Ethephon treatment caused SPCP3 gene expression (Chen et al., 2006), reduction of chlorophyll content, decrease of Fv/Fm value, increase of H2O2 amount, and more cell death, and accelerated leaf senescence in detached sweet potato leave. Exogenous application of modulators such as reduced glutathione, EGTA or cycloheximide delay leaf senescence and cell death caused by ethephon. These data suggest that oxidative stress, calcium influx and de novo synthesized protein may influence ethephon-mediated leaf senescence and cell death. When ethephon induced leaf senescence and cell death, granulin-containing cysteine protease SPCP3 gene was induced. Transgenic Arabidopsis system was used to explore the possible physiological role and function of SPCP3. The results showed that ectopic expression of SPCP3 in transgenic Arabidopsis plants caused earlier flowering, less rosette leaves when flowering, higher yellowing silique percentage during harvest, and lower germination percentage than that in control. During drought treatment, transgenic plants also exhibited reduction of Fv/Fm value and relative water content, but an increase in H2O2 content and cell death. These data suggest that ecopic expression of SPCP3 caused altered developmental characteristics and drought stress sensitivity. Previous report suggests that granulin-like domain may play a role in regulating enzymatic activity of granulin-containing cysteine protease (Yamada et al., 2001). In this report we demonstrate that pre-removal of granulin-like domain of SPCP3 does not affect significantly drought stress sensitivity compared to full-length SPCP3 in transgenic Arabidopsis plants. Based on these data we conclude that oxidative stress, calcium influx, and de novo synthesized proteins may be involved in ethylene signaling leading to leaf senescence and SPCP3 gene expression in detached sweet potato leaves, and ectopic SPCP3 expression in transgenic Arabidopsis plants caused altered developmental characteristics and enhanced drought sensitivity. Granulin-like domain may have no significant influence on SPCP3-mediated effect on drought stress sensitivity.

Reversion-inducing cysteine-rich with Kazal motif (RECK) is a cell surface anchoring protein, which known for the ability to inhibit matrix metalloproteinases (MMPs) and participate in angiogenesis regulation. The inhibition of membrane type-1 matrix metalloproteinase (MT1-MMP), MMP-2, MMP-7 and, MMP-9 by RECK has been demonstrated. Our previous studies show that RECK expression is suppressed by Ras and Her-2/neu oncogene. In addition, oncogenic Ras activates downstream ERK signaling pathway to increase Sp1/HDAC promoter binding affinity which results in reduction of RECK gene transcription and increase of tumor progression and metastasis. From the clinical investigation, RECK expression is down-regulated in a number of cancer types. In breast cancer, RECK expression is associated with the prognosis of the patients. Recently, single nucleotide polymorphisms (SNPs) of RECK promoter have been suggested to be linked with survival rate and prognosis of breast cancer patients. Whether SNP of the RECK promoter has any effect on RECK expression and its clinical significance is still unclear. . In this study, we investigate -402 SNP at RECK promoter and find this SNP directly affects RECK expression through progesterone receptor binding. Additionally, we also address the -402 SNP in the sample collected from patients and analyze its association with clinicopathological parameters to clarify its clinical significance. Our results suggest that RECK SNP may be an valuable prognosis factor for breast cancer.

The disulfide bond in a protein is a single covalent bond formed from the oxidation of two cysteines. It plays an important role in the folding and structure stability, and may regulate protein functions. The connectivity prediction problem is difficult because the number of possible patterns grows rapidly with respect to the number of cysteines. We discover some rules to discriminate the patterns with high accuracy in many methods. We implement multiple SVM methods, and utilize the BKS to fuse these classifiers. We apply the hybrid method to SP39 dataset with 4-fold cross-validation for the comparison with the previous works. We raise the accuracy to 71.5%, which improves significantly that of the best previous work, 65.9%.

Iron-sulfur (Fe-S) clusters are critical protein cofactors found in all life forms. In eukaryotes, a well-conserved biosynthetic pathway located in the mitochondria is used to assemble Fe-S clusters. Although proteins required for Fe-S cluster biosynthesis have been identified, their precise function and mechanism remain elusive. In this study, biochemical and biophysical methods are applied to understand molecular details for the core components of the human Fe-S cluster biosynthesis: Nfs1, Isd11, Isu2, and frataxin (Fxn). Nfs1 is a cysteine desulfurase that converts cysteine into alanine and transfers the sulfur to a scaffold protein Isu2 for Fe-S clusters. Fxn depletion is associated with the neurodegenerative disease Friedreich’s ataxia (FRDA), and results in a complicated phenotype that includes loss of Fe-S clusters. The results presented here provide the first in vitro evidence for a stable protein complex that exists in at least two forms: an inactive complex with Nfs1, Isd11, and Isu2 (SDU) components and an active form that also includes Fxn (SDUF). Fxn binding dramatically changes the catalytic efficiency (kcat/KM) of Nfs1 from 25 to 10,100 M-1s-1 and enhances the rate of Fe-S cluster biosynthesis 25 fold. Oxidizing conditions diminish the levels of both complex formation and Fxn-based activation, whereas Fe2 further stimulates Nfs1 activity. Mutagenesis coupled to enzyme kinetics indicate that one of the three conserved cysteines (C104) on Isu2 accepts the sulfane sulfur from Nfs1 and that this transfer event likely requires prior binding of Fxn. In vitro interrogation of FRDA I154F and W155R and related Fxn variants revealed the binding affinity to SDU followed the trend Fxn ~ I154F > W155F > W155A ~ W155R. The Fxn variants also have diminished ability to facilitate both sulfur transfer and Fe-S cluster assembly. Fxn crystallographic structures reveal specific rearrangements associated with the loss of function. Importantly, the weaker binding and lower activity of the W155R variant compared to I154F explains the earlier onset and more severe disease progression. Finally, these experimental results coupled with computational docking studies suggest a model for how human Fxn functions as an allosteric activator and triggers sulfur transfer and Fe-S cluster assembly.

