Thermal Shift Assay as a Tool to Evaluate the Release of Breakdown Peptides from Cowpea β-Vignin during Seed Germination

The present work aimed to characterize the molecular relationships between structure and function of the seed storage protein β-vignin, the vicilin storage protein of cowpea (Vigna unguiculata, l. Walp) seeds. The molecular characterization of β-vignin was carried out firstly by assessing its thermal stability, under different conditions of pH and ionic strength, by thermal shift assay (TSA) using SYPRO Orange fluorescent dye. Secondly, its aggregation propensity was evaluated using a combination of chromatographic and electrophoretic techniques. Two forms of β-vignin were considered: the native form purified from mature quiescent seeds, and a stable breakdown intermediate of 27 kDa produced while seeds germinate. TSA is a useful tool for determining and following over time the structural changes that occur to the protein during germination. The main result was the molecular characterization of the 27 kDa intermediate breakdown polypeptide, which, to the best of our knowledge, has never been described before. β-vignin seems to retain its trimeric conformation despite the evident degradation of its polypeptides.

Fluconazole and Curcumin Loaded Nanoemulsion Against Multiple Drug Resistance Dermatophytes

Purpose: Topical nanoemulsion comprising of fluconazole and curcumin was developed to target multiple drug resistance dermatophytes infection and to facilitate cutaneous delivery of these poorly water soluble drugs. Methods: Almond oil, sesame oil and paraffin light were used to formulate nanoemulsions and screened for the stability. The solubility of fluconazole and curcumin in surfactants, co-surfactants and oils was screened to decide the various components of the nanoemulsion. The oil phase was light paraffin whereas tween 80 and span 80 were the surfactants and ethanol was used as a co-surfactant. To identify the area of nanoemulsion existence, a pseudoternary diagram was drawn and optimum systems were developed. Drug-loading efficiency was assessed and the developed nanoemulsions were characterized for globule size, stability, robustness to dilution and pH. The optimized nanoemulsion was further evaluated for drug content, viscosity, skin permeation study (ex vivo) and assay of antifungal activity. Results: The globule size was below 200 nm and uniform for the optimized nanoemulsion formulation. It showed enhanced skin permeation (ex vivo) and better antifungal efficacy as compared to the native form of fluconazole and curcumin suspensions. Antimicrobial assay confirmed the synergistic effect of fluconazole and curcumin combination against multiple drug resistance Trychophytum rubrum and Trichophyton metagrophytes as compared to the fluconazole alone. Conclusion: The results clearly indicate an optimized delivery of fluconazole and curcumin in a synergistic way from the nanoemulsion formulation. This resulted in better penetration of these poorly soluble molecules and overall enhanced antifungal activity as compared to these drugs as such against multiple drug resistance dermatophytes.

Expression and purification of a native Thy1-single-chain variable fragment for use in molecular imaging

Molecular imaging using singlechain variable fragments (scFv) of antibodies targeting cancer specific antigens have been considered a non-immunogenic approach for early diagnosis in the clinic. Usually, production of proteins is performed within Escherichia coli. Recombinant proteins are either expressed in E. coli cytoplasm as insoluble inclusion bodies, that often need cumbersome denaturation and refolding processes, or secreted toward the periplasm as soluble proteins that highly reduce the overall yield. However, production of active scFvs in their native form, without any heterologous fusion, is required for clinical applications. In this study, we expressed an anti-thymocyte differentiation antigen-scFv (Thy1-scFv) as a fusion protein with a N-terminal sequence including 3 × hexa-histidines, as purification tags, together with a Trx-tag and a S-tag for enhanced-solubility. Our strategy allowed to recover ~ 35% of Thy1-scFv in the soluble cytoplasmic fraction. An enterokinase cleavage site in between Thy1-scFv and the upstream tags was used to regenerate the protein with 97.7 ± 2.3% purity without any tags. Thy1-scFv showed functionality towards its target on flow cytometry assays. Finally, in vivo molecular imaging using Thy1-scFv conjugated to an ultrasound contrast agent (MBThy1-scFv) demonstrated signal enhancement on a transgenic pancreatic ductal adenocarcinoma (PDAC) mouse model (3.1 ± 1.2 a.u.) compared to non-targeted control (0.4 ± 0.4 a.u.) suggesting potential for PDAC early diagnosis. Overall, our strategy facilitates the expression and purification of Thy1-scFv while introducing its ability for diagnostic molecular imaging of pancreatic cancer. The presented methodology could be expanded to other important eukaryotic proteins for various applications, including but not limited to molecular imaging.

