Spelling suggestions: "subject:"1protein chemistry"" "subject:"2protein chemistry""
11 |
Protein complexes in the gas phase : structural insights from ion mobility-mass spectrometry and computational modellingHall, Zoe Lauren January 2013 (has links)
Structure determination of macromolecular protein assemblies remains a challenge for well-established experimental methods. In this thesis, an emerging structural technique, ion mobility-mass spectrometry (IM-MS) is explored. An assessment of collision cross section (CCS) measurement accuracy using travelling-wave IM (TWIMS) instrumentation was carried out (Chapter 3). Through the collation of a protein complex CCS database and the development of a calibration framework for TWIMS, significant improvements to CCS measurement accuracy have been achieved. Next, the advantages and limitations of using IM-MS to generate restraints for structure characterisation were explored. Computational tools designed to exploit IM-MS data for structural modelling were developed and tested on a training set of systems (Chapter 4). These include two heteromeric protein complexes, and an oligomeric intermediate involved in beta-2-microglobulin aggregation. Further structural information can be attained by using gas-phase dissociation techniques, such as collision-induced dissociation (CID). The effects of charge state on CCS and the gas-phase dissociation pathway of complexes were investigated (Chapter 5). This highlighted the possibility of using CID in conjunction with supercharging to manipulate dissociation pathways to achieve more useful structural information. Finally, the gas-phase structures of globular and intrinsically disordered protein complexes were probed by IM-MS and molecular dynamics (MD) simulations (Chapter 6). Experimental observations were recapitulated remarkably closely by simulations, including gas-phase structural collapse and the ejection of monomer subunits when the energy of the system was increased sufficiently. Overall, this research has contributed to the IM-MS field by providing the framework for improved CCS measurements of large protein complexes and the use of restraints from IM-MS for structural modelling. Significantly, IM-MS has been used in combination with charge manipulation, CID and MD simulations to reveal further insights into the gas-phase structures, stabilities and dissociation pathways of multimeric protein complexes.
|
12 |
Single-molecule studies of transcription initiationDuchi Llumigusin, Diego Armando January 2014 (has links)
Single-molecule Förster resonance energy transfer (smFRET) has emerged as an important tool for studying biological reactions. This thesis describes smFRET investigations into the mechanism of bacterial transcription initiation. We developed protocols to immobilize RNAP-DNA initiation complexes using vesicles and antibodies. We used these techniques to show that the transcription bubble conformation in immobilized complexes exhibits inter-molecular heterogeneity. We observed large FRET changes that we attribute to transcription bubble opening and closing dynamics. We found that σ<sup>70</sup> region 3.2 (σR3.2) influences the kinetics of the bubble dynamics, which supports proposals that σR3.2 interacts with the transcription bubble template strand. We extended our investigations to RNA synthesis and were able to observe abortive initiation cycles directly. We observed RNAP pausing and backtracking for the first time in transcription initiation. We obtained data suggesting that σR3.2 stabilises short RNAs at the active centre and forms a barrier to the extension of RNAs longer than 5-nt in length. We extended our abortive initiation assay to observe signal changes that we attribute to promoter escape. Our data revealed the number of abortive cycles that occur prior to escape, the kinetics of promoter escape, and pausing events that may have some regulatory function. We investigated the conformational dynamics of the RNAP β clamp and observed dynamic conformational changes between clamp-open and clamp-closed states. Our work confirms proposals that the clamp remains stably closed once the open complex (RPO) is formed. We investigated what affect the antibiotics Myxopyronin and Lipiarmycin have on the clamp conformation. Our results revealed that Myxopyronin traps the clamp in a closed conformation, while Lipiarmycin traps it in an open conformation. Overall, we made a number of novel observations that we believe advance our understanding of the mechanism of transcription. We hope that the discoveries reported here will direct future research efforts into RNAP function.
