Proximal and distal control for ligand binding in neuroglobin: role of the CD loop and evidence for His64 gating

Neuroglobin (Ngb) is predominantly expressed in neurons of the central and peripheral nervous systems and it clearly seems to be involved in neuroprotection. Engineering Ngb to observe structural and dynamic alterations associated with perturbation in ligand binding might reveal important structural determinants, and could shed light on key features related to its mechanism of action. Our results highlight the relevance of the CD loop and of Phe106 as distal and proximal controls involved in ligand binding in murine neuroglobin. We observed the effects of individual and combined mutations of the CD loop and Phe106 that conferred to Ngb higher CO binding velocities, which we correlate with the following structural observations: the mutant F106A shows, upon CO binding, a reduced heme sliding hindrance, with the heme present in a peculiar double conformation, whereas in the CD loop mutant "Gly-loop", the original network of interactions between the loop and the heme was abolished, enhancing binding via facilitated gating out of the distal His64. Finally, the double mutant, combining both mutations, showed a synergistic effect on CO binding rates. Resonance Raman spectroscopy and MD simulations support our findings on structural dynamics and heme interactions in wild type and mutated Ngbs

Proximal and distal control for ligand binding in neuroglobin: role of the CD loop and evidence for His64 gating

Neuroglobin (Ngb) is predominantly expressed in neurons of the central and peripheral nervous systems and it clearly seems to be involved in neuroprotection. Engineering Ngb to observe structural and dynamic alterations associated with perturbation in ligand binding might reveal important structural determinants, and could shed light on key features related to its mechanism of action. Our results highlight the relevance of the CD loop and of Phe106 as distal and proximal controls involved in ligand binding in murine neuroglobin. We observed the effects of individual and combined mutations of the CD loop and Phe106 that conferred to Ngb higher CO binding velocities, which we correlate with the following structural observations: the mutant F106A shows, upon CO binding, a reduced heme sliding hindrance, with the heme present in a peculiar double conformation, whereas in the CD loop mutant “Gly-loop”, the original network of interactions between the loop and the heme was abolished, enhancing binding via facilitated gating out of the distal His64. Finally, the double mutant, combining both mutations, showed a synergistic effect on CO binding rates. Resonance Raman spectroscopy and MD simulations support our findings on structural dynamics and heme interactions in wild type and mutated Ngbs

Evaluation and Diagnostic Potential of Serum Ghrelin in Feline Hypersomatotropism and Diabetes Mellitus

BACKGROUND: Ghrelin is a growth hormone secretagogue. It is a potent regulator of energy homeostasis. Ghrelin concentration is down‐regulated in humans with hypersomatotropism (HS) and increases after successful treatment. Additionally, ghrelin secretion seems impaired in human diabetes mellitus (DM). HYPOTHESIS: Serum ghrelin concentration is down‐regulated in cats with HS‐induced DM (HSDM) compared to healthy control cats or cats with DM unrelated to HS and increases after radiotherapy. ANIMALS: Cats with DM (n = 20) and with HSDM (n = 32), 13 of which underwent radiotherapy (RT‐group); age‐matched controls (n = 20). METHODS: Retrospective cross‐sectional study. Analytical performance of a serum total ghrelin ELISA was assessed and validated for use in cats. Differences in serum ghrelin, fructosamine, IGF‐1 and insulin were evaluated. RESULTS: Ghrelin was significantly higher (P < .001) in control cats (mean ± SD: 12.9 ± 6.8 ng/mL) compared to HSDM‐ (7.9 ± 3.3 ng/mL) and DM‐cats (6.7 ± 2.3 ng/mL), although not different between the HSDM‐ and DM‐cats. After RT ghrelin increased significantly (P = .003) in HSDM‐cats undergoing RT (from 6.6 ± 1.9 ng/mL to 9.0 ± 2.2 ng/mL) and the after RT ghrelin concentrations of HSDM cats were no longer significantly different from the serum ghrelin concentration of control cats. Serum IGF‐1 did not significantly change in HSDM‐cats after RT, despite significant decreases in fructosamine and insulin dose. CONCLUSION AND CLINICAL IMPORTANCE: Ghrelin appears suppressed in cats with DM and HSDM, although increases after RT in HSDM, suggesting possible presence of a direct or indirect negative feedback system between growth hormone and ghrelin. Serum ghrelin might therefore represent a marker of treatment effect

Dissecting the cytochrome P450 OleP substrate specificity: evidence for a preferential substrate

