Structural Investigation of Helical Intermediates in the Misfolding Pathway of Amyloid Peptides Associated With Type II Diabetes and HIV.

A variety of aging related diseases including Alzheimer’s, Type II diabetes, Huntington’s, Parkinson’s, and Creutzfeldt-Jakob are characterized by the formation of abnormal proteinaceous deposits. These deposits, known as amyloid, are abnormal accumulations of misfolded proteins with a characteristic cross β-sheet conformation. The formation of amyloid fibers can occur through many pathways, of which one of the most important pathway is catalyzed by binding to the cell membrane. The peptide-membrane interaction can disrupt the integrity of the cell membrane, causing disruption of calcium homeostasis and eventual cell death. In membrane-mediated aggregation, the protein is thought to initially bind with the membrane in an α-helical conformation before undergoing a conformational change during aggregation to a β-sheet form characterstic of the amyloid fiber. Therefore, it is important to determine atomic-level structures of these intermediates, as they can provide insights into the toxic mechanism exhibited by these amyloidogenic proteins and into the design of drugs that can suppress these intermediate helical species to stop further progression into toxic states. This dissertation reports high-resolution NMR structural studies of two different membrane bound amyloid proteins: (1) IAPP (an amyloidogenic peptide related to Type II diabetes), in order to understand the role of α-helical intermediate structures in causing membrane disruption, (2) PAP248-286 (the corresponding amyloid fiber (SEVI) enhances the infectivity of the HIV virus), in order to understand the bridging interactions it exhibits between the host and viral cell membranes. Our studies on the membrane bound α-helical intermediates of rat and human IAPP/IAPP1-19 reveal a pH dependent membrane orientation for IAPP1-19, which correlates well with its ability to disrupt synthetic membrane vesicles and β-cells, and that the position of the disulfide bridge with respect to the hydrophobic interface of the N-terminal helix could be one of the factors that modulate the membrane disruptive behaviour of these peptides. Our study on membrane bound PAP248-286, reveals an unusual amount of structural disorder that, in combination with high positive charge at the N-terminus could play an important role in the fusion of host and viral cell membranes by weakening the electrostatic interactions that repel similarly charged membranes.Ph.D.ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/86258/1/nrpreddy_1.pd

In vivo Magnetic Resonance Imaging of Tumor Protease Activity

Increased expression of cathepsins has diagnostic as well as prognostic value in several types of cancer. Here, we demonstrate a novel magnetic resonance imaging (MRI) method, which uses poly-L-glutamate (PLG) as an MRI probe to map cathepsin expression in vivo, in a rat brain tumor model. This noninvasive, high-resolution and non-radioactive method exploits the differences in the CEST signals of PLG in the native form and cathepsin mediated cleaved form. The method was validated in phantoms with known physiological concentrations, in tumor cells and in an animal model of brain tumor along with immunohistochemical analysis. Potential applications in tumor diagnosis and evaluation of therapeutic response are outlined

Expression, purification and characterization of C2 domain of milk fat globule-EGF-factor 8-L

Milk fat globule-EGF-factor 8-L (MFG-E8L) is secreted by activated macrophages and functions as a linker protein or opsonin between the dying cells and phagocytes. MFG-E8L recognizes the apoptotic or dying cells by specifically binding to Phosphatidylserine (PS) exposed on the outer cell surface and enhances the engulfment of the apoptotic cells by phagocytes, thereby preventing the inflam¬mation and autoimmune response against intracellular antigens that can be released from the dying cells. MFG-E8L contains two EGF¬like domains, P/T (proline/threonine) rich domain followed by two discoidin-like domains (C1 and C2). Recent studies have shown that the C2 domain of MFG-E8L is specifically involved in interaction with PS exposed on the apoptotic cells. Towards understanding this specific molecular interaction between the MFG-E8L C2 domain and PS, we expressed, purified the C2 domain of MFG-E8L and per¬formed the binding studies with phospholipids by 31P NMR experiment. We demonstrated that our recombinant construct and expres¬sion system were effective and allowed us to obtain the C2 domain and also showed that the purified C2 domain was stable and properly folded, and our 31P NMR studies indicated that the C2 domain had specific binding with PS.Accepted versio

High quality three-dimensional gagCEST imaging of in vivo human knee cartilage at 7 Tesla

\u3cp\u3ePurpose: To develop a new faster and higher quality three-dimensional (3D) gagCEST MRI technique for reliable quantification of glycosaminoglycan (GAG) present in the human knee cartilages. Methods: A new magnetization-prepared 3D gradient echo-based MRI pulse sequence has been designed to obtain the B\u3csub\u3e0\u3c/sub\u3e inhomogeneity, B\u3csub\u3e1\u3c/sub\u3e inhomogeneity, and CEST Z-spectra images. Results: The gagCEST values of different compartments of knee cartilage are calculated using a newly developed technique for healthy subjects and a symptomatic knee cartilage degenerated subject. The effect of the acquired CEST saturation frequency offset step-size was investigated to establish the optimal step-size to obtain reproducible gagCEST maps. Our novel 3D gagCEST technique demonstrates markedly higher gagCEST contrast value than the previously reported 3D gagCEST studies. This study demonstrates the need for separate B\u3csub\u3e0\u3c/sub\u3e and B\u3csub\u3e1\u3c/sub\u3e inhomogeneity estimation and correction. Conclusion: The new technique provided high quality gagCEST maps with clearer visualization of different layers of knee cartilage with reproducible results. Magn Reson Med 77:1866–1873, 2017.\u3c/p\u3

