Remote Control Electrodeposition: Patterning on Substrates without Direct Electrical Connections

Thesis (Ph.D.)--University of Washington, 2016-06

Phosphotyrosyl peptides and analogues as substrates and inhibitors of purple acid phosphatases

Purple acid phosphatases are metal-containing hydrolases. While their precise biological role(s) is unknown, the mammalian enzyme has been linked in a variety of biological circumstances (e.g., osteoporosis) with increased bone resorption. Inhibition of the human enzyme is a possible strategy for the treatment of bone-resorptive diseases such as osteoporosis. Previously, we determined the crystal structure of pig purple acid phosphatase to 1.55 Angstrom and we showed that it is a good model for the human enzyme. Here, a study of the pH dependence of its kinetic parameters showed that the pig enzyme is most efficient at pH values similar to those encountered in the osteoclast resorptive space. Based on the observation that phosphotyrosine-containing peptides are good substrates for pig purple acid phosphatase, peptides containing a range of phosphotyrosine mimetics were synthesized. Kinetic analysis showed that they act as potent inhibitors of mammalian and plant purple acid phosphatases, with the best inhibitors exhibiting low micromolar inhibition constants at pH 3-5. These compounds are thus the most potent organic inhibitors yet reported for the purple acid phosphatases. (C) 2004 Published by Elsevier Inc.

Crystalline lanthanum hydroxycarbonates with controlled phases and varied morphologies prepared on non-crystalline substrates

Lanthanum hydroxycarbonate crystals with controlled phases and varied morphologies were prepared on the surface of a non-crystalline substrate, glass. The phases and morphologies of the crystals were controlled conveniently by varying the reaction temperature and the quantity of starting materials. Orthorhombic crystals were obtained at 160 degreesC, distributed individually on the substrate and had a flaky rhombic shape. Hexagonal crystals were obtained at 180 degreesC. The crystals had a rhomboidal shape, were uniform and continuous enough to form a solid film on the substrate. The substrates were corroded under the hydrothermal conditions and offered a coarse surface for the crystal growth. The hexagonal lanthanum hydroxycarbonate was discovered to show significant second harmonic generation, which would be of interest for developing novel optical materials. (C) 2004 Elsevier Inc. All rights reserved.

Substrate specifity of protein kinases and computational prediction of substrates

To ensure signalling fidelity, kinases must act only on a defined subset of cellular targets. Appreciating the basis for this substrate specificity is essential for understanding the role of an individual protein kinase in a particular cellular process. The specificity in the cell is determined by a combination of peptide specificity of the kinase (the molecular recognition of the sequence surrounding the phosphorylation site), substrate recruitment and phosphatase activity. Peptide specificity plays a crucial role and depends on the complementarity between the kinase and the substrate and therefore on their three-dimensional structures. Methods for experimental identification of kinase substrates and characterization of specificity are expensive and laborious, therefore, computational approaches are being developed to reduce the amount of experimental work required in substrate identification. We discuss the structural basis of substrate specificity of protein kinases and review the experimental and computational methods used to obtain specificity information. (c) 2005 Elsevier B.V. All rights reserved.

Structural, mutagenic, and kinetic analysis of the binding of substrates and inhibitors of human phenylethanolamine N-methyltransferase

The X-ray structure of human phenylethanolamine N-methyltransferase (hPNMT) complexed. with its product, S-adenoSyl-L-homocysteine (4), and the most potent inhibitor reported to date, SK&F 64139 (7), was used to identify the residues involved in inhibitor binding. Four of these residues, Va153, Lys57, Glu219 and Asp267, were replaced, in turn, with alanine. All variants had increased K-m values for phenylethanolamine (10), but only D267A showed a noteworthy (20-fold) decrease in its k(cat) value. Both WT hPNMT and D267A had similar k(cat) values for a rigid analogue, anti-9-amino-6-(trifluoromethyl)benzonorbornene (12), suggesting that Asp267 plays an important role in positioning the substrate but does not participate directly in catalysis. The K-i values for the binding of inhibitors such as 7 to the E219A and D267A variants increased by 2-3 orders of magnitude. Further, the inhibitors were shown to bind up to 50-fold more tightly in the presence of S-adenoSyl-(L)-methionine (3), suggesting that the binding of the latter brings about a conformational change in the enzyme.

