Дисертації з теми "Protein Biology"
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1
Robinson, Ross Alexander. "Structural biology of protein - protein interactions." Thesis, University of Oxford, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.504517.
2
Li, Wei. "Protein-protein interaction specificity of immunity proteins for DNase colicins." Thesis, University of East Anglia, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302033.
3
Song, Hong Chang. "The role of protein structure and heat shock protein 70 molecules in the import of peroxisomal proteins /." Thesis, McGill University, 1997. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=20867.
Анотація:
Almost all peroxisomal proteins, which play significant roles in peroxisome biogenesis, are synthesized in the cytosol and imported into peroxisomes post-translationally. Failure to assemble normal peroxisomes causes detrimental disorders of peroxisome biogenesis known as peroxisomopathies. In the present study we examined (1) the peroxisomal import competency of unfolded proteins, and (2) the essential amino acid sequence of peroxisomal targeting signal type I (PTS-1) peptide. In the first part, human serum albumin (HSA) with seventeen disulfide bonds was fully reduced at 37°C and partially reduced at the room temperature. Microinjection of HSA that was unfolded, biotinylated and cross-linked with PTS-1 peptides demonstrated colocalization with peroxisomes, indicating the peroxisomal import competency. Furthermore, they induced heat shock protein (hsp) 72 which is a part of hsp 70 molecules. In the second part of our studies, we examined the essential amino acid sequence of PTS-1 by observing the peroxisomal import competency of mutated PTS-1 peptides and their inducibility of hsp 70 molecules. The hydrophobicity and basicity of the last seven amino acids from the carboxy-terminal were shown to be essential in the peroxisomal import. Furthermore, all the mutated PTS-1 peptides induced hsp 72 response. These results suggest that the ability of hsp 70 molecules to interact with the last seven amino acid sequence of the PTS-1 peptide plays a crucial role in the peroxisomal import.
4
Laos, Roberto, and Steven A. Benner. "Linking chemistry and biology: protein sequences." Revista de Química, 2016. http://repositorio.pucp.edu.pe/index/handle/123456789/99314.
Анотація:
En los últimos veinte años el número de genomas completos que han sido secuenciados y depositados en bancos de datos ha crecido dramáticamente. Esta abundancia de información de secuencias ha servido de base para la creación de una disciplina llamada paleogenética. En este artículo, sin ahondar en algoritmos complejos, presentamos algunos conceptos clave para comprender cómo las proteínas han evolucionado con el tiempo. Luego ilustraremos como la paleogenética es utilizada en biotecnología. Estos ejemplos resaltan la conexión entre la química y la biología, dos disciplinas que quizás veinte años atrás parecían ser mucho más distintas que lo que parecen ser hoy.In the last twenty years, the number of complete genomes that have been sequenced and deposited in data banks has grown dramatically. This abundance in sequence information has supported the creation of the discipline known as paleogenetics. In this article, without going into complex algorithms, we present some key concepts for understanding how proteins have evolved in time. We then illustrate how paleogenetic analysis can be used in biotechnology. These examples highlight the connection between chemistry and biology, two disciplines that twenty years ago seemed to be more different than what they seem to be today.
5
Strasser, Rona. "Protein-protein interactions of receptors LdPEX5 and LPEX7 with PTS1 and PTS2 cargo proteins, and with glycosomal docking protein LdPEX14 for protein import into «Leishmania donovani»." Thesis, McGill University, 2014. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=122960.
Анотація:
A unique subcellular structure found in Leishmania donovani is the glycosome. This organelle compartmentalizes the enzymatic machinery required for multiple metabolic pathways, including glycolysis. Correct targeting of glycosomal enzymes is essential for parasite viability. Proteins targeted to the glycosome have either a C-terminal PTS1 or N-terminal PTS2 topogenic signal sequence, which is recognized by cytosolic receptors LdPEX5 or LPEX7, respectively. These cargo-loaded receptors interact with the peroxin protein LdPEX14, located on the cytosolic face of the glycosomal membrane, an event required for import of the cargo proteins into the glycosomal lumen. However, the complete glycosomal protein import pathway has not been fully elucidated. This work has been undertaken to better understand the protein-protein interactions involved in the trafficking of cargo proteins across the glycosomal membrane.The cytosolic fraction from L. donovani parasites was used to determine protein-protein interactions of receptors LdPEX5 and LPEX7. Size exclusion chromatography, isoelectric focusing, and affinity pull-downs showed that in the cytosol these receptors form large heterologous PTS1-LdPEX5-LPEX7-PTS2 complexes. Purified glycosomes were used to evaluate the effects of receptor-cargo complexes on glycosomal LdPEX14 conformation. Limited trypsin proteolysis showed that interaction of receptor-cargo complexes with native LdPEX14 protected this protein from digestion, whereas native LdPEX14 alone was highly sensitive to proteolysis. Protection was not dependent on membrane integrity as disruption of the lipid bilayer did not alter the effect of trypsin on these proteins. Native gel electrophoresis showed that native LdPEX14 forms large ~800 kDa complexes; however, when associated with receptor-cargo complexes the molecular weight of LdPEX14 complexes increased to ~1200 kDa. Alkaline carbonate extractions showed that native LdPEX14 acts like a peripheral glycosomal membrane protein; however loading with receptor-cargo complexes caused LdPEX14 to behave like an integral membrane protein. Furthermore, membrane insertion of LdPEX14 drove insertion of LdPEX5 and LPEX7 into the glycosomal membrane. Receptor-cargo complex association causes LdPEX14 to undergo a conformational change resulting in deeper membrane insertion and increase in complex size.Purification of recombinant LPEX7 was hampered by its association with bacterial chaperone protein GroEL. A refolding technique was developed to purify LPEX7 from inclusion bodies free of bacterial proteins. Far Western and protein-protein affinity assays showed that refolded LPEX7 specifically bound PTS2 proteins as both a monomer and a dimer, the co-receptor LdPEX5, and LdPEX14. Mapping of the interaction domains on LPEX7 showed that LPEX7-PTS2 interaction required the entire receptor protein, while LdPEX5 and LdPEX14 interaction motifs were situated in the N-terminal region of LPEX7.There are metabolites in glycosomes that are not imported via the peroxin based glycosomal import pathway but by glycosomal membrane transporters. L-arginine is one such metabolite; it is the substrate for the PTS1 glycosomal enzyme arginase, which catalyses the first step in the polyamine biosynthetic pathway. L-arginine is scavenged from the extracellular milieu and by the L-arginine transporter, amino acid permease 3 (LdAAP3). Subcellular fractionation showed that LdAAP3 localized to both the plasma and glycosomal membranes. Furthermore, L. donovani promastigotes were capable of sensing the L-arginine levels in the media and upregulated LdAAP3 expression on the plasma and glycosome membrane in the absence of L-arginine. These studies provide evidence that metabolite specific transporters are present on the glycosomal membrane.Together these studies contribute to the elucidation of glycosomal function in Leishmania donovani, and a better understanding of some of the mechanisms required for glycosomal import.Le glycosome est une structure subcellulaire unique qui se trouve dans le parasite Leishmania donovani. Cette organelle compartimente la machinerie enzymatique requise pour de multiples voies métaboliques, y compris la glycolyse. Le bon ciblage des enzymes du glycosome est essentiel pour la viabilité du parasite. Les protéines ciblées pour le glycosome ont une séquence signal topogénique, un PTS1 C-terminale ou un PTS2 N-terminale, qui est reconnue par les récepteurs cytosoliques, le LdPEX5 ou le LPEX7, respectivement. Ces complexes de récepteurs chargés s'interagissent avec la protéine LdPEX14, située du côté cytosolique de la membrane glycosomale, un événement requis pour le transport des protéines à travers la membrane du glycosome. Cependant, la voie complète d'importation de protéines glycosomales n'a pas été totalement élucidée. Ce travail a été entrepris pour mieux comprendre ces interactions protéine-protéine.La fraction cytosolique des parasites L.donovani a été utilisée pour déterminer les interactions protéine-protéine des récepteurs LdPEX5 et LPEX7. La chromatographie d'exclusion de taille, la focalisation isoélectrique, et les interactions d'affinité proteine-proteine ont montré que, dans les cytosols, ces récepteurs forment des grands complexes hétérologues. Les glycosomes purifiés ont été utilisés pour évaluer l'effet des complexes récepteur sur la conformation du LdPEX14. Une protéolyse limitée a montré que l'interaction du LdPEX14 chargé avec les complexes récepteur l'à protèger de la digestion à la surface de la membrane. L'électrophorèse sur gel natif a montré que le LdPEX14 forme des grands complexes de ~ 800 kDa et que lorsqu'il est associé à des complexes récepteur, le poids moléculaire des complexes LdPEX14 passe à ~ 1200 kDa. Les extractions avec le carbonate alcalin a déterminé que le LdPEX14 seul s'agit comme une protéine périphérique; mais son chargement avec des complexes récepteur l'entrainer à s'agir comme une protéine membranaire intégrale. L'insertion de LdPEX14 dans la membrane du glycosome conduit à l'insertion du LdPEX5 et LPEX7 dans la membrane aussi. L'association des complexes récepteur à causer LdPEX14 à subir un changement de conformation causant l'insertion profonde dans la membrane et l'augmentation de la taille des complexes.La purification du récepteur LPEX7 recombinante été entravée par son association avec la protéine chaperonne bactérienne GroEL. Une technique de repliement a été développé pour purifier LPEX7 en évitant l'association de protéines bactériennes. Les techniques de Far Western et d'affinité protéine-protéine ont montré que ce LPEX7 replier est spécifiquement associé à des protéines PTS2, le co-récepteur LdPEX5, et le LdPEX14. La cartographie des domaines d'interaction de LPEX7 a montré que l'interaction LPEX7-PTS2 nécessit le LPEX7 entière, alors que les motifs d'interaction avec LdPEX5 et LdPEX14 étaient situés dans sa région N-terminale.Il y a des métabolites glycosomal qui ne sont pas importés par la voie de l'importation glycosomale, mais par des transporteurs membranaires du glycosome. L-arginine est un de ces métabolites, substrat de l'enzyme glycosomale PTS1 arginase. L-arginine est récupéré dans le milieu extracellulaire par son transporteur, LdAAP3. Un fractionnement subcellulaire a été utilisés pour séparer les membranes plasmiques des glycosomes, et LdAAP3 a été localisé sur les deux membranes. De plus, des promastigotes de L. donovani sont capable de detecter le niveau de L-arginine dans le millieu, ce qui provoque une régulation positive de l'expression de LdAAP3 à la fois dans la membrane plasmique et dans la membrane du glycosome. Ces études fournissent des preuves que des transporteurs de métabolites spécifique sont présent dans la membrane du glycosome.Ensemble, ces études contribuent à l'élucidation de la fonction glycosomale de Leishmania donovani, et une meilleure compréhension de certains mécanismes nécessaires pour l'importation glycosomale.
