A-B family of toxins consists of plant toxins such as ricin and bacterial toxins such as cholera. The A subunit is the enzymatic domain and the B subunit is the receptor binding domain. Commonly, these toxins bind to the target cell plasma membrane receptors through their B subunit followed by endocytosis and a transport to the endoplasmic reticulum (ER). Inside the ER, the A subunit dissociates from the rest of the toxin, unfolds and triggers the ER quality control mechanism of ER-associated degradation (ERAD). Most ERAD substrates are purged out of the ER into the cytosol for proteasomal degradation. However, the low content of lysine amino acid residues allows the toxin to evade polyubiquitination and subsequent proteasomal degradation. The toxin A subunit refolds into an active conformation in the cytosol, setting off downstream toxic events. In the first part of my thesis, the hypothesis was tested that inhibiting the unfolding of the toxin A subunit inside the ER will prevent ERAD activation, toxin export to the cytosol and intoxication. The chemical chaperones glycerol and sodium 4-phenyl butyrate (PBA) were used to inhibit the toxin A chain unfolding. In vitro biophysical experiments indicated that both chemical chaperones indeed stabilize the cholera toxin A subunit and prevent cytotoxicity. In case of ricin, both chaperones stabilized the toxin A chain but only glycerol prevented cytotoxicity. Additional experiments showed that PBA-treated ricin A chain is destabilized when exposed to anionic lipid membranes mimicking the properties of the ER membrane. In contrast, anionic lipid did not prevent ricin A chain stabilization by glycerol. This explains why glycerol but not PBA blocked ricin intoxication, as only glycerol stabilizes ricin A chain in the presence of ER membranes. Cholera toxin in contrast, remained either unaffected or slightly stabilized in presence of anionic lipids both in presence and absence of PBA. This shows that destabilization by anionic lipids is a toxin-specific rather than a general effect. In the second part of my thesis, the effect of inner leaflet of plasma membrane on the structure of cholera toxin A chain (CTA1) was studied. Since CTA1 refolds into an active conformation in the cytosol in association with unidentified host factors, I hypothesized that inner leaflet of the plasma membrane might play a role to stabilization and/or refolding of CTA1. CTA1 was shown to be a membrane interacting protein, and membranes mimicking lipid rafts had a significant stabilizing effect on its structure. Lipid rafts helped in the regaining of the tertiary and secondary structure of CTA1, while non-raft lipids had a smaller stabilizing effect on CTA1 structure. In the next part of my thesis, I studied the effect of membrane binding on the structure and function of human pancreatic phospholipase A₂ (PLA₂). Lipid thermal phase transition was found to have a dramatic effect on PLA₂ activity. It was also established that although membrane binding and insertion was essential for of PLA₂ activity, lipid structural heterogeneity was more important than the depth of membrane insertion for enzyme activation. Most importantly, significant changes in PLA₂ secondary and tertiary structures were identified that evidently contribute to the interfacial activation of PLA₂. Overall, we conclude that the function of membrane binding enzymes can be significantly modulated via conformational changes induced by interactions with membranes. Thus, we have elucidated various roles of membrane lipids from unfolding and refolding to activation and modulation of membrane binding enzymes. Physical properties of lipids help in regulating various aspects of protein structure and function and their analysis helped us in appreciating the influence wielded by the membrane lipids in the enzyme's surrounding environment.