Are branched chain fatty acids the natural substrates for P450(BM3)?

Branched chain fatty acids are substrates for cytochrome P450(BM3) (CYP102) from Bacillus megaterium; oxidation of C-15 and C-17 iso and anteiso fatty acids by P450(BM3) leads to the formation of hydroxylated products that possess high levels of regiochemical and stereochemical purity.

Uses for JNK: the many and varied substrates of the c-Jun N-terminal kinases

The c-Jun N-terminal kinases (JNKs) are members of a larger group of serine/ threonine (Ser/Thr) protein kinases from the mitogen-activated protein kinase family. JNKs were originally identified as stress-activated protein kinases in the livers of cycloheximide-challenged rats. Their subsequent purification, cloning, and naming as JNKs have emphasized their ability to phosphorylate and activate the transcription factor c-Jun. Studies of c-Jun and related transcription factor substrates have provided clues about both the preferred substrate phosphorylation sequences and additional docking domains recognized by JNK There are now more than 50 proteins shown to be substrates for JNK These include a range of nuclear substrates, including transcription factors and nuclear hormone receptors, heterogeneous nuclear ribonucleoprotein K and the Pol I-specific transcription factor TIF-IA, which regulates ribosome synthesis. Many nonnuclear substrates have also been characterized, and these are involved in protein degradation (e.g., the E3 ligase Itch), signal transduction (e.g., adaptor and scaffold proteins and protein kinases), apoptotic cell death (e.g., mitochondrial Bcl2 family members), and cell movement (e.g., paxillin, DCX, microtubule-associated proteins, the stathmin family member SCG10, and the intermediate filament protein keratin 8). The range of JNK actions in the cell is therefore likely to be complex. Further characterization of the substrates of JNK should provide clearer explanations of the intracellular actions of the JNKs and may allow new avenues for targeting the JNK pathways with therapeutic agents downstream of JNK itself.

Cleavage of caspases-1, -3, -6, -8 and -9 substrates by proteases in skeletal muscles from mice undergoing cancer cachexia

A prominent feature of several type of cancer is cachexia. This syndrome causes a marked loss of lean body mass and muscle wasting, and appears to be mediated by cytokines and tumour products. There are several proteases and proteolytic pathways that could be responsible for the protein breakdown. In the present study, we investigated whether caspases are involved in the proteolytic process of skeletal muscle catabolism observed in a murine model of cancer cachexia (MAC16), in comparison with a related tumour (MAC13), which does not induce cachexia. Using specific peptide substrates, there was an increase of 54% in the proteolytic activity of caspase-1, 84% of caspase-8, 98% of caspase-3 151% to caspase-6 and 177% of caspase-9, in the gastrocnemius muscle of animals bearing the MAC16 tumour (up to 25% weight loss), in relation to muscle from animals bearing the MAC13 tumour (1-5% weight loss). The dual pattern of 89 kDa and 25 kDa fragmentation of poly (ADP-ribose) polymerase (PARP) occurred in the muscle samples from animals bearing the MAC16 tumour and with a high amount of caspase-like activity. Cytochrome c was present in the cytosolic fractions of gastrocnemius muscles from both groups of animals, suggesting that cytochrome c release from mitochondria may be involved in caspase activation. There was no evidence for DNA fragmentation into a nucleosomal ladder typical of apoptosis in the muscles of either group of mice. This data supports a role for caspases in the catabolic events in muscle involved in the cancer cachexia syndrome. © 2001 Cancer Research Campaign.