6
Le, Min. "Protein coimmobilization: Reactions of vicinal thiol groups of proteins /." The Ohio State University, 1997. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487946776021788.
7
Rassadi, Roozbeh. "The effect of stress on nuclear protein transport : classical nuclear protein transport versus the nuclear transport of heat shock proteins." Thesis, McGill University, 1999. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=33476.
Анотація:
The stress response is conserved among eukaryotes and affects different cellular functions including protein transport. Here, we have investigated the effect of different types of stress on classical nuclear protein import as well as nuclear import of Ssa4p family of heat shock proteins in Saccharomyces cerevisiae.Under normal conditions, Aequorea victoria green fluorescent protein (GFP), carrying a classical nuclear localization sequence (cNLS-GFP) is nuclear. However, cNLS-GFP equilibrates throughout the cell upon exposure to heat, ethanol, H2O2 or starvation. Redistribution of the small GTPase Gsp1p, a soluble nuclear transport factor, correlates with cNLS-GFP equilibration. This suggests that a collapse of the Gsp1p gradient underlies the inhibition of classical nuclear protein import. In contrast to cNLS-GFP, the cytoplasmic heat shock protein Ssa4p accumulates in nuclei when classical nuclear import is inhibited. The N-terminal 236 amino acid residues of Ssa4p are sufficient for nuclear localization of Ssa4p-GFP upon heat and ethanol stress. The nuclear localization of Ssa4p(1--236)-GFP requires components of Gsp1-GTPase system, but is independent of Srp1p, the cNLS receptor.
Ssa4p(16--642)-GFP accumulates in nuclei of starving cells, mediated by a hydrophobic stretch of amino acid residues in its N-terminal domain. This nuclear localization is reversible upon addition of fresh medium and its export is sensitive to oxidants and temperature-dependent.
8
Field, James Edward John. "Engineering protein cages with synthetic biology." Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/45404.
Анотація:
Nanotechnology has the potential to revolutionise every facet of human life. One particularly exciting branch of nanotechnology involves the construction of nanodevices using protein cages. Protein cages are spherically shaped structures with large internal cavities. The research described in this thesis was conducted with the aim of rationalising the design and fabrication of protein cage-based nanodevices. Protein-based nanodevices are typically constructed by re-engineering naturally occurring protein chassis (e.g. ferritin). To rationalise the process of chassis selection, an online registry of protein cages, rings and tubes was designed and populated by computationally mining the Protein Data Bank. The resulting registry was made publically available to the research community through the website – www.nanodevice.build. The functionality of protein cage-based nanodevices can be augmented by packaging inorganic nanoparticles inside their internal cavities. The methods currently used to achieve this typically involve exposure to harsh conditions, which can cause irreversible damage to the protein cage. To address this, a strategy for efficiently packaging inorganic nanoparticles into protein cages under mild conditions was formulated and tested. These experiments were conducted using gold nanoparticles and a number of different protein cages (e.g. Bfr, FtnH and FtnL). Cholangiocarcinoma (CCA) is a deadly liver cancer for which current treatment options are limited. Therefore a CCA-targeting protein cage-based nanodevice was designed, constructed and experimentally evaluated. CCA-targeting was achieved in the context of the CCA cell line TFK-1 using an anti-mesothelin antibody as a targeting agent. Collectively, these three outputs provide a rational framework for selecting a protein cage chassis, loading it with a pre-fabricated inorganic nanoparticle and targeting the resulting device to a particular cell-type. It is hoped that by leveraging these three tools, synthetic biologists will be able to engineer a new generation of nanodevices.
9
Sonnen, Andreas Franz-Peter. "Structural biology of protein-membrane interactions and membrane protein function." Thesis, University of Oxford, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.514997.
10
Lite, Thúy-Lan Võ. "The genetic landscape of protein-protein interaction specificity." Thesis, Massachusetts Institute of Technology, 2020. https://hdl.handle.net/1721.1/129035.
Анотація:
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2020Cataloged from student-submitted PDF of thesis.
Includes bibliographical references.
Protein-protein interaction specificity is often encoded at the primary sequence level, and by just a few interfacial residues. Collectively, these residues have both positive and negative roles, promoting a desired, cognate interaction and preventing non-cognate interactions, respectively. However, for most protein-protein interactions, the contributions of individual specificity residues are poorly understood and often obscured by robustness and degeneracy of protein interfaces. Using bacterial toxin-antitoxin systems as a model, we use a variant of deep mutational scanning to dissect the positive and negative contributions of antitoxin residues that dictate toxin specificity. By screening a combinatorially complete library of antitoxin variants, we uncover a distribution of fitness effects for individual interface mutations measured across hundreds of genetic backgrounds. We show that positive and negative contributions to specificity are neither inherently coupled nor mutually exclusive. Further, we argue that the wild-type antitoxin may be optimized for specificity, because mutations that further destabilize the non-cognate interaction also weaken the cognate interaction. No mutations strengthen the cognate interaction. By comparing crystal structures of paralogous complexes, we provide a structural rationale for all of these observations. Finally, we use a library approach to identify hundreds of novel systems that are insulated from their parental systems, and that carry only two mutations - a negative specificity element on the toxin, and one on the antitoxin. This result demonstrates that highly similar (and in this case, nearly identical) complexes can be insulated using compensatory mutations of individually large effect. Collectively, this work provides a generalizable approach to understanding the logic of molecular recognition.
by Thúy-Lan Võ Lite.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biology
11
Batal, Rami. "RNA and protein interactions of the measles virus nucleocapsid protein." Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=55437.
Анотація:
We are interested in studying the RNA and protein binding activities of the measles virus (MV) NP. MV is one of the members of Paramyxoviridae, a family of non-segmented negative-stranded RNA viruses family. We have expressed the MV NP in procaryotic systems and by in vitro translation. We have created a number of carboxy-terminal deletions of NP to use in mapping the domains involved in RNA and protein binding. We have transcribed the 5$ sp prime$ end antigenome sequences (positive leader) in vitro. We have metabolically labeled viral and MV-infected cellular proteins. We have applied different RNA-protein and protein-protein binding assays in order to study the postulated binding. We were not able to unequivocally detect specific binding between NP and the RNA. However, we have observed significant binding between NP and each of three MV-specific proteins NP, P, and M. Furthermore, we have found that the carboxy terminus of NP is important in this binding. Deletions in that domain will abolish M binding, and further deletions towards the amino terminus will abolish P binding, and eventually NP binding. Successively larger carboxy-terminal deletions first abolish NP binding to M, then to P, and then eventually to itself.
12
Papadopoulos, Maria. "The prion protein interacts with Bcl-2 and Bax proteins." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0026/MQ50849.pdf.