Skin cancer is the most common type of cancer in the US. The American Academy of Dermatology estimated that more than 3.5 million new cases of skin cancer are diagnosed in the US each year and 1 in 5 Americans will likely to develop skin cancer in their life time. Most cases of skin cancer are caused by exposure to ultraviolet (UV) radiation from the sun. Some of the most common sunscreen ingredients are unstable and can form harmful radicals upon exposure to UV radiation. There is a strong clinical need for a more stable and effective sunscreen ingredient such as bovine milk phospholipids (MP). Phospholipids were shown to have beneficial health effects such as regulation of the inflammatory reactions, protective effects against colon cancer, and reducing cardiovascular risk factors. Previous histology and MTT tissue viability research studies suggested that MP act upon skin cells in a protective manner against UV radiation. This thesis aims to further investigate the protective effects of bovine milk phospholipids by evaluating the expression of a UV-induced DNA damage marker, cyclin-dependent kinase inhibitor, p21WAF1/CIP1. Western blots were used to quantify p21 expression in human keratinocytes in four categories of samples: No-UV, UV, UV+MP, MP and in HeLa (p21 positive control). In the No-UV samples, cells were not irradiated by UV light. However, in the UV samples, keratinocytes were exposed to a UV dosage of 10 mJ/cm2. In the UV+MP samples, keratinocytes were first treated with 1% MP solution (w/v) in their culture media for 24 hours then exposed to a UV dosage of 10 mJ/cm2. In MP, keratinocytes were treated with 1% MP solution in their culture media for 24 hours. Total cell proteins were extracted 24 hours post-UV irradiation. The same amount of protein from each sample (determined by BCA assay) was loaded into a 4-12% Bis-Tris SDS-PAGE gel, run under denaturing, non-reducing conditions then blotted and treated with antibodies for the quantitative detection of p21 proteins. Finally, intensities of p21 protein bands were analyzed by using ImageJ software. Under non-reducing conditions, three p21 proteins covalently bonded with each other showed up as 63 KDa molecules on the PVDF membrane. The UV, and HeLa samples showed a 2.28 fold, and 1.23 fold increase in p21 expression, respectively, compared with the No-UV samples control. The MP samples showed a 0.948 fold decrease in p21 compared with the No-UV samples, and the UV+MP samples showed only a 1.13 fold increase in p21. When comparing with the UV sample, the UV+MP sample has 50.4% less p21 expression. Less p21 expression in the UV+MP sample compare with the UV sample suggested that less DNA damage occurred in the sample that was treated with milk phospholipids. This result strongly suggests that 1% bovine milk phospholipids can protect skin cells from UV induced DNA damage.

To reduce reliance on fossil fuels, sustainable biofuels are being pursued, especially advanced biofuels like 1-butanol that have higher energy content and greater compatibility with existing infrastructure than ethanol. A persistent challenge is the yield-limiting toxicity of biofuels and process solvents, such as tetrahydrofuran, to the microbes that ferment biomass into biofuel. The cell membrane is a focal point of toxicity, and understanding how it interacts with fuels and solvents is key to improving yield. Phospholipid bilayers are the core of biomembranes, and model biomembranes of defined composition provide the ideal platform for biophysical studies. To this end, glycerophospholipids characteristic of Bacillus subtilis, a model producer organism, were synthesized. Two fatty acids (iso- and anteisopentadecanoic acids) characteristic of Bacilli were synthesized and incorporated into representative phosphatidic acid, phosphatidylethanolamine and phosphatidylglycerol lipids. The validated synthetic approach opens the door to future studies on the interaction of biofuels and solvents with biomembranes.

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No description available.

