Peptidylglycine α-amidating monooxygenase is required for atrial secretory granule formation.

The discovery of atrial secretory granules and the natriuretic peptides stored in them identified the atrium as an endocrine organ. Although neither atrial nor brain natriuretic peptide (ANP, BNP) is amidated, the major membrane protein in atrial granules is peptidylglycine α-amidating monooxygenase (PAM), an enzyme essential for amidated peptide biosynthesis. Mice lacking cardiomyocyte PAM (PamMyh6-cKO/cKO) are viable, but a gene dosage-dependent drop in atrial ANP and BNP content occurred. Ultrastructural analysis of adult PamMyh6-cKO/cKO atria revealed a 13-fold drop in the number of secretory granules. When primary cultures of Pam0-Cre-cKO/cKO atrial myocytes (no Cre recombinase, PAM floxed) were transduced with Cre-GFP lentivirus, PAM protein levels dropped, followed by a decline in ANP precursor (proANP) levels. Expression of exogenous PAM in PamMyh6-cKO/cKO atrial myocytes produced a dose-dependent rescue of proANP content; strikingly, this response did not require the monooxygenase activity of PAM. Unlike many prohormones, atrial proANP is stored intact. A threefold increase in the basal rate of proANP secretion by PamMyh6-cKO/cKO myocytes was a major contributor to its reduced levels. While proANP secretion was increased following treatment of control cultures with drugs that block the activation of Golgi-localized Arf proteins and COPI vesicle formation, proANP secretion by PamMyh6-cKO/cKO myocytes was unaffected. In cells lacking secretory granules, expression of exogenous PAM led to the accumulation of fluorescently tagged proANP in the cis-Golgi region. Our data indicate that COPI vesicle-mediated recycling of PAM from the cis-Golgi to the endoplasmic reticulum plays an essential role in the biogenesis of proANP containing atrial granules.

Multiple roles for peptidylglycine α-amidating monooxygenase in the response to hypoxia.

The biosynthesis of many of the peptides involved in homeostatic control requires peptidylglycine α-amidating monooxygenase (PAM), an ancient, highly conserved copper- and ascorbate-dependent enzyme. Using the production of amidated chromogranin A to monitor PAM function in tumor cells, physiologically relevant levels of hypoxia were shown to inhibit this monooxygenase. The ability of primary pituitary cells exposed to hypoxic conditions for 4 h to produce amidated chromogranin A was similarly inhibited. The affinity of the purified monooxygenase for oxygen (Km = 99 ± 19 µM) was consistent with this result. The ability of PAM to alter secretory pathway behavior under normoxic conditions required its monooxygenase activity. Under normoxic conditions, hypoxia-inducible factor 1a levels in dense cultures of corticotrope tumor cells expressing high levels of PAM exceeded those in control cells; expression of inactive monooxygenase did not have this effect. The effects of hypoxia on levels of two PAM-regulated genes (activating transcription factor 3 [Atf3] and FK506 binding protein 2 [Fkbp2]) differed in cells expressing high versus low levels of PAM. Putative hypoxia response elements occur in both human and mouse PAM, and hPAM has consistently been identified as one of the genes upregulated in response to hypoxia. Expression of PAM is also known to alter gene expression. A quarter of the genes consistently upregulated in response to hypoxia were downregulated following increased expression of PAM. Taken together, our data suggest roles for PAM and amidated peptide secretion in the coordination of tissue-specific responses to hypoxia.

Mass spectrometric evidence for neuropeptide-amidating enzymes in Caenorhabditis elegans.

