Indiana University-Purdue University Indianapolis (IUPUI) / The nuclear protein SETMAR has been reported to be involved in many processes such as non-homologous end joining (NHEJ), di-methylation (arguably) of K36 of histone H3, restart of stalled replication forks, chromosome decatenation, enhancing of TOPII inhibitors which results in resistance to chemotherapeutics in cancer patients, etc. All these purported functions are impossible to execute without interaction with other proteins. It is established that SETMAR binds specifically to DNA at terminal inverted repeat sequences and can loop DNA. This DNA sequence specific pull-down exploits this attribute to identify possible protein interactors of SETMAR. As a result of this experiment several proteins have been identified for further research: BAG2, c12orf45, PPIA, XRCC5/6, and ZBTB43, all of which are found in higher statistical abundances in full length SETMAR samples.

Indiana University-Purdue University Indianapolis (IUPUI) / The obligate intracellular protozoan parasite Toxoplasma gondii is a medically relevant pathogen that has infected a third of the world’s population. Toxoplasma is the causative agent of toxoplasmosis, which can have severe repercussions such as encephalitis and even death in immunocompromised patients. Current treatments for toxoplasmosis only target acute infection and can be toxic to patients, resulting in complications including allergy and bone marrow suppression. Thus, the identification of novel drug targets and therapies for toxoplasmosis is vital. Epigenetic modulators of lysine acetylation, including ‘writers,’ ‘erasers,’ and ‘readers,’ have been identified as promising drug targets for protozoan parasites. The lysine acetyltransferase (KAT) GCN5b appears to be an essential gene for Toxoplasma viability. The KAT domain of GCN5b is essential to GCN5b function and is targetable by small molecule inhibitors. While the acetyltransferase activity of this gene is well-characterized, the functionality of its C-terminal bromodomain (BRD) remains to be understood. Bromodomains are readers of lysine acetylation, and recently, bromodomain inhibitors have shown promise in a number of human diseases, as well as in protozoan parasites. We hypothesized that the GCN5b bromodomain is critical for Toxoplasma viability. The data reported herein suggest that the GCN5b bromodomain is important for tachyzoite viability and may serve as a novel therapeutic target in Toxoplasma.

The human Absent, small, or homeotic disc1 (ASH1L) is a member of the Trithorax group (TrxG) proteins that play a role in epigenetic gene activation of developmental HOX genes via H3K36me2 methylation mark. ASH1L contains the evolutionarily conserved SET domain responsible for catalyzing monomethylated and dimethylated lysine formation. The crystal structure of the SET domain of ASH1L revealed a substrate-binding pocket blockage caused by an autoinhibitory loop (AI-loop) that undergoes dynamic changes during catalysis and could be exploited for inhibitor development. Studies have shown that the AI-loop regulates the SET domain, thus the KMTase activity of ASH1L. The SET domain adopts an autoinhibited state where the AI-loop blocks the entry of substrate to the active site, have made it a difficult target for the development of inhibitors. The emerging ASH1L's role in multiple oncogenic processes leading to cancer makes it a viable therapeutic target. Effective targeted inhibition of ASH1L enzymatic activity would be a potential therapeutic approach in cancers driven by high HOX gene expression. We employed the state-of-the-art 1H and 13C-detected solution NMR to better understand the ASH1L regulatory mechanism. We investigated the AI-loop's dynamic structure and conformational mobility of backbone and side chains in the absence and presence of the first- in-class small molecule inhibitors. Numerous backbone amide signals across the AI loop and the catalytic cleft of the SET domain are being broadened, indicating the complex interplay of fast local to slow segmental dynamics across the ASH1L SET domain. The binding of the first-in-class inhibitors perturbs the signals around the AI-loop and SAM binding cleft, validating the inhibitor binding site in the solution. The recently published crystal structures of the MRG domain bound to the ASH1L SET domain revealed disordered conformations of the AI-loop and rearrangement in the SAM binding site compared to the apo ASH1L SET domain. It has been proposed that MRG15 allosterically activates ASH1L by releasing the AI loop. Therefore, we performed extensive studies in an aqueous solution to understand the role of MRG15 in stimulating the catalytic activity of ASH1L. We found that the full-length MRG15 is necessary to induce histone methyltransferase activity of the catalytic SET domain of ASH1L. In contrast, the MRG domain alone cannot enhance the catalytic activity. Furthermore, we found that only the complex of ASH1L SET domain with MRG15 but not with isolated MRG domain can interact with nucleosomes. In summary, I have established the direct link between the structural dynamics of the ASH1L SET domain and its enzymatic activity. Moreover, I have defined the adaptor role of the complete MRG15 protein as the substrate recognition factor for the ASH1L protein without perturbing the AI loop or SAM binding site. The atomic level studies mentioned above, supported by the detailed structure and dynamics studies of the first-in-class inhibitor complex with ASH1L, establish the solid foundations for further drug candidate development, selectively targeting the ASH1L and potentially other H3K36me2 methyltransferases.

