intriguingly, binding of inhibitors affected water accessibility of residues at the membrane border of TMD1, suggesting the possibility of a dynamic motion of TMD1 during the catalytic process. Our results provide mechanistic insights into the functional role of TMD1 of PS1 in the intramembrane-cleaving activity of the γ-secretase.

Functional analysis of the transmembrane domains of presenilin 1: participation of transmembrane domains 2 and 6 in the formation of initial substrate-binding site of gamma-secretase.

gamma-Secretase is a multimeric membrane protein complex composed of presenilin (PS), nicastrin, Aph-1, and Pen-2, which mediates intramembrane proteolysis of a range of type I transmembrane proteins. We previously analyzed the functional roles of the N-terminal transmembrane domains (TMDs) 1-6 of PS1 in the assembly and proteolytic activity of the gamma-secretase using a series of TMD-swap PS1 mutants. Here we applied the TMD-swap method to all the TMDs of PS1 for the structure-function analysis of the proteolytic mechanism of gamma-secretase. We found that TMD2- or -6-swapped mutant PS1 failed to bind the helical peptide-based, substrate-mimic gamma-secretase inhibitor. Cross-linking experiments revealed that both TMD2 and TMD6 of PS1 locate in proximity to the TMD9, the latter being implicated in the initial substrate binding. Taken together, our data suggest that TMD2 and the luminal side of TMD6 are involved in the formation of the initial substrate-binding site of the gamma-secretase complex.

The C-terminal PAL motif and transmembrane domain 9 of presenilin 1 are involved in the formation of the catalytic pore of the gamma-secretase.

Gamma-secretase is an unusual membrane-embedded protease, which cleaves the transmembrane domains (TMDs) of type I membrane proteins, including amyloid-beta precursor protein and Notch receptor. We have previously shown the existence of a hydrophilic pore formed by TMD6 and TMD7 of presenilin 1 (PS1), the catalytic subunit of gamma-secretase, within the membrane by the substituted cysteine accessibility method. Here we analyzed the structure of TMD8, TMD9, and the C terminus of PS1, which encompass the conserved PAL motif and the hydrophobic C-terminal tip, both being critical for the catalytic activity and the formation of the gamma-secretase complex. We found that the amino acid residues around the PAL motif and the extracellular/luminal portion of TMD9 are highly water accessible and located in proximity to the catalytic pore. Furthermore, the region starting from the luminal end of TMD9 toward the C terminus forms an amphipathic alpha-helix-like structure that extends along the interface between the membrane and the extracellular milieu. Competition analysis using gamma-secretase inhibitors revealed that the TMD9 is involved in the initial binding of substrates, as well as in the subsequent catalytic process as a subsite. Our results provide mechanistic insights into the role of TMD9 in the formation of the catalytic pore and the substrate entry, crucial to the unusual mode of intramembrane proteolysis by gamma-secretase.

Effects of expression and inhibition of negative emotions on health, mood states, and salivary secretory immunoglobulin A in Japanese mildly depressed undergraduates.

Previous studies have indicated that expression of negative emotions facilitates mental and physical health and inhibition of negative emotions increases susceptibility to illness. This study was conducted to examine whether those findings can be expanded to populations with non-Western cultural backgrounds. Specifically, we explored effects of expression and inhibition of negative emotions on health, mood states, and mucosal immune function in mildly depressed Japanese individuals. 16 depressed and 16 nondepressed female undergraduates were required either to write about their unpleasant experiences and superficial topics or to suppress any emotional responses and thoughts about them. Secretory immunoglobulin A (s-IgA) in saliva and psychological indices were measured at an experimental session and at a follow-up 1 wk. later. Beneficial effects of expression of emotions on subjective health were indicated in the nondepressed group, whereas harmful effects of inhibition on subjective health were shown in the depressed group. Emotional expression by writing improved mood states both in the depressed and nondepressed groups but induced elevation of salivary s-IgA only in the depressed group.