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Mechanizmy regulace inhibičního faktoru IF1 / Mechanisms of regulation of inhibitory factor IF1Sklenář, Filip January 2020 (has links)
Inhibitory factor 1 (IF1) is one of the major regulators of mitochondrial ATP synthase activity, a key enzyme of energy metabolism. Its inhibitory effects are known in conditions such as hypoxia or starvation, but the hypothesis that IF1 inhibits ATP synthase activity even under physiological conditions is still not entirely accepted. Disorders of ATP synthase regulation can be fatal to the cell and have been described, for example, in carcinogenesis and ischemia. It has also been found that silencing of the IF1 gene in pancreatic β-cells increases insulin secretion, and thus, IF1 may be important in the pathogenesis of type 2 diabetes. The goal of this work is to summarize the current knowledge about the IF1 protein and to obtain new results that will help elucidate the mechanism by which this protein regulates mitochondrial ATP synthase. Specifically, this work deals with the ratio of IF1 protein to ATP synthase in pancreatic β-cells, depending on different culture conditions. It further investigates the occurrence of post-translational modifications of the IF1 protein in pancreatic β-cells (INS- 1E model cells), which may play a role in the regulation of IF1 activity. It also deals with the cellular ATP/ADP ratio, which is one of the key factors for insulin secretion by pancreatic β-cells. An...
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Strukturní hmotnostní spektrometrie faktorů virulence rodu Bordetella / Structural mass spectrometry of Bordetella virulence factorsJurnečka, David January 2020 (has links)
The Bordetellae are aerobic Gram-negative coccobacilli colonizing the upper respiratory tract of mammals and thereby causing diseases with similar symptoms but different host specificity. The bacteria produce a variety of adhesins and toxins that facilitate their ability to promote infection and evade the innate immune system. Among them, the filamentous hemagglutinin (FHA) and the adenylate cyclase toxin (CyaA) are the major virulence factors providing the adherence to the host epithelial cells and the protection against bactericidal activity of phagocytic cells, respectively. Moreover, CyaA along with the Escherichia coli α-hemolysin (HlyA) and the Kingella kingae cytotoxin (RtxA) represent a prominent group of Repeats in ToXin (RTX) cytotoxins/hemolysins that undergo post-translational acylation on conserved lysine residues. Here, different mass spectrometry approaches were employed to analyze the structural features of FHA and to characterize the acylation status of the RTX toxins and their various hybrid molecules. First, the differential 16O/18O labeling revealed that the mature FHA proteins of B. pertussis (Bp-FHA) and the B. bronchiseptica (Bb-FHA) are processed at different sites, after Ala2348 and Lys2479 of the FhaB precursor, respectively. Second, the bottom-up proteomics of the...
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Cardiac sodium channel palmitoylation regulates channel function and cardiac excitability with implications for arrhythmia generationPei, Zifan 09 December 2016 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / The cardiac voltage-gated sodium channels (Nav1.5) play a specific and critical role in regulating cardiac electrical activity by initiating and propagating action potentials in the heart. The association between Nav1.5 dysfunctions and generation of various types of cardiac arrhythmia disease, including long-QT3 and Brugada syndrome, is well established. Many types of post-translational modifications have been shown to regulate Nav1.5 biophysical properties, including phosphorylation, glycosylation and ubiquitination. However, our understanding about how post-translational lipid modification affects sodium channel function and cellular excitability, is still lacking. The goal of this dissertation is to characterize Nav1.5 palmitoylation, one of the most common post-translational lipid modification and its role in regulating Nav1.5 function and cardiac excitability. In our studies, three lines of biochemistry evidence were shown to confirm Nav1.5 palmitoylation in both native expression background and heterologous expression system. Moreover, palmitoylation of Nav1.5 can be bidirectionally regulated using 2-Br-palmitate and palmitic acid. Our results also demonstrated that enhanced palmitoylation in both cardiomyocytes and HEK293 cells increases sodium channel availability and late sodium current activity, leading to enhanced cardiac excitability and prolonged action potential duration. In contrast, blocking palmitoylation by 2-Br-palmitiate increases closed-state channel inactivation and reduces myocyte excitability. Our computer simulation results confirmed that the observed modification in Nav1.5 gating properties by protein palmitoylation are adequate for the alterations in cardiac excitability. Mutations of potential palmitoylation sites predicted by CSS-Palm bioinformatics tool were introduced into wild-type Nav1.5 constructs using site-directed mutagenesis. Further studies revealed four cysteines (C981, C1176, C1178, C1179) as possible Nav1.5 palmitoylation sites. In particular, a mutation of one of these sites(C981) is associated with cardiac arrhythmia disease. Cysteine to phenylalanine mutation at this site largely enhances of channel closed-state inactivation and ablates sensitivity to depalmitoylation. Therefore, C981 might be the most important site that regulates Nav1.5 palmitoylation. In summary, this dissertation research identified novel post-translational modification on Nav1.5 and revealed important details behind this process. Our data provides new insights on how post-translational lipid modification alters cardiomyocyte excitability and its potential role in arrhythmogenesis.
