*2.2. Mechanisms Underlying the Biological Effects of CgA Fragments in Angiogenesis and Tumor Growth*

The findings described above raise a series of questions. First, which proteases can switch on this pro-angiogenic mechanism in tumors? Second, which receptor mediates the biological activity of CgA1-373? Considering that thrombin and plasmin are known to be activated in tumors, and that these enzymes can efficiently cleave the R373R374 peptide bond [17,43], both enzymes are good candidates for cleaving CgA in tumors. Although other proteases might also be involved (discussed below), it is interesting to note that full-length CgA1-439 can induce, in endothelial cells, the production of protease-nexin 1 (a potent inhibitor of plasmin, plasminogen activators, and thrombin [21]), and that the plasminogen activator inhibitor-1 inhibits CgA cleavage to CgA1-373 by cultured endothelial cells [43]. It is therefore tempting to speculate that changes in the relative levels of these protease/anti-protease molecules in tumor lesions represent a major mechanism for the regulation of the CgA-dependent angiogenic switch.

Regarding the second question, the results of biochemical studies suggest that CgA1-373 can bind to neuropilin-1 (NRP-1) with high affinity (Kd = 3.49 ± 0.73 nM), via its C-terminal PGPQLR373 sequence [37]. The functional role of this ligand-receptor interaction is suggested by the fact that the pro-angiogenic effects of this fragment are blocked by antineuropilin-1 or anti-PGPQLR373 antibodies. No interaction of full-length CgA and CgA1-372 with neuropilin-1 occurs, indicating that the PGPQLR373 binding site is cryptic in the fulllength precursor and that the C-terminal arginine residue (R373, which is absent in CgA372) is necessary for the binding [37].

Studies on the topology of the NRP-1-binding site showed that CgA1-373 and short peptides containing the PGPQLR sequence interact with a pocket of the *b1* domain of the receptor, a site that recognizes peptides and polypeptides ending with the so-called C-end Rule (CendR) motif (R/K-X-X-R/K, as in the prototypical CendR peptide RPARPAR). Remarkably, this binding pocked can also accommodate and bind the C-terminal sequence of VEGF165, a pro-angiogenic factor that contains a CendR motif (CDKPRR) [44,45]; thus,

CgA1-373, VEGF165, and even the short PGPQLR and RPARPAR peptides, all with a C-terminal arginine, compete for the same binding pocket of the *b1* domain of NRP-1. Although the PGPQLR373 sequence cannot be fully considered a CendR motif, as it lacks the first R/K residue of the consensus sequence, it is interesting to note this sequence, such as the CendR motif, ends with an arginine that is crucial for NRP-1 recognition.

The importance of the C-terminal arginine of CgA1-373 for neuropilin-1 recognition is also supported by the results of molecular docking and molecular dynamics experiments, performed with CgA352-372 (catestatin) and CgA352-373 (catestatin-R). Despite these compounds differ only for the presence of an arginine (C-terminal sequence: PGPQL in catestatin; PGPQLR in catestatin-R), a clear difference in the interaction with NRP-1 was observed [46]. The interaction of catestatin-R with neuropilin-1 showed strong similarity with that of the compound EG00229, a small inhibitor of NRP-1 that contains an arginine with a free carboxyl group and whose structure in the complex with NRP-1 has been resolved by crystallography studies. In both cases, complex formation is driven by salt bridges between the guanidine moiety of the C-terminal arginine of the ligand and the carboxyl group of an aspartate residue of neuropilin-1 (D48) [46]. Remarkably, despite the presence of other positively charged arginine residues and amino-groups in catestatin-R, the best binding mode was obtained with the interaction of the C-terminal R373 of the ligand with D48 of the receptor.

Another important question raised by these findings concerns the possible involvement of co-receptors. Experimental evidence showed that the pro-angiogenic activity of CgA1-373 in assays based on endothelial cell spheroids can be inhibited by mecamylamine and α-bungarotoxin, two antagonists of nicotinic acetylcholine receptors [37]. Considering that (a) the nicotinic acetylcholine receptors are expressed on endothelial cells and are known to contribute to the regulation of angiogenesis [47–50], and (b) catestatin (CgA352-372) is known to bind nicotinic acetylcholine receptors [51–53], this class of receptors may represent important co-receptors for CgA1-373 signaling in endothelial cells. It cannot be excluded, however, that other receptor systems are also involved.

A final point that should be discussed concerns the issue of counterregulatory mechanisms. Experimental evidence suggests that R373 is rapidly removed from CgA1-373 by plasma carboxypeptidases, when this fragment is released in circulation [37]. Given the importance of R373 for neuropilin-1 recognition, the cleavage of CgA1-373 to form CgA1-372 may represent an important mechanism to limit the CgA1-373 activity at the site of its production (for example, in cancer lesions) and to avoid systemic effects.

Thus, the results obtained so far support a model in which cleavage of the R373R374 bond of circulating CgA, followed by neuropilin-1 engagement in tumors, and the subsequent removal of R373 in plasma, represent a sort of "*off*/*on*/*off* " switch for the spatiotemporal regulation of angiogenesis in tumor lesions (see Figure 1A for a schematic representation of the model).

Interestingly, it is well known that pro-hormone convertases can cleave proteins at dibasic sites (R/K-R/K), and that carboxypeptidase H/E remove the C-terminal R or K. It is possible that also these enzymes are brought into play in the regulation of the CgAdependent angiogenic switch. Indeed, it is possible that a lower expression, or a reduced activity, of carboxypeptidase H/E in tissues in which CgA1-373 is overproduced (see below) may contribute to activate the pro-angiogenic switch. On the other hand, the normal expression/function of carboxypeptidase H/E in other tissues might have a role in the generation of the anti-angiogenic vasostatin-1 fragment (by removal of K77 after cleavage of the K77/K78 dibasic site) and CgA1-372 (by removal of R373 after cleavage of the R373/R374 dibasic site), thereby promoting an anti-angiogenic effect, a hypothesis that deserves to be tested.

**Figure 1.** Hypothetical model of the CgA-dependent "*off*/*on*/*off* " switch for the regulation of angiogenesis in tumor and its inhibition by anti-PGPQLR antibodies. (**A**) Mechanisms of activation/deactivation of the NRP-1 binding site of chromogranin A. According to this model cleavage of the R373R374 peptide bond of full-length CgA (CgA1-439) leads to exposure of the PGPQLR sequence, a site that can recognize the CendR-binding pocket of the *b1* domain of neuropilin-1 (NRP-1) on endotal cells. Removal of the R373 residue by carboxypeptidases causes loss of NRP-1 recognition [37]. (**B**) Mechanism of anti-tumor activity of anti-PGPQLR antibodies. Cleavage of bloodborne full-length CgA (CgA1-439) in tumors, e.g., by plasmin or thrombin, causes loss of anti-angiogenic CgA1-439 and generates the pro-angiogenic CgA1-373 fragment, which may interact with NRP-1 and contribute to promote angiogenesis and tumor growth. Antibodies against the NRP-1 binding site of CgA1-373 (anti-PGPQLR antibodies) block the CgA1-373/NRP-1 interaction and, consequently inhibit angiogenesis and tumor growth [37]. This schematic representation has been prepared using the BioRender software.
