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Sharma, Khushbu. "Studying sigma family of transcription factors in mycobacteria: In-silico and functional analyses of conserved sigma proteins of mip by in-vitro and in-vivo methods". Thesis, IIT Delhi, 2019. http://eprint.iitd.ac.in:80//handle/2074/8065.
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Lee, S. F. K. "Understanding protein tyrosine phosphatase sigma function : dimer formation and interacting proteins". Thesis, University College London (University of London), 2007. http://discovery.ucl.ac.uk/1444958/.
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Cytoplasmic and transmembrane protein tyrosine phosphatases (PTPs) provide the enzymatic counterbalance to protein tyrosine kinase activity. PTP sigma (PTPa) is an adhesion molecule-like receptor PTP (RPTP) that is expressed on the growth cones of developing axons. PTPa binds ligands located in the basement membrane (heparan sulphate proteoglycans: agrin and collagen 18) and developing muscle (Nucleolin). Disruption of ligand-PTPa interactions affects axon guidance, although neither the role of PTPa in neurons nor the effect of ligand binding on PTPa activity is well understood. Further characterisation of PTPa function remains difficult in the absence of an understanding of PTPa biochemistry that would allow a functional assay to measure the effects of an experimental manipulation on PTPa function. PTPa was shown to be dimeric using a combination of disulphide cross-linking and co-immunoprecipitation techniques. This dimeric form of PTPa was principally cell surface localised according to its accessibility to trypsinisation. However, neither co-immunoprecipitation nor glutathione S-transferase (GST) pull-down techniques allowed the identification of proteins that interact with wild type or recombinant substrate-trapping PTPa. Secondly, H202 treatment of PTPa-expressing cells induced the formation of reduction-sensitive, high molecular weight species. In contrast to the formation of PTPa dimers under the same conditions, this did not require the tyrosine phosphatase domain catalytic site cysteines. It may be possible to utilise the formation of high molecular weight species on non-reducing SDS-PAGE analysis as a proxy measure of PTPa oligomerisation. Moreover, unlike co-immunoprecipitation it can be used on wild type and even endogenously expressed proteins. Finally, the type Ila RPTP family consists of PTPa, PTPS and LAR. To allow the simultaneous disruption of type Ila RPTPs, chicken LAR was cloned from embryonic chick whole body mRNA and characterised.
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Donà, Valentina. "Caratterizzazione dei fattori sigma micobatterici SigE e SigF Caratterizzazione del dominio PPE della proteina PPE17 di Mycobacterium tuberculosis". Doctoral thesis, Università degli studi di Padova, 2010. http://hdl.handle.net/11577/3422362.
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Abstract
Mycobacterium tuberculosis (MTB) is the causative of tuberculosis, a disease, which causes 2 millions of death every year, with a dramatic incidence especially in developing countries.
To find new drug and vaccine strategies against MTB, it is of fundamental importance to study the mechanisms, that allow its survival to environmental stresses, to which it is subjected during the period of infection and latency in the host macrophages. The fine transcriptional regulation of specific genes in response to stress conditions and the peculiar structure of its wall play a key role on this.
In the first part of the PhD project two mycobacterial sigma factors, SigE and SigF, which regulate the transcription of specific genes in response to various environmental stresses such as surface stress, oxidative stress, alkaline pH and thermal shock, have been characterized.
First the transcriptional regulation, translational and post-translational regulation of the extracytoplasmic function (ECF) transcription factor SigE were studied.
Regarding the study of the transcriptional regulation, it was possible to confirm by 5'RACE PCR and RT-PCR experiments the presence of three promoters of sigE, and to determine the contribution of each promoter in the transcription of this gene, depending on the environmental conditions of bacterial growth.
The fact, that the transcriptional start codon of one of these promoters is located 63 base pairs downstream of the start codon annotated in MTB genome opened the possibility of the existence of two isoforms of Sige.
By translational fusions between specific sequences of sigE with lacZ, deprived of its own translational initiation codon, and subsequent site-specific mutagenesis, it was possible to confirm, based on further beta-galactosidase activity detection, the existence of two alternative start codons, an ATC and a TTG, coding for an isoform of respectively 218 and 215 of amino acids, in addition to the ATG already annotated in MTB genome, which encodes for an isoform of 257 amino acids.
Finally, it was possible to confirm, that the gene downstream sigE encodes for the anti-sigma factor of SigE, called RseA, capable of binding both isoforms of SigE.
In a second project also the role of the factor SigF M. smegmatis in the biosynthesis of carotenoid pigments, resistance to hydrogen peroxide and in the efficiency of bacterial transformation was studied.
By RT-PCR it has been shown, that SigF controls the transcription of genes involved in the biosynthesis of carotenoid pigments, and, assuming that they serve as protection against free radicals, it was verified that the sigF mutant strain is actually more sensitive compared to the wild type strain to treatment with hydrogen peroxide. Finally, we also demonstrated, that the mutant strain has a higher transformation efficiency than the wild type strain, indicating that SigF regulates the transcription of genes possibly involved in the permeability of the cell wall.
In the second part of the project, the localization of the protein on the surface PPE17 mycobacteria was characterized. Like other members of the PPE family, the PPE17 has a highly conserved N-terminal domain, which, based on different evidences in literature, is assumed to play an important role in their translocation to the mycobacterial surface. Moreover, it was investigated the possible influence of the presence of PE11 in the translocation process or in the stability of PPE17, as the PE11 coding sequence is in tandem and co-transcribed with that encoding the PPE17, and there is a specific interaction between these two proteins.
The data obtained by proteinase K sensitivity assays performed on M. smegmatis strains, expressing the entire PPE17 or only its domain PPE (dPPE17) fused with the HA epitope, confirm, that the entire PPE17 is exposed on the surface, both in the presence and absence of PE11.
According to data obtained, the possibility to translocate the MTB model antigen (Mpt64) on the surface of the vaccine strain M. bovis BCG, by fusing them with the dPPE17 was tested. Proteinase K and whole cell ELISA assays performed on cultures of M. bovis BCG expressing this chimeric protein indicate, that it is indeed localized at the mycobacterial surface. Similarly, another two fusions with dPPE17 were constructed to express on the mycobacterial surface the multimeric MTB antigen AG85-ESAT6 of MTB and the Csp C3 antigen of Plasmodium bergii. According to the proteinase K sensitivity assays carried out on strains of M. smegmatis expressing the two chimeric proteins indicate that also in this case both are localized at the surface.
The strains of M. bovis BCG expressing these antigens on their surface will be tested in future in the mouse model to measure any increase in protection compared to the wild type strain.Riassunto Mycobacterium tuberculosis (MTB) è l’agente eziologico della tubercolosi, patologia che nel mondo causa ogni anno due milioni di morti, con un’incidenza drammatica specie nei Paesi in via di sviluppo. Per poter trovare nuove strategie farmacologiche e vaccinali contro MTB è di fondamentale importanza lo studio dei meccanismi, che permettono la sua sopravvivenza ai vari stress ambientali, ai quali è sottoposto durante il periodo di infezione e latenza nei macrofagi dell’ospite. La fine regolazione della trascrizione di geni specifici in risposta a condizioni di stress e la peculiare struttura della sua parete giocano in merito un ruolo fondamentale. Nella prima parte del progetto di dottorato sono stati caratterizzati due fattori di trascrizione sigma micobatterici, SigE e SigF, che regolano la trascrizione di geni specifici in risposta a vari tipi di stress ambientali, come lo stress di superficie, lo stress ossidativo, il pH alcalino e lo shock termico. Anzitutto è stata studiata la regolazione trascrizionale, traduzionale e posttraduzionale del fattore di trascrizione con funzione extracitoplasmatica (ECF) SigE. Per quanto riguarda lo studio della regolazione trascrizionale, è stato possibile confermare tramite esperimenti di 5’RACE PCR e RT-PCR la presenza di tre promotori di sigE, e a dosare, a seconda delle condizioni ambientali di crescita batterica, il contributo di ciascun promotore nella trascrizione di questo gene. Dato che l’inizio della trascrizione di uno di questi promotori è sito 63 paia di basi a valle del codone di start annotato nel genoma, si è aperta l’ipotesi dell’esistenza di due isoforme di SigE. Mediante fusioni traduzionali tra specifiche sequenze di sigE con lacZ, private del proprio codone di inizio della traduzione, e successive mutagenesi sito-specifiche, è stato possibile confermare, in base all’attività beta-galattosidasica rilevata, l’esistenza di due codoni di start alternativi, un ATC ed un TTG, che codificano per un’isoforma di rispettivamente 218 e 215 di amminoacidi, oltre all’ATG già annotato nel genoma di MTB, che codifica per un’isoforma di 257 amminoacidi. Infine è stato possibile confermare, che il gene a valle di sigE codifica per il fattore anti-sigma di SigE, denominato RseA, in grado di legare entrambe le isoforme di SigE. In un secondo progetto è stato studiato anche il ruolo del fattore SigF di M. smegmatis nella biosintesi di pigmenti carotenoidi, nella resistenza a perossido d’idrogeno e nell’efficienza di trasformazione batterica. Tramite RT-PCR è stato dimostrato che SigF controlla la trascrizione di geni coinvolti nella biosintesi dei pigmenti carotenoidi, e, partendo dal presupposto che essi fungono da protezione contro i radicali liberi, è stato verificato che il mutante per il gene sigF è effettivamente più sensibile rispetto al ceppo selvatico al trattamento con perossido d’idrogeno. Infine è stato dimostrato anche, che il ceppo mutante possiede una maggiore efficienza di trasformazione rispetto al ceppo selvatico, indicando che SigF regola probabilmente la trascrizione di geni coinvolti nella permeabilità della parete. Nella seconda parte del progetto è stata caratterizzata la localizzazione della proteina PPE17 sulla superficie micobatterica. Come altri membri della famiglia PPE, la PPE17 presenta un dominio N-terminale altamente conservato, il quale, in base a diverse evidenze in letteratura, si suppone svolgere un ruolo importante per la loro traslocazione in superficie. Inoltre, si è voluto verificare un’eventuale influenza della presenza della proteina PE11 nel processo di traslocazione in o nella stabilità della PPE17, in quanto la sequenza codificante la PE11 è in tandem e co-trascritta con quella codificante la PPE17, e vi è un’interazione specifica tra queste due proteine. I dati ottenuti mediante saggi di sensibilità alla proteinasi K su ceppi di M. smegmatis, esprimenti la PPE17 intera o solo il suo dominio PPE (dPPE17) fuse all’epitopo HA, confermano che la PPE17 intera sia esposta in superficie, sia in presenza che in assenza di PE11. In base ai dati ottenuti si è infine tentato di veicolare un antigene modello (Mpt64) di MTB sulla superficie del ceppo vaccinale M. bovis BCG fondendolo con il dPPE17. Saggi di sensibilità alla proteinasi K e ELISA su cellule intere effettuati su culture di M. bovis BCG esprimenti questa proteina chimerica indicano, che essa sia effettivamente localizzata a livello superficiale. Allo stesso modo sono state costruite due ulteriori fusioni con il dPPE17 per esprimere sulla superficie micobatterica l’antigene multimerico Ag85-ESAT6 di MTB e l’antigene Csp C3 di Plasmodium berghii. In base a saggi di sensibilità alla proteinasi K svolti su ceppi di M. smegmatis esprimenti le due fusioni anche in questo caso entrambe localizzano in superficie. I ceppi di M. bovis BCG esprimenti questi antigeni sulla loro superficie saranno testati in futuro nel modello del topo per misurare un eventuale aumento della protezione rispetto al ceppo selvatico.