Neuropeptides constitute a vast and functionally diverse family of neurochemical signaling molecules and are widely involved in the regulation of various physiological processes. The nematode Caenorhabditis elegans is well-suited for the study of neuropeptide biochemistry and function, as neuropeptide biosynthesis enzymes are not essential for C. elegans viability. This permits the study of neuropeptide biosynthesis in mutants lacking certain neuropeptide-processing enzymes. Mass spectrometry has been used to study the effects of proprotein convertase and carboxypeptidase mutations on proteolytic processing of neuropeptide precursors and on the peptidome in C. elegans However, the enzymes required for the last step in the production of many bioactive peptides, the carboxyl-terminal amidation reaction, have not been characterized in this manner. Here, we describe three genes that encode homologs of neuropeptide amidation enzymes in C. elegans and used tandem LC-MS to compare neuropeptides in WT animals with those in newly generated mutants for these putative amidation enzymes. We report that mutants lacking both a functional peptidylglycine α-hydroxylating monooxygenase and a peptidylglycine α-amidating monooxygenase had a severely altered neuropeptide profile and also a decreased number of offspring. Interestingly, single mutants of the amidation enzymes still expressed some fully processed amidated neuropeptides, indicating the existence of a redundant amidation mechanism in C. elegans All MS data are available via ProteomeXchange with the identifier PXD008942. In summary, the key steps in neuropeptide processing in C. elegans seem to be executed by redundant enzymes, and loss of these enzymes severely affects brood size, supporting the need of amidated peptides for C. elegans reproduction.

PAM50 for prediction of response to neoadjuvant chemotherapy for ER-positive breast cancer.

PURPOSE: There is an urgent need for the development of a predictor of response to chemotherapy for ER-positive breast cancer which is less chemosensitive than for ER-negative breast cancer in order to avoid unnecessary chemotherapy. In the present study, intrinsic subtyping by PAM50 was evaluated for its ability to predict a response to chemotherapy. PATIENTS AND METHODS: For this study, 124 patients with ER-positive breast cancer treated with neoadjuvant sequential paclitaxel and FEC (NAC) were evaluated. Tumor biopsy specimens obtained before NAC were subjected to intrinsic subtyping (IS) by gene expression (GE) using PAM50 (PAM50-IS) or immunohistochemistry (IHC-IS). RESULTS: Of the PAM50-ISs (Luminal A, Luminal B, HER2-enriched, and Basal-like), GE-Luminal A showed the lowest pCR rate (1.9%), and multivariate analysis revealed that GE-Luminal A was a significant (P = 0.031) predictor of non-pCR independently of other clinicopathological parameters, including Ki67, and tumor-infiltrating lymphocytes. Of the IHC-ISs, on the other hand, IHC-Luminal A was not significantly associated with pCR. We also found that breast tumors with low ER levels (1-9%), like ER-negative tumors, were mostly GE-HER2-enriched and GE-Basal-like, and more sensitive to NAC than those with high ER levels (≥ 10%). CONCLUSIONS: GE-Luminal A intrinsically subtyped by PAM50 was the least sensitive to NAC and very unlikely to attain pCR. IHC-Luminal A identified by IHC, on the other hand, was not significantly predictive of pCR. In addition, PAM50 revealed that tumors with low ER (1-9%) were more like ER-negative tumors than ER-positive tumors, and most such cases should therefore would better be treated with chemotherapy.

Islet prohormone processing in health and disease.

Biosynthesis of peptide hormones by pancreatic islet endocrine cells is a tightly orchestrated process that is critical for metabolic homeostasis. Like neuroendocrine peptides, insulin and other islet hormones are first synthesized as larger precursor molecules that are processed to their mature secreted products through a series of proteolytic cleavages, mediated by the prohormone convertases Pc1/3 and Pc2, and carboxypeptidase E. Additional posttranslational modifications including C-terminal amidation of the ß-cell peptide islet amyloid polypeptide (IAPP) by peptidyl-glycine α-amidating monooxygenase (Pam) may also occur. Genome-wide association studies (GWAS) have showed genetic linkage of these processing enzymes to obesity, ß-cell dysfunction, and type 2 diabetes (T2D), pointing to their important roles in metabolism and blood glucose regulation. In both type 1 diabetes (T1D) and T2D, and in the face of metabolic or inflammatory stresses, islet prohormone processing may become impaired; indeed elevated proinsulin:insulin (PI:I) ratios are a hallmark of the ß-cell dysfunction in T2D. Recent studies suggest that genetic or acquired defects in proIAPP processing may lead to the production and secretion of incompletely processed forms of proIAPP that could contribute to T2D pathogenesis, and additionally that impaired processing of both PI and proIAPP may be characteristic of ß-cell dysfunction in T1D. In islet α-cells, the prohormone proglucagon is normally processed to bioactive glucagon by Pc2 but may express Pc1/3 under certain conditions leading to production of GLP-1(7-36NH2 ). A better understanding of how ß-cell processing of PI and proIAPP, as well as α-cell processing of proglucagon, are impacted by genetic susceptibility and in the face of diabetogenic stresses, may lead to new therapeutic approaches for improving islet function in diabetes.

