Principles and Determinants of G-Protein Coupling by the Rhodopsin-Like Thyrotropin Receptor

In this study we wanted to gain insights into selectivity mechanisms between G-protein-coupled receptors (GPCR) and different subtypes of G-proteins. The thyrotropin receptor (TSHR) binds G-proteins promiscuously and activates both Gs (cAMP) and Gq (IP). Our goal was to dissect selectivity patterns for both pathways in the intracellular region of this receptor. We were particularly interested in the participation of poorly investigated receptor parts.We systematically investigated the amino acids of intracellular loop (ICL) 1 and helix 8 using site-directed mutagenesis alongside characterization of cAMP and IP accumulation. This approach was guided by a homology model of activated TSHR in complex with heterotrimeric Gq, using the X-ray structure of opsin with a bound G-protein peptide as a structural template.We provide evidence that ICL1 is significantly involved in G-protein activation and our model suggests potential interactions with subunits Gα as well as Gβγ. Several amino acid substitutions impaired both IP and cAMP accumulation. Moreover, we found a few residues in ICL1 (L440, T441, H443) and helix 8 (R687) that are sensitive for Gq but not for Gs activation. Conversely, not even one residue was found that selectively affects cAMP accumulation only.Together with our previous mutagenesis data on ICL2 and ICL3 we provide here the first systematically completed map of potential interfaces between TSHR and heterotrimeric G-protein. The TSHR/Gq-heterotrimer complex is characterized by more selective interactions than the TSHR/Gs complex. In fact the receptor interface for binding Gs is a subset of that for Gq and we postulate that this may be true for other GPCRs coupling these G-proteins. Our findings support that G-protein coupling and preference is dominated by specific structural features at the intracellular region of the activated GPCR but is completed by additional complementary recognition patterns between receptor and G-protein subtypes.     

Tritium isotope effect in high-pressure liquid chromatography of vitamin D metabolites

A significant chromatographic isotope effect is reported for 1,25-dihydroxyvitamin D3 in a wide variety of HPLC separation systems. The effect is also observed for 24,25-dihydroxyvitamin D3. Retention times differ from less than 1% up to 4% depending on the separation system and the degree and position of tritium substitution. Such an effect must be corrected for whenever both labeled and unlabeled vitamin D metabolites are used in HPLC cochromatography or assay recovery studies.     

Molecular Dynamics of the Ha-ras Protein: Nucleotide Atom-Centred Charges within the AMBER Force Field

Molecular dynamics simulations have become an essential tool for the study of biological systems. The Ha-ras protein, is a system suitable for such studies. Despite much recent progress, it is still not known exactly how the protein functions in the cell growth cycle. In this work atom-centred point charges for the guanosine nucleotide ligands are calculated and tested. To be compatible with the other AMBER force field parameters these are fitted to a molecular electrostatic potential derived from an ab initio wavefunction. The smallest basis set able to produce a stable wavefunction for the negatively charged GDP and GTP molecule ions was 3-21G* with diffuse functions added on the phosphate groups. To maintain force field integrity these charges were scaled to be equivalent to STO-3G derived values. This procedure is seen to produce a good magnesium-phosphate interaction potential when compared to 6-31++G* ab initio calculations. With the nucleotides fixed in the binding site conformation, it was found essential to include the electrostatics of the binding site in the calculation of the charges. It was also found to be inappropriate to divide the nucleotide into constituent parts for the calculations. From the calculated charges and experimental data, the nucleotide protonation states in the protein are deduced. It is unlikely that GDP is protonated, GTP probably binds one proton. The charges were tested in MD simulations of a protein modelled on the crystal structure of Tong et al., during which the dynamics of the nucleotide and binding site residues were in good agreement with the crystal structure data. The model is seen to be sensitive, not only to the inclusion of explicit solvent, but to the number of waters ligating the magnesium ion and the conformation of the loop between residues 60 and 66; both pieces of information are lacking in the crystal structure data.     

