Catabolic attacks of membrane-bound angiotensin-converting enzyme on the N-terminal part of species-specific amyloid-beta peptides.

Catabolic processes play a crucial role in the steady state of the amyloid-beta peptide (Abeta). Neprilysin (NEP) and angiotensin-converting enzyme (ACE), two transmembranal enzymes with greatest importance in peptide pharmacology, are known to play a role in Abeta catabolism. This paper focuses on the N-terminal part of Abeta. This region contains the three amino acid residues that determine the differences between human (hAbeta) and murine Abeta (mAbeta). Moreover, the N-terminal part of Abeta contains the zinc-binding site of the molecule. Consequently, all hydrolytic attacks on this part of the Alzheimer peptide should be of exceptional interest. We investigated domain-selective forms of ACE in HPLC-monitored peptide degradation studies and used mass spectrometry for product analyses. We found that ACE-evoked a hydrolysis of the N-terminal part of m- and hAbeta. The hAbeta sequence hAbeta (4-15) was found to be a better substrate for ACE compared to the corresponding murine form. Moreover, we localized the corresponding cleavage sites in the N-terminal part of Abeta as well as in the full-length molecule and identified new sites of endopeptidolytic attack by ACE. Finally, we demonstrate that both catalytic domains of mACE have similar hydrolytic activity on the N-terminal part of Abeta. Our results show that ACE besides its typical function as a dipeptidyl-carboxypeptidase has also unequivocal endopeptidolytic activities.

Overexpression of claudin-5 but not claudin-3 induces formation of trans-interaction-dependent multilamellar bodies.

Tight junctions (TJs) regulate paracellular barriers and claudins (Cld) form the backbone of TJ strands. To elucidate the molecular mechanism of claudin polymer formation, TJs were reconstituted by claudin transfection of TJ-free HEK293 cells. Therewith, typical TJ stands can be found at cell-cell contacts. In addition, overexpression of Cld5-YFP induces formation of huge intracellular multilamellar bodies. In contrast, Cld3 does not induce similar structures. Inhibition of trans-interaction of Cld5 by Y148A substitution diminished formation of multilamellar bodies. These results demonstrate claudin subtype-specific oligomerization. Cld3 and Cld5 localize to the plasma membrane differentially. Phosphorylation at T207 of Cld5 was suggested to participate in regulation of Cld5 internalization. However, prevention of potential phosphorylation by T207A substitution did not increase Cld5 amount in the plasma membrane of transfected cells. Taken together, if carefully evaluated, transfection of claudin constructs in nonpolar cells is a powerful strategy to improve understanding of subcellular targeting and assembly of TJ proteins.