A "neural" enzyme in nonbilaterian animals and algae: preneural origins for peptidylglycine α-amidating monooxygenase.

Secreted peptides, produced by enzymatic processing of larger precursor molecules, are found throughout the animal kingdom and play important regulatory roles as neurotransmitters and hormones. Many require a carboxy-terminal modification, involving the conversion of a glycine residue into an α-amide, for their biological activity. Two sequential enzymatic activities catalyze this conversion: a monooxygenase (peptidylglycine α-hydroxylating monooxygenase or PHM) and an amidating lyase (peptidyl-α-hydroxyglycine α-amidating lyase or PAL). In vertebrates, these activities reside in a single polypeptide known as peptidylglycine α-amidating monooxygenase (PAM), which has been extensively studied in the context of neuropeptide modification. Bifunctional PAMs have been reported from some invertebrates, but the phylogenetic distribution of PAMs and their evolutionary relationship to PALs and PHMs is unclear. Here, we report sequence and expression data for two PAMs from the coral Acropora millepora (Anthozoa, Cnidaria), as well as providing a comprehensive survey of the available sequence data from other organisms. These analyses indicate that bifunctional PAMs predate the origins of the nervous and endocrine systems, consistent with the idea that within the Metazoa their ancestral function may have been to amidate epitheliopeptides. More surprisingly, the phylogenomic survey also revealed the presence of PAMs in green algae (but not in higher plants or fungi), implying that the bifunctional enzyme either predates the plant/animal divergence and has subsequently been lost in a number of lineages or perhaps that convergent evolution or lateral gene transfer has occurred. This finding is consistent with recent discoveries that other molecules once thought of as "neural" predate nervous systems.

Extracellular ammonia at sites of pulmonary infection with Coccidioides posadasii contributes to severity of the respiratory disease.

Coccidioides is the causative agent of a potentially life-threatening respiratory disease of humans. A feature of this mycosis is that pH measurements of the microenvironment of pulmonary abscesses are consistently alkaline due to ammonia production during the parasitic cycle. We previously showed that enzymatically active urease is partly responsible for elevated concentrations of extracellular ammonia at sites of lung infection and contributes to both localized host tissue damage and exacerbation of the respiratory disease in BALB/c mice. Disruption of the urease gene (URE) of Coccidioides posadasii only partially reduced the amount of ammonia detected during in vitro growth of the parasitic phase, suggesting that other ammonia-producing pathways exist that may also contribute to the virulence of this pathogen. Ureidoglycolate hydrolase (Ugh) expressed by bacteria, fungi and higher plants catalyzes the hydrolysis of ureidoglycolate to yield glyoxylate and the release CO2 and ammonia. This enzymatic pathway is absent in mice and humans. Ureidoglycolate hydrolase gene deletions were conducted in a wild type (WT) isolate of C. posadasii as well as the previously generated Δure knock-out strain. Restorations of UGH in the mutant stains were performed to generate and evaluate the respective revertants. The double mutant revealed a marked decrease in the amount of extracellular ammonia without loss of reproductive competence in vitro compared to both the WT and Δure parental strains. BALB/c mice challenged intranasally with the Δugh/Δure mutant showed 90% survival after 30 days, decreased fungal burden, and well-organized pulmonary granulomas. We conclude that loss of both Ugh and Ure activity significantly reduced the virulence of this fungal pathogen.

