Integrative genomic, epigenetic and metabolomic characterization of beef from grass-fed Angus steers

Beef constitutes a main component of the American diet and still represent the principal source of protein in many parts of the world. Currently, the meat market is experiencing an important transformation; consumers are increasingly switching from consuming traditional beef to grass-fed beef. People recognized products obtained from grass-fed animals as more natural and healthy. However, the true variations between these two production systems regarding various aspects remain unclear. This dissertation provides information from closely genetically related animals, in order to decrease confounding factors, to explain several confused divergences between grain-fed and grass-fed beef. First, we examined the growth curve, important economic traits and quality carcass characteristics over four consecutive years in grain-fed and grass-fed animals, generating valuable information for management decisions and economic evaluation for grass-fed cattle operations. Second, we performed the first integrated transcriptomic and metabolomic analysis in grass-fed beef, detecting alterations in glucose metabolism, divergences in free fatty acids and carnitine conjugated lipid levels, and altered β-oxidation. Results suggest that grass finished beef could possibly benefit consumer health from having lower total fat content and better lipid profile than grain-fed beef. Regarding animal welfare, grass-fed animals may experience less stress than grain-fed individuals as well. Finally, we contrasted the genome-wide DNA methylation of grass-fed beef against grain-fed beef using the methyl-CpG binding domain sequencing (MBD-Seq) method, identifying 60 differentially methylated regions (DMRs). Most of DMRs were located inside or upstream of genes and displayed increased levels of methylation in grass-fed individuals, implying a global DNA methylation increment in this group. Interestingly, chromosome 14, which has been associated with large effects on ADG, marbling, back fat, ribeye area and hot carcass weight in beef cattle, allocated the largest number of DMRs (12/60). The pathway analysis identified skeletal and muscular system as the preeminent physiological system and function, and recognized carbohydrates metabolism, lipid metabolism and tissue morphology among the highest ranked networks. Therefore, although we recognize some limitations and assume that additional examination is still required, this project provides the first integrative genomic, epigenetic and metabolomics characterization of beef produced under grass-fed regimen.

Crystal structure of Bn IV in complex with myristic acid: A Lys49 myotoxic phospholipase A(2) from Bothrops neuwiedi venom

The LY549-PLA(2)s myotoxins have attracted attention as models for the induction of myonecrosis by a catalytically independent mechanism of action. Structural studies and biological activities have demonstrated that the myotoxic activity of LYS49-PLA(2) is independent of the catalytic activity site. The myotoxic effect is conventionally thought to be to due to the C-terminal region 111-121, which plays an effective role in membrane damage. In the present study, Bn IV LYS49-PLA(2) was isolated from Bothrops neuwiedi snake venom in complex with myristic acid (CH3(CH2)(12)COOH) and its overall structure was refined at 2.2 angstrom resolution. The Bn IV crystals belong to monoclinic space group P2(1) and contain a dimer in the asymmetric unit. The unit cell parameters are a = 38.8, b = 70.4, c = 44.0 angstrom. The biological assembly is a "conventional dimer" and the results confirm that dimer formation is not relevant to the myotoxic activity. Electron density map analysis of the Bn IV structure shows clearly the presence of myristic acid in catalytic site. The relevant structural features for myotoxic activity are located in the C-terminal region and the Bn IV C-terminal residues NKKYRY are a probable heparin binding domain. These findings indicate that the mechanism of interaction between Bn IV and muscle cell membranes is through some kind of cell signal transduction mediated by heparin complexes. (C) 2010 Elsevier Masson SAS. All rights reserved.

Análise da interação entre moléculas desenhadas a partir do ácido anacárdico e a proteína PPAR usando ferramentas in silico

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Molecular, 2016.

The actinin family of actin crosslinking proteins: natural functions and potential applications in synthetic biology

Actinin and spectrin proteins are members of the Spectrin Family of Actin Crosslinking Proteins. The importance of these proteins in the cytoskeleton is demonstrated by the fact that they are common targets for disease causing mutations. In their most prominent roles, actinin and spectrin are responsible for stabilising and maintaining the muscle architecture during contraction, and providing shape and elasticity to the red blood cell in circulation, respectively. To carry out such roles, actinin and spectrin must possess important mechanical and physical properties. These attributes are desirable when choosing a building block for protein-based nanoconstruction. In this study, I assess the contribution of several disease-associated mutations in the actinin-1 actin binding domain that have recently been linked to a rare platelet disorder, congenital macrothrombocytopenia. I investigate the suitability of both actinin and spectrin proteins as potential building blocks for nanoscale structures, and I evaluate a fusion-based assembly strategy to bring about self-assembly of protein nanostructures. I report that the actinin-1 mutant proteins display increased actin binding compared to WT actinin-1 proteins. I find that both actinin and spectrin proteins exhibit enormous potential as nano-building blocks in terms of their stability and ability to self-assemble, and I successfully design and create homodimeric and heterodimeric bivalent building blocks using the fusion-based assembly strategy. Overall, this study has gathered helpful information that will contribute to furthering the advancement of actinin and spectrin knowledge in terms of their natural functions, and potential unnatural functions in protein nanotechnology.

