Divergent Regulatory Roles of Transcriptional Variants of the Chicken LDB3 Gene in Muscle Shaping.

LIM domain binding 3 (LDB3) serves as a striated muscle-specific Z-band alternatively spliced protein that plays an important role in mammalian skeletal muscle development, but its regulatory role and molecular mechanism in avian muscle development are still unclear. In this study, we reanalyzed RNA sequencing data sets of 1415 samples from 21 chicken tissues published in the NCBI GEO database. First, three variants (LDB3-X, LDB3-XN1, and LDB3-XN2) generated by alternative splicing of the LDB3 gene were identified in chicken skeletal muscle, among which LDB3-XN1 and LDB3-XN2 are novel variants. LDB3-X and LDB3-XN1 are derived from exon skipping in chicken skeletal muscle at the E18-D7 stage and share three LIM domains, but LDB3-XN2 lacks a LIM domain. Our results preliminarily suggest that the formation of three variants of LDB3 is regulated by RBM20. The three splice isomers have divergent functions in skeletal muscle according to in vitro and in vivo assays. Finally, we identified the mechanism by which different variants play different roles through interactions with IGF2BP1 and MYHC, which promote the proliferation and differentiation of chicken myoblasts, in turn regulating chicken myogenesis. In conclusion, this study revealed the divergent roles of three LDB3 variants in chicken myogenesis and muscle remodeling and demonstrated their regulatory mechanism through protein-protein interactions.

Integrated landscape of chicken egg chalaza proteomics.

Chicken egg chalaza (CLZ) is a natural colloidal structure in eggs that exists as an egg yolk stabilizer and is similar in composition to egg white. In this study, the proteome, phosphoproteome, and N-glycoproteome of CLZ were characterized in depth. We hydrolyzed the CLZ proteins and enriched the phosphopeptides and glycopeptides. We identified 45 phosphoproteins and 80 N-glycoproteins, containing 59 phosphosites and 203 N-glycosylation sites, respectively. Typically, the ovalbumin in CLZ was both phosphorylated and N-glycosylated, with 4 phosphosites and 4 N-glycosylation sites. Moreover, we identified 2 N-glycosylated subunits of ovomucin, mucin-5B and mucin-6, with 32 and nine N- glycosylation sites, respectively. Analysis of the phosphorylation and N-glycosylation status of CLZ proteins could provide novel insights into the structural and functional characteristics of CLZ.

Repeated MDA5 Gene Loss in Birds: An Evolutionary Perspective.

Two key cytosolic receptors belonging to the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family sense the viral RNA-derived danger signals: RIG-I and melanoma differentiation-associated protein 5 (MDA5). Their activation establishes an antiviral state by downstream signaling that ultimately activates interferon-stimulated genes (ISGs). While in rare cases RIG-I gene loss has been detected in mammalian and avian species, most notably in the chicken, MDA5 pseudogenization has only been detected once in mammals. We have screened over a hundred publicly available avian genome sequences and describe an independent disruption of MDA5 in two unrelated avian lineages, the storks (Ciconiiformes) and the rallids (Gruiformes). The results of our RELAX analysis confirmed the absence of negative selection in the MDA5 pseudogene. In contrast to our prediction, we have shown, using multiple dN/dS-based approaches, that the MDA5 loss does not appear to have resulted in any compensatory evolution in the RIG-I gene, which may partially share its ligand-binding specificity. Together, our results indicate that the MDA5 pseudogenization may have important functional effects on immune responsiveness in these two avian clades.

Identification of Chicken Transglutaminase 1 and In Situ Localization of Transglutaminase Activity in Avian Skin and Esophagus.

Transglutaminase 1 (TGM1) is a membrane-anchored enzyme that cross-links proteins during terminal differentiation of epidermal and esophageal keratinocytes in mammals. The current genome assembly of the chicken, which is a major model for avian skin biology, does not include an annotated region corresponding to TGM1. To close this gap of knowledge about the genetic control of avian cornification, we analyzed RNA-sequencing reads from organotypic chicken skin and identified TGM1 mRNA. By RT-PCR, we demonstrated that TGM1 is expressed in the skin and esophagus of chickens. The cysteine-rich sequence motif required for palmitoylation and membrane anchorage is conserved in the chicken TGM1 protein, and differentiated chicken keratinocytes display membrane-associated transglutaminase activity. Expression of TGM1 and prominent transglutaminase activity in the esophageal epithelium was also demonstrated in the zebra finch. Altogether, the results of this study indicate that TGM1 is conserved among birds and suggest that chicken keratinocytes may be a useful model for the study of TGM1 in non-mammalian cornification.