E3 ligases Arf-bp1 and Pam mediate lithium-stimulated degradation of the circadian heme receptor Rev-erb alpha.

The metazoan circadian clock mechanism involves cyclic transcriptional activation and repression by proteins whose degradation is highly regulated via the ubiquitin-proteasome pathway. The heme receptor Rev-erb alpha, a core negative component of the circadian network, controls circadian oscillation of several clock genes, including Bmal1 Rev-erb alpha protein degradation can be triggered by inhibitors of glycogen synthase kinase 3beta, such as lithium, and also by serum shock, which synchronizes circadian rhythms in cultured cells. Here we report that two E3 ligases, Arf-bp1 and Pam (Myc-bp2), are copurified with Rev-erb alpha and required for its ubiquitination. RNA-interference-mediated depletion of Arf-bp1 and Pam stabilizes the Rev-erb alpha protein and protects Rev-erb alpha from degradation triggered by either lithium or serum shock treatment. This degradation pathway modulates the expression of Rev-erb alpha-regulated Clock gene and circadian function in mouse hepatoma cells. Thus, Arf-bp1 and Pam are novel regulators of circadian gene expression that target Rev-erb alpha for degradation.

Histone removal in sperm protects paternal chromosomes from premature division at fertilization.

The global replacement of histones with protamines in sperm chromatin is widespread in animals, including insects, but its actual function remains enigmatic. We show that in the Drosophila paternal effect mutant paternal loss (pal), sperm chromatin retains germline histones H3 and H4 genome wide without impairing sperm viability. However, after fertilization, pal sperm chromosomes are targeted by the egg chromosomal passenger complex and engage into a catastrophic premature division in synchrony with female meiosis II. We show that pal encodes a rapidly evolving transition protein specifically required for the eviction of (H3-H4)2 tetramers from spermatid DNA after the removal of H2A-H2B dimers. Our study thus reveals an unsuspected role of histone eviction from insect sperm chromatin: safeguarding the integrity of the male pronucleus during female meiosis.

Role of Magmas in protein transport and human mitochondria biogenesis.

Magmas, a conserved mammalian protein essential for eukaryotic development, is overexpressed in prostate carcinomas and cells exposed to granulocyte-macrophage colony-stimulating factor (GM-CSF). Reduced Magmas expression resulted in decreased proliferative rates in cultured cells. However, the cellular function of Magmas is still elusive. In this report, we have showed that human Magmas is an ortholog of Saccharomyces cerevisiae Pam16 having similar functions and is critical for protein translocation across mitochondrial inner membrane. Human Magmas shows a complete growth complementation of Deltapam16 yeast cells at all temperatures. On the basis of our analysis, we report that Magmas localizes into mitochondria and is peripherally associated with inner mitochondrial membrane in yeast and humans. Magmas forms a stable subcomplex with J-protein Pam18 or DnaJC19 through its C-terminal region and is tethered to TIM23 complex of yeast and humans. Importantly, amino acid alterations in Magmas leads to reduced stability of the subcomplex with Pam18 that results in temperature sensitivity and in vivo protein translocation defects in yeast cells. These observations highlight the central role of Magmas in protein import and mitochondria biogenesis. In humans, absence of a functional DnaJC19 leads to dilated cardiac myophathic syndrome (DCM), a genetic disorder with characteristic features of cardiac myophathy and neurodegeneration. We propose that the mutations resulting in decreased stability of functional Magmas:DnaJC19 subcomplex at human TIM23 channel leads to impaired protein import and cellular respiration in DCM patients. Together, we propose a model showing how Magmas:DnaJC19 subcomplex is associated with TIM23 complex and thus regulates mitochondrial import process.

A network of hydrogen bonds on the surface of TLR2 controls ligand positioning and cell signaling.

