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Exigências de metionina + cistina e treonina para manutenção de aves /Bonato, Melina Aparecida. January 2010 (has links)
Orientador: Nilva Kazue Sakomura / Banca: Robert Mervyn Gous / Banca: Izabelle Auxiliadora Molina de Almeida Teixeira / Resumo: A mantença pode ser definida como o estado em que o animal se encontra em equilíbrio de nitrogênio, no qual a ingestão de N é igual à soma das perdas, permanecendo constante o conteúdo de N do corpo. E esta pode ser definida com base em estudos de metabolismo com aves adultas improdutivas, pelo fato das exigências totais de aminoácidos dessas aves estarem associadas apenas às perdas inevitáveis (mantença), não incluindo necessidades específicas de aminoácidos para o crescimento e/ou produção. Porém, há alguns problemas na determinação da exigência de mantença: primeiro é como comparar a mantença entre genótipos de diferentes tamanhos à maturidade, o segundo é como comparar a mantença entre animais de um mesmo genótipo em diferentes estágios de crescimento, e o terceiro é como lidar com a variação no conteúdo de gordura corporal, uma vez que não existe demanda de aminoácidos para a manutenção das reservas lipídicas. Assim, as diferenças de valores entre níveis de exigências para mantença de aminoácidos encontrados na literatura, tem sido a diretriz para o desenvolvimento de novos estudos, visando à obtenção de metodologias padronizadas e estimativas de valores condizentes com as necessidades das aves. Este estudo teve como objetivo estimar as exigências de metionina+cistina e treonina digestíveis para a mantença de aves adultas utilizando e comparando galos de diferentes pesos e composições corporais / Abstract: The maintenance can be defined as the state where the animal is in nitrogen balance, in which the intake of N is equal to the sum of the losses, stabilizing the N content in the body. And this can be defined based on studies of metabolism in adult birds unproductive, because the total amino acid requirements of these birds are associated only to the inevitable losses (maintenance), not including the specific amino acids for growth and/or production. However, there are some problems in determining the requirement for maintenance: first is to compare the maintenance among genotypes of different sizes at maturity, the second is like comparing the maintenance of animals of the same genotype at different stages of growth, and the third is how to deal with the variation in body fat content, since there is no demand for amino acids for the maintenance of lipid reserves. Thus, differences in values between levels of requirements for maintenance of amino acids found in the literature has been the guideline for the development of new studies aiming to produce standardized methodologies and estimates of amounts consistent with the needs of birds. This study aimed to estimate the methionine+cystine and threonine digestible for the maintenance of adult birds using and comparing roosters of different weights and body composition / Mestre
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Identification of a novel interaction partner of serine-arginine protein kinase 2 and studies on their roles in transcriptional regulation.January 2014 (has links)
