Loss of Nmp4 optimizes osteogenic metabolism and secretion to enhance bone quality

A goal of osteoporosis therapy is to restore lost bone with structurally sound tissue. Mice lacking the transcription factor Nuclear Matrix Protein 4 (Nmp4, Zfp384, Ciz, ZNF384) respond to several classes of osteoporosis drugs with enhanced bone formation compared to wild type (WT) animals. Nmp4-/- mesenchymal stem/progenitor cells (MSPCs) exhibit an accelerated and enhanced mineralization during osteoblast differentiation. To address the mechanisms underlying this hyper-anabolic phenotype, we carried out RNA-sequencing and molecular and cellular analyses of WT and Nmp4-/- MSPCs during osteogenesis to define pathways and mechanisms associated with elevated matrix production. We determined that Nmp4 has a broad impact on the transcriptome during osteogenic differentiation, contributing to the expression of over 5,000 genes. Phenotypic anchoring of transcriptional data was performed for the hypothesis-testing arm through analysis of cell metabolism, protein synthesis and secretion, and bone material properties. Mechanistic studies confirmed that Nmp4-/- MSPCs exhibited an enhanced capacity for glycolytic conversion- a key step in bone anabolism. Nmp4-/- cells showed elevated collagen translation and secretion. Expression of matrix genes that contribute to bone material-level mechanical properties were elevated in Nmp4-/- cells, an observation that was supported by biomechanical testing of bone samples from Nmp4-/- and WT mice. We conclude that loss of Nmp4 increases the magnitude of glycolysis upon the metabolic switch, which fuels the conversion of the osteoblast into a super-secretor of matrix resulting in more bone with improvements in intrinsic quality

Regulation of 3β-Hydroxysteroid Dehydrogenase/∆5-∆4 Isomerase: A Review

This review focuses on the expression and regulation of 3β-hydroxysteroi ddehydrogenase/Δ5-Δ4 isomerase (3β-HSD), with emphasis on the porcine version. 3β-HSD is often associated with steroidogenesis, but its function in the metabolism of both steroids and xenobiotics is more obscure. Based on currently available literature covering humans,rodents and pigs, this review provides an overview of the present knowledge concerning the regulatory mechanisms for 3β-HSD at all omic levels. The HSD isoenzymes are essential in steroid hormone metabolism, both in the synthesis and degradation of steroids. They display tissue-specific expression and factors influencing their activity, which therefore indicates their tissue-specific responses. 3β-HSD is involved in the synthesis of a number of natural steroid hormones, including progesterone and testosterone, and the hepatic degradation of the pheromone androstenone. In general, a number of signaling and regulatory pathways have been demonstrated to influence 3β-HSD transcription and activity, e.g., JAK-STAT, LH/hCG, ERα, AR, SF-1 and PPARα. The expression and enzymic activity of 3β-HSD are also influenced by external factors, such as dietary composition. Much of the research conducted on porcine 3β-HSD is motivated by its importance for the occurrence of the boar taint phenomenon that results from high concentrations of steroids such as androstenone. This topic is also examined in this review

Prevalence of the EH1 Groucho interaction motif in the metazoan Fox family of transcriptional regulators

<p>Abstract</p> <p>Background</p> <p>The Fox gene family comprises a large and functionally diverse group of <it>forkhead</it>-related transcriptional regulators, many of which are essential for metazoan embryogenesis and physiology. Defining conserved functional domains that mediate the transcriptional activity of Fox proteins will contribute to a comprehensive understanding of the biological function of Fox family genes.</p> <p>Results</p> <p>Systematic analysis of 458 protein sequences of the metazoan Fox family was performed to identify the presence of the engrailed homology-1 motif (eh1), a motif known to mediate physical interaction with transcriptional corepressors of the TLE/Groucho family. Greater than 50% of Fox proteins contain sequences with high similarity to the eh1 motif, including ten of the nineteen Fox subclasses (A, B, C, D, E, G, H, I, L, and Q) and Fox proteins of early divergent species such as marine sponge. The eh1 motif is not detected in Fox proteins of the F, J, K, M, N, O, P, R and S subclasses, or in yeast Fox proteins. The eh1-like motifs are positioned C-terminal to the winged helix DNA-binding domain in all subclasses except for FoxG proteins, which have an N-terminal motif. Two similar eh1-like motifs are found in the zebrafish FoxQ1 and in FoxG proteins of sea urchin and amphioxus. The identification of eh1-like motifs by manual sequence alignment was validated by statistical analyses of the Swiss protein database, confirming a high frequency of occurrence of eh1-like sequences in Fox family proteins. Structural predictions suggest that the majority of identified eh1-like motifs are short α-helices, and wheel modeling revealed an amphipathicity that supports this secondary structure prediction.</p> <p>Conclusion</p> <p>A search for eh1 Groucho interaction motifs in the Fox gene family has identified eh1-like sequences in greater than 50% of Fox proteins. The results predict a physical and functional interaction of TLE/Groucho corepressors with many members of the Fox family of transcriptional regulators. Given the functional importance of the eh1 motif in transcriptional regulation, our annotation of this motif in the Fox gene family will facilitate further study of the diverse transcriptional and regulatory roles of Fox family proteins.</p

