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1
Takeda, Armelle-Natsuo. "Role of intracellular cysteines in ENaC function." Paris 6, 2009. http://www.theses.fr/2009PA066110.
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Le canal épithélial sodique ENaC est responsable de la réabsorption du sodium dans les cellules principales du tubule collecteur rénal. La cristallisation d'ASIC, un autre membre de la famille ENaC/Deg, a été publiée mais la constitution du pore interne reste indéterminée. La mutation aS589C du filtre de sélectivité d'ENaC permet le passage du Cd2+ externe capable d'inhiber le canal muté. Nous avons montré que la chaine latérale de la aSer589 n'est pas orientée vers l'intérieur du pore, permettant au Cd2+ de traverser le canal et d'interagir avec la cysteine gCys546. Puis nous nous sommes intéressés au mécanisme d'inhibition d'ENaC par les réactifs sulfhydryl (MTS) internes. Nous avons mis en place une nouvelle approche couplant la méthode d'analyse par substitution de cystéines avec des perfusions internes de MTSEA-biotine. Ainsi, nous pouvons mettre en corrélation les acides aminés accessibles aux MTS internes et ceux qui sont importants dans la fonction du canal.
2
Du, Aiguo. "Prediction of Oxidation States of Cysteines and Disulphide Connectivity." Digital Archive @ GSU, 2007. http://digitalarchive.gsu.edu/cs_diss/28.
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Knowledge on cysteine oxidation state and disulfide bond connectivity is of great importance to protein chemistry and 3-D structures. This research is aimed at finding the most relevant features in prediction of cysteines oxidation states and the disulfide bonds connectivity of proteins. Models predicting the oxidation states of cysteines are developed with machine learning techniques such as Support Vector Machines (SVMs) and Associative Neural Networks (ASNNs). A record high prediction accuracy of oxidation state, 95%, is achieved by incorporating the oxidation states of N-terminus cysteines, flanking sequences of cysteines and global information on the protein chain (number of cysteines, length of the chain and amino acids composition of the chain etc.) into the SVM encoding. This is 5% higher than the current methods. This indicates to us that the oxidation states of amino terminal cysteines infer the oxidation states of other cysteines in the same protein chain. Satisfactory prediction results are also obtained with the newer and more inclusive SPX dataset, especially for chains with higher number of cysteines. Compared to literature methods, our approach is a one-step prediction system, which is easier to implement and use. A side by side comparison of SVM and ASNN is conducted. Results indicated that SVM outperform ASNN on this particular problem. For the prediction of correct pairings of cysteines to form disulfide bonds, we first study disulfide connectivity by calculating the local interaction potentials between the flanking sequences of the cysteine pairs. The obtained interaction potential is further adjusted by the coefficients related to the binding motif of enzymes during disulfide formation and also by the linear distance between the cysteine pairs. Finally, maximized weight matching algorithm is applied and performance of the interaction potentials evaluated. Overall prediction accuracy is unsatisfactory compared with the literature. SVM is used to predict the disulfide connectivity with the assumption that oxidation states of cysteines on the protein are known. Information on binding region during disulfide formation, distance between cysteine pairs, global information of the protein chain and the flanking sequences around the cysteine pairs are included in the SVM encoding. Prediction results illustrate the advantage of using possible anchor region information.
3
Hall, Christopher. "The synthesis, enzymic and chemical reactivity of S-glycosyl cysteines." Thesis, University of Surrey, 2005. http://epubs.surrey.ac.uk/842924/.
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A number of N-acetyl-S-glycosyl cysteine derivatives have been prepared through the development of a general and simply applicable synthetic pathway, by modifying existing literature methods. The coupling of N-acetyl-L-cysteine and a carbohydrate is desirable as it may improve the efficacy of the labile N-acetyl-L-cysteine as a drug. The S-glycosyl cysteines prepared are as follows: N-acetyl-S-D-glucopyranosyl-L-cysteine, alpha and beta-anomers, N-acetyl-S-beta-D-ribopyranosyl-L-cysteine, N-acetyl-S-alpha-D-mannopyranosyl-L-cysteine and N-acetyl-O-methyl-S-(2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl)-L-cysteine. The coupling reaction was designed to yield both a and beta-anomers in the same step, and this was observed in the synthesis of the glucose derivatives. However, the other carbohydrates chosen appear to couple more selectively. The preparation of N-acetyl-O-methyl-S-alpha-D-glucopyranosyl-L-cysteine was also carried out by a different method, but this proved to be more involved and resulted in lower yields. The stability of N-acetyl-S-beta-D-glucopyranosyl-L-cysteine towards the thioglycosidase enzyme myrosinase was studied. N-acetyl-S-beta-D-glucopyranosyl-L- cysteine was found to be stable to hydrolysis by myrosinase, but some inhibition of a standard sinigrin-myrosinase hydrolysis was observed. The thioglycosidic linkage (-SH) of N-acetyl-S-beta-D-glucopyranosyl-L-cysteine is stable to hydrolysis in acidic media, which is contrary to previous work reported in the literature. However, the hydrolysis of the amide group of the cysteine side chain occurred in acidic solution giving the apparently acid-stable S-beta-D-glucopyranosyl-L-cysteine. A kinetic study of this acetyl cleavage was undertaken and a second order rate constant of 5.96 +/- 0.24 x 10-4 dm3 mol-1 hours-1 was obtained.
4
Du, Aiguo. "Prediction of oxidation states of cysteines and disulphide bridges in proteins." unrestricted, 2007. http://etd.gsu.edu/theses/available/etd-11272007-024411/.
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Thesis (Ph. D.)--Georgia State University, 2007.Title from file title page. Y. Pan, committee chair; G. Qin, A. Bourgeois, A. Zelikovski, committee members. Electronic text (124 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed June 3, 2008. Includes bibliographical references (p. 111-124).
5
Lacey, Brian. "Investigation Into the Role of the C-Terminal Vicinal Cysteine Residues in High MR Thioredoxin Reductases." ScholarWorks @ UVM, 2008. http://scholarworks.uvm.edu/graddis/130.
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Mammalian thioredoxin reductase (TR) contains the rare amino acid selenocysteine (Sec), which is essential for the enzyme’s catalytic activity. Substitution of the catalytic Sec residue for a cysteine (Cys) residue, results in a drop in kcat of 100- fold. Homologous high molecular weight TRs from other eukaryotes such as D. melanogaster and C. elegans, have naturally evolved a Sec to Cys substitution in their active sites and these enzymes function with high catalytic activity without the need for a Sec residue. Thus, various TRs can catalyze an identical reaction with either a Cys or Sec residue. A natural assumption in the field has always been that the lower nucleophilicity of a Cys thiol, relative to the selenol of Sec, is the reason for the much lower activity of the mammalian Cys-containing mutant. However, here I provide an alternative explanation. High Mr TRs contain either a Cys-Cys or Cys-Sec dyad that forms an eight-membered ring in the oxidized state during the redox cycle of the enzyme. These eight-membered ring structures are rare in protein structures, presumably due to the strain induced in the intervening peptide bond between the Cys residues. Here I take a “chemical approach” to studying the enzyme mechanism of TR by breaking it into two pieces. This approach is possible because of TR’s structural and mechanistic similarity to glutathione reductase (GR). In comparison to GR, TR contains an additional thiol-disulfide exchange step resulting from the presence of a sixteen amino acid C-terminal extension containing either a vicinal disulfide bond or vicinal selenylsulfide bond. This additional thiol-disulfide exchange step is in the form of the reduction and opening of the eight-membered ring motif. I have constructed a truncated version of the enzyme lacking the amino acid sequence possessing the ring motif so that I could isolate this ring-opening step from the rest of the catalytic cycle by using peptide disulfides/selenylsulfides as substrates. The results of this study using peptide substrates show that the ring opening step is the step of the catalytic cycle that is most effected by Sec to Cys substitution because the higher pKa of the Cys thiolate in comparison to the Sec selenolate means that the Cys residue must be protonated in this step.
6
Heckler, Erin J. "Human quiescin-sulhydryl oxidase 1b role of CxxC motif cysteines in catalysis /." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file, 174 p, 2009. http://proquest.umi.com/pqdweb?did=1818417401&sid=5&Fmt=2&clientId=8331&RQT=309&VName=PQD.
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Cutrera, Jason Lewis. "Insights into the Structure and Function of PrgW and its Conserved Cysteines." Diss., Temple University Libraries, 2014. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/299645.
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Microbiology and ImmunologyPh.D.
