Reporter gene transactivation by human p53 is inhibited in budding yeast lacking the TRR1 gene encoding thioredoxin reductase. Thioredoxin reductase specifically catalyzes the NADPH-dependent reduction of thioredoxin. Thioredoxin provides a source of electrons for disulfide reduction in various cellular processes. Reduction of disulfides within the cell can be accomplished by the separate but partially overlapping glutathione reductase - glutathione - glutaredoxin pathway. The basis for p53 inhibition was investigated by measuring the redox state of thioredoxin and glutathione in wild-type and Δtrr1 yeast lacking the gene encoding thioredoxin reductase. The Δtrr1 mutation caused an increased in oxidation in both molecules. Highcopy expression of the GLR1 gene encoding glutathione reductase in Δtrr1 yeast restored the redox state of glutathione to wild-type levels, but did not restore p53 activity. Also, p53 activity was unaffected by be a Δglr1 mutation, even though the mutation was known to result in glutathione oxidation. These results indicate that p53 activity has a specific requirement for an intact thioredoxin system, rather than a general dependence on the intracellular reducing environment. In order to test if p53 activity requires an intact thioredoxin system in mammalian cells, dominant-negative and RNAi approaches designed to suppress thioredoxin reductase activity were used in a breast adenocarcinoma cell which contains an endogenous wild-type p53. In cells stably transformed with a plasmid encoding a dominant-negative form of thioredoxin reductase, thioredoxin reductase activity was inhibited 4.3-fold and p53 reporter gene expression was inhibited by 2-fold. In cells stably transformed with a RNAi plasmid designed to target thioredoxin reductase mRNA, thioredoxin reductase activity was inhibited by 1.7-fold and p53 reporter gene expression was inhibited by 1.6-fold. A decrease in the protein levels of the p53 endogenous target genes p21 and Bax was also observed in both dominant-negative and RNAi transformants. Additionally, thioredoxin was shown to bind p53 in vitro (Kd=0.9 μM), and a LexA-thioredoxin fusion protein was shown to bind p53 in vivo. These results suggest that p53 activity is regulated by thioredoxin reductase in mammalian cells through a direct interaction with thioredoxin. / Graduation date: 2004

This dissertation examines the interaction of the linker histone with DNA and with nucleosomes. The first goal of the project was to characterize the interaction of the linker histone with DNA. Three factors previously reported to influence the linker histone's interaction with DNA were examined: ratio of linker histone to DNA sites of binding, monovalent ions in the local environment, and conformation of the DNA molecules. Evidence obtained through gel mobility shift assays demonstrates the strong preference by the linker histone for DNA with superhelical torsion, i.e., supercoiling, and the negative cooperative mode of binding that the linker histone exhibits in association with supercoiled DNA. The second part of the dissertation examines the location of linker histone binding on the nucleosome, and documents the pronounced tendency of the linker histone to bind to two DNA duplex strands. A preparation of homogeneous nucleosome core particles, consisting of a defined 238 base pair DNA fragment and the core histone octamer positioned precisely on this DNA, was used as a substrate for the UV-induced crosslinking of the linker histone to the DNA of this nucleosome. By site-specific labeling of a single site on the DNA of the nucleosome, the linker histone was observed crosslinked at that labeled site, confirming that the linker histone binds at the pseudo-dyad axis of the nucleosome. This evidence was used to support a model of linker histone binding to the nucleosome that invokes the association of the linker histone with no fewer than two duplex strands of DNA of the nucleosome. / Graduation date: 2004

In order to better characterize linker histone interactions with DNA, avian erythrocyte-specific linker H5 and the trypsin-resistant globular domain of H5 (GH5) were used in DNA binding studies. To begin, H5 displayed a considerably higher binding affinity for DNA than the isolated globular domain (GH5), supporting the importance of the terminal tail domains in binding. This conclusion is based upon binding curves conducted in low-salt solution, and on the considerably-higher salt concentration required to prevent protein-DNA contact. Linker histones also induce DNA-protein aggregation in a process that was found to result in protein insolubility in 2% SDS, and included protein-protein interactions that did not require the terminal tail domains. In addition, DNA supercoiling appeared to impede the aggregation process; this that may be attributable to binding of linker histones in isolated clusters, as gauged by a limit in the number of observed dithiobis (succinimidyl) propionate (DSP)-crosslinkable contacts. In a related study, the property of GH5 to bind, then organize onto DNA was investigated. GH5 crosslinked onto DNA with dithiobis (succinimidyl propionate), then cleaved with chymotrypsin, displayed highly uniform contacts that appeared to involve the C-terminal four amino acids, and suggests protein-protein interactions are important for binding. This finding may be relevant since GH5 (and H5) were observed to self-associate free in solution in an arguably specific manner. Finally, the exposure of Phe 93 to chymotrypsin was used to identify the surface of the globular domain that contacts DNA for the binding of intact H5. Results suggests that the side of the protein opposite to the recognition helix preferentially binds to DNA, supporting a novel winged-helix protein DNA-binding mechanism. Furthermore, parallel studies with octamers reconstituted onto a DNA fragment with twelve copies of the 208 b.p. rDNA 5s gene from Lytechinus variegatus, shows that H5 had a high binding affinity with all detectable protein binding to the reconstituted complex. H5 binding conferred protection to a site located near the dyad axis from endonuclease digestion, supporting the contention that H5 binds near or at the nucleosome dyad axis. H5 binding also was observed to condense fibers as observed from agarose gel electrophoresis, although velocity analytical sedimentation studies indicate that H5 in itself was not sufficient to fully compact chromatin fibers; rather H5 and 30 mM NaCl, in combination, were required. Results indicate that the chromatin-reconstituted "208-12 DNA" makes an excellent model for analyzing the effect of linker proteins on chromatin morphology. / Graduation date: 1998