Molecular characterization of the recombinant A-chain of a Type II ribosome-inactivating protein (RIP) from viscum album coloratum and structural basis on its ribosome-inactivating activity and the sugar-binding properties of the B-chain

Mistletoe (Viscum album) lectins, which are classified as a type II ribosome-inactivating protein (RIP) due to their unique biological function and the potential medical and therapeutic application in cancer cells, receive a rising attention. The heterodimeric glycoproteins contain the Achain with catalytic activity and the B-chain with sugar binding properties. In recent years, studies involving the lectins from the white berry European mistletoe (Viscum album) and the yellow berry Korean mistletoe (Viscum album coloratum) have been described. However, the detailed mechanism in exerting unique cytotoxic effect on cancer cells still remains unclear. Here, we aim to understand and define the molecular basis and biological effects of the type II RIPs, through the studies of the recombinant Korean mistletoe lectin. To this end, we expressed, purified the recombinant Korean mistletoe lectin (rKML), and investigated its molecular characteristics in vitro, its cytotoxicity and ability to induce apoptotic cell death in cancer cells. To gain structural basis for its catalytic activity and sugar binding properties, we performed homology modeling studies based on the high degree of sequence identity and conserved secondary structure prediction between Korean and European, Himalayan mistletoe lectins, and Ricin.Accepted versio

High resolution mapping of modafinil induced changes in glutamate level in rat brain.

Modafinil is marketed in the United States for the treatment of narcolepsy and daytime somnolence due to shift-work or sleep apnea. Investigations of this drug in the treatment of cocaine and nicotine dependence in addition to disorders of executive function are also underway. Modafinil has been known to increase glutamate levels in rat brain models. Proton magnetic resonance spectroscopy (1HMRS) has been commonly used to detect the glutamate (Glu) changes in vivo. In this study, we used a recently described glutamate chemical exchange saturation transfer (GluCEST) imaging technique to measure Modafinil induced regional Glu changes in rat brain and compared the results with Glu concentration measured by single voxel 1HMRS. No increases in either GluCEST maps or 1HMRS were observed after Modafinil injection over a period of 5 hours. However, a significant increase in GluCEST (19 ± 4.4%) was observed 24 hours post Modafinil administration, which is consistent with results from previous biochemical studies. This change was not consistently seen with 1HMRS. GluCEST mapping allows regional cerebral Glu changes to be measured and may provide a useful clinical biomarker of Modafinil effects for the management of patients with sleep disorders and addiction

In vivo B1+ enhancement of calf MRI at 7 T via optimized flexible metasurfaces

Purpose: Ultrahigh field (≥7 T) MRI is at the cutting edge of medical imaging, enabling enhanced spatial and spectral resolution as well as enhanced susceptibility contrast. However, transmit ((Formula presented.)) field inhomogeneity due to standing wave effects caused by the shortened RF wavelengths at 7 T is still a challenge to overcome. Novel hardware methods such as dielectric pads have been shown to improve the (Formula presented.) field inhomogeneity but are currently limited in their corrective effect by the range of high-permittivity materials available and have a fixed shelf life. In this work, an optimized metasurface design is presented that demonstrates in vivo enhancement of the (Formula presented.) field. Methods: A prototype metasurface was optimized by an empirical capacitor sweep and by varying the period size. Phantom temperature experiments were performed to evaluate potential metasurface heating effects during scanning. Lastly, in vivo gradient echo images and (Formula presented.) maps were acquired on five healthy subjects on a 7 T system. Dielectric pads were also used as a comparison throughout the work as a standard comparison. Results: The metasurfaces presented here enhanced the average relative SNR of the gradient echo images by a factor of 2.26 compared to the dielectric pads factor of 1.61. Average (Formula presented.) values reflected a similar enhancement of 27.6% with the metasurfaces present versus 8.9% with the dielectric pads. Conclusion: The results demonstrate that metasurfaces provide superior performance to dielectric padding as shown by (Formula presented.) maps reflecting their direct effects and resulting enhancements in image SNR at 7 T.</p

Application of glutamate weighted CEST in brain imaging of nicotine dependent participants in vivo at 7T.

IntroductionWith nicotine dependence being a significant healthcare issue worldwide there is a growing interest in developing novel therapies and diagnostic aids to assist in treating nicotine addiction. Glutamate (Glu) plays an important role in cognitive function regulation in a wide range of conditions including traumatic brain injury, aging, and addiction. Chemical exchange saturation transfer (CEST) imaging via ultra-high field MRI can image the exchange of certain saturated labile protons with the surrounding bulk water pool, making the technique a novel tool to investigate glutamate in the context of addiction. The aim of this work was to apply glutamate weighted CEST (GluCEST) imaging to study the dorsal anterior cingulate cortex (dACC) in a small population of smokers and non-smokers to determine its effectiveness as a biomarker of nicotine use.Methods2D GluCEST images were acquired on 20 healthy participants: 10 smokers (ages 29-50) and 10 non-smokers (ages 25-69), using a 7T MRI system. T1-weighted images were used to segment the GluCEST images into white and gray matter tissue and further into seven gray matter regions. Wilcoxon rank-sum tests were performed, comparing mean GluCEST contrast between smokers and non-smokers across brain regions.ResultsGluCEST levels were similar between smokers and non-smokers; however, there was a moderate negative age dependence (R2 = 0.531) in smokers within the cingulate gyrus.ConclusionFeasibility of GluCEST imaging was demonstrated for in vivo investigation of smokers and non-smokers to assess glutamate contrast differences as a potential biomarker with a moderate negative age correlation in the cingulate gyrus suggesting reward network involvement