Exocytosis, the fusion of intracellular vesicles with the plasma membrane, is fundamental to intercellular communication in multicellular organisms. This pathway facilitates the release or secretion of molecules from the cell. In addition, exocytosis is essential for delivery of resident proteins to the plasma membrane. There are two different pathways of exocytosis, constitutive and regulated exocytosis. Constitutive exocytosis occurs without regulation, e.g. pathways regulating the delivery of lipids and ‘house-keeping’ proteins to the plasma membrane or the secretion of antibodies and extra-cellular matrix components from the cell. In contrast, regulated exocytosis facilitates the controlled release of extra-cellular molecules or insertion of new membrane components only in response to a physiological signal. The most common signal for regulated exocytosis is an increase in intracellular Ca2+ concentration. Several proteins function in exocytosis, and the membrane fusion step is widely believed to result from an interaction between SNARE (SNAP receptor) proteins on the vesicle membrane and plasma membrane. In neuroendocrine cells, these SNARE proteins are VAMP2, which is bound to vesicle membranes and syntaxin1A and SNAP25, which are associated with the plasma membrane. Several proteins have been implicated as SNARE regulators, such as NSF (N-ethylmaleimide-sensitive factor) and its cofactor α-SNAP, Munc18 and synaptotagmin. Another possible SNARE regulator is the cysteine string protein (CSP). CSPα was first identified in Drosophila melanogaster and was later identified in Torpedo as a possible Ca2+-channel regulator. Inactivation of the CSPα gene in Drosophila is lethal at an embryonic stage and in embryos synaptic vesicle exocytosis was decreased by ~50% at 22°C and was abolished at higher temperatures. These results provided strong evidence that CSPα has an important role in presynaptic neurotransmission. However, more recent work on CSPα null mice uncovered an important neuroprotective function for CSPα in brain, but also challenged the proposed function of CSPα in neuronal exocytosis, as no defect in this pathway was evident, at least in young animals. The only reported developmental abnormality of CSPα null mice was bilateral cryptorchidism, a failure of testicular descent during development. Interestingly, two additional CSP isoforms were recently identified in mouse and human testis, CSPβ and CSPγ. One consequence of the identification of CSPβ and CSPγ is that they may complicate analysis of CSPα knockout mice. Here, we have used a combination of techniques, cell systems and human brain samples to examine the function of CSPα in exocytosis, the expression of novel CSPα isoforms in testis, and expression changes of CSPα and its partner proteins in neurological disorders. Furthermore, we have initiated studies to examine how CSPα function is linked to cryptorchidism at the molecular level. My results show that CSPα depletion perturbs regulated exocytosis in neuroendocrine cells, but has no consistent effect on constitutive exocytosis. CSPα has been reported to have an important neuroprotective function; however, no significant changes in CSPα expression were detected in brain samples for schizophrenia, depression and bipolar disorder. Nevertheless the expression of specific CSPα binding partners was found to be significantly changed in some of these disorders. In addition to these studies focussing on CSPα function and expression in neuronal and neuroendocrine cells, studies were undertaken to analyse expression profiles of CSP isoforms in testis. This analysis found that CSPβ and CSPγ are exclusively expressed in testis, and that mRNA transcription of both isoforms is initiated with sexual maturation. Furthermore expression of both isoforms is restricted to germ cells, whereas CSPα is expressed throughout testes. Previous work has shown that the secretory hormone INSL3, which is exclusively expressed in testicular Leydig cells, is involved in the development of cryptorchidism. Confocal microscopic analysis revealed that CSPα and INSL3 colocalise on vesicles in Leydig cells, suggesting the intriguing possibility that CSPα inactivation might cause cryptorchidism due to a loss of INSL3 secretion.

No description available.

No description available.

