As the major protein of the lens, α-crystallin is a molecular chaperone that stabilises lens proteins to prevent their precipitation into solution. In this role it is vital in maintaining lens transparency. The chaperone ability of α-crystallin and its individual subunits, αΑ- and αB-crystallin, has been shown to be sensitive to a variety of environmental and intrinsic factors, including temperature, denaturation and post-translational modification. The effect of pH on α-crystallin chaperone ability, however, has not been thoroughly investigated. There is limited evidence to suggest that the chaperone ability of α-crystallin is pH-sensitive such that α- crystallin is a significantly worse chaperone at pH 6.0 than at pH 8.0. This is of physiological significance since in the lens there is a measurable pH gradient of pH 7.2 in outer lens cells, compared to pH 6.7 in the lens nucleus. A loss of α-crystallin chaperone function in the lens nucleus, as a consequence of decreased pH, may compromise lens transparency. Similarly, extra-lenticular fibrillar aggregation of some disease-related target proteins (Aβ-peptide, for example) is promoted by acidic pH. This study investigates the effect of pH on the chaperone ability of α-crystallin and its subunits. Further, this study characterises the structural changes to α-crystallin accompanying pH variation in an attempt to explain the structural basis for the observed pH sensitivity. In addition, this study examines the chaperone function of cyclodextrins, a class of chemical chaperones that may act in conjunction with α-crystallin as part of a two-step protein refolding pathway. This study demonstrated that the chaperone activity of α-crystallin is pH sensitive between pH 6.0 and 8.0; the ability of α-crystallin to protect against temperature- and reduction-stress induced amorphous aggregation is significantly reduced at pH 6.0 and 6.5 compared to pH 7.0 and above. The decreased chaperone ability of α-crystallin at pH 6.0 and 6.5 was accompanied by partial unfolding of the protein, and a loss of secondary structure, while α-crystallin quaternary structure remained unchanged. Interestingly, α-crystallin was found to have significant chaperone ability below pH 4.0, conditions under which α-crystallin is largely unfolded. The unfolding of α-crystallin at pH 6.0 and 6.5 is comparatively minor, and it is difficult to say whether this unfolding is directly responsible for the observed pH sensitivity of α-crystallin chaperone ability. The thermal stability of α-crystallin was compromised at pH 6.0 and 6.5, which may partially explain its decreased chaperone ability at these pH values in heat-stress assays conducted at temperatures above 50oC. However, α-crystallin chaperone activity remained pH sensitive at 37°C and 45°C, at which temperatures it is thermally stable. Blocking exposed αB-crystallin histidine residues by chemical modification removed, to a large extent, the pH-sensitivity of its chaperone activity. This suggests that the protonation of an exposed histidine residue(s) at pH 6.0 and 6.5 is responsible for the observed pH sensitivity of α-crystallin chaperone ability. Inhibiting the protonation of a specific histidine residue, H83, by site-directed mutagenesis (H83A) did not remove the pH sensitivity of αB-crystallin chaperone activity, and suggests that protonation of this residue alone does not explain the decreased chaperone ability of α-crystallin at mildly acidic pH. This residue lies within the putative chaperone-binding region of αB-crystallin, and is highly conserved between species and between the human small heat shock proteins. It appears that the protonation of several histidine residues, or residues other than H83, is primarily responsible for the influence of pH on α-crystallin chaperone ability observed in this study. The observed decrease in α-crystallin chaperone function below pH 7.0 partially explains the preferential formation of age-related cataract in the lens nucleus, as the chaperone ability of α-crystallin would be compromised under the mildly acidic conditions characteristic of the nucleus. Additionally, the pH sensitivity of α-crystallin chaperone ability may be significant in the ability of extra-lenticular αB-crystallin to inhibit amyloid-related disease at sites of localised acidosis. Cyclodextrins are a family of cyclic oligosaccharides that have been shown to function as chemical chaperones under specific protein aggregation conditions. Cyclodextrins have been demonstrated to facilitate the refolding of chemicallystressed target proteins that have already bound to synthetic nanogels, which act in a manner reminiscent of small heat shock proteins. In this study, cyclodextrins were unable to act in conjunction with α-crystallin to facilitate the refolding of thermallystressed target proteins. β-Cyclodextrin (βCD) demonstrated little or no ability to inhibit the amorphous aggregation of target proteins, but was able to significantly inhibit the fibrillar aggregation of a number of target proteins, including the diseaserelated A53T α-synuclein mutant. Characterisation of the binding of βCD to target proteins during fibrillar aggregation via circular dichroism, intrinsic and extrinsic fluorescence and competitive chaperone assays provided a model of the cyclodextrin chaperone mechanism. In this model, cyclodextrins interact with already partially unfolded, pre-fibrillar protein intermediates via the insertion of aromatic residues into the cyclodextrin anulus, and by doing so inhibit intra-fibrillar π-bonding and protofilament assembly. This suggests the potential for cyclodextrins as therapeutic molecular chaperones in vivo that may be able to inhibit the pathogenic aggregation of target proteins. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1368102 / Thesis (Ph.D.) - University of Adelaide, School of Chemistry and Physics, 2009