<p>The comprehensive analysis of intact lipids (called lipidomics) can provide information about the presence of microbial communities in an ecosystem and assist in understanding the biogeochemistry in that system. In previous work we had developed a method to determine the profiles of eight phospholipid classes in a soil microorganism by direct-infusion electrospray mass spectroscopy using tandem mass spectrometry. The work done in this study encompasses first the optimization of previous methodology for use with water and sediment samples containing low concentration of phospholipids and large amounts of organic contaminants and secondly the application of this method to the analysis of phospholipids within composite tailings and recycled process water using a triple quadrupole mass spectrometer to determine the intact lipids in the bacterial community. The results are presented illustrating the phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipids present in composite tailing samples and recycled process water. This thesis begins with the optimization of a direct infusion mass spectrometry method, which allowed the analysis of intact phospholipids within both water and sediment samples. This method allows for high through-put analysis using both the separation afforded by neutral loss and precursor ion scanning modes and a database containing all possible adduct masses to identify and quantify unknown phospholipids. This method was then applied to water and sediment samples obtained from the Syncrude Sandhill Fen composite tailings site. This analysis discovered multiple differences within the water samples attributed to changes both in well temperature and the ongoing reclamation projects resulting in the change in phospholipid profiles. This thesis also outlines the shortcomings of the direct infusion lipidomics method when used for the analysis of complex samples such as composite tailings sediment samples. In summary, this thesis has demonstrated that direct infusion lipidomics can be successfully applied to the analysis of water samples and yield statistically significant differences within the microbial lipidome.</p> / Master of Science (MSc)

Ferroptosis, an oxidative cell death mechanism, is driven by iron-dependent lipid peroxidation. Despite being generally associated with lipid peroxidation that overwhelms endogenous repair systems, ferroptosis mechanisms and regulators in various pathological contexts remain elusive. Identifying novel modulators of the ferroptosis pathway is essential for cell-death marker development and drug discovery to target this process. Small molecule drugs and dietary intervention of metabolites and lipids can modulate ferroptosis sensitivity in diverse disease contexts. In this thesis, I investigated lipid metabolism involving ferroptosis in cancer models and an infectious lung disease model. I dissected the different roles of PUFA-containing phospholipids in dietary modulation of ferroptosis and discovered a specific phospholipid class, phosphatidylcholine with diacyl-polyunsaturated fatty acid tails (PC-PUFA2; diacyl-PUFA-PC) that promote ferroptosis. Exogenous PC-PUFA2 or free PUFA enriches PC-PUFA2 abundance in cancer cells and accounts for the ferroptosis-sensitizing effects. I also discovered the accumulation of PC-PUFA2 in the mitochondria, which disrupts mitochondrial redox homeostasis and initiates lipid peroxidation in the endoplasmic reticulum. These findings unveil the essential roles of diacyl-PUFA phospholipids during ferroptosis. Utilizing biomarkers of ferroptosis, I studied the pathogenic mechanism of COVID-19-associated pulmonary diseases. Elevated ferroptosis markers including transferrin receptor 1 and lipid peroxidation products were detected in human COVID-19 lung autopsies. Dysregulation in lipid profile, including a significant decrease in PUFA phospholipids and accumulation of lysophospholipids, further suggests dysregulation of lipid metabolism and ferroptosis that may contribute to inflammation and acute lung injury in COVID-19 lungs. Iron metabolism is affected in the COVID-19 lung and is associated with ferroptosis activation. We further discovered a strong correlation of ferroptosis markers with lung injury severity in a COVID-19 model using Syrian hamsters. These findings provide the fundament for targeting ferroptosis as a novel therapeutic and diagnostic strategy for various diseases.