Thesis (Ph. D.)--University of Rhode Island, 2006. / Typescript. Includes bibliographical references (leaves 136-153).

Neuronal pentraxin with chromo domain (NPCD) belongs to a family of neuronally-expressed pentraxin proteins thought to be involved in synaptic refinement and plasticity. One isoform of Npcd, neuronal pentraxin receptor (NPR), is a type-II transmembrane protein responsible for the clustering of related neuronal pentraxins 1 and 2. However, recently identified cytosolic NPCD isoforms with no known function were discovered through their interaction with the intracellular domain of a receptor protein tyrosine phosphatase PTPRO. PTPRO is a signaling molecule known to be involved in the development of the nervous system. Additionally, upregulated expression of neuronal pentraxins has been implicated in neuronal cytotoxicity and associated with neurodegenerative diseases. Here, we demonstrate that a novel cytosolic NPCD isoform interacts with the BTB-Kelch protein Mayven/KLHL2. This interaction was identified through a yeast two-hybrid screen using the C-terminal pentraxin domain region of NPCD and confirmed through mammalian cell colocalization and co-immunoprecipitation studies. Domain truncation analysis suggests that the kelch domains of Mayven/KLHL2 are responsible for this interaction with NPCD. We also show that Mayven/KLHL2 is capable of interacting with Cullin 3, an integral protein in the Cullin-RING ubiquitin ligase complex. An in-vivo ubiquitylation assay demonstrates that overexpression of Mayven/KLHL2 increases NPCD ubiquitylation, and suggests a novel E3 ubiquitin ligase function of Mayven/KLHL2 with NPCD as its substrate. Furthermore, we observed an increased propensity of overexpressed NPCD to form aggresomes with coexpression of Mayven/KLHL2. As the formation of aggresomes is often associated with protein aggregation and deposition diseases, including a multitude of neurodegenerative diseases, we tested NPCD overexpression and the effects of Mayven/KLHL2 coexpression on neuronal cytotoxicity and apoptosis. Overexpressed NPCD in hippocampal neuron cultures resulted in increased cytotoxicity and apoptosis, further exacerbated by Mayven/KLHL2 coexpression. Our findings report an interaction between NPCD and Mayven/KLHL2, demonstrate a novel role of Mayven/KLHL2 as an E3 ubiquitin ligase, and explore a possible intersection between the ubiquitin-proteasome degradation pathway, neuronal pentraxins, and neurodegenerative disease.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 55-57). Also available in electronic version.

Undergraduate honors paper--Mount Holyoke College, 2006. Dept. of Chemistry. / Includes bibliographical references (leaves 106-109).

Given an amino acid sequence with the £\-carbon 3D coordinates on its backbone, the all-atom protein backbone reconstruction problem (PBRP) is to rebuild the 3D coordinates of all atoms (N, C and O atoms) on the backbone. In this thesis, we propose a method for solving PBRP based on the homology modeling. First, we extract all consecutive 4-residue fragments from all protein structures in PDB. Each fragment is identified by its second, third and fourth residues. Thus, the fragments are classified into 8000 residue groups. In each residue group, the fragments with similar structures are clustered together. And one typical fragment is used to represent one cluster. These typical fragments form our fragment library. Then, we find out possible candidates in the fragment library to reconstruct the backbone of the target protein. To test the performance of our method, we use two testing sets of target proteins, one was proposed by Maupetit et al. [20] and the other is a subset extracted from CASP7. We compare the experimental results of our method with three previous works MaxSprout, Adcock¡¦s method, and SABBAC proposed by Maupetit et al.. The reconstruction accuracy of our method is comparable to these previous works. And the solution of our method is more stable than the previous works in most target proteins. The time efficiency of our method is also satisfactory.