13
McArdle, Bernadette. "Natural Product Interactions with Biology Space." Thesis, Griffith University, 2007. http://hdl.handle.net/10072/366724.
Анотація:
Natural products have withstood the test ot time as therapeutics but new lead generation strategies have focused away from natural products. This study reports a new approach that uses natural product recognition to drive an understanding of biology space which may provide an impetus for renewed focus on natural product starting points.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Eskitis Institute for Cell and Molecular Therapies
Science, Environment, Engineering and Technology
Full Text
14
Nobrega, Robert P. "Early Folding Biases in the Folding Free-Energy Surface of βα-Repeat Proteins: A Dissertation". eScholarship@UMMS, 2014. https://escholarship.umassmed.edu/gsbs_diss/723.
Анотація:
Early events in folding can determine if a protein is going to fold, misfold, or aggregate. Understanding these deterministic events is paramount for de novo protein engineering, the enhancement of biopharmaceutical stabilities, and understanding neurodegenerative diseases including amyotrophic lateral sclerosis and Alzheimer's disease. However, the physicochemical and structural biases within high energy states of protein biopolymers are poorly understood.
A combined experimental and computational study was conducted on the small β/α-repeat protein CheY to determine the structural basis of its submillisecond misfolding reaction to an off-pathway intermediate. Using permutations, we were able to discriminate between the roles of two proposed mechanisms of folding; a nucleation condensation model, and a hydrophobic collapse model driven by the formation of clusters of isoleucine, leucine, and valine (ILV) residues. We found that by altering the ILV cluster connectivity we could bias the early folding events to either favor on or off-pathway intermediates.
Structural biases were also experimentally observed in the unfolded state of a de novo designed synthetic β/α-repeat protein, Di-III_14. Although thermodynamically and kinetically 2-state, Di-III_14 has a well structured unfolded state that is only observable under native-favoring conditions. This unfolded state appears to retain native-like structure, consisting of a hydrophobic 7 core (69% ILV) stabilized by solvent exposed polar groups and long range electrostatic interactions.
Together, these results suggest that early folding events are largely deterministic in these two systems. Generally, low contact order ILV clusters favor local compaction and, in specific cases, long range electrostatic interactions may have stabilizing effects in higher energy states.
15
Nobrega, Robert P. "Early Folding Biases in the Folding Free-Energy Surface of βα-Repeat Proteins: A Dissertation". eScholarship@UMMS, 2007. http://escholarship.umassmed.edu/gsbs_diss/723.
Анотація:
Early events in folding can determine if a protein is going to fold, misfold, or aggregate. Understanding these deterministic events is paramount for de novo protein engineering, the enhancement of biopharmaceutical stabilities, and understanding neurodegenerative diseases including amyotrophic lateral sclerosis and Alzheimer's disease. However, the physicochemical and structural biases within high energy states of protein biopolymers are poorly understood.
A combined experimental and computational study was conducted on the small β/α-repeat protein CheY to determine the structural basis of its submillisecond misfolding reaction to an off-pathway intermediate. Using permutations, we were able to discriminate between the roles of two proposed mechanisms of folding; a nucleation condensation model, and a hydrophobic collapse model driven by the formation of clusters of isoleucine, leucine, and valine (ILV) residues. We found that by altering the ILV cluster connectivity we could bias the early folding events to either favor on or off-pathway intermediates.
Structural biases were also experimentally observed in the unfolded state of a de novo designed synthetic β/α-repeat protein, Di-III_14. Although thermodynamically and kinetically 2-state, Di-III_14 has a well structured unfolded state that is only observable under native-favoring conditions. This unfolded state appears to retain native-like structure, consisting of a hydrophobic 7 core (69% ILV) stabilized by solvent exposed polar groups and long range electrostatic interactions.
Together, these results suggest that early folding events are largely deterministic in these two systems. Generally, low contact order ILV clusters favor local compaction and, in specific cases, long range electrostatic interactions may have stabilizing effects in higher energy states.
16
McElroy, Kyle A. "Balancing transcriptional activity in Drosophila through protein-protein interactions on chromatin." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493492.
Анотація:
Chromatin plays a vital role in the implementation of gene expression programs. Several disparate groups of regulatory proteins alter chromatin state through post-translational modification of histone proteins, nucleosome remodeling, and higher order chromatin structure in order to affect gene expression. Several of these key groups, such as the Male-Specific Lethal complex and Polycomb Group have been well characterized in Drosophila. Yet aspects of their biology at the molecular level, such as the means by which they are faithfully targeted to regulated loci throughout the genome and the molecular mechanisms they employ to alter transcriptional state, still remain unexplained. In this dissertation I explore how identifying protein-protein interactions on chromatin reveals insights into these unanswered questions critical to chromatin biology. My results highlight the importance of balancing active and repressive chromatin states for the proper maintenance of gene expression.
The Male-Specific Lethal complex is the dosage compensation complex in Drosophila, which upregulates gene expression on the male X chromosome approximately two-fold. The MSL complex catalyzes an acetyl mark which may create a uniquely permissive chromatin state to promote transcriptional elongation. A proteomic screen for MSL-interacting proteins identified UpSET, the Drosophila homolog of yeast SET3 and mammalian MLL5. Interestingly, SET3 and UpSET have been characterized to assemble into histone deacetylase complexes. I employed genetic, genomic, and proteomic techniques to assess whether UpSET plays a role in dosage compensation. UpSET appears to play a role in limiting the level of activation of the MSL complex. Surprisingly, UpSET appears to play a more important role in the maintenance of heterochromatin.
The Polycomb Group is comprised of a well characterized set of developmental repressors. The PcG assembles into several multiprotein complexes to maintain the repressed state. The PcG is opposed by a group of activators known as the Trithorax group. Although the PcG and TrxG often appear to be recruited to the same genomic elements in different tissues, whether they might interact directly was not known. In a collaboration with Dr. Hyuckjoon Kang, I characterized the TrxG protein Female sterile (1) homeotic and found that it interacts specifically with PRC1. The data support a model that bivalency, a poised state observed in mammalian stem cells, may be critical, perhaps transiently, in the developing Drosophila embryo. The mechanism of coordination amongst the various PcG complexes on chromatin is not well understood. We also identified the Sex comb on midleg protein, a known member of the PcG, as a potential physical bridge between PRC1 and PRC2.
In these sets of experiments, I have characterized instances of crosstalk between activating and repressing regulators which are critical for the proper maintenance of chromatin state. Perturbations of these interactions may lead to an imbalance of regulators on chromatin and aberrant transcriptional activity. These findings highlight the need for tuning gene expression state and suggest chromatin-based mechanisms by which this can be accomplished.Biology, Molecular and Cellular
17
Diks, Sander Henricus. "Analysis of protein superhighways in cell biology." [S.l. : [Groningen : s.n.] ; University Library Groningen] [Host], 2006. http://irs.ub.rug.nl/ppn/298197421.
18
Larocque, Gabrielle. "Cell biology of tumor protein D54 (TPD54)." Thesis, University of Warwick, 2017. http://wrap.warwick.ac.uk/107000/.
Анотація:
The expression of Tumor protein D52 (TPD52) family members is deregulated in many types of cancer. When overexpressed, it is suggested that they increase cell proliferation and migration/invasion as well as avoid apoptosis. Deregulation in the expression of the TPDs is therefore linked to poor prognosis. Little characterisation has been carried out to date, but it is known that the TPDs are found in association with components of the membrane trafficking pathway. The aim of this work is to uncover how the least studied member of the family, TPD54, affects cellular processes involved in carcinogenesis, such as cell migration and invasion. By using the knocksideways method, we have been able to map the cellular localisation of TPD54 and have identified association partners. These associations have been confirmed by immunoprecipitation and mass spectrometry analysis. Amongst these was the small GTPase Rab14. We have also found that TPD54 is involved in the trafficking of receptors containing a dileucine motif in their cytosolic tail, but not a tyrosine-based or NPXY motif. With the mapping of the localisation of TPD54, we hypothesise that TPD54 is on the recycling route following the Golgi apparatus, and in association with Rab14, regulates the trafficking of receptors containing a dileucine motif. Integrins are receptors controlling cell migration. They can be trafficked through the Golgi apparatus before being recycled back to the plasma membrane. This recycling route is not well characterised. We therefore hypothesise that TPD54 regulates this route with Rab14, and that this is the reason why TPD54 is important for cell migration, and that a defect in its function can cause cancer.
19
Joachimiak, Lukasz A. "In silico evolution of protein-protein interactions : from altered specificities to de novo complexes /." Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/9211.
20
Kane, Émilie. "Protein-protein regulation of calsequestrin expression in cardiomyocytes." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=107782.