|
13 |
Modulation of the hypoxic response in cancer : inhibition of the HIF-1α/p300 protein-protein interactionJayatunga, Madura Kelum Perera January 2014 (has links)
Hypoxia inducible factor (HIF)-1α is a heterodimerically-activated transcription factor central to the cellular response to hypoxic environments and is often upregulated in cancer. Binding of HIF-1α to the co-activator p300 is necessary for the hypoxia-induced transcription of many oncogenic proteins. The aim of this project was to develop novel small molecule inhibitors of the HIF-1α/p300 protein-protein interaction (PPI). Initial work focused on designing, validating and optimising two high-throughput competition binding assays to screen for inhibitors of the PPI (Chapter 2). Alongside these, zinc ejector assays for both p300 and KDM4A proteins were developed to probe the mechanism of action and selectivity. Analysis of hits from a natural product high-throughput screen (HTS) revealed two compound classes; benzoquinones and 2-substituted indandiones, which modulate the PPI. The potency of these series correlated with the reactivity of the core functional groups, which act as electrophiles to covalently modify reactive cysteines, ejecting structural zinc and disrupting the p300/KDM4A protein fold (Chapter 3). Conjugating electrophilic groups to putative HIF-1α/p300 inhibitors did not replicate the activity of the zinc ejecting HTS hits (Chapter 4). Further work focused on non-covalent inhibitors of the HIF-1α/p300 interaction, first with peptide truncates, and then rationally designed α-helix peptidomimetics. An 11mer truncate showed encouraging activity (IC50 ≈ 70 μM), and corresponded to a key α-helix in the HIF-1α C-terminal transactivation domain. Three distinct double-sided scaffolds were used to imitate up to five hot-spot ampiphilic residues on this α-helix (Chapter 6 and 7). Of the 35 compounds screened, only modest inhibition was observed (IC50 ≈ 200-500 μM). Future work will look to conjugate electrophilic functionality onto the 11mer peptide in an attempt to gain potency from zinc ejection, while maintaining selectivity for p300.
|
14 |
Regulation of the MEK/ERK signaling cascade by ADAM12 in triple-negative breast cancer cellsHodge, Jacob G. January 1900 (has links)
Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program / Anna Zolkiewska / Mitogen-activated protein kinase (MAPK) signaling plays an important role in the proliferation, survival, and therapy resistance of breast cancer cells. Two important protein kinases involved in the MAPK pathway are MEK and ERK. The MEK/ERK signaling cascade can be stimulated by activation of the epidermal growth factor receptor (EGFR) upon binding of EGF-like ligands, which are released from cells by ADAM proteases. EGFR is frequently overexpressed in triple-negative breast cancer (TNBC), a particularly aggressive form of breast cancer. Our analysis of clinical data revealed that high expression of ADAM12, but not other ADAMs, in TNBC is associated with poor patient survival. Thus, we hypothesized that ADAM12 plays a critical role in the progression of TNBC, possibly by stimulating MEK/ERK activity in an EGFR-dependent manner. To test this hypothesis, ADAM12 was knocked-down (KD) in SUM159PT TNBC cells, which express high levels of the endogenous ADAM12 protein. An antibody array assay indicated a significant decrease in the activation of the MAPK pathway in SUM159PT cells after ADAM12 KD. The decrease in MAPK activity was further confirmed by Western blotting using phospho-MEK and phospho-ERK specific antibodies. Additionally, conditioned media from ADAM12-deficient SUM159PT cells failed to support the survival of MCF10A cells, suggesting that ADAM12 KD reduced the release of pro-survival growth factors from SUM159PT cells. Based upon this data, we propose that ADAM12 is a novel regulator of the MAPK pathway and a potential therapeutic target in breast cancer.