Enterococcus faecalis is a Gram-positive bacterial species that is typically a member of the human gastrointestinal tract microbiota. However, E. faecalis is also a nosocomial pathogen, which is involved in urinary tract infections, soft tissue infections and endocarditis. In recent times, the occurrence of antibiotic resistance has complicated the treatment of these infections. One of the major differences between commensal and pathogenic strains of E. faecalis is that pathogens contain multiple mobile elements such as plasmids, transposons and integrative conjugative elements (ICE). These elements allow for the acquisition and transfer of virulence factors and resistance genes. Conjugative plasmids are a class of plasmids present in E. faecalis whose transfer to host cells is induced by a small pheromone peptide, cCF10 (LVTLVFV). This peptide is initially encoded as a 22-amino acid precursor (pre-cCF10) from the signal sequence of the chromosomal ccfA gene and is then proteolytically cleaved by signal peptidase II and Eep. Once pCF10 has been transferred a host E. faecalis cell, it is exceptionally stable. A replicon clone is maintained in greater than 85% of host cells over 100 generations in the absence of selection, suggesting the stability of pCF10 is intrinsic to the replicon. Three unique features of the replication initiation protein PrgW may contribute to this stability: (a) the interaction of PrgW with pre-cCF10, (b) disulfide bond formation at three conserved cysteines (C78, C275, and C307) in PrgW, and (c) processing of the nascent PrgW protein. Replication initiation proteins associated with theta replicons, such as pCF10, are often self-contained units. To initiate plasmid replication, the replication initiation protein (PrgW) binds to direct repeats (oriV) in its own coding sequence (prgW). In silico analysis of PrgW suggests the existence of three distinct domains within the protein. The first 122 amino acids are homologous to a conserved domain present in related replication initiation proteins, which includes a Helix-Turn-Helix (HTH) DNA binding domain. This suggests that this domain of PrgW has a DNA-binding function and binds to the oriV site in prgW. The following 61 amino acids are not similar to any known sequence, and are encoded by the DNA sequence containing the direct repeats in the oriV site. This domain may or may not have a distinct function. The remaining sequence forms a domain that contains cysteines C275 and C307, and is also similar to no known structure. It is hypothesized that this domain is related to the stability of pCF10. C307 appears to be critical, as previous experiments indicate that its mutation alone affects plasmid stability. Secondary structure analysis of this domain revealed the presence of multiple alpha-helices that contain distinct hydrophobic domains that possibly contribute to pre-cCF10 binding and PrgW tertiary structure. The positions of the conserved cysteines within these alpha-helices may stabilize a hydrophobic binding pocket that could potentially facilitate interaction with pre-cCF10. PrgW has a predicted molecular weight of 38.6 KDa and can be detected in Western blots as a band with an apparent approximate molecular weight (mw) of 36,000. Previous data from our lab indicates that, when overexpressed in E. faecalis, four bands of PrgW are present with observed molecular weights of 40,000, 36,000, 24,000 and 18,000. Time course experiments revealed that the 40,000 mw form is converted to a 36,000 mw form independent. The 40,000 mw form is unstable (with a complete turnover in 30 minutes) while the 36,000 mw form has a half-life of greater than 4 hours. The 24,000 mw band does not have a DNA binding motif and is likely a turnover product. When the three conserved cysteines (and only cysteines) in PrgW are replaced with alanine, the 40,000 mw form is still processed to the 36,000 mw form. However, the cysteine to alanine mutants accumulate the 36,000 mw form.
Temple University--Theses
8
Hagedorn, Tara Dawn. "Reactive Sulfur: Redox Reactions of Cysteines and Methionines in the Cytoskeletal Protein Tubulin." W&M ScholarWorks, 2011. https://scholarworks.wm.edu/etd/1539626916.
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Lin, Liwen. "Comprehensive identification of nitroxyl-reactive cysteines in human platelet proteins by quantitative mass spectrometry." Thesis, University of British Columbia, 2011. http://hdl.handle.net/2429/37089.
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HNO, a small compound which can be produced upon hydrolysis of Angeli’s Salt, is
known to be able to inhibit platelet aggregation. An important step to elucidate the
mechanism of the antiaggregatory effect of HNO and define the drug’s targets is the
identification of HNO-reactive proteins in platelets. The sulfhydryl group is considered
the major target of HNO. It can be modified into either sulfinamide or disulfide. The
sulfinamide modification is directly detectable by mass spectrometry; however,
cysteines converted into disulfide groups by HNO cannot be monitored this way,
because the origin of the other cysteine it reacts with is unknown. The goal of this thesis
was to identify HNO-reactive cysteines in human platelet proteins regardless of the
eventual modification states and discover cysteines which might play key roles in
platelet inhibition by HNO.
In the first series of studies, a differential alkylation method was developed, evaluated
on a protein model and applied on human platelets to identify HNO-reactive cysteines.
The results show that 32 HNO-reactive cysteines from 18 proteins were identified.
Moreover, utilizing the detectable mass shift derived from sulfinamide, the
modification states (i.e., sulfinamide or disulfide) of the 32 HNO-reactive cysteines
were further investigated.
In the second series of studies, isotope-coded affinity tag enrichment was combined
with the differential alkylation method to increase the number of HNO-reactive
cysteines discovered. The results show that 159 HNO-reactive cysteines from 78
proteins were identified. Studies have shown that platelet inhibition by HNO was
time-dependent; the effect was not evident after 60 minutes of incubation with HNO.
Therefore, a time-dependent study was performed to identify cysteines whose
HNO-induced modifications were reversed after 60 minutes of incubation. The results
show that the modifications of 83 cysteines out of the 159 HNO-reactive cysteines were reversible. Based on the reactivity toward HNO, the reversibility of HNO-induced
modifications and the biological functions, talin, filamin, α-actinin and integrin αIIbβ3
are proposed as potential drug targets that may play key roles in platelet inhibition by
HNO.
10
Guo, Xiaodan. "The role of C-terminal cysteines in regulating human proteinase-activated receptor-1 function." Thesis, University of Hull, 2010. http://hydra.hull.ac.uk/resources/hull:4483.
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Proteinase-activated receptors (PARs) are a novel group of G-protein coupled receptors (GPCRs). The most striking evidence to distinguish them from other GPCRs is that they carry their own tethered ligand within extracellular N-terminus. To activate the receptors, the tethered ligand is exposed by proteolytic cleavage which subsequently binds to the extracellular loop 2 to trigger receptor function. Four members have been identified so far in this group—PAR1, PAR2, PAR3 and PAR4. Palmitoylation is the reversible covalent attachment of fatty acids to the cysteine residues of some GPCRs via a thioester linkage; resulting in a fourth intracellular loop. Although recent evidence has suggested that palmitoylation can have a profound effect on GPCR function such as cell surface expression and receptor signalling, the role of palmitoylation in regulating PAR function is currently unknown. This study focused on the role of putative palmitoylation region of hPAR1--C-terminal cysteines (C387and C388) in regulating receptor function. Wild type hPAR1 (wt-hPAR1) and hPAR1 mutants (hPAR1C387A, hPAR1C388A and hPAR1C387AC388A) were constructed and permanently expressed in Kirsten virus sarcoma transformed rat kidney epithelial cells (KNRK). hPAR1C387A and hPAR1C388A displayed similar cell surface expression (~80%) to that of wt-hPAR1, but hPAR1C387AC388A displayed only ~40% cell surface expression. hPAR1C387A, hPAR1C388A and wt-hPAR1 displayed similar sensitivity in calcium signalling towards selective PAR1 agonists—thrombin and TFLLR-NH2. Surprisingly, hPAR1C387AC388A failed to generate a calcium signal to either PAR1 agonists. The reduced cell surface expression of hPAR1C387A388A was not responsible for the lack of calcium signal since a wt-hPAR1 cell line with similar cell surface expression to hPAR1C387AC388A displayed robust responses to both thrombin and TFLLR-NH2. hPAR1C387AC388A was also unable to trigger ERK1/2 phosphorylation in response to either PAR1 agonists. In agonist triggered internalisation experiments all mutant receptors internalised in response to thrombin and TFLLR, except for wt-hPAR1 which only internalised in response to thrombin. Therefore, we conclude that putative palmitoylation sites within hPAR1 regulate receptor expression, agonist triggered internalisation and are critical for hPAR1 coupling to calcium and ERK1/2.
11
Qi, Shuang, and 亓爽. "UreE-Hpn/Hpnl interaction in H. pylori, and the role of cysteines in Hpn." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B45962753.
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Haeussler, Dagmar Johanna Franziska. "Regulation of HRAS reactive cysteines in endothelial cell migration - HRAS signaling from the endomembranes." Thesis, Boston University, 2013. https://hdl.handle.net/2144/12774.
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Thesis (Ph.D.)--Boston University
PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.The small GTPase HRas is a major molecular switch, regulating various cellular responses including proliferation, survival, migration and apoptosis. After binding GTP, Ras undergoes a conformational change, and activates downstream signaling partners by recruiting them to distinct cellular membrane localizations. Thus activation through Ras occurs by interaction with effector Ras-binding-domains (RBD) such as that found in Raf and the catalytic subunit p110 of phosphatidylinositol 3-kinase (PI3K). HRas subcellular localization and therefore effector interaction is regulated by its state of S-palmitoylation. Biotin switch assays are a molecular tool for the detection of reversible protein oxidation and S-palmitoylation. To assess the state of H as cysteine modifications during VEGF signaling in EC in the second aim of this thesis, various adaptations were introduced to the protocol allowing accurate detection and differential assessment of reversible oxidative modifications from S-palmitoylation when using the biotin switch assay. By depleting HAECs of endogenous HRas, I provided evidence that HRas was essential in VEGF-mediated activation of the PI3K/Akt/eNOS (endothelial nitric oxide synthase) pathway and EC migration. However, using optimized thiol labeling strategies and immune-fluorescent cell staining demonstrated that only 31% of total HRas is S-palmitoylated, tethering the small GTPase to the plasma membrane but leaving the function of the large majority of endomembrane localized HRas unexplained. Utilizing a knock-down/reconstitution approach, I showed for the first time that endomembrane-delimited HRas mediates VEGF-induced phosphorylation of Akt and activation of eNOS, leading subsequently to EC migration in response to VEGF. The loss of migratory response in cells lacking endogenous HRas was fully restored overexpression of an endomembrane-delimited HRas palmitoylation by modest mutant. These studies define a newly recognized role for endomembrane localized HRAs in mediating nitric oxide-dependent proangiogenic signaling.