Анотація:
Heart failure is the leading cause of death in both men and women of Western countries. The pathophysiology of heart failure is associated with abnormalities in intracellular calcium control. Calsequestrin (CSQ2), a calcium storage protein in cardiomyocytes, is negatively regulated by the transcription factor Egr-1 thus altering calcium availability for cardiac contraction/relaxation. Here, we tested the hypothesis that the proteins complexed to Egr-1 and/or their post-translational modifications would affect regulation of CSQ2 expression. Egr-1 and Sp1 compete for binding at the CSQ2 promoter, but also bind one another. In fact, together they form a complex with another ubiquitous transcription factor YBX-1. This complex was identified in vivo and in vitro by a series of co-immunoprecipitations. To test the idea that complex formation and CSQ2 expression could be affected by acetylation, histone acetyltransferase (HAT) inhibitors and histone deacetylase (HDAC) inhibitors were used to respectively decrease or increase acetylation within cells. We found that acetylation did not impact the formation of the Egr-1: Sp1: YBX-1 complex thought to regulate CSQ2 expression. However, changes in CSQ2 expression were observed when acetylation was modified by HAT and HDAC inhibitors. We conclude that acetylation modifies CSQ2 expression although not by means of the Egr-1: Sp1: YBX-1 complex even though Egr-1 is known to be acetylated.La maladie du coeur est la cause principale de mortalité chez les femmes et les hommes en Occident. L'arrêt cardiac est souvent associé à des anomalies en control de calcium intracellulaire. Le facteur de transcription Egr-1 est connu pour son contrôle négatif de l'expression de calsequestrine (CSQ2), une protéine réservoir de calcium. Un manque de CSQ2 impacte les quantités de calcium disponible pour un bon fonctionnement cardiaque. Ici nous avons examiné l'hypothèse que les protéines liées à Egr-1 et que ses modifications post-translationelles. Egr-1 et Sp1 sont en compétition pour le promoteur de CSQ2 mais sont aussi liés l'un à l'autre. En fait, ensemble ils forment un complexe avec le facteur de transcription universel YBX-1. Ce complexe a été identifié en vivo et en vitro par co-immunoprécipitations. Considérant que la formation de ce complexe ainsi que l'expression de CSQ2 peuvent être affectés par l'acétylation, des inhibiteurs d'acétyltransferase d'histone (ATH) et de déacétylase d'histone (DACH) ont été respectivement utilisés pour réduire et augmenter l'acétylation en cellules. Nous avons trouvés que la formation du complexe Egr-1: Sp1: YBX-1 qui est possiblement responsable de l'expression de CSQ2 n'est pas affecté par les changements en acétylation. Par contre, des changements ont été aperçus dans l'expression de CSQ2 quand l'acétylation a été modifiée par ATH ou par DACH. Nous croyons que l'acétylation impacte l'expression de CSQ2 mais pas par entremise du complexe Egr-1 : Sp1 : YBX-1 malgré l'acétylation de la protéine Egr-1 elle-même.
21
Louie, Brenton E. "Modeling uncertainty in data integration for improving protein function assignment /." Thesis, Connect to this title online; UW restricted, 2008. http://hdl.handle.net/1773/7154.
22
Feng, Bochen. "Specific DNA-Protein and Protein-Protein interactions determine the operation of the Nitrogen regulatory circuit of Neurospora Crassa /." The Ohio State University, 2000. http://rave.ohiolink.edu/etdc/view?acc_num=osu1488192447431226.
23
Grigoryan, Gevorg Ph D. Massachusetts Institute of Technology. "Computational approaches for the design and prediction of protein-protein interactions." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/38997.
Анотація:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2007.Includes bibliographical references (leaves 167-187).
There is a large class of applications in computational structural biology for which atomic-level representation is crucial for understanding the underlying biological phenomena, yet explicit atomic-level modeling is computationally prohibitive. Computational protein design, homology modeling, protein interaction prediction, docking and structure recognition are among these applications. Models that are commonly applied to these problems combine atomic-level representation with assumptions and approximations that make them computationally feasible. In this thesis I focus on several aspects of this type of modeling, analyze its limitations, propose improvements and explore applications to the design and prediction of protein-protein interactions.
by Gevorg Grigoryan.
Ph.D.
24
Chen, Huiling Zhou Huan Xiang Ferrone Frank A. "Prediction of protein structures and protein-protein interactions : a bioinformatics approach /." Philadelphia, Pa. : Drexel University, 2005. http://dspace.library.drexel.edu/handle/1860/481.
25
Chan, Man Kid. "The interaction between Y box binding protein 1 and DNA replication proteins." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=66802.
Анотація:
A coordinated response to DNA damage is vital to maintain cellular viability and prevent the onset of disease. In mammalian cells, the intra-S phase checkpoint regulator, ATR (Ataxia-telangiectasia mutated and RAD3-related) kinase coordinates the response to DNA damage to ensure the genome is accurately and completely replicated before the cell enters mitosis. Y box Binding Protein 1 (YB-1), a transcription and translation factor, has previously been implicated in cell proliferation and the development of chemotherapeutic resistance. YB-1 has been linked to a wide variety of cellular stresses, but has not been studied in the context of DNA replication. In this study, we determined that YB-1 associates to both the β-globin replication origin (origin-containing) and the control (origin-lacking) DNA regions. This observation suggested that YB-1 may be involved in DNA replication elongation instead of initiation. By immunoprecipiating YB-1, we identified that PCNA and MCM7 preferentially interact with YB-1 during S phase. The examination of the spatial and temporal dynamics of these interactions by immunofluorescence microscopy, however, did not reveal nuclear colocalization of these proteins. By treating cells with hydroxyurea to stall the replication fork, we re-examined the protein-protein interaction between YB-1 and MCM7 and found that following 8 hours of hydroxyurea treatment, YB-1 and MCM7 exhibited diffuse colocalization in the cell nucleus. This finding may implicate YB-1 in exerting a late-onset response to prolonged replication fork arrest either directly at stalled replication forks or at "dormant" origins bound by MCM complexes. A number of roles for YB-1 can be postulated, such as the requirement of YB-1 in facilitating the resumption of DNA replication, or the activation of additional origins to duplicate the genome in the presence of a replication stress. This finding may in turn account foUne réponse coordonnée lors de dommages à l'ADN est vitale pour le maintien de la viabilité cellulaire et pour éviter l'installation de maladies. Dans les cellules de mammifères, le point de contrôle de la phase S, la protéine kinase ATR (Ataxia-telangiectasia mutated and RAD3-related), coordonne la réponse aux dommages de l'ADN afin d'assurer une réplication complète et fidèle du génome avant l'entrée en mitose. La protéine YB-1 (Y box Binding Protein 1), un facteur de transcription et de traduction, est impliqué dans la prolifération cellulaire et la résistance aux chimiothérapies. YB-1 est également lié à une large variété de stress cellulaires but aucune donnée n'est disponible quant à son rôle dans la réplication de l'ADN. Lors de mon travail de Master, j'ai pu montrer qu'YB-1 s'associe à la fois à l'origine de réplication de la β-globine et dans les régions contrôles de l'ADN. Ce résultat suggère qu'YB-1 pourrait être impliqué dans la phase d'élongation de la réplication de l'ADN plutôt que dans celui de l'initiation. Par immunoprécipitation, j'ai identifié PCNA et MCM7 comme interacteurs préférentiels d'YB-1 en phase S. Par contre, en immunofluorescence, je n'observe pas de colocalisation nucléaire entre ces protéines. Lors du blocage de la fourche de réplication par un traitement à l'hydroxyurée, l'interaction entre YB-1 et MCM7 a été réexaminée et j'ai mis en évidence que 8h après le traitement, ces deux protéines présentent une co-localisation diffuse dans le noyau. Ces données indiquent qu'YB-1 pourrait être impliqué dans une réponse tardive suite à un arrêt prolongé de la fourche de réplication soit directement au point d'arrêt soit au niveau d'origines « dormantes » liées aux complexes MCM. YB-1 peut donc avoir plusieurs rôles tels que l'aide à la reprise de la réplication de l'ADN ou l'activation d'origines de réplicatio
26
Ler, Lian Wee. "Identification and characterization of novel mammalian eIF4E-Homologous Protein (4EHP) interacting proteins." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=66760.