Bermudagrass (Cynodon dactylon (L) Pers.), one of the predominant forages in the southeastern US, varies in nutritive value in response to management and environmental factors. Beef cattle supplementation decisions are complicated by this variability. Therefore, our objective was to determine the effect of four protein supplementation levels (0, 82, 119 and 155 mg N/kg BW) on the utilization of three bermudagrass hays (5.6, 6.3, and 8.1 percent CP).Thirteen ruminally fistulated Angus x Hereford steers (BW = 330 plus/minus 19 kg) were used in a 13 x 4 incomplete Latin square design with 13 treatments. Treatments were arranged as a 3 x 4 factorial plus a control bermudagrass hay (10.8 percent CP). Hay was provided ad libitum and protein supplements were offered as range cubes once daily. Periods were 15 d with intake determinations made on d 10 through d 13 to correspond with fecal grab samples collected from d 11 through d 14. Acid detergent insoluble ash was used as an internal marker for determination of fecal output. Hay OM intake of unsupplemented steers increased linearly (P < 0.01) as hay nutritive value increased from 75 to 77, 96 and 94 g/kg BW^0.75 for 5.6, 6.3, 8.1 and 10.8 percent CP hays, respectively. A cubic increase (P = 0.03) in OM digestibility for unsupplemented hays was observed with values ranging from 46 to 65 percent. This resulted in a linear increase (P < 0.01) in total digestible OM intake in response to hay nutritive value from 35 to 45, 51, and 60 g/kg BW^0.75 for 5.6, 6.3, 8.1, and 10.8 percent CP hays, respectively. No significant effects on total digestible OM intake were observed when hays were supplemented with protein. There was a tendency for forage OM intake of the 6.3 percent CP hay to increase linearly with supplemental protein (P = 0.08). Total OM intake increased linearly (P < 0.01) when CP was supplemented to the 6.3 percent CP hay from 77 to 88, 92, and 98 g/kg BW^0.75 for 0, 82, 119, and 155 mg N/kg BW, respectively. We conclude that forage CP content was the primary driver in determining total digestible OM intake, and the effects of protein supplementation on utilization of bermudagrass hay were varied.

Objectives of the study were to evaluate if calves fed 6 L of high protein milk replacer (HPMR; 1135 g/d, 28% crude protein (CP), 20% fat) had improved performance and health as compared to calves fed 4 L of a conventional milk replacer (CMR; 454 g/d, 20% CP, 20% fat) in heat stress and non heat stress environments. Holstein bull calves (n=52) <3 d of age were assigned to a 2 x 2 factorial trial based on initial BW, physical health score, and total serum protein levels. One half of each nutrition group was housed indoors with temperature control, non-heat stress (NHS) environment and one half was housed outside under a shaded barn in subjecting them to a heat stress (HS) environment. The study was conducted for 56 d from June 18 to August 13, 2010. Average thermal heat index (THI) was calculated for each day using the average of 24 recorded temperatures and relative humidity (RH%). The 56 d average, low, and high range THI for the HS was 79, 67, and 86, respectively, while THI for the NH was 69, 66, and 74, respectively. Weekly measurements of body weight (BW) in kg, body length (BL), hip width (HW), wither height (WH), heart girth (HG), and hip height (HH) in cm were collected and average daily gain (ADG) was calculated. Water consumption (WC) in mL and starter intake (SI) in grams was measured daily. Feed conversion (FC) was also calculated for each nutritional treatment and environment. Fecal scores (FS) of 1 to 4 (1=hard, firm, 2=soft, firm, 3=no form, and 4=watery) were recorded daily. Calves with a FS of >3 were considered to have diarrhea and required treatment. Respiration rates (RR) were recorded at 0630 (AM) and 1830 (PM) to monitor respiratory challenges while rectal temperatures (RT) were also measured using a digital thermometer daily in AM and PM to monitor febrile events. If RT was greater than 39.2 degrees C for NHS calves and 39.7 degrees C for HS calves, they were treated for febrile events (FE). Data was analyzed using PROC MIXED (SAS 9.2). HPMR had a greater (P &lt; 0.01) WH, HG, BL, HH, ADG, WC, and FS than the CMR (0.15 vs. 0.11, 0.37 vs. 0.28, 0.27 vs. 0.22, 0.21 vs. 0.14, 0.82 vs. 0.58, 4235 vs. 2656, and 2.05 vs. 1.73, respectively). HS had a greater (P &lt; 0.01) WC than NHS (4365 vs. 2526, respectively). CMR had a greater SI and FC (P &lt; 0.05) than HPMR (0.942 vs. 0.437, and 1.99 vs. 1.78, respectively). HS had a higher RT AM, RT PM, RR AM, and RR PM (P&lt;0.01) than NHS (38.87 vs. 38.77, 39.03 vs. 38.79, 35.79 vs. 32.77, and 55.73 vs. 38.57, respectively. Calves in NHS had a higher FE (P&lt;0.01) than the HS calves (6.24 vs. 2.33). There was no significant difference in growth parameters in HS or NHS in calves of like feeding strategies. The results show calves in HS experienced higher RT AM, RT PM, RR AM, and RR PM. The increased protein and energy fed to the HPMR calves resulted in greater FS and increased growth.