13
Qin, Xiaoyan. "Oxidative and electrophilic structural modification and catalytic regulation of human hydroxysteroid sulfotransferase 2a1 (hsult2a1)." Diss., University of Iowa, 2012. https://ir.uiowa.edu/etd/3514.
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Human hydroxysteroid sulfotransferase (hSULT2A1) catalyzes the sulfation of a broad range of endogenous (e.g., hormones, neurotransmitters, bile acids) as well as xenobiotic (e.g, drugs, environmental pollutants) compounds. Alteration in the catalytic activity of hSULT2A1 can lead to outcomes like endocrine disruptions or aberrant drug metabolism and xenobiotic toxicity. Oxidative and electrophilic stresses are known to cause physiological damage and be implicated as possible underlying pathologic mechanisms of a wide range of diseases. To examine the oxidative as well as electrophilic regulation of hSULT2A1, model oxidants (glutathione disulfide (GSSG), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), diamide, tert-butyl hydroperoxide (TBHP)) and electrophiles such as quinone metabolites of polychlorinated biphenyls (PCB-quinones) and phenyl-p- benzoquinone were chosen for this study. Mechanistic studies correlating the enzyme structural modifications with alteration in the catalytic properties were performed to elucidate the catalytic regulative mechanism of an individual oxidant or electrophile.
Thiol oxidants including GSSG, DTNB, and diamide showed catalytic regulation of hSULT2A1. Changes in protein intrinsic fluorescence indicated conformational alterations in hSULT2A1 following the reaction with diamide. Binding properties of hSULT2A1 for its substrates were also altered after reaction with these thiol oxidants, which could be one major reason for the kinetic alteration due to oxidative modification. Formation of mixed disulfides with cysteines in hSULT2A1 was also identified as a result of reaction with GSSG and DTNB.
TBHP was chosen as a model for lipid peroxides, and reaction with this hydroperoxide decreased the catalytic function of hSULT2A1. The dissociation constant for binding of dehydroepiandrosterone (DHEA) was significantly altered with TBHP-pretreatment, but this did not affect the binding of 3',5'-adenosine diphosphate (PAP) to the enzyme. Structural analysis identified cysteine sulfonic acids and methionine sulfoxide formation after reaction of hSULT2A1 with TBHP, which could account for the alterations in the binding properties and the catalytic activity.
Both PCB-quinones and PBQ could regulate the catalytic activity of hSULT2A1. Although PCB-quinones only caused decreases in the catalytic activity at all concentrations tested, pretreatment with PBQ indicated that lower concentrations resulted in an increase in the catalytic activity of hSULT2A1 that was followed by a decrease in the catalytic activity of hSULT2A1 upon increasing the concentration of PBQ in the pretreatment. Differences in the dissociation constants of PAP after PBQ-pretreatment were also observed, indicating the key role played by these PCB-quinones in altering the binding of either PAP or the sulfuryl donors, PAPS. Adducts at cysteines in hSULT2A1 were formed following reactions with PCB-quinones and PBQ. Small amounts of cysteine sulfonic acids and methionine sulfoxides were also formed following reaction of the protein with PCB-quinones and PBQ.
Therefore, alterations in both the catalytic function as well as the structural properties of hSULT2A1 by interaction with oxidants and electrophiles may lead to changes in the metabolism of xenobiotics, as well as alterations in the endogenous balance of various steroid hormones. Such changes may be an important component in physiological damage that occurs under oxidative and electrophilic stress.
14
Unal, Hamiyet. "Ligand-induced conformations of extracellular loop 2 of AT1R." Cleveland State University / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=csu1282068466.
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Osborne, Leisa Jane. "Characterisation of Thioredoxin Dimers: A Biochemical Study." Thesis, Griffith University, 2011. http://hdl.handle.net/10072/365531.
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In addition to the conserved active site cysteines that are responsible for the classical
redox activity of thioredoxins (Trx’s), vertebrate Trx’s contain an additional three
conserved cysteines at position 62, 69 and 73. These structural cysteines are known to
be subjected to a variety of post translational modifications including dimerisation that
are believed to contribute to the regulation and diversity of function of vertebrate Trx’s.
Reports of the formation of “disulphide linked dimers” have been a long standing
observation since the earliest studies on vertebrate Trx’s, however detailed studies on
dimerisation have been limited in number and the extent of characterisation achieved.
Despite the potential for a diversity of dimeric forms with different structural and
functional properties there has been a common assumption arising from the literature
(despite some evidence to the contrary) that all dimers so far described are much the
same and all are redox inactive.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Science
Science, Environment, Engineering and Technology
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Botham, Andrew Michael. "The role of intracellular and extracellular cysteines in regulating human proteinase-activated receptor-2 (hPAR2) function." Thesis, University of Hull, 2007. http://hydra.hull.ac.uk/resources/hull:1372.
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hPAR2, a member of the novel family of proteolytically-activated G-protein coupled receptors termed Proteinase-Activated Receptors (PAR), has recently been implicated in cardiovascular disease. Previous pharmacological studies have found that activation of hPAR2 by mast cell tryptase, (the major PAR2 activator outside the gastrointestinal tract) can be regulated by receptor N-terminal glycosylation. In order to elucidate other post-translational modifications of hPAR2 that can regulate function, we have explored the functional role of three extracellular receptor cysteines (C22, C148, and C226) and one intracellular cysteine (C361).The putative disulphide bridging site (C148 in ECL1 and C226 in ECL2) of hPAR2 were mutagenically removed both individually and together. Mutagenic removal of C226 resulted in ablation of receptor cell surface expression and intracellular retention of receptor. Mutagenic removal of C148 resulted a receptor successfully expressed at the plasma membrane with only a small reduction in cell surface expression over wildtype. The hPAR2C148A mutant still retained the ability to signal through ERK MAP kinase and internalise post-activation with trypsin but was incapable of agonist mediated Ca2+ mobilisation. Removal of C22 resulted in a receptor with similar ERK signalling to wt-hPAR2 but altered agonist-mediated Ca2+ mobilisation. hPAR2C22A showed no change in sensitivity toward SLIGKV-NH2, but a slight decrease in sensitivity towards trypsin, increasing to similar to wt-hPAR2 on pre-treatment with thrombin.The role of the putative palmitoylation site (C361) in regulating hPAR2 function was explored. We demonstrated, using autoradiography, that C361 is the primary palmitoylation site of hPAR2. hPAR2C361A displayed greater cell surface expression compared to wt-hPAR2. The hPAR2C361A also showed a decreased sensitivity and efficacy (intracellular calcium signalling) towards both trypsin and SLIGKV-NH2. In stark contrast hPAR2C361A triggered greater and more prolonged ERK phosphorylation compared to that of wt-hPAR2. Inhibitor studies revealed that hPAR2C361A triggered the majority of the ERK signal through Gi, since pertussis toxin completely inhibited this receptors ability to activate ERK. Finally, flow cytometry was utilised to assess the rate, and extent of receptor internalisation following agonist challenge. hPAR2C361A displayed faster internalisation kinetics following trypsin activation, compared to wt-hPAR2, whilst SLIGKV-NH2 had negligible effect on internalisation for either receptor.This study has highlighted the importance of post-translational modifications in regulating PAR2 function. More specifically we have shown that palmitoylation of C361 on hPAR2 plays a pivotal role in regulating the ability of the receptor to signal to Gq and Gi. Thus, we have identified a potential target site within PAR2 that maybe useful in the design of novel therapeutic agents for cardiovascular disease.
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Thorpe, Chevon N. "The Role of Phospholamban Cysteines in the Activation of the Cardiac Sarcoplasmic Reticulum Ca2+ Pump by Nitroxyl (HNO)." Diss., Virginia Tech, 2012. http://hdl.handle.net/10919/28047.
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Phospholamban (PLN) is an integral membrane protein that regulates the sarco(endo)plasmic Ca2+-ATPase (SERCA2a) within the cardiac sarcoplasmic reticulum (CSR). SERCA2a regulates intracellular Ca2+- handling and thus plays a critical role in initiating cardiac contraction and relaxation. It is believed that dysregulation of SERCA2a is a contributing factor in human heart failure patients. Even though there have been substantial advancements in understanding heart failure pharmacological therapies, patient prognosis remains poor. Nitroxyl (HNO), a new candidate heart failure drug therapy, has been shown to enhance overall cardiovascular function in both healthy and failing hearts, at least in part, by increasing Ca2+ re-uptake into the CSR. Previous research has shown that activation of SERCA2a by HNO is PLN-dependent; however, the mechanism of action of HNO remains unknown. We propose that HNO, a thiol oxidant, modifies one or more of the three PLN cysteine residues (C36, C41, C46) affecting the regulatory potency of PLN toward SERCA2a. To test this hypothesis, a series of PLN mutants were constructed containing single, double and triple cysteine substitutions. Using the baculovirus expression insect cell system, each PLN cysteine mutant was expressed alone and co-expressed with SERCA2a in insect cells and isolated in cellular endoplasmic reticulum (ER) microsomes. Samples were treated with Angeliâ s salt (an HNO donor) to determine the role of each PLN cysteine residue in the mechanism of SERCA2a activation by nitroxyl. Using a standard phosphate activity assay and SDS-PAGE/immunoblot techniques, we determined that the PLN cysteine residues at positions 41 and 46 are important in HNO activation of SERCA2a. Both SERCA2a + 41C PLN and SERCA2a + 46C PLN microsomal samples showed a Î K0.5 of ~0.33 μM and evidence of reversible HNO induced disulfide bond formation. These studies provide important new insight into the mechanism of action of HNO on cardiac SR and thereby help evaluate the drug as a candidate therapy for congestive heart failure.Ph. D.