SR蛋白在前體信使核糖核酸(pre-mRNA)的組成性剪接和選擇性剪接中扮演者重要的角色,在這個過程中它需要被SR蛋白激酶(SRPK) 燐酸化才能正常行使功能。經典的SR蛋白是由N端一到二個RNA識別基序(RRM) 以及C端一串精氨酸-絲氨酸(RS) 二肽所構成。SR蛋白的燐酸化調控它的亞細胞定位以及生理功能。此外,SR 蛋白激酶1(SRPK1) 和SR蛋白原型ASF/SF2的復合物結構顯示底物的結合需要第二個非標準的RRM結構域以及在N端可以被燐酸化的RS結構域,但是,第一個標準的RRM結構域對於SR 蛋白激酶1的結合卻是可以或缺的。 / 在這裡,我們展示了SR蛋白激酶2(SRPK2) 結合並且燐酸化SRp20的RS結構域,SRp20是另外一個只包含一個RNA識別基序(RRM) 的SR蛋白。與ASF/SF2相似的是,SRp20中的標準RNA識別基序對於SRPK2的結合並不是必要的。與此同時,我們發現錨定槽對於底物的識別作用在SRPK2中也是保守的,因為,錨定槽中四個關鍵氨基酸的突變會削弱它對SRp20的結合。 / 此外,現在認為SRPK2的功能已經不限於對前體信使核糖核酸(pre-mRNA) 的剪接調控。最近發現,SRPK2也可以燐酸化Tau蛋白並且介導阿爾茨海默疾病中的認知性缺陷。組成性的激活是SR蛋白激酶中的一個固有特性,然而人們對於它的調控機制還不是很清楚。因此, 為了更好的瞭解SRPK2,我們采用酵母雙雜交的方法並且最終發現一個新的SRPK2相互作用蛋白: ZNF187。 / ZNF187是一個可以結合血清反應元件(SRE) 的轉綠因子。我們的研究發現,它可以正向調控SRE的轉錄激活。然而,SRPK2在EGF的刺激下卻起着抑制的效果,其中EGF的刺激會促使SRPK2進入細胞核。進一步證實,通過RNAi干擾的方法敲掉SRPK2可以增加ZNF187誘導的SRE活性。在共轉染實驗中,SRPK2可以把ZNF187誘導的SRE活性逆轉到本底水平。對於可以和EGF刺激的SRPK2有着相似細胞定位的缺失或者突變研究發現,它們都可以產生相一致的抑制現象。於此相反,對於和SRPK2有着不同細胞定位的突變,它卻不能產生抑制效果。因此,我們認為在EGF的刺激下,SRPK2進入細胞核並且負向的調控ZNF187激活的SRE。令人驚訝的是,如果細胞在FBS的刺激下,SRPK2卻上調SRE活性,並且它可以協同增加ZNF187對於SRE的誘導。這些結果表明SRPK2對於ZNF187誘導的SRE轉綠調控是刺激物依賴的。 / SR proteins are critical players in regulating both constitutive and alternative pre-mRNA splicing, during which the phosphorylation by SR Protein Kinases (SRPKs) is required. Classical SR proteins contain one or two RNA Recognition Motifs (RRM) in their N terminus and a stretch of Arginine-Serine (RS) dipeptides in C terminus. Phosphorylation status of SR proteins regulates their subcellular localization as well as physiological function. In addition, complex structure of SRPK1 with ASF/SF2, a prototype of SR protein, shows that substrate binding requires non-canonicalRRM2 domain and RS domain, which can be extensivelyphosphorylated. However, the canonical RRM1 domain is dispensable for such interaction. / Here we show that SRPK2 binds and phosphorylates SRp20, a classical single RRM domain-containing SR protein, at its RS domain. Similarly with ASF/SF2, the canonical RRM domain of SRp20 is dispensable for interacting with SRPK2. Meanwhile, we also find that a docking groove that iscritical for substrate binding in SRPK1 is also conserved in SRPK2, since mutations on four key residues in docking groove impair its binding affinity with SRp20. / In addition, SRPK2 is now known to function more then regulating mRNA splicing, such as cell proliferation and cell apoptosis. Recently, SRPK2 is also shown to be a kinase phosphorylating Tau and mediate the cognitive defects in Alzheimer’s disease (AD). Besides, an intrinsic character of SRPKs lies in that they are constitutively active, but the regulation mechanism is not well understood. Therefore, in order to obtain a better recognition about SRPK2, we applied yeast two-hybrid assay and eventually anew interaction partner called ZNF187 was identified. / ZNF187 is a transcriptional factor that binds with Serum Response Element (SRE). Our studies showed that it isa positive regulator of SRE activity. However, SRPK2 showed inhibiting effect on SRE activation with the treatment of EGF, which could induce its nucleus entry, when co-transfected, it reversed the stimulating effect on SRE by ZNF187 to basal level. Furthermore, knockdown of SRPK2 by RNAi would enhance ZNF187-stimuated SRE activation. Studies on truncation and mutations that have the similar effect with EGF-induced subcellular localization of SRPK2 also generated the same inhibiting phenomenon. In contrast, mutant that has distinct localization with SRPK2 wild type failed to exert suppression. Therefore, we conclude that with the treatment of EGF, SRPK2 moves into nucleus and negatively regulates ZNF187-stimulated transactivation of SRE. Surprisingly, when cells were treated with FBS, SRPK2 showed stimulation on SRE activity and it synergized ZNF187-stimulated effect on SRE, indicating that transcriptional regulation of SRPK2 on ZNF187-stimulated SRE activity is stimuli-dependent. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Shang, Yong. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 113-137). / Abstracts also in Chinese.
