Probing the Production of Amidated Peptides following Genetic and Dietary Copper Manipulations

Amidated neuropeptides play essential roles throughout the nervous and endocrine systems. Mice lacking peptidylglycine α-amidating monooxygenase (PAM), the only enzyme capable of producing amidated peptides, are not viable. In the amidation reaction, the reactant (glycine-extended peptide) is converted into a reaction intermediate (hydroxyglycine-extended peptide) by the copper-dependent peptidylglycine-α-hydroxylating monooxygenase (PHM) domain of PAM. The hydroxyglycine-extended peptide is then converted into amidated product by the peptidyl-α-hydroxyglycine α-amidating lyase (PAL) domain of PAM. PHM and PAL are stitched together in vertebrates, but separated in some invertebrates such as Drosophila and Hydra. In addition to its luminal catalytic domains, PAM includes a cytosolic domain that can enter the nucleus following release from the membrane by γ-secretase. In this work, several glycine- and hydroxyglycine-extended peptides as well as amidated peptides were qualitatively and quantitatively assessed from pituitaries of wild-type mice and mice with a single copy of the Pam gene (PAM+/−) via liquid chromatography-mass spectrometry-based methods. We provide the first evidence for the presence of a peptidyl-α-hydroxyglycine in vivo, indicating that the reaction intermediate becomes free and is not handed directly from PHM to PAL in vertebrates. Wild-type mice fed a copper deficient diet and PAM+/− mice exhibit similar behavioral deficits. While glycine-extended reaction intermediates accumulated in the PAM+/− mice and reflected dietary copper availability, amidated products were far more prevalent under the conditions examined, suggesting that the behavioral deficits observed do not simply reflect a lack of amidated peptides

Genetic and nutritional reductions in peptide amidation result in neuroendocrine deficits in mice

Genetic and pharmacological manipulation demonstrated the neurological and metabolic importance of specific amidated peptides and highly conserved enzymes involved in peptide synthesis. Deletion of the only mammalian peptide amidating enzyme, peptidylglycine a-amidating monooxyenase (PAM), resulted in developmental lethality. The goal of this dissertation was to investigate the neurophysiological importance of haplioinsufficient reductions in PAM in mice following genetic and nutritional manipulation. It was hypothesized that physiological functions requiring multiple amidated peptides would be most sensitive to genetic and nutritional reductions in the posttranslational peptide amidation which is necessary to confer their bioactivity: namely temperature regulation, vasoconstriction, seizure sensitivity, and anxiety-like behavior. PAM requires copper to amidate peptides; copper is also critical for many physiological functions which require multiple amidated peptides. As a result, it was hypothesized that supplementation of dietary copper availability would improve peptide amidation and physiological functions requiring multiple amidated peptides. Conversely, it was hypothesized that reductions in dietary copper availability would impair peptide amidation and physiological functions requiring multiple amidated peptides. To address these hypotheses, dietary copper availability was manipulated in mice heterozygous for the PAM gene (PAM+/-) and their wild-type (WT) littermates. ^ Impairments in physiological function were identified in PAM+/- mice compared to their WT littermates at multiple levels. Temperature regulation in PAM+/- mice was impaired with acute cold exposure due to a lack of peripheral vasoconstriction. Neurological function was also impaired in PAM+/--mice at two levels: (1) increased sensitivity to acute pharmacologically-induced seizures; and (2) increased anxiety-like behavior in the elevated zero maze. Variations in dietary copper availability altered peptide amidation and physiological function. Dietary copper supplementation improved temperature regulation and vasoconstriction, and reduced anxiety-like behavior in PAM+/- mice. Copper deficient WT mice exhibited the following deficits: (1) impaired peptide amidation; (2) impaired temperature regulation and vasoconstriction; (3) increased seizure sensivity; (4) increased anxiety-like behavior. Surprisingly, copper metabolism was altered at multiple levels in PAM+/- mice compared to their WT littermates maintained on a copper-adequate control diet. Together, these results demonstrate a novel gene-nutrient interaction between PAM and copper whose balance alters multiple physiological functions.

Effects of PAM haploinsufficiency and dietary copper (Cu) on the amidation of joining peptide (JP) and arginine-vasopressin (AVP).

<p>Pituitary samples were taken from WT and PAM^+/− mice maintained on normal, copper-deficient or copper-supplemented diets. In order to take into account differences in the sample preparation and mass spectrometry, for each peptide in each sample, the ratio of glycine-extended to amidated peptide was calculated, with higher numbers indicating an accumulation of glycine-extended peptide. The effects of (<b>A</b>) haploinsufficiency, (<b>B</b>) copper deficiency and (<b>C</b>) copper supplementation were then assessed by comparing the ratios between the pairs of samples to indicate the extent of the glycine-extended peptide accumulation. For example, (A) reports the WT normal diet value divided by the PAM^+/− normal diet value. N is the number of biological replicate pairs. Error bar, SEM. Student's <i>t</i>-test: ***, <i>p</i><0.0005; **, <i>p</i><0.05; *, <i>p</i><0.1.</p

Analysis of mouse pituitary extracts using capillary LC coupled to ESI-IT MS².

<p>Mass spectra of amidated (top) and glycine-extended (bottom) forms of (<b>A</b>) synthetic adrenocorticotropic hormone (ACTH) (1-13), and (<b>B</b>) endogenous joining peptide (JP). The assignment of b- and y-ions matches expected fragments within 0.2 Da.</p

Amidated peptides identified in adult male mouse pituitary extracts.

<p>All masses listed here are monoisotopic. Abbreviations: MSH, melanocyte stimulating hormone; JP, joining peptide.</p

Conversion of ACTH(1-13)-Gly-OH to its amidated form ACTH(1-13)-NH₂ during the measurement process when analyzed by MALDI-TOF MS and ESI-IT MS².

<p>(<b>A</b>) The table shows the assessment of the amidated and hydroxyglycine-extended ACTH(1-13) peaks in MALDI-TOF MS. (<b>B</b>) With manual ESI-IT MS², ACTH(1-13)-NH₂ and ACTH(1-13)-Gly-OH were respectively isolated, and then fragmented. The MS² of ACTH(1-13)-NH₂ matches the MS³ of ACTH(1-13)-Gly-OH.</p

Long-term neurological symptoms after acute COVID-19 illness requiring hospitalization in adult patients: insights from the ISARIC-COVID-19 follow-up study

in this study we aimed to characterize the type and prevalence of neurological symptoms related to neurological long-COVID-19 from a large international multicenter cohort of adults after discharge from hospital for acute COVID-19