TLR2 is a pattern recognition receptor that functions in association with TLR1 or TLR6 to mediate innate immune responses to a variety of conserved microbial products. In the present study, the ectodomain of TLR2 was extensively mutated, and the mutants were assessed for their ability to bind and to mediate cellular responses to triacylated lipopeptide Pam(3)CSK(4). This analysis provides evidence that the recently published crystal structure of the TLR2-TLR1-Pam(3)CSK(4) complex represents a functional signal-inducing complex. Furthermore, we report that extended H-bond networks on the surface of TLR2 are critical for signaling in response to Pam(3)CSK(4) and to other di- and tri-acylated TLR2-TLR6 and TLR2-TLR1 ligands. Based on this finding, we suggest a dynamic model for TLR2-mediated recognition of these ligands in which TLR2 fluctuates between a conformation that is more suitable for binding of the fatty acyl moieties of the ligands and a conformation that favors, via a specific orientation of the ligand head group, formation of a signal-inducing ternary complex.

An alternative pathway for ureide usage in legumes: enzymatic formation of a ureidoglycolate adduct in Cicer arietinum and Phaseolus vulgaris.

Ureidoglycolate is an intermediate in the degradation of the ureides, allantoin and allantoate, found in many organisms. In some leguminous plant species these compounds are used to transport recently fixed nitrogen in the root nodules to the aerial parts of the plant. In the present study, it was demonstrated that purified ureidoglycolases from chickpea (Cicer arietinum) and French bean (Phaseolus vulgaris) do not produce glyoxylate, and can use phenylhydrazine as a substrate with K(m) values of 4.0 mM and 8.5 mM, respectively. Furthermore, these enzymes catalyse the transfer of the ureidoglycolyl group to phenylhydrazine to produce ureidoglycolyl phenylhydrazide, which degrades non-enzymatically to glyoxylate phenylhydrazone and urea. This supports their former classification as ureidoglycolate urea-lyases. The enzymatic reaction catalysed by the characterized ureidoglycolases uncovered here can be viewed as a novel type of phenylhydrazine ureidoglycolyl transferase. The implications of these findings for ureide metabolism in legume nitrogen metabolism are discussed.

PAM staining intensity of primary neuroendocrine neoplasms is a potential prognostic biomarker.

Neuroendocrine neoplasms (NENs) are rare epithelial tumors with heterogeneous and frequently unpredictable clinical behavior. Available biomarkers are insufficient to guide individual patient prognosis or therapy selection. Peptidylglycine α-amidating monooxygenase (PAM) is an enzyme expressed by neuroendocrine cells that participates in hormone maturation. The objective of this study was to assess the distribution, clinical associations and survival implications of PAM immunoreactivity in primary NENs. Of 109 primary NENs, 7% were PAM-negative, 25% were PAM-low and 68% were PAM-high. Staining intensity was high in small bowel (p = 0.04) and low in stomach (p = 0.004) NENs. PAM staining was lower in higher grade tumors (p < 0.001) and patients who died (p < 0.001) but did not vary by tumor size or stage at surgery. In patients who died, time to death was shorter in patients with reduced PAM immunoreactivity: median times to death were 11.3 (PAM-negative), 29.4 (PAM-low) and 61.7 (PAM-high) months. Lower PAM staining was associated with increased risk of death after adjusting for disease stage [PAM negative, HR = 13.8 (CI: 4.2-45.5)]. PAM immunoreactivity in primary NENs is readily assessable and a potentially useful stage-independent predictor of survival.

Involvement of metals in enzymatic and nonenzymatic decomposition of C-terminal alpha-hydroxyglycine to amide: an implication for the catalytic role of enzyme-bound zinc in the peptidylamidoglycolate lyase reaction.