In Vitro and in Vivo Characterisation of P. Aeruginosa Oxidoreductase Enzymes in Pathogenesis and Therapy

<p>Pseudomonas aeruginosa, an increasingly multi-drug resistant human pathogen, is now one of the top three causes of opportunistic infection and there is much interest in identifying novel therapeutic targets for treatment. As a bacterial pathogen, P. aeruginosa encounters innate immune system defences and must continue to adapt to its defence strategies to accommodate the ever-changing environment. Though P. aeruginosa virulence determinants have been heavily characterised over the last several decades, most recent work acknowledges the complex interaction between the human host and the pathogen as an on-going dialogue of virulence factors adapting to the continuum that is the immune response. A major challenge that P. aeruginosa must overcome are reactive oxygen species (ROS) that are released at all stages of infection. Based on previous work which demonstrated a role for soluble nitro- and quinone oxidoreductase (NQOR) enzymes in protecting a related bacterium (Pseudomonas putida) from oxidative stress, we hypothesized that P. aeruginosa would similarly utilize NQORs to withstand ROS. This thesis seeks to understand the role of ROS-protecting enzymes in pathogenesis as well as their potential applications in a therapeutic context. Several NQORs of P. aeruginosa were identified to possess biochemical characteristics consistent with the enzymatic capacity to indirectly reduce reactive species like H₂O₂. However, when individual genes encoding NQORs were deleted from P. aeruginosa, no apparent H₂O₂ sensitivity was seen. In contrast, when candidate genes were over-expressed, certain NQOR enzymes conferred the ability to tolerate H₂O₂ challenge at low concentrations; indicating that these NQORs may play a protective role whose effects are masked in vitro by genetic redundancy as well as a highly active endogenous catalase. By developing a novel in vivo cell culture infection model, the survival of P. aeruginosa post exposure to immunocompetent murine macrophages was also assessed. This not only demonstrated that several putative NQORs were activated in the presence of macrophages but also that an in vivo modelling system is likely to be more appropriate for discovering virulence determinants. In a different aspect of this study it was investigated whether the reductive capacity of the P. aeruginosa-derived NQORs might hold potential for gene-directed enzyme-prodrug therapy (GDEPT). Prodrugs, such as 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) or the nitro-chloromethyl benzindoline SN 26438, are nontoxic in their native form, but become highly toxic upon reduction of their nitro functional groups. The P. aeruginosa NQORs, were tested to identify enzymes capable of efficient activation of CB1954 or SN 26438. Although none of these enzymes exhibited greater activity with CB1954 than the “best in class” Eschericha coli enzymes NfsA or NfsB, the P. aeruginosa NfsB orthologue (PA5190) demonstrated greater than 20-fold improved activity over NfsB from Escherichia coli in its ability to sensitise human cells to SN 26438. This finding offers promise for development of PA5190 and SN 26438 as a novel enzyme-prodrug paradigm for GDEPT.</p>

In Vitro and in Vivo Characterisation of P. Aeruginosa Oxidoreductase Enzymes in Pathogenesis and Therapy

<p>Pseudomonas aeruginosa, an increasingly multi-drug resistant human pathogen, is now one of the top three causes of opportunistic infection and there is much interest in identifying novel therapeutic targets for treatment. As a bacterial pathogen, P. aeruginosa encounters innate immune system defences and must continue to adapt to its defence strategies to accommodate the ever-changing environment. Though P. aeruginosa virulence determinants have been heavily characterised over the last several decades, most recent work acknowledges the complex interaction between the human host and the pathogen as an on-going dialogue of virulence factors adapting to the continuum that is the immune response. A major challenge that P. aeruginosa must overcome are reactive oxygen species (ROS) that are released at all stages of infection. Based on previous work which demonstrated a role for soluble nitro- and quinone oxidoreductase (NQOR) enzymes in protecting a related bacterium (Pseudomonas putida) from oxidative stress, we hypothesized that P. aeruginosa would similarly utilize NQORs to withstand ROS. This thesis seeks to understand the role of ROS-protecting enzymes in pathogenesis as well as their potential applications in a therapeutic context. Several NQORs of P. aeruginosa were identified to possess biochemical characteristics consistent with the enzymatic capacity to indirectly reduce reactive species like H₂O₂. However, when individual genes encoding NQORs were deleted from P. aeruginosa, no apparent H₂O₂ sensitivity was seen. In contrast, when candidate genes were over-expressed, certain NQOR enzymes conferred the ability to tolerate H₂O₂ challenge at low concentrations; indicating that these NQORs may play a protective role whose effects are masked in vitro by genetic redundancy as well as a highly active endogenous catalase. By developing a novel in vivo cell culture infection model, the survival of P. aeruginosa post exposure to immunocompetent murine macrophages was also assessed. This not only demonstrated that several putative NQORs were activated in the presence of macrophages but also that an in vivo modelling system is likely to be more appropriate for discovering virulence determinants. In a different aspect of this study it was investigated whether the reductive capacity of the P. aeruginosa-derived NQORs might hold potential for gene-directed enzyme-prodrug therapy (GDEPT). Prodrugs, such as 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) or the nitro-chloromethyl benzindoline SN 26438, are nontoxic in their native form, but become highly toxic upon reduction of their nitro functional groups. The P. aeruginosa NQORs, were tested to identify enzymes capable of efficient activation of CB1954 or SN 26438. Although none of these enzymes exhibited greater activity with CB1954 than the “best in class” Eschericha coli enzymes NfsA or NfsB, the P. aeruginosa NfsB orthologue (PA5190) demonstrated greater than 20-fold improved activity over NfsB from Escherichia coli in its ability to sensitise human cells to SN 26438. This finding offers promise for development of PA5190 and SN 26438 as a novel enzyme-prodrug paradigm for GDEPT.</p>

