Chronic kidney disease (CKD), defined as a decrease in renal function, is a global issue. The treatment of CKD and its comorbidities imparts a costly burden on the American healthcare system, therefore the need for therapeutics that prevent the progression of chronic kidney disease is urgent. Microarray studies have shown that the serine protease inhibitor clade A member 3 (SERPINA3) is transcriptionally upregulated in kidney injury. SERPINA3 is an extracellular protease inhibitor that maintains the homeostasis of extracellular matrix proteins. Our lab hypothesizes that SERPINA3 might not only be a transcriptional biomarker for kidney injury, but the SERPINA3 protein might act as a key upstream regulator in the advancement of renal inflammation and fibrosis. Our research characterizes the expression patterns of SERPINA3 in models of acute and chronic kidney injury through immunoblotting and immunohistochemistry. Our unilateral ureteral obstruction (UUO) model of chronic renal injury displays significant glomerular localization of SERPINA3. The adenine diet model of chronic kidney injury and the renal ischemic reperfusion injury (RIRI) model of acute kidney injury both display tubular upregulation of SERPINA3. The DOCA-salt hypertension model of chronic kidney injury was imposed on two strains of mice, C57BL/6 and 129/sv, both of which display tubular and glomerular upregulation of SERPINA3. However, the C57BL/6 strain, which is known for its resistance to glomerular sclerosis, displays higher renal localization of SERPINA3 when exposed to DOCA-salt hypertension, than does the 129/sv strain. In conclusion, our data suggests that SERPINA3 protein is upregulated in both acute and chronic kidney injury. The role of SERPINA3 in these models remains unknown, however, our lab theorizes that SERPINA3 protein may be renoprotective in certain instances of kidney injury. Functional assays must be performed to elucidate the role of SERPINA3 in these models of kidney injury. Characterizing the function of SERPINA3 in chronic and acute kidney injury might aid in the development of novel therapeutics to prevent the advancement of CKD.

<p>Inhibitors belonging to the serpin (serine protease inhibitor) family control proteases involved in various physiological processes. All serpins have a common tertiary structure based on the dominant b-sheet A, but they have different inhibitory specificity. The specificity of a serpin is determined by the Pl-Pl’ peptide bond acting as a bait for the target protease which is made up of an exposed reactive centre loop (RCL). The serpin plasminogen activator inhibitor type-1 (PAI-1) is the main physiological inhibitor of urokinase-type and tissue-type plasminogen activators (uPA and tPA, respectively). Elevated plasma levels of PAI-l have been correlated with a higher risk of deep venous thrombosis, and PAI-1 is a risk factor for recurrent myocardial infarction. Furthermore, PAI-1 has a role in cell migration and has been suggested to regulate tumor growth and angiogenesis. PAI-1 is unique among the serpins in that it can spontaneously and rapidly convert into its latent form. This involves full insertion of the RCL into b-sheet A. </p><p>There were two partially overlapping goals for this thesis. The first was to use latent PAI-1 as model for development of a fluorescence-based method, Donor-Donor Energy Migration for intramolecular distance measurements. The second goal was to use DDEM, together with other biochemical methods, to reveal the structure of the PAI-1/uPA complex, the conformation of the RCL in active PAI-1, and molecular determinants responsible for the conversion of PAI-1 from the active to the latent form.</p><p>The use of molecular genetics for introduction of fluorescent molecules enables the use of DDEM to determine intramolecular distances in a variety of proteins. This approach can be applied to examin the overall molecular dimensions of proteins and to investigate structural changes upon interactions with specific target molecules. In this work, the accuracy of the DDEM method has been evaluated by experiments with the latent PAI-1 for which X-ray structure is known. Our data show that distances approximating the Förster radius (57±1 Å) obtained by DDEM are in good agreement (within 5.5 Å) with the distances obtained by X-ray crystallography.</p><p>The molecular details of the inhibitory mechanism of serpins and the structure of the serpin/protease complex have remained unclear. To obtain the structural insights required to discriminate between different models of serpin inhibition, we used fluorescence spectroscopy and cross-linking techniques to map sites of PAI-1/uPA interaction, and distance measurement by DDEM to triangulate the position of the uPA in the complex. The data have demonstrated clearly that in the covalent PAI-1/uPA complex, the uPA is located at the distal end of the PAI-1 molecule relative to the initial docking site. This indicates that serpin inhibition involves reactive center cleavage followed by full loop insertion, whereby the covalently linked protease is translocated from one pole of the inhibitor to the opposite one. </p><p>To search for molecular determinants that could be responsible for conversion of PAI-1 to the latent form, we studied the conformation of the RCL in active PAI-1 in solution. Intramolecular distance measurements by DDEM, the newly a developed method based on probe quenching and biochemical methods revealed that the RCL in PAI-1 is located much closer to the core of PAI-1 than has been suggested by the recently resolved X-ray structures of stable PAI-1 mutants, and it can be partially inserted. This possibly explains for the ability of PAI-1 to convert spontaneously to its latent form. </p>