Анотація:
Translation of an mRNA begins with the recruitment of the eIF4F complex to the 5' cap of the mRNA and is completed upon start codon recognition by the preinitiation complex. Regulation of translation initiation is a major mechanism for the control of gene expression. The focus of this thesis is human 4EHP (h4EHP), a homolog of the cap-binding translation initiation factor eIF4E. 4EHP can bind to the cap structure but cannot initiate cap-dependent translation. Drosophila 4EHP functions as a translational repressor of specific mRNAs by forming a "closed loop", in which the binding partners of 4EHP determine the mRNA specificity. This study sought to investigate the function of h4EHP by identifying its interacting proteins. In total, three novel binding proteins of h4EHP have been identified: PERQ1, PERQ2, and eIF4E-Transporter (4E-T). All these proteins utilize a similar motif for h4EHP-binding. Experiments demonstrate that h4EHP:PERQ1/2 complexes are involved in translational repression. In addition, this study shows that the protein levels of PERQ1, PERQ2 and h4EHP are co-regulated. Among them, PERQ2 undergoes CUL7-mediated degradation upon h4EHP depletion. 4E-T has been shown to be involved in translational repression, eIF4E nuclear import, P-body formation and mRNA turnover. This study demonstrates that h4EHP can be localized to the nucleus. However, its nuclear localization is not affected by 4E-T depletion. The depletion of h4EHP results in a six-fold increase in the number of P-bodies but has no effect on the rate of turnover of a reporter mRNA. In conclusion, this study identifies PERQ1 and PERQ2 as two translational repressors; their function and stability are dependent on h4EHP. The function of h4EHP:4E-T interaction, however, remains elusive.La traduction d'un ARNm commence par le recrutement du complexe eIF4F au niveau de la coiffe (ou cap) en 5' de l'ARNm et se complète lors de la reconnaissance du codon d'initiation par le complexe de pré-initiation. La régulation de l'initiation de la traduction est une étape majeure dans le contrôle de l'expression génique. Cette thèse s'intéresse à la protéine humaine 4EHP (h4EHP), un homologue du facteur d'initiation de la traduction se liant à la coiffe, eIF4E. 4EHP peut d'ailleurs s'associer à la coiffe de l'ARNm mais ne peut initier la traduction dépendante de la coiffe. L'homologue chez la drosophile, d4EHP, est un inhibiteur de la traduction de certain ARNm et agit en formant une boucle 5'-3' avec l'ARNm. Les partenaires protéiques de 4EHP définissent alors la spécificité pour l'ARNm. Cette thèse s'intéresse à la fonction de h4EHP en recherchant ses partenaires protéiques. Les trois nouveaux partenaires identifiés (PERQ1, PERQ2 et 4E-T) utilisent un même motif de liaison à h4EHP. Mes expériences démontrent non seulement, que les complexes h4EHP:PERQ1/2 sont impliqués dans la répression de la traduction, mais aussi qu'il existe une co-régulation des niveaux relatifs des protéines h4EHP, PERQ1 et PERQ2. Ce dernier est d'ailleurs dégradé par CUL7 après la déplétion de h4EHP. La protéine 4E-T est connu pour son rôle dans l'inhibition de la traduction, l'import nucléaire d'eIF4E, la formation des « P-Bodies » et le renouvellement des ARNm. Cette étude démontre que h4EHP a une localisation nucléaire et indépendante de 4E-T. La suppression de 4E-T augmente cependant de six fois le nombre de « P-Bodies », sans pour autant affecter le taux de renouvellement d'un ARNm reporteur. En conclusion, cette étude identifie PERQ1 et PERQ2 comme deux inhibiteurs de la traduction dont la fonction et la stabilité sont dépendantes de h4EHP. Cependant, le rôle de l'inter
27
Liu, Huanting. "Molecular biology of maize streak virus movement in maize." Thesis, University of East Anglia, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.361478.
28
Peterson, Mark Erik. "Evolutionary constraints on the structural similarity of proteins and applications to comparative protein structure modeling." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3339202.
29
Gilker, Eva Adeline Gilker. "INTERACTIONS AND LOCALIZATION OF PROTEIN PHOSPHATASES, YWHA PROTEINS AND CELL CYCLE CONTROL PROTEINS IN MEIOSIS." Kent State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=kent1532699317257539.
30
Delacour, Quentin. "Light-induced protein degradation : a chemical biology approach." Thesis, Paris 6, 2015. http://www.theses.fr/2015PA066347/document.
Анотація:
La régulation de la protéolyse est un outil efficace pour le contrôle de la fonction d'une protéine dans des cellules. Nous présentons dans ce travail une stratégie générique permettant d'activer la protéolyse de façon conditionnelle par la lumière, améliorant ainsi la résolution spatio-temporelle. Notre approche repose sur un système de dégradation inductible par l'auxine (AID), mis au point en transposant des composants de la voie de dégradation contrôlée par l'auxine existant chez les plantes dans des cellules de mammifères. Nous présentons une version optimisée du système AID qui a permis de diminuer de façon significative la stabilité de protéines cibles en présence d'auxine. Nous avons en parallèle développé un déclencheur de dégradation photo-activable sous la forme d'une auxine cagée. Une illumination courte et locale permet la libération efficace de l'auxine dans les cellules et induit la dégradation de protéine d'intérêt avec un bon contrôle spatiotemporel. Cette méthode générique a été utilisée dans des contextes nucléaires et cytoplasmiquesThe regulation of proteolysis is an efficient way to control protein function in cells. Here, we present a general strategy enabling to increase the spatiotemporal resolution of conditional proteolysis by using light activation as trigger. Our approach relies on the auxin-inducible degradation (AID) system obtained by transposing components of the plant auxin-dependent degradation pathway in mammalian cells. We developed an optimized version of the AID which enables to significantly destabilize target proteins in presence of auxin. Parallely, we developed a photoactivatable auxin that acts as a photoactivatable inducer of degradation. Upon local and short light illumination, auxin is released in cells and triggers the degradation of a protein of interest with spatiotemporal control. This generic method was implemented in nuclear and cytoplasmic contexts
31
Tan, C. H. "The cell biology of the mitochondrial protein IF1." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1302546/.
Анотація:
Mitochondria are important organelles that regulate processes such as energy homeostasis and apoptosis. Dysregulation of mitochondrial function contributes to pathophysiology of diseases including ischaemia-reperfusion injury and cancer. When mitochondrial respiration is disturbed during oxygen deprivation, the mitochondrial ATP synthase runs ‘backwards’ as an ATPase, consuming ATP whilst maintaining mitochondrial membrane potential. The depletion of ATP can drive cell death. In this thesis, I have studied the roles of the endogenous inhibitor of ATPase, IF1, in regulating progression and outcome of cellular injury in models of necrosis and apoptosis. I first studied localisation and expression profiles of IF1 in HeLa and HL-1 cells. IF1 localised exclusively to mitochondria in both cell types. In HL-1 cells, IF1 expression profile changed dramatically with confluency, correlating with changes in cell differentiation. IF1 overexpression increased viability of HL-1 cardiomyocytes following oxygen and glucose deprivation, a model of ischaemia-reperfusion. A pivotal step in triggering apoptosis is the release of cytochrome c from mitochondrial intracristal space, activating cytosolic enzymatic cascades which cause cell death. It has been suggested that cristae remodelling facilitates cytochrome c release. As IF1 increases cristae density, I studied the impact of IF1 overexpression on cytochrome c release following exposure of HeLa cells to staurosporine. IF1 upregulation delayed cytochrome c release, possibly as a result of altered cristae structure. It has been suggested that cytochrome c release increases calcium release from endoplasmic reticulum (ER), promoting a positive feedback system that accelerates complete cytochrome c release. I found that cytochrome c release correlated with ER calcium efflux, depletion of ER calcium and with mitochondrial depolarisation, mediated by permeability transition pore opening. These processes were reduced following IF1 overexpression, suggesting that IF1 plays a significant role in progression of apoptosis. Thus, IF1 modulates progression and outcome of cell injury and death through necrotic and apoptotic pathways.
32
Forghani, Farnaz. "Protein engineering of bacteriophage Mu transposase." Thesis, McGill University, 1990. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=60444.
Анотація:
Bacteriophage Mu is an ideal system to study DNA transposition. The 70-KDa protein product of the phage early gene A, termed transposase, is absolutely required for transposition. Transposase binds specifically at sites located at both ends of the phage genome, termed attL and attR, and at an enhancer-like element at the left end of the genome, called IAS (internal activation sequence). It then nicks at these ends, and nicks a random target DNA sequence in a 5 base pair staggered fashion with 5$ sp prime$ extensions and promotes strand transfer between the Mu ends and the target DNA. The transposase protein can be roughly divided into three domains. The other activities of the protein have not been mapped even at the domain level. To further define the different functional domains of this complex enzyme, a series of insertion mutants at 8 different sites along the transposase protein were constructed using TAB linker mutagenesis. In this study, 1 and 2 TAB linkers were inserted into 8 HpaII sites in the Mu A gene, generating a set of 2 and 4 amino acid insertion mutants. Examination of these mutants for specific DNA-binding activity of transposase to the ends of the phage genome in vitro revealed temperature sensitive proteins. Transpositional activity of the mutant proteins revealed that the mutant proteins, which are temperature sensitive in specific DNA-binding activity, are deficient in transpositional activity.