Early origin of sweet perception in the songbird radiation.

Early events in the evolutionary history of a clade can shape the sensory systems of descendant lineages. Although the avian ancestor may not have had a sweet receptor, the widespread incidence of nectar-feeding birds suggests multiple acquisitions of sugar detection. In this study, we identify a single early sensory shift of the umami receptor (the T1R1-T1R3 heterodimer) that conferred sweet-sensing abilities in songbirds, a large evolutionary radiation containing nearly half of all living birds. We demonstrate sugar responses across species with diverse diets, uncover critical sites underlying carbohydrate detection, and identify the molecular basis of sensory convergence between songbirds and nectar-specialist hummingbirds. This early shift shaped the sensory biology of an entire radiation, emphasizing the role of contingency and providing an example of the genetic basis of convergence in avian evolution.

The B-type natriuretic peptide of the Congo and Timneh grey parrot.

In captivity, cardiovascular diseases are common in grey parrots. The diagnosis of these diseases in living birds is difficult, and new diagnostic possibilities would be desirable. The heart is an important endocrine organ in which cardiomyocytes synthetise B-type natriuretic peptide (BNP) and release it into the bloodstream. This hormone has a significant role in cardiovascular and body fluid regulation. The blood concentration of BNP is used in human medicine and small animal medicine as a diagnostic tool in the identification of heart diseases and as a prognostic marker for the risk of mortality. The nucleotide and amino acid sequence of BNP was described in Congo (n = 4) and Timneh (n = 3) grey parrots by PCR after RNA isolation from the atria and ventricles. The results showed a high similarity between the nucleotide sequences of the grey parrots' BNP and the already known sequence of this hormone in chickens. The amino acid sequence of the mature peptide region is consistent in these three species. BNP plasma concentration could be a possible blood parameter for identifying clinically manifest cardiovascular diseases in grey parrots as it is in other species.

An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography.

Serial femtosecond crystallography has opened up many new opportunities in structural biology. In recent years, several approaches employing light-inducible systems have emerged to enable time-resolved experiments that reveal protein dynamics at high atomic and temporal resolutions. However, very few enzymes are light-dependent, whereas macromolecules requiring ligand diffusion into an active site are ubiquitous. In this work we present a drop-on-drop sample delivery system that enables the study of enzyme-catalyzed reactions in microcrystal slurries. The system delivers ligand solutions in bursts of multiple picoliter-sized drops on top of a larger crystal-containing drop inducing turbulent mixing and transports the mixture to the X-ray interaction region with temporal resolution. We demonstrate mixing using fluorescent dyes, numerical simulations and time-resolved serial femtosecond crystallography, which show rapid ligand diffusion through microdroplets. The drop-on-drop method has the potential to be widely applicable to serial crystallography studies, particularly of enzyme reactions with small molecule substrates.

Evolution of single gyroid photonic crystals in bird feathers.

Vivid, saturated structural colors are conspicuous and important features of many animals. A rich diversity of three-dimensional periodic photonic nanostructures is found in the chitinaceous exoskeletons of invertebrates. Three-dimensional photonic nanostructures have been described in bird feathers, but they are typically quasi-ordered. Here, we report bicontinuous single gyroid ß-keratin and air photonic crystal networks in the feather barbs of blue-winged leafbirds (Chloropsis cochinchinensis sensu lato), which have evolved from ancestral quasi-ordered channel-type nanostructures. Self-assembled avian photonic crystals may serve as inspiration for multifunctional applications, as they suggest efficient, alternative routes to single gyroid synthesis at optical length scales, which has been experimentally elusive.

Molecular characterization of Gyps africanus (African white-backed vulture) organic anion transporter 1 and 2 expressed in the kidney.