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Study of GCN2 in Arabidopsis thaliana.January 2009 (has links)
Li, Man Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 109-119). / Abstracts in English and Chinese. / Thesis Committee --- p.I / Statement --- p.II / Abstract --- p.III / 摘要 --- p.V / Acknowledgements --- p.VI / Abbreviations --- p.VIII / Abbreviations of Chemicals --- p.X / List of Tables --- p.XI / List of Figures --- p.XII / Table of Contents --- p.XIII / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- General amino acid control in yeast --- p.1 / Chapter 1.2 --- Mammalian eIF2α kinases --- p.7 / Chapter 1.2.1 --- Heme-regulated inhibitor kinase (EIF2AK1/HRI) --- p.7 / Chapter 1.2.2 --- Protein kinase dsRNA-dependent (EIF2AK2/PKR) --- p.8 / Chapter 1.2.3 --- PKR-like ER kinase (EIF2AK3/PERK) --- p.9 / Chapter 1.2.4 --- General control non-repressible 2 (EIF2AK4/GCN2) --- p.10 / Chapter 1.2.5 --- Activating transcription factor 4 (ATF4) --- p.11 / Chapter 1.3 --- Plant General Amino Acid Control --- p.12 / Chapter 1.3.1 --- Studies of the homolog of GCN2 in Arabidopsis thaliana --- p.12 / Chapter 1.3.2 --- Studies of the homolog of other eIF2a kinase in plant --- p.14 / Chapter 1.3.3 --- Studies of the homolog of other GAAC components --- p.14 / Chapter 1.4 --- Previous works in our lab --- p.15 / Chapter 1.5 --- Hypothesis and Objectives --- p.17 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.18 / Chapter 2.1.1 --- "Bacterial cultures, plant materials and vectors" --- p.18 / Chapter 2.1.2 --- Primers --- p.21 / Chapter 2.1.3 --- Commercial kits --- p.25 / Chapter 2.1.4 --- "Buffer, solution, gel and medium" --- p.25 / Chapter 2.1.5 --- "Chemicals, reagents and consumables" --- p.25 / Chapter 2.1.6 --- Enzymes --- p.25 / Chapter 2.1.7 --- Antibodies --- p.25 / Chapter 2.1.8 --- Equipments and facilities --- p.25 / Chapter 2.2 --- Methods --- p.26 / Chapter 2.2.1 --- Growth conditions of Arabidopsis thaliana --- p.26 / Chapter 2.2.1.1 --- Surface sterilize of Arabidopsis thaliana seed --- p.26 / Chapter 2.2.1.2 --- Growing of Arabidopsis thaliana --- p.26 / Chapter 2.2.1.3 --- Treatment of Arabidopsis seedling --- p.26 / Chapter 2.2.2 --- Basic molecular techniques --- p.27 / Chapter 2.2.2.1 --- Liquid culture of Escherichia coli --- p.27 / Chapter 2.2.2.2 --- Preparation of plasmid DNA --- p.27 / Chapter 2.2.2.3 --- Restriction digestion --- p.27 / Chapter 2.2.2.4 --- DNA purification --- p.28 / Chapter 2.2.2.5 --- DNA gel electrophoresis --- p.28 / Chapter 2.2.2.6 --- DNA ligation --- p.29 / Chapter 2.2.2.7 --- CaCl2 mediated E. coli transformation --- p.29 / Chapter 2.2.2.8 --- Preparation of DNA fragment for cloning --- p.29 / Chapter 2.2.2.9 --- PCR reaction for screening positive E. coli transformants --- p.30 / Chapter 2.2.2.10 --- DNA sequencing --- p.30 / Chapter 2.2.2.11 --- RNA extraction from plant tissue with tRNA --- p.31 / Chapter 2.2.2.12 --- Extraction of RNA without tRNA --- p.31 / Chapter 2.2.2.13 --- cDNA synthesis --- p.32 / Chapter 2.2.2.14 --- SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.33 / Chapter 2.2.2.15 --- Western blotting --- p.33 / Chapter 2.2.3 --- Sub-cloning of AtGCN2 --- p.34 / Chapter 2.2.3.1 --- Sub-cloning full length AtGCN2 into pMAL-c2 --- p.36 / Chapter 2.2.3.2 --- Sub-cloning of the N-terminal sequence of AtGCN2 into pMAL-c2 --- p.38 / Chapter 2.2.3.3 --- Sub-cloning of the C-terminal sequence of AtGCN2 into pMAL-c2 --- p.38 / Chapter 2.2.4 --- Cloning of the eIF2α candidates for the in vitro assay --- p.41 / Chapter 2.2.4.1 --- Cloning of At2g40290 (putative eIF2α candidate) --- p.41 / Chapter 2.2.4.2 --- Cloning of At5g05470 (putative eIF2α candidate) into pBlueScript KS II + --- p.43 / Chapter 2.2.4.3 --- Sub-cloning of At5g05470 into pGEX-4T-1 --- p.43 / Chapter 2.2.4 --- Expression and purification of fusion proteins --- p.45 / Chapter 2.2.5 --- Expression of fusion proteins in E. coli --- p.45 / Chapter 2.2.5.2 --- Extraction of E. coli soluble proteins --- p.45 / Chapter 2.2.5.3 --- Purification of GST tagged fusion protein --- p.46 / Chapter 2.2.5.4 --- Purification of MBP tagged fusion protein --- p.46 / Chapter 2.2.5.5 --- Concentration of purified fusion proteins --- p.46 / Chapter 2.2.5.6 --- MS/MS verification of purified fusion proteins --- p.47 / Chapter 2.2.6 --- Gel mobility shift assay --- p.47 / Chapter 2.2.6.1 --- Synthesis of short biotinylated RNA --- p.47 / Chapter 2.2.6.2 --- Ligation of short biotinylated RNA with tRNA --- p.48 / Chapter 2.2.6.3 --- Gel mobility shift assay --- p.48 / Chapter 2.2.6.4 --- Blotting of the sample on to nitrocellulose membrane --- p.48 / Chapter 2.2.6.5 --- Detection of the tRNA on the membrane --- p.49 / Chapter 2.2.6.6 --- Detection of the MBP fusion proteins on the membrane --- p.49 / Chapter 2.2.7 --- In vitro kinase assay of AtGCN2 --- p.49 / Chapter 2.2.8 --- In vitro translation inhibition assay --- p.50 / Chapter 2.2.8.1 --- In vitro transcription of HA mRNA --- p.50 / Chapter 2.2.8.2 --- In vitro translation --- p.51 / Chapter 2.2.8.3 --- Detection of the protein dot blot --- p.51 / Chapter 2.2.9 --- Gene expression analysis by real time PCR --- p.52 / Chapter 2.2.10 --- Total seed nitrogen analysis --- p.53 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Blast search results suggested that AtGCN2 may be the sole eIF2α kinase in Arabidopsis thaliana --- p.54 / Chapter 3.2 --- Existence of two eIF2α candidates in Arabidopsis thaliana genome --- p.59 / Chapter 3.3 --- Fusion proteins were successfully expressed and purified --- p.63 / Chapter 3.4 --- C-terminal of AtGCN2 has a higher affinity toward tRNA than rRNA --- p.67 / Chapter 3.5 --- Both eIF2α candidates can be phosphorylated by full length AtGCN2 in vitro --- p.70 / Chapter 3.6 --- AtGCN2 can inhibit translation in vitro --- p.72 / Chapter 3.7 --- Overexpression of