18
Dayalan, Saravanan, and saravanan dayalan@rmit edu au. "On the Structure Differences of Short Fragments and Amino Acids in Proteins with and without Disulfide Bonds." RMIT University. Computer Science and Information Technology, 2008. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20081128.122615.
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Of the 20 standard amino acids, cysteines are the only amino acids that have a reactive sulphur atom, thus enabling two cysteines to form strong covalent bonds known as disulfide bonds. Even though almost all proteins have cysteines, not all of them have disulfide bonds. Disulfide bonds provide structural stability to proteins and hence are an important constraint in determining the structure of a protein. As a result, disulfide bonds are used to study various protein properties, one of them being protein folding. Protein structure prediction is the problem of predicting the three-dimensional structure of a protein from its one-dimensional amino acid sequence. Ab initio methods are a group of methods that attempt to solve this problem from first principles, using only basic physico-chemical properties of proteins. These methods use structure libraries of short amino acid fragments in the process of predicting the structure of a protein. The protein structures from which these structure libraries are created are not classified in any other way apart from being non-redundant. In this thesis, we investigate the structural dissimilarities of short amino acid fragments when occurring in proteins with disulfide bonds and when occurring in those proteins without disulfide bonds. We are interested in this because, as mentioned earlier, the protein structures from which the structure libraries of ab initio methods are created, are not classified in any form. This means that any significant structural difference in amino acids and short fragments when occurring in proteins with and without disulfide bonds would remain unnoticed as these structure libraries have both fragments from proteins with disulfide bonds and without disulfide bonds together. Our investigation of structural dissimilarities of amino acids and short fragments is done in four phases. In phase one, by statistically analysing the phi and psi backbone dihedral angle distributions we show that these fragments have significantly different structures in terms of dihedral angles when occurring in proteins with and without disulfide bonds. In phase two, using directional statistics we investigate how structurally different are the 20 different amino acids and the short fragments when occurring in proteins with and without disulfide bonds. In phase three of our work, we investigate the differences in secondary structure preference of the 20 amino acids in proteins with and without disulfide bonds. In phase four, we further investigate and show that there are significant differences within the same secondary structure region of amino acids when they occur in proteins with and without disulfide bonds. Finally, we present the design and implementation details of a dihedral angle and secondary structure database of short amino acid fragments (DASSD) that is publicly available. Thus, in this thesis we show previously unknown significant structure differences in terms of backbone dihedral angles and secondary structures in amino acids and short fragments when they occur in proteins with and without disulfide bonds.
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Wang, Yibing. "Study of cysteines in the stalk region of CD3 proteins : evolutionarily conserved residues critical for T cell development and function /." Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2008.
Find full textAbstract:
Thesis (Ph.D. in Immunology) -- University of Colorado Denver, 2008.Typescript. Includes bibliographical references (leaves 138-153). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;
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Joe, Patrick Allen. "A structure-function study of the cysteines and carboxy-terminal tail domain of the beta-III tubulin isotype a dissertation /." San Antonio : UTHSC, 2008. http://learningobjects.library.uthscsa.edu/cdm4/item_viewer.php?CISOROOT=/theses&CISOPTR=43&CISOBOX=1&REC=18.
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Morssing, Vilén Eric. "Studies of Disulfide Bridge Formation in Human Carbonic Anhydrase Between Engineered Cysteines in Non Ideal Conformations Under Equilibrium and Kinetic Conditions." Thesis, Linköping University, The Department of Physics, Chemistry and Biology, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-9120.
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Stabilization of proteins is of great interest for the biotechnological society, industrial as well as research areas. Proteins with high stability are more suitable as reagents, easier to handle, store, transport and use in industrial processes. One way to stabilize a protein is to introduce a disulfide bridge into the structure by protein engineering. In this report the formation of a disulfide bridge between engineered cysteines in non ideal conformations in human carbonic anhydrase has been investigated. The disulfide bridge is not formed when the protein is in its native state. It is shown that when the protein is exposed to mild concentrations of urea in the presence of DTTox the disulfide bridge is formed. Also upon refolding in vitro, in a non oxidative environment, disulfide bridges are formed. This observation is worth to notice, since the disulfide bridge does not form to any appreciable extent when the protein is expressed and folded in vivo in Escherichia coli.
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Bagchi, Pritha. "Expanding the metallomics toolbox: Development of chemical and biological methods in understanding copper biochemistry." Diss., Georgia Institute of Technology, 2013. http://hdl.handle.net/1853/52160.
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Copper is an essential trace element and required for various biological processes, but free copper is toxic. Therefore, copper is tightly regulated in living cells and disruptions in this homeostatic machinery are implicated in numerous diseases. The current understanding of copper homeostasis is substantial but incomplete, particularly in regard to storage and exchange at the subcellular level. Intracellular copper is primarily present in the monovalent oxidation state. Therefore, copper(I) selective fluorescent probes can be utilized for imaging exchangeable copper ions in live cells, but these probes are often lipophilic and hence poorly water soluble. To address this problem, water-soluble fluorescent probes with greatly improved contrast ratio and fluorescence quantum yield are characterized in this work. This work also describes a novel application of water-soluble fluorescent probes, in-gel detection of copper proteins with solvent accessible Cu(I) sites under non-denaturing conditions. Knowledge of copper(I) stability constants of proteins is important to elucidate the mechanisms of cellular copper homeostasis. Due to the high affinity of most Cu(I)-binding proteins, the stability constants cannot be determined directly by titration of the apo-protein with Cu(I). Therefore, accurate determination of Cu(I) stability constants of proteins critically depends on the Cu(I) affinity standards. However, the previously reported binding affinity values of the frequently used Cu(I) affinity standards are largely inconsistent impeding reliable data acquisition for the Cu(I) stability constants of proteins. To solve this problem, a set of water-soluble ligands are developed in this work that form colorless, air-stable copper(I)-complexes with 1:1 stoichiometry. These ligands can be applied as copper(I) buffering agents and affinity standards in order to study copper biochemistry. Copper(I) binding proteins are an integral part of the copper homeostatic machinery and they work in conjunction to regulate copper uptake, distribution, and excretion. However, available evidence indicates the existence of putative copper-binding proteins that are yet to be characterized. Therefore, several proteomics-based methods are developed in this work by employing the strategy to label Cu(I)-binding cysteines in a copper-dependent manner which lays the foundation for the identification of new copper proteins from cellular extracts.
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Hitron, John Andrew. "AN OPTIMIZED SOLID-PHASE REDUCTION AND CAPTURE STRATEGY FOR THE STUDY OF REVERSIBLY-OXIDIZED CYSTEINES AND ITS APPLICATION TO METAL TOXICITY." UKnowledge, 2018. https://uknowledge.uky.edu/toxicology_etds/22.
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The reversible oxidation of cysteine by reactive oxygen species (ROS) is both a mechanism for cellular protein signaling as well as a cause of cellular injury and death through the generation of oxidative stress. The study of cysteine oxidation is complicated by the methodology currently available to isolate and enrich oxidized-cysteine containing proteins. We sought to simplify this process by reducing the time needed to process samples and reducing sample loss and contamination risk.
We accomplished this by eliminating precipitation steps needed for the protocol by (a) introducing an in-solution NEM-quenching step prior to reduction and (b) replacing soluble dithiothreitol reductant with a series of newly-developed high-capacity polyacrylamide-based solid-phase reductants that could be easily separated from the lysate through centrifugation. These modifications, collectively called resin-assisted reduction and capture (RARC), reduced the time needed to perform the RAC method from 2-3 days to 4-5 hours, while the overall quality and quantity of previously-oxidized cysteines captured was increased.
In order to demonstrate the RARC method’s utility in studying complex cellular oxidants, the optimized methodology was used to study cysteine oxidation caused by the redox-active metals arsenic, cadmium, and chromium. As(III), Cr(VI), and Cd(II) were all found to increase cysteine oxidation significantly, with As(III) and Cd(II) inducing more oxidation than Cr(VI) following a 24-hour exposure to cytotoxic concentrations. Label-free proteomic analysis and western blotting of RARC-isolated oxidized proteins found a high degree of commonality between the proteins oxidized by these metals, with cytoskeletal, translational, stress response, and metabolic proteins all being oxidized. Several previously-unreported redox-active cysteines were also identified.