Hair cell structures are one of the most common forms of sensing elements found in nature. In humans, approximately 16,000 auditory hair cells can be found in the cochlea of the ear. Each hair cell contains a stereocilia, which is the primary structure for sound transduction. This study looks to develop and characterize a bilayer lipid membrane (BLM) operated artificial hair cell sensor that resembles the stereocilia of the human ear. To develop this sensor, a flexible substrate with internal compartments for hosting the biomolecules and mating cap are constructed and experimentally characterized. The regulated attachment method (RAM) is used to form bilayers within the sealed device. Capacitance measurements of the encapsulated bilayer show that the sealing cap slightly compresses the bottom insert and reduces the size of the enclosed bilayer. Single channel measurements of alamethicin peptides further verify that the encapsulated device can be used to detect the gating activity of transmembrane proteins in the membrane. The flexible substrate was incorporated into a low-noise, portable test fixture. The response of the sensor and tip velocity of the hair were measured with respect to an impulse input on the test fixture and several frequency response functions (FRFs) were created. The FRF between the sensor and the tip velocity was used to show that the hair vibration was transmitted to the bilayer for certain hair lengths. The transfer function between the sensor and the input was used to show the effect of membrane potential on sensor response. / Master of Science

In plants, calcium is a ubiquitous signaling molecule and changes in cytosolic calcium levels reported in response to various abiotic and biotic stresses like salt stress, drought, pathogen attack and phytohormone signaling. Any calcium- mediated signal transduction process involves the establishment of a signal-specific change in the cytosolic calcium concentration termed as ‗calcium signature‘ which is decoded by the specific group of proteins called ‗calcium sensors‘ (eg: Calmodulin (CaM) and Ca2+ - regulated kinases). Plants have a novel group of kinases designated as Ca2+- dependent protein kinases (CDPK; EC 2.7.1.37). CDPKs are biochemically distinct from other Ca2+- dependent kinases, such as Ca2+- and phospholipid- dependent protein kinases, as they are activated directly by Ca2+-and are independent of CaM. They exist as monomeric serine/threonine protein kinases and consist of four domains namely an amino-terminal variable domain, a kinase domain, an autoinhibitory domain and a calmodulin-like domain (CaM-LD). CDPKs represent a unique class of Ca2+ sensors, having protein kinase as well as CaM-LD in a single polypeptide chain, enabling them to couple the calcium sensor directly to its responder (kinase). In the absence of calcium signature, CDPKs activity is inhibited by the autoinhibitory domain, which acts as a pseudo-substrate of kinase domain and thus blocks the active site of the enzyme. In the presence of calcium signature, CDPKs undergo conformational changes leading to removal of the inhibition. Besides plants, CDPKs are also reported in few protozoans viz Plasmodium falciparam, Paramecium and Taxoplasma. However, CDPKs are not found in the eukaryotic genome of yeast, nematodes, fruitflies and humans. In the current study, we have cloned CDPK1 gene from Cicer arietinum (CaCDPK1) in pRSET-A expression vector and expressed it in Escherichia coli BL21pLysS strain. However, while expressing the recombinant CaCDPK1 in E.coli, most of the recombinant CaCDPK1 protein was expressed as insoluble form. Therefore, we focused our efforts on optimizing the culture conditions for achieving the maximum yield of soluble recombinant CaCDPK1. Expression of the soluble CaCDPK1 was achieved by optimizing the different conditions like IPTG concentrations, temperature and growth time after induction. Maximum amount of soluble expression of recombinant CaCDPK1 was achieved by inducing the bacterial culture with 0.1 mM IPTG at 0.6 OD and growing it further for 4 h at 25°C. As with several other CDPKs, CaCDPK1 was found to get autophosphorylated in a calcium-dependent manner. To find the significance of autophosphorylation, we measured the substrate phosphorylation activity of the native and autophosphorylated CaCDPK1, which revealed that the autophosphorylation enhances the kinase activity of CaCDPK1 by 2-fold. Autophosphorylation was linearly dependant on concentrations of the enzyme suggesting that the autophosphorylation in CaCDPK1 occurs via an intra-molecular mechanism. Further analysis of autophosphorylation shows that autophosphorylation happens before substrate phosphorylation and provides calcium -independent substrate phosphorylation property. It also reduces the lag phase for activation of the enzyme and utilizes both ATP and GTP as phosphor-donor, but ATP is preferred over GTP. Autophosphorylation was found to occur at serine and threonine residues. The MALDI MS/MS analysis of the cold ATP autophosphorylated CaCDPK1 showed Thr- 339, Ser- 357, and Ser- 367 residues could be the potential autophosphorylation sites in CaCDPK1. Phospholipids, the major structural components of membranes, can also have functions in regulating signaling pathways in plants under biotic and abiotic stress conditions. The effects of adding phospholipids on the activity of stress-induced calcium dependent protein kinase (CaCDPK1) from chickpea are reported in this study. Both