Target sequence recognition by the calmodulin superfamily: implications from light chain binding to the regulatory domain of scallop myosin.

Some of the rules for how members of the calmodulin (CaM) superfamily bind to target peptides are revealed by the crystal structure of the regulatory domain of scallop myosin. The structure shows that the IQ motif of the heavy chain in this invertebrate myosin imposes constraints on both the positioning and conformation of the individual lobes of the light chains. In contrast, analysis of the contact residues in the targets bound by Ca(2+)-CaM reveals how the structure of CaM accommodates a broader range of sequences consonant with this protein's functional diversity.

Role of Neurogranin in the regulation of calcium binding to Calmodulin

Role of Neurogranin in the regulation of calcium binding to Calmodulin Anuja Chandrasekar, B.S Advisor: M. Neal Waxham, Ph.D The overall goal of my project was to gain a quantitative understanding of how the interaction between two proteins neurogranin (RC3) and calmodulin (CaM) alters a fundamental property of CaM. CaM, has been extensively studied for more than four decades due to its seminal role in almost all biological functions as a calcium signal transducer. Calcium signals in cardiac and neuronal cells are exquisitely precise and enable activation of some processes while down-regulating others. CaM, with its four calcium binding sites, serves as a central component of calcium signaling in these cells. It is aided in this role as a regulatory hub that differentially activates targets in response to a calcium flux by proteins that alter its calcium binding properties. Neurogranin, also known as RC3, is a member of a family of small neuronal IQ (SNIQ) domain proteins that was originally thought to play a ‘capacitive’ role by sequestering CaM until a calcium influx of sufficient intensity arrived. However, based on earlier work in our lab on neurogranin, we believe that this protein plays a more nuanced role in neurons than simply acting as a CaM buffer. We believe that neurogranin is one of the proteins which, by altering the kinetics of calcium binding allow CaM to decode a variety of signals with fine precision. To quantify the interaction between CaM, neurogranin and calcium, I used biophysical techniques and computational simulations. From my results, I conclude that neurogranin finely regulates the proportion of calcium-saturated CaM and thereby directs CaM’s target specificity.

Giant protein kinases: Domain interactions and structural basis of autoregulation

The myosin-associated giant protein kinases twitchin and titin are composed predominantly of fibronectin- and immunoglobulin-like modules, We report the crystal structures of two autoinhibited twitchin kinase fragments, one from Aplysia and a larger fragment from Caenorhabditis elegans containing an additional C-terminal immunoglobulin-like domain, The structure of the longer fragment shoes that the immunoglobulin domain contacts the protein kinase domain on the opposite side from the catalytic cleft, laterally exposing potential myosin binding residues, Together, the structures reveal the cooperative interactions between the autoregulatory region and the residues from the catalytic domain involved in protein substrate binding, ATP binding, catalysis and the activation loop, and explain the differences between the observed autoinhibitory mechanism and the one found in the structure of calmodulin-dependent kinase I.

Cutting Edge: Nucleotide-Binding Oligomerization Domain 1-Dependent Responses Account for Murine Resistance against Trypanosoma cruzi Infection

An effective innate immune recognition of the intracellular protozoan parasite Trypanosoma cruzi is critical for host resistance against Chagas disease, a severe and chronic illness that affects millions of people in Latin America. In this study, we evaluated the participation of nucleotide-binding oligomerization domain (Nod)like receptor proteins in host response to T cruzi infection and found that Nod1-dependent, but not Nod2-dependent, responses are required for host resistance against infection. Bone marrow-derived macrophages from Nod1(-/-) mice showed an impaired induction of NF-kappa B-dependent products in response to infection and failed to restrict T cruzi infection in presence of IFN-gamma. Despite normal cytokine production in the sera, Nod1(-/-) mice were highly susceptible to T cruzi infection, in a similar manner to MyD88(-/-) and NO synthase 2(-/-) mice. These studies indicate that Nod1-dependent responses account for host resistance against T cruzi infection by mechanisms independent of cytokine production. The Journal of Immunology, 2010, 184: 1148-1152.