This thesis was focused on the identification and characterization of the salivary proteins from Ixodes ricinus, the European vector of Lyme disease and tick-borne encephalitis causative agents. In the first part of this work, the the transcriptomic approach was used in order to identify and describe I. ricinus salivary proteins. The second part is dealing with functional and structural characterization of the salivary protein named IRS-2 (I. ricinus serpin-2).

>Magister Scientiae - MSc / Human African trypanosomiasis is the world’s third most important parasitic disease affecting human health after malaria and schistosomiaisis. The world health organization estimates approximately 60 million people at risk in sub-Saharan Africa and up to 50,000 deaths per year caused by trypanosomiasis. Current management of human African trypanosomiasis relies on active surveillance and chemotherapy of infected patients. Efforts to develop a vaccine to immunize the human host have been hampered by antigenic variation of the parasites cell coat. The advent of the genome era has opened up opportunities for developing novel strategies for interrupting the transmission cycle of trypanosomes, specifically using any of the three players,the human host, the tsetse fly vector and/or the parasite. The human genome has been deciphered and the genomes of several trypanosome species have been sequenced. Sequencing of additional neglected trypanosome species is in progress. The tsetse fly genome is currently being sequenced as part of the genomic activities of the International Glossina genome initiative (IGGI). In an attempt to support the tsetse fly sequencing effort, expressed sequence tags (ESTs) from various tissues and developmental stages of Glossina morsitans have been generated.In this study, tsetse fly EST data was analyzed using bioinformatics approaches, focusing on transcripts encoding serpin genes implicated in the immune defenses of tsetse flies. Glossina morsitans homologues to Drosophila melanogaster serpin4, serpin5, and serpin27A and Anopheles gambiae serpin10 were identified in the tsetse fly EST contigs. Comparison of the reactive center loop of tsetse fly serpins with human α-1-antitrypsin suggests that these tsetse serpins are inhibitory. Preliminary EST clustering did not succeed in assembling 3564 Tsal encoded ESTs into one contig. In this study, these ESTs were assembled together with three published Tsal cDNAs. A total of 29 Tsal-encoded contigs were generated. An analysis of the sequence variation within the Tsal EST assembled contigs identified five single base mismatches namely A-T, T-A, G-T and T-G.Results from this study form a basis onto which genetic and biochemical experimental studies can be designed, a process that will be successfully carried out once we have a reference genome. Specifically, studies aimed at genetic modification of tsetse flies towards populations that are inhabitable to trypanosomes. Ultimately, this will supplement current vector control strategies towards elimination of human African trypanosomiasis.

Vaspin is a glycoprotein with three predicted glycosylation sites at asparagine residues located in proximity to the reactive center loop and close to domains that play important roles in conformational changes underlying serpin function. In this study, we have investigated the glycosylation of human vaspin and its effects on biochemical properties relevant to vaspin function. We show that vaspin is modified at all three sites and biochemical data demonstrate that glycosylation does not hinder inhibition of the target protease kallikrein 7. Although binding affinity to heparin is slightly decreased, the protease inhibition reaction is still significantly accelerated in the presence of heparin. Glycosylation did not affect thermal stability.