|
15 |
Kinetic and mechanistic studies of oxygen sensing Fe(II)/2-oxoglutarate dependent oxygenasesTarhonskaya, Hanna January 2014 (has links)
The Fe(II)/2-oxoglutarate (2OG) dependent oxygenases are a widespread enzyme family, which are characterised by structurally similar active sites and proposed to employ a common reaction mechanism. The work described in this thesis concerned kinetic and biophysical studies on 2OG oxygenases, with a particular focus on the hypoxia-inducible transcription factor (HIF) hydroxylases and mechanistic aspects of their reaction with oxygen. The four human HIF hydroxylases regulate cellular levels and transcriptional activity of HIF by catalysing its post-translational hydroxylation in response to changes in oxygen availability. The three prolyl hydroxylase domain enzymes (PHDs1-3) and factor inhibiting HIF (FIH) are proposed to act as cellular oxygen sensors and provide a direct link between oxygen availability and the hypoxic response. Previous transient kinetic studies have shown that PHD2 (the most important human PHD isoform) reacts slowly with oxygen, a factor proposed to be related to its oxygen-sensing role. The molecular mechanisms for the slow PHD2 reaction with oxygen were investigated using a range of kinetic and biophysical techniques to probe the effects of key active site substitutions. The studies reveal that a conservative substitution to an Fe(II)/H<sub>2</sub>O binding residue results in 5-fold faster reaction with oxygen, suggesting a role for H<sub>2</sub>O release from the active site in limiting the ability of oxygen to react with PHD2. This thesis also describes the first transient kinetic studies of FIH. The obtained results show that the rate of the FIH reaction with oxygen was significantly faster than for PHD2. Further, FIH catalyses hydroxylation not only of HIF-α, but also of proteins containing ankyrin repeat domains (ARD). The rate of the FIH reaction with oxygen was shown to be substrate dependent; faster oxygen activation of the reaction in the presence of ARD compared with HIF substrates was observed. Mechanistic studies were performed to investigate a report that PHD2 is involved in the enzymatic oxidation of an oncometabolite (R)-2-hydroxyglutarate (2HG) to give 2OG, in what would be an unprecedented reaction for a 2OG oxygenase. This work found that 2HG does not substitute for 2OG in PHD2 catalysis. Instead, the non-enzymatic transformation of 2HG to 2OG was observed, which could potentially contribute to the reported 2HG-dependent PHD activation in vivo. The biophysical and transient kinetic techniques used for studying the HIF hydroxylases were also applied to study the mechanism of deacetoxycephalosporin C synthase (DAOCS, the enzyme catalysing penicillin N ring expansion). Previously, it has been suggested that the DAOCS mechanism differs from the consensus 2OG oxygenase mechanism. The results described in this thesis provide strong evidence that DAOCS employs the consensus ordered mechanism characteristic of 2OG oxygenases, supporting the proposal that the consensus mechanism is a common feature of the 2OG oxygenase family. Overall, the work described in this thesis is supportive of the proposal that most, if not all, 2OG oxygenases employ a common mechanism. However, the differences in the kinetics of their reaction with oxygen, presented throughout the thesis, suggest that different 2OG oxygenases have different rate-limiting steps. Thus, the kinetics of specific oxygenases may be adapted to their biological function, in particular that of PHD2 as the key cellular O<sub>2</sub> sensor.
|
16 |
Structure and function of AMPK: subunit interactions of the AMPK heterotrimeric complexIseli, Tristan J. Unknown Date (has links) (PDF)
AMP-activated protein kinase (AMPK) is an important metabolic stress-sensing protein kinase responsible for regulating metabolism in response to changing energy demand and nutrient supply. Mammalian AMPK is a stable aß? heterotrimer comprising a catalytic a subunit and two non-catalytic subunits, ß and ?. The ß subunit targets AMPK to membranes via an N-terminal myristoyl group and to glycogen via a mid-molecule glycogen-binding domain. Here I show that the conserved C-terminal 85-residue sequence of the ß subunit, ß1(186-270), is sufficient to form an active AMP-dependent heterotrimer a1ß1(186-270)?1, whereas the 25-residue ß1 C-terminal (246-270) sequence is sufficient to bind ?1, ?2, or ?3 but not the a subunit. Within this sequence (246-270), two residues were essential for ß? association based on Ala scanning mutagenesis. / Substitution of ß1 Tyr-267 for Ala precludes ß? but not aß association suggesting independent binding requirements. Substitution of Tyr-267 for Phe or His but not Ala or Ser can rescue ß? binding. Substitution of Thr-263 for Ala also resulted in decreased ß? but not aß association. Truncation of the a subunit reveals that ß1 binding requires the a1(313-473) sequence while the remainder of the a C-terminus is required for ? binding. The conserved C-terminal 85-residue sequence of the ß subunit (90% between ß1 and ß2) is the primary a? binding sequence responsible for the formation of the AMPK aß? heterotrimer. The ? subunits contain four repeat CBS sequences with variable N-terminal extensions and the ?1 isoform is N-terminally acetylated. The ?2 subunit can be multiply phosphorylated by protein kinase C (PKC) in vitro, with Ser-32 identified as a minor site. A detailed understanding of the structure and regulation of AMPK will enable rational drug design for treatment of such linked diseases as obesity, insulin resistance and type 2 diabetes.
