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.