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Regulation of Protein Arginine Methyl Transferase 5 by Novel Serine 15 Phosphorylation in Colorectal CancerHartley, Antja-Voy Anthoneil 01 1900 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / The overexpression of protein arginine methyltransferase 5 (PRMT5) is strongly correlated to poor clinical outcomes for colorectal cancer (CRC) patients. Previously, we demonstrated that PRMT5 overexpression could substantially augment activation of NF-κB via methylation of arginine 30 (R30) on its p65 subunit, while knockdown of PRMT5 showed the opposite effect on the transcriptional competence of p65. However, the precise mechanisms governing this PRMT5/NF-κB axis are still largely unknown. We report a novel finding that PRMT5 is phosphorylated on serine 15 (S15) in response to interleukin-1β (IL-1β) stimulation. Overexpression of the serine-to-alanine mutant of PRMT5 (S15A-PRMT5), in either HEK293 cells or HT29, DLD1 and HCT116 CRC cells attenuated NF-κB activation compared to wild type (WT)-PRMT5, confirming that S15 phosphorylation is critical for the activation of NF-κB by PRMT5. Furthermore, we found that overexpression of S15A-PRMT5 mutant attenuated the expression of a subset of NF-κB target genes through decreased p65 occupancy at their respective promoters. Importantly, the S15A-PRMT5 mutant also reduced IL-1β-induced methyltransferase activity of PRMT5 as well as its ability to form a complex with p65. Finally, we observed that the S15A-PRMT5 mutant diminished the growth, migratory and colony-forming abilities of CRC cells compared to the WT-PRMT5. Collectively, our findings provide strong evidence that novel phosphorylation of PRMT5 at S15 is critical to its regulation of NF-κB and plays an essential role in promoting the cancer-associated functions exerted by the PRMT5/NF-κB axis. Therefore, development of inhibitors to block phosphorylation of PRMT5 at S15 could become a potential novel therapeutic approach to treat CRC. / 2020-10-15
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Potencies of the resistant maize genotypes against biotic stresses and understanding their strategiesAnkala, Arunkanth 01 May 2010 (has links)
Maize is an important food crop in most parts of the world including the United States. The plants growing in the field are constantly challenged with various biotic stresses like insect herbivores and fungal pathogens. The physical wounds produced on the growing crops by the insects render the plants more vulnerable to the fungal pathogens. Hence developing both insect and fungal resistant maize varieties is crucial to benefit more from the harvest. Several studies have been in advance in this direction and as a consequence insect, in particular lepidopteran larve resistant maize genotype Mp708 and Aspergillus flavus resistant genotype Mp313E were developed. This study particularly focuses on understanding the functional involvement of the major phytohormones in the signal transduction and expression of the unique defense protein, Maize insect resistance 1-cysteine protease (Mir1-CP) shown to accumulate in response to herbivory by lepidopteran larvae, Spodoptera frugiperda (Fall armyworm, FAW) as a defense mechanism. Using a pharmacological approach involving exogenous hormone and hormone inhibitor treatments and analyzing the expression and accumulation of Mir1-CP protein and mir1 transcript by immunoblot and qRT-PCR analysis respectively, both JA and ET were found to be involved in mediating Mir1-CP accumulation with JA acting upstream of ET. Results also indicate that Mir1-CP accumulation involves both transcriptional and post-transcriptional (or post-translational) regulations. A different part of the study involved in understanding and evaluating the performance of Aspergillus flavus on the resistant and susceptible maize genotypes during infection. As of part of this study I also analyzed and compared the defense response offered by the resistant maize genotype, Mp313E and the susceptible genotype, Va35 by looking at the expression levels of the various defense related genes. The potency of the resistant maize genotype in sustaining the fungal infection in the field was of particular focus. Resistant maize genotype Mp313E was found to potentially oppose A.flavus proliferation and colonization and also delay aflatoxin biosynthesis unlike Va35. The up regulation of the maize defense genes during the early time points of infection, in Mp313E, indicate the potential role of these genes in conferring resistance against fungal pathogens.