Gyps species have been previously shown to be highly sensitive to the toxic effects of diclofenac, when present in their food sources as drug residues following use as a veterinary medicine. Vultures exposed to diclofenac soon become depressed and die with signs of severe visceral gout and renal damage on necropsy. The molecular mechanism behind toxicity and renal excretion of uric acid is still poorly understood. With the clinical pictures suggesting renal uric acid excretion as the target site for toxicity, as a first step the following study was undertaken to determine the uric acid excretory pathways present in the African white-backed vulture (Gyps africanus) (AWB), one of the species susceptible to toxicity. Using transcriptome analysis, immunohistochemistry and functional predictions, we demonstrated that AWB makes use of the organic anion transporter 2 (OAT2) for their uric acid excretion. RT-qPCR analysis subsequently demonstrated relatively similar expression of the OAT2 transporter in the vulture and chicken. Lastly docking analysis, predicted that the non-steroidal drugs induce their toxicity through an allosteric binding.

Characterization and biological role of cysteine-rich venom protein belonging to CRISPs from turkey seminal plasma†.

Turkey semen contains cysteine-rich secretory proteins (CRISPs) that belong to the dominant seminal plasma proteins. We aimed to isolate and characterize CRISP from turkey seminal plasma and evaluate its possible involvement in yellow semen syndrome (YSS). YSS, which is well characterized, causes reduced fertility and hatchability. The protein was purified using hydrophobic interaction, gel filtration, and reverse phase chromatography. It then was subjected to identification by mass spectrometry, analysis of physicochemical properties, and specific antibody production. The biological function of the isolated protein was tested and included its effects on sperm motility and migration and sperm-egg interactions. Sperm motility was measured with the CASA system using Hobson Sperm Tracker. The reproductive tract of turkey toms was analyzed for gene expression; immunohistochemistry was used for protein localization in the male reproductive tract, spermatozoa, and inner perivitelline layer. The isolated protein was identified as cysteine-rich venom protein-like isoform X2 (CRVP X2; XP_010706464.1) and contained feature motifs of CRISP family proteins. Turkey CRVP X2 was present in both spermatozoa and seminal plasma. The extensive secretion of CRVP X2 by the epithelial cells of the epididymis and ductus deferens suggests its involvement in post-testicular sperm maturation. The internally localized CRVP X2 in the proximal part of the sperm tail might be responsible for stimulation of sperm motility. CRVP X2 on the sperm head might be involved in several events prior to fusion and may also participate in gamete fusion itself. Although the mechanisms by which CRVP X2 mediates fertilization are still unknown, the involvement of complementary sites cannot be excluded. The disturbance of CRVP X2 expression can serve as an etiologic factor of YSS in the turkey. This study expands the understanding of the detailed mechanism of fertilization in birds by clarifying the specific role of CRVP X2.

Different fluorescence emitting copper nanoclusters protected by egg white and double-emission fluorescent probe for fast detection of ethanol.

Green emitting copper nanoclusters (G-Cu NCs), yellow emitting Cu NCs (Y-Cu NCs), orange emitting Cu NCs (O-Cu NCs) and red emitting Cu NCs (R-Cu NCs) were prepared using chicken egg white as the stabilizer by changing the reaction conditions. This is a green, facile and cheap method to explore different color emitting CuNCs by the same precursor and stabilizers. The G-Cu NCs were employed for the detection of ethanol due to their aggregation induced emission enhancement (AIEE) effect. The fluorescence emission of Cu NCs at 526 nm under the excitation of 444 nm can be effectively enhanced in the presence of ethanol due to AIEE effect, thus realizing the quantitative determination of ethanol content in the range 5-60%. In addition, a visual dual-emission fluorescence probe with the combination of G-Cu NCs and silicon nanoparticles (Si NPs/G-Cu NCs) was designed to evaluate ethanol content conveniently and rapidly. Desirable linear relationship is observed between ratio of fluorescence intensity (I525/I441) and ethanol content under the excitation of 383 nm. Visible color transformation of this probe is observed in the ethanol content range 2-20%. Moreover, the ethanol sensing platforms were applied to the detection and evaluation of the alcohol content of liquor, and the recoveries in liquor were in the range 99.7% to 113%, broadening the applications of Cu NCs and providing a sensitive detection method for ethanol.