33
Morris, Zachary James. "Actin Binding Proteins Regulate the Localization of the Fission Yeast Hippo Pathway Protein Mob1p." University of Toledo / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1533229650651521.
34
Saleh, Maya. "Protein-protein interactions and cell signaling in the regulation of HOX.PBX functions." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=37620.
Анотація:
HOX proteins are homeodomain-containing transcription factors essential for embryonic patterning. Despite amino acid differences, all HOX homeodomains recognize highly similar sites on DNA. One mechanism by which HOX proteins achieve specificity is through interaction with cofactors of the PBX and MEIS/PREP1 families. Higher order complexes between HOX, PBX and MEIS/PREP1 proteins form in vivo and are essential for target recognition and transcriptional regulation. Another level of control of HOX function is the nuclear availability of its cofactors. This thesis addresses the regulation of the nuclear availability of the PBX protein by MEIS/PREP1 family members. We identified two nuclear localization signals (NLS) in the PBX homeodomain and showed that the NLS are masked in the absence of MEIS/PREP1. Upon a conformational change in PBX induced by MEIS/PREP1 binding, the NLS are exposed and a receptor-mediated active transport of PBX into the nucleus is allowed. This thesis also investigates the mechanisms of transcriptional regulation by the HOX·PBX complexes. We show that HOX·PBX complexes repress transcription and are switched to transcriptional activators in response to cell signaling. We demonstrate that PBX mediates the repression function by recruiting histone deacetylases (HDACs) to HOX target promoters. Inhibition of HDAC activity or stimulation of protein kinase A (PKA) signaling converts the HOX·PBX complex into a net activator of transcription. The activation function is mediated by the HOX protein through its recruitment of CREB-binding protein (CBP), a coactivator with histone acetyl-transferase (HAT) activity. We propose a model whereby HOX·PBX transcriptional activity is determined by cell signaling, and is mediated by the local modification of chromatin structure in the promoter of downstream targets.
35
Lau, Tsz Cham Derek. "Dissecting Protein-Protein Interactions that Regulate the Spindle Checkpoint in Budding Yeast." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10558.
Анотація:
Errors in segregation of genetic materials are detrimental to all organisms. The budding yeast ensures accurate chromosome segregation by employing a system called the spindle checkpoint. The spindle checkpoint, which consists of proteins such as Mad1, Mad2, Mad3, Bub1, and Bub3, monitors the attachment of microtubules to the chromosomes and prevents cell cycle progression until all chromosomes are properly attached. To understand how the spindle checkpoint arrests cells in response to attachment errors at the chromosomes, we recruited different checkpoint proteins to an ectopic site on the chromosome by taking advantage of the binding of the lactose repressor (LacI) to the lactose operator (LacO). We found that cells expressing Bub1-LacI arrest in metaphase. The phenotype is in fact caused by dimerization of Bub1 when it is fused to LacI rather than the recruitment of Bub1 to chromosome. The cell cycle arrest by the Bub1 dimer depends on the presence of other checkpoint proteins, suggesting that the dimerization of Bub1 represents an upstream event in the spindle checkpoint pathway. The results with the Bub1 dimer inspired us to fuse checkpoint proteins to each other to mimic protein interactions that may contribute to checkpoint activation. We showed that fusing Mad2 and Mad3 arrests cells in mitosis and that this arrest is independent of other checkpoint proteins. We believe that combining Mad2 and Mad3 arrests cells because both proteins can bind weakly to Cdc20, the main target of the spindle checkpoint, and the sum of these two weak bindings creates a hybrid protein that binds tightly to Cdc20. We reasoned that if Mad3's role is to make Mad2 bind tightly, artificially tethering Mad2 directly to Cdc20 should also arrest cells and this arrest should not depend on any other checkpoint components. Our experiments confirmed these predictions, suggesting that Mad3 is required for the stable binding of Mad2 to Cdc20 in vivo, that this binding is sufficient to inhibit APC activity, and that this reaction is the most downstream event in spindle checkpoint activation. The interactions among spindle checkpoint proteins thus play an important role in cell cycle arrest and must be carefully regulated.
36
Levey, Kristian. "Protein-Protein interactions of ExbD in the complex TonB- ExbB-ExbD." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=67043.
Анотація:
A major feature of Gram-negative bacteria is that the outer-membrane (OM) does not have an energy source to regulate active transport of nutrients; instead it relies on energy from the cytoplasmic membrane (CM) to accomplish this. The energy-transducing complex of TonB–ExbB–ExbD in the CM helps overcome this obstacle in TonB-dependent transport systems. In order to further understanding of this multi-component complex, ExbD was purified for use in phage display (PD) experiments to predict interacting partners and to identify sites of interaction with other members of the complex. Surface plasmon resonance (SPR) was carried out to biochemically validate PD predictions. Affinity-selected peptides from two commercial PD libraries predicted interactions between ExbD–ExbD and ExbD–TonB. No interaction between ExbD–ExbB was predicted. Notably, an interaction was predicted between ExbD and the periplasmic binding protein BtuF of the vitamin B12 transport system. SPR experiments validated the prediction of interactions between ExbD and TonB. Interaction between ExbD–ExbB was not apparent. Additionally, TonB–ExbB and ExbB–ExbB interactions were observed. Multi-component SPR analyses suggested that ExbB and ExbD bind to unique sites on TonB suggesting the role of primary scaffold for TonB in the complex.L'un des traits distinctifs de la membrane externe des bactéries gram-négatives est son dénuement de toute source d'énergie permettant le transport actif des nutriments. Ces bactéries font plutôt appel à l'énergie disponible à la membrane cytoplasmique. Localisé à la membrane cytoplasmique, le complexe de transduction de l'énergie formé des protéines ExbB, ExbD et TonB permet de surmonter cet obstacle dans les systèmes de transport TonB-dépendants. Dans le but d'avancer notre compréhension de ce complexe multi-protéique, nous avons purifié ExbD et l'avons soumis à des expériences de présentation de phage (PP) avec pour objectif de prédire certains de ses partenaires d'interaction et aussi afin d'identifier ses sites de contact avec les autres membres du complexe. Nous avons fait appel à la résonance plasmon de surface (RPS) pour valider nos résultats de PP.Nous fîmes usage de deux librairies de phages disponibles commercialement, grâce auxquelles nous pûmes prédire qu'ExbD interagit avec TonB ainsi qu'avec lui-même. Il nous a cependant été impossible de prédire une interaction entre ExbD et ExbB. Fait digne de mention, nos résultats prédisent une interaction entre ExbD et la protéine périplasmique BtuF impliquée dans le transport de la vitamine B12. Par ailleurs, la RPS permit de confirmer l'interaction entre ExbD et TonB prédite par PP. Par contre, aucune interaction ne put être observée à l'interface ExbB-ExbD. En outre, il nous a été possible d'observer des interactions entre ExbB et ExbB ainsi qu'entre ExbB et TonB. Finalement, une analyse RPS multi-composants a permis de suggérer que les interactions observées entre TonB et ses partenaires ExbB et ExbD ont lieu par l'intermédiaire de points de contact différents, appuyant par la même l'hypothèse d'un rôle central de protéine-échafaudage pour TonB dans le complexe.
37
Deme, Justin. "Novel protein-protein interactions involved in siderophore uptake in «Escherichia coli»." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=66873.