These results indicate that cysteine oxidation by As(III), Cr(VI), and Cd(II) may play a significant role in these metals’ cytotoxicity and demonstrates the utility of the RARC method as a strategy for studying reversible cysteine oxidation by oxidants in oxidative signaling and disease. The RARC method is a simplification and improvement upon the current state of the art which decreases the barrier of entry to studying cysteine oxidation, allowing more researchers to study this modification. We predict that the RARC methodology will be critical in expanding our understanding of reactive cysteines in cellular function and disease.
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CORNET, BRUNO. "Structure et activites de petites proteines riches en cysteines : defensines d'insecte et toxines de scorpion. modelisation sur la base de donnees rmn et modelisation par homologie." Paris 6, 1996. http://www.theses.fr/1996PA066096.
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La structure tridimensionnelle de la defensine a, extraite de l'hemolymphe de phormia terranovae (mouche), a ete determinee sur la base de donnees rmn. La structure de cet antibiotique de 4 kda, stabilisee par trois ponts disulfure, comporte une helice, un feuillet de deux brins anti-paralleles et une grande boucle n-terminale. Cette structure particuliere, baptise csalpha-beta pour cystein stabilized alpha-beta motif, se retrouve egalement dans les defensines de plantes et les toxines de scorpion. Le modele a une bonne resolution sauf la grande boucle n-terminale qui presente une dynamique particuliere. Par homologie avec la defensine a, les structures de defensines extraites d'autres insectes ont ete construites. L'analyse et la comparaison des potentiels tridimensionnelles (electrostatique et lipophilie) montre une grande homogeneite et permet d'etablir quelques hypotheses de relations structures-activite pour cette famille de proteines. La structure de la toxine lqqiii, extraite du venin d'un scorpion du soudan (leiurus quinquestriatus quinquestriatus), a egalement ete determinee sur la base de donnees rmn. La structure de cette neurotoxine de classe alpha de 6 kda presente le motif csalpha-beta. Par contre, la partie n-terminale forme un troisieme brin anti-parallele avec le deuxieme brin du feuillet et une grande boucle c-terminale la complete. Elle possede une double activite anti-insecte et anti-mammifere en se fixant specifiquement sur les canaux sodium de ces deux types d'organismes. La comparaison des potentiels electrostatiques et de lipophilie de plusieurs toxines de scorpion montre une tres grande diversite en rapport avec le large spectre d'activite
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Doyen, Denis. "Régulation de l'échangeur Na[+]/[H+] 1 par les espèces réactives de l'oxygène." Electronic Thesis or Diss., Université Côte d'Azur, 2023. http://www.theses.fr/2023COAZ6007.
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L'échangeur NHE1 Na[+]/H[+] joue un rôle clef dans la régulation du pH et du volume intracellulaire. Son rôle est paradoxal au cours des phénomènes d'ischémie-reperfusion où son activation provoque une surcharge en sodium et indirectement en calcium ce qui peut provoquer des troubles lors de la reperfusion notamment dans les tissus excitables comme le coeur. Cet échangeur est régulé par le pH intracellulaire, le volume cellulaire et par une multitude de voies de signalisation. Plusieurs études et résultats préliminaires ont indiqué une possible régulation par les espèces réactives de l'oxygène qui sont produites lors de l'ischémie-reperfusion. L'objectif de cette étude est d'analyser par quels mécanismes ces espèces réactives de l'oxygène et en particulier l'anion superoxyde O2.- régulent l'activité de NHE1 et de comprendre quelles régions et quels acides-aminés de NHE1 sont importants pour cette régulation.L'activité de NHE1 wild type a été mesurée en présence de donneurs d'O2.- (Ménadione) ou en inhibition de production d'O2.- (Diphényliodonium (DPI) inhibiteur de la NaDPH oxydase). L'activité NHE1 a été quantifiée par flux de lithium en fonction du pH intracellulaire. La Ménadione a ainsi entraîné une activation de NHE1 tandis que le DPI a diminué l'activité de NHE1, démontrant que NHE1 est donc régulé par O2.-. Cette activation par Ménadione et inhibition par DPI sont perdues chez le mutant Cysless de NHE1 dépourvu de toutes ses cystéines. Les cystéines jouent donc un rôle clef dans la régulation de NHE1 par O2.-. Nous avons ensuite testé l'inhibition par le DPI sur des mutants de NHE1 sur chacune de ses cystéines. Cela a permis d'identifier plus précisément 3 cystéines clefs dans ce mécanisme.Afin d'approfondir les groupements structurels des cystéines potentiellement impliqués dans cette régulation, une analyse des modifications covalentes possibles a été menée incluant la recherche de nitrosylation par biotin switch, la recherche de ponts disulfure par exposition à l'iodoacétamide, et la glutathionylation. Cela a permis de montrer la présence de nitrosylation des cystéines, mais pas de ponts disulfures.Parallèlement, nous avons constaté un profil d'adhésion et de migration très différent des cellules selon le type de mutant cystéine de NHE1 exprimé. Nous avons donc analysé l'expression de protéines d'adhésion et la liaison de NHE1 au cytosquelette. Par coimmunoprécipitation de NHE1 avec le complexe ERM (Ezrine/Radixine/Moesine) nous avons pu montrer que les cystéines de ce transporteur sont cruciales pour sa liaison avec le complexe ERM et donc à l'actine corticale.Cette thèse a donc permis de montrer que l'activité de NHE1 est régulée par l'anion superoxyde O2.- et que cette régulation met en jeu certaines cystéines de NHE1 que nous avons identifiées. Nous avons aussi pu montrer que certaines des cystéines de NHE1 sont cruciales pour l'interaction avec le cytosquelette d'actine et jouent un rôle clef dans l'adhésion et la migration cellulaire. L'ensemble de ce travail identifie une connexion clef entre production d'espèces réactives de l'oxygène, la régulation du pH intracellulaire et la motilité et l'adhésionThe NHE1 Na[+]/H[+] exchanger plays a key role in regulating pH and intracellular volume. Its role is paradoxical during ischemia-reperfusion where its activation causes an overload in sodium and indirectly in calcium which can cause disorders during reperfusion, particularly in excitable tissues such as the heart. This exchanger is regulated by intracellular pH, cell volume and by a multitude of signaling pathways. Several studies and preliminary results have indicated a possible regulation by reactive oxygen species that are produced during ischemia-reperfusion. The objective of this study is to analyze by which mechanisms these reactive oxygen species and in particular the superoxide anion O2.- regulate the activity of NHE1 and to understand which regions and which amino acids of NHE1 are important for this regulation.The activity of NHE1 wild type was measured in the presence of O2.- donors (Menadione) or in inhibition of O2.- production (Diphenyliodonium (DPI) NaDPH oxidase inhibitor). NHE1 activity was quantified by lithium flux as a function of intracellular pH. Menadione thus caused an activation of NHE1 while DPI decreased the activity of NHE1, demonstrating that NHE1 is therefore regulated by O2.-. This activation by Menadione and inhibition by DPI are lost in the Cysless mutant of NHE1 deprived of all its cysteines. Cysteines therefore play a key role in the regulation of NHE1 by O2.-. We then tested the inhibition by DPI on mutants of NHE1 on each of its cysteines, which made it possible to identify more precisely 3 key cysteines in this mechanism.In order to deepen the structural groups of cysteines potentially involved in this regulation, an analysis of possible covalent modifications was carried out including the search for nitrosylation by biotin switch, the search for disulfide bridges by exposure to iodoacetamide, and glutathionylation. This showed the presence of nitrosylation of the cysteines, but no disulphide bridges.At the same time, we observed a very different adhesion and migration profile of cells depending on the type of NHE1 cysteine mutant expressed. We therefore analyzed the expression of adhesion proteins and the binding of NHE1 to the cytoskeleton. By coimmunoprecipitation of NHE1 with the ERM complex (Ezrin/Radixin/Moesin) we showed that the cysteines of this transporter are crucial for its binding to the ERM complex and therefore to cortical actin.This thesis has therefore made it possible to show that the activity of NHE1 is regulated by the superoxide anion O2.- and that this regulation involves certain cysteines of NHE1 that we have identified. We also showed that some of the cysteines of NHE1 are crucial for the interaction with the actin cytoskeleton and play a key role in cell adhesion and migration. All of this work identifies a key connection between the production of reactive oxygen species, the regulation of intracellular pH and motility and adhesion
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Zhang, Chi Ph D. Massachusetts Institute of Technology Department of Chemistry. "Cysteine arylation." Thesis, Massachusetts Institute of Technology, 2017. http://hdl.handle.net/1721.1/112448.
Full textAbstract:
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2017.Cataloged from PDF version of thesis.
Includes bibliographical references.