autophosphorylation as well as phosphorylation of the added substrate were enhanced specifically by phosphatidylcholine and to a lesser extent by phosphatidic acid, but not by phosphatidylethanolamine. Diacylgylerol, the neutral lipid known to activate mammalian PKC, stimulated CaCDPK1 but at higher concentrations. Increase in Vmax of the enzyme activity by these phospholipids significantly decreased the Km indicating that phospholipids enhance the affinity towards its substrate. In the absence of calcium, addition of phospholipids had no effect on the negligible activity of the enzyme. Intrinsic fluorescence intensity of the CaCDPK1 protein was quenched on adding PA and PC. Higher binding affinity was found with PC (K½ = 1.3 nM) when compared to PA (K½ = 56 nM). We also found that the concentration of PA increased in chickpea plants under salt stress. The stimulation by PA and PC suggests regulation of CaCDPK1 by these phospholipids during stress response. In the current study we also investigated CaCDPK1 interactions with calcium ions to address the Ca2+ -induced conformational changes in CaCDPK1 by using circular dichroism (CD), fluorescence spectroscopy and isothermal titration (ITC). Isothermal calorimetric analysis of calcium binding to CaCDPK1 shows a biphasic curve with two Kd of 27 nM and 1.72 µM respectively. The fluorescence measurements showed quenching in fluorescence intensity with a 5 nm red shift. The plot of changes in intensity against calcium concentrations again showed a biphasic curve, indicating that there may be more than one kind of Ca2+ binding sites. 8-anilinonaphthalene-1-sulfonic acid (ANS) binding showed that calcium bound form of CaCDPK1 exposes hydrophobic surfaces which may act as binding sites for other proteins. CD analysis of CaCDPK1 showed that it‘s an alpha helical rich protein and its helical content increases after binding to calcium. Taken all together this study describes the successful heterologous expression of Cicer arietinum CDPK isoform 1 in E.coli. and demonstrates that the autophoshorylation happens via an intra-molecular mechanism and it increases the kinase activity of CaCDPK1 at least by 2-fold. We also report here that CaCDPK1 prefers ATP as phosphodonor over GTP. The present study also shows the activation of CaCDPK1 by PC and PA, but not by PE or diacylglycerol. Both phospholipids were able to bind to CaCDPK1 and increased its Vmax and affinity towards the exogenous substrate, histone III-S. The current study also shows that calicum binding induces conformational changes in CaCDPK1 and the all four EF hand motifs of CaCDPK1 do not function in an equivalent manner.

Bile is a biofluid synthesized by liver and concentrated in the gallbladder. Interference with the bile flow may cause cholestasis. Primary sclerosing cholangitis (PSC) is an inflammatory cholestatic disorder which eventually may result in liver cirrhosis and failure. The management of PSC is controversial. The only effective treatment for end stage disease is orthotopic liver transplantation (OLT). However, cholangiocarcinoma (CC), which is the major complication of this long-lasting disease, is an absolute contraindication for the surgery. Therefore, early diagnosis of the disease can not only improve the outcome of PSC, but also facilitate the allocation of donated livers to those who can benefit from transplantation. Unfortunately, the diagnosis of CC is challenging. Endoscopic retrograde cholangiopancreatography (ERCP), the gold standard technique, is highly invasive. Non-invasive alternatives such as magnetic resonance cholangiopancreatography (MRCP) have lower accuracy. Therefore, it is essential to develop more accurate and less invasive diagnostic techniques. Magnetic resonance spectroscopy (MRS) is an evolving technique with potential to detect disease-related metabolic changes. In vitro studies have proven the capacity of MRS in the early detection of hepatopancreatobiliary (HPB) disorders based on the metabolic analysis of bile obtained invasively. An in vivo alternative has been attempted by others on human bile within the gallbladder. However, due to the poor quality of the acquired spectra, quantification of most major bile metabolites was not possible, except for choline-containing phospholipids (chol-PLs). In the current study, the quality of the in vivo 1D spectra has been greatly improved, and we have obtained the first 2D L-COSY spectra from bile within the gallbladder. Spectral data from healthy controls and PSC patients were compared. Statistically significant differences in the concentrations of chol-PLs, and glycine- and taurine-conjugated bile acids were revealed in the 1D analysis. Our 2D spectra also demonstrated potential for the detection of metabolic differences between the two groups. The success of these studies indicates a strong potential of in vivo bile MRS techniques to characterize and diagnose a wide variety of HPB disorders. / May 2014