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Morikawa, Kazuya. "Regulation of Plastid Gene Transcription by Sigma Factors and Sigma Factor Binding Proteins". Kyoto University, 2001. http://hdl.handle.net/2433/150743.
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Kyoto University (京都大学)0048
新制・課程博士
博士(人間・環境学)
甲第9028号
人博第121号
12||123(吉田南総合図書館)
新制||人||30(附属図書館)
UT51-2001-F358
京都大学大学院人間・環境学研究科文化・地域環境学専攻
(主査)教授 豊島 喜則, 教授 藤堂 剛, 助教授 瀬戸口 浩彰
学位規則第4条第1項該当
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Chadsey, Meggen Shepherd. "Regulation of the flagellar specific sigma factor, sigma28, of Salmonella typhimurium by the anti-sigma factor FlgM /". Thesis, Connect to this title online; UW restricted, 1998. http://hdl.handle.net/1773/11490.
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Ruan, Qingguo. "Aire regulates central and peripheral tolerance through direct control of autoantigens and other key genes in thymus epithelial cells and dendritic cells". [Gainesville, Fla.] : University of Florida, 2004. http://purl.fcla.edu/fcla/etd/UFE0006464.
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Thesis (Ph.D.)--University of Florida, 2004.Typescript. Title from title page of source document. Document formatted into pages; contains 100 pages. Includes Vita. Includes bibliographical references.
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Yeung, Shu-wai. "Analysis of 14-3-3 [sigma] protein in nasopharyngeal tissues". Click to view the E-thesis via HKUTO, 2003. http://sunzi.lib.hku.hk/hkuto/record/B31971398.
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Smillie, David Andrew. "Genes encoding sigma cross-reacting proteins of Escherichia coli". Thesis, University of Edinburgh, 1994. http://hdl.handle.net/1842/14435.
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In the course of work aimed at discovering new sigma transcription initiation factors in Escherichia coli, two unknown sigma cross-reacting proteins (SCRP-23 and SCRP-27A) were identified by cross-reaction with antibodies raised against region 2.2 of sigma 70. This thesis describes the mapping, sequencing and characterisation of the corresponding genes. The gene encoding SCRP-23 was located near 652kb on the physical map of the E. coli chromosomes. Its sequence and that of part of a downstream open reading frame were determined, and found to be closely similar to the ahpC and F genes (respectively) of Salmonella typhimurium. These encode the C22 and F52a subunits of the anti-oxidant enzyme, alkylhydroperoxide reductase. The identity of the E. coli genes was further established by their ability when introduced on plasmids into an ahp deletion strain to restore cumene hydroperoxide resistance. Transcription of ahpCF was found to be driven by two promoters: ahpP1 is dependent on activation by the OxyR transcriptional regulator, whilst ahpP2 is independent of this factor. Indeed ahpP2 is located within the OxyR target site, and is repressed when OxyR is activated by oxidation. The gene encoding SCRP-27A, scrP, was located near 3416 kb on the physical map, just 250 bp downstream of arcB. arcB encodes a transmembrane sensor-regulator of respiratory functions.
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楊舒瑋 i Shu-wai Yeung. "Analysis of 14-3-3 [sigma] protein in nasopharyngeal tissues". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2003. http://hub.hku.hk/bib/B31971398.
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Chagnon, Mélanie J. 1977. "Physiological and molecular functions of the murine receptor protein tyrosine phosphatase sigma (RPTP[sigma])". Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=115661.
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The control of cellular tyrosine phosphorylation levels is of great importance in many biological systems. Among the kinases and phosphatases that modulate these levels, the LAR-RPTPs have been suggested to act in several key aspects of neural development, and in a dysfunctional manner in various pathologies from diabetes to cancer. The aim of this thesis is to describe the physiological functions of one of the members of this subfamily of RPTPs, namely RPTPsigma. First, we showed that glucose homeostasis is altered in RPTPsigma null mice. They are hypoglycemic and more sensitive to exogenous insulin and we proposed that the insulin hypersensitivity observed in RPTPsigma-null mice is likely secondary to their neuroendocrine dysplasia and GH/IGF-1 deficiency. In addition to regulating nervous system development, RPTPsigma was previously shown to regulate axonal regeneration after injury. In the absence of RPTPsigma, axonal regeneration in the sciatic, facial and optical nerves was enhanced following nerve crush. However, myelin-associated growth inhibitory proteins and components of the glial scar such as CSPGs (chondroitin sulfate proteoglycans) have long been known to inhibit axonal regeneration in the CNS, making spinal cord injury irreversible. In collaboration with Dr Samuel David, we unveiled that RPTPsigma null mice are able to regenerate their corticospinal tract following spinal cord hemisections as opposed to their WT littermates. We then isolated primary neurons from both sets of animals and found that the absence of RPTPsigma promotes the ability of the neurons to adhere to certain inhibitory substrates. Finally, in order to better understand the physiological role of RPTPsigma, we used a yeast substrate-trapping approach, to screen a murine embryonic library for new substrates. This screen identified the RhoGAP p250GAP as a new substrate, suggesting a downstream role for RPTPsigma in RhoGTPase signaling. We also identified p130Cas and Fyn as new binding partners. All these proteins have clear functional links to neurite extension. The characterization of RPTPsigma and its signaling partners is essential for understanding its role in neurological development and may one day translate into treatments of neural diseases and injuries.
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Ferguson, Anna Louise. "Interactions of bacterial sigma subunits with core RNA polymerase". Thesis, University of York, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341839.
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Reeves, Adam J. "Signaling and interaction of the Bacillus subtilis physical stress pathway regulators of sigma B : a dissertation /". San Antonio : UTHSC, 2007. http://proquest.umi.com/pqdweb?did=1390290691&sid=1&Fmt=2&clientId=70986&RQT=309&VName=PQD.
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Lee, Chung-Sheng Brian. "Studies of SpoIIAA, the anti-anti-#sigma#'F factor of Bacillus subtilis". Thesis, University of Oxford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365810.
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Rynda, Agnieszka. "Low dose tolerance vaccine platform, reovirus protein sigma 1 and treatment of autoimmunity". Thesis, Montana State University, 2008. http://etd.lib.montana.edu/etd/2008/rynda/RyndaA0808.pdf.
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Effective treatments for multiple sclerosis (MS) are problematic due to its unknown etiology. Experimental autoimmune encephalomyelitis (EAE) in rodents mimics MS. Mucosal treatment of EAE with antigens to induce tolerance is effective, but requires large and/or multiple administrations, which introduces an allergy risk. We utilized reovirus adhesin, protein sigma 1 (p sigma1), to improve mucosal auto-antigen delivery and show that a single low-dose of pσ1-based vaccines induces tolerance and prevents autoimmunity when administered nasally. We engineered three pσ1-based vaccines carrying chicken ovalbumin (OVA-pσ1) and/or myelin antigens (PLP:OVA-pσ1, MOG-pσ1). When mice were nasally immunized with OVA-pσ1, tolerance to OVA was established. This tolerance resisted co-administration of mucosal adjuvants and peripheral challenge with OVA. Pσ1-mediated tolerance relied upon specific IL-10- producing regulatory T (Treg) cells, which inhibited OVA-specific CD4+ T cell proliferation. OVA-pσ1 did not generate tolerance in IL-10-deficient mice presumably by a failure to induce Treg cells. Mucosal, but not systemic pσ1 delivery, induced tolerance, while mice lacking mucosal inductive tissues were resistant to pσ1-mediated tolerance. Likewise, PLP:OVA-pσ1 and MOG-pσ1 protected mice against relapsing-remitting or acute EAE, respectively. Protection against PLP 139-151-induced EAE was accomplished by PLP:OVA-pσ1, but not OVA-pσ1, implicating antigen-specificity of pσ1-mediated tolerance. Moreover, MOG-pσ1, but not PLP:OVA-pσ1, ameliorated MOG 35-55-induced EAE via apoptosis of encephalitogenic CD4+ T cells. The PLP:OVA-pσ1- or MOG-pσ1-mediated protection against EAE depends on specific IL-10+ Treg cells and is supported by IL-4+ Th2-type cells. Adoptive transfer of PLP:OVA-pσ1-primed Treg cells entirely prevented EAE development in mice; however, transfer of PLP:OVA-pσ1-specific CD25-CD4 + Th2 cells significantly reduced and delayed clinical EAE. Aggressive EAE, due to the TGF-β which induced activation of Th17 cells, was observed in mice dosed with PLP:OVA-pσ1 and were functionally depleted of Treg cells. Concomitant inactivation of TGF-β and Treg cells induced Th2 cells bias and re-established PLP:OVA-pσ1-mediated protection against EAE. IL-10-producing B cells supported MOG-pσ1-mediated protection against EAE, as MOG-pσ1-dosed B cell-deficient mice developed attenuated disease. Adoptive transfer of Treg cells, but not Th2 or B cells from MOG-pσ1-dosed B6 mice to diseased IL-10-/- mice, significantly accelerated recovery from EAE. These data demonstrate the feasibility of using pσ1-based single-dose delivery system to prevent and/or treat autoimmunity.