Characterization of a glycine receptor domain that controls the binding and gating mechanisms of the beta-amino acid agonist, taurine

The beta -amino acid, taurine, is a full agonist of the human glycine receptor al subunit when recombinantly expressed in a mammalian (HEK293) cell line, but a partial agonist of the same receptor when expressed in Xenopus oocytes. Several residues in the Ala101-Thr112 domain have previously been identified as determinants of beta -amino acid binding and gating mechanisms in Xenopus oocyte-expressed receptors. The present study used the substituted cysteine accessibility method to investigate the role of this domain in controlling taurine-specific binding and gating mechanisms of glycine receptors recombinantly expressed in mammalian cells. Asn102 and Glu103 are identified as taurine and glycine binding sites, whereas Ala101 is eliminated as a possible binding site. The N102C mutation also abolished the antagonistic actions of taurine, indicating that this site does not discriminate between the putative agonist- and antagonist-bound conformations of beta -amino acids. The effects of mutations from Lys104-Thr112 indicate that the mechanism by which this domain controls beta -amino acid-specific binding and gating processes differs substantially depending on whether the receptor is expressed in mammalian cells or Xenopus oocytes. Thr112 is the only domain element in mammalian cell-expressed GlyRs which was demonstrated to discriminate between glycine and taurine.

Synthesis and structural analysis of the N-terminal domain of the thyroid hormone-binding protein transthyretin

Transthyretin (TTR) is a 55 kDa protein responsible for the transport of thyroid hormones and retinol in human serum. Misfolded forms of the protein are implicated in the amyloid diseases familial amyloidotic polyneuropathy and senile systemic amyloidosis. Its folding properties and stabilization by ligands are of current interest due to their importance in understanding and combating these diseases. To assist in such studies we developed a method for the solid phase synthesis of the monomeric unit of a TTR analogue and its folding to form a functional 55 kDa tetramer. The monomeric unit of the protein was chemically synthesized in three parts, comprising amino acid residues 151, 5499 and 102127, and ligated using chemoselective thioether ligation chemistry. The synthetic protein was folded and assembled to a tetrameric structure in the presence of the TTRs native ligand, thyroxine, as shown by gel filtration chromatography, native gel electrophoresis, TTR antibody recognition and thyroid hormone binding. In the current study the solution structure of the first of these fragment peptides, TTR(151) is examined to determine its intrinsic propensity to form beta-sheet structure, potentially involved in amyloid fibril formation by TTR. Despite the presence of extensive beta-structure in the native form of the protein, the Nterminal fragment adopts an essentially random coil conformation in solution.

Regulation of the DNA-binding and transcriptional activities of Xenopus laevis NFI-X by a novel C-terminal domain.

The nuclear factor I (NFI) family consists of sequence-specific DNA-binding proteins that activate both transcription and adenovirus DNA replication. We have characterized three new members of the NFI family that belong to the Xenopus laevis NFI-X subtype and differ in their C-termini. We show that these polypeptides can activate transcription in HeLa and Drosophila Schneider line 2 cells, using an activation domain that is subdivided into adjacent variable and subtype-specific domains each having independent activation properties in chimeric proteins. Together, these two domains constitute the full NFI-X transactivation potential. In addition, we find that the X. laevis NFI-X proteins are capable of activating adenovirus DNA replication through their conserved N-terminal DNA-binding domains. Surprisingly, their in vitro DNA-binding activities are specifically inhibited by a novel repressor domain contained within the C-terminal part, while the dimerization and replication functions per se are not affected. However, inhibition of DNA-binding activity in vitro is relieved within the cell, as transcriptional activation occurs irrespective of the presence of the repressor domain. Moreover, the region comprising the repressor domain participates in transactivation. Mechanisms that may allow the relief of DNA-binding inhibition in vivo and trigger transcriptional activation are discussed.

Cutting edge: influence of the TCR V beta domain on the avidity of CD1d:alpha-galactosylceramide binding by invariant V alpha 14 NKT cells.

CD1d tetramers loaded with alpha-galactosylceramide (alpha-GalCer) bind selectively to mouse invariant Valpha14 (Valpha14i) NKT cells and their human counterparts. Whereas tetramer binding strictly depends on the expression of a Valpha14-Jalpha18 chain in murine NKT cells, the associated beta-chain (typically expressing Vbeta8.2 or Vbeta7) appears not to influence tetramer binding. In this study, we describe novel alpha-GalCer-loaded mouse and human CD1d-IgG1 dimers, which revealed an unexpected influence of the TCR-beta chain on the avidity of CD1d:alpha-GalCer binding. A subset of Valpha14i NKT cells clearly discriminated alpha-GalCer bound to mouse or human CD1d on the basis of avidity differences conferred by the Vbeta domain of the TCR-beta chain, with Vbeta8.2 conferring higher avidity binding than Vbeta7.

Pathogenic Vibrio activate NLRP3 inflammasome via cytotoxins and TLR/nucleotide-binding oligomerization domain-mediated NF-kappa B signaling.