The objectives of this project were to investigate the effects of dietary lysine restriction on (1) the plasma concentrations of selected metabolites, free AAs, and three growth-related hormones, (2) the gene expression profile in longissimus dorsi muscle growing pigs. Twelve individually penned young barrows (Yorkshire × Landrace; 22.6 ± 2.04 kg) were randomly assigned to two dietary treatments (a lysine-deficient, LDD, and a lysineequate, LAD) diets, respectively. Pigs had ad libitum access to water and their respective diets for 8 weeks, and the ADG, ADFI, and G:F were determined. At the end of the trial, jugular vein blood was collected, and plasma was separated for the analysis of plasma parameters. Also, the longissimus dorsi muscle samples were collected from each pig for gene expression profile analysis. The ADG of LDD pigs was lower than that of LAD pigs, and so was the G:F since there was no difference in the ADFI between the two groups of pigs. The plasma concentrations of lysine, methionine, leucine, and tyrosine were lower, while that of β-alanine was higher, in the LDD pigs. The total plasma protein concentration was lower in the LDD pigs, whereas no differences were observed for the other metabolites, and the plasma concentrations of GF, insulin, and IGF-1 between the two groups. While gene expression profile results showed that sixty-nine genes were found differentially expressed (Benjamin-Hochberg corrected P-value < 0.05) in Diet I vs. Diet II pigs, of which 29 genes were down-regulated (Log₂ fold change (FC) < - 0.58) and 40 genes were up-regulated (Log₂ FC > 0.58). Furthermore, bioinformatic analyses revealed that the upregulation and downregulation of the DEGs may negatively affect the muscle protein turnover, structural development, and lipid metabolism of skeletal muscle. In conclusion, these results indicate that the lack of lysine including other two EAA as protein building block must be the primary reason for the compromised G:F ratio and ADG. The changes in the performance might be associated with some cell signaling and metabolic pathways suggested from the Bioinformatic results; however, may not involve the GH/IGF-1 axis.

<p> This thesis describes the ionic strength and ion binding effects on the oxidation reduction properties of cytochrome c and its lysine modified derivatives.</p> <p> Cytochrome c has been modified in two different ways: a complete modification of all lysine residues and specific modification of one lysine residue. Some properties of the modified derivatives are described.</p> <p> Ion binding properties of cytochrome c and its lysine modified derivatives were studied by measuring the apparent equilibrium constant of the reaction between the ferri- form of the protein and potassium ferrocyanide. It was found that unmodified cytochrome c binds one cation (K+, Na+) per molecule, and binding is much stronger to the reduced form of the protein. Binding of cations is not changed upon modification of the lysine residues. For binding of the chloride, there are two binding sites on the cytochrome c molecule, and the binding is much stronger to the oxidized form of the protein. It was shown that upon the modification of the lysine residues in either way the binding of chloride was considerably changed. It was concluded that one of these two binding sites for chloride on cytochrome c involves lysine residue, probably the residue number 13.</p> / Thesis / Master of Science (MSc)