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Mason, S. "Studies on the cell-binding properties of the retrovirus #sigma#1 protein". Thesis, University of Warwick, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.357840.
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Laskos, Lina 1973. "Characterisation of alternative sigma factors and the heat shock rsponse in Neisseria gonorrhoeae". Monash University, Dept. of Microbiology, 2003. http://arrow.monash.edu.au/hdl/1959.1/5617.
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Xu, Rong. "The effects of structural modifications on sigma receptor binding". Diss., Columbia, Mo. : University of Missouri-Columbia, 2007. http://hdl.handle.net/10355/4742.
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Thesis (Ph. D.)--University of Missouri-Columbia, 2007.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on December 18, 2007) Vita. Includes bibliographical references.
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Gilmore, Ross. "Sigma 1 protein of reovirus as a model system for the study of protein folding within the mammalian cell cytosol". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp02/NQ38469.pdf.
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Thibault, Stéphanie. "Biochemical and genetic approaches to understanding the functions of the protein tyrosine phosphatase-sigma". Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=79146.
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PTP-sigma, a developmentally regulated receptor protein tyrosine phosphatase of the LAR family, is highly expressed in the nervous system and is involved in axon guidance. In this study, two potential substrates of PTP-sigma have been detected by substrate trapping and two-dimensional electrophoresis. In addition, an expression pattern of PTP-sigma in adult non-neural mouse tissues has been established and opens new research avenues for the function of PTP-sigma. The high levels of PTP-sigma in fat led us to look into a possible role for this enzyme in insulin signaling. Insulin stimulated PTP-sigma knockout adipose tissue showed an hyperphosphorylation of the insulin receptor (IR). GH-deficiency is predominantly responsible for glucose homeostasis defects of PTP-sigma knockout mice and could mask the effect of the absence of PTP-sigma in insulin signaling in vivo. To overcome this, adipocytes were isolated. Upon insulin stimulation of PTP-sigma knockout adipocytes, an increase in IR and a decrease in Akt phosphorylation were observed, suggesting that the IR could be a substrate of PTP-sigma and that PTP-sigma could be involved in the regulation of the activation of Akt. The elucidation of the mechanisms of action of PTP-sigma in insulin signaling may lead to the development of new treatments in diabetes and obesity.
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Cunning, Christofer Lee. "Regulation of the synthesis and protein stability of the alternative sigma factor RpoS in Salmonella typhimurium". Morgantown, W. Va. : [West Virginia University Libraries], 1999. http://etd.wvu.edu/templates/showETD.cfm?recnum=533.
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Ambler, Linda. "Investigation of the potential use of reovirus sigma 1 proteins for drug delivery across the gastrointestinal tract". Thesis, Open University, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.278430.
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Thompson, Katherine M. "Receptor protein tyrosine phosphatase sigma (RPTPơ) inhibits axonal regeneration and the rate of axon extension". Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=29479.
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Transgenic mice lacking RPTPsigma, a type IIa receptor protein tyrosine phosphatase, exhibit severe neural-developmental deficits. Continued expression of RPTPsigma in the adult suggests that it plays a functional role in the mature nervous system. To determine if RPTPsigma might influence axonal regeneration, the time course of regeneration following facial nerve crush in wild type and RPTPsigma (-/-) mice was compared. Mice lacking RPTPsigma exhibited an accelerated rate of functional recovery, suggesting that RPTPsigma slows the extension of regenerating axons. We detected a decrease in RPTPsigma expression by facial motoneurons following nerve crush in wild type mice. Consistent with this, we show that the rate of axon extension is enhanced in neurons obtained from RPTPsigma (-/-) mice. Furthermore, in wild type mice. RPTPsigma is enriched in axonal growth cones. These findings indicate that RPTPsigma slows axon growth via a mechanism intrinsic to the neuron and identify a role for RPTPsigma in regulating axonal regeneration.
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Polychronidou, Maria. "Studying the role of dimerization in regulating the activity of Receptor Protein Tyrosine Phosphatase Sigma". Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=99198.
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RPTP-sigma is a member of the LAR-RPTP subfamily that has been associated with neurite outgrowth and neuronal regeneration. In this study, it is reported that dimenzation, seems to play a role in the regulation of RPTP-sigma activity. RPTP-sigma forms homodimers in cells under physiological conditions and antibody induced dimerization of HA-tagged RPTP-sigma leads to changes in the localization of the protein from the plasma membrane to vesicles in the cytoplasm. Furthermore, when N1E-115 cells were used as an in vitro model for studying the effect of RPTP-sigma-dimerization on neurite outgrowth, it was observed that induction of dimerization of the phosphatase allows neurite outgrowth in conditions that are normally non permissive for differentiation of the cells, indicating that dimer formation probably inhibits RPTP-sigma activity. In order to provide useful tools for studying the signaling cascades regulated by the phosphatase, a series of tagged constructs of potential RPTP-sigma interacting proteins were made. Finally, potential interactions between RPTP-sigma and c-MET were investigated.
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Perrody, Elsa. "The viral protein Rki : an atypical cochaperone partner of Hsp70". Toulouse 3, 2012. http://thesesups.ups-tlse.fr/1870/.
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Les chaperons moléculaires de la famille Hsp70 sont des protéines ubiquitaires qui interviennent dans de nombreux processus cellulaires liés au repliement des protéines. Les fonctions d'Hsp70 sont indissociables de la présence des cochaperons JDP (J-domain protein). Ces protéines, grâce à leur J-domaine caractéristique, stimulent l'activité ATPasique de Hsp70, facilitent leur prise en charge des substrats et leur confèrent leur localisation cellulaire. L'analyse de la séquence du génome de l'entérobactriophage RB43, membre de la famille du bacteriophage T4, a révélé la présence d'un gène codant une JDP putative (l'orfan 057w). Dans ce travail, nous avons montré que le J-domaine de cette JDP, nommée Rki, est fonctionnel in vivo et que Rki interagit spécifiquement avec le chaperon multifonctionnel Hsp70/DnaK de l'hôte Escherichia coli. Cependant, à la différence des trois cochaperons JDP de DnaK présents chez E. Coli, la protéine Rki ne fonctionne pas comme un cochaperon de type général de DnaK in vivo et in vitro. Au contraire, l'expression de Rki est fortement toxique dans une souche sauvage d'E. Coli. De façon remarquable, cette toxicité est totalement abolie en l'absence de DnaK endogène ou quand le J-domaine de Rki est inactivé. D'autres expériences in vivo ont ensuite révélé que Rki est exprimée précocement durant l'infection par RB43 et que la délétion du gène rki diminue significativement la prolifération du bactériophage. De plus, nous avons trouvé que des mutations dans le gène dnaK de l'hôte suppriment efficacement le phénotype de retard de croissance du phage muté pour le gène rki, indiquant que Rki interfère spécifiquement avec les fonctions cellulaires de DnaK. Enfin, nous avons montré que l'interaction de Rki avec le chaperon DnaK de l'hôte stabilise rapidement le facteur s32 de réponse au choc thermique, qui est normalement adressé à la dégradation par DnaKThe highly conserved molecular chaperone Hsp70 is involved in a plethora of cellular processes associated with protein folding. To function as a molecular chaperone, Hsp70 requires the presence of its obligate J-domain cochaperone partners (JDP). These proteins, thanks to their J-domain signature, stimulate Hsp70's ATPase activity, facilitate substrate delivery and confer specific cellular localization to Hsp70. Genome analysis of the T4-like enterobacteriophage RB43, revealed a gene encoding a putative JDP (orfan 057w). In this work, we first show that the J-domain of this JDP, named Rki, is functional in vivo. Moreover, we show that Rki specifically interacts with the E. Coli host multifunctional Hsp70/DnaK chaperone. However, in sharp contrast with the three known host JDP cochaperones of DnaK encoded by E. Coli, Rki full-length does not act as a generic cochaperone in vivo or in vitro. Expression of Rki alone is highly toxic for wild-type E. Coli, but toxicity is abolished in the absence of endogenous DnaK or when the conserved J-domain of Rki is mutated. Further in vivo analyses revealed that Rki is expressed early after infection by RB43 and that deletion of the rki gene significantly impairs RB43 proliferation. Furthermore, we show that mutations in the host dnaK gene efficiently suppress the growth phenotype of the RB43 rki deletion mutant, thus indicating that Rki specifically interferes with DnaK cellular function. Finally, we show that the interaction of Rki with the host DnaK chaperone rapidly results in the stabilization of the heat-shock factor s32, which is normally targeted for degradation by DnaK
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Herrera, Yelenis. "Modulation of ASIC1a Function by Sigma-1 Receptors: Physiological and Pathophysiological Implications". [Tampa, Fla.] : University of South Florida, 2009. http://digital.lib.usf.edu/?e14.2855.