Vaspin expression is increased in white adipose tissue (WAT) of diet-induced obese mice and rats and is supposed to compensate HFD-induced inflammatory processes and insulin resistance in adipose tissue by counteracting pro-inflammatory gene expression in obesity. Multiple studies have also demonstrated strong anti-inflammatory effects in vascular and skin cells. Here, we used vaspin treated 3T3-L1 murine adipocytes as well as 3T3-L1 cells with stable vaspin expression to investigate the effect of exogenous and endogenous vaspin on inflammatory processes and insulin signaling in adipocytes. Our stably transfected cells secreted significant amounts of vaspin which was in the physiological range of ∼0.5 ng/ml in cell supernatants. Adipocyte differentiation was not affected by vaspin as expression of adipogenic marker genes as well as lipid accumulation after full differentiation was similar to control cells. We found that IL-1β induced expression and secretion of pro-inflammatory cytokines, such as IL-6, MCP1 and TNFα was significantly blunted in vaspin expressing 3T3-L1 cells. Treatment of 3T3-L1 cells with exogenous vaspin resulted in reduced cytokine-induced activation of the intracellular and pro-inflammatory NFκB signaling cascades (IKKα/β, IκB and NFκB). Moreover, endogenous vaspin positively affected insulin signaling by increasing insulin-stimulated phosphorylation of the key mediator protein kinase B (AKT). Together, we demonstrate anti-inflammatory effects of vaspin in 3T3-L1 adipocytes as well as increased insulin signaling by endogenous expression or exogenous treatment. The results provide evidence for potent anti-inflammatory action of vaspin not only in vascular cells but also in adipose tissue.

Vaspin is a serine protease inhibitor that protects against adipose tissueinflammation and insulin resistance, two key drivers of adipocyte dysfunc-tion and metabolic disorders in obesity. Inhibition of target proteases suchas KLK7 has been shown to reduce adipose tissue inflammation in obesity,while vaspin binding to cell surface GRP78 has been linked to reducedobesity-induced ER stress and insulin resistance in the liver. However, themolecular mechanisms by which vaspin directly affects cellular processes inadipocytes remain unknown. Using fluorescently labeled vaspin, we foundthat vaspin is rapidly internalized by mouse and human adipocytes, but lessefficiently by endothelial, kidney, liver, and neuronal cells. Internalizationoccurs by active, clathrin-mediated endocytosis, which is dependent on vas-pin binding to the LRP1 receptor, rather than GRP78 as previously thought.This was demonstrated by competition experiments and RNAi-mediatedknock-down in adipocytes and by rescuing vaspin internalization in LRP1-deficient Pea13 cells after transfection with a functional LRP1 minireceptor.Vaspin internalization is further increased in mature adipocytes after insulin-stimulated translocation of LRP1. Although vaspin has nanomolar affinityfor LRP1 clusters II-IV, binding to cell surface heparan sulfates is requiredfor efficient LRP1-mediated internalization. Native, but not cleaved vaspin,and also vaspin polymers are efficiently endocytosed, and ultimately targetedfor lysosomal degradation. Our study provides mechanistic insight into theuptake and degradation of vaspin in adipocytes, thereby broadening ourunderstanding of its functional repertoire. We hypothesize the vaspin-LRP1axis to be an important mediator of vaspin effects not only in adipose tissuebut also in other LRP1-expressing cells.

Vaspin is an adipokine which improves glucose metabolism and insulin sensitivity in obesity. Kallikrein 7 (KLK7) is the first known protease target inhibited by vaspin and a potential target for the treatment of metabolic disorders. Here, we tested the hypothesis that inhibition of KLK7 in adipose tissue may beneficially affect glucose metabolism and adipose tissue function. Therefore, we have inactivated the Klk7 gene in adipose tissue using conditional gene-targeting strategies in mice. Klk7-deficient mice (ATKlk7 −/−) exhibited less weight gain, predominant expansion of subcutaneous adipose tissue and improved whole body insulin sensitivity under a high fat diet (HFD). ATKlk7 −/− mice displayed higher energy expenditure and food intake, most likely due to altered adipokine secretion including lower circulating leptin. Pro-inflammatory cytokine expression was significantly reduced in combination with an increased percentage of alternatively activated (anti-inflammatory) M2 macrophages in epigonadal adipose tissue of ATKlk7 −/−. Taken together, by attenuating adipose tissue inflammation, altering adipokine secretion and epigonadal adipose tissue expansion, Klk7 deficiency in adipose tissue partially ameliorates the adverse effects of HFD-induced obesity. In summary, we provide first evidence for a previously unrecognized role of KLK7 in adipose tissue with effects on whole body energy expenditure and insulin sensitivity.