Proteins are the chemical and biological foundation of life. The longstanding goals of chemical biology are to understand the structure and function of proteins and to use these biomolecules for applications in chemistry, biology, medicine, and material science. To achieve such goals, highly efficient, selective, and robust chemical reactions are desired to modify proteins. For decades, cysteine-based reactions with maleimides and alkyl halides are the primary methods for selectively tagging proteins with fluorescent dyes, affinity and radio labels, drug molecules, and polymers and nanocomposites. These traditional reactions generate sulfur-sp³ carbon bonds between the cysteine thiol and the labeling reagents. The goal of this thesis is to develop new cysteine arylation reactions to generate sulfur-sp² carbon bonds on proteins. These reactions are used to make novel peptide and protein therapeutics. Two mechanistically complementary approaches are developed to arylate cysteine thiol. First, through a nucleophilic aromatic substitution (SNAr) mechanism, fluorinated aromatic reagents are used for the regioselective arylation of a single cysteine in the presence of many. An enzyme-tag pair (glutathione S-transferase (GST) and glutathione (GSH)) and a self-labeling short peptide (Phe-Cys-Pro-Phe, the 7-clamp) are respectively developed to recognize the fluorinated aromatic reagents and to promote the arylation reaction in aqueous solution. The second approach utilizes organometallic palladium reagents to chemoselectively install electron-neutral and electron-rich aryls on cysteine thiols. These cysteine arylation reactions are applied to the synthesis of macrocyclic peptides and antibody-drug conjugates (ADCs). Long unprotected macrocyclic peptides up to forty residues are efficiently synthesized using the GST enzyme. Using bispalladium reagents, macrocyclic peptides bearing aryl linkers are synthesized via crosslinking of two cysteine thiols. [pi]-Clamp antibodies enable a one-step synthesis of site-specific antibody-drug conjugates to selectively kill cancer cells. Organometallic palladium reagents are used to synthesize linker-free ADCs where the drug molecules are directly linked to cysteine thiols in antibodies.
by Chi Zhang.
Ph. D.
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Ólafsson, Ísleifur. "Molecular biology and clinical studies of human cystatin C." Lund : Dept. of Clinical Chemistry, University of Lund, University Hospital, 1994. http://catalog.hathitrust.org/api/volumes/oclc/39761834.html.
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Ramos, Tania. "Cysteine biosynthesis in Leishmania." Thesis, University of Glasgow, 2014. http://theses.gla.ac.uk/5156/.
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Every year 2 million people are diagnosed with leishmaniasis and 350 million are at risk of becoming infected. Spread throughout 88 countries in the world, leishmaniasis is a group of diseases comprising visceral, mucocutaneous and cutaneous leishmaniasis as the main forms. Visceral leishmaniasis is the most severe form of the disease and is caused by Leishmania donovani. The parasite, Leishmania, is transmitted to humans by a sandfly vector. The absence of vector-control procedures and effective vaccines for humans makes chemotherapy the only weapon against leishmaniasis. Great efforts have been made to develop new drugs against these diseases, however toxic side effect and the constant cases of resistance call for new drug targets and drugs to be discovered and developed. Cysteine is a key building block of trypanothione, an antioxidant unique to trypanosomatids that plays a pivotal role for the survival of the parasites. Leishmania can obtain cysteine in two ways, using the sulphydrylation and trans-sulphuration pathways. Humans lack the sulphydrylation pathway, thus this, and especially cysteine synthase (CS), of Leishmania could be a potential drug target. In order to determine the relative importance of these pathways, the levels of thiols at different stages of promastigote growth of wild-type, mutants lacking CS (deltacs), and CS episomal re-expressor parasite lines were determined. It was found that during logarithmic phase the mutant parasites have significantly reduced levels of thiols, which is reversed in the CS re-expressing parasite line. The mRNA and protein levels of cystathionine β-synthase (CBS) were increased in deltacs L. donovani, while this decrease was reversed in the CS re-expessing line. These data suggest that the reverse trans-sulphuration pathway compensates for the loss of CS to some extent but that this is not sufficient to maintain thiol levels during logarithmic growth. It was further found that ornithine decarboxylase mRNA is up-regulated while the protein levels appear to be reduced in the deltacs parasite line. The latter was supported by the finding that the deltacs mutant parasites have low sensitivity to alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase. The studies on the recombinant L. major cysteine synthase (CS) have shown that it can be inhibited by small peptides based on the C-terminal of L. major serine acetyltransferase. The CPM assay showed promising results to be used in high throughput screening of CS inhibitors. The data overall suggests that the levels of thiols are dependent on an adequate supply of cysteine through the sulphydrylation pathway. How this affects polyamine biosynthesis is yet to be investigated.
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Shiu, Hoi-yan Fiona, and 邵凱恩. "Cleavable cysteine modification by electron-deficient alkynes and molecular imaging of cysteine and homocysteine." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B45996325.
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Kingsbury, Oliver William. "The inhibition of cysteine proteinases." Thesis, University of Oxford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360390.
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Watson, Mark Wayne. "Cysteine-rich proteins of chlamydia." Thesis, University of Southampton, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.385342.
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Evans, Ethan Daniel. "Long peptides for cysteine arylation." Thesis, Massachusetts Institute of Technology, 2018. http://hdl.handle.net/1721.1/118258.
Full textAbstract:
Thesis: Ph. D. in Biological Chemistry, Massachusetts Institute of Technology, Department of Chemistry, 2018.Cataloged from PDF version of thesis.
Includes bibliographical references.
Biological reactivity is typically carried out by large enzymes. There are few examples of reactive, amino-acid-based polymers shorter than 100 residues in length. Of those that do exist, the majority are very short tags (<15 amino acids). Here, we attempted to first discover peptides roughly 30 amino acids in length that promote a nucleophilic aromatic substitution reaction and then understand the features and properties that emerge. Using the 20 canonical amino acids, there are 20³⁰ different peptide sequences possible in this size realm. To isolate a portion of the space capable of reacting with a perfluoroaromatic small molecule, we performed an mRNA display selection. This uncovered a host of putative reactive peptides with little similarity at the sequence level. The primary isolate (MP01) displayed reactivity confirming the success of the selection and was sensitive to truncation and denaturants. We next set out to study the reactivity of an expanded portion of the isolated peptides and look for shared structural or mechanistic themes. Analyzing an additional 26 peptides with almost no sequence similarity, we discovered diverse levels of reactivity along with sequences capable of undergoing multiple reactions. Using computational structure prediction and circular dichroism spectroscopy we discovered that both mixed alpha-helical and random coil as well as beta-sheet-based reactive peptides existed. Studying their structural properties revealed that many of the peptides undergo significant structural alterations upon reaction with the perfluoroaromatic. Returning to MP01 we studied its mutational tolerance as well as its structural and mechanistic properties. Alanine scanning mutagenesis revealed mutations that diminished reactivity in addition to others that improved its function. Computational structure prediction suggested a mixed helical and random coil structure. Combining the beneficial mutations with insights from modeling initiated an iterative process that ultimately led to a 100-fold improved reaction rate. This sequence (MP01-Gen4) was six mutations different from MPG 1 and was more reactive than any other peptide discovered. MP01-Gen4 displayed flexibility and lacked a defined three-dimensional structure, however, it was significantly more helical than its progenitor. This sequence also displayed structural alterations, becoming more helical in the presence of its small molecule reaction partner, when either covalently reacted or noncovalently interacting.
by Ethan Daniel Evans.
Ph. D. in Biological Chemistry
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Nathani, R. I. "Novel approaches for cysteine bioconjugation." Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1431698/.
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This thesis describes and investigates novel strategies for cysteine modification to achieve protein bioconjugation. Chapter 1 provides an introduction to the research project with an overview of protein modification techniques. Chapter 2 describes the development of a site selective dual labelling strategy based on substrate controlled cysteine modification. The application of this strategy to green fluorescent protein is also detailed. Chapter 3 describes the development and evaluation of thiophosphonium as a platform for protein modification. An in-depth discussion on reaction mechanisms, pathways, stability of thiophosphonium and the utility of this platform is also included. Chapter 4 describes the development of bromomaleimide based reversible cysteine modification, with particular focus on development of improved strategies for conversion of thiomaleimides back to free cysteines. The utility of the approach was demonstrated using a proof of concept experiment.
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Creasy, Blaine. "Inflammatory Regulation of Cysteine Cathepsins." VCU Scholars Compass, 2008. http://scholarscompass.vcu.edu/etd/1586.
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Cysteine cathepsins B, L and S are endosomal/lysosomal proteases that participate in numerous physiological systems. Cathepsin expression and activity are altered during various inflammatory diseases, including rheumatoid arthritis, atherosclerosis, neurodegenerative diseases and cancers. Early immune responses to microbial pathogens are mediated by pattern-recognition receptors, including Toll-like receptors (TLR). Signaling through TLR causes cell activation and release of inflammatory mediators, which can contribute to the severity of chronic inflammatory diseases. The impact of TLR cell activation on cathepsins B, L and S activities was investigated using live-cell enzymatic assays. Individual ligands of TLR4, TLR2 and TLR3 increased intracellular activities of the three cathepsins indicating the involvement of both MyD88-dependent and -independent pathways. To investigate the role of inflammatory cytokines in regulating these proteases, a lipopolysaccharide (LPS) non-responsive cell line was utilized. LPS non-responsive cells co-cultured with LPS responsive macrophages upregulated cathepsin activities. Furthermore, culture supernatants from LPS-stimulated macrophages increased cathepsin activities in LPS non-responsive cells, which could be reduced by neutralizing antibodies to TNF-α or IL-1β. These findings indicate cytokines regulate cathepsin activities during macrophage responses to TLR stimulation. Using LPS as a model for inflammation, the ability of the cannabinoids, delta9-tetrahydrocannabinol (THC), and CP55940 to suppress cysteine cathepsins during an inflammatory response was investigated. Cannabinoids, including the major psychoactive component of marijuana THC, modulate a variety of immune responses and have been proposed as possible therapeutics to control chronic inflammation. Cannabinoids may mediate their effects through receptor-dependent or independent mechanisms. Cannabinoid receptor subtype 1 (CB1) and receptor subtype 2 (CB2) have differential expression in leukocytes. Dose response studies showed that 1 nM THC was sufficient to inhibit cathepsin enhancement in LPS-stimulated cells. P388D1 macrophages expressed CB2 mRNA, but had no detectable CB1 mRNA indicating a role for the CB2 receptor. Utilizing a CB2-/- macrophage cell line, the role of CB2 receptor participation in THC inhibition of cysteine cathepsin upregulation was explored. THC did not affect cathepsin activity in LPS-stimulated cells lacking CB2 expression. These findings support the possibility of receptor selective agonists as therapeutic treatment during inflammatory diseases to prevent cathepsin involvement in pathological tissue destruction.
