Isoaspartate formation in extracellular matrix proteins, by aspartate isomerization or asparagine deamidation, is generally viewed as a degradation reaction occurring in vivo during tissue aging. For instance, non-enzymatic isoaspartate formation at RGD-integrin binding sites causes loss of cell adhesion sites, which in turn can be enzymatically "repaired" to RGD by protein-l-isoAsp-O-methyltransferase. We show here that isoaspartate formation is also a mechanism for extracellular matrix activation. In particular, we show that deamidation of Asn263 at the Asn-Gly-Arg (NGR) site in fibronectin N-terminal region generates an alpha(v)beta3-integrin binding site containing the L-isoDGR sequence, which is enzymatically "deactivated" to DGR by protein-L-isoAsp-O-methyltransferase. Furthermore, rapid NGR-to-isoDGR sequence transition in fibronectin fragments generates alpha(v)beta3 antagonists (named "isonectins") that competitively bind RGD binding sites and inhibit endothelial cell adhesion, proliferation, and tumor growth. Time-dependent generation of isoDGR may represent a sort of molecular clock for activating latent integrin binding sites in proteins.

A growing body of evidence suggests that peptides containing the Asn-Gly-Arg (NGR) motif can selectively recognize tumor neovasculature and can be used, therefore, for ligand-directed targeted delivery of various drugs and particles to tumors or to other tissues with an angiogenesis component. The neovasculature binding properties of these peptides rely on the interaction with an endothelium-associated form of aminopeptidase N (CD13), an enzyme that has been implicated in angiogenesis and tumor growth. Recent studies have shown that NGR can rapidly convert to isoaspartate-glycine-arginine (isoDGR) by asparagine deamidation, generating αvβ3 ligands capable of affecting endothelial cell functions and tumor growth. This review focuses on structural and functional properties of the NGR motif and its application in drug development for angiogenesis-dependent diseases. Furthermore, we discuss the time-dependent transition of NGR to isoDGR in natural proteins, such as fibronectins, and its potential role of as a “molecular timer” for generating new binding sites for integrins impli-cated in angiogenesis.

Cyclic and linear peptides containing the Asn-Gly-Arg (NGR) motif have proven useful for delivering various anti-tumor compounds and viral particles to tumor vessels. We have investigated the role of cyclic constraints on the structure and tumor-homing properties of NGR peptides using tumor necrosis factor-α (TNF) derivatives containing disulfide-bridged (CNGRC-TNF) and linear (GNGRG-TNF) NGR domains. Experiments carried out in animal models showed that both GNGRG and CNGRC can target TNF to tumors. However, the anti-tumor activity of CNGRC-TNF was >10-fold higher than that of GNGRG-TNF. Molecular dynamic simulation of cyclic CNGRC showed the presence of a bend geometry involving residues Gly³-Arg⁴. Molecular dynamic simulation of the same peptide without disulfide constraints showed that the most populated and thermodynamically favored configuration is characterized by the presence of a β-turn involving residues Gly³-Arg⁴ and hydrogen bonding interactions between the backbone atoms of Asn² and Cys⁵. These results suggest that the NGR motif has a strong propensity to form β-turn in linear peptides and may explain the finding that GNGRG peptide can target TNF to tumors, albeit to a lower extent than CNGRC. The disulfide bridge constraint is critical for stabilizing the bent conformation and for increasing the tumor targeting efficiency.

Aims High chromogranin-A (CgA) levels were observed in patients with heart failure but its source remained uncertain. We evaluated whether CgA is produced by myocardium and might affect myocardial function. Methods and results We measured plasma CgA levels and performed immunohistochemistry with anti-CgA antibodies on myocardial biopsies in 40 patients with dilated cardiomyopathy (DCM) and 20 patients with hypertrophic cardiomyopathy (HCM). Surgical myocardial specimens from DCM and HCM patients were used for PCR and ELISA. The presence of CgA-derived fragments in plasma was evaluated by gel-filtration HPLC and their effects on cardiac performance assessed in isolated perfused rat heart. All patients showed increased CgA plasma levels (DCM 153.7 ± 158.5 ng/mL; HCM 150.2 ± 86.7 ng/mL vs. 64.1 ± 17.9 ng/mL of controls) with a positive correlation between CgA and left ventricular end-diastolic pressure (DCM, R = 0.86; HCM, R = 0.83), and plasma brain natriuretic peptide (BNP) levels (DCM, R = 0.88; HCM, R = 0.85) ( P < 0.001). Immunohistochemistry showed cytoplasmic expression of CgA and co-localization with BNP in all patients, but not in controls. PCR detected CgA mRNA in both pathologic and normal myocardium, while ELISA showed a significant amount of CgA only in pathologic myocardium (>0.5 µg/g of tissue); gel-filtration HPLC of plasma samples identified immunoreactive N-terminal CgA fragments containing vasostatin-1. Administration of vasostatin-1 to perfused rat heart produced negative inotropic and lusitropic effects counteracting isoproterenol actions. Conclusion We demonstrate for the first time that CgA is produced by human myocardium and exerts negative inotropic and lusitropic effects on mammalian heart. CgA may represent a key player in neuroendocrine regulation of cardiac function and a potential therapeutic target in heart failure.

Abnormal tumor vasculature impairs T lymphocyte adhesion to endothelial cells and lymphocyte extravasation into neoplastic tissues, limiting the therapeutic potential of both active and adoptive immunotherapies. We have found that treatment of tumor-bearing mice with NGR-TNF, a Cys-Asn-Gly-Arg-Cys peptide-TNF fusion product capable of altering the endothelial barrier function and improving drug penetration in tumors, associated with the intratumor upregulation of leukocyte-endothelial cell adhesion molecules, the release of proinflammatory cytokines and chemokines, and the infiltration of tumor-specific effector CD8⁺ T cells. As a result, NGR-TNF enhanced the therapeutic activity of adoptive and active immunotherapy, delaying tumor growth and prolonging survival. Furthermore, we have found that therapeutic effects of these combinations can be further increased by the addition of chemotherapy. Thus, these findings might be relevant for the design of novel immunotherapeutic approaches for cancer patients.