New insights into copper monooxygenases and peptide amidation: structure, mechanism and function

Many bioactive peptides must be amidated at their carboxy terminus to exhibit full activity. Surprisingly, the amides are not generated by a transamidation reaction. Instead, the hormones are synthesized from glycine-extended intermediates that are transformed into active amidated hormones by oxidative cleavage of the glycine N-C alpha bond. In higher organisms, this reaction is catalyzed by a single bifunctional enzyme, peptidylglycine alpha-amidating monooxygenase (PAM). The PAM gene encodes one polypeptide with two enzymes that catalyze the two sequential reactions required for amidation. Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3) catalyzes the stereospecific hydroxylation of the glycine alpha-carbon of all the peptidylglycine substrates. The second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL; EC 4.3.2.5), generates alpha-amidated peptide product and glyoxylate. PHM contains two redox-active copper atoms that, after reduction by ascorbate, catalyze the reduction of molecular oxygen for the hydroxylation of glycine-extended substrates. The structure of the catalytic core of rat PHM at atomic resolution provides a framework for understanding the broad substrate specificity of PHM, identifying residues critical for PHM activity, and proposing mechanisms for the chemical and electron-transfer steps in catalysis. Since PHM is homologous in sequence and mechanism to dopamine beta-monooxygenase (DBM; EC 1.14.17.1), the enzyme that converts dopamine to norepinephrine during catecholamine biosynthesis, these structural and mechanistic insights are extended to DBM.     

‘Classical’ and ‘new’ diabetogens—comparison of their effects on isolated rat pancreatic islets in vitro

This study compares functional and morphological alterations caused by application of alloxan, streptozotocin, xanthine oxidase/hypoxanthine (generation of reactive oxygen species), or S-nitroso-N-acetyl-D,L-penicillamine (SNAP, liberation of nitric oxide) to isolated rat pancreatic islets in vitro. In perifusion experiments, membrane leakage—detected by non-stimulated insulin release—was found after application of all drugs, but showed a substance-specific time pattern. Twenty-four hours after application of the classical diabetogens (alloxan or streptozotocin), potassium chloride- and glucose-stimulated insulin secretion were markedly reduced, while a persistent reduction was observed neither after exposure to xanthine oxidase/hypoxanthine, nor to SNAP. Morphological analysis of the islets revealed that nearly all β-cells were destroyed following alloxan or streptozotocin treatment, while the majority of β-cells were configured regularly after application of xanthine oxidase/hypoxanthine or SNAP. Necrotic cells found after xanthine oxidase/hypoxanthine usually differed in morphology from those observed after application of the classical diabetogens. While the former cells were characterised by swollen nuclei, the latter had shrunken nuclei with irregular condensed chromatin. Apoptosis was found only following nitric oxide exposure. Due to these differences, it seems unlikely that alloxan, streptozotocin, xanthine oxidase/hypoxanthine, and nitric oxide have a common major feature in their toxic action. Received 16 September 1999; received after revision 15 November 1999; accepted 26 November 1999     

Insertion of Inhbb into the Inhba locus rescues the Inhba-null phenotype and reveals new activin functions

The activins (dimers of betaA or betaB subunits, encoded by the genes Inhba and Inhbb, respectively) are TGF-beta superfamily members that have roles in reproduction and development. Whereas mice homozygous for the Inhba-null allele demonstrate disruption of whisker, palate and tooth development, leading to neonatal lethality, homozygous Inhbb-null mice are viable, fertile and have eye defects. To determine if these phenotypes were due to spatiotemporal expression differences of the ligands or disruption of specific ligand-receptor interactions, we replaced the region of Inhba encoding the mature protein with Inhbb, creating the allele Inhbatm2Zuk (hereafter designated InhbaBK). Although the craniofacial phenotypes of the Inhba-null mutation were rescued by the InhbaBK allele, somatic, testicular, genital and hair growth were grossly affected and influenced by the dosage and bioactivity of the allele. Thus, functional compensation within the TGF-beta superfamily can occur if the replacement gene is expressed appropriately. The novel phenotypes in these mice further illustrate the usefulness of insertion strategies for defining protein function.     

Nf1;Trp53 mutant mice develop glioblastoma with evidence of strain-specific effects

Astrocytomas are the leading cause of brain cancer in humans. Because these tumours are highly infiltrative, current treatments that rely on targeting the tumour mass are often ineffective. A mouse model for astrocytoma would be a powerful tool for dissecting tumour progression and testing therapeutics. Mouse models of astrocytoma have been designed to express oncogenic proteins in astrocytes, but have had limited success due to low tumour penetrance or limited tumour progression. We present here a mouse model of astrocytomas involving mutation of two tumour-suppressor genes, Nf1 and Trp53. Humans with mutations in NF1 develop neurofibromatosis type I (NF1) and have increased risk of optic gliomas, astrocytomas and glioblastomas. The TP53 tumour suppressor is often mutated in a subset of astrocytomas that develop at a young age and progress slowly to glioblastoma (termed secondary glioblastomas, in contrast to primary glioblastomas that develop rapidly de novo). This mouse model shows a range of astrocytoma stages, from low-grade astrocytoma to glioblastoma multiforme, and may accurately model human secondary glioblastoma involving TP53 loss. This is the first reported mouse model of astrocytoma initiated by loss of tumour suppressors, rather than overexpression of transgenic oncogenes.     

A transgenic insertion upstream of sox9 is associated with dominant XX sex reversal in the mouse

In most mammals, male development is triggered by the transient expression of the Y-chromosome gene, Sry, which initiates a cascade of gene interactions ultimately leading to the formation of a testis from the indifferent fetal gonad. Several genes, in particular Sox9, have a crucial role in this pathway. Despite this, the direct downstream targets of Sry and the nature of the pathway itself remain to be clearly established. We report here a new dominant insertional mutation, Odsex (Ods), in which XX mice carrying a 150-kb deletion (approximately 1 Mb upstream of Sox9) develop as sterile XX males lacking Sry. During embryogenesis, wild-type XX fetal gonads downregulate Sox9 expression, whereas XY and XX Ods/+ fetal gonads upregulate and maintain its expression. We propose that Ods has removed a long-range, gonad-specific regulatory element that mediates the repression of Sox9 expression in XX fetal gonads. This repression would normally be antagonized by Sry protein in XY embryos. Our data are consistent with Sox9 being a direct downstream target of Sry and provide genetic evidence to support a general repressor model of sex determination in mammals.