The cytochrome P450 OleP catalyzes the epoxidation of aliphatic carbons on both the aglycone 8.8a-deoxyoleandolide (DEO) and the monoglycosylated L-olivosyl-8.8a-deoxyoleandolide (L-O-DEO) intermediates of oleandomycin biosynthesis. We investigated the substrate versatility of the enzyme. X-ray and equilibrium binding data show that the aglycone DEO loosely fits the OleP active site, triggering the closure that prepares it for catalysis only on a minor population of enzyme. The open-to-closed state transition allows solvent molecules to accumulate in a cavity that forms upon closure, mediating protein–substrate interactions. In silico docking of the monoglycosylated L-O-DEO in the closed OleP–DEO structure shows that the L-olivosyl moiety can be hosted in the same cavity, replacing solvent molecules and directly contacting structural elements involved in the transition. X-ray structures of aglycone-bound OleP in the presence of L-rhamnose confirm the cavity as a potential site for sugar binding. All considered, we propose L-O-DEO as the optimal substrate of OleP, the L-olivosyl moiety possibly representing the molecular wedge that triggers a more efficient structural response upon substrate binding, favoring and stabilizing the enzyme closure before catalysis. OleP substrate versatility is supported by structural solvent molecules that compensate for the absence of a glycosyl unit when the aglycone is bound

Structural insights into the DNA recognition mechanism by the bacterial transcription factor PdxR

Specificity in protein-DNA recognition arises from the synergy of several factors that stem from the structural and chemical signatures encoded within the targeted DNA molecule. Here, we deciphered the nature of the interactions driving DNA recognition and binding by the bacterial transcription factor PdxR, a member of the MocR family responsible for the regulation of pyridoxal 5 &amp; PRIME;-phosphate (PLP) biosynthesis. Single particle cryo-EM performed on the PLP-PdxR bound to its target DNA enabled the isolation of three conformers of the complex, which may be considered as snapshots of the binding process. Moreover, the resolution of an apo-PdxR crystallographic structure provided a detailed description of the transition of the effector domain to the holo-PdxR form triggered by the binding of the PLP effector molecule. Binding analyses of mutated DNA sequences using both wild type and PdxR variants revealed a central role of electrostatic interactions and of the intrinsic asymmetric bending of the DNA in allosterically guiding the holo-PdxR-DNA recognition process, from the first encounter through the fully bound state. Our results detail the structure and dynamics of the PdxR-DNA complex, clarifying the mechanism governing the DNA-binding mode of the holo-PdxR and the regulation features of the MocR family of transcription factors

Height and body-mass index trajectories of school-aged children and adolescents from 1985 to 2019 in 200 countries and territories: a pooled analysis of 2181 population-based studies with 65 million participants

Summary Background Comparable global data on health and nutrition of school-aged children and adolescents are scarce. We aimed to estimate age trajectories and time trends in mean height and mean body-mass index (BMI), which measures weight gain beyond what is expected from height gain, for school-aged children and adolescents. Methods For this pooled analysis, we used a database of cardiometabolic risk factors collated by the Non-Communicable Disease Risk Factor Collaboration. We applied a Bayesian hierarchical model to estimate trends from 1985 to 2019 in mean height and mean BMI in 1-year age groups for ages 5–19 years. The model allowed for non-linear changes over time in mean height and mean BMI and for non-linear changes with age of children and adolescents, including periods of rapid growth during adolescence. Findings We pooled data from 2181 population-based studies, with measurements of height and weight in 65 million participants in 200 countries and territories. In 2019, we estimated a difference of 20 cm or higher in mean height of 19-year-old adolescents between countries with the tallest populations (the Netherlands, Montenegro, Estonia, and Bosnia and Herzegovina for boys; and the Netherlands, Montenegro, Denmark, and Iceland for girls) and those with the shortest populations (Timor-Leste, Laos, Solomon Islands, and Papua New Guinea for boys; and Guatemala, Bangladesh, Nepal, and Timor-Leste for girls). In the same year, the difference between the highest mean BMI (in Pacific island countries, Kuwait, Bahrain, The Bahamas, Chile, the USA, and New Zealand for both boys and girls and in South Africa for girls) and lowest mean BMI (in India, Bangladesh, Timor-Leste, Ethiopia, and Chad for boys and girls; and in Japan and Romania for girls) was approximately 9–10 kg/m2. In some countries, children aged 5 years started with healthier height or BMI than the global median and, in some cases, as healthy as the best performing countries, but they became progressively less healthy compared with their comparators as they grew older by not growing as tall (eg, boys in Austria and Barbados, and girls in Belgium and Puerto Rico) or gaining too much weight for their height (eg, girls and boys in Kuwait, Bahrain, Fiji, Jamaica, and Mexico; and girls in South Africa and New Zealand). In other countries, growing children overtook the height of their comparators (eg, Latvia, Czech Republic, Morocco, and Iran) or curbed their weight gain (eg, Italy, France, and Croatia) in late childhood and adolescence. When changes in both height and BMI were considered, girls in South Korea, Vietnam, Saudi Arabia, Turkey, and some central Asian countries (eg, Armenia and Azerbaijan), and boys in central and western Europe (eg, Portugal, Denmark, Poland, and Montenegro) had the healthiest changes in anthropometric status over the past 3·5 decades because, compared with children and adolescents in other countries, they had a much larger gain in height than they did in BMI. The unhealthiest changes—gaining too little height, too much weight for their height compared with children in other countries, or both—occurred in many countries in sub-Saharan Africa, New Zealand, and the USA for boys and girls; in Malaysia and some Pacific island nations for boys; and in Mexico for girls. Interpretation The height and BMI trajectories over age and time of school-aged children and adolescents are highly variable across countries, which indicates heterogeneous nutritional quality and lifelong health advantages and risks