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 238-308).

Many proteins can easily attain a non-native fold and be of no use or even a detriment to the host. The host cell has a myriad of molecules dedicated to assisting nascent and existing proteins in folding properly and maintaining the native fold. Of these molecular chaperones, the small Heat Shock Proteins (sHSP’s) are an important group and worthy of study. The sHSP’s are a diverse group of proteins that have in common an a-crystallin domain and generally display a chaperone activity. A sHSP (HSP20) isolated from the cattle parasite Babesia bovis has similar activities, and limited sequence homology to other a-crystallins. The gene encoding HSP20 was cloned into an expression system where the gene product was induced and purified for study. It was shown that HSP20 inhibits thermally induced aggregation of alcohol dehydrogenase at equimolar ratios. HSP20 was also used to significantly reduce amyloid formation of the b-Amyloid (1-40) Peptide in vitro at the sub-stoichiometric ratio of 1:10. A study of the oligomeric forms of HSP20 using size exclusion chromatography and gel electrophoresis revealed a broad range of multimers present in solution. The distribution of oligomers was affected by altering the solution conditions and concentration of the protein. The domains responsible for multimerization of HSP20 were mapped via sequence homology with known a-crystallins. These regions correspond to 12 carboxy-terminal amino acids and 50 amino-terminal amino acids. Truncated versions of HSP20 lacking these proposed oligomerization domains were created using PCR of the original gene and cloning into an expression vector as before. Using size exclusion chromatography, gel electrophoresis and analytical centrifugation, we show that the deleted domains alter the multimeric population of the protein in solution. The carboxy-terminal domain has a slight effect on multimerization while the amino-terminal deletion results in a drastic reduction in any multimers above a dimer under the conditions tested. Despite this drastic change in the multimerization of HSP20, there were no changes in the activities observed when compared to the full-length form. From this we conclude that the regions responsible for multimerization play little role in the observed activities of HSP20.

No description available.

Protein nanofibres, commonly known as amyloid fibrils, are emerging as potential biological nanomaterials in a number of applications. Protein nanofibres are a highly ordered insoluble form of protein, which results when a normally soluble protein aggregates via a self-association process. However, researchers are currently faced with several challenges such as finding a cheap source of proteins that can be obtained without expensive purification and optimizing a scalable method of the manufacturing of protein nanofibres. This thesis has identified crude mixtures of fish lens crystallins as a cheap protein source and has optimized methods for large scale production of protein nanofibres of varying morphologies. Results show that by varying the conditions of fibre formation, individual protein fibres can be used as building blocks to form higher order structures. This ability to control the morphology and form higher ordered structures is a crucial step in bottom up assembly of bionanomaterials and opens possibilities for applications of protein nanofibres. The method of formation of protein nanofibres was optimized on a bench scale (1.5 mL Eppendorf tubes) and successfully scaled-up to 1 L volume. For larger scale-up volume (i.e. greater than 10 ml), internal surface area was important for the formation of protein nanofibres. The crude crystallin mixture prepared at 10 mg/mL was heated at 80oC in the presence of 10% v/v TFE at pH 3.8 for 24 hours and stored for an additional of 24 hours at room temperature for storage process. Aggregation and precipitation of proteins were observed as the protein solution was added to the pre-heated TFE. The resulting protein nanofibres were characterised using ThT dye binding, TEM and SEM. The TEM images show a network of long and criss-crossing protein nanofibres with individual fibres of approximately 10 to 20 nm in diameter and 0.5 to 1 μm long. These protein nanofibres were prepared in 1 mL centrifuge tubes and were left on the laboratory bench at room temperature. After 5 months, fresh TEM grids of the sample were prepared and visualized using TEM. Interestingly, TEM images show that a number of individual fibres had self-assembled in an intertwining fashion to form large bundles and higher order structures containing bundles of nanofibres up to 200 nm thick.