The peptide C-terminal amide group essential for the full biological activity of many peptide hormones is produced by consecutive actions of peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidylamidoglycolate lyase (PAL); PHM catalyzes the hydroxylation of C-terminal glycine, and PAL decomposes the peptidyl-alpha-hydroxyglycine to an amidated peptide and glyoxylate. PAL contains 1 mol of zinc, but its role, catalytic or structural, has not yet been clarified. In this study, we found that a series of transition metals, Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), and Cd(2+), catalyze the nonenzymatic decomposition of the hydroxyglycine intermediate in a concentration-dependent manner. The second-order rate constant of the metal catalysis increased with elevation of pH, indicating that the hydrated metal acts as a general base. Extensive removal of the enzyme-bound metals remarkably diminished the PAL activity; k(cat) of the metal-depleted enzyme retaining 0.1 mol of zinc decreased to 3.2 s(-1) from 25.7 s(-1) of the wild-type enzyme. Among a series of divalent metals tested, Zn(2+), Co(2+), and Cd(2+) could fully restore the PAL activity of the metal-depleted enzyme. Especially, Zn substitution reproduced the steady-state parameters of the wild-type enzyme. On the other hand, Co and Cd substitution largely altered the kinetic parameters; the k(cat) increased 3- and 5-fold and the K(m) for the substrate increased 2.5- and 4-fold, respectively. These observations support that the enzyme-bound zinc plays a catalytic role, rather than a structural role, in the PAL reaction through the action of zinc-bound water as a general base.

Intermittent hypoxia activates peptidylglycine alpha-amidating monooxygenase in rat brain stem via reactive oxygen species-mediated proteolytic processing.

Intermittent hypoxia (IH) associated with sleep apneas leads to cardiorespiratory abnormalities that may involve altered neuropeptide signaling. The effects of IH on neuropeptide synthesis have not been investigated. Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the alpha-amidation of neuropeptides, which confers biological activity to a large number of neuropeptides. PAM consists of O(2)-sensitive peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) activities. Here, we examined whether IH alters neuropeptide synthesis by affecting PAM activity and, if so, by what mechanisms. Experiments were performed on the brain stem of adult male rats exposed to IH (5% O(2) for 15 s followed by 21% O(2) for 5 min; 8 h/day for up to 10 days) or continuous hypoxia (0.4 atm for 10 days). Analysis of brain stem extracts showed that IH, but not continuous hypoxia, increased PHM, but not PAL, activity of PAM and that the increase of PHM activity was associated with a concomitant elevation in the levels of alpha-amidated forms of substance P and neuropeptide Y. IH increased the relative abundance of 42- and 35-kDa forms of PHM ( approximately 1.6- and 2.7-fold, respectively), suggesting enhanced proteolytic processing of PHM, which appears to be mediated by an IH-induced increase of endoprotease activity. Kinetic analysis showed that IH increases V(max) but has no effect on K(m). IH increased generation of reactive oxygen species in the brain stem, and systemic administration of antioxidant prevented IH-evoked increases of PHM activity, proteolytic processing of PHM, endoprotease activity, and elevations in substance P and neuropeptide Y amide levels. Taken together, these results demonstrate that IH activates PHM in rat brain stem via reactive oxygen species-dependent posttranslational proteolytic processing and further suggest that PAM activation may contribute to IH-mediated peptidergic neurotransmission in rat brain stem.

The endocytic pathways of a secretory granule membrane protein in HEK293 cells: PAM and EGF traverse a dynamic multivesicular body network together.

Peptidylglycine α-amidating monooxygenase (PAM) is highly expressed in neurons and endocrine cells, where it catalyzes one of the final steps in the biosynthesis of bioactive peptides. PAM is also expressed in unicellular organisms such as Chlamydomonas reinhardtii, which do not store peptides in secretory granules. As for other granule membrane proteins, PAM is retrieved from the cell surface and returned to the trans-Golgi network. This pathway involves regulated entry of PAM into multivesicular body intralumenal vesicles (ILVs). The aim of this study was defining the endocytic pathways utilized by PAM in cells that do not store secretory products in granules. Using stably transfected HEK293 cells, endocytic trafficking of PAM was compared to that of the mannose 6-phosphate (MPR) and EGF (EGFR) receptors, established markers for the endosome to trans-Golgi network and degradative pathways, respectively. As in neuroendocrine cells, PAM internalized by HEK293 cells accumulated in the trans-Golgi network. Based on surface biotinylation, >70% of the PAM on the cell surface was recovered intact after a 4h chase and soluble, bifunctional PAM was produced. Endosomes containing PAM generally contained both EGFR and MPR and ultrastructural analysis confirmed that all three cargos accumulated in ILVs. PAM containing multivesicular bodies made frequent dynamic tubular contacts with younger and older multivesicular bodies. Frequent dynamic contacts were observed between lysosomes and PAM containing early endosomes and multivesicular bodies. The ancient ability of PAM to localize to ciliary membranes, which release bioactive ectosomes, may be related to its ability to accumulate in ILVs and exosomes.