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Epigenetic regulations in cell wall degradation and regeneration in Oryza sativaTan, Feng 06 August 2011 (has links)
It is well known that chromatin components are key players in establishing and maintaining spatial and temporal gene expression in plants, however, little is known about the epigenetic regulation on cell wall degradation and regeneration. This study aimed to 1) investigate the global proteome and phosphoproteome of rice chromatin, and 2) characterize changes in chromatin components and chromatin structure associated with cell wall degradation and regeneration, and 3) characterize the differentially regulated proteins and eventually explore the mechanism. In this dissertation, we examine proteins copurified with chromatin using both 2-DE gel and shotgun approaches from rice (Oryza sativa) suspension cells. Nine hundred seventy-two distinct protein spots were resolved on 2-DE gels and 509 proteins were identified by MALDI-MS/MS following gel excision, these correspond to 269 unique proteins. When the chromatin copurified proteins are examined using shotgun proteomics, a large number of histone variants in addition to the four common core histones were identified. Furthermore, putative phosphoproteins copurified with chromatin were examined using Pro-Q Diamond phosphoprotein stain and followed by MALDI-MS/MS. Our studies provided new insights into the chromatin composition in plants. To study the epigenetic regulation of the cell wall degradation and regeneration, we examined cellular responses to the enzymatic removal of the cell wall in rice suspension cells using proteomic approaches. We found that removal of cell wall stimulates cell wall synthesis from multiple sites in protoplasts instead of from a single site as in cytokinesis. Microscopy examination and chromatin decondensation assay further showed that removal of the cell wall is concomitant with substantial chromatin reorganization. Histone post-translational modification studies using both Western blots and isotope labeling assisted quantitative mass spectrometry analyses revealed substantial histone modification changes, particularly H3K18AC and H3K23AC, are associated with the degradation and regeneration of the cell wall. Labelree comparative proteome analyses further revealed that chromatin associated proteins undergo dramatic changes upon removal of the cell wall, particularly cytoskeleton, cell wall metabolism, and stress-response proteins. This study demonstrates that cell wall removal is associated with substantial chromatin change and may lead to stimulation of cell wall synthesis using a novel mechanism.
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Characterization of the Role of Testis-Specific Serine Kinase1 (tssk1) in Human Sperm CapacitationChicoine, Kay-ellen 01 January 2013 (has links) (PDF)
The ability of a sperm to fertilize an egg involves a complex series of events, referred to as capacitation. Capacitation involves sperm acquiring a hyperactive state of motility and the ability to undergo an exocytotic process called the acrosome reaction (AR). Molecular intervention of cell signaling pathways involved in sperm capacitation has been proposed as a way for developing novel male contraceptives.
Testis-specific serine kinase1 (TSSK1) is a novel protein in the calcium/calmodulin superfamily of human kinases, and has the potential for targeted inhibition to provide a highly safe and effective form of male contraception. It is expressed post-meiotically during spermatogenesis, and is localized to the head and tail of the mature sperm. Tssk1/Tssk2 knockout (KO) mouse models have demonstrated that these proteins are necessary for sperm function as deletion of these genes have rendered male mice infertile. Studies presented here involve the investigation of the biochemical regulation of TSSK1 and its role in human sperm capacitation.
Posttranslational modification of TSSK1 was analyzed in capacitated human sperm in the presence or absence of AR inducers in vitro. Furthermore, experimental conditions were tested for measuring TSSK1 enzymatic activity in capacitated human sperm, and TSSK1 kinase activity was further correlated to the observed phosphorylation/dephosphorylation.
Lastly, a computer-aided sperm analysis (CASA) protocol for assessing human sperm motility was standardized. Human sperm cell subpopulations were evaluated under different sperm capacitating conditions, and a Boolean argument was created to quantify progressive and non-progressive hyperactive cells. Determining TSSK1’s regulation by posttranslational modifications will help further characterize this protein as a potential target for inhibition and, ultimately, for use as a novel technique of male contraception. Improved conditions for human sperm analysis by CASA may provide a tool for assessing a potential role of TSSK1 in human sperm motility and hyperactivation.