αB-crystallin is a small heat-shock chaperone protein (sHSP) required for the homeostasis of multiple tissues including eye lens, retina, heart and brain. Correspondingly, mutation or altered levels of αB-crystallin are associated with multiple degenerative diseases including cataract, retinal degeneration, cardiomyopathy and Lewy body disease. Based on its wide-ranging importance understanding the protective and homeostatic properties of α B-crystallin is critical for understanding degenerative diseases and could lead to the development of therapies to treat these diseases. αB-crystallin is localized to the mitochondria suggesting a direct effect on mitochondrial function. My thesis work has examined those molecular pathways required for translocation of αB-crystallin to the mitochondria and to identify the downstream pathways controlled by mitochondrial translocation of αB-crystallin that could be important for cellular protection and differentiation. My results point to a novel role of αB-crystallin in regulation of key apoptotic pathways that mediate the balance between cell survival and differentiation. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

L-lactate dehydrogenase (LDH) cDNAs encoding for LDH-A4 (muscle) and LDH-B4 (heart) isozymes from caiman (Caiman crocodilus apaporiensis) belonging to the order Crocodilia were sequenced. The phylogenetic relationships of the newly determined cDNA and their deduced protein sequences, as well as the previously published sequences of vertebrate LDH isozymes were analyzed by various phylogenetic tree construction methods. These results indicated that Chelonia is indeed more closely related to Crocodilia. The divergent times between caiman and alligator, Chelonia and Crocodilia, were estimated to be approximately 36, 177 million years, respectively. £`-crystallin/Lactate dehydrogenase B cDNA from caiman (Caiman crocodilus apaporiensis), Pekin duck (Anas platyrhynchos), Muscovy duck (Cairina moschata) and Greylag goose (Anser anser) eye lens were sequenced. Accorcding to cDNA sequences, duck lens £`¡Vcrystallin and heart LDH-B are the products of the same gene. In amino acid sequences, two residues Asn-114 and Phe-118 are well conserved in£`-crystallin/ LDH-B among caiman, Muscovy duck and Greylag goose except in Pekin duck which are replaced by glycine residues. The lens protein composition, LDH activity and£`-crystallin/ LDH B4 protein structure of caiman and three avians were analyzed and compared. The results show no significant differences in conformational or enzymatic properties between Pekin duck £`-crystallin and caiman, Muscovy duck and Greylag goose £`-crystallin. The unique replacement of both Asn-114 and Phe-118 by Gly residues in Pekin duck £`-crystallin amino acid sequence might therefore be due to the selective pressure during the recruitment processes of active enzyme into avian lens£`-crystallins.

