Thrombospondin 1 (TSP1) is a 450 kDa homotrimeric multidomain glycoprotein with fundamental roles in many cell-cell and cell-matrix interactions. These varied, and sometimes conflicting, functions are mediated by specific domains in TSP1. One region with diverse biological roles is the Ca2+ binding loops (or type 3 repeats). The biological activity of this region is determined through a complex assembly of disulfide bonds linking structure and function. Disulfide interchange in a protein is usually very specific and quite slow, unless catalysed. I have found that protein disulfide isomerase (PDI) is expressed on the surface of platelets and endothelial cells in a reduced active conformation. The presence of enzymatically active PDI on the surface of TSP1-secreting cells suggests PDI is well positioned to catalyse disulfide interchange in, and regulate the structure/function relationships of, TSP1. PDI was observed to form disulfide-linked complexes with TSP1. Moreover, incubation of platelet or fibroblast TSP1 with PDI enhanced binding of an isomer-specific anti-TSP1 antibody whose epitope is in the Ca2+ binding loops. These findings suggest that PDI may mediate disulfide bond rearrangement in both the soluble and extracellular matrix-bound forms of TSP1. TSP1 is a tight-binding competitive inhibitor of neutrophil cathepsin G; however, incubation with PDI increased the Ki for the interaction ???10-14-fold. TSP1 bound platelet-derived growth factor (PDGF) tightly in the region of the Ca2+ binding loops and supported binding of PDGF to its receptor. PDI-mediated disulfide interchange in TSP1 ablated PDGF binding, indicating that PDI-catalysed disulfide interchange in TSP1 may modulate PDGF-TSP1 complex formation and the biological activity of PDGF. Finally, PDI-catalysed isomerization of TSP1 potently affected its cell adhesive properties. Treatment of TSP1 with PDI enhanced adhesion and spreading of endothelial cells through the ??v??3 integrin receptor to TSP1, by exposure of a cryptic RGD sequence. Thus, endothelial cell surface PDI may be a physiological regulator of RGD-dependent binding to TSP1. These data suggest that cell-surface PDI may regulate the disulfide-bonded structure and certain biological functions of TSP1. In conclusion, I propose a novel mechanism for the post-translational regulation of TSP1 structure/function, which in turn may regulate certain aspects of TSP1 in vascular biology.

Protein disulfide isomerase (PDI), a member of the thioredoxin superfamily, contains two domains with significant sequence homology to the active sites in thioredoxin. PDI facilitates the folding of nascent proteins in the endoplasmic reticulum (ER), binds hormones and Ca�����, catalyzes the glutathione dependent reduction of dehydroascorbate, serves as a major chaperone molecule in the ER and serves as a subunit for prolyl-4-hydroxylase and microsomal triglyceride transferase. Because of its abundance in the ER and association with disease and chemically induced toxicity, the goal of this research was to investigate the relative susceptibility of PDI thiols to alkylation. The sensitivity of PDI to 1-chloro-2,4-dinitrobenzene (CDNB), iodoacetamide (IAM) and biotinoylated iodoacetamide (BIAM) was explored. The relative susceptibility of the thiolate anions present in the two active sites of PDI each containing the -CGHC- sequence was investigated with mass spectrometric techniques. PDI was inactivated by CDNB but was not found as sensitive as thioredoxin reductase as shown by Amer and coworkers (1995). IAM and BIAM were used as model alkylating agents to explore the two active sites of PDI and determine the residues most susceptible to alkylation. Alkylation by IAM and BIAM was first detected at the N-terminal cysteine in each active site (-C*GHC-) followed by alkylation at the second cysteine residue (-C*GHC*-) as shown by tandem mass spectrometry. Mass spectroscopy showed that the episulfonium ion derived from the glutathione conjugate of 1,2-dichloroethane, S-(2-chloroethyl)glutathione (CEG), decreased activity and protein thiols of PDI. CEG produced two protein adducts at very low excesses of CEG over PDI; however, higher concentrations resulted in several protein adducts. Only one modification in each active site at the N-terminal cysteine residue can be identified, indicating that while these thiolate anions of PDI are susceptible, it would appear that the episulfonium ion may present itself to other sites as well. This may have important toxicologic significance regarding the mechanism of 1,2-dichloroethane toxicity and the role of PDI in the redox status of the cell. / Graduation date: 2003

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Thesis (M.S.)--University of Texas at El Paso, 2008. / Title from title screen. Vita. CD-ROM. Includes bibliographical references. Also available online.

Thesis (M.S.)--University of Texas at El Paso, 2009. / Title from title screen. Vita. CD-ROM. Includes bibliographical references. Also available online.