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Exploring the Role of Endogenous TDP-43 SUMOylation in Mice: Implications for Amyotrophic Lateral Sclerosis and Frontotemporal DementiaPart, Caroline 20 February 2024 (has links)
As the most common motor neuron disease, Amyotrophic lateral sclerosis (ALS) affects around 4 in every 100,000 people worldwide with reports of increasing prevalence over the years. Characterized by progressive degeneration of motor neurons, ALS patients suffer impairments of movement and typically die from respiratory failure 2-5 years after diagnosis. Curiously, ALS exists on a disease continuum with Frontotemporal Dementia (FTD) where 30-50% of patients will be diagnosed with both diseases. In FTD, degeneration of cortical neurons results in diverse behavioural changes including deficits in executive and social skills as well as language and memory. A central connection between ALS and FTD is TDP-43 (encoded by TARDBP), an essential DNA/RNA binding protein known to serve critical functions in numerous cellular processes. Despite mutations in TARDBP constituting a small percentage of familial cases, TDP 43 nuclear-to-cytoplasmic mislocalization is a pathological hallmark of most ALS-FTD cases. Therefore, therapeutic targets to rectify pathology and disease may be uncovered by identifying factors that regulate TDP-43. While it is currently established TDP-43 is ubiquitinated and phosphorylated in diseased states, our lab recently found TDP-43 is SUMOylated in response to stress. Of note, perturbations in the stress response are becoming increasingly implicated in neurodegenerations. Furthermore, TDP-43 plays critical roles in the stress response which become perturbed in ALS/FTD. We developed a TDP-43 "SUMO dead" mouse allele to gain an understanding of how disrupting this may contribute to the pathogenesis of ALS-FTD. Longitudinal characterization of the model explored behavioural and histological in vivo consequences following loss of TDP-43 SUMOylation. However, the phenotypes observed in the mutant mice were less robust in comparison to established ALS/FTD mouse models. Mutant mice did not have consistent differences in tests for similar outcomes, trials of the same test, or across age. Female mutant mice presented with early hyperactivity and disinhibition along with altered social grooming behaviour. At later age, these female mice developed impairments in spatial working memory. Male mice developed apathetic behaviour and motor deficits at the middle age timepoint. Histologically, various forms of pathological TDP-43 were observed in the absence of neurodegeneration. These data reveal that TDP-43 SUMOylation may play an important role in ALS/FTD pathogenesis.
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Regulation of Rice Flowering Time and Seed DevelopmentMeng, Xiaoxi 10 August 2018 (has links)
Rice is one of the most important cereal crops for the world population. Flowering time and seed development of rice are directly related to plant regional and ecological adaptions, and productive yield. In this dissertation, to gain knowledge of seed development in rice, the status of post-translational modifications (PTMs) in developing rice seeds was investigated. Numerous modified lysine sites in developing rice seeds were identified utilizing antibody-based affinity enrichment approaches and nano-HPLC/MS/MS analyses of acetylated, succinylated, crotonylated and 2-hydroxyisobutyrylated peptides. Functional annotation analyses indicated that a wide variety of vital biological processes were targeted by lysine PTMs. A number of modified histone and non-histone proteins were found to harbor multiple PTMs, and our findings showed that many modified histone sites were conserved across plant, human, and animal systems. Comprehensive analyses of lysine modification sites illustrated that the sites were highly sequence-specific for distinct motifs. Overall, this study provides a systematic analysis of lysine PTM proteome in plants, which will serve as the basis for future investigations of the regulatory mechanisms and functions of lysine PTMs. The mechanisms of flowering time variances in response to different photoperiods were further studied in the rice mutant, HSS. QTL-seq analysis identified a major effect on chromosome 6 responsible for the phenotypic divergence between Nipponbare (wild-type) and HSS rice. Sequence and mRNA expression analyses confirmed that allelic variants of Hd1 make HSS plants less sensitive to photoperiod by altering expression level of Hd3a. Diurnal expression pattern analyses revealed that DTH8 transcripts were largely affected by Hd1 expression level in both LD and SD. This result suggested that Hd1 may able to regulate DTH8 and DTH8-Hd1 complex abundance in response to day length in rice flowering time regulation. In addition, we discussed the functions of PTMs in flowering time regulation in rice.
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Ribosomally Synthesized and Post-Translationally Modified Peptides as Potential Scaffolds for Peptide EngineeringBursey, Devan 01 March 2019 (has links)
Peptides are small proteins that are crucial in many biological pathways such as antimicrobial defense, hormone signaling, and virulence. They often exhibit tight specificity for their targets and therefore have great therapeutic potential. Many peptide-based therapeutics are currently available, and the demand for this type of drug is expected to continue to increase. In order to satisfy the growing demand for peptide-based therapeutics, new engineering approaches to generate novel peptides should be developed. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a group of peptides that have the potential to be effective scaffolds for in vivo peptide engineering projects. These natural RiPP peptides are enzymatically endowed with post-translational modifications (PTMs) that result in increased stability and greater target specificity. Many RiPPs, such as microcin J25 and micrococcin, can tolerate considerable amino acid sequence randomization while still being capable of receiving unique post-translational modifications. This thesis describes how we successfully engineered E. coli to produce the lasso peptide microcin J25 using a two-plasmid inducible expression system. In addition, we characterized the protein-protein interactions between PTM enzymes in the synthesis of micrococcin. The first step in micrococcin synthesis is the alteration of cysteines to thiazoles on the precursor peptide TclE. This step is accomplished by three proteins: TclI, TclJ, and TclN. We found that a 4-membered protein complex is formed consisting of TclI, TclJ, TclN, and TclE. Furthermore, the TclI protein functions as a central adaptor joining two other enzymes in the Tcl pathway with the substrate peptide.
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