The role of the sonic hedgehog signalling pathway in patients with midline defects and congenital hypopituitarism.

INTRODUCTION: The Gli family of zinc finger (GLI) transcription factors mediates the sonic hedgehog signalling pathway (HH) essential for CNS, early pituitary and ventral forebrain development in mice. Human mutations in this pathway have been described in patients with holoprosencephaly (HPE), isolated congenital hypopituitarism (CH) and cranial/midline facial abnormalities. Mutations in Sonic hedgehog (SHH) have been associated with HPE but not CH, despite murine studies indicating involvement in pituitary development. OBJECTIVES/METHODS: We aimed to establish the role of the HH pathway in the aetiology of hypothalamo-pituitary disorders by screening our cohort of patients with midline defects and/or CH for mutations in SHH, GLI2, Shh brain enhancer 2 (SBE2) and growth-arrest specific 1 (GAS1). RESULTS: Two variants and a deletion of GLI2 were identified in three patients. A novel variant at a highly conserved residue in the zinc finger DNA-binding domain, c.1552G > A [pE518K], was identified in a patient with growth hormone deficiency and low normal free T4. A nonsynonymous variant, c.2159G > A [p.R720H], was identified in a patient with a short neck, cleft palate and hypogonadotrophic hypogonadism. A 26·6 Mb deletion, 2q12·3-q21·3, encompassing GLI2 and 77 other genes, was identified in a patient with short stature and impaired growth. Human embryonic expression studies and molecular characterisation of the GLI2 mutant p.E518K support the potential pathogenicity of GLI2 mutations. No mutations were identified in GAS1 or SBE2. A novel SHH variant, c.1295T>A [p.I432N], was identified in two siblings with variable midline defects but normal pituitary function. CONCLUSIONS: Our data suggest that mutations in SHH, GAS1 and SBE2 are not associated with hypopituitarism, although GLI2 is an important candidate for CH.

SufC hydrolyzes ATP and interacts with SufB from Thermotoga maritima.

Genetic experiments in bacteria have shown the suf operon is involved in iron homeostasis and the oxidative stress response. The sufB and sufC genes that always occur together in bacteria are also found in plants, and even the malaria parasite, associated with the plastid organelle. Although the suf operon is believed to encode an iron-dependent ABC-transporter there is no direct evidence. By immunolocalization we show here that SufB and SufC are associated with the membrane of Escherichia coli. We also present kinetic studies with a recombinant version of SufC from Thermotoga maritima that shows it is an ATPase and that it interacts with SufB in vitro.

The extracellular regions of PSMA and the transferrin receptor contain an aminopeptidase domain: implications for drug design.

The Aeromonas proteolytica aminopeptidase (AMP), Pseudomonas sp. (RS-16) carboxypeptidase G2 (CPG2), and Streptomyces griseus aminopeptidase (SGAP) are zinc dependent proteolytic enzymes with cocatalytic zinc ion centers and a conserved aminopeptidase fold. A BLAST search with the sequence of the solved AMP structure indicated that a similar domain could be found in prostate-specific membrane antigen (PSMA) and the transferrin receptor (TfR). When the PSMA or TfR sequence was input into the THREADER program, the top structural matches were SGAP and AMP confirming that these are structurally conserved domains. Optimal sequence alignment of PSMA and TfR using the known three-dimensional structures of AMP, CPG2, and SGAP shows that the critical amino acids involved in forming the catalytic pocket are conserved in PSMA but absent in the TfR. The specificity pocket in AMP is formed from four aromatic side chains and the equivalent region in CPG2/PSMA has a changed sequence pattern. Since CPG2 and PSMA are folate hydrolases, the changed specificity pocket leaves space to accommodate the large pteroate moiety of folic acid. In contrast, no enzyme function has been ascribed to the TfR.

Structural role of extracellular domain 1 of alpha-platelet-derived growth factor (PDGF) receptor for PDGF-AA and PDGF-BB binding.

The purpose of this study was to bacterially express, purify, and refold combinations of the extracellular immunoglobulin (Ig)-like domains (2-3, 1-3, and 1-5) of the human alpha-platelet-derived growth factor receptor (alpha PDGFR) to characterize molecular interactions with its ligand, platelet-derived growth factor (PDGF). The far UV circular dichroism spectroscopy of the alpha-PDGFR extracellular domains (ECDs) revealed a predominantly beta-sheet protein, with a structure consistent with folded Ig-like domains. The addition of PDGF-BB to these ECD types changed the conformation of all three types with a decrease in mean residue ellipticity in the following rank order: 1-5 = 1-3 > 2-3. In striking contrast, addition of PDGF-AA to these ECD types markedly changed the conformation of ECD 2-3, by an increased mean residue ellipticity but no changes were observed for ECDs 1-3 and 1-5. PDGF-AA bound to the immobilized ECD types 2-3, 1-3, and 1-5 at concentrations of 20, 11, and 7.5 nM, respectively. In contrast, PDGF-BB bound the ECD types 2-3, 1-3, and 1-5 at concentrations of 3, 3, and 2.2 nM, respectively. Scatchard analysis of binding studies using labeled ECDs indicated that PDGF-BB bound ECD 1-3 and ECD 2-3 with KD values of 74 and 72 nM, respectively. While, PDGF-AA bound ECD 1-3 and ECD 2-3 with KD values of 33 and 87 nM, respectively. Therefore, our results indicated that the loss of ECD 1 impaired the binding affinity of alpha PDGFR ECD 1-3 toward PDGF-AA without having a similar effect on PDGF-BB binding. Together all of our data suggest that ECD 1 is differentially required for proper orientation of PDGF-AA but not PDGF-BB binding determinant within ECDs 2 and 3.