Thesis advisor: Eranthie Weerapana / Disulfide-bonding cysteine residues perform critical roles in the structural stabilization and redox regulation of protein function. Secreted proteins are often enriched for structural disulfide bonds conferring conformational stability in the oxidizing extracellular environment. The controlled formation of disulfide bonds in secreted proteins is regulated in the endoplasmic reticulum (ER) by the protein disulfide isomerase (PDI) family. To investigate disulfide-bond formation in the ER, quantitative chemical-proteomic methods were coupled to subcellular-fractionation-based ER enrichment. Cysteine reactivity studies identified highly reactive post-translationally modified cysteine residues including disulfide-bonding cysteines. Upon discovering a highly reactive population of traditionally oxidized cysteines, the percentage of oxidation for cysteines localizing to the ER was determined. Next, ER function was chemically perturbed to evaluate changes to cysteine oxidation following upregulation of the unfolded protein response (UPR). Disulfide bond formation was specifically disrupted in the ER by CRISPR-Cas9-mediated PDIA1 and PDIA4 knockout. The effects of PDI knockout on cancer cell phenotype and changes to cysteine oxidation states were evaluated. Finally, in vitro studies were performed to evaluate PDIA4 oxidase activity and identify potential PDIA4-selective inhibitors. In the future, the platforms developed within may be applied to profiling changes to cysteine oxidation in other biological systems such as other organelles and disease states. / Thesis (PhD) — Boston College, 2019. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Cholera toxin (CT) is a classic A-B type protein toxin that has an A subunit (A1 + A2) and a pentameric B subunit. The catalytic A1 domain is linked to the A2 domain via a disulfide linkage. CTA1 must be dissociated from the rest of the toxin to cause a cytopathic effect. Protein disulfide isomerase (PDI) can reduce the CTA1/CTA2 disulfide bond, but disassembly of the reduced toxin requires the partial unfolding of PDI that occurs when it binds to CTA1. This unfolding event allows PDI to push CTA1 away from the rest of the toxin. My research question is whether the efficiency of PDI in disassembling CT would be affected by molecular crowding, where a dense internal cell environment is recreated in vitro by the use of chemical agents such as Ficoll. This will give insight on how CT behaves inside a cell. Our hypothesis was that molecular crowding would make CTA1 disassembly more efficient by recreating the tight packing of macromolecules in cells, which provides an extra nudge to enhance toxin disassembly. We then used enzyme-linked immunosorbent assays (ELISAs), a pull-down assay and a biochemical assay to determine how molecular crowders affect the binding, reduction, and disassembly of CT by PDI. Our results will bring about a deeper understanding of the cellular events that may affect the course of a cholera infection. From the preliminary results, molecular crowders increased PDI's ability to bind to CTA1 and did not prevent PDI from cleaving the CTA1/CTA2 disulfide bond. Based off the disassembly results, molecular crowders reduced PDI's ability to displace CTA1 from the rest of the toxin. This contradicts our original hypothesis. Our new hypothesis is that crowders block PDI unfolding, which is required for CT disassembly. Biophysical experiments using Fourier Transform Infrared Spectroscopy will test this prediction in future work.

Cholera, caused by the secretion of cholera toxin (CT) by Vibrio cholerae within the intestinal lumen, triggers massive secretory diarrhea which may lead to life-threatening dehydration. CT is an AB5-type protein toxin that is comprised of an enzymatically active A1 chain, an A2 linker, and a cell-binding B pentamer. Once secreted, the CT holotoxin moves from the cell surface to the endoplasmic reticulum (ER) of a host target cell. To cause intoxication, CTA1 must be displaced from CTA2/CTB5 in the ER and is then transferred to the cytosol where it induces a diarrheal response by stimulating the efflux of chloride ions into the intestinal lumen. Protein disulfide isomerase (PDI), a resident ER oxidoreductase and chaperone, is involved in detaching CTA1 from the holotoxin. The PDI domain(s) that binds to CTA1 and precisely how this interaction is involved in CTA1 dissociation from the holotoxin are unknown. The goal of this project is to identify which domain(s) of PDI is responsible for binding to and dislodging CTA1 from the CT holotoxin. Through incorporation of ELISA, surface plasmon resonance (SPR), and Fourier transform infrared (FTIR) spectroscopy techniques in conjunction with a panel of purified PDI deletion constructs, this project aims to provide important molecular insight into a crucial interaction of the CT intoxication process.