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Rogers, Angela. "The role of the sigma subunit of adaptor protein-2, AP2σ2, in the regulation of calcium homeostasis". Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:743b6eb9-ddcd-46bd-aee0-78ff4b8b6b2c.
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The calcium sensing receptor (CaSR), a G-protein coupled receptor (GPCR), and its downstream signalling pathways are essential for calcium homeostasis. Loss- or gain-of-function (LOF and GOF, respectively) mutations in components of these pathways result in disorders of calcium homeostasis including Familial hypocalciuric hypercalcaemia (FHH) and Autosomal dominant hypocalcaemia (ADH). FHH type 3 (FHH3) is due to LOF mutations in the s2 subunit (AP2s2) of adaptor protein-2 (AP-2). The FHH3-associated AP2s2 mutations identified to date affect the Arg15 residue and comprise heterozygous Arg15Cys, Arg15Leu, and Arg15His mutations. AP-2 is a ubiquitously expressed heterotetrameric protein, with a central role in clathrin-mediated endocytosis of transmembrane proteins, such as GPCRs. This thesis demonstrates that AP2s2 mutations account for ~7% of all FHH mutations, only affect the Arg15 residue and show evidence of mutation bias, with only AP2s2 Arg15Cys, Arg15Leu, and Arg15His mutations identified. Additionally, the study has identified de novo AP2s2 mutations and revealed that FHH3 probands may present with a broader phenotype not seen in FHH1. The heterozygous missense AP2s2 mutation was demonstrated to have no effect on the stability of the AP-2 complex in vivo. This thesis revealed that gain-of-function AP2s2 mutations are unlikely to be a cause of ADH, which is due to GOF mutations in CaSR in >70% of cases. The CaSR activates its downstream signalling pathways, including the mitogen-activated protein kinase (MAPK) pathway, through coupling with its associated G-proteins. The AP2s2 mutations resulted in loss of activating phosphorylation events in the MAPK pathway suggesting a role for AP2s2 in CaSR signalling. Additionally, the AP2s2 Arg15Cys, Arg15His and Arg15Leu mutations were revealed to exert differential effects on the Gaq/11 and MAPK signalling pathways, suggesting mutation-bias signalling. Clathrin-mediated endocytosis is crucial for embryological development. This thesis has demonstrated that mice homozygous for an Ap2s1 splice site mutation, predicted to produce a functional knock-out, were lethal before embryonic day 12.5 (E12.5), suggesting AP2s2 is essential for cell viability in the developing embryo. Thus, the work of this thesis has further elucidated the role of AP2s2 in the biological pathways of CaSR signalling and clathrin-mediated endocytosis, and in the molecular pathology of disorders of calcium homeostasis.
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Bortoluzzi, Alessio. "Structural characterization of Mycobacterium tuberculosis RNA polymerase binding protein A (RbpA) and its interactions with sigma factors". Thesis, University of Leicester, 2013. http://hdl.handle.net/2381/28401.
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The RNA polymerase binding protein A (RbpA) is a 13 kDa protein, encoded by the gene Rv2050, that was shown to be essential for the growth and survival of the important human pathogen Mycobacterium tuberculosis. Although is not clear yet why RbpA is essential in M. tuberculosis, significant progress has been made in the characterization of the protein. For instance, it was shown that RbpA binds to the β-subunit of the RNA polymerase (RNAP) and activates transcription. Interestingly, it was reported that RbpA can enhance the transcription activity of the RNAP containing the primary σ-subunit σ[superscript A] but does not have any detectable effect if the RNAP is associated with the alternative σ-subunit σ[superscript F]. Moreover, it was also shown that RbpA might influence the response of M. tuberculosis to the current frontline anti-tuberculosis drug rifampicin. The research project described in this thesis contributes to the ongoing efforts to characterize RbpA by providing the structure of the protein and identifying the principle σ-subunit σ[superscript A], and the principle-like σ-subunit σ[superscript B], as interaction partners. The solution structure of RbpA reveals the presence of a central structured region and highly dynamic N- and C- termini. Both termini are involved in the formation of a tight complex with the σ-subunit but only the C-terminal region appears to be essential for this interaction. The finding that RbpA also binds to the RNAP σ-subunit suggests new possibilities for the mechanism of action used by RbpA to activate transcription. Furthermore, preliminary data obtained using a ΔRv2050 conditional mutant strain of M. tuberculosis suggest that the interaction with the σ-subunit is essential for the functionality of RbpA.
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Crottès, David. "Rôle du récepteur Sigma-1 sur la régulation des canaux ioniques impliqués dans la carcinogenèse". Thesis, Nice, 2014. http://www.theses.fr/2014NICE4032/document.
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Le récepteur sigma-1 est une protéine chaperonne active dans des tissus lésés. Le récepteur sigma-1 est principalement exprimé dans le cerveau et joue un rôle neuroprotecteur dans l’ischémie ou les maladies neurodégénératives. Le récepteur sigma-1 est également exprimé dans des lignées cellulaires cancéreuses et des travaux récents suggèrent sa participation dans la prolifération et l’apoptose. Cependant, son rôle dans la carcinogenèse reste à découvrir. Les canaux ioniques sont impliqués dans de nombreux processus physiologiques (rythme cardiaque, influx nerveux, …). Ces protéines membranaires émergent actuellement comme une nouvelle famille de cibles thérapeutiques dans les cancers. Au cours de ma thèse, j’ai montré que le récepteur sigma-1 régule l’activité du canal potassique voltage-dépendent hERG et du canal sodique voltage-dépendent Nav1.5 respectivement dans des cellules leucémiques et des cellules issues de cancer du sein. J’ai également montré que le récepteur sigma-1, à travers son action sur l’adressage du canal hERG, augmente l’invasivité des cellules leucémiques en favorisant leur interaction avec le microenvironnement tumoral. Ces résultats mettent en évidence le rôle du récepteur sigma-1 sur la plasticité électrique des cellules cancéreuses et suggèrent l’intérêt de cette protéine chaperonne comme cible thérapeutique potentielle pour limiter la progression tumoraleThe sigma-1 receptor is a chaperone protein active in damaged tissues. The sigma-1 receptor is mainly expressed into brain and have a neuroprotective role in ischemia and neurodegenerative diseases. The sigma-1 receptor is also expressed into cancer cell lines and recent investigations suggest its involvement into proliferation and apoptosis. However, its role in carcinogenesis remains to delineating. Ion channels are involved in numerous physiological processes (heart beating, nervous influx, …). These membrane proteins currently emerge as a new class of therapeutic targets in cancer. During my thesis, I observed that the sigma-1 receptor regulates voltage-dependent potassium channel hERG and voltage-dependent sodium channel Nav1.5 activities respectively into leukemic and breast cancer cell lines. I also demonstrated that the sigma-1 receptor, through its action on hERG channel, increases leukemia invasiveness by promoting interaction with tumor microenvironment. These results highlight the role of the sigma-1 receptor on cancer cell electrical plasticity and suggest this chaperone protein as a potential therapeutic target to limit tumor progression
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Howles, Sarah Anne. "The role of the G-protein subunit, G-α-11, and the adaptor protein 2 sigma subunit, ap2-σ-2, in the regulation of calcium homeostasis". Thesis, University of Oxford, 2015. http://ora.ox.ac.uk/objects/uuid:9be3d447-4f5c-4782-8d6a-4cb25a8ac6d8.
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The calcium sensing receptor (CaSR) is a G-protein coupled receptor (GPCR) that plays a central role in calcium homeostasis. Loss-of-function mutations of the CaSR cause familial hypocalciuric hypercalcaemia type 1 (FHH1), whilst gain-of-function mutations are associated with autosomal dominant hypocalcaemia (ADH). However, 35% of cases of FHH and 60% of cases of ADH are not due to CaSR mutations. This thesis demonstrates that FHH type 2 (FHH2) and the new clinical disorder, ADH type 2 (ADH2), are due to loss- and gain-of-function mutations in the G-protein subunit, Gα11, respectively. The CaSR signals through Gα11 and FHH2-associated mutations are shown to exert their effects through haploinsufficiency. Three-dimensional modelling of ADH2-associated Gα11 mutations predicts impaired GTPase activity and increases in the rate of GDP/GTP exchange. Furthermore, mouse models of FHH2 and ADH2 have been identified and re-derived to enable in vivo studies of the role of Gα11 in calcium homeostasis. I also demonstrate that FHH3 is due to loss-of-function mutations in the adaptor protein 2 sigma subunit, AP2σ2, which exert dominant-negative effects. AP2σ2 is a component of the adaptor protein 2 (AP2), which is a crucial component of clathrin-coated vesicles (CCV) and facilitates clathrin-mediated endocytosis of plasma membrane components such as GPCRs. All of the identified FHH3-associated mutations affect the Arg15 residue of AP2σ2, which forms key polar contacts with CCV cargo proteins. This thesis proposes that FHH3-associated AP2σ2 mutations impair CaSR internalisation and thus negatively impact on CaSR signalling. In addition, these studies show that these signalling defects can be rectified by the use of the CaSR allosteric modulator cinacalcet, which may represent a useful therapeutic modality for FHH3 patients. In summary, FHH2 is due to loss-of-function mutations in Gα11 causing haploinsufficiency, whilst FHH3 is due to loss-of-function mutations in AP2σ2, which exert dominant-negative effects. In contrast, ADH2 is due to gain-of-function mutations in Gα11.