AtGCN2 did not affect expression of selected genes --- p.74 / Chapter 3.8 --- Overexpression of AtGCN2 did not affect seed nitrogen content and C:N ratio under normal growth conditions --- p.83 / Chapter Chapter 4 --- Discussion --- p.85 / Chapter 4.1 --- Existing evidence supported that AtGCN2 is the sole eIF2α kinase in Arabidopsis thaliana --- p.85 / Chapter 4.2 --- Kinase activities of AtGCN2 and its two substrates in Arabidopsis --- p.86 / Chapter 4.3 --- C-terminal binds tRNA in the gel mobility shift assay --- p.88 / Chapter 4.4 --- Overexpression of AtGCN2 did not affect gene expression of the transgenic lines under nitrogen starvation and azerserine treatment --- p.90 / Chapter 4.5 --- Overexpression of AtGCN2 did not alter the seed nitrogen content --- p.91 / Chapter 4.6 --- Existence of GCN4 and ATF4 in plant --- p.92 / Chapter 4.7 --- Alternative model without GCN4 and ATF4 homolog --- p.93 / Chapter 4.8 --- Possible application of the in vitro kinase assay --- p.94 / Chapter 4.9 --- Possible application of the in vitro translation inhibition analysis platform in future study --- p.95 / Chapter Chapter 5 --- Conclusion and Future Prospective --- p.97 / Appendices / Appendix I Commercial kits used in this project --- p.98 / "Appendix II Buffer, solution, gel and medium" --- p.99 / "Appendix III Chemicals, reagents and consumables" --- p.102 / Appendix IV Enzymes --- p.103 / Appendix V Antibodies --- p.104 / Appendix VI Equipments and facilities --- p.105 / Appendix VII Supplementary Data --- p.106 / Appendix VIII Amplification efficiency of real time primers --- p.108 / References --- p.109
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Characterization of PknB, a Putative Eukaryotic-type Serine/threonine Protein Kinase in Streptococcus mutansDel Re, Deanna 13 January 2010 (has links)
PknB is a putative transmembrane eukaryotic-type serine/threonine protein kinase (STPK) in the cariogenic bacterium Streptococcus mutans that affects biofilm formation, genetic competence and acid tolerance. PknB contains extracellular penicillin-binding and serine/threonine kinase associated (PASTA) domains predicted to bind the D-alanyl-D-alanine (D-ala-D-ala) dipeptide of unlinked peptidoglycan. D-ala-D-ala elicits responses dependent and independent of the presence of pknB. Biofilm-derived cells of a pknB-deficient mutant (PKNB) exhibited concentration-dependent growth enhancement with D-ala-D-ala, which was not a nutrient response as addition of L-alanine or D-alanine did not give the same results. A total of 77 genes were differentially expressed in PKNB, including 7 with putative functions in fatty acid biosynthesis. PKNB was more sensitive to cell wall- and membrane-targeting antibiotics compared to wild-type. Based on these results, PknB in S. mutans appears to play an important role in cell wall biosynthesis, response to membrane stress and/or regulation of cell membrane composition.