Cancer cell metabolism is reorganized around the needs of proliferating cells, particularly the management of organic metabolites and the balance of redox state. Here, we show that pancreatic cancer requires exogenous sources of cysteine for tumor growth and maintenance due to its critical role in redox balance. Using a multidisciplinary approach, we find that cancer cells rely on imported cystine (oxidized cysteine) to detoxify lipid reactive oxygen species (ROS) and avert ferroptosis, a form of non-apoptotic cell death. Cystine–derived glutathione was necessary for this protection, but its depletion was not sufficient to induce ferroptosis. Correspondingly, genetic inactivation of system xc–, the cystine/glutamate antiporter, in established pancreatic tumors induced stabilization or regression, extending survival in an autochthonous mouse model. We observed distinctive lesions of non-apoptotic cell death that may represent an in vivo manifestation of ferroptosis, highlighting a novel, cancer-specific dependency on a potentially druggable membrane channel.

Rainbow trout cystatin C cDNA has been isolated from trout liver. The full-length cystatin cDNA (674 bp) included the 5'untranslated region and the polyadenylation signal sequence AATAAA in the 3' region. Translation of the cDNA defines 132 amino acid residues. Comparison of the amino acid sequence with those of family 2 cystatins indicates that the 21 amino acids at the N-terminal end is a signal peptide necessary for cystatin secretion, and the remaining 111 amino acids represent mature cystatin. Four cysteine residues in the cystatin may form two disulfide bonds producing a molecule with the properties of a family 2 cystatin. Trout cystatin C gene expression was analyzed by Northern blot. This gene is expressed at various levels in all tissues examined. This difference may reflect differences in degree of regulation of cysteine proteinase activities. A high level of trout cystatin C expressed in trout hepatic tissue or cell cultures suggested that cystatin C expression might be related to tumorigenesis. Southern blot of trout genomic DNA showed that the copy number of the trout cystatin gene is probably one per haploid genome. Trout cystatin C was expressed in E. coli at a yield of 3-5 mg/L culture, but no inhibitory activity was detected for the untreated recombinant protein. However, after refolding, recombinant cystatin C displayed inhibitory activity against papain. The dissociation constant of recombinant cystatin C against papain is 1.2 x 10������ nM, similar to that of human cystatin C. Trout cystatin C was also expressed in yeast cells, but no inhibitory activity was detected either. No cystatin C was secreted in the yeast expression system using either the trout cystatin C secretion signal, or the yeast invertase secretion signal. The expression levels of trout cystatin C in our expression systems are still low for industrial requirements. Therefore, further investigation will be needed to construct more efficient expression systems and vectors for trout cystatin C heterologous production. / Graduation date: 1999

Disulfide bonds are special covalent cross links between two cysteines in a protein. This kind of bonding state plays an important role in protein folding and stabilization. For connectivity pattern prediction, it is a very difficult problem because of the fast growth of possible patterns with respect to the number of cysteines. In this thesis, we propose a new approach to address this problem. The method is based on hybrid models with SVM. Via this strategy, we can improve the prediction accuracies by selecting appropriate models. In order to evaluate the performance of our method, we apply the method by 4-fold cross-validation on SP39 dataset, which contains 446 proteins. We achieve accuracies with 70.8% and 65.9% for pair-wise and pattern-wise prediction respectively, which is better than the previous works.

Disulfide bond information can help the prediction of protein secondary structure, tertiary structure and all-atom coordinates. Most of previous works focused on either state classification or connectivity prediction with some assumption that some constraints were added to make the problem solvable in reality. In this thesis, we propose a multi-phase approach to solve the problem. Our method can export the number of bonds and achieve 90.7% accuracy in the state classification. For the connectivity prediction problem, we use the number of bonds we predict as a base to decide bond pairs. For overcoming the ratio imbalance of samples, we propose a down-sampling method to reducing processing time. Finally, we perform the weighted graph matching algorithm to obtain the bonding pattern, which achieves 63.5% accuracy. We also achieve 48% accuracy for the thorough prediction. Our method is validated by the datasets derived from SWISS-PROT and PDB. The results are better than the previous works.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 74-91).

Thesis (Ph. D.)--School of Chemistry and Biochemistry, Georgia Institute of Technology, 2004. Directed by James C. Powers. / Includes bibliographical references (leaves 142-153).

Thesis (Ph. D.)--School of Chemistry and Biochemistry, Georgia Institute of Technology, 2004. Directed by James C. Powers. / Vita. Includes bibliographical references (114-120).