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Lang, Bradley Thomas. "THE ROLE OF PTPs IN REGENERATION FAILURE FOLLOWING SPINAL CORD INJURY". Case Western Reserve University School of Graduate Studies / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=case1417619755.
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Rayner, Lucy Jane. "Exploring inhibition of the bacterial enhancer binding protein PspF and transcription factor sigma 54 using peptide-based chemical genetic approaches". Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/10957.
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AAA+ proteins (ATPases Associated with various cellular Activities) are a sub-family of P-loop NTPases which convert chemical energy into physical motion to carry out a multitude of functions. The AAA+ protein phage shock protein F (PspF) is a bacterial enhancer binding protein (bEBP) for the major variant transcription factor sigma 54 (σ54) in E. coli. PspF binds to and remodels the promoter DNA bound σ54-RNA polymerase holoenzyme (Eσ54), inducing isomerisation from a transcriptionally silent closed complex to an open complex. Without binding to and concurrent ATP hydrolysis by the bEBP, Eσ54 is unable to spontaneously form the transcriptionally competent open complex, thus maintaining tight control over its associated genes. Conformational changes in the PspF ATP binding site upon hydrolysis are communicated through a pathway of conformational changes in PspF, resulting in the reorganisation of two mobile loops – termed Loop 1 and Loop 2. Loop 1 contains the GAFTGA motif, highly conserved across bEBPs, which forms a stable interaction with σ54. Thus far, traditional approaches such as site-directed mutagenesis and cryo-EM have been used to probe the conformational changes produced at each stage of nucleotide hydrolysis. In this work, the PspF/ σ54 interaction is investigated using a chemical genetic approach. The interactions of designed synthetic peptides with σ54 are investigated using in vitro techniques including a dansyl-based fluorescence binding assay, isothermal titration calorimetry (ITC) and traditional activity based transcription assays. Maltose binding protein fusions of the PspF fragments carrying Loop 1 were also investigated using an in vivo β-galactosidase assay. Finally, Split-Intein Mediated Circular Ligation of Peptides and Proteins (SICLOPPS) was used to create two plasmid-borne cyclic peptide libraries, one random and the other containing the GAFTGA motif. To select for peptides inhibitory to the PspF-σ54 interaction, a selection system was developed based on a promoter-lacZ fusion and the conditionally toxic lactose analogue phenyl-β-D-galactose (Pgal). To confirm hits, a secondary screen based on a structurally and functionally related σ54-bEBP interaction was developed and several peptides active in vivo were identified.
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Stanga, Daniela. "The role of LAR RPTPs family Receptor Protein Tyrosine Phosphatases RPTP Sigma, RPTP Delta and RPTP Lar in mouse embryonic development". Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=117158.
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RPTP_Sigma, RPTP_Delta and RPTP_Lar are the three members of the LAR_RPTPs family. These members share similar structures and superposed expression patterns in different tissues. Although they are key players in different cellular functions their role in embryonic development is little known. We show that RPTP_Sigma, RPTP_Delta and RPTP_Lar are redundant in the heart and liver. Triple mutant (SDLKO) embryos show lethality starting with E12.5 and have defect in erythrocytes maturation translated by a delay in erythroblasts enucleation and persistent truncus arteriosus that leads to a failure in separation of aorta from pulmonary artery.RPTP_Sigma, RPTP_Delta et RPTP_Lar sont trois membres de la famille des LAR_RPTPs. Ces membres partagent un patron d'expression dans différents tissus et des structures similaires. Malgré le fait qu'ils sont des acteurs important de différentes fonctions cellulaires, leur rôle dans le développement embryonnaire reste mal compris. Nos résultats montrent que RPTP_Sigma, RPTP_Delta et RPTP_Lar agissent de façon redondante dans le cœur et le foie. Les embryons triple mutants (SDLKO) commencent à présenter une létalité à partir E12.5 et un défaut dans la maturation des érythrocytes se traduisant par un délai d'énucléation des érythroblastes. De plus, ces embryons montrent un truncus arteriosus persistant causant un défaut dans la séparation entre l'aorte et l'artère pulmonaire.
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Reiß, Kerstin [Verfasser], i Thilo [Akademischer Betreuer] Stehle. "Structural Analyses of the T1L Reovirus Attachment Protein sigma 1 and the Phytophthora sojae Transglutaminase GP42 / Kerstin Reiß ; Betreuer: Thilo Stehle". Tübingen : Universitätsbibliothek Tübingen, 2013. http://d-nb.info/1163396796/34.
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Tran, Amanda P. "Modulation of Receptor Protein Tyrosine Phosphatase Sigma Enhances Protease Activity to Relieve Chondroitin Sulfate Proteoglycan Inhibition of Peripheral Axons and Oligodendrocytes". Case Western Reserve University School of Graduate Studies / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=case1528450936899252.
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Gris, Ormo Georgia. "Selective blockade of the sigma-1 receptor for the treatment of pain of different aetiology: Preclinical studies". Doctoral thesis, Universitat de Barcelona, 2015. http://hdl.handle.net/10803/361398.
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The present Doctoral Thesis focuses on the study of the Sigma-1 receptor (σ1R) in the field of pain. This research has been a part of the preclinical σ1R project focusing on drug discovery of σ1R ligands for the treatment of pain of different aetiologies at the pharmaceutical company ESTEVE.
The main goal of this Doctoral Thesis was to explore the therapeutic interest of σ1R blockade in the pharmacological management of neuropathic, inflammatory and postoperative pain. Neuropathic pain was the main indication at the beginning of this Doctoral Thesis, but inflammatory and postoperative pain had never been explored. The efficacy of the selective σ1R antagonist S1RA (E-58262) in these different types of pain was evaluated, and its potency and efficacy was compared to other marketed analgesic drugs. To this end, two species (rat and mouse), different pain-related behavioural endpoints (hind paw withdrawal response to thermal and mechanical stimulation), and different pharmacological strategies (systemic acute and repeated E-52862 administration), were evaluated. σ1R knockout mice were also used to study the in vivo specificity of E-52862 and the involvement of σ1R in the spinal modulation of several pain-related molecular markers in order to ascertain the mechanism of action of σ1R.
Taken together, the results of this Doctoral Thesis provide new knowledge about σ1R and support the clinical development of selective σ1R antagonists as a suitable therapeutic intervention to achieve analgesia in pain conditions of different aetiology.La presente Tesis Doctoral se centra en el estudio del receptor sigma-1 (σ1) en el campo del dolor. Esta investigación ha sido parte de un proyecto de la empresa farmacéutica ESTEVE centrado en el descubrimiento de fármacos con afinidad por el receptor σ1 para el tratamiento de dolor de diferente etiología. El objetivo principal de esta Tesis fue explorar el interés terapéutico del bloqueo del receptor σ1 para el manejo farmacológico del dolor neuropático, inflamatorio y postoperatorio. Se evaluó la potencia y eficacia del antagonista selectivo del receptor σ1, S1RA (E-52862) en estos diferentes tipos de dolor, y se comparó con otros fármacos analgésicos comercializados. Con este fin, se emplearon dos especies (rata y ratón), diferentes evaluaciones comportamentales relacionadas con el dolor (respuesta de retirada de la pata trasera a la estimulación térmica y mecánica), y diferentes estrategias farmacológicas (administración sistémica aguda y repetida del antagonista E-52862). También se utilizaron ratones knockout por el receptor σ1 para estudiar la especificidad in vivo del E-52862 y la participación del receptor σ1 en la modulación espinal de varios marcadores moleculares relacionados con el dolor con el fin de determinar el mecanismo de acción del receptor. En resumen, los resultados de esta Tesis Doctoral proporcionan nuevos conocimientos sobre el receptor σ1 y apoyan el desarrollo clínico de antagonistas selectivos por este receptor como una intervención terapéutica adecuada para lograr analgesia en condiciones de dolor de diferente etiología.
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Ibrahim, Mahmoud Arafat Abd el-hamid. "Developments and applications in computer-aided drug discovery". Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/developments-and-applications-in-computeraided-drug-discovery(eb57dde8-6190-4ea6-8fa8-219693788daf).html.
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Noncovalent interactions are of great importance in studies on crystal design and drug discovery. One such noncovalent interaction, halogen bonding, is present between a covalently bound halogen atom and a Lewis base. A halogen bond is a directional interaction caused by the anisotropic distribution of charge on a halogen atom X covalently bound to A, which in turn forms a positive region called σ-hole on the A–X axis. Utilization of halogen bonds in lead optimization have been rarely considered in drug discovery until recently and yet more than 50% of the drug candidates are halogenated. To date, the halogen bond has not been subjected to practical molecular mechanical-molecular dynamics (MM-MD) study, where this noncovalent interaction cannot be described by conventional force fields because they do not account for the anisotropic distribution of the charge density on the halogen atoms. This problem was solved by the author and, for the first time, an extra-point of positive charge was used to represent the σ-hole on the halogen atom. This approach is called positive extra-point (PEP) approach. Interestingly, it was found that the performance of the PEP approach in describing halogen bond was better than the semiempirical methods including the recent halogen-bond corrected PM6 (PM6-DH2X) method. The PEP approach also gave promising results in describing other noncovalent halogen interactions, such as C–X···H and C–X···π-systems. The PEP resulted in an improvement in the accuracy of the electrostatic-potential derived charges of halogen-containing molecules, giving in turn better dipole moments and solvation free energies compared to high-level quantum mechanical and experimental data.With the aid of our PEP approach, the first MM-molecular dynamics (MM-MD) study of inhibitors that form a halogen bond with a receptor was performed for tetrahalobenzotriazole inhibitors complexed to cyclin-dependent protein kinase (CDK2). When the PEP approach was used, the calculated MM-generalized Born surface area (MM-GBSA)//MM-MD binding energies for halobenzimidazole and halobenzotriazole inhibitors complexed with protein kinase CK2 were found to correlate well with the corresponding experimental data, with correlation coefficients R2 of greater than 0.90. The nature and strength of halogen bonding in halo molecule···Lewis base complexes were studied in terms of molecular mechanics using our PEP approach. The contributions of the σ-hole (i.e., positively charged extra-point) and the halogen atom to the strength of this noncovalent interaction were clarified using the atomic parameter contribution to the molecular interaction approach. The molecular mechanical results revealed that the halogen bond is electrostatic and van der Waals in nature. The strength of the halogen bond increases with increasing the magnitude of the extra-point charge. The van der Waals interaction’s contribution to the halogen bond strength is most favorable in chloro complexes, whereas the electrostatic interaction is dominant in iodo complexes.The failure of the PM6 semiempirical method in describing noncovalent halogen interactions —not only halogen bonds, but also hydrogen bonds involving halogen atoms— was reported and corrected by the introduction of a second and third generation of noncovalent halogen interactions correction. The developed correction yielded promising results for the four examined noncovalent halogen interactions, namely: C–X···O, C–X···N, C–X···π-system, and C–X···H interactions.
