Peptide inhibitors of angiotensin-I converting enzyme based on angiotensin (1-7) with selectivity for the C-terminal domain.

The renin-angiotensin system (RAS) is a key regulator of human arterial pressure. Several of its effects are modulated by angiotensin II, an octapeptide originating from the action of angiotensin-I converting enzyme (ACE) on the decapeptide angiotensin-I. ACE possess two active sites (nACE and cACE) that have their own kinetic and substrate specificities. ACE inhibitors are widely used as the first-line treatment for hypertension and other heart-related diseases, but because they inactivate both ACE domains, their use is associated with serious side effects. Thus, the search for domain-specific ACE inhibitors has been the focus of intense research. Angiotensin (1-7), a peptide that also belongs to the RAS, acts as a substrate of nACE and an inhibitor of cACE. We have synthetized 15 derivatives of Ang (1-7), sequentially removing the N-terminal amino acids and modifying peptides extremities, to find molecules with improved selectivity and inhibition properties. Ac-Ang (2-7)-NH2 is a good ACE inhibitor, resistant to cleavage and with improved cACE selectivity. Molecular dynamics simulations provided a model for this peptide's selectivity, due to Val3 and Tyr4 interactions with ACE subsites. Val3 has an important interaction with the S3 subsite, since its removal greatly reduced peptide-enzyme interactions. Taken together, our findings support ongoing studies using insights from the binding of Ac-Ang (2-7)-NH2 to develop effective cACE inhibitors.

Alternative splicing of P2X6 receptors in developing mouse brain and during in vitro neuronal differentiation.

Adenosine triphosphate acts as a fast excitatory neurotransmitter by binding to and activating seven structurally related subtypes of purinergic P2X receptors, which act as ligand-gated ion channels. Besides its role in neurotransmission, ATP also has trophic functions during development of the neuronal system. P2X receptor expression, mainly of P2X(4) and P2X(6) subtypes, has been detected in adult brain and also during neuronal development. We have used the mouse teratocarcinoma P19 cell line as an in vitro model to study P2X(6) receptor expression during early neuronal differentiation. We have detected a full-length and an alternatively spliced form of the mouse P2X(6) receptor gene in P19 cells using reverse transcriptase-polymerase chain reaction. The alternatively spliced form was already present at the stage of pluripotent undifferentiated P19 cells, and was predominant compared to the full-length form during the whole course of neuronal differentiation of P19 cells. Alternative splicing of P2X(6) receptor subunits was also confirmed during postnatal development of mouse brain. During postnatal development, however, the full-length form was predominant compared to the spliced form. Alternative splicing is suggested to regulate P2X(6) receptor function during neuronal differentiation.