X-ray crystallography is a popular method for resolving protein structures. Protein crystals need to be used for X-ray crystallography, but most naturally occurring proteins do not readily crystallize. The Hunt lab performed computational analyses showing that arginine is the most overrepresented amino acid in crystal-packing interfaces in the Protein Data Bank. Given the similar physicochemical characteristics of arginine and lysine, we hypothesized that multiple lysine-to-arginine (KR) substitutions should improve crystallization. To test this hypothesis, we developed software that ranks lysine sites in a target protein based on the redundancy-corrected KR substitution frequency in homologs. We demonstrate that three unrelated single-domain proteins can tolerate 5-11 KR substitutions with at most minor destabilization and that these substitutions consistently enhance crystallization propensity. This approach rapidly produced a 1.9 Å crystal structure of a human protein domain refractory to crystallization with its native sequence. Structures from bulk-KR-substituted domains show the engineered arginine residues frequently make high-quality hydrogen-bonds across crystal-packing interfaces. We thus demonstrate that bulk KR substitution represents a rational and efficient method for probabilistic engineering of protein surface properties to improve protein crystallization. This stands in direct contrast to earlier work and dogmas that posited that surface entropy reduction was the clear path forward to crystallzing proteins. Arginine is a high-entropy sidechain, yet it helps drive protein crystallization. To understand which structure and dynamical features of arginine give rise to crystal packing propensity, we performed 60 Molecular Dynamics (MD) simulations to measure the sidechain order parameter of arginine and compare it against crystal packing propensity. This work found that surface-exposed arginines with low order parameters are most likely to participate in crystal packing interactions. This is evidence against earlier thinking that high entropy surface sidechains oppose crystallization. Entropic barriers to protein crystallization can be enthalpically overcome.

Currently accepted optimum protein levels for growth in the horse may be reduced with amino acid supplementation. This study investigated the effects on growth and protein status of Thoroughbred foals offered a supplement with a typical CP level to a supplement that had a lower CP level fortified with limiting amino acids. The control supplement (CS) contained 14% CP, 3.0 Mcal/kg DM, 10% corn oil, 22% soybean meal, 1.4% calcium, and three sources of fiber. The experimental supplement (LTS) contained 9% CP, 3.0 Mcal/kg DM, 10% corn oil, 3% soybean meal, 1.4% calcium, three sources of fiber, and was fortified with .6% lysine and .4% threonine. Lysine and threonine are the first two limiting amino acids in common diets of the horse. Mares and foals were fed twice daily (0700 and 1400) and kept on 30-acre pastures (mixed grass/white clover) until weaning (7 months). Weanlings continued on specified supplements and pastures for seven additional months. Physical measurements and blood samples were taken monthly. Measurements included weight, average daily gain (ADG), body condition (BC), wither height, hip height, length, girth, forearm length, front and hind cannon length, physis and fetlock circumference. Blood analysis included total protein (TP), albumin (ALB), creatinine (CREA), and plasma urea nitrogen (PUN). Effect of diet and time were evaluated by analysis of variance with repeated measures. No differences were found in physical measurements between the CS and LTS groups (ADG 0.8(0.4 kg/day, BC 4.9(0.05) for the observational period. Blood data also showed no difference for the period (ALB 2.9 ( 0.03 g/dl, TP 5.7 ( 0.10 g/dl, CREA 1.1 ( 0.02 mg/dl). These results suggest that the foals offered the LTS faired as well or better than foals on the CS. Lower levels of crude protein (CP) in the diet will result in less nitrogen pollution of pasture lands due to better utilization in the horse. By fortifying a low protein diet with the first two limiting amino acids, the protein is used more efficiently for growth and development, thereby benefiting both the horse and the land. / Master of Science

Dissertation (MSD) --Boston University, Goldman School of Dental Medicine, 2012 (Department of Oral Biology). / Includes bibliographic references: leaves 71-77. / Lysyl oxidases constitute a family of enzymes responsible for the formation of crosslinks in collagen and elastin. These enzymes have also been linked to pathological fibrosis. The importance of collagen in the structural and mechanical properties of bone led us to investigate the hypothesis that the absence of one or more of these enzymes could lead to a significant bone phenotype. This phenotype could resemble osteoporosis or diabetic bone disease. In addition, we tried to overexpress lysyl oxidase proenzyme in vitro. The ability to produce enough amounts of lysyl oxidase proenzyme and the ability to process it and activate it could facilitate the development of drugs that control its activity in pathological fibrosis. [TRUNCATED]

