**8. Dipeptidyl peptidase 3**

hydrophobic residues and cleaves multiple peptide substrates. In regard to the RAS, thimet oligopeptidase hydrolyzes Ang I exclusivity at the Pro-Phe bond to form Ang-(1-7) (**Figure 1**). We recently showed that both neprilysin and thimet oligopeptidase contributed to the processing of Ang I to Ang-(1-7) within isolated mitochondria [21]. In the human proximal tubule HK-2 cell line, cytosolic thimet oligopeptidase was the sole activity responsible for the generation of Ang-(1-7) from exogenous Ang I [57]. Moreover, thimet oligopeptidase activity in isolated nuclei processed Ang I exclusively to Ang-(1-7) and may be a potential candidate that contributes to the nuclear levels of Ang-(1-7) within the cell [22]. The RAS was originally characterized as a classic endocrine or circulating system, but there is very compelling evidence for the intracellular expression of the RAS peptides, receptors, and synthetic components [7, 54, 58–60]. It is presently unclear the cellular mechanisms for the intracellular expression of Ang II or Ang-(1-7), although the intracellular peptidase thimet oligopeptidase may play a role

ACE2 is a membrane-bound monocarboxypeptidase [EC 3.4.17.23; 120 kDa] that converts Ang II directly to Ang-(1-7) (**Figure 1**). ACE2 was initially characterized as a homolog to ACE and exhibits approximately ~40% homology with ACE [60–62]. ACE2 was initially reported to cleave Ang I not to Ang II, but to the nonapeptide Ang-(1-9) [63]. Subsequent studies found that Ang II exhibits far better kinetic values for ACE2 that would argue that the endogenous substrate is Ang II, not Ang I [64]. Among a number of peptide substrates (>100) that were screened for ACE2, Vickers et al. reported that only apelin 13 exhibited comparable kinetic values to that of Ang II [65]. In the murine heart, Ang II was primarily converted to Ang-(1-7) by ACE2 and that in the presence of the ACE2 inhibitor MLN-4760 or in ACE2 null mice there was essentially no metabolism of Ang II in the heart [66]. By contrast, under identical kinetic conditions, Ang I was primarily converted to Ang-(1-9) by carboxypeptidase A and not ACE2

In comparison to ACE, the circulating levels of ACE2 are typically quite low. Whether this reflects a reduced degree of shedding, lower vascular expression or both is not presently clear. Rice et al. reported that the molar concentration of ACE in human serum averaged 7 nM in over 500 subjects while ACE2 content was *200-fold lower* (33 pM) and was detectable in <10% of the patient population [67]. Circulating NEP content (290 pM) was also lower than ACE and evident in <30% of these patients [67]. Serum and urinary ACE2 activities are elevated in diabetes, heart failure, and hypertension [68–70]. Circulating ACE2 activity increased approximately threefold in the diabetic hypertensive mRen2.Lewis rat; however, serum ACE activity also increased in the diabetic rats. As measured under identical kinetic conditions, serum ACE was far higher than ACE2 suggesting that the capacity to generate Ang II (or metabolize Ang- (1-7) and bradykinin) remains greater than the capability to form Ang-(1-7) from Ang II [68].

Similar to ACE, ACE2 has a potentially significant role in the RAS pathway as a single catalytic step metabolizes Ang II to attenuate the Ang II-AT1 receptor pathway, and generate Ang-(1-7)

in the cellular Ang-(1-7) axis.

8 Enzyme Inhibitors and Activators

**7. Angiotensin-converting enzyme 2**

in both the wild-type and ACE2 knockout mice.

We demonstrated a role for ACE in the metabolism of Ang-(1-7), but there are other potential pathways that may regulate endogenous levels of the peptide [95]. Marshall and colleagues reported that ACE and a second peptidase activity in the sheep cerebrospinal fluid degraded Ang-(1-7) [95–97]. Interestingly, the non-ACE-degrading activity accounted for a greater contribution of metabolism than ACE [96]. Moreover, this activity was inversely correlated to CSF levels of Ang-(1-7) in control and betamethasone-exposed sheep, a model of fetal programming that exhibits elevated blood pressure and an attenuated baroreflex [7]. Subsequent

**Figure 2.** Kinetics of human DPP 3 hydrolysis of Ang-(1-7) and Ang-(3-7). (A) Chromatographs reveal that human DPP 3 cleaves Ang-(1-7) to the tripeptide Ang-(5-7). (B) Chromatograph reveals that the JMV-390 inhibitor attenuates Ang- (1-7) metabolism by human DPP 3. (C) Kinetic analysis of the hydrolysis of Ang-(1-7) and Ang-(3-7) by human DPP 3. Data adapted from Cruz-Diaz et al. [99].

studies found the Ang-(1-7)-degrading activity in sheep brain and kidney cortex, as well as in the human proximal tubule HK-2 cell line [57, 98]. The enzyme activity exhibited unusual characteristics as Ang I and other peptides equal to or greater than 10 residues were not substrates for the peptidase [57, 98]. In addition, the peptidase was sensitive to both chelating agents such as o-phenanthroline and EDTA, and the sulfhydryl inhibitors APMA and PCMB [57, 98]. Additional studies identified the inhibitor JMV-390, originally developed to block the metalloendopeptidases neprilysin, thimet oligopeptidase, and neurolysin, that potently inhibited the Ang-(1-7)-degrading activity in the brain and kidney [IC50 <1 nM] [57, 98]. Conversely, specific inhibitors against these endopeptidases did not attenuate the Ang-(1-7) degrading activity [98]. Interestingly, the peptidase activity accounted for the sole degradative pathway in the cytosolic fraction and in the media of the HK-2 cells [57]. Utilizing the HK-2 cells as the source of the Ang-(1-7)-degrading activity, we recently purified the peptidase from the cell cytosol by ion exchange and hydrophobic interaction chromatography and identified the enzyme as dipeptidyl peptidase 3 [EC 3.4. 14.4, DPP 3] [99].