Rising rural body-mass index is the main driver of the global obesity epidemic in adults

Body-mass index (BMI) has increased steadily in most countries in parallel with a rise in the proportion of the population who live in cities(.)(1,2) This has led to a widely reported view that urbanization is one of the most important drivers of the global rise in obesity(3-6). Here we use 2,009 population-based studies, with measurements of height and weight in more than 112 million adults, to report national, regional and global trends in mean BMI segregated by place of residence (a rural or urban area) from 1985 to 2017. We show that, contrary to the dominant paradigm, more than 55% of the global rise in mean BMI from 1985 to 2017-and more than 80% in some low- and middle-income regions-was due to increases in BMI in rural areas. This large contribution stems from the fact that, with the exception of women in sub-Saharan Africa, BMI is increasing at the same rate or faster in rural areas than in cities in low- and middle-income regions. These trends have in turn resulted in a closing-and in some countries reversal-of the gap in BMI between urban and rural areas in low- and middle-income countries, especially for women. In high-income and industrialized countries, we noted a persistently higher rural BMI, especially for women. There is an urgent need for an integrated approach to rural nutrition that enhances financial and physical access to healthy foods, to avoid replacing the rural undernutrition disadvantage in poor countries with a more general malnutrition disadvantage that entails excessive consumption of low-quality calories.Peer reviewe

Heterogeneous contributions of change in population distribution of body mass index to change in obesity and underweight NCD Risk Factor Collaboration (NCD-RisC)

From 1985 to 2016, the prevalence of underweight decreased, and that of obesity and severe obesity increased, in most regions, with significant variation in the magnitude of these changes across regions. We investigated how much change in mean body mass index (BMI) explains changes in the prevalence of underweight, obesity, and severe obesity in different regions using data from 2896 population-based studies with 187 million participants. Changes in the prevalence of underweight and total obesity, and to a lesser extent severe obesity, are largely driven by shifts in the distribution of BMI, with smaller contributions from changes in the shape of the distribution. In East and Southeast Asia and sub-Saharan Africa, the underweight tail of the BMI distribution was left behind as the distribution shifted. There is a need for policies that address all forms of malnutrition by making healthy foods accessible and affordable, while restricting unhealthy foods through fiscal and regulatory restrictions

Cryo-EM structure of PdxR from Bacillus clausii in complex with its target DNA

PdxR is a bacterial transcription factor belonging to the MocR family, which is involved in the regulation of the de novo biosynthesis of pyridoxal 5’-phosphate (PLP). PdxR has been extensively studied at the functional level and its target regulon has been characterized. However, a structural characterization of this transcription factor, and of MocRs in general, is missing. In fact, to date no structure of MocRs in complex with DNA is available, and a mechanism of their regulatory activity has been hypothesised on the basis of functional and computational data. In this thesis I report the structure of PdxR from Bacillus clausii in complex with its target DNA sequence of 48-bp, determined by cryo-electron microscopy (cryo-EM). Our data revealed that in PdxR the DNA-binding winged helix-turn-helix domain (wHTH) and the aspartate aminotransferase (AAT)-like domain are arranged in a domain-swap homodimeric assembly and are connected by a long flexible linker. Single particle analysis allowed us to isolate two different conformational states of the PdxR-DNA complex, an “open” and a “closed” one, differently bound to the target oligonucleotide, and to identify residues on the protein and bases on the DNA involved in sequence recognition and complex formation. Moreover, we analysed the role of both the DNA sequence and conformation in the specific recognition by PdxR, evaluating the complex stability using rationally designed DNA sequences. Our results provide relevant information on the structure and the dynamics of PdxR-DNA complex and represent a fundamental step to clarify the mechanism governing the DNA-binding mode and the transcriptional regulation of MocRs transcription factors

Lack of orientation selectivity of the heme insertion in murine neuroglobin revealed by resonance Raman spectroscopy

Different murine neuroglobin variants showing structural and dynamic alterations which are associated with perturbation of ligand binding have been studied. The CD loop mutants characterized by an enhanced flexibility (Gly-loop40-48 and Gly-loop44-47 ), the F106A mutant, and the double Gly-loop44-47 /F106A mutant. Their ferric resonance Raman spectra in solution and in crystals are almost identical. In the high frequency region, the identification of a double set of core size marker bands indicates the presence of two 6-coordinate low spin species. The resonance Raman data, together with the corresponding crystal structures, indicate the presence of two neuroglobin conformers with a reversed (A conformer) or a canonical (B conformer) heme insertion orientation. With the identification of the marker bands corresponding to each conformer, the data indicates that the B conformer increases at the expense of the A form, predominantly in the Gly-loop44-47 /F106A double mutant, as confirmed by X-ray crystallography. This is the first time that a reversed heme insertion has been identified by resonance Raman in a native 6-coordinate low spin heme protein. This diagnostic tool could be extended to other heme proteins in order to detect heme orientational disorder, which are likely to be correlated to functionally relevant heme dynamics