Interferon-α (IFN-α) is essential for antiviral immunity, but in the absence of matrix metalloproteinase-12 (MMP-12) or IκBα (encoded by NFKBIA) we show that IFN-α is retained in the cytosol of virus-infected cells and is not secreted. Our findings suggest that activated IκBα mediates the export of IFN-α from virus-infected cells and that the inability of cells in Mmp12(-/-) but not wild-type mice to express IκBα and thus export IFN-α makes coxsackievirus type B3 infection lethal and renders respiratory syncytial virus more pathogenic. We show here that after macrophage secretion, MMP-12 is transported into virus-infected cells. In HeLa cells MMP-12 is also translocated to the nucleus, where it binds to the NFKBIA promoter, driving transcription. We also identified dual-regulated substrates that are repressed both by MMP-12 binding to the substrate's gene exons and by MMP-12-mediated cleavage of the substrate protein itself. Whereas intracellular MMP-12 mediates NFKBIA transcription, leading to IFN-α secretion and host protection, extracellular MMP-12 cleaves off the IFN-α receptor 2 binding site of systemic IFN-α, preventing an unchecked immune response. Consistent with an unexpected role for MMP-12 in clearing systemic IFN-α, treatment of coxsackievirus type B3-infected wild-type mice with a membrane-impermeable MMP-12 inhibitor elevates systemic IFN-α levels and reduces viral replication in pancreas while sparing intracellular MMP-12. These findings suggest that inhibiting extracellular MMP-12 could be a new avenue for the development of antiviral treatments.

Intracellular aminopeptidases endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2), and as well as insulin-regulated aminopeptidase (IRAP) process antigenic epitope precursors for loading onto MHC class I molecules and regulate the adaptive immune response. Their activity greatly affects the antigenic peptide repertoire presented to cytotoxic T lymphocytes and as a result can regulate cytotoxic cellular responses contributing to autoimmunity or immune evasion by viruses and cancer cells. Therefore, pharmacological regulation of their activity is a promising avenue for modulating the adaptive immune response with possible applications in controlling autoimmunity, in boosting immune responses to pathogens, and in cancer immunotherapy. In this study we exploited recent structural and biochemical analysis of ERAP1 and ERAP2 to design and develop phosphinic pseudopeptide transition state analogs that can inhibit this family of enzymes with nM affinity. X-ray crystallographic analysis of one such inhibitor in complex with ERAP2 validated our design, revealing a canonical mode of binding in the active site of the enzyme, and highlighted the importance of the S2' pocket for achieving inhibitor potency. Antigen processing and presentation assays in HeLa and murine colon carcinoma (CT26) cells showed that these inhibitors induce increased cell-surface antigen presentation of transfected and endogenous antigens and enhance cytotoxic T-cell responses, indicating that these enzymes primarily destroy epitopes in those systems. This class of inhibitors constitutes a promising tool for controlling the cellular adaptive immune response in humans by modulating the antigen processing and presentation pathway. molecular structure | adaptive immunity | major histocompatibility molecules | specificity | kinetics

Abstract-Somatic angiotensin-converting enzyme (ACE) contains two homologous domains, each bearing a functional active site. The in vivo contribution of each active site to the release of angiotensin II (Ang II) and the inactivation of bradykinin (BK) is still unknown. To gain insights into the functional roles of these two active sites, the in vitro and in vivo effects of compounds able to selectively inhibit only one active site of ACE were determined, using radiolabeled Ang I or BK, as physiological substrates of ACE. In vitro studies indicated that a full inhibition of the Ang I and BK cleavage requires a blockade of the two ACE active sites. In contrast, in vivo experiments in mice demonstrated that the selective inhibition of either the N-domain or the C-domain of ACE by these inhibitors prevents the conversion of Ang I to Ang II, while BK protection requires the inhibition of the two ACE active sites. Thus, in vivo, the cleavage of Ang I and BK by ACE appears to obey to different mechanisms. Remarkably, in vivo the conversion of Ang I seems to involve the two active sites of ACE, free of inhibitor. Based on these findings, it might be suggested that the gene duplication of ACE in vertebrates may represent a means for regulating the cleavage of Ang I differently from that of BK. Key Words: angiotensin-converting enzyme Ⅲ angiotensin Ⅲ bradykinin Ⅲ phosphinic peptide inhibitors S omatic angiotensin-converting enzyme in vertebrates (ACE, EC 3.4.15.1) is a zinc metallopeptidase involved in the release of angiotensin II (Ang II) and the inactivation of bradykinin (BK), two peptide hormones that play a key role in the regulation of blood pressure, renal and cardiovascular functions. [1][2][3][4][5] These peptides are end products of two highly regulated systems, respectively, the renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS). It has been suggested that the plasma KKS could be the physiological counterbalance to the RAS. 6 As ACE is strategically poised between the RAS and KKS systems, the question may arise as to whether the ACE activity is controlled to regulate the balance between the RAS and KKS systems and, if so, by which mechanism.Somatic ACE is a two-domain protein resulting from a tandem gene duplication, with each domain possessing a functional active site. 7,8 Since the discovery that ACE has two active sites, there has been much speculation about the functional significance of the presence of two active sites in the same enzyme. Although the two domains of ACE display in vitro a relatively broad substrate specificity, such as the ability to cleave Ang I and BK, there are some biochemical features that differentiate between the two active sites. 9 -13 For example, the hematoregulatory peptide, Ac-S-D-K-P, was proved in vitro to be specifically cleaved by the N-domain of ACE. 14 The N-domain and C-domain have different in vitro patterns of chloride activation 10 and can be differentiated by the relative potencies of some inhibitors. 12,15 Among these, RXP407, a phosphinic peptide...

Several phosphinic pseudo-tripeptides of general formula R-XaaPsi(PO(2)-CH(2))Xaa'-Yaa'-NH(2) were synthesized and evaluated for their in vitro activities to inhibit stromelysin-3, gelatinases A and B, membrane type-1 matrix metalloproteinase, collagenases 1 and 2, and matrilysin. With the exception of collagenase-1 and matrilysin, phosphinic pseudo-tripeptides behave as highly potent inhibitors of matrix metalloproteinases, provided they contain in P(1)' position an unusual long aryl-alkyl substituent. Study of structure-activity relationships regarding the influence of the R and Xaa' substituents in this series may contribute to the design of inhibitors able to block only a few members of the matrix metalloproteinase family.