Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on Sept. 16, 2009). Includes bibliographical references.

Indiana University-Purdue University Indianapolis (IUPUI) / GCN5 is a histone acetyltransferase (HAT) that remodels chromatin by acetylating lysine residues of histones. The GCN5 HAT identified in Toxoplasma gondii (TgGCN5) contains a unique N-terminal “extension” that bears no similarity to known proteins and is devoid of known protein motifs. The hypothesis of this thesis is the N-terminal extension is critical to the function of TgGCN5. Three possible roles of the N-terminus were investigated: nuclear localization, protein-protein interactions, and substrate recognition. Subcellular localization was determined via immunocytochemistry using parasites expressing recombinant forms of TgGCN5 fused to a FLAG tag. Initial studies performed with parasites expressing full length FLAG-TgGCN5 were positive for nuclear localization. Without the N-terminal extension (FLAG-ΔNT-TgGCN5) the protein remains cytoplasmic. Additional studies mapped a six amino acid motif (RKRVKR) as the nuclear localization signal (NLS). When RKRVKR is fused to a cytoplasmic protein, it gains access to the nucleus. Furthermore, we have established the NLS interacts with Toxoplasma importin α, a protein involved in nuclear trafficking. Interaction with importin α provides evidence that the TgGCN5 N-terminal extension is involved in mediating protein-protein interactions. In order to identify additional interacting proteins, FLAG affinity purification was performed on parasites expressing full length FLAG-TgGCN5 and FLAG-ΔNT-TgGCN5. Upon comparing the results of the two purifications, proteins captured with only full length TgGCN5 may be interacting with the N-terminal extension. Full length TgGCN5 affinity purification indicates an interaction with histone proteins, two different homologues of Ada2 (adapter protein reported to interact with GCN5 homologues), and several heat shock proteins. With regard to substrate recognition, the N-terminal extension of TgGCN5 is dispensable for the acetylation of non-nucleosomal histones in vitro. However, the lysine acetylated by TgGCN5 is surprisingly unique. Other GCN5 homologues preferentially acetylate lysine 14 in histone H3, but TgGCN5 exclusively acetylates lysine 18 in histone H3 and has no activity on lysine 14. Taken together, these results argue that the N-terminal extension of TgGCN5 is critical for mediating protein-protein interactions, including those responsible for trafficking the HAT to the parasite nucleus but does not appear to be required for the acetylation of non-nucleosomal histones.

No description available.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on April 6, 2010). Vita. Thesis advisor: Shivendra D. Shukla. "August 2008" Includes bibliographical references

Compaction of DNA into chromatin is an important feature of every living cell. This compaction phenomenon is brought about and maintained by a variety of DNA binding proteins, which have evolved to suit the specific needs of the different cell types spanning the three kingdoms of life; the eukaryotes, prokaryotes and archaea. Sulfolobus solfataricus, a member of the crenarchaeal subdivision of the archaea, has two prominent DNA binding proteins known as Alba (1&2) and Sso7d. Alba1 is acetylated in vivo at two positions and this modification lowers its’ affinity for binding DNA. Acetylation levels impact many cellular processes and in higher organisms play a critical role in the development of many cancers and other diseases. This thesis documents the finding and characterisation of the N-terminal acetyltransferase (ssArd1) of SsoAlba1, based on its’ sequence homology to the catalytic subunits Ard1, Nat3 and Mak3 belonging to the larger eukaryal Nat complexes NatA, NatB and NatC, respectively. Mutagenesis studies revealed that ssArd1 preferentially acetylates N-termini bearing a serine or alanine residue at position 1 (after methionine cleavage). It is also capable of acetylating other proteins with very different physical structures. These findings allow classification of ssArd1 as a promiscuous acetyltransferase belonging to the Gcn5-N-acetyltransferase (GNAT) superfamily. The active site of the enzyme was examined through mutagenesis studies, revealing that the mechanism of acetylation is likely to proceed through a direct acetyl transfer involving a tetrahedral intermediate. Structural studies provided some insight into the molecular structure of ssArd1.