Thesis advisor: Eranthie Weerapana / The 1,3,5-triazine is a privileged scaffold in that it is planar and has three-fold symmetry which allows for controlled modification around the ring structure with various substituents. In this thesis, we report on two modular inhibitor libraries that center around a 1,3,5-triazine core scaffolding system, which have been shown to target protein disulfide isomerase A1 (PDIA1), glutaredoxin-3 (GLRX3), and 6-phosphofructo-1-kinase (PFKP). Protein disulfide isomerase A1 (PDIA1) is a thiol-disulfide oxidoreductase localized in the lumen of the endoplasmic reticulum (ER), and is an important folding catalyst and chaperone for proteins in the secretory pathway. PDIA1 contains two active-site domains (a and a’), each containing a Cys-Gly-His-Cys (CGHC) active-site motif. Here, we synthesize a targeted library o second-generation triazine-based inhibitors to optimize the potency and selectivity of our lead compound, RB-11-ca. Characterization of this targeted library afforded an optimized PDIA1 inhibitor, KSC-34, which covalently modifies C53 in the a site of PDIA1 and demonstrates time-dependent inhibition of the reductase activity of PDIA1 in vitro with a kinact/KI = 9.66 x 103 M-1s-1. Interestingly, KSC-34 treatment demonstrated that a-site inhibition led to decreased secretion of amyloidogenic antibody light chain, thus illustrating that site-selective inhibitors like KSC-34 provide useful tools for delineating the pathological role and therapeutic potential of PDIA1. In 2014, our lab first reported on RB7, a dichlorotriazine-based electrophilic small molecule which displayed extremely high reactivity and selectivity toward lysine residues in the proteome. Herein, we further on this study by investigating the unique reactivity of RB7 through the synthesis of a second-generation small molecule electrophile library and investigating proteome-wide reactivity in vitro and in situ. This library afforded KSC-46, an RB-7 analogue with p-chlorothiophenol tuning element, which provided optimal proteome reactivity to use as a scaffold for the generation of a targeted library. To take advantage of the tuned reactivity of KSC-46, a second-generation targeted library was generated to target react residues in the proteome. This library yielded two molecules, KSC-56 and KSC-65, which were identified to target glutaredoxin-3 (GLRX3) and 6-phosphofructo-1-kinase (PFKP), respectively. GLRX3 is a cytosolic, monothiol iron-sulfur cluster chaperon protein which relies on two nucleophilic cysteine residues to bind and transfer iron clusters. PFKP is known to catalyze the first irreversible step in glycolysis and regulates the flux of glucose metabolism in the cell, which makes PFKP an attract therapeutic target. KSC-56 was further characterized to bind to Cys261 in the C-terminal glutaredoxin domain of GLRX3. / Thesis (PhD) — Boston College, 2018. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

A Dissulfeto Isomerase Proteína (PDI) é uma chaperona redox essencial responsável pela inserção correta das ligações dissulfeto em proteínas nascentes no retículo endoplasmático. Nesta localização celular, bem como em outras regiões, como na superfície celular, a PDI atua na manutenção da homeostase redox e sinalização. Houve substanciosa evolução no conhecimento sobre a estrutura e funções da PDI, graças a estudos in vitro que utilizam a PDI purificada, quimeras ou seus domínios isolados. Nestas abordagens experimentais, as medidas das atividades redutase e chaperona da PDI são realizadas de forma relativamente simples. Entretanto, medir a atividade isomerase, que é a atividade autêntica da família das PDIs, é tecnicamente bastante complexo. Em células e tecidos, o papel da PDI tem sido descrito com base principalmente em estratégias experimentais de ganho e perda de função. Todavia, ainda há pouca informação na correlação entre os resultados funcionais com a medida das atividades da PDI. Este trabalho compila os principais métodos descritos para medir as quatro atividades da PDI: tiol redutase, tiol oxidase, tiol isomerase e chaperona, com ênfase na descrição de controles e interferentes críticos, como os tampões que contém surfactantes. Ainda, discutir-se-á criticamente os resultados obtidos quando da transposição destes métodos para amostras de homogenatos (celular ou tecidual) / Protein disulfide isomerase is an essential redox chaperone from endoplasmic reticulum, responsible for correct disulfide bond insertion in nascent proteins. At the endoplasmic reticulum and other locations including the cell surface, PDI accounts for redox homeostasis and signaling. Knowledge about PDI structure and function evolved substantially from in vitro studies using purified PDI and chimeras. In these experimental scenarios, PDI reductase and chaperone are readily approachable. However, isomerase activity, the hallmark of PDI family, is significantly complex. Assessment of PDI roles in cells and tissues mainly relies on gain- or loss-of-function experiments. However, there is limited information regarding correlation of these results with PDI activities. In this manuscript, we put together the main methods described for measuring the four PDI activities: thiol reductase, thiol oxidase, thiol isomerase and chaperone, with emphasis on controls and critical interferents, such as detergent-containing buffers. We also discuss the transposition of these methods from purified PDI to cellular or in vivo samples, with critical thoughts about the interpretation of results