pH-regulated metal-ligand switching in the HM loop of ATP7A: a new paradigm for metal transfer chemistry.

Cuproproteins such as PHM and DBM mature in late endosomal vesicles of the mammalian secretory pathway where changes in vesicle pH are employed for sorting and post-translational processing. Colocation with the P1B-type ATPase ATP7A suggests that the latter is the source of copper and supports a mechanism where selectivity in metal transfer is achieved by spatial colocation of partner proteins in their specific organelles or vesicles. In previous work we have suggested that a lumenal loop sequence located between trans-membrane helices TM1 and TM2 of the ATPase, and containing five histidines and four methionines, acts as an organelle-specific chaperone for metallation of the cuproproteins. The hypothesis posits that the pH of the vesicle regulates copper ligation and loop conformation via a mechanism which involves His to Met ligand switching induced by histidine protonation. Here we report the effect of pH on the HM loop copper coordination using X-ray absorption spectroscopy (XAS), and show via selenium substitution of the Met residues that the HM loop undergoes similar conformational switching to that found earlier for its partner PHM. We hypothesize that in the absence of specific chaperones, HM motifs provide a template for building a flexible, pH-sensitive transfer site whose structure and function can be regulated to accommodate the different active site structural elements and pH environments of its partner proteins.

60 YEARS OF POMC: From POMC and α-MSH to PAM, molecular oxygen, copper, and vitamin C.

A critical role for peptide C-terminal amidation was apparent when the first bioactive peptides were identified. The conversion of POMC into adrenocorticotropic hormone and then into α-melanocyte-stimulating hormone, an amidated peptide, provided a model system for identifying the amidating enzyme. Peptidylglycine α-amidating monooxygenase (PAM), the only enzyme that catalyzes this modification, is essential; mice lacking PAM survive only until mid-gestation. Purification and cloning led to the discovery that the amidation of peptidylglycine substrates proceeds in two steps: peptidylglycine α-hydroxylating monooxygenase catalyzes the copper- and ascorbate-dependent α-hydroxylation of the peptidylglycine substrate; peptidyl-α-hydroxyglycine α-amidating lyase cleaves the N-C bond, producing amidated product and glyoxylate. Both enzymes are contained in the luminal domain of PAM, a type 1 integral membrane protein. The structures of both catalytic cores have been determined, revealing how they interact with metals, molecular oxygen, and substrate to catalyze both reactions. Although not essential for activity, the intrinsically disordered cytosolic domain is essential for PAM trafficking. A phylogenetic survey led to the identification of bifunctional membrane PAM in Chlamydomonas, a unicellular eukaryote. Accumulating evidence points to a role for PAM in copper homeostasis and in retrograde signaling from the lumen of the secretory pathway to the nucleus. The discovery of PAM in cilia, cellular antennae that sense and respond to environmental stimuli, suggests that much remains to be learned about this ancient protein.

Neuronal differentiation is associated with a redox-regulated increase of copper flow to the secretory pathway.