The matrisome contributes to the increased rigidity of the bovine ovarian cortex and provides a source of new bioengineering tools to investigate ovarian biology

The gonadotoxic effects of some cancers significantly increase the risk of developing infertility and cessation of ovary hormones (premature ovarian insufficiency, POI). Fertility preservation in the form of ovarian tissue cryopreservation (OTC) is offered to pediatric and adolescent cancer patients who cannot undergo oocyte retrieval and egg cryopreservation. The cryopreserved ovarian tissue can be transplanted back and has been found to restore fertility in 20 - 40% of transplants and restore hormone function for an average of 3 to 5 years. However, some individuals have primary or metastatic disease within their ovarian tissue and would not be able to transplant it back in its native form. Therefore, there is a need for additional methods for hormone and fertility restoration that would support a safe transplant with increased successful livebirths and long-term hormone restoration. To support this goal, we sought to understand the contribution of the ovarian microenvironment to its physical and biochemical properties to inform bioprosthetic ovary scaffolds that would support isolated follicles. Using atomic force microscopy (AFM), we determined that the bovine ovarian cortex was significantly more rigid than the medulla. To determine if this difference in rigidity was maintained in isolated matrisome proteins from bovine ovarian compartments, we cast, and 3D printed hydrogels created from decellularized bovine ovarian cortex and medulla slices. The cast gels and 3D printed bioprosthetic ovary scaffolds from the cortex was still significantly more rigid than the medulla biomaterials. To expand our bioengineering toolbox that will aide in the investigation of how biochemical and physical cues may affect folliculogenesis, we sought to confirm the concentration of matrisome proteins in bovine ovarian compartments. The matrisome proteins, COL1, FN, EMILIN1 and AGRN were more abundant in the bovine ovarian cortex than the medulla. Whereas VTN was more abundant in the medulla than the cortex and COL4 was present in similar amounts within both compartments. Finally, we removed proteins of interest, EMILIN1 and AGRN, from decellularized bovine ovarian cortex materials and confirmed that this specifically depleted these proteins without affecting the rigidity of cast or 3D printed hydrogels. Taken together our results indicate the existence of a rigidity gradient in the bovine ovary, that this rigidity gradient is maintained in resulting engineered materials strongly implicating a role for matrisome proteins in contributing to the physical properties of the bovine ovary. By establishing additional engineering tools, we will continue to explore mechanisms behind matrisome-follicle interactions.

Structural Mechanics of the Alpha-2-Macroglobulin Transformation

SummaryAlpha-2-Macroglobulin (A2M) is the critical pan-protease inhibitor of the innate immune system. When proteases cleave the A2M bait region, global structural transformation of the A2M tetramer is triggered to entrap the protease. The structural basis behind the cleavage-induced transformation and the protease entrapment remains unclear. Here, we report cryo-EM structures of native- and intermediate-forms of the Xenopus laevis egg A2M homolog (A2Moo or ovomacroglobulin) tetramer at 3.7-4.1 Å and 6.4 Å resolution, respectively. In the native A2Moo tetramer, two pairs of dimers arrange into a cross-like configuration with four 60 Å-wide bait-exposing grooves. Each bait in the native form threads into an aperture formed by three macroglobulin domains (MG2, MG3, MG6). The bait is released from the narrowed aperture in the induced protomer of the intermediate form. We propose that the intact bait region works as a “latch-lock” to block futile A2M transformation until its protease-mediated cleavage.