Arginase 2 Suppresses Renal Carcinoma Progression via Biosynthetic Cofactor Pyridoxal Phosphate Depletion and Increased Polyamine Toxicity

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Structural biology and bioinformatics in drug design: opportunities and challenges for target identification and lead discovery

Impressive progress in genome sequencing, protein expression and high-throughput crystallography and NMR has radically transformed the opportunities to use protein three-dimensional structures to accelerate drug discovery, but the quantity and complexity of the data have ensured a central place for informatics. Structural biology and bioinformatics have assisted in lead optimization and target identification where they have well established roles; they can now contribute to lead discovery, exploiting high-throughput methods of structure determination that provide powerful approaches to screening of fragment binding.     

Shared alterations in NK cell frequency, phenotype, and function in chronic human immunodeficiency virus and hepatitis C virus infections

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) cause clinically important persistent infections. The effects of virus persistence on innate immunity, including NK cell responses, and the underlying mechanisms are not fully understood. We examined the frequency, phenotype, and function of peripheral blood CD3⁻ CD56⁺ NK subsets in HIV⁺ and HCV⁺ patients and identified significantly reduced numbers of total NK cells and a striking shift in NK subsets, with a marked decrease in the CD56^dim cell fraction compared to CD56^bright cells, in both infections. This shift influenced the phenotype and functional capacity (gamma interferon production, killing) of the total NK pool. In addition, abnormalities in the functional capacity of the CD56^dim NK subset were observed in HIV⁺ patients. The shared NK alterations were found to be associated with a significant reduction in serum levels of the innate cytokine interleukin 15 (IL-15). In vitro stimulation with IL-15 rescued NK cells of HIV⁺ and HCV⁺ patients from apoptosis and enhanced proliferation and functional activity. We hypothesize that the reduced levels of IL-15 present in the serum during HIV and HCV infections might impact NK cell homeostasis, contributing to the common alterations of the NK pool observed in these unrelated infections.     

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.     

Infant feeding in the context of HIV: a qualitative study of health care workers’ knowledge of recommended infant feeding options in Papua New Guinea

Background

Interventions to prevent mother to child transmission of human immunodeficiency virus (HIV) during childbirth and breastfeeding can reduce HIV infections in infants to less than 5% in low and middle income countries. The World Health Organization (WHO) recommends all mothers, regardless of their HIV status, practice exclusive breastfeeding for the first six months of an infant’s life. In line with these recommendations and to protect, promote and support breastfeeding, in 2009 the PNG National Department of Health revised their National HIV infant feeding guidelines, reinforcing the WHO recommendation of exclusive breastfeeding for the first six months followed by the introduction of other food and fluids, while continuing breastfeeding.The overall aim of this paper is to explore health care workers’ knowledge regarding infant feeding options in PNG, specifically as they relate to HIV exposed infants.

Methods

As part of a study investigating women’s and men’s experiences of prevention of mother to child transmission (PMTCT) services in two sites in PNG, 28 key informant interviews were undertaken. This paper addresses one theme that emerged from thematic data analysis: Health care workers’ knowledge regarding infant feeding options, specifically how this knowledge reflects the Papua New Guinea National HIV Care and Treatment Guidelines on HIV and infant feeding (2009).

Results

Most informants mentioned exclusive breastfeeding, the majority of whom reflected the most up-to-date National Guidelines of exclusive breastfeeding for six months. The importance of breastfeeding continuing beyond this time, along with the introduction of food and fluids was less well understood. The most senior people involved in PMTCT were the informants who most accurately reflected the national guidelines of continuing breastfeeding after six months.

Conclusion

Providing advice on optimal infant feeding in resource poor settings is problematic, especially in relation to HIV transmission. Findings from our study reflect those found elsewhere in identifying that key health care workers are not aware of up-to-date information relating to infant feeding, especially within the context of HIV. Greater emphasis needs to be placed on ensuring the most recent feeding guidelines are disseminated and implemented in clinical practice in PNG.