ABSTRACT SENSITIVE AND QUANTITATIVE DETERMINATION OF CYSTEINE BY SURFACE ENHANCED RAMAN SPECTROMETRY BASED ON THEIR INDUCED AGGREGATES OF GOLD AND SILVER NANOSTRUCTURES Y&uuml / ksel, Recep M.Sc., Department of Chemistry Supervisor: Prof. Dr. M&uuml / rvet Volkan September 2011, 76 pages The synthesis of noble metal nanostructures are an active research area and controlling the shape and the size is a challenging task. In this study, nanostructures with different morphologies were prepared using wet chemical synthesis techniques in the aqueous solutions. Gold and silver nanospheres were produced by reducing and capping agent trisodium citrate. Gold nanorods were synthesized by chemical reduction of HAuCl4 by ascorbic acid in the presence of cetyltrimethylammonium bromide (CTAB), AgNO3, and gold nanoseeds (in 1.5 nm diameter) and gold silver core shell nanorods were prepared by addition of silver atoms on the surface of gold nanorods in the presence of CTAB. Parameters that were critical to obtain homogeneous nanostructures were optimized. The characterization of the nanoparticles was performed by UV-VIS spectrometry, High Resolution - Transition Electron Microscopy (HR-TEM), Field Emission - Scanning Electron Microscopy (FE-SEM) and Energy Dispersive X-ray Spectroscopy (EDX). Besides, their electromagnetic enhancement properties were demonstrated through SERS measurement of cysteine. Self-assembly or assisted assembly of nanorods or nanospheres into organized arrays allows the realization of their collective properties that arise from the coupling of the optical and electronic properties of the neighbouring individual nanoparticles. In this study cysteine molecule was used as a linker molecule. The controlled addition of cysteine into the gold nanorod solution resulted in their preferential binding to the two ends of the gold nanorods and the formation of gold nanochains. In the usage of gold nanospheres on the other hand, cooperative hydrogen bonding of the cysteine molecules, resulted in the formation of gold nanoclusters. The assembly formation was demonstrated by UV&ndash / vis spectrometry and FE-SEM. Cysteine is a thiol group containing amino acid and deficiency of cysteine results in serious health problems. Therefore, determination of cysteine is an important issue. Herein we report two strategies for the quantitative determination of micromolar concentrations of cysteine by exploiting the interplasmon coupling in Au nanoparticles. One of them is a recently published colorimetric method and the other is a novel SERS method.

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Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "August 2001." Typescript. Vita. Includes bibliographical references (leaves 172-183). Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 109-123).

Hedgehog signaling is a pathway essential in embryonic development, adult stem cell maintenance, and is implicated in the formation and progression of cancer. Signaling in this pathway is triggered when the secreted protein Hedgehog binds to its membrane receptor, Patched. Patched normally inhibits the seven-spanner transmembrane protein Smoothened (Smo). Binding of Hedgehog inhibits Patched resulting in Smo derepression. Active Smo then triggers the activation of the cytoplasmic steps of the signaling pathway.

The function of the metalloenzymes is mainly determined by four structural features: the metal core, the metal binding motif, the second sphere residues in the active site and the electronic statistics. Cysteamine dioxygenase (ADO) and cysteine dioxygenase (CDO) are the only known enzymes that oxidize free thiol containing molecules in mammals by inserting of a dioxygen molecue. Both ADO and CDO are known as non-heme iron dependent enzymes with 3-His metal binding motif. However, the mechanistic understanding of both enzymes is obscure. The understanding of the mechanistic features of the two thiol dioxygenases is approached through spectroscopic and metal substitution in this dissertation. Another focus of the dissertation is the understanding of the function of a second sphere residue His228 in a 3-His-1-carboxyl zinc binding decarboxylase α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD). ACMSD catalyzes the decarboxylation through a hydrolase-like mechanism that is initialized by the deprotonation of metal bounded water molecule. Our study reveled that the second sphere residue His228 is responsible for the water deprotonation through hydrogen bonding. The spectroscopic and crystallographic data showed the H228Y mutation binds ferric iron instead of native zinc metal and the active site water is replaced by the Tyr228 residue ligation. Thus, we concluded that, H228Y not only plays a role of stabilizing and deprotonating the active site water but also is an essential residue on metal selectivity.