Objective: P300/CBP Associated Factor (PCAF) is a histone acetyltransferase, and has been reported to interact with nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and to stimulate cyclooxygenase-2 (COX-2) transcriptional activation. The aim of this study was to determine the role of PCAF in chronic pain modulation. / Method: In an in vitro experiment, PCAF small-interfering RNA (siRNA) was used to knock down PCAF. Interleukin-1 β (IL1β) was applied as COX-2 inducer in SK-N-SH neuroblastoma cells. Luciferase assay and chromatin immunoprecipitation (ChIP) were performed regarding COX-2 promoter region. / In an in vivo experiment, PCAF was examined for its distribution in dorsal horn of Spraque-Dawley rats. COX-2 level in the spinal cord was determined after inhibition of PCAF in rats with Complete Freund's Adjuvant (CFA)-induced pain. ChIP was also performed. / Finally, we tested whether genetic variations in the PCAF gene affected the risk of chronic pain in a gene association study of 267 surgical patients. The associations of pain with genotypes (58 single nucleotide polymorphisms, SNPs)/haplotypes were analyzed by χ² and Fisher exact tests. / Results: Knock-down of PCAF reduced COX-2 level and NF-κB activity. PCAF and acetylated histone 3 lysine 14 (H3K14) were enriched at COX-2 promoter when IL1β was applied. / PCAF was expressed in neurons at superficial layers of rat dorsal horn. In the in vivo experiment, COX-2 was reduced with the inhibition of PCAF in CFA rats. PCAF was enhanced at COX-2 promoter when CFA was injected. Anacardic acid and PCAF siRNA significantly alleviated thermal nociception and mechanical allodynia. / In the gene association study, 6 SNPs and 5 haplotypes were significantly associated with higher risk of severe chronic postsurgical pain. Multivariable analysis showed that patients with a SNP rs6763504 had a higher risk of developing severe chronic postsurgical pain (p = 0.001). / Conclusion: PCAF regulated COX-2 transcription and reduction or inhibition in PCAF resulted in a decrease in COX-2 and less chronic inflammatory pain. Genetic variations in the PCAF gene increased risk of severe chronic post-surgical pain in patients. / 實驗目的：P300/CBP相關蛋白（PCAF）是一種組蛋白乙酰化轉移酶。這種蛋白已被報道可以和核因子活化B細胞κ輕鏈增強子(NF-κB)相互作用，以及增進環氧合酶-2（COX-2）的轉錄激活。本實驗的目的在於研究PCAF在疼痛調節中的作用。 / 實驗方法：在細胞實驗中，我們使用了小干擾RNA（siRNA）來降低PCAF的含量。同時我們使用了白細胞介素-1β（IL1β）來作為SK-N-SH神經母細胞瘤細胞中COX-2的誘導劑。我們使用了熒光素酶試劑和染色質免疫沉澱來研究COX-2啟動子區域。 / 在體內實驗中，我們檢測了PCAF在大鼠脊椎背角部位的分佈。在弗氏完全佐劑（CFA）致痛的大鼠模型中，我們在PCAF抑制的情況下檢測脊髓中COX-2的水平。我們還進行了染色質免疫沉澱。 / 最後，在招募了267位手術患者的基因關聯試驗中，我們對PCAF基因中的基因變異對慢性疼痛風險的影響進行了分析。我們運用卡方檢驗和費雪精確性檢驗對疼痛與基因型（58個單核苷酸多態性）和單倍型的關係進行分析。 / 實驗結果：減少的PCAF降低了COX-2的水平以及NF-κB的活性。當添加了IL1β時PCAF和乙酰化第三組蛋白14號賴胺酸（H3K14）在COX-2啟動子處富集。 / PCAF在大鼠脊椎背角部位的淺表層（第一層和第二層）的神經細胞里表達。在動物實驗中，注射了CFA的大鼠顯示COX-2會隨著PCAF的抑制而下降。大鼠注射了CFA后PCAF在COX-2啟動子處有所增加。漆樹酸和PCAF siRNA顯著地減輕了熱痛和機械痛提高了機械痛閾值。 / 在該基因關聯試驗中，我們鑒定出六個單核苷酸多態性和五種單倍型與較高風險的嚴重術後慢性痛有顯著的相關性。多元回歸分析表明在PCAF基因上具有rs6763504遺傳變異的病人在術後發展嚴重慢性痛的幾率會較高(p = 0.001)。 / 結論：PCAF調節COX-2的轉錄，而且PCAF的減少或者抑制導致了COX-2的降低同時慢性炎症痛的減少。PCAF基因上的遺傳變異提高了術後病人嚴重慢性痛的風險。 / Meng, Zhaoyu. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 117-134). / Abstracts also in Chinese. / Title from PDF title page (viewed on 30, December, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Histone acetyltransferases (HATs) are important enzymes in transcriptional control and potential targets for chemotherapeutic intervention in malignant diseases. Among different HAT members, the yeast Esa1 and human Tip60 (the HIV-1 Tat interactive protein, 60KDa) play multiple roles in normal cellular processes including transcription, cell cycle and checkpoint machinery, double strand DNA break repair, apoptosis, and cell cycle progression. Tip60 is also implicated in several human diseases such as prostate cancer, and gastric cancer. These studies suggest that Tip60 is a potential therapeutic target for new cancer treatment. So, we designed experimental work to synthesize and investigate organic inhibitors of Tip60 using different strategies, including substrate analogs, small molecule screening, and modification of the natural product anacardic acid. These studies provide important chemical agents for basic biology research of HAT function, and produce potential lead compounds for future pharmacologic intervention of HAT deregulation in cancer. Currently, of the methods used for the measurement of acetyltransferase activities, many comprise tedious separation procedures and involve enzyme-coupled steps or radioactive materials. These shortcomings have limited their applications in high-throughput screening (HTS) of HAT inhibitors. To circumvent these problems, a homogenous fluorescent HAT assay based on engineered H4 peptide was designed, synthesized, and evaluated. The data showed that these fluorescent reporters can be used to detect the acetyltransferase activities.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 69-77).