Phosphoproteomic Analysis of Thermomorphogenic Responses in Arabidopsis

Plants rapidly adapt to elevated ambient temperature by adjusting their growth and developmental programs. To date, a number of experiments have been carried out to understand how plants sense and respond to warm temperatures. However, how warm temperature signals are relayed from thermosensors to transcriptional regulators is largely unknown. To identify new early regulators of plant thermo-responsiveness, we performed phosphoproteomic analysis using TMT (Tandem Mass Tags) labeling and phosphopeptide enrichment with Arabidopsis etiolated seedlings treated with or without 3h of warm temperatures (29°C). In total, we identified 13,160 phosphopeptides in 5,125 proteins with 10,700 quantifiable phosphorylation sites. Among them, 200 sites (180 proteins) were upregulated, while 120 sites (87 proteins) were downregulated by elevated temperature. GO (Gene Ontology) analysis indicated that phosphorelay-related molecular function was enriched among the differentially phosphorylated proteins. We selected ATL6 (ARABIDOPSIS TOXICOS EN LEVADURA 6) from them and expressed its native and phosphorylation-site mutated (S343A S357A) forms in Arabidopsis and found that the mutated form of ATL6 was less stable than that of the native form both in vivo and in cell-free degradation assays. Taken together, our data revealed extensive protein phosphorylation during thermo-responsiveness, providing new candidate proteins/genes for studying plant thermomorphogenesis in the future.

Enzyme Aggregation and Fragmentation Induced by Catalysis Relevant Species

It is usually assumed that enzymes retain their native structure during catalysis. However, the aggregation and fragmentation of proteins can be difficult to detect and sometimes conclusions are drawn based on the assumption that the protein is in its native form. We have examined three model enzymes, alkaline phosphatase (AkP), hexokinase (HK) and glucose oxidase (GOx). We find that these enzymes aggregate or fragment after addition of chemical species directly related to their catalysis. We used several independent techniques to study this behavior. Specifically, we found that glucose oxidase and hexokinase fragment in the presence of D-Glucose but not L-glucose, while hexokinase aggregates in the presence of Mg2+ ion and either ATP or ADP at low pH. Alkaline phosphatase aggregates in the presence of Zn2+ ion and inorganic phosphate. The aggregation of hexokinase and alkaline phosphatase does not appear to attenuate their catalytic activity. Our study indicates that specific multimeric structures of native enzymes may not be retained during catalysis and suggests pathways for different enzymes to associate or separate over the course of substrate turnover.

Nano-Curcumin Prevents Cardiac Injury, Oxidative Stress and Inflammation, and Modulates TLR4/NF-κB and MAPK Signaling in Copper Sulfate-Intoxicated Rats

Copper (Cu) is essential for a plethora of biological processes; however, its high redox reactivity renders it potentially toxic. This study investigated the protective effect of curcumin (CUR) and nano-CUR (N-CUR) against Cu cardiotoxicity, emphasizing the role of oxidative stress, TLR4/NF-κB and mitogen-activated protein kinase (MAPK) signaling and cell death in rats. Rats received 100 mg/kg copper sulfate (CuSO4), a pesticide used for repelling pests, and were concurrently treated with CUR or N-CUR for 7 days. Cu caused cardiac injury manifested by elevated serum cardiac troponin I (cTnI), creatine kinase (CK)-MB, and lactate dehydrogenase (LDH), as well as histopathological alterations. Cardiac malondialdehyde (MDA), NF-κB p65, TNF-α, and IL-6 were increased, and reduced glutathione (GSH), superoxide dismutase (SOD) and catalase were decreased in Cu-treated rats. CUR and N-CUR prevented cardiac tissue injury, decreased serum cTnI, CK-MB, and LDH, and cardiac MDA, NF-κB p65, TNF-α, and IL-6, and enhanced cellular antioxidants. CUR and N-CUR downregulated TLR4 and AP-1, and decreased the phosphorylation levels of p38 MAPK, JNK, and ERK1/2. In addition, CUR and N-CUR increased cardiac Bcl-2 and BAG-1, decreased Bax and caspase-3, and prevented DNA fragmentation. In conclusion, N-CUR prevents Cu cardiotoxicity by attenuating oxidative injury, inflammatory response, and apoptosis, and modulating TLR4/NF-κB and MAPK signaling. The cardioprotective effect of N-CUR was more potent than the native form.