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Creasy, Blaine Madison. "Inflammatory regulation of cysteine cathepsins /." Unavailable until 8/19/2013, 2008. http://hdl.handle.net/10156/2273.
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Håkansson, Katarina. "Cystatin C functions in vitro and in vivo studies on target enzyme inhibition by cystatin C variants and cystatin C deficient mice /." Lund : Dept. of Clinical Chemistry, University of Lund, University Hospital, 1998. http://catalog.hathitrust.org/api/volumes/oclc/40343026.html.
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Adcock, Harriet Jane. "Novel sources of cysteine conjugate #beta#-lyase for the study of halogenated cysteine conjugate toxicity." Thesis, De Montfort University, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.391328.
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Ismail, Ihab. "Function and Regulation of Xylem Cysteine Protease 1 and Xylem Cysteine Protease 2 in Arabidopsis." Diss., Virginia Tech, 2003. http://hdl.handle.net/10919/11243.
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A functional water-conducting system, the tracheary elements of the xylem, is required to sustain plant growth and development. Tracheary element formation is dependent on many biological processes terminated by programmed cell death and cellular autolysis. The final two processes are probably dependent on the activity of hydrolytic enzymes such as XCP1 and XCP2 known to be expressed in tracheary elements during these final two processes. Thus, the transcriptional regulation of XCP1 and the function of XCP2 were investigated. Qualitative and quantitative assessments of GUS activity as directed by various fragments of the XCP1 promoter showed that a 237-bp internal region was able to drive GUS expression in a tracheary element-specific manner in Arabidopsis. A 25-bp deletion at the 3' end of this region abolished GUS expression. The 237-bp region served as bait in a yeast one-hybrid analysis. Screening of yeast colonies retrieved 109 putative positive interactions, which included a potential transcriptional regulator, indole acetic acid-induced protein 8 (IAA8). An auxin responsive element that potentially binds auxin responsive transcription factors was found within the 25-bp deletion. Cis-elements were predicted by Genomatix and Athamap computer programs. The cis-elements form pyrimidine and gibberellic acid responsive elements that can potentially bind Dof and Myb transcription factors, respectively. In an independent effort, attempts to develop a mapping population to isolate upstream regulators of XCP1 expression did not succeed. Functionally, tracheary element-specific expression of XCP2 in Arabidopsis suggested a specialized role for XCP2 in final phases of tracheary element differentiation. The function of XCP2 was assessed using T-DNA insertional mutants, post-transcriptional gene silencing, and through tracheary element-specific expression of the cysteine protease inhibitor, soyacystatin N in Arabidopsis. Our findings revealed that the absence of XCP2 expression due to T-DNA insertional mutagenesis did not affect plant growth and development in the laboratory. Soyacystatin N was an effective in vitro inhibitor of cysteine proteases. Plants expressing 35S-driven cytosolic form of soyacystatin exhibited stunting and reduced apical dominance. Plants expressing pXCP1-driven cytosolic soyacystatin did not differ from wild type plants. Additionally, transgenic plants expressing pXCP1- and 35S-directed XCP2-double-stranded RNA for the silencing of XCP2 showed no unusual phenotypes compared to their wild type counterpartsPh. D.
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Ho, Han Kiat. "An investigation of cellular responses to tetrafluoroethylcysteine-induced mitochondrial dysfunction /." Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/8175.
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Wang, Xiaoguang Stanbury David McNeill. "Mechanism of the outer-sphere oxidation of aqueous L-Cysteine and of iodide in acetonitrile by a series of iron (III) complexes." Auburn, Ala., 2007. http://repo.lib.auburn.edu/Send%2011-10-07/WANG_XIAOGUANG_13.pdf.
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Pace, Nicholas. "Peptide-Based Probes To Monitor Cysteine-Mediated Protein Activities." Thesis, Boston College, 2015. http://hdl.handle.net/2345/bc-ir:104128.
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Thesis advisor: Jianmin GaoThesis advisor: Eranthie Weerapana
Cysteine residues are known to perform an array of functional roles in proteins, including nucleophilic and redox catalysis, regulation, metal binding, and structural stabilization, on proteins across diverse functional classes. These functional cysteine residues often display hyperreactivity, and electrophilic chemical probes can be utilized to modify reactive cysteines and modulate their protein functions. A particular focus was placed on three peptide-based cysteine-reactive chemical probes (NJP2, NJP14. and NJP15) and their particular biological applications. NJP2 was discovered to be an apoptotic cell-selective inhibitor of glutathione S-transferase omega 1 and shows additional utility as an imaging agent of apoptosis. NJP14 aided in the development of a chemical-proteomic platform to detect Zn2+-cysteine complexes. This platform identified both known and unknown Zn2+-cysteine complexes across diverse protein classes and should serve as a valuable complement to existing methods to characterize functional Zn2+-cysteine complexes. Finally, NJP15 was part of a panel of site-selective cysteine-reactive inhibitors of protein disulfide isomerase A1 (PDIA1). These inhibitors show promise in clarifying the unique and redundant properties of PDIA1's dual active-sites, as well as interrogating the protein's role in cancer. Together, these case studies illustrate the potential of cysteine-reactive chemical probes to modulate protein activities, interrogate biological systems, and aid in the development of powerful therapeutic drugs
Thesis (PhD) — Boston College, 2015
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
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Pol, Ewa. "Mechanism of interaction of the mammalian cysteine protease inhibitors, cystatin A and B, with target proteases /." Uppsala : Swedish Univ. of Agricultural Sciences (Sveriges lantbruksuniv.), 2001. http://epsilon.slu.se/avh/2001/91-576-5927-3.pdf.
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Nycander, Maria. "Interaction of cystatins with cysteine proteinases /." Uppsala : Swedish Univ. of Agricultural Sciences (Sveriges lantbruksuniv.), 1998. http://epsilon.slu.se/avh/1998/91-576-5428-X.gif.
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Qusti, Safa Yousef. "Cysteine metabolism : modulation and cellular effects." Thesis, University of Birmingham, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.396065.
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Dai, Peng Ph D. Massachusetts Institute of Technology. "Site-selective modification of cysteine residues." Thesis, Massachusetts Institute of Technology, 2018. http://hdl.handle.net/1721.1/115793.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2018.Cataloged from PDF version of thesis.
Includes bibliographical references.
Proteins are biomolecules that carry out essential work in cells and are required for the structure, function, and regulation of biological systems. It is desired to install designer modifications on proteins of interest, in a site-selective manner mimicking nature's post-translational modifications, for various biological and therapeutic applications. Towards this goal, efforts were devoted to develop site-selective protein modification methods. The [pi]-clamp-mediated cysteine perfluoroarylation, a novel sequence-selective protein modification strategy, enabled regioselective cysteine modification in the presence of competing cysteines on protein surface. The major goals of this thesis include improving and understanding the [pi]-clamp-mediated reaction, and discovering new sequence-selective protein modification chemistries. Aiming to improve the n-clamp-mediated site-selective protein modification, the effect of inorganic salts is discovered. Salt in aqueous solution significantly changes the reaction rate of [pi]clamp-mediated cysteine bioconjugation over four orders of magnitude, enabling fast and sitespecific modification of proteins including antibodies. The salt effect on n-clamp-mediated arylation is concentration-dependent and ion-specific following the Hofmeister series. Salts composed of early member ions in the Hofmeister series accelerate the reaction, while late members decrease the rate. To understand the unique self-labelling reactivity of the n-clamp tetrapeptide (Phe-Cys-Pro-Phe), mutational, computational, and structural investigations are carried out. Our findings suggest several structural and chemical features contribute to the unique reactivity of [pi]-clamp including the trans prolyl amide bond, lowered reaction enthalpy of activation ([delta]H**), reduced cysteine pKa and side chain-perfluoroaryl electrophile interactions. A sequence-selective thiol-yne reaction is discovered. The seven-residue peptide tag (DBCO-tag, Leu-Cys-Tyr-Pro-Trp-Val-Tyr) can selectively react with various azadibenzocyclooctyne (DBCO) reagents. To demonstrate the sequence-selectivity, this reaction is applied to site-selective cysteine modificaition in proteins bearing more than one cysteine residue. In addition, organometallic palladium oxidative addition complexes bearing O-phenyl carbamate are developed for protein crosslinking between cysteine and lysine residues. A glutathione S-transferase (GST) catalyzed site-selective macrocyclization reaction for peptides up to 40 amino acids in length is also reported.
by Peng Dai.