Tumors are composed not only of malignant cells, but also of various types of normal cells, including vascular cells and infiltrating immune cells, which drive tumor development and progression. The tumor vasculature is abnormal and dysfunctional due to sustained tumor angiogenesis driven by high levels of pro-angiogenic factors. Proteins differentially expressed in tumor vessels affect vascular function and the tumor microenvironment and may serve as targets for therapy. The tumor is also a site of sustained chronic inflammation. The recruitment and activation of inflammatory cells significantly influence tumor progression and regression. Targeting molecules regulating tumor angiogenesis and inflammation in the tumor microenvironment is therefore a promising strategy for the treatment of cancer. This thesis is aiming to understand and investigate the molecular regulation of these two processes in tumors. αB-crystallin is a heat shock protein previously proposed as a target for cancer therapy due to its role in increasing survival of tumor cells and enhancing tumor angiogenesis. In this thesis, we demonstrate a novel role of αB-crystallin in limiting expansion of CD11b+Gr1+ immature myeloid cells in pathological conditions, including tumor development. In addition, we show that αB-crystallin regulates leukocyte recruitment by promoting expression of adhesion molecules ICAM-1, VCAM-1 and E-selectin during TNF-α-induced endothelial activation. Therefore, targeting of αB-crystallin may influence tumor inflammation by regulating immature myeloid cell expansion and leukocyte recruitment. Abnormal, dysfunctional vessels are characteristic of glioblastomas, which are aggressive malignant brain tumors. We have identified the orphan G-protein coupled receptor ELTD1 as highly expressed in glioblastoma vessel and investigated its role in tumor angiogenesis. Interestingly, deficiency of ELTD1 was associated with increased growth of orthotopic GL261 glioma and T241 fibrosarcoma, but did not affect vessel density in any model. Further investigation is warranted to evaluate whether ELTD1 serves a suitable vascular target for glioblastoma treatment. Anti-angiogenic drugs targeting VEGF signaling is widely used in the clinic for various types of cancer. However, the influences of anti-angiogenic treatment on tumor inflammation have not been thoroughly investigated. We demonstrate that VEGF inhibits TNF-α-induced endothelial activation by repressing NF-κB activation and expression of chemokines involved in T-cell recruitment. Sunitinib, a small molecule kinase inhibitor targeting VEGF/VEGFR2 signaling increased expression of chemokines CXCL10, CXCL11, and enhanced T-lymphocyte infiltration into tumors. Our study suggests that anti-angiogenic therapy may improve immunotherapy by enhancing endothelial activation and facilitating immune cell infiltration into tumors.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19517号 / 理博第4177号 / 新制||理||1600(附属図書館) / 32553 / 京都大学大学院理学研究科化学専攻 / (主査)教授 藤井 紀子, 教授 三木 邦夫, 教授 杉山 弘 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

The three projects utilized polymer physics theories to investigate polymer aggregation mechanics. Dynamic light scattering (DLS), static light scattering (SLS) and small angle light scattering (SALS) were the primary characterization tools. The goal of the first project was to study the aggregation of bovine βL-crystallin and apply that knowledge towards cataract formation, which is caused by aggregation of the crystallins. The first series of experiments characterized the kinetics of α-crystallin and βL-crystallin in water at room temperature. α-crystallin’s equilibrium hydrodynamic radius value was kinetically independent. βL-crystallin formed an aggregate with an Rh that was kinetically dependent. The packing structure of the aggregate formed by βL-crystallin was determined to be loosely packed using SLS. α -crystallin was uniquely demonstrated to be a chaperone in a way that indicated electrostatics played a significant role in aggregation. The role of electrostatics led to an investigation into sodium chloride. Sodium chloride proved to reduce the βL-crystallin aggregate size. The next series of experiments simulated biological conditions using a phosphate buffered saline (PBS). The experiments were performed at 35oC. α -crystallin and βL-crystallin were shown to be kinetically independent and demonstrate equilibrium Rh values on the time scale that the experiments were performed. A pH study revealed that multiple size-scales were present only at physiological pH. Above and below physiological pH, only two aggregate size-scales existed. A charge model was made of βL-crystallin to compare theory with experimental results. The future goal of project is to reproduce these experiments with human crystallins. In the second project, by changing the order and arrangement of β-spiral elastin (E) and α -helical COMPcc (C) the macroscopic structure was controlled. The EC diblock exhibited a fast and slow mode below the transition temperature of 25oC and single mode behavior above the transition. Phase separation occurred above the transition. CE showed three different size-scales below the transition of 15oC and demonstrated spinodal decomposition above the transition. The ECE triblock demonstrated bimodal behavior below the transition of 25oC and one micellar size above the transition. α-helical COMPcc has the ability to bind to small molecules, making the findings from this project instrumental in creating a drug delivery vehicle. The third project investigated sodium polystyrene sulfonate and polyethylene oxidepolypropylene oxide-polyethylene oxide in solution. Both systems self-assemble into aggregate structures at specific conditions. The significant difference between these two polymers is that sodium polystyrene sulfonate is a polyelectrolyte. It is well known that aggregate structures can be formed by variation in temperature and concentration. However, by having a charged polymer in solution with a neutral polymer the aggregate structure can also be controlled by changing the pH and adding salt to the solution, as was performed in the first project. The third project is an excellent conclusion to the previous two because it allows for the aggregate structure to be controlled even more so than in the previous projects by mediating the polydispersity index, molecular weight and concentration of each component. Each project focused on a different method of mediating the aggregate structure. A better understanding of aggregation has applications in industry and medicine. Polymer physics theory is instrumental in understanding aggregation mechanics.