Gut microbiota functions: metabolism of nutrients and other food components

The diverse microbial community that inhabits the human gut has an extensive metabolic repertoire that is distinct from, but complements the activity of mammalian enzymes in the liver and gut mucosa and includes functions essential for host digestion. As such, the gut microbiota is a key factor in shaping the biochemical profile of the diet and, therefore, its impact on host health and disease. The important role that the gut microbiota appears to play in human metabolism and health has stimulated research into the identification of specific microorganisms involved in different processes, and the elucidation of metabolic pathways, particularly those associated with metabolism of dietary components and some host-generated substances. In the first part of the review, we discuss the main gut microorganisms, particularly bacteria, and microbial pathways associated with the metabolism of dietary carbohydrates (to short chain fatty acids and gases), proteins, plant polyphenols, bile acids, and vitamins. The second part of the review focuses on the methodologies, existing and novel, that can be employed to explore gut microbial pathways of metabolism. These include mathematical models, omics techniques, isolated microbes, and enzyme assays

Structural Insights into Pseudokinase Domains of Receptor Tyrosine Kinases

Despite their apparent lack of catalytic activity, pseudokinases are essential signaling molecules. Here, we describe the structural and dynamic properties of pseudokinase domains from the Wnt-binding receptor tyrosine kinases (PTK7, ROR1, ROR2, and RYK), which play important roles in development. We determined structures of all pseudokinase domains in this family and found that they share a conserved inactive conformation in their activation loop that resembles the autoinhibited insulin receptor kinase (IRK). They also have inaccessible ATP-binding pockets, occluded by aromatic residues that mimic a cofactor-bound state. Structural comparisons revealed significant domain plasticity and alternative interactions that substitute for absent conserved motifs. The pseudokinases also showed dynamic properties that were strikingly similar to those of IRK. Despite the inaccessible ATP site, screening identified ATP-competitive type-II inhibitors for ROR1. Our results set the stage for an emerging therapeutic modality of "conformational disruptors" to inhibit or modulate non-catalytic functions of pseudokinases deregulated in disease.Peer reviewe

Direct Regulation of CLOCK Expression by REV-ERB

Circadian rhythms are regulated at the cellular level by transcriptional feedback loops leading to oscillations in expression of key proteins including CLOCK, BMAL1, PERIOD (PER), and CRYPTOCHROME (CRY). The CLOCK and BMAL1 proteins are members of the bHLH class of transcription factors and form a heterodimer that regulates the expression of the PER and CRY genes. The nuclear receptor REV-ERBα plays a key role in regulation of oscillations in BMAL1 expression by directly binding to the BMAL1 promoter and suppressing its expression at certain times of day when REV-ERBα expression levels are elevated. We recently demonstrated that REV-ERBα also regulates the expression of NPAS2, a heterodimer partner of BMAL1. Here, we show that REV-ERBα also regulates the expression another heterodimer partner of BMAL1, CLOCK. We identified a REV-ERBα binding site within the 1st intron of the CLOCK gene using a chromatin immunoprecipitation – microarray screen. Suppression of REV-ERBα expression resulted in elevated CLOCK mRNA expression consistent with REV-ERBα's role as a transcriptional repressor. A REV-ERB response element (RevRE) was identified within this region of the CLOCK gene and was conserved between humans and mice. Additionally, the CLOCK RevRE conferred REV-ERB responsiveness to a heterologous reporter gene. Our data suggests that REV-ERBα plays a dual role in regulation of the activity of the BMAL1/CLOCK heterodimer by regulation of expression of both the BMAL1 and CLOCK genes

Fuzzy oil drop model to interpret the structure of antifreeze proteins and their mutants

Mutations in proteins introduce structural changes and influence biological activity: the specific effects depend on the location of the mutation. The simple method proposed in the present paper is based on a two-step model of in silico protein folding. The structure of the first intermediate is assumed to be determined solely by backbone conformation. The structure of the second one is assumed to be determined by the presence of a hydrophobic center. The comparable structural analysis of the set of mutants is performed to identify the mutant-induced structural changes. The changes of the hydrophobic core organization measured by the divergence entropy allows quantitative comparison estimating the relative structural changes upon mutation. The set of antifreeze proteins, which appeared to represent the hydrophobic core structure accordant with “fuzzy oil drop” model was selected for analysis

HDX reveals the conformational dynamics of DNA sequence specific VDR co-activator interactions

The vitamin D receptor/retinoid X receptor-α heterodimer (VDRRXRα) regulates bone mineralization via transcriptional control of osteocalcin (BGLAP) gene and is the receptor for 1α,25-dihydroxyvitamin D3 (1,25D3). However, supra-physiological levels of 1,25D3 activates the calcium-regulating gene TRPV6 leading to hypercalcemia. An approach to attenuate this adverse effect is to develop selective VDR modulators (VDRMs) that differentially activate BGLAP but not TRPV6. Here we present structural insight for the action of a VDRM compared with agonists by employing hydrogen/deuterium exchange. Agonist binding directs crosstalk between co-receptors upon DNA binding, stabilizing the activation function 2 (AF2) surfaces of both receptors driving steroid receptor co-activator-1 (SRC1) interaction. In contrast, AF2 of VDR within VDRM:BGLAP bound heterodimer is more vulnerable for large stabilization upon SRC1 interaction compared with VDRM:TRPV6 bound heterodimer. These results reveal that the combination of ligand structure and DNA sequence tailor the transcriptional activity of VDR toward specific target genes.The vitamin D receptor/retinoid X receptor-α heterodimer (VDRRXRα) regulates bone mineralization. Here the authors employ hydrogen/deuterium exchange (HDX) mass spectrometry to study the conformational dynamics of VDRRXRα and give mechanistic insights into how VDRRXRα controls the transcriptional activity of specific genes.Jie Zheng, Mi Ra Chang, Ryan E. Stites, Yong Wang, John B. Bruning, Bruce D. Pascal, Scott J. Novick, Ruben D. Garcia-Ordonez, Keith R. Stayrook, Michael J. Chalmers, Jeffrey A. Dodge & Patrick R. Griffi