Анотація:
The level of bioavailable ferric iron (10-24 M Fe3+) in human serum is limited by its low solubility at physiological pH and by iron-binding host proteins. This situation poses a dilemma for bacteria; for survival, they must maintain an intracellular free iron concentration of 10-6 M. Gram-negative bacteria overcome iron limitation by producing siderophores that chelate iron with high affinity. Escherichia coli imports the siderophore ferrichrome via the TonB-dependent ferric hydroxamate uptake (Fhu) system. Extracellular ferrichrome is transported across the outer membrane (OM) through FhuA, a β-barrel receptor. The TonB–ExbD–ExbB complex provides energy for transport by harnessing the proton motive force across the cytoplasmic membrane (CM) and delivering it to the OM receptor. After passing through FhuA, ferrichrome is captured by the periplasmic binding protein FhuD and shuttled to the CM-embedded ABC transporter complex FhuB–FhuC. We previously demonstrated a novel interaction between FhuD and the energy transducer TonB. By phage display, we identified three well-defined regions of interaction on TonB to which FhuD was predicted to bind. To extend these studies, we utilized PSIPRED and SERp bioinformatics servers to identify areas on TonB that were unstructured and flexible. Redundant residues within the proline-rich region of TonB (residues 66-100) fit these criteria and were also not predicted to be necessary for complex formation with FhuD. A TonB construct lacking its proline-rich region, yet retaining its three predicted binding regions for FhuD, was engineered and purified to homogeneity. By ELISA, TonBΔ66-100 bound FhuD in a concentration-dependent manner, comparable to binding by full-length TonB. Surface plasmon resonance measured a binding affinity between FhuD and TonBΔ66-100 of 9 nM, consistent with data obtained for FhuD binding full-length TonB. Furthermore, presence of boundLa disponibilité du fer dans le sérum humain est limitée à 10-24 M de Fe3+. Ceci pose un dilemme pour les bactéries, car elles doivent en maintenir une concentration intracellulaire de 10-6 M pour survivre. Les bactéries Gram-négatives peuvent éviter cette limitation en produisant des sidérophores, capables de chélater le Fe3+ avec une grande affinité. Le ferrichrome, un sidérophore, est importé dans Escherichia coli par le système 'ferric hydroxamate uptake' (Fhu), qui dépend de la protéine TonB. Le ferrichrome extracellulaire est transporté à travers la membrane externe par FhuA, un tonneau β. Le complexe TonB–ExbD–ExbB fournit l'énergie nécessaire au transport via la force protomotrice. Le ferrichrome est capturé dans le périplasme par FhuD, pour être ensuite transloqué au complexe FhuB–FhuC, un transporteur de type ABC localisé dans la membrane interne. Notre groupe était la première à découvrir une interaction entre FhuD et TonB. Par la technique génétique moléculaire phage display, on a identifié trois régions sur TonB donc FhuD est prédit d'interagir. Nous avons généré un plasmide contenant TonB avec une délétion interne (résidus 66-100), qui est identifiée comme non structurée et très entropique par les programmes bioinformatiques PSIPRED et SERp. Une forme de TonB sans cette région (TonBΔ66-100) a été purifiée à l'homogenéité par chromatographie d'affinité. Nous avons observé que TonBΔ66-100 se lie à FhuD, comme sa contrepartie complète, par ELISA et par résonance plasmonique de surface (SPR), avec une affinité de liaison de 9 nM. Aussi, cette liaison n'était pas dépendente sur la présence de sidérophore. Cependant, la liaison entre FhuD et TonB n'est pas affectée par l'élimination de la région flexible riche en prolines de cette dernière. Ces résultats approfondissent la description de cette interaction critique de prot
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Deme, Justin. "Protein-protein interactions for early intracellular vitamin B12 metabolism in mammals." Thesis, McGill University, 2014. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=123014.
Анотація:
Vitamin B12, or cobalamin, is a water soluble vitamin required as cofactor for two mammalian enzymatic processes: homocysteine remethylation to methionine in the cytoplasm using methionine synthase (MS), and fatty acid/amino acid metabolism in the mitochondrion using methylmalonyl-CoA mutase (MCM). Whereas the molecular nature of intracellular cobalamin metabolism in mammals remains poorly understood, the proteins MMACHC, MMADHC, LMBD1 and ABCD4 are implicated in its early uptake and processing. Due to the inherent challenges associated with the cellular utilization of cobalamin, we propose that these proteins mediate its early intracellular channeling; the objective of this thesis was to characterize the protein-protein interactions that coordinate this process.To gain insight into the function of MMADHC, recombinant isoforms were purified and low-resolution structural features were determined. MMADHC is monomeric and, in solution, adopts an extended conformation, with regions of disorder identified at the N-terminal domain. Panning combinatorial phage libraries against recombinant MMADHC allowed the mapping of putative sites of interaction on MMACHC. Kinetic analyses using surface plasmon resonance (SPR) confirmed a sub-micromolar affinity for the MMACHC–MMADHC interaction. Based on these studies, we propose that the function of MMADHC is exerted through its structured C-terminal domain via interactions with MMACHC in the cytoplasm.Clinical phenotypes and subcellular location of MS and MCM dictate that MMACHC functions in the cytoplasm while MMADHC functions at a branch point in the pathway in both the cytoplasm and the mitochondrion. To demonstrate that the MMACHC–MMADHC interaction is physiologically plausible, we used immunofluorescence and subcellular fractionation to confirm that MMACHC is cytoplasmic while MMADHC is dual-localized to the cytoplasm and mitochondria. Protein interaction analyses were extended by describing the recombinant production of the lysosomal membrane proteins LMBD1 and ABCD4. Detergent-solubilized LMBD1 and ABCD4 each formed homodimers in solution. SPR provided direct in vitro binding data for an LMBD1–ABCD4 interaction with low nanomolar affinity. Consistent with our phage display predictions, MMACHC interacted with LMBD1 and ABCD4 with high affinity.Our results support a model whereby membrane-bound LMBD1 and ABCD4 regulate the vectorial delivery of lysosomal cobalamin to cytoplasmic MMACHC, preventing cofactor dilution to the cytoplasmic milieu and protecting against inactivating side reactions. Subsequent formation of a cytoplasmic MMACHC–MMADHC complex then processes and partitions this cofactor to the downstream enzymes MCM and MS. These studies identify and characterize multiprotein complexes, advancing our basic understanding of early intracellular cobalamin metabolism.La vitamine B12, ou bien la cobalamine, est une vitamine soluble requise pour deux processus enzymatiques distincts chez les mammifères; la production de l'acide aminée méthionine par la méthionine synthase (MS), et le métabolisme d'acides gras et d'acides aminées par la méthylmalonyl-CoA mutase (MCM). Malgré le fait que les procédées métaboliques intracellulaires de la cobalamine restes peu bien caractérisés, les protéines dont MMACHC, MMADHC, LMBD1, et ABCD4 jouent un rôle dans l'acquisition et le traitement de ce cofacteur. Vu les difficultés intrinsèques de l'utilisation cellulaire de la cobalamine, nous proposons que ces protéines assurent l'efficacité de son canalisation. Cette thèse avait pour objectif de caractériser les interactions protéine-protéine impliquées dans ce processus.Pour pouvoir caractériser la fonction de MMADHC, des isoformes protéiques ont été purifiées et leurs traits structurales ont étés déterminés à basse résolution. MMADHC se trouve à être monomérique et adopte une conformation étendue en solution, avec des régions non structurées dans la terminaison aminée de la protéine. Ensuite, des librairies combinatoires de phages ont été utilisées comme substrats pour tracer des sites d'interactions potentiels avec MMADHC. Les analyses kinésiques des interactions MMACHC–MMADHC ont été faites à l'aide de la résonance plasmonique de surface (SPR) et ont confirmées une intéraction d'affinité sub-micromolaire. Avec ces résultats, nous proposons que la fonction de MMADHC se fasse par sa terminaison acidique en interagissant avec MMACHC dans le cytoplasme.Les phénotypes cliniques et la localisation subcellulaire de MS et de MCM envisagent que MMACHC joue un rôle dans le cytoplasme et que MMADHC se trouve à être impliquée dans le processus au niveau de la mitochondrie et du milieu cytoplasmique. Pour démontrer que l'interaction MMACHC–MMADHC est physiologique, nous avons utilisé l'immunofluorescence et la fractionnement subcellulaire pour confirmer que MMACHC est cytoplasmique et que MMADHC se retrouvent au cytoplasme et au mitochondrie.Des analyses protéiques ont également engendré LMBD1 et ABCD4. Solubilisés à l'aide de détergent, ces deux protéines prennent la conformation d'homodimères en solution. Une interaction d'affinité nanomolaire entre LMBD1 et ABCD4 a été confirmée en SPR. En lien avec nos analyses de phages, MMACHC interagit avec haute affinité avec LMBD1 et ABCD4.Nos résultats supportent un modèle dans lequel LMBD1 et ABCD4, tous deux liés dans la membrane, régularisent l'octroi de la cobalamine lysosomale à MMACHC en prévenant la dilution de ce cofacteur dans le milieu cytoplasmique et en protégeant contre des réactions inactivant. La dissociation et le recrutement de la MMADHC cytoplasmique à MMACHC facilitent le transfert de la cobalamine vers les réactions enzymatiques catalysées par MCM et MS. L'identification et la caractérisation de ces complexes multiprotéiques font en sorte d'avancer nos connaissances générales sur le métabolisme de la cobalamine.
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Prevodnik, Andreas. "The use of protein biomarkers in ecotoxicology : Studies of oxidative and genotoxic stress in the blue mussel (Mytilus edulis)." Doctoral thesis, Stockholm : Department of Systems Ecology, Stockholm University, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-6755.
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Chen, Tsan-Chou Scott. "Design of protein-protein interaction specificity using computational methods and experimental library screening." Thesis, Massachusetts Institute of Technology, 2012. http://hdl.handle.net/1721.1/70386.
Анотація:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2012.Cataloged from PDF version of thesis.
Includes bibliographical references.