Inhibitors belonging to the serpin (serine protease inhibitor) family control proteases involved in various physiological processes. All serpins have a common tertiary structure based on the dominant b-sheet A, but they have different inhibitory specificity. The specificity of a serpin is determined by the Pl-Pl’ peptide bond acting as a bait for the target protease which is made up of an exposed reactive centre loop (RCL). The serpin plasminogen activator inhibitor type-1 (PAI-1) is the main physiological inhibitor of urokinase-type and tissue-type plasminogen activators (uPA and tPA, respectively). Elevated plasma levels of PAI-l have been correlated with a higher risk of deep venous thrombosis, and PAI-1 is a risk factor for recurrent myocardial infarction. Furthermore, PAI-1 has a role in cell migration and has been suggested to regulate tumor growth and angiogenesis. PAI-1 is unique among the serpins in that it can spontaneously and rapidly convert into its latent form. This involves full insertion of the RCL into b-sheet A. There were two partially overlapping goals for this thesis. The first was to use latent PAI-1 as model for development of a fluorescence-based method, Donor-Donor Energy Migration for intramolecular distance measurements. The second goal was to use DDEM, together with other biochemical methods, to reveal the structure of the PAI-1/uPA complex, the conformation of the RCL in active PAI-1, and molecular determinants responsible for the conversion of PAI-1 from the active to the latent form. The use of molecular genetics for introduction of fluorescent molecules enables the use of DDEM to determine intramolecular distances in a variety of proteins. This approach can be applied to examin the overall molecular dimensions of proteins and to investigate structural changes upon interactions with specific target molecules. In this work, the accuracy of the DDEM method has been evaluated by experiments with the latent PAI-1 for which X-ray structure is known. Our data show that distances approximating the Förster radius (57±1 Å) obtained by DDEM are in good agreement (within 5.5 Å) with the distances obtained by X-ray crystallography. The molecular details of the inhibitory mechanism of serpins and the structure of the serpin/protease complex have remained unclear. To obtain the structural insights required to discriminate between different models of serpin inhibition, we used fluorescence spectroscopy and cross-linking techniques to map sites of PAI-1/uPA interaction, and distance measurement by DDEM to triangulate the position of the uPA in the complex. The data have demonstrated clearly that in the covalent PAI-1/uPA complex, the uPA is located at the distal end of the PAI-1 molecule relative to the initial docking site. This indicates that serpin inhibition involves reactive center cleavage followed by full loop insertion, whereby the covalently linked protease is translocated from one pole of the inhibitor to the opposite one. To search for molecular determinants that could be responsible for conversion of PAI-1 to the latent form, we studied the conformation of the RCL in active PAI-1 in solution. Intramolecular distance measurements by DDEM, the newly a developed method based on probe quenching and biochemical methods revealed that the RCL in PAI-1 is located much closer to the core of PAI-1 than has been suggested by the recently resolved X-ray structures of stable PAI-1 mutants, and it can be partially inserted. This possibly explains for the ability of PAI-1 to convert spontaneously to its latent form.

The serpin antiplasmin (APL) is the primary inhibitor of plasmin, a proteinase that digests fibrin, the main component of blood clots. Most serpins are serine protease inhibitors, which undergo dramatic conformational change in forming a tight covalent complex with the target protease. Plasmin has been shown to be angiogenic through its protease activity, but it is also angiostatic, being the source of angiostatin, which inhibits angiogenesis. The main objective of our study is to obtain antiplasmin in large amounts, for crystallization and structure determination of APL and of its complex with plasmin, and for solution studies of the complex. Bacterially expressed APL will not be glycosylated, an advantage in crystallization trials.Bacterial expression of rAPL has been problematic. We have found that it can be greatly enhanced through the use of host E.coli cells that carry extra copies of genes for tRNAs coding for rarely used codons in E.coli that occur in high frequency in eukaryotic genes. Several vectors were screened for rAPL expression (pET19b, pET20b and pET28b). rAPL is expressed in high yield from a pET28b construct in host BL-21 RIPL codon plus cells. rAPL thus expressed accumulates as inclusion bodies, but can be solubilized using N-lauroyl sarcosine at pH11. Refolding and purification of rAPL is achieved by using a sizing column followed by a Nickel His-tag affinity column with an imidazole gradient. rAPL fractions thus obtained are stable at 4°C in the presence of EDTA. However, no inhibitor activity of this rAPL towards trypsin was observed, nor did it form inhibition complex with trypsin. The presence of trace protease and/or failure to fold correctly may be preventing recovery of inhibitory activity. A screen of various refolding buffers failed to yield soluble, stable APL.