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Characterization of PknB, a Putative Eukaryotic-type Serine/threonine Protein Kinase in Streptococcus mutansDel Re, Deanna 13 January 2010 (has links)
PknB is a putative transmembrane eukaryotic-type serine/threonine protein kinase (STPK) in the cariogenic bacterium Streptococcus mutans that affects biofilm formation, genetic competence and acid tolerance. PknB contains extracellular penicillin-binding and serine/threonine kinase associated (PASTA) domains predicted to bind the D-alanyl-D-alanine (D-ala-D-ala) dipeptide of unlinked peptidoglycan. D-ala-D-ala elicits responses dependent and independent of the presence of pknB. Biofilm-derived cells of a pknB-deficient mutant (PKNB) exhibited concentration-dependent growth enhancement with D-ala-D-ala, which was not a nutrient response as addition of L-alanine or D-alanine did not give the same results. A total of 77 genes were differentially expressed in PKNB, including 7 with putative functions in fatty acid biosynthesis. PKNB was more sensitive to cell wall- and membrane-targeting antibiotics compared to wild-type. Based on these results, PknB in S. mutans appears to play an important role in cell wall biosynthesis, response to membrane stress and/or regulation of cell membrane composition.
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USING THE INDICATOR AMINO ACID OXIDATION TECHNIQUE TO STUDY THREONINE REQUIREMENTS IN HORSES FED DIFFERENT FEED COMPOSITIONSMok, ChanHee 01 January 2015 (has links)
Threonine has been reported to be the second limiting amino acid in typical equine diets, but its actual requirement has not been determined in horses. The indicator amino acid oxidation (IAAO) method has been successfully used for evaluating amino acid metabolism and requirements in animals and humans. The objective of this research was to use the IAAO method to estimate threonine requirements in mature horses fed two different feed compositions. In the first study, 6 Thoroughbred mares (4 – 16 years old) received each of 6 levels of threonine intake in a high concentrate diet, in a randomly determined order. The experimental diets consisted of concentrate at 0.95% of body weight (BW), and chopped timothy hay at 0.95% of BW, and met or exceeded NRC recommendations for all nutrients. Threonine intakes for the 6 treatments were 45, 56, 67, 79, 90 and 102 mg/kg BW/d and were generated by mixing the two experimental concentrates, containing 2.9 and 14.8 g threonine/kg diet, in different ratios. In the second study, horses received each of 6 levels of threonine intake, 41, 51, 61, 70, 80, and 89 mg/kg BW/d, in a high forage diet, in a randomly determined order. The experimental diet was concentrate at 0.4% of BW, and chopped timothy hay at 1.6% of BW. Study periods for each study were 7-d long and on d 6, blood samples were collected before and 90 min after feeding to measure amino acid concentrations using HPLC. On d 7, horses underwent IAAO procedures, which included a two hour primed, constant intravenous infusion of [13C]sodium bicarbonate to measure total CO2 production and a four hour primed, constant oral administration of [1-13C]phenylalanine to estimate phenylalanine oxidation to CO2. Blood and breath samples were collected to measure blood [13C]phenylalanine, using GC-MS analysis, and breath 13CO2 enrichment, using an infrared isotope analyzer. Experimental data were analyzed using a one-way ANOVA, including orthogonal linear and quadratic contrasts, in the mixed procedures of SAS version 9.3, with treatment as the fixed effect and horse nested in treatment as the random effect. Statistical significance was declared at P0.05). Increasing threonine intake levels in a high forage diet did not affect plasma phenylalanine oxidation by the ANOVA test (P>0.05) but resulted in a linear decrease in phenylalanine oxidation (P=0.04) without a breakpoint by the orthogonal linear contrast. Threonine requirements are still unknown in mature horses fed either high concentrate or high forage diet. The present studies were the first attempt to evaluate threonine requirements in horses by the indicator amino acid oxidation method.