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Chen, Yuemnmu. "Characterization of the sigma receptor : a cocain binding protein /". 2001. http://www.library.wisc.edu/databases/connect/dissertations.html.
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Wang, Ta-Yuan, i 王大元. "characterization of extracytoplasmic function sigma factor (mab4459c) and sigma-associated protein (mab4454c) in Mycobacterium abscessus CS1c". Thesis, 2009. http://ndltd.ncl.edu.tw/handle/46q472.
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碩士國立陽明大學
微生物及免疫學研究所
97
Mycobacterium abscessus is an increasingly important Gram-positive bacterium that can cause a wide spectrum of human infections, such as lung, skin, soft tissue and limbs infections. In earlier study by Thomas, it had been observed that Mycobacterium abscessus clinical strain 390 can be isolated as smooth and rough colony. Both of morphologies can reverse during serial passages. The spontaneously morphologic reversion rate was about 10-5~10-6 and the rough colony was more virulent than the smooth colony. In earlier study by our laboratory, we also analyzed the colony morphology variation of clinical strain CS1c. The CS1c colony morphology can switch from smooth transparent (SmT) to rough (R1), from rough (R1) to smooth opaque (SmO), and from smooth opaque (SmO) to rough (RR2) again. The CS1c morphologic reversion rate was about 10-4~10-5. The major difference between smooth and rough morphology was glycopeptidolipid (GPL) content. GPL was synthesized and regulated by GPL locus. It was predicted that two genes in GPL locus, ecf (mab_4459c) and sap (mab_4454c) may regulate GPL synthesis. The ecf that is thought as extracytoplasmic function sigma factor, may express in optimal stress conditions and growth phase, then transcribed other genes. The sap that is thought as sigma-associated protein may participate in initiation step of transcription. In this study, we demonstrated that Ecf can interact with Sap and that exogenous Ecf can enhance the transcription of ecf, sap and pks promoter but inhibit the transcription of mmpS4 promoter. In the presence of Sap, the transcriptional function of Ecf may be inhibited. In order to analyze the mechanism of colony morphology variation, we over-express exogenous Ecf in Mycobacterium abscessus R1, and found the rough colony could not switch to smooth one. Ecf and Sap protein can regulate parts of GPL locus without being involved in morphological switch. It was known that extracytoplasmic function sigma factor can be up-regulated in optimal growth condition and stress stimulation. In this study transcription from the ecf promoter can be enhanced in log phase in Mycobacterium smegmatis, whereas the optimal stress conditions remained investigated.
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Lo, Yin-Hsiu, i 羅尹秀. "Characterization of extracytoplasmic function sigma factor (MSMEG_0405) and sigma-associated protein (MSMEG_0404) in Mycobacterium smegmatis MC2155". Thesis, 2010. http://ndltd.ncl.edu.tw/handle/16218034266672715711.
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碩士國立陽明大學
微生物及免疫學研究所
98
M. smegmatis as Mycobacterium tuberculosis belongs to the family Mycobacteriaceae. It is generally considered a non-pathogenic microorganism. Therefore, Mycobacterium smegmatis is useful in laboratory experiments for the research analysis of other Mycobacteria species. Glycopeptidolipids（GPLs）are major components present on the outer layers of the cell walls of mycobacteria. Current research has focused on the role of GPL in colony morphology, sliding motility, biofilm formation, and virulence. Two genes in GPL locus, ecf （MSMEG_0405）and sap （MSMEG_0404）may regulate GPL synthesis. The ecf is predicted as an extracytoplasmic function sigma factor and the sap is predicted as a sigma–associated protein. Both may participate in the initiation step of transcription. In this study, we used bacterial two-hybrid assay to demonstrate that Ecf can interact with Sap. In addition, we generated the ecf and ecf sap knockout strain and the reporter construct of ecf, sap, mmpS4, pks, mmpS4, mbtH, fadE5, gtf2 and gtf3 promoter to analyze the promoter activity of these genes in knockout strain and wide-type. According to the previous study, activation of extracytoplasmic function sigma factor is only inducteed under optimal condition or stress stimulation. Here we employed two conditions, 0.02% SDS and 0.5M NaCl for analysis and no significant differences between the ecf or ecf sap deletion strain and the wide-type strain were observed. The previous studies showed that Lsr2 can regulate the genes in the GPL locus. We generated the lsr2 and lsr2 ecf deletion strain to analyze the promoter acitivity in ∆lsr2 knockout strain. The expression level of mbtH, gtf3, rv0926, ecf can be down-regulated; however, the expression level of fadE5, gtf2 and mmpS4 can be up-regulated in ∆lsr2 strain. In this study we demonstrate that Ecf can interact with Sap and the expression of ecf was drived by two promoters. Whether the expression of GPL genes can be regulated by Ecf and Sap remain unknown. The stress to induce Ecf expression and whether Ecf participated in Lsr2 regulation needed to be futher investigation.
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Guglielmi, Kristen Marie. "Structure-function analysis of mammalian orthoreovirus attachment protein [sigma]1". Diss., 2008. http://etd.library.vanderbilt.edu/ETD-db/available/etd-03242008-110238/.
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Gopal, Krishan. "Structural And Biophysical Analysis Of The Regulatory Mechanism Of Mycobacterium Tuberculosis Sigma Factors". Thesis, 2009. https://etd.iisc.ac.in/handle/2005/958.
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Mycobacterium tuberculosis has one ribosomal RNA operon. The survival of this bacillus thus depends on a transcription mechanism that can effectively couple gene expression to changes in the environment. σ factors are transcription proteins that bind to the RNA polymerase (RNAP) and dictate gene expression. Extra Cytoplasmic Function σ factors (ECF) are a subset of σ factors that coordinate environment-induced changes in transcription. The environment specific binding of ECF σ factors to the RNAP presents an effective mechanism for the bacillus to modulate gene expression. ECF σ factors, in turn, are regulated by their interaction with an anti-σ factor. The active σ factor is released from this complex upon specific cellular or environmental stimuli. The aim of this study was to understand the structural and mechanistic aspects of σ factor activation. Towards this goal, two ECF σ factors, σC and σL, were examined. Structural and biophysical studies on M. tuberculosis σC provided a novel insight into ECF σ factor regulation. Inter-domain interactions in σC were sufficient to occlude the DNA recognition regions even in the absence of an interacting protein. The structure of M. tuberculosis σL in complex with the anti-σ factor RslA provides a structural basis to rationalize the release of active σL under oxidative stress. The other chapters of this thesis include a description of the structure and biochemical features of a hypothetical protein Rv2704 that is co-transcribed with the primary σ factor σA. In an effort to understand the collaboration-competition-redundancy model of prokaryotic σ factors, we performed a computational analysis of this system compiling experimental data from the E. coli and B. subtilis model systems. These results are also presented in this thesis. Put together, the structural and biochemical characteristics of the σ factors presented in this thesis suggest substantial variations in the regulatory mechanisms of the M. tuberculosis σ factors when compared to the canonical E. coli or B. subtilis model systems.
This thesis is organized as follows:
Chapter 1: The introductory chapter of this thesis is organized to frame the pertinent mechanistic issues involved in the σ factor-regulatory protein interactions in the context of the underlying biology of M. tuberculosis. The first part of this chapter provides an overview of σ factors and a summary of the classification of these proteins and their roles in different prokaryotes. The latter part of this chapter is a summary of the pathogen M. tuberculosis in terms of its genetic composition, gene expression as well as aspects of virulence and pathogenecity.
Chapter 2: This chapter describes the characterization of the ECF σ factor, σC. Here we report the structure of an ECF σ factor σC from M. tuberculosis. σC is essential for the lethality of M. tuberculosis in a mouse model of infection. Our studies suggest that M. tuberculosis σC differs from the canonical ECF σ factors as it has an N-terminal domain comprising of 126 amino acids that precedes the σC2 and σC4 domains. In an effort to understand the regulatory mechanism of this protein, the crystal structures of the σC2 and C4 domains of σC were determined. These promoter recognition domains are structurally similar to the corresponding domains of E. coli σA despite the low sequence similarity. Fluorescence experiments using the intrinsic tryptophan residues of σC2 as well as surface plasmon resonance measurements reveal that the σC2 and σC4 domains interact with each other. Mutational analysis suggests that the Pribnow box-binding region of σC2 is involved in this inter-domain interaction. Interactions between the promoter recognition domains in M. tuberculosis σC are thus likely to regulate the activity of this protein even in the absence of an anti-σ factor.