Purified brush border and basolateral membranes were isolated from homogenized intestinal enterocytes of Holstein steers by divalent cation precipitation followed by differential and sucrose density gradient centrifugation. Membrane marker enzymes were used to determine the effectiveness of the fractionation procedure. Alkaline phosphatase and sodium-potassium adenosine triphosphatase served as the marker enzymes for the brush border and basolateral membranes, respectively. The brush border fraction was enriched 5.1-fold over the cellular homogenate. Purification of 10.1-fold over cellular homogenate was obtained for the basolateral membrane fraction. Electronmicrographs and osmotic response data were used to confirm the vesicular nature of the membrane preparations. Brush border membrane vesicles from bovine jejunal and ileal tissue were used to evaluate lysine (LYS) and methionine (MET) transport. Total transport of LYS and MET was divided into mediated and diffusion components. Mediated uptake was further divided into sodium-dependent (Na⁺) and sodium independent (Na⁻) systems. Total LYS and MET uptake by ileal tissue tended to be higher than jejunal tissue at all concentrations evaluated but differences were significant (P<.O5) at 2.5 and 7.5 mM for LYS and 5, 12.5 and 15 mM for MET. The greater capacity of ileal tissue appeared to be due to the Na⁺ component of LYS uptake and the diffusion component of MET uptake. Methionine transporters had lower affinities and higher capacities than the corresponding LYS transporters in both ileal and jejunal tissue. Methionine transport was greater (P<.O5) than LYS transport in both ileal and jejunal tissue when initial amino acid concentration was 7.5 mM. When initial amino acid concentration was 1.25 mM, MET uptake was greater (P<.13) than LYS uptake in jejunal but not ileal tissue. The relative contribution of mediated and diffusion uptake systems to total MET and LYS uptake was found to be dependent of substrate concentration. / Ph. D.

L'immobilisation de biomacromolécules à la surface de polymères peu réactifs est une voie de synthèse de nanomatériaux qui fait actuellement l'objet de nombreuses recherches pour le développement d'applications biologiques et médicales. Nous avons synthétisé de nouveaux nanomatériaux à base de polypropylène (PP) greffé par des dendrimères de lysine (DGL). Les DGL sont parfaitement solubles dans l'eau, biocompatibles, polycationiques à pH neutre et leur structure dendritique particulière font d'eux des macromolécules de plus en plus étudiées en interactions avec les milieux biologiques. Différents traitements par plasma ont permis de fonctionnaliser la surface du PP et plusieurs stratégies ont été adoptées pour greffer les DGL sous forme de monocouche, multicouche ou à partir de brosses de polymères : le greffage direct, les polymérisations non contrôlée et contrôlée de type RAFT associées au plasma d'iode et à la chimie click de surface. L'aptitude des matériaux PP fonctionnalisés par le DGL à interagir avec les milieux biologiques a été étudiée, en particulier l'immobilisation de l'ATP et le comportement vis-à-vis des bactéries et des virus. Les propriétés de ces nanomatériaux sont liées à la réactivité des groupements amine des DGL ainsi qu'à la structure régulière et sphérique des dendrimères. Les résultats obtenus ouvrent de nombreuses applications potentielles pour le traitement des eaux, le diagnostic et la prévention du développement des micro-organismes. / Great attention has been focused these last years on tailoring polymer surfaces by immobilizationof suitable molecules for biological and medical applications such as tissue engineering, drug delivery systems, antibacterial supports, and biosensors. In that context, we report the preparation of an original hybrid material based on polypropylene and poly-L-Lysine dendrigrafts (DGL) which are perfectly water soluble, and biocompatible. First, activation of the polypropylene surface (PP) was achieved using plasma treatment. Then, several strategies have been developed to graft DGL onto the PP surface such as (i) direct grafting of DGL after surface activation, (ii) the use of conventional radical polymerization or (iii) RAFT polymerization of monomers from the PP surface. The last methodology favored the increase of the DGL grafts density onto the surface. The ability of PP surface functionalized with DGL to interact with biological media was studied and the modified surfaces open the way to many potential applications in water treatment, diagnosis and prevention of the development of microorganisms.