Ph. D.
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Baumann, Alice Leonie. "Electrophilic Phosphonothiolates for Cysteine-selective Bioconjugations." Doctoral thesis, Humboldt-Universität zu Berlin, 2020. http://dx.doi.org/10.18452/22194.
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In dieser Arbeit wurden ungesättigte Vinyl- und Ethynylphosphonothiolate hergestellt und als Linker für Cystein-selektive Proteinmodifikationen verwendet. Zunächst wurde, ausgehend von ungesättigten Phosphoniten und elektrophilen Disulfiden als Startmaterialien, eine Syntheseroute zur Herstellung von ungesättigten Phosphonothiolaten entwickelt. Die hohe Chemoselektivität dieser Reaktion ermöglichte die Einführung von Vinylphosphonothiolaten auf ungeschützten Modellpeptiden und dem Protein Ubiquitin. Es konnte gezeigt werden, dass ungesättigte Phosphonothiolate unter neutralen bis leicht basischen Bedingungen selektiv mit Thiolen reagieren und sich daher als Linker für Cystein-selektive Proteinmodifikationen eignen. Die Vielseitigkeit der hier entwickelten Biokonjugationsmethode wurde in drei Anwendungen demonstriert. Ausgehend von dem Vinylphoshonothiolat-modifizierten Ubiquitin konnten homogene Ubiquitin-Protein-Konjugate erzeugt werden; zum einen ein nicht hydrolysierbares Diubiquitin-Konjugat und zum anderen ein Ubiquitin-α–Synuclein-Konjugat. Des Weiteren wurden ungesättigte Phosphonothiolate als Linker in Antikörper-Wirkstoff-Konjugaten getestet. Hier zeigte sich, dass insbesondere Vinylphosphonothiolat-Linker Potential zur Herstellung von stabilen Antikörper-Konjugaten aufweisen. Schließlich wurden Vinylphosphonothiolate als Linker verwendet, um sowohl Chaperon-bindende Antikörper als auch Deubiquitinasen (DUBs) mit photoreaktiven Crosslinkern auszustatten um damit dynamische Protein-Interaktionen zu untersuchen.
Insgesamt ermöglicht das hier entwickelte Verfahren die chemoselektive Umwandlung von elektrophilen Disulfiden in elektrophile Vinyl- und Ethynylphosphonothiolate, wodurch Reaktivität für eine Thioladdition induziert wird. Dadurch können zwei komplexe, thiolhaltige Moleküle selektiv konjugiert werden, was insbesondere für die Herstellung von homogen modifizierten Peptid- und Proteinkonjugaten von Bedeutung ist.In this work, unsaturated vinyl- and ethynylphosphonothiolates were synthesised and used as linkers for cysteine-selective protein modifications. First, a synthetic route for the generation of unsaturated phosphonothiolates was developed, using unsaturated phosphonites and electrophilic disulfides as starting materials. The high chemoselectivity of this reaction enabled the introduction of vinylphosphonothiolates on unprotected model peptides and the protein ubiquitin. It could then be shown that unsaturated phosphonothiolates react selectively with thiols under neutral to slightly basic conditions and are therefore suitable as linkers for cysteine-selective protein modifications. The versatility of the herein developed bioconjugation method was demonstrated in three applications. First, starting from the vinylphosphonothiolate-modified ubiquitin, homogeneous ubiquitin-protein conjugates could be generated, in particular a non-hydrolyzable diubiquitin conjugate and a ubiquitin-α-synuclein conjugate. Second, the suitability of unsaturated phosphonothiolates as linkers for the generation of stable antibody-drug conjugates was tested. Vinylphosphonothiolate linkers thereby showed potential to produce stable antibody conjugates. Finally, vinylphosphonothiolates were used as linkers to conjugate both chaperone-binding antibodies and deubiquitinases (DUBs) to photo-reactive crosslinkers in order to investigate dynamic protein interactions. Overall, the herein developed methodology enables the chemoselective conversion of electrophilic disulfides into electrophilic vinyl- and ethynylphosphonothiolates, which in turn react selectively with thiols. As a result, two complex, thiol-containing molecules can be selectively conjugated, which is particularly important for the production of homogeneously modified peptide and protein conjugates.
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Rawat, Reetika. "Characterization of the promoter of SmCP, the gene encoding Solanum melongena cysteine proteinase." Thesis, Click to view the E-thesis via HKUTO, 2004. http://sunzi.lib.hku.hk/hkuto/record/B34740156.
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Leung, Donmienne Doen Mun. "Studies of serine and cysteine protease inhibitors /." St. Lucia, Qld, 2001. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe16491.pdf.
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Witheford, Richter Miranda. "Olfactory ensheathing cell mediated mechanisms of neurite outgrowth and axon regeneration." Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/963.
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The capacity of the olfactory neuraxis to undergo neuronal replacement and axon targeting following injury, has led to scrutiny concerning the molecular and physical determinants of this growth capacity. This is because injury to the central nervous system, in contrast, leads to permanent disconnection of neurons with targets. Olfactory ensheathing cells (OECs), a specialized glial cell, may contribute to olfactory repair, and have been used to promote recovery from spinal cord injury. However, there mechanisms underlying OEC-induced regeneration are poorly appreciated.
To understand these mechanisms, OECs from the lamina propria (LP OECs) or olfactory bulb (OB OECs) were transplanted into a lesion of the dorsolateral funiculus. While both cells demonstrated reparative capacities, LP and OB OECs differentially promoted spinal fibre growth; large-diameter neurofilament-positive, CGRP-positive, and serotonergic fibres sprouted in response to both LP and OB OEC transplantation, whereas substance-P and tyrosine hydroxylase-positive neurons grew more extensively following OB or LP OEC transplantation, respectively.
To further understand the growth of spinal cord neurons in response to OECs, a proteomic analysis of OEC secreted factors was performed, identifying secreted protein acidic and rich in cysteines (SPARC) as a mediator of OEC-induced outgrowth in vitro. To test the contributions of SPARC to spinal cord repair after OEC transplantation, cultures of LP OECs from SPARC null and wildtype (WT) mice were transplanted into a crush of the dorsolateral funiculus. Substance P and tyrosine hydroxylase positive axon sprouting was significantly reduced in SPARC null OEC-treated animals, suggesting that individual factors may contribute to OEC-promoted regeneration.
To investigate the effect of OECs on corticospinal (CST) neurons, an in vitro assay was developed using postnatal day 8 CST neurons. Coculture of CST neurons with OB OECs produced extensive axon elongation. Application of OB OEC secreted factors increased CST neurite branching, but did not increase axon elongation. In contrast, plating of CST neurons on OB OEC plasma membrane resulted in extensive axon elongation. Furthermore, the OB OEC plasma membrane could overcome CST neurite outgrowth inhibition induced by an outgrowth inhibitor. Together these findings provide insight into OEC mechanisms of neurite outgrowth and axon regeneration.
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Ovat, Asli. "Design, synthesis and evaluation of cysteine protease inhibitors." Diss., Georgia Institute of Technology, 2009. http://hdl.handle.net/1853/33822.
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Cysteine proteases are important drug targets due to their involvement in many biological processes such as protein turnover, digestion, blood coagulation, apoptosis, cell differentiation, cell signaling, and the immune response. In this thesis, we have reported the design, synthesis and evaluation of clan CA and clan CD cysteine protease inhibitors.
Aza-peptidyl Michael acceptor and epoxide inhibitors for asparaginyl endopeptidases (legumains) from the bloodfluke, Schistosoma mansoni (SmAE) and the hard tick, Ixodes ricinus (IrAE) were designed and synthesized. SARs were similar, but with some notable exceptions. Both enzymes prefer disubstituted amides to monosubstituted amides in the P1' position and potency increased as we increased the hydrophobicity of the inhibitor in this position. Extending the inhibitor to P5 resulted in increased inhibitory potency, especially against IrAE, and both enzymes prefer small over large hydrophobic residues in the P2 position. Aza-peptide Michael acceptor inhibitors are more potent than aza-peptide epoxide inhibitors and, for some of these compounds, second order inhibition rate constants are the fastest yet discovered.
We have also synthesized aza-peptidyl Michael acceptor and epoxide inhibitors for the parasitic cysteine proteases; cruzain, rhodesain. We have found that monosubstituted amides were favored over disubstituted amides indicating the involvement of the amide hydrogen in a H-bond network. We have shown that aza-peptide epoxides were as potent as Michael acceptors and we have obtained compounds with IC50 values as low as 20 nM.
We have worked on the synthesis of heterocyclic peptidyl α-ketoamides, peptidyl ketones and aza-peptidyl ketones as calpain inhibitors. We have synthesized peptidyl α-ketoamides with nucleotide bases in the primed region to create compounds that can cross the blood-brain barrier. We have improved the potency by introducing a hydrophobic group on the adenine ring. We have obtained compounds with Ki values in the nanomolar range. We have designed peptidyl aminoketones as a new class of inhibitors for calpain. Peptidyl aminoketones were less potent than peptidyl α-ketoamides but still reasonable inhibitors of calpain that have the potential to cross the BBB.