Molecular cloning and characterization of HSP60 gene in domestic pigeons (Columba livia) and differential expression patterns under temperature stress.

Heat shock protein 60 (HSP60) is a well-recognized multifunctional protein, playing a substantial role in protecting organisms from environmental stress. The domestic pigeon (Columba livia) is a promising model organism, with important economic and ecological value, and its health is susceptible to temperature stress. To explore the molecular characteristics, tissue expression profile, and response to temperature stress for HSP60 of Columba livia (ClHSP60), we firstly cloned and characterized the complete cDNA sequence and investigated its expression profile under optimal conditions and acute temperature stress. The cDNA of ClHSP60 contained 2257 nucleotides, consisting of 12 exons with length ranging from 65 to 590 bp. The open reading frame (ORF) encoded 573 amino acids with calculated molecular weight of 60.97 kDa that contained a number of structurally prominent domains or motifs. Under optimal temperature conditions, levels of ClHSP60 expression differed between all the tested tissues (the highest was noted in liver and the lowest in pectoralis major muscle). Under acute temperature stress, five patterns of change were detected in the tested tissues, suggesting that different tissues in domestic pigeons differentially responded to various temperature stress conditions. Upregulation of ClHSP60 expression was highest in the lung and pectoralis major muscle, reflecting the crucial role of these two tissues in temperature regulation. However, the crop, cerebrum, and heart showed little change or decreased ClHSP60 expression. The results indicate that ClHSP60 may be sensitive to and play pivotal roles in responding to acute temperature stress.

Parakeet Hemoglobin - Its Crystal Structure and Oxygen Affinity in Relation to Some Avian Hemoglobins.

BACKGROUND: "Avians" often show efficient oxygen management to meet the demands of their metabolism. Hemoglobin, a transporter protein consists of four non-covalently linked subunits contain haem binding hydrophobic pocket serves as a site of allosteric cooperativity. The physiology and anatomy of both mammals and avian are functionally different, in birds, the respiratory system formed by small air sacs that serve as tidal ventilation for the lungs and have no significant exchange across their cells. Parakeet (Psittacula krameri) a tropical and non-migrating species and it is easily adapted to living in disturbed habitat. The sequence analysis reveals that α and ß chain of parakeet hemoglobin highly similar grey lag goose and bar headed goose hemoglobin respectively. Thus it has been tempted us to study in to analyzing the sequence and structural comparison of this hemoglobin to find out the physiological capabilities of parakeet hemoglobin. OBJECTIVE: The structure determination studies of parakeet hemoglobin by X-ray diffraction. The sequence and structure are compared with goose, chicken and human Hb, emphasizing the role of amino acids in the subunit contacts that facilitate survival by low oxygen demand. METHODS: The Hb was purified and crystallized by hanging drop vapor diffusion method using poly ethylene glycol (PEG) 3350 and sodium phosphate buffer. X-ray diffracted data set was collected at 3Å resolution, the data was processed in Automar and molecular replacement, refinements, model building was carried out in CCP4i program package. The final refined model was deposited in protein data bank with accession id 2zfb. RESULTS: The tertiary structure of Parakeet Hb is compared with the met form of BHG Hb (1c40) and oxy form of GLG (1faw) and oxy form of human Hbs (1hho). Superimposing parakeet Hb α1ß1 subunit with 'R' state human Hb shows an r.m.s.d of 0.98 Å and for BHG and GLG Hb, the r.m.s.d shows 0.72 and 0.61 Å. The replacement of α115Asp in parakeet Hb as against the α115Glu in human Hb results in the movement of GH corners. The amino acid proline at α50 present only in Parakeet Hb and Chicken HbD and not present in any other avian family which includes human Hb. The residue α78Thr located in EF corner loop region, which slightly diverge when superimposing with human and BHG Hb and also replacement of α113Asn present only in Parakeet Hb placed near the FG helix corner. CONCLUSION: The present study describes the structure determination of parakeet hemoglobin and its structural features to understand its oxygen affinity characteristics. The crystals were obtained by buffered low-salt conditions, like those of chicken HbD, carbonmonoxy and cyanomet human Hb. The present study reveals several interesting and unique modifications in the finer aspects of the quaternary structure of parakeet Hb, which are involved in oxygen affinity characteristics and the α1ß1 subunit contacts. Crystallization of parakeet Hb with allosteric effectors like Inositol pentaphosphate may bring further understanding of the influence of physiological and environmental factors on the quaternary structure.