|
17 |
The Protein Binding Potential of C2H2 Zinc Finger DomainsBrayer, Kathryn Jo January 2008 (has links)
Cys2-His2 (C2H2) zinc finger domains were originally identified as DNA binding domains, and uncharacterized domains are typically assumed to bind DNA. However, a growing body of evidence suggests an important and widespread role for these domains in protein binding. Over 100 C2H2 zinc finger-protein interactions have been described. This study uses common bioinformatics tools to identify sequence features that predict a DNA- or protein-binding function. Several issues, including uncertainties about the full functional capabilities of the zinc fingers, complicated these efforts. Therefore, an unbiased approach which directly examined the potential for zinc fingers to facilitate DNA or protein interactions was used to determine the full functional capabilities of the C2H2 domains in two model proteins, human OLF-1/EBF associated zinc finger (OAZ) protein and Zif268. OAZ contains 30 zinc fingers in six clusters, some of which have been previously indicated in DNA or protein interactions. Zif268 is a well-known DNA binding protein with three C2H2 domains. DNA binding was assessed using a target site selection (CAST) assay, and protein binding was assessed using a yeast two-hybrid assay. Results indicate that clusters known to bind DNA could facilitate specific protein interactions, but clusters known to bind protein did not facilitate specific DNA interactions, indicating that DNA binding is a more restricted function of zinc fingers than has previously been recognized. These results also suggest that the role of C2H2 zinc finger domains in protein interactions has probably been underestimated. The implication of these findings for the prediction of zinc finger function is discussed.
|
18 |
Structure and function of AMPK: subunit interactions of the AMPK heterotrimeric complexIseli, Tristan J. Unknown Date (has links) (PDF)
AMP-activated protein kinase (AMPK) is an important metabolic stress-sensing protein kinase responsible for regulating metabolism in response to changing energy demand and nutrient supply. Mammalian AMPK is a stable aß? heterotrimer comprising a catalytic a subunit and two non-catalytic subunits, ß and ?. The ß subunit targets AMPK to membranes via an N-terminal myristoyl group and to glycogen via a mid-molecule glycogen-binding domain. Here I show that the conserved C-terminal 85-residue sequence of the ß subunit, ß1(186-270), is sufficient to form an active AMP-dependent heterotrimer a1ß1(186-270)?1, whereas the 25-residue ß1 C-terminal (246-270) sequence is sufficient to bind ?1, ?2, or ?3 but not the a subunit. Within this sequence (246-270), two residues were essential for ß? association based on Ala scanning mutagenesis. / Substitution of ß1 Tyr-267 for Ala precludes ß? but not aß association suggesting independent binding requirements. Substitution of Tyr-267 for Phe or His but not Ala or Ser can rescue ß? binding. Substitution of Thr-263 for Ala also resulted in decreased ß? but not aß association. Truncation of the a subunit reveals that ß1 binding requires the a1(313-473) sequence while the remainder of the a C-terminus is required for ? binding. The conserved C-terminal 85-residue sequence of the ß subunit (90% between ß1 and ß2) is the primary a? binding sequence responsible for the formation of the AMPK aß? heterotrimer. The ? subunits contain four repeat CBS sequences with variable N-terminal extensions and the ?1 isoform is N-terminally acetylated. The ?2 subunit can be multiply phosphorylated by protein kinase C (PKC) in vitro, with Ser-32 identified as a minor site. A detailed understanding of the structure and regulation of AMPK will enable rational drug design for treatment of such linked diseases as obesity, insulin resistance and type 2 diabetes.