Vibrio vulnificus and Vibrio cholerae are Gram-negative pathogens that cause serious infectious disease in humans. The beta form of pro-IL-1 is thought to be involved in inflammatory responses and disease development during infection with these pathogens, but the mechanism of beta form of pro-IL-1 production remains poorly defined. In this study, we demonstrate that infection of mouse macrophages with two pathogenic Vibrio triggers the activation of caspase-1 via the NLRP3 inflammasome. Activation of the NLRP3 inflammasome was mediated by hemolysins and multifunctional repeat-in-toxins produced by the pathogenic bacteria. NLRP3 activation in response to V. vulnificus infection required NF-kappaB activation, which was mediated via TLR signaling. V. cholerae-induced NLRP3 activation also required NF-kappaB activation but was independent of TLR stimulation. Studies with purified V. cholerae hemolysin revealed that toxin-stimulated NLRP3 activation was induced by TLR and nucleotide-binding oligomerization domain 1/2 ligand-mediated NF-kappaB activation. Our results identify the NLRP3 inflammasome as a sensor of Vibrio infections through the action of bacterial cytotoxins and differential activation of innate signaling pathways acting upstream of NF-kappaB.

Characterization of HbpR binding by site-directed mutagenesis of its DNA-binding site and by deletion of the effector domain.

In the presence of 2-hydroxybiphenyl, the enhancer binding protein, HbpR, activates the sigma54-dependent P(hbpC) promoter and controls the initial steps of 2-hydroxybiphenyl degradation in Pseudomonas azelaica. In the activation process, an oligomeric HbpR complex of unknown subunit composition binds to an operator region containing two imperfect palindromic sequences. Here, the HbpR-DNA binding interactions were investigated by site-directed mutagenesis of the operator region and by DNA-binding assays using purified HbpR. Mutations that disrupted the twofold symmetry in the palindromes did not affect the binding affinity of HbpR, but various mutations along a 60 bp region, and also outside the direct palindromic sequences, decreased the binding affinity. Footprints of HbpR on mutant operator fragments showed that a partial loss of binding contacts occurs, suggesting that the binding of one HbpR 'protomer' in the oligomeric complex is impaired whilst leaving the other contacts intact. An HbpR variant, devoid of its N-terminal sensing A-domain, was unable to activate transcription from the hbpC promoter while maintaining protection of the operator DNA in footprints. Wild-type HbpR was unable to activate transcription from the hbpC promoter when delta A-HbpR was expressed in the same cell, suggesting the formation of (repressing) hetero-oligomers. This model implies that HbpR can self-associate on its operator DNA without effector recognition or ATP binding. Furthermore, our findings suggest that the N-terminal sensing domain of HbpR is needed to activate the central ATPase domain rather than to repress a constitutively active C domain, as is the case for the related regulatory protein XylR.

PDZ domain-binding motif regulates cardiomyocyte compartment-specific NaV1.5 channel expression and function.

BACKGROUND: Sodium channel NaV1.5 underlies cardiac excitability and conduction. The last 3 residues of NaV1.5 (Ser-Ile-Val) constitute a PDZ domain-binding motif that interacts with PDZ proteins such as syntrophins and SAP97 at different locations within the cardiomyocyte, thus defining distinct pools of NaV1.5 multiprotein complexes. Here, we explored the in vivo and clinical impact of this motif through characterization of mutant mice and genetic screening of patients. METHODS AND RESULTS: To investigate in vivo the regulatory role of this motif, we generated knock-in mice lacking the SIV domain (ΔSIV). ΔSIV mice displayed reduced NaV1.5 expression and sodium current (INa), specifically at the lateral myocyte membrane, whereas NaV1.5 expression and INa at the intercalated disks were unaffected. Optical mapping of ΔSIV hearts revealed that ventricular conduction velocity was preferentially decreased in the transversal direction to myocardial fiber orientation, leading to increased anisotropy of ventricular conduction. Internalization of wild-type and ΔSIV channels was unchanged in HEK293 cells. However, the proteasome inhibitor MG132 rescued ΔSIV INa, suggesting that the SIV motif is important for regulation of NaV1.5 degradation. A missense mutation within the SIV motif (p.V2016M) was identified in a patient with Brugada syndrome. The mutation decreased NaV1.5 cell surface expression and INa when expressed in HEK293 cells. CONCLUSIONS: Our results demonstrate the in vivo significance of the PDZ domain-binding motif in the correct expression of NaV1.5 at the lateral cardiomyocyte membrane and underline the functional role of lateral NaV1.5 in ventricular conduction. Furthermore, we reveal a clinical relevance of the SIV motif in cardiac disease.