Proteins associated with quality deterioration of prepared chicken breast based on differential proteomics during refrigerated storage.

BACKGROUND: Prepared chicken breast deterioration is a complex biochemical process, of which protein change is one of the main features. The present research focuses on the analysis of proteins related to the deterioration in quality of prepared chicken breast through differential proteomics analysis. RESULTS: The physicochemical indexes of prepared chicken breast showed that quality gradually decreased at the second week of refrigerated storage, while the deterioration of chicken breast meat was obvious at the third week. Three key time points of quality change were determined to be at 0th, 2th and 5th week, respectively. In addition, 39 differential proteins were successfully identified using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Most of the identified proteins showed significant differences in expression at the three key points of storage, of which actin, myosin, α-1,4-glucan phosphorylase, phosphoglucomutase 1, heat shock protein ß-1, tubulin ß-7 chain and skeletal muscle type tropomodulin (fragment) were closely related to the quality deterioration of prepared chicken breast, and thus potential indicator proteins to evaluate the quality of chicken breast. CONCLUSION: The current study indicated that the physicochemical quality of prepared breast notably changed during refrigerated storage. Three key time points of quality change in the storage process of prepared chicken breast were determined. Furthermore, differential proteomics identified the key proteins related to freshness, which provides a theoretical basis for exploring the mechanism of chicken breast deterioration. © 2020 Society of Chemical Industry.

Cloning and structural analysis of complement component 3d in wild birds provides insight into its functional evolution.

Complement component 3 d (C3d) is the final cleavage product of the complement component C3 and serves as a crucial role in link innate and adaptive immunity, and increase B-cell sensitivity to an antigen by 1000-10000 fold. The crystal structure of human C3d revealed there are two distinct surfaces, a convex surface containing the thioester-constituting residues that mediate covalent binding to the target antigen, and a concave surface with an acidic pocket responsible for interaction with CR2. In this study, we cloned and sequenced cDNA fragment encoding C3d region from 15 wild bird species. Then, the C3d sequences from wild birds, chicken and mammals were aligned to construct phylogenetic trees. Phylogenetic tree displayed two main branches, indicating mammals and birds, but the bird C3d branch was divided into two main parts, with five wild birds (Ardeola bacchus, Zoothera, Bubo, Crossoptilon mantchuricum and Caprimulgus europaeus) clustering much closer to mammals. In addition, the C3d proteins of Ardeola bacchus, Bubo, Crossoptilon mantchuricum and Caprimulgus europaeus contained a Glu163 residue at the position at which Lys163 was found in other birds. However, Glu163 have the same charge polarity as Asp163, which is the key amino acid residue comprising the acidic pocket combined with CR2 found at this position in mammals, and Zoothera also possessed Asp163 at this position. Structure modeling analyses also verified that the C3ds of these five wild bird species exhibited the amino acid sequence and structure comprising the typical acidic pocket found in mammals that is required for combination with B cell surface receptors, which contribute electrostatic forces to interact with CR2. Our investigations indicate that some bird C3ds may already have the ability to bind with CR2 by electrostatic force, like mammals. As Ardeola bacchus, Zoothera, Bubo, Crossoptilon mantchuricum and Caprimulgus europaeus have more typical C3d concave acid pockets and thus a stronger ability to bind CR2, we speculate that these five wild birds may have a solider immunity against pathogens. Our phylogenetic and structural analyses of bird C3ds provide insights on the evolutionary divergence in the function of immune factors of avian and mammalian.

Characterization of intra- and inter-species hybrid tetramers of pyruvate carboxylase: Biotin and the BCCP domain play a crucial role in determination of the kinetics and thermodynamics of catalysis.

