Biophysical and structural investigation of the regulation of human GTP cyclohydrolase I by its regulatory protein GFRP.

GTP Cyclohydrolase I (GCH1) catalyses the conversion of guanosine triphosphate (GTP) to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). BH4 functions as co-factor in neurotransmitter biosynthesis. BH4 homeostasis is a promising target to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). Dependent on the relative cellular concentrations of effector ligands, BH4 and phenylalanine, GFRP binds GCH1 to form inhibited or activated complexes, respectively. We determined high-resolution structures of the ligand-free and -bound human GFRP and GCH1-GFRP complexes by X-ray crystallography. Highly similar binding modes of the substrate analogue 7-deaza-GTP to active and inhibited GCH1-GFRP complexes confirm a novel, dissociation rate-controlled mechanism of non-competitive inhibition to be at work. Further, analysis of all structures shows that upon binding of the effector molecules, the conformations of GCH1 or GFRP are altered and form highly complementary surfaces triggering a picomolar interaction of GFRP and GCH1 with extremely slow koff values, while GCH1-GFRP complexes rapidly disintegrate in absence of BH4 or phenylalanine. Finally, comparing behavior of full-length and N-terminally truncated GCH1 we conclude that the disordered GCH1 N-terminus does not have impact on complex formation and enzymatic activity. In summary, this comprehensive and methodologically diverse study helps to provide a better understanding of the regulation of GCH1 by GFRP and could thus stimulate research on GCH1 modulating drugs.

Donor site morbidity and quality of life after microvascular head and neck reconstruction with free fibula and deep-circumflex iliac artery flaps.

PURPOSE: Bone defects after resective tumor surgeries often require the use of microvascular reanastomized bone grafts for reconstruction. The decision as to which specific flap is most suitable for the particular patient is dependent on various factors. The aspects donor site morbidity and quality of life are rarely taken into account in this connection. The aim of this study was to analyze whether these factors, in the future, should influence the choice of donor site. MATERIAL AND METHODS: In this study, the donor sites of 46 patients with respect to deep-circumflex iliac artery (DCIA) and fibula flaps were analyzed using subjective and objective parameters. The primary outcome was postoperative pain measured by VAS. Postoperative complication evaluations as well as 2 orthopedic scores were implemented (American Orthopedic Foot and Ankle Society (AOFAS) Ankle-Hindfoot Score and Harris Hip Score) and the patients' quality of life was assessed using the 36-Item Short Form (SF-36) questionnaire to quantify donor site impairment. RESULTS: Postoperative pain was rated with a mean value of 42.4 mm (SD 34.8) for the DCIA group and 36.9 mm (SD 37.1) for the fibula group (p = 0.617). After a mean period of 13.72 months, pain was rated with a mean value of 15.3 mm (SD 21.7) for DCIA and 13.3 mm (SD 22.6 mm) for the fibula (p = 0.763). Persistent pain, however, was recorded only in 11.11% of DCIA patients and 5.26% of fibula patients. Furthermore pain intensity was higher in the DCIA group. A changed gait pattern was observed in 59.26% of DCIA patients and 21.05% of fibula patients. DCIA patients required walking aids for walking and stair climbing more often. Looking at the results of the 2 orthopedic scores, fibula patients showed slightly better results. Concerning quality of life, patients after reconstructive surgery with DCIA flaps showed slight better results than patients in the fibula group. CONCLUSIONS: Taking the results of this study into account, the outcome in terms of pain, morbidity and quality of life did not show a significant superiority of any donor site.

Crystal structure of the human liver X receptor beta ligand-binding domain in complex with a synthetic agonist.

LXRbeta belongs to the nuclear hormone receptor superfamily of ligand-activated transcription factors. Its natural ligands are supposed to be oxidised derivatives of cholesterol. Stimulation of LXRbeta by agonists activates a number of genes that are involved in the regulation of lipid metabolism and cholesterol efflux from cells. Therefore, LXRbeta may represent a novel therapeutic target for the treatment of dyslipidemia and atherosclerosis.Here, we report the X-ray crystal structure of the LXRbeta ligand-binding domain in complex with a synthetic agonist, T-0901317. This compound occupies the ligand-binding pocket of the receptor, forms numerous lipophilic contacts with the protein and one crucial hydrogen bond to His435 and stabilises the agonist conformation of the receptor ligand-binding domain. The recruitment of the AF2-region of the protein is not achieved via direct polar interactions of the ligand with protein side-chains of this helical segment, but rather via few hydrophobic contacts and probably more importantly via indirect effects involving the pre-orientation of side-chains that surround the ligand-binding pocket and form the interface to the AF2-helix. On the basis of these results we propose a binding mode and a mechanism of action for the putative natural ligands, oxidised derivatives of cholesterol.

Frigocyclinone, a novel angucyclinone antibiotic produced by a Streptomyces griseus strain from Antarctica.

A new angucyclinone antibiotic, frigocyclinone, was isolated from Streptomyces griseus strain NTK 97, consisting of a tetrangomycin moiety attached through a C-glycosidic linkage with the aminodeoxysugar ossamine. Frigocyclinone showed antibacterial activities against Gram-positive bacteria.