Acknowledgments
We thank the many lab members and colleagues over several decades whose insights and hard work contributed to these studies. Supported by: NIH R01-DK032948 and R01-DK032949 (BAE, REM); NIH R01-GM051293 (BAE, Stephen M. King); The Daniel Schwartzberg Fund (BAE, REM); NIH R21-GM140390 and R15-GM073659 (DJM); Eppley Foundation for Research (DJM); Milheim Foundation for Cancer Research (DJM); Unigene Laboratories, Inc. (DJM).Figure Legends
Figure 1. Peptide processing basics, the amidation reaction and amidation enzymes. A. The preprotein peptide precursor is processed by endoproteases (prohormone convertases such as PCSK1, PCSK2, PCSK3) and an exoprotease (such as CPE or CPD) to present to PAM the immediate precursor (peptide-Gly), which yields the final amidated peptide product. B. The two-step PAM reaction. Step 1 is performed by the first enzyme, PHM, and Step 2 is performed by PAL. The two copper residues bound to PHM are reduced by ascorbate, peptide and dioxygen bind to the reduced enzyme, a proton is abstracted and the peptide is hydroxylated by PHM. The N-C bond is then cleaved by PAL, liberating the α-amidated peptide plus glyoxalate. C. Proteins used for enzymology and structural determination. In mammals, two major forms of PAM are the bifunctional membrane enzymes PAM1 and PAM2, each with a transmembrane domain (TMD) and short cytoplasmic domain (CD). The proteins used thus far for structural analyses are the recombinant catalytic cores, PHMcc and PALcc. The reactions have been studied with purified natural proteins from various sources, recombinant pure rat PHMcc and PALcc, and with two pure recombinant bifunctional proteins, rat PHM-PAL820s and Type A a-AE. References given in the text. Residues (using NP_037132.2): PHMcc (42-356), PALcc (498-820, lacking N-glycosylation [S767A]), PHM-PAL820s (42-820 lacking Exon 16 [residues 393-498], lacking N-glycosylation [S767A]), Type A PAM (27-820 lacking Exon 16).
Figure 2. The Reactions Catalyzed by PHM and PAL. Bifunctional PAM is compromised of the two separate catalytic units, PHM and PAL (Reaction A). Steps 4 and 5 in reaction A represent a collection of steps in the PHM mechanism (Cowley et al., 2016; Prigge et al., 2000; Wu et al., 2019). Reactions B and C catalyzed by PHM, any involvement of PAL in reactions B and C have not been specifically addressed. TheS -dealkylation reaction, shown in reaction B, is consistent with the finding of glyoxylate as a minor product during the sulfoxidation reaction. A sulfoxide/glyoxylate ratio of 8 was reported, but no mercaptan was found (A.G. Katopodis & S.W. May, 1990). The formation of glyoxylate from the imino-oxy acetate (reaction D, bottom reaction) is PAL-independent.
Figure 3. Ligands that Bind to PHM and PAL. A.Structures for the inhibitors and activators discussed in this review.B. In silico model for the interaction of PHMcc withN -hydrocinnamoyl-L-Phe-L-homocysteine, IC50 = 10 nM (compound #22 from (Erion et al., 1994)). C. In silico model for the interaction of PHMcc and mimosine. D.In silico model for the interaction better PHMcc and mimosine or ascorbate. E. In silico model of the interaction of PHMcc with N-α-acetyl-3,5-diodotyrosylglycine and ascorbate. Our model is based on the published model of the PHMcc complexed with the same dipeptide (Prigge et al., 2000). The copper atoms are shown in brown (B-E) and the PHMcc backbone is either gray (B) or light green (C-E). For Panels C-E, the amino acid ligands for the copper atoms are in blue and binding site amino acid side chains are in dark green. The colors for the ligands are as follows: N -hydrocinnamoyl-L-Phe-L-Phe is purple (B), mimosine is purple (C and D), ascorbate is brownish-yellow (D and E), and N -α-acetyl-3,5-diiodotyrosylglycine is gray (E). The in silico models were generated using AutoDock Vina.N -Hydrocinnamoyl-L-Phe-L-homocysteine was docked using the flexible side chain method, with the ligand covalently attached and then modeled as a flexible residue (Bianco, Forli, Goodsell, & Olson, 2016).
Figure 4. A. PAM trafficking in neuroendocrine cells. As newly synthesized PAM and soluble cargo proteins exit the trans -Golgi, they accumulate in immature secretory granules. Granule maturation involves vesicular trafficking and acquisition of the cytosolic proteins needed to respond to secretagogues. Upon fusion of the secretory granule membrane with the plasma membrane, soluble content proteins are released and membrane PAM appears on the cell surface. Clathrin-mediated endocytosis means that less than 5% of the PAM protein in a cell typically resides on the plasma membrane. PAM retrieved from the cell surface can be degraded or returned to the secretory pathway for re-use.B. In C. reinhardtii , PAM is localized to the Golgi Complex, small vesicular structures and the ciliary membrane. The ciliary budding process that generates ectosomes is illustrated, with the localization of CrPAM and one of its amidated products (Cre03.g20450) illustrated (Luxmi et al., 2018; Luxmi et al., 2019).C. The movement of PAM through late endosomes and into the intraluminal vesicles (ILVs) that form in multivesicular bodies (MVBs) was determined using ectodomain antibodies (Bäck et al., 2017; Rajagopal, Mains, & Eipper, 2012). Upon fusion with the plasma membrane, PAM-containing exosomes are released. D. The species-specific roles of PAM in ciliogenesis are summarized.E. The non-catalytic effects of PAM are summarized. The ability of PAM to alter gene expression is thought to require the generation of sfCD through γ-secretase-catalyzed regulated intramembrane proteolysis (RIP) (Rajagopal, Stone, Mains, & Eipper, 2010). Neither the ability of PAM to support the formation of atrial granules or its ability to interact with actin require its catalytic activity.