The formation, kinetics and thermodynamic activation parameters of hybrid tetramers of pyruvate carboxylase (PC) formed between wild-type Rhizobium etli pyruvate carboxylase (WTRePC) and mutant forms of this enzyme, as well as between Aspergillus nidulans PC and mutant forms of RePC have been characterized in a previous study. In this current work, we aim to extend the previous study by forming hybrid tetramers between WTRePC or chicken liver PC (CLPC) with single or double mutant RePCs. By forming hybrid tetramers between WTRePC with either K1119A or ΔBCCP RePC, the biotin moiety and BCCP (biotin carboxyl carrier protein) domain appear to play a crucial role in determination of thermodynamic activation parameters, especially the activation entropy, and the order of tetrameric structure. Using E218A:K1119A hybrid tetramers, an alternative pathway of biotin carboxylation occurred only in the absence of acetyl CoA. In this pathway, the biotin of the E218A subunits is carboxylated in the BC domain of the K1119A subunits, since the E218A mutation destroys the catalytic activity of the BC domain. Transfer of the carboxyl group to pyruvate could then occur in the CT domain of either E218A or K1119A. Part of the reduction of activity in hybrid tetramers of WTRePC and double mutant, E218A.K1119A could result from the loss of this pathway. Previously, D1018A mutant RePC homotetramers exhibited a 12-fold increase in the rate constant for catalysis in the absence of acetyl CoA. This was taken to indicate that inter-residue interactions involving D1018 inhibit the interconversion between the symmetrical and asymmetrical forms of the tetramer in the absence of acetyl CoA. The mutation, D1018A, in hybrid tetramers of WTRePC:D1018A.K1119A (D1018A.K1119A is a double mutant form of RePC) had no such effect on the rate constant, suggesting that in hybrid tetramers obligatory oscillation between asymmetrical and symmetrical conformers of the tetramer is not required to drive the catalytic cycle. Finally, K1119A or E218A RePC mutant can form hybrid tetramers with PC subunits from an evolutionarily distant species, chicken, that have stability characteristics that lie between those of the homotetramers of the two enzymes. This work provides insights into the how the PC tetramer functions to perform catalysis and is regulated by acetyl CoA. The ability to form hybrid tetrameric PCs composed of PC subunits from widely varying species that have a mixture of characteristics of the two source enzymes may also provide ways of developing novel PCs for biotechnological purposes.

Chicken bromodomain-containing protein 2 interacts with the Newcastle disease virus matrix protein and promotes viral replication.

Bromodomain-containing protein 2 (BRD2) is a nucleus-localized serine-threonine kinase that plays pivotal roles in the transcriptional control of diverse genes. In our previous study, the chicken BRD2 (chBRD2) protein was found to interact with the Newcastle disease virus (NDV) matrix (M) protein using a yeast two-hybrid screening system, but the role of the chBRD2 protein in the replication of NDV remains unclear. In this study, we first confirmed the interaction between the M protein and chBRD2 protein using fluorescence co-localization, co-immunoprecipitation and pull-down assays. Intracellular binding studies indicated that the C-terminus (aa 264-313) of the M protein and the extra-terminal (ET) domain (aa 619-683) of the chBRD2 protein were responsible for interactions with each other. Interestingly, although two amino acids (T621 and S649) found in the chBRD2/ET domain were different from those in the human BRD2/ET domain and in that of other mammals, they did not disrupt the BRD2-M interaction or the chBRD2-M interaction. In addition, we found that the transcription of the chBRD2 gene was obviously decreased in both NDV-infected cells and pEGFP-M-transfected cells in a dose-dependent manner. Moreover, small interfering RNA-mediated knockdown of chBRD2 or overexpression of chBRD2 remarkably enhanced or reduced NDV replication by upregulating or downregulating viral RNA synthesis and transcription, respectively. Overall, we demonstrate for the first time that the interaction of the M protein with the chBRD2 protein in the nucleus promotes NDV replication by downregulating chBRD2 expression and facilitating viral RNA synthesis and transcription. These results will provide further insight into the biological functions of the M protein in the replication of NDV.

Rapid Oxidation Following Photoreduction in the Avian Cryptochrome4 Photocycle.