DPP 3 belongs to a family of metalloaminopeptidases that sequentially cleave two residues from the N-terminus of peptides no more than eight residues in length and this likely explains our previous results that Ang I, apelin-13, and neurotensin were not substrates for the Ang- (1-7)-degrading activity in the CSF and brain [98]. We obtained a human recombinant form of Peptidases and the Renin-Angiotensin System: The Alternative Angiotensin-(1-7) Cascade http://dx.doi.org/10.5772/65949 11

studies found the Ang-(1-7)-degrading activity in sheep brain and kidney cortex, as well as in the human proximal tubule HK-2 cell line [57, 98]. The enzyme activity exhibited unusual characteristics as Ang I and other peptides equal to or greater than 10 residues were not substrates for the peptidase [57, 98]. In addition, the peptidase was sensitive to both chelating agents such as o-phenanthroline and EDTA, and the sulfhydryl inhibitors APMA and PCMB [57, 98]. Additional studies identified the inhibitor JMV-390, originally developed to block the metalloendopeptidases neprilysin, thimet oligopeptidase, and neurolysin, that potently inhibited the Ang-(1-7)-degrading activity in the brain and kidney [IC50 <1 nM] [57, 98]. Conversely, specific inhibitors against these endopeptidases did not attenuate the Ang-(1-7) degrading activity [98]. Interestingly, the peptidase activity accounted for the sole degradative pathway in the cytosolic fraction and in the media of the HK-2 cells [57]. Utilizing the HK-2 cells as the source of the Ang-(1-7)-degrading activity, we recently purified the peptidase from the cell cytosol by ion exchange and hydrophobic interaction chromatography and identified

**Figure 2.** Kinetics of human DPP 3 hydrolysis of Ang-(1-7) and Ang-(3-7). (A) Chromatographs reveal that human DPP 3 cleaves Ang-(1-7) to the tripeptide Ang-(5-7). (B) Chromatograph reveals that the JMV-390 inhibitor attenuates Ang- (1-7) metabolism by human DPP 3. (C) Kinetic analysis of the hydrolysis of Ang-(1-7) and Ang-(3-7) by human DPP 3.

DPP 3 belongs to a family of metalloaminopeptidases that sequentially cleave two residues from the N-terminus of peptides no more than eight residues in length and this likely explains our previous results that Ang I, apelin-13, and neurotensin were not substrates for the Ang- (1-7)-degrading activity in the CSF and brain [98]. We obtained a human recombinant form of

the enzyme as dipeptidyl peptidase 3 [EC 3.4. 14.4, DPP 3] [99].

Data adapted from Cruz-Diaz et al. [99].

10 Enzyme Inhibitors and Activators

**Figure 3.** The JMV-390 inhibitor reduces DPP 3 activity and alters Ang-(1-7) cellular content of HK-2 cells. (A) Increasing doses of the JMV-390 inhibitor reduced DPP 3 activity (125I-Ang-(3-4) generation—fmol/min/mg protein) in HK-2 cells. (B) Low-dose JMV-390 (20 nM) tended to increase the cellular content of Ang-(1-7) (fmols/mg protein), but high dose (200 nM) significantly reduced Ang-(1-7). \* *P* < 0.05 versus control; # *P* < 0.05 versus 20 nM JMV. Data adapted from Cruz-Diaz et al. [99].

DPP 3 to show that the enzyme metabolized Ang-(1-7) in two cleavage steps [99]. DPP 3 initially hydrolyzes Ang-(1-7) at the Arg2 -Val3 bond to form Ang-(3-7) and the dipeptide Arg1 -Asp2 . Ang-(3-7) is then very rapidly cleaved at Tyr4 -Ile5 to form Ang-(5-7) and Val3 -Tyr4 . The kinetic analysis of DPP 3 hydrolysis revealed a higher efficiency constant (kcat/km) for Ang-(3-7) than Ang-(1-7) (**Figure 2**) [99]. The preferred hydrolysis of Ang-(3-7) by DPP 3 would account for our inability to demonstrate the accumulation of Ang-(3-7) following the initial metabolism of Ang-(1-7). In regard to an *in vivo* role for DPP 3 to modulate Ang-(1-7), human HK-2 cells were treated with varying doses of the JMV-390 inhibitor and we assessed both the endogenous content of Ang-(1-7) and the intracellular DPP-3/Ang-(1-7)-degrading activity in the cells. As shown in **Figure 3**, 20- and 200-nM JMV-390 reduced DPP3 activity by >30 and >80%, respectively, as compared to control suggesting that the inhibitor effectively penetrates the cells [99]. The lower dose of JMV increased the cellular content Ang-(1-7) approximately twofold, although this did not reach statistical significance. The higher dose of JMV, however, significantly reduced the intracellular levels of the peptide [99]. We interpret the latter results that the high dose may spill over to block other peptidases including thimet oligopeptidase that may be involved in the generation of Ang-(1-7) in the renal cells [57]. Thus, the blockade of Ang-(1-7)-forming enzymes by the high-dose JMV may override any protective effects of DPP 3 inhibition. We are currently assessing alternative approaches to block DPP 3 within the HK-2 cells and assess both secreted and intracellular levels of Ang-(1-7), as well as Ang-(1-7) dependent actions on these cells.