Chapter 3 provides an account of the regulatory features of the ECF σ factor, σL. ECF σ factors are often regulated by their interactions with an anti-σ factor that can sense diverse environmental stimuli. Transcriptional responses to changes in the oxidation state are particularly important for M. tuberculosis as it adapts to the environment of the host alveoli and macrophages. Here we demonstrate that the protein RslA binds Zinc and can sequester σL in a reducing environment. Our data suggests that the cytosolic domain at the N-terminus of RslA alone is involved in binding σL. Under oxidizing conditions, the σL/RslA complex undergoes substantial conformational rearrangements that coincide with the release of the Zinc cofactor. In the absence of Zinc, the affinity of RslA for σL reduces by ca 8 fold compared to the holo form. The CXXC motif of RslA acts as a redox sensor. In response to oxidative stimuli, the proximal cysteines in this motif can form a disulfide bond with the release of the bound Zn2+ ion. This observation could be rationalized based on the crystal structure of the σL4/RslA complex. Put together, RslA is a distinct variant of the Zinc binding anti-σ factor (ZAS) family. The structural and biophysical parameters that control σL/RslA interactions demonstrate how variations in the rate of Zinc release and associated conformational changes in RslA could regulate the release of free σL in a measured response to oxidative stress.
Chapter 4 is based on the biochemical and structural characterization of a hypothetical protein Rv2704. The gene for M. tuberculosis Rv2704 is located in the same operon as the principal σ factor σA. The biochemical and structural features of Rv2704 were thus examined to identify its role, if any, in the regulation of σA. This protein is a trimer in solution and adopts a chorismate mutase-like fold. The crystal structure reveals that Rv2704 is a member of the functionally diverse YjgF family of proteins. The important structural differences between Rv2704 and other YjgF proteins lie in the arrangement of secondary structural elements and the putative functional clefts between the subunit interface. Although Rv2704 does not interact with σA in vitro, the structural similarities to the YjgF family suggests that this protein could interact with a variety of metabolites, potentially influencing its function.
Chapter 5 of this thesis is based on a computational analysis of σ factors. Four conformational segments of σ factors, referred to as σ1, σ2, σ3 and σ4 interact with specific regions of promoter DNA. ECF σ factors are a subset of σ factors that coordinate environment-induced transcription. ECF σ factors are minimalist σ factors with two DNA binding domains viz., σ2 and σ4 that recognize the –10 and –35 promoter elements and are unable to interact with either upstream-activating regions or the extended –10 element of the promoter. There are several ECF σ factors in a typical bacterium often characterized by substantial overlap in function. Here we present an analysis of B. subtilis ECF σ factors and their cognate promoters to understand functional overlap and redundancy in this class of proteins. As expected, conserved bases in the –10 element appear more critical for promoter selectivity than the –35 element. However, we note distinct conformational features in the –35 promoter interaction with the helix-turn-helix (HTH) motif when compared to a data-set of known HTH-DNA complexes. Furthermore, we note differences in –35 element interaction between σ factors that act alone and those that overlap in function. The σ factor promoter interactions were then examined vis-à-vis the estimated cellular concentration of these proteins and their affinity to bind the core RNAP. Put together, this analysis suggests that while the cellular protein concentration dictates the choice of an ECF σ factor to form a complex with the RNAP, conformational features of the –35 element serve to select potential collaborative members, a subset of which eventually initiate transcription. Collaborative arrangements and functional redundancy in ECF σ factors are thus possible within the limits placed by these two parameters.
Chapter 6 is a summary of the work reported in this thesis and the conclusions that can be drawn based on these studies.
The appendix section of this thesis comprises of technical details that were not included in the main text of this thesis. Appendix I describes the initial characterization of the M. tuberculosis σD/anti-σD complex. Appendix II provides the experimental protocols as well as some of the supplementary data to the work reported in Chapters 2-5 of this thesis.
42
Gopal, Krishan. "Structural And Biophysical Analysis Of The Regulatory Mechanism Of Mycobacterium Tuberculosis Sigma Factors". Thesis, 2009. http://hdl.handle.net/2005/958.
Pełny tekst źródłaStreszczenie:
Mycobacterium tuberculosis has one ribosomal RNA operon. The survival of this bacillus thus depends on a transcription mechanism that can effectively couple gene expression to changes in the environment. σ factors are transcription proteins that bind to the RNA polymerase (RNAP) and dictate gene expression. Extra Cytoplasmic Function σ factors (ECF) are a subset of σ factors that coordinate environment-induced changes in transcription. The environment specific binding of ECF σ factors to the RNAP presents an effective mechanism for the bacillus to modulate gene expression. ECF σ factors, in turn, are regulated by their interaction with an anti-σ factor. The active σ factor is released from this complex upon specific cellular or environmental stimuli. The aim of this study was to understand the structural and mechanistic aspects of σ factor activation. Towards this goal, two ECF σ factors, σC and σL, were examined. Structural and biophysical studies on M. tuberculosis σC provided a novel insight into ECF σ factor regulation. Inter-domain interactions in σC were sufficient to occlude the DNA recognition regions even in the absence of an interacting protein. The structure of M. tuberculosis σL in complex with the anti-σ factor RslA provides a structural basis to rationalize the release of active σL under oxidative stress. The other chapters of this thesis include a description of the structure and biochemical features of a hypothetical protein Rv2704 that is co-transcribed with the primary σ factor σA. In an effort to understand the collaboration-competition-redundancy model of prokaryotic σ factors, we performed a computational analysis of this system compiling experimental data from the E. coli and B. subtilis model systems. These results are also presented in this thesis. Put together, the structural and biochemical characteristics of the σ factors presented in this thesis suggest substantial variations in the regulatory mechanisms of the M. tuberculosis σ factors when compared to the canonical E. coli or B. subtilis model systems.
This thesis is organized as follows:
Chapter 1: The introductory chapter of this thesis is organized to frame the pertinent mechanistic issues involved in the σ factor-regulatory protein interactions in the context of the underlying biology of M. tuberculosis. The first part of this chapter provides an overview of σ factors and a summary of the classification of these proteins and their roles in different prokaryotes. The latter part of this chapter is a summary of the pathogen M. tuberculosis in terms of its genetic composition, gene expression as well as aspects of virulence and pathogenecity.
Chapter 2: This chapter describes the characterization of the ECF σ factor, σC. Here we report the structure of an ECF σ factor σC from M. tuberculosis. σC is essential for the lethality of M. tuberculosis in a mouse model of infection. Our studies suggest that M. tuberculosis σC differs from the canonical ECF σ factors as it has an N-terminal domain comprising of 126 amino acids that precedes the σC2 and σC4 domains. In an effort to understand the regulatory mechanism of this protein, the crystal structures of the σC2 and C4 domains of σC were determined. These promoter recognition domains are structurally similar to the corresponding domains of E. coli σA despite the low sequence similarity. Fluorescence experiments using the intrinsic tryptophan residues of σC2 as well as surface plasmon resonance measurements reveal that the σC2 and σC4 domains interact with each other. Mutational analysis suggests that the Pribnow box-binding region of σC2 is involved in this inter-domain interaction. Interactions between the promoter recognition domains in M. tuberculosis σC are thus likely to regulate the activity of this protein even in the absence of an anti-σ factor.
Chapter 3 provides an account of the regulatory features of the ECF σ factor, σL. ECF σ factors are often regulated by their interactions with an anti-σ factor that can sense diverse environmental stimuli. Transcriptional responses to changes in the oxidation state are particularly important for M. tuberculosis as it adapts to the environment of the host alveoli and macrophages. Here we demonstrate that the protein RslA binds Zinc and can sequester σL in a reducing environment. Our data suggests that the cytosolic domain at the N-terminus of RslA alone is involved in binding σL. Under oxidizing conditions, the σL/RslA complex undergoes substantial conformational rearrangements that coincide with the release of the Zinc cofactor. In the absence of Zinc, the affinity of RslA for σL reduces by ca 8 fold compared to the holo form. The CXXC motif of RslA acts as a redox sensor. In response to oxidative stimuli, the proximal cysteines in this motif can form a disulfide bond with the release of the bound Zn2+ ion. This observation could be rationalized based on the crystal structure of the σL4/RslA complex. Put together, RslA is a distinct variant of the Zinc binding anti-σ factor (ZAS) family. The structural and biophysical parameters that control σL/RslA interactions demonstrate how variations in the rate of Zinc release and associated conformational changes in RslA could regulate the release of free σL in a measured response to oxidative stress.
Chapter 4 is based on the biochemical and structural characterization of a hypothetical protein Rv2704. The gene for M. tuberculosis Rv2704 is located in the same operon as the principal σ factor σA. The biochemical and structural features of Rv2704 were thus examined to identify its role, if any, in the regulation of σA. This protein is a trimer in solution and adopts a chorismate mutase-like fold. The crystal structure reveals that Rv2704 is a member of the functionally diverse YjgF family of proteins. The important structural differences between Rv2704 and other YjgF proteins lie in the arrangement of secondary structural elements and the putative functional clefts between the subunit interface. Although Rv2704 does not interact with σA in vitro, the structural similarities to the YjgF family suggests that this protein could interact with a variety of metabolites, potentially influencing its function.
Chapter 5 of this thesis is based on a computational analysis of σ factors. Four conformational segments of σ factors, referred to as σ1, σ2, σ3 and σ4 interact with specific regions of promoter DNA. ECF σ factors are a subset of σ factors that coordinate environment-induced transcription. ECF σ factors are minimalist σ factors with two DNA binding domains viz., σ2 and σ4 that recognize the –10 and –35 promoter elements and are unable to interact with either upstream-activating regions or the extended –10 element of the promoter. There are several ECF σ factors in a typical bacterium often characterized by substantial overlap in function. Here we present an analysis of B. subtilis ECF σ factors and their cognate promoters to understand functional overlap and redundancy in this class of proteins. As expected, conserved bases in the –10 element appear more critical for promoter selectivity than the –35 element. However, we note distinct conformational features in the –35 promoter interaction with the helix-turn-helix (HTH) motif when compared to a data-set of known HTH-DNA complexes. Furthermore, we note differences in –35 element interaction between σ factors that act alone and those that overlap in function. The σ factor promoter interactions were then examined vis-à-vis the estimated cellular concentration of these proteins and their affinity to bind the core RNAP. Put together, this analysis suggests that while the cellular protein concentration dictates the choice of an ECF σ factor to form a complex with the RNAP, conformational features of the –35 element serve to select potential collaborative members, a subset of which eventually initiate transcription. Collaborative arrangements and functional redundancy in ECF σ factors are thus possible within the limits placed by these two parameters.