Arylamine N-acetyltransferases (NATs) were partially purified from human breast tumor tissues with complete separation of the isoforms in DEAE-Cellulose ion-exchange step. NAT with activity towards p-aminobenzoic acid (PABA) was isolated and purified from human breast tumor with 77 % yield and a purification factor of 5-fold. NAT with activity towards sulfamethazine (SMZ) was isolated and purified from human breast tumor with 21 % yield and a purification factor of 3-fold. Further purification attempts by Blue Sepharose affinity column chromatography resulted in the complete loss of both enzyme activities. The NAT1 purified from human breast tumor tissues had a molecular weight (Mr) value of about 27600 and an isoelectric point (pI) around 4.8, as confirmed by SDS-PAGE, IEF and Western blotting analysis. With immunohistochemical analysis, level of intensity of NAT1 immunostaining was observed to be going from weak in reduction mammoplasty samples to strongest in malignant breast tissue. The interindividual variation in the conjugation of p-aminobenzoic acid (PABA) and of sulfamethazine (SMZ) by cytosolic arylamine N-acetyltransferases (NATs) were investigated in 30 human breast tumor and matched samples. The average specific activity against PABA was calculated as 13&amp / #61617 / 2 pmole/min/mg protein for breast control NATs, and 20&amp / #61617 / 3 pmole/min/mg protein for breast tumor NATs. The average specific activity against SMZ was calculated as 12&amp / #61617 / 2 pmole/min/mg protein for breast control NATs, and 34&amp / #61617 / 6 pmole/min/mg protein for breast tumor NATs. Wilcoxon test revealed that the difference between the control and tumor groups is statistically significant with respect to the NAT1 activities as well as NAT2 activities. In three (3/30, 10%) patients tumor and tumor-free breast tissue NAT1 activity was not detectable. Among control tissues, the percentage of measurable NAT2 activity was 77% (23/30), while in tumor tissues it increased to 91%. Chemotherapy treatment was observed to have a slight inhibitory effect on mean NAT1 and NAT2 activities. There was an indication of a possible negative association with mean NAT1 activity and estrogen receptor status, while mean tumor NAT2 activity was observed to increase among estrogen receptor positive patients. Grade of malignancy seems to be positively associated with NAT1, but no such association could be suggested for NAT2 enzyme. Menopausal state of the patient was suggested to have a significant effect on NAT2 activity. Genotype determination of NATs revealed that NAT1*4 and NAT2*5A allele being most common among 10 breast cancer patients. NAT1*11 allele was prevalent among postmenopausal women. The putative rapid NAT1 genotypes was found to display lower control and tumor mean NAT1 activities compared to normal NAT1 genotypes. Among slow NAT2 acetylators, mean tumor NAT2 activities was found to be significantly higher than respective controls.