Investigation of the Expression of Glucose Transporter Proteins in Human Cancer Cells

Cancer cells are known to display increased glucose uptake and consumption. The glucose transporter (GLUT) proteins facilitate glucose uptake, however, their exact role in cancer metabolism remains unclear. The present study examined mRNA and protein expression of GLUT1, GLUT3, GLUT4 and GLUT12 in lung, breast and prostate cancer cells and corresponding noncancerous cells. Additionally, GLUT expression was determined in tumours from mice xenografted with human cancer cells. Differences in the mRNA and protein expression of GLUTs were found between cancerous and corresponding noncancerous cells. These findings demonstrate abundant expression of GLUT1 in cancer and highlight the importance of GLUT3 as it was expressed in several cancer cells and tumours. GLUT expression patterns in vitro were supported by the in vivo findings. The study of GLUT protein expression in cancer is important for understanding cancer metabolism and may lead to identification of biomarkers of cancer progression and development of target therapies.

Investigation of single tryptophan proteins encapsulated in TEOS-derived sol-gel matrices by fluorescence spectroscopy /

In the work reported here, optically clear, ultrathin TEOS derived sol-gel slides which were suitable for studies of tryptophan (Trp) fluorescence from entrapped proteins were prepared by the sol-gel technique and characterized. The monitoring of intrinsic protein fluorescence provided information about the structure and environment of the entrapped protein, and about the kinetics of the interaction between the entrapped protein and extemal reagents. Initial studies concentrated on the single Trp protein monellin which was entrapped into the sol-gel matrices. Two types of sol-gel slides, termed "wet aged", in which the gels were aged in buffer and "dry-aged", in which the gels were aged in air , were studied in order to compare the effect of the sol-gel matrix on the structure of the protein at different aging stages. Fluorescence results suggested that the mobility of solvent inside the slides was substantially reduced. The interaction of the entrapped protein with both neutral and charged species was examined and indicated response times on the order of minutes. In the case of the neutral species the kinetics were diffusion limited in solution, but were best described by a sum of first order rate constants when the reactions occurred in the glass matrix. For charged species, interactions between the analytes and the negatively charged glass matrix caused the reaction kinetics to become complex, with the overall reaction rate depending on both the type of aging and the charge on the analyte. The stability and conformational flexibility of the entrapped monellin were also studied. These studies indicated that the encapsulation of monellin into dry-aged monoliths caused the thermal unfolding transition to broaden and shift upward by 14°C, and causedthe long-term stability to improve by 12-fold (compared to solution). Chemical stability studies also showed a broader transition for the unfolding of the protein in dry-aged monoliths, and suggested that the protein was present in a distribution of environments. Results indicated that the entrapped proteins had a smaller range of conformational motions compared to proteins in solution, and that entrapped proteins were not able to unfold completely. The restriction of conformational motion, along with the increased structural order of the internal environment of the gels, likely resulted in the improvements in themial and long-term stability that were observed. A second protein which was also studied in this work is the metal binding protein rat oncomodulin. Initially, the unfolding behavior of this protein in aqueous solution was examined. Several single tryptophan mutants of the metal-binding protein rat oncomodulin (OM) were examined; F102W, Y57W, Y65W and the engineered protein CDOM33 which had all 12 residues of the CD loop replaced with a higher affinity binding loop. Both the thermal and the chemical stability were improved upon binding of metal ions with the order apo < Ca^^ < Tb^"^. During thermal denaturation, the transition midpoints (Tun) of Y65W appeared to be the lowest, followed by Y57W and F102W. The placement of the Trp residue in the F-helix in F102W apparently made the protein slightly more thermostable, although the fluorescence response was readily affected by chemical denaturants, which probably acted through the disruption of hydrogen bonds at the Cterminal end of the F-helix. Under both thermal and chemical denaturation, the engineered protein showed the highest stability. This indicated that increasing the number of metal ligating oxygens in the binding site, either by using a metal ion with a higher coordinatenumber (i.e. Tb^*) which binds more carboxylate ligands, or by providing more ligating groups, as in the CDOM33 replacement, produces notable improvements in protein stability. Y57W and CE)OM33 OM were chosen for further studies when encapsulated into sol-gel derived matrices. The kinetics of interaction of terbium with the entrapped proteins, the ability of the entrapped protein to binding terbium, as well as thermal stability of these two entrapped protein were compared with different levels of Ca^"*^ present in the matrix and in solution. Results suggested that for both of the proteins, the response time and the ability to bind terbium could be adjusted by adding excess calcium to the matrix before gelation. However, the less stable protein Y57W only retained at most 45% of its binding ability in solution while the more stable protein CDOM33 was able to retain 100% binding ability. Themially induced denaturation also suggested that CDOM33 showed similar stability to the protein in solution while Y57W was destabilized. All these results suggested that "hard" proteins (i.e. very stable) can easily survive the sol-gel encapsulation process, but "soft" proteins with lower thermodynamic stability may not be able to withstand the sol-gel process. However, it is possible to control many parameters in order to successfully entrap biological molecules into the sol-gel matrices with maxunum retention of activity.

The study of ligand binding specificities of the lipid binding proteins : recombinant human a-tocopherol transport protein (a-ttp), supernatant protein factor (spf) and S. cerevisiae Sec 14p for vitamin e (rrr-a-tocopherol) and other hydrophobic ligands.

One of the various functions of proteins in biological systems is the transport of small molecules, for this purpose proteins have naturally evolved special mechanisms to allow both ligand binding and its subsequent release to a target site; a process fundamental to many biological processes. Transport of Vitamin E (a-tocopherol), a lipid soluble antioxidant, to membranes helps in the protection of polyunsaturated fatty acids against peroxidative damage. In this research, the ligand binding characteristics of several members of the CRALTRIO family of lipid binding proteins was examined; the recombinant human a-Tocopherol Transfer Protein (a-TIP), Supernatant Protein Factor (SPF)ffocopherol Associated Protein (TAP), Cellular Retinaldehyde Binding Protein (CRALBP) and the phosphatidylinositol transfer protein from S. cerevisiae Sec 14p. Recombinant Sec 14p was expressed and purified from E. coli for comparison of tocopherol binding to the two other recombinant proteins postulated to traffic a-tocopherol. Competitive binding assays using [3H]-a-tocopherol and Lipidex-l000 resin allowed determination of the dissociation constants ~) of the CRAL-TRIO proteins for a-tocopherol and - 20 hydrophobic ligands for evaluation of the possible biological relevance of the binding interactions observed. The KIs (nM) for RRR-a-tocopherol are: a-TIP: 25.0, Sec 14p: 373, CRALBP: 528 and SPFffAP: 615. This indicates that all proteins recognize tocopherol but not with the same affinity. Sec 14p bound its native ligand PI with a KI of381 whereas SPFffAP bound PI (216) and y-tocopherol (268) similarly in contrast to the preferential binding ofRRR-a-tocopherol by a-TIP. Efforts to adequately represent biologically active SPFff AP involved investigation of tocopherol binding for several different recombinant proteins derived from different constructs and in the presence of different potential modulators (Ca