Chapter 6 is a summary of the work reported in this thesis and the conclusions that can be drawn based on these studies.
The appendix section of this thesis comprises of technical details that were not included in the main text of this thesis. Appendix I describes the initial characterization of the M. tuberculosis σD/anti-σD complex. Appendix II provides the experimental protocols as well as some of the supplementary data to the work reported in Chapters 2-5 of this thesis.
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Dvořáková, Pavla. "Vlastnosti expresních vektorů pro Corynebacterium glutamicum a jejich využití při studiu faktorů sigma RNA polymerasy". Master's thesis, 2017. http://www.nusl.cz/ntk/nusl-355666.
Pełny tekst źródłaStreszczenie:
The aim of the thesis was to characterize chosen expression vectors used in biotechnologically important bacterial species, Corynebacterium glutamicum, and to test their use in studies of promoter activity control by sigma factors of RNA polymerase. Different properties of these vectors (level of expression of the cloned gene, leaky expression without inducer, dependence of expression level on inducer concentration and cell population homogeneity) were found by determination of expression level of the model gfpuv gene by fluorescence intensity assay of the produced protein and by gfpuv-expressing C. glutamicum cell population analysis using flow cytometry. The vector pEC-XT99A was chosen for testing the bi-plasmid system for assignment of a sigma factor to the chosen promoter. Although the level of expression provided by pEC-XT99A was not high, the vector showed no leaky expression, expression from the vector was comparable for a wide range of IPTG concentrations and the cell population was homogenous concerning the gene expression. Using pEC-XT99A from which individual stress sig genes were expressed, the σD factor was clearly assigned to the up-to-now unknown Pcg0420 promoter. Another vector for isolation and purification of C. glutamicum proteins was used to express the C. glutamicum sigM gene and to...
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Jané-Valbuena, Judit. "Analysis of the structural and dsRNA binding activities of reovirus protein [sigma]3". 2002. http://www.library.wisc.edu/databases/connect/dissertations.html.
Pełny tekst źródła
45
Wang, Shu-Mei, i 王淑美. "The effects of nucleoid-associated proteins on the core-independent promoter interaction of Bacillus subtilis sigma-A". Thesis, 2015. http://ndltd.ncl.edu.tw/handle/61609577386935207595.
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碩士國立中興大學
生物化學研究所
103
The nucleoid-associated proteins (NAPs) possess DNA-binding activity and the ability to alter DNA molecule by bending, wrapping, and bridging. In addition, NAPs are capable of regulating transcription in a positive or negative manner. It has been reported that the Escherichia coli H-NS protein belongs to the family of NAPs and can improve the functions of some specific regulator proteins. Recently, it has been reported that H-NS can facilitate specific DNA-binding by RNA polymerase σ70 in AT-rich gene regulatory regions. Our lab also found that E. coli H-NS seems to be able to facilitate the binding of Bacillus subtilis σA and E. coli σ70 to their cognate promoter DNA as analyzed by Electrophoretic Mobility Shift Assay (EMSA). The present study is aimed to confirm the previous EMSA results and to answer whether the B. subtilis NAPs such as DnaD, HBsu and LrpC are able to assist σA binding to its cognate promoter DNA in core-independent manner as analyzed by DNA-affinity assay. To fulfill this goal, plasmids which are able to overexpress the three NAPs were first constructed and all of them were purified by column chromatography after overexpression in E. coli BL21 (DE3). The purified NAPs were confirmed to be correct by MALDI-TOF Mass Spectrometry. Furthermore, the results of DNA-affinity assays show that LrpC seems to be able to facilitate the binding of B. subtilis σA to the G3b promoter DNA, but H-NS and HBsu are not.
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Clarke, Marcie B. "The functional characterization of the quorum sensing E. coli regulators B and C in EHEC". 2005. http://edissertations.library.swmed.edu/pdf/ClarkeM121905/ClarkeMarcie.pdf.
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He, Cheng. "14-3-3 [sigma] is a p37 AUF1 binding protein that facilitates AUF1-mediated AU-rich mRNA decay /". 2006. http://wwwlib.umi.com/dissertations/gateway.
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Thesis (Ph.D.)--New York University, Graduate School of Arts and Science, 2006.Typescript. Includes bibliographical references (leaves 112-127). Also available in electronic format on the World Wide Web. Access restricted to users affiliated with the licensed institutions.
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Hsu, Shaoyen, i 許紹彥. "Improve solubility and activity of a recombinant enzyme protein by co-expressing sigma 32 factor in Escherichia coli". Thesis, 2012. http://ndltd.ncl.edu.tw/handle/31355460428258494537.
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碩士國立中正大學
化學工程研究所
100
We used Escherichia coli BL21 overexpressing recombinant enzyme protein, N-acetyl-D-neuraminic acid aldolase (GST-Neu5Ac aldolase-5R or Neu5Ac aldolase), this enzyme protein overexpressed with glutathione S-transferase (GST, Glutathione S-transferase) and polyionic peptide (5R, 5 Arginine) tags became two tags fusion enzyme protein. By genetic engineering technique, we add a new cutting site into the existed E. coli strain (E. coli BL21 pGEX-2TK nana5R), to become E. coli BL21 pGEX-2TK nana5R-XhoI (abbr. XhoI). At the same time we add σ32 factor gene (rpoH) into pGEX-2TK with shine-dalgrno ribosome binding site, right after Neu5Ac aldolase sequence, to become E. coli BL21 pGEX-2TK nana5R-rpoH (abbr. rpoH). During E. coli culture, we add Isopropyl β-D-1-thiogalactopyranoside (IPTG) or arabinose to induce these engineered enzymatic protein expression. Compare with E. coli BL21 pGEX-2TK nana5R and pBAD33 rpoH (abbr. dual, control group), we studied for the changes of proteomics and the activity or solubility of target enzymatic protein. We add rpoH gene to overexpressing σ32 factor, that offsets the conditions of heat-shock. In this condition, E. coli will express heat-shock proteins to improve the defects of low solubility and activity of overexpressed proteins while inclusion body forming. This way will reduce the cost of artificial protein production. Then we extract the protein of E. coli of each group. Through 2-dimensional SDS-PAGE to study the proteomics changes of heat-shock proteins and σ32 factor. The results indicated that XhoI, rpoH and dual groups showed a significant increase of ibpA/B after 3 hours of IPTG or arabinose induction compare with control group (p < 0.05). And a significant decrease of ibpA/B protein in rpoH group compare with XhoI group after 3 hours of IPTG induction (p < 0.05). In western blot, that also indicated σ32 protein increased 3.5 times, 2.1 times and 2.1times in XhoI group after 1, 2, 3 hours of IPTG induction compare with 0 hour. And σ32 protein increased 12.6 times, 11 times and 12.8times in dual group after 1, 2, 3 hours of IPTG and arabinose induction compare with 0 hour. In rpoH group, σ32 protein increased 15.3 times, 16.4 times and 19.1 times after 1, 2, 3 hours of IPTG or arabinose induction compare with 0 hour. The solubility of Neu5AC aldolase had a significant 27% increase in rpoH group compare to XhoI group after 1 hour of IPTG induction, but decrease with induction time. The solubility of Neu5AC aldolase had a significant 10% increase in dual group compare to XhoI group after 1 hour of IPTG and arabinose induction, but decrease with induction time. Another results showed that whole cell activity in rpoH group increased 1.6 times, 1.42 times and 1.41 times after 1, 2, 3 hours of IPTG induction compare with XhoI group. And the whole cell activity in dual group also increased 3.23 times, 2.63 times and 2.29 times after 1, 2, 3 hours of IPTG and arabinose induction compare with XhoI group. The specific activity of supernatant after lysis in rpoH group increased 1.6 times, 3 times and 2.5 times after 1, 2, 3 hours of IPTG induction compare with XhoI group. And the specific activity of dual group also increased 2 times, 1.5 times and 1.7 times after 1, 2, 3 hours of IPTG and arabinose induction compare with XhoI group. The specific activity in the rest insoluble in rpoH group increased 1.4 times 1.8 times and 2.4 times after 1, 2, 3 hours of IPTG induction compare with XhoI group. And in dual group also increased 5 times, 4 times and 2.7 times after 1, 2, 3 hours of IPTG and arabinose induction compare with XhoI group.
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Tsodikov, Oleg Vyacheslav. "Novel quantitative approaches to studies of protein-DNA interactions lac repressor-lac operator and E[sigma]⁷⁰ RNA polymerase-[lambdal]PR promoter complex formation /". 1998. http://catalog.hathitrust.org/api/volumes/oclc/41131217.html.
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Kirchner, Eva [Verfasser]. "Structural and functional studies of the reovirus attachment protein σ1 [sigma-1] and its interaction with the receptor JAM-A = Strukturelle und funktionelle Studien am Zelladsorptionsprotein-σ1 [Zelladsorptionsprotein-sigma-1] des Reovirus und seiner Interaktion mit dem Rezeptor JAM-A / vorgelegt von Eva Kirchner". 2009. http://d-nb.info/994687494/34.
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