|
19 |
Role of posttranslational modifications of histone proteins in epigeneticsRaj, Ritu January 2016 (has links)
Nature has evolved an additional level of genetic regulation by-passing direct changes in genetic code through the means of posttranslational modifications (PTMs) of nucleobases and histone proteins. Acetylation, methylation, phosphorylation, O-GlcNAcylation, ubiquitination, sumoylation, and ADP ribosylation are few common examples of various histone modifications. Identification of these modifications and subsequent access to homogeneously modified histone proteins are key for understanding the functional consequence of these PTMs. In this doctoral thesis, the role of PTMs of histone proteins in epigenetics was investigated with emphasis on understanding the role of O-GlcNAcylation in particular. In the second chapter, the functional consequence of O-GlcNAcylation at histone protein, H2B-Ser112 was explored. Homogeneously GlcNAcylated histones and nucleosomes were synthesized using protein chemical reactions. Mass Spectrometry (MS) based quantitative interaction proteomics revealed a direct interaction between GlcNAcylated nucleosomes and the Facilitates Chromatin Transcription (FACT) complex. Preferential binding of FACT to GlcNAcylated nucleosomes provides a molecular mechanism for FACT-driven transcriptional control. In the third chapter, the physical effect of O-GlcNAcylation on the nucleosome structure is described. Homogeneously GlcNAcylated histone protein, H2A-Thr101 was synthesized. The modified protein was used to reconstitute histone sub-complexes and nucleosomes. Various biophysical studies involving circular dichroism and native mass spectrometry revealed that H2A-T101 GlcNAcylation regulates the stability of the nucleosome structure, suggesting a role in transcriptional activation. In the fourth chapter, we discuss an interesting scenario where two PTMs - O-GlcNAcylation and phosphorylation - can compete for the same modification site of histone protein, H2B-Ser36. The resulting outcome is possibly a competitive antagonism or cross-talk, which can modulate the overall control of chromatin regulation. Using a "Tag-and-modify" approach, modified histone proteins bearing both modifications was synthesized, and was later used for nucleosome reconstitution. Quantitative interaction proteomics experiments with the modified nucleosome revealed key interacting protein partners for both the modifications.
|
20 |
Roles of ADAM12 in triple-negative breast cancer: regulation of cancer stem cellsQi, Yue January 1900 (has links)
Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Anna Zolkiewska / ADAM12 (A Disintegrin and Metalloprotease 12) is a cell surface protease, which is deregulated in many human diseases. High expression of ADAM12 in triple-negative breast cancers (lacking estrogen receptor, progesterone receptor, and HER2 expression) is associated with poor patient prognosis. My dissertation focused on the understanding of the biological functions of ADAM12 in triple-negative breast cancers. I found that ADAM12 is significantly upregulated in the claudin-low molecular subtype of breast cancer. Claudin-low tumors are typically triple-negative and are enriched in cancer stem cells. Here, I demonstrated that the loss of ADAM12 expression not only decreased the number of cancer stem-like cells in vitro but also significantly compromised the tumor-initiating capabilities of breast cancer cells in vivo. This is the first evidence showing that ADAM12 might regulate the cancer stem cell-like phenotype in triple-negative breast cancers. I also discovered a novel mechanism of ADAM12-regulated signaling by transforming growth factor β (TGFβ) through modulation of TGFBR1 mRNA expression in breast cancer cells. Lastly, I characterized the effects of six different somatic mutations in the ADAM12 gene found in human breast cancers on the intracellular trafficking, post-translational processing, and function of ADAM12 protein. Collectively, the findings of this study support the notion that ADAM12 with catalytically active metalloprotease domain is required for the progression of triple-negative breast cancers.
|
Page generated in 0.067 seconds