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Serine/threonine phosphorylation in mycobacterium tuberculosis : identification of protein kinase B (PknB) substratesLee, Guinevere Kwun Wing Queenie 05 1900 (has links)
Tuberculosis, caused by the intracellular pathogen Mycobacterium tuberculosis, is one of the most prevalent infectious diseases in our world today. In order to survive within the host the bacteria need to sense and respond to changes in the environment; however, signal transduction in this bacterium is poorly understood. PknB is a serine/threonine kinase essential for the in vitro survival of M. tuberculosis and therefore a potential drug target against the bacteria. It is the goal of the current study to elucidate downstream substrates of PknB. We have found that PknB shares in vitro substrates with another serine/threonine kinase, PknH, implying the potential complexity of the signaling pathways in the bacteria. We have also provided the first description of the coupling between serine/threonine kinases PknB and PknH with a two-component system response regulator DevR, and further proposed Ser/Thr phosphorylation as the negative regulator of DevR transcription activator activity based on LC-MS/MS analysis. Finally, we have identified a previously unknown phosphoprotein glyceraldehyde 3-phosphate dehydrogenase encoded by the ORF Rv1436, which demonstrates autophosphorylation activity and which phosphorylation is independent of PknB. Overall, the current study has contributed to advance our understanding of the signal transduction pathways and phosphoproteome in Mycobacterium tuberculosis.
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Structural basis for the recruitment of the SerThr kinase Mnk1 by the scaffolding proteins DAP5 and elF4GTalje, Lama. January 2008 (has links)
Scaffolding proteins control the localization of protein kinases. During translation, the scaffolding proteins eIF4G and DAP5 recruit the Ser/Thr kinase Mnk1 to phosphorylate the mRNA cap-binding protein eIF4E and modulate translation. Biochemical deletion analysis previously showed that the interaction between Mnk1 and eIF4G/DAP5 is mediated by N-terminal residues in Mnk1 and C-terminal residues in eIF4G/DAP5. Using X-ray crystallography I have determined the structure (1.5 A) of the C-terminal domain of DAP5 (DAP5C). This structure reveals that DAP5C contains two atypical HEAT domains similar to the ones seen previously in the structure of the C-terminal region of eIF4G (4GC). Using ITC I showed that the Kd for the interaction between the N-terminus ofMnk1 and 4GCIDAPSC is 20 muM and 10 muM, respectively. Using NMR chemical shifts we have mapped the residues on both Mnk1 and 4GC/DAP5C which are important for maintaining this interaction. Finally, using SAXS a low resolution configuration of the hMnk1-4GC complex was modeled. It is hoped that an understanding of the structural basis for the recruitment of protein kinases to their sites of action will allow the design of small-molecule compounds that can be used to modulate the location of the kinase and hence its activity.
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Serine/threonine phosphorylation in mycobacterium tuberculosis : identification of protein kinase B (PknB) substratesLee, Guinevere Kwun Wing Queenie 05 1900 (has links)
Tuberculosis, caused by the intracellular pathogen Mycobacterium tuberculosis, is one of the most prevalent infectious diseases in our world today. In order to survive within the host the bacteria need to sense and respond to changes in the environment; however, signal transduction in this bacterium is poorly understood. PknB is a serine/threonine kinase essential for the in vitro survival of M. tuberculosis and therefore a potential drug target against the bacteria. It is the goal of the current study to elucidate downstream substrates of PknB. We have found that PknB shares in vitro substrates with another serine/threonine kinase, PknH, implying the potential complexity of the signaling pathways in the bacteria. We have also provided the first description of the coupling between serine/threonine kinases PknB and PknH with a two-component system response regulator DevR, and further proposed Ser/Thr phosphorylation as the negative regulator of DevR transcription activator activity based on LC-MS/MS analysis. Finally, we have identified a previously unknown phosphoprotein glyceraldehyde 3-phosphate dehydrogenase encoded by the ORF Rv1436, which demonstrates autophosphorylation activity and which phosphorylation is independent of PknB. Overall, the current study has contributed to advance our understanding of the signal transduction pathways and phosphoproteome in Mycobacterium tuberculosis.
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Linking PAR polarity proteins to cell fate regulation : analysis of MEX-5 localization in Caenorhabditis elegans embryos /Tenlen, Jennifer R. January 2007 (has links)
Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 89-100).
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