Avian magnetoreception is assumed to occur in the retina. Although its molecular mechanism is unclear, magnetic field-dependent formation and the stability of radical-containing photointermediate(s) are suggested to play key roles in a hypothesis called the radical pair mechanism. Chicken cryptochrome4 (cCRY4) has been identified as a candidate magnetoreceptive molecule due to its expression in the retina and its ability to form stable flavin neutral radicals (FADH●) upon blue light absorption. Herein, we used millisecond flash photolysis to investigate the cCRY4 photocycle, in both the presence and absence of dithiothreitol (DTT); detecting the anion radical form of FAD (FAD●-) under both conditions. Using spectral data obtained during flash photolysis and UV-visible photospectroscopy, we estimated the absolute absorbance spectra of the photointermediates, thus allowing us to decompose each spectrum into its individual components. Notably, in the absence of DTT, approximately 37% and 63% of FAD●- was oxidized to FADOX and protonated to form FADH●, respectively. Singular value decomposition analysis suggested the presence of two FAD●- molecular species, each of which was destined to be oxidized to FADOX or protonated to FADH●. A tyrosine neutral radical was also detected; however, it likely decayed concomitantly with the oxidation of FAD●-. On the basis of these results, we considered the occurrence of bifurcation prior to FAD●- generation, or during FAD●- oxidization, and discussed the potential role played by the tyrosine radical in the radical pair mechanism.

Structural Analysis of the Regulatory GAF Domains of cGMP Phosphodiesterase Elucidates the Allosteric Communication Pathway.

Regulation of photoreceptor phosphodiesterase (PDE6) activity is responsible for the speed, sensitivity, and recovery of the photoresponse during visual signaling in vertebrate photoreceptor cells. It is hypothesized that physiological differences in the light responsiveness of rods and cones may result in part from differences in the structure and regulation of the distinct isoforms of rod and cone PDE6. Although rod and cone PDE6 catalytic subunits share a similar domain organization consisting of tandem GAF domains (GAFa and GAFb) and a catalytic domain, cone PDE6 is a homodimer whereas rod PDE6 consists of two homologous catalytic subunits. Here we provide the x-ray crystal structure of cone GAFab regulatory domain solved at 3.3 Šresolution, in conjunction with chemical cross-linking and mass spectrometric analysis of conformational changes to GAFab induced upon binding of cGMP and the PDE6 inhibitory γ-subunit (Pγ). Ligand-induced changes in cross-linked residues implicate multiple conformational changes in the GAFa and GAFb domains in forming an allosteric communication network. Molecular dynamics simulations of cone GAFab revealed differences in conformational dynamics of the two subunits forming the homodimer and allosteric perturbations on cGMP binding. Cross-linking of Pγ to GAFab in conjunction with solution NMR spectroscopy of isotopically labeled Pγ identified the central polycationic region of Pγ interacting with the GAFb domain. These results provide a mechanistic basis for developing allosteric activators of PDE6 with therapeutic implications for halting the progression of several retinal degenerative diseases.

Characterization of the different oligomeric states of the DAN family antagonists SOSTDC1 and SOST.

The DAN (differential screening-selected gene aberrative in neuroblastoma) family are a group of secreted extracellular proteins which typically bind to and antagonize BMP (bone morphogenetic protein) ligands. Previous studies have revealed discrepancies between the oligomerization state of certain DAN family members, with SOST (a poor antagonist of BMP signaling) forming a monomer while Grem1, Grem2, and NBL1 (more potent BMP antagonists) form non-disulfide linked dimers. The protein SOSTDC1 (Sclerostin domain containing protein 1) is sequentially similar to SOST, but has been shown to be a better BMP inhibitor. In order to determine the oligomerization state of SOSTDC1 and determine what effect dimerization might have on the mechanism of DAN family antagonism of BMP signaling, we isolated the SOSTDC1 protein and, using a battery of biophysical, biochemical, and structural techniques, showed that SOSTDC1 forms a highly stable non-covalent dimer. Additionally, this SOSTDC1 dimer was shown, using an in vitro cell based assay system, to be an inhibitor of multiple BMP signaling growth factors, including GDF5, while monomeric SOST was a very poor antagonist. These results demonstrate that SOSTDC1 is distinct from paralogue SOST in terms of both oligomerization and strength of BMP inhibition.