Computational design of protein-protein interaction specificity is a powerful tool to examine and expand our understanding about how protein sequence determines interaction specificity. It also has many applications in basic bioscience and biotechnology. One of the major challenges for design is that current scoring functions relying on general physical principles do not always make reliable predictions about interaction specificity. In this thesis I described application of two approaches to address this problem. The first approach sought to improve scoring functions with experimental interaction specificity data related to the protein family of design interest. I used this approach to design inhibitor peptides against the viral bZIP protein BZLF 1. Specificity against design self-interaction was considered in the study. The second approach exploited the power of experimental library screening to characterize a large number of designed sequences at once, increasing the overall probability of identifying successful designs. I presented a novel framework for such library design approach and applied it to the design of anti-apoptotic Bcl-2 proteins with novel interaction specificity toward BH3 peptides. Finally I proposed how these two approaches can be combined together to further enhance our design capabilities.
by Tsan-Chou Scott Chen.
Ph.D.
41
Zhu, Lingyu. "Transcriptional regulation of the gene for the human retinoblastoma protein-related p107 protein /." The Ohio State University, 1997. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487945744572295.
42
Stamler, Robin Jacob. "Structural biology of two proteins from two eye-related protein families : ABCR and TSP36." Thesis, Birkbeck (University of London), 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414721.
43
Shurte, Leah A. "Determining Protein-Protein Interactions of ALS-Associated SOD1." Wright State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=wright1464283630.
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Taylor, Tanya. "Protein purification, sequencing, and cDNA cloning of P68, a sperm surface protein." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ36745.pdf.
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Zhang, Xinyi. "PROTEIN ENGINEERING IN THE STUDY OF PROTEIN LABELING AND DEGRADATION." UKnowledge, 2018. https://uknowledge.uky.edu/chemistry_etds/93.
Анотація:
Proteins are large macromolecules that play important roles in nature. With the development of modern molecular biology techniques, protein engineering has emerged as a useful tool and found many applications in areas ranging from food industry, environmental protection, to medical and life science. Biomimetic membrane incorporates biological elements, such as proteins, to form membranes that mimic the high specificity and conductance of natural biological membranes. For any application involving the usage of proteins, the first barrier is always the production of proteins with sufficient stability, and the incorporation of proteins into the artificial matrix. This thesis contains two major parts, the first part is focused on the development and testing of method to immobilize active enzymes. The second part is devoted to study the degradation of membrane proteins in E. coli cells.
In the immobilization study, Pyrophosphatase (PpaC) was chose as a model enzyme. A dual functional tag consist of histidine and methionine has been developed, in which histidine is used for purification while methionine is metabolically replaced with azidohomoalanine (AHA) for immobilization. We found that the addition of the tag and the incorporation of AHA did not significantly impair the properties of proteins, and the histidine–AHA tag can facilitate protein purification, immobilization, and labeling. This tag is expected to be useful in general for many proteins.
Degradation of soluble protein has been well characterized, but the membrane protein degradation process remains elusive. SsrA tag is a well-known recognition sequence for soluble protein degradation, which marks prematurely terminated protein products translated from damaged mRNA. SsrA tagged membrane proteins was found to be substrate of a cytosolic protease complex ClpXP, which mediated complete degradation.
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Lowery, Drew M. "Modulation of mitotic progression and cell cycle checkpoints by phosphorylation-dependent protein-protein interactions." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/40953.
Анотація:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2007.Includes bibliographical references.
Alteration of mitotic gene function has recently been discovered to play a key role in tumor formation and cancer progression through the induction of chromosomal aberrations and genomic instability. Polo-like-kinase-1 is a critical mitotic regulator, overexpressed in human tumors, that functions in mitotic entry after cellular stress, centrosome maturation, mitotic spindle control, and cytokinesis, which are all disregulated in cancer cells. To study the role of Polo-like kinases we took advantage of the recent discovery that the polo-box domain of Polo-like kinases is a phosphorylation-dependent binding module that regulates targeting of Polo-like kinases to their substrates. To identify the interactors of Polo-box domains we developed and performed a mitotic-specific yeast two hybrid and a pulldown mass spectrometry screen. This yielded a large number of specific interactors known to be involved in a vast variety of mitotic processes including those previously described to be involved in tumor progression. We demonstrate that Polo-like kinase regulation of cytokinesis-specific guanine-nucleotide exchange factors for the small G-protein Rho is necessary for proper actomyosin ring contraction and cytokinesis. Additionally we demonstrate that Polo-like-kinase-1 directly regulates the activity of the Rho-effector-kinase ROCK2, and thus Polo-like kinases modulate Rho signaling both upstream and downstream of Rho during cytokinesis. In addition to Polo-box domains we also worked on two other phosphorylation-dependent binding domains involved in cell cycle checkpoints that become disregulated in cancer cells, tandem BRCT domains and WW domains.
(cont.) We examined the molecular basis for phosphorylation-dependent recognition by the tandem BRCT domains of BRCA1 through oriented-peptide-library screening and determination of an X-ray crystal structure of the domain bound to a phosphopeptide. This allowed us to rationalize why inherited mutations within the tandem BRCT domains of BRCA1 promote breast and ovarian cancer in humans. Secondly, we assayed WW domains that were generated from in silicon determined sequences for natural-like function to more fully understand the folding and binding requirements of this domain class. All three domains (tandem BRCT domains, Polo-box domains, and WW domains) are attractive targets for cancer therapeutics as they participate in control of processes necessary for genomic stability that become disregulated in cancer.
by Drew M. Lowery.
Ph.D.
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Hetti, Arachchilage Madara Dilhani. "Coevolution of epitopes in HIV-1 pre-integration complex proteins: protein-protein interaction insights." Kent State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=kent1530646538935895.
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Weber, Christine. "Cdx-Hox protein interactions." Thesis, University of Ottawa (Canada), 2008. http://hdl.handle.net/10393/27656.
Анотація:
Cdx and Hox gene families both code for homeodomain-containing transcription factors and both are required for proper patterning of the anterior-posterior axis. Although Cdx is known to be upstream of certain Hox genes, preliminary data indicated Cdx1 interacts in vitro with Hoxd4 but not Hoxa9, and that this interaction is localized to the C-terminal region of both proteins. GST-pulldown assays with in vitro radiolabelled proteins or transfected cell lysates were used to investigate the extent of the Cdx-Hox interactions. The interaction was narrowed down to specific residues in the first half of the homeodomain, but an inhibitory N-terminus can abrogate interaction in vitro. A larger group of Hox proteins interact with GST-Cdx when they are expressed in a cellular environment. This could mean that the interaction requires other cofactors, such as Hox cofactors Pbx and Meis that also interact with GST-Cdx1 in vitro. This study is the first example of Cdx-Hox protein interactions and suggests that the Cdx-Hox complex could be involved in the regulation of downstream Hox target genes.
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Sarkar, Mohosin M. "Engineering Proteins with GFP: Study of Protein-Protein Interactions In vivo, Protein Expression and Solubility." The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1261418776.
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Jiménez, Roldán José Emilio. "Rigidity analysis of protein structures and rapid simulations of protein motion." Thesis, University of Warwick, 2012. http://wrap.warwick.ac.uk/49629/.
Анотація:
It is a common goal in biophysics to understand protein structural properties and their relationship to protein function. I investigated protein structural properties using three coarse graining methods: a rigidity analysis method First, a geometric simulation method Froda and normal mode analysis as implemented in Elnemo to identify the protein directions of motion. Furthermore, I also compared the results between the coarse graining methods with the results from molecular dynamics and from experiments that I carried out. The results from the rigidity analysis across a set of protein families presented in chapter 3 highlighted two different patterns of protein rigidity loss, i.e. "sudden" and "gradual". It was found that theses characteristic patterns were in line with the rigidity distribution of glassy networks. The simulations of protein motion by merging flexibility, rigidity and normal mode analyses presented in chapter 4 were able to identify large conformational changes of proteins using minimal computational resources. I investigated the use of RMSD as a measure to characterise protein motion and showed that, despite it is a good measure to identify structural differences when comparing the same protein, the use of extensive RMSD better captures the extend of motion of a protein structure. The in-depth investigation of yeast PDI mobility presented in chapter 5 confirmed former experimental results that predicted a large conformational change for this enzyme. Furthermore, the results predicted: a characteristic rigidity distribution for yeast PDI, a minimum and a maximum active site distance and a relationship between the energy cutoff, i.e. the number of hydrogen bonds part of the network of bonds, and protein mobility. The results obtained were tested against molecular dynamics simulations in chapter 6. The MD simulation also showed a large conformational change for yeast PDI but with a slightly different minimum and maximum inter-cysteine distance. Furthermore, MD was able to reveal new data, i.e. the most likely inter-cysteine distance. In order to test the accuracy of the coarse graining and MD simulations I carried out cross-linking experiments to test the minimum inter-cysteine distance predictions. The results presented in chapter 7 show that human PDI minimum distance is below 12Å whereas the yeast PDI minimum distance must be above 12Å as no cross-linking structures where found with the available (12Å long) cross-linkers.