Brain development requires a fine-tuned copper homoeostasis. Copper deficiency or excess results in severe neuro-pathologies. We demonstrate that upon neuronal differentiation, cellular demand for copper increases, especially within the secretory pathway. Copper flow to this compartment is facilitated through transcriptional and metabolic regulation. Quantitative real-time imaging revealed a gradual change in the oxidation state of cytosolic glutathione upon neuronal differentiation. Transition from a broad range of redox states to a uniformly reducing cytosol facilitates reduction of the copper chaperone Atox1, liberating its metal-binding site. Concomitantly, expression of Atox1 and its partner, a copper transporter ATP7A, is upregulated. These events produce a higher flux of copper through the secretory pathway that balances copper in the cytosol and increases supply of the cofactor to copper-dependent enzymes, expression of which is elevated in differentiated neurons. Direct link between glutathione oxidation and copper compartmentalization allows for rapid metabolic adjustments essential for normal neuronal function.

Enzymes involved in purine metabolism--a review of histochemical localization and functional implications.

Many enzymes are involved in the biosynthesis, interconversion, and degradation of purine compounds. The exact function of these enzymes is still unknown, but they seem to play important roles other than in purine metabolism. To elucidate their functional roles, it is imperative to clarify their tissue distribution at the cellular or subcellular level. The present review summarizes the currently available information about their histochemical localization and proposed functions. In general, 5'-nucleotidase has been considered as a marker enzyme for the plasma membrane, and is considered to be a key enzyme in the generation of adenosine, a potential vasodilator. However, from its wide range of localization in tissues it is also considered to be related to the membrane movement of cells in the transitional epithelium, cellular motile response, transport process, cellular growth, synthesis of fibrous protein and calcification, lymphocyte activation, neurotransmission, and oxygen sensing mechanism. Adenosine deaminase (ADA) is present in all tissues in mammals. Although the main function of ADA is the development of the immune system in humans, it seems to be associated with the differentiation of epithelial cells and monocytes, neurotransmission, and maintenance of gestation. Purine nucleoside phosphorylase (PNP) is generally considered as a cytosolic enzyme, but recently, mitochondrial PNP, a different protein from cytosolic PNP, was reported. PNP is also widely expressed in human tissues. It is found in most tissues of the body, but the highest activity is in peripheral blood granulocyte and lymphoid tissues. It is also related to the development of T-cell immunity in humans as is ADA. Moreover, its contribution to centriole replication and/or regulation of microtubule assembly has been suggested. Immunohistochemical localization of xanthine oxidase has been reported in various tissues from various animal species. Xanthine oxidase has been suggested to be involved in the pathogenesis of post-ischemic reperfusion tissue injury through the generation of reactive oxygen species, while the extensive tissue localization of xanthine dehydrogenase/oxidase suggests several other roles for this enzyme, including a protective barrier against bacterial infection by producing either superoxide radicals or uric acid. Furthermore, an involvement in cellular proliferation and differentiation has been suggested. Urate oxidase is generally considered a liver-specific enzyme, except for bovines which possess this enzyme in the kidney. Urate oxidase is exclusively located in the peroxisomes of fish, frogs, and rats, but was lost in birds, some reptiles, and primates during evolution. A histochemical demonstration of allantoin-degrading enzymes has not been performed, but these enzymes have been located in peroxisomes by sucrose density gradient centrifugation. AMP deaminase activity is higher in skeletal muscle than in any other tissues. AMP deaminase may be involved in a number of physiological processes, such as the conversion of adenine nucleotide to inosine or guanine nucleotide, stabilizing the adenylate energy charge, and the reaction of the purine nucleotide cycle. There are three distinct isozymes (A, B, C) with different kinetic, physical, and immunological properties. Isozymes A, B, C have been isolated from muscle, liver (kidney), and heart tissue, respectively. In the muscle, AMP deaminase isozymes exist in a different part, suggesting a multiple functional role of this enzyme. High hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity is found in some regions of a normal adult human brain. However, very little is known regarding the histochemical tissue localization of HGPRT. Immunohistochemical localization of its developmental expression suggests that HGPRT may not be essential for purine nucleotide supplement in the segmentation of brain cells, but may play a significant role in the developing hippocampus.