Ubiquitin and ubiquitin-derived peptides as substrates for peptidylglycine α-amidating monooxygenase (original) (raw)
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Peptidylglycine α-amidating monooxygenase as a therapeutic target or biomarker
2021
Peptides play a key role in controlling many physiological and neurobiological pathways. Many bioactive peptides require a C-terminal α-amide for full activity. The bifunctional enzyme catalyzing α-amidation, peptidylglycine α-amidating monooxygenase (PAM), is the sole enzyme responsible for amidated peptide biosynthesis, from Chlamydomonas reinhardtii to Homo sapiens. Many neuronal and endocrine functions are dependent upon amidated peptides; additional amidated peptides are growth promoters in tumors. The amidation reaction occurs in two steps, glycine α-hydroxylation followed by dealkylation to generate the α-amide product. Currently, most potentially useful inhibitors target the first reaction, which is rate-limiting. PAM is a membrane-bound enzyme that visits the cell surface during peptide secretion. PAM is then used again in the biosynthetic pathway, meaning that cell-impermeable inhibitors or inactivators could have therapeutic value for the treatment of cancer or psychiatri...
Structure of the Precursor to an Enzyme Mediating COOH-Terminal Amidation in Peptide Biosynthesis
Molecular Endocrinology, 1987
Many bioactive peptides terminate with an amino acid a-amide at their COOH terminus. The enzyme responsible for this essential posttranslational modification is known as peptidyl-glycine a-amidating monooxygenase or PAM. We identified cDNAs encoding the enzyme by using antibodies to screen a bovine intermediate pituitary Agt11 expression library. Antibodies to a 0-galactosidase/PAM fusion protein removed PAM activity from bovine pituitary homogenates. The 108,207 dalton protein predicted by the complete cDNA is approximately twice the size of purified PAM. An NH 2 -terminal signal sequence and short propeptide precede the NH 2 terminus of purified PAM. The sequences of several PAM cyanogen bromide peptides were localized in the NHz-terminal half of the predicted protein. The cDNA encodes an additional 430 amino acid intragranular domain followed by a putative membrane spanning domain and a hydrophilic cytoplasmic domain. The forms of PAM purified from bovine neurointermediate pituitary may be generated by endoproteolytic cleavage at a subset of the 10 pairs of basic amino acids in the precursor. High levels of PAM mRNA were found in bovine pituitary and cerebral cortex. In corticotropic tumor cells, levels of PAM mRNA and pro-ACTH/endorphin mRNA were regulated in parallel by glucocorticoids and CRF.
Signaling Mediated by the Cytosolic Domain of Peptidylglycine α-Amidating Monooxygenase
Molecular Biology of the Cell, 2001
The luminal domains of membrane peptidylglycine α-amidating monooxygenase (PAM) are essential for peptide α-amidation, and the cytosolic domain (CD) is essential for trafficking. Overexpression of membrane PAM in corticotrope tumor cells reorganizes the actin cytoskeleton, shifts endogenous adrenocorticotropic hormone (ACTH) from mature granules localized at the tips of processes to the TGN region, and blocks regulated secretion. PAM-CD interactor proteins include a protein kinase that phosphorylates PAM (P-CIP2) and Kalirin, a Rho family GDP/GTP exchange factor. We engineered a PAM protein unable to interact with either P-CIP2 or Kalirin (PAM-1/K919R), along with PAM proteins able to interact with Kalirin but not with P-CIP2. AtT-20 cells expressing PAM-1/K919R produce fully active membrane enzyme but still exhibit regulated secretion, with ACTH-containing granules localized to process tips. Immunoelectron microscopy demonstrates accumulation of PAM and ACTH in tubular structures a...
Mechanistic studies of peptidylglycine alpha-amidating monooxygenase (PAM)
2008
Peptide hormones are responsible for cellular functions critical to the survival of an organism. Approximately 50% of all known peptide hormones are post-translationally modified at their C-terminus. Peptidylglycine alpha-amidating monooxygenase (PAM) is a bi-functional enzyme which catalyzes the activation of peptide pro-hormones. PAM also functionalizes long chain N-acylglycines suggesting a potential role in signaling as their respective fatty acid amides.
The Journal of cell biology, 1992
Peptidylglycine alpha-amidating monooxygenase (PAM: EC 1.14.17.3) is a bifunctional protein which catalyzes the COOH-terminal amidation of bioactive peptides; the NH2-terminal monooxygenase and mid-region lyase act in sequence to perform the peptide alpha-amidation reaction. Alternative splicing of the single PAM gene gives rise to mRNAs generating PAM proteins with and without a putative transmembrane domain, with and without a linker region between the two enzymes, and forms containing only the monooxygenase domain. The expression, endoproteolytic processing, storage, and secretion of this secretory granule-associated protein were examined after stable transfection of AtT-20 mouse pituitary cells with naturally occurring and truncated PAM proteins. The transfected proteins were examined using enzyme assays, subcellular fractionation, Western blotting, and immunocytochemistry. Western blots of crude membrane and soluble fractions of transfected cells demonstrated that all PAM prote...
Archives of Biochemistry and Biophysics, 2003
The C-terminal a-amide moiety of most peptide hormones arises by the posttranslational cleavage of a glycine-extended precursor in a reaction catalyzed by bifunctional peptidylglycine a-amidating monooxygenase (PAM). Glutathione and the S-alkylated glutathiones have a C-terminal glycine and are, thus, potential substrates for PAM. The addition of PAM to glutathione, a series of S-alkylated glutathiones, and leukotriene C 4 results in the consumption of O 2 and the production of the corresponding amidated peptide and glyoxylate. This reaction proceeds in two steps with the intermediate formation of a C-terminal a-hydroxyglycineextended peptide. Amidated glutathione (cGlu-Cys-amide) is a relatively poor substrate for glutathione S-transferase with a V =K value that is 1.3% of that for glutathione. Peptide substrates containing a penultimate hydrophobic or sulfur-containing amino acid exhibit the highest ðV =KÞ app values for PAM-catalyzed amidation. The S-alkylated glutathiones incorporate both features in the penultimate position with S-decylglutathione having the highest (V =KÞ app of the substrates described in this report.
The Journal of biological chemistry, 1993
The production of alpha-amidated peptides is accomplished through the sequential action of two enzymes, peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), that are contained within the bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) protein. Tissue-specific alternative splicing and endoproteolysis are known to generate both soluble and integral membrane mono- and bifunctional PAM proteins. In order to investigate the functional consequences of these differences we purified PAM-3, a soluble 95-kDa bifunctional form of the enzyme, from the spent medium of stably transfected hEK-293 cells. Using NH2-terminal sequence analysis of products of limited endoproteolysis and antibody cross-reactivity we identified protease-sensitive regions at the NH2 terminus, between the 35-kDa PHM and 42-kDa PAL domains and at the COOH terminus of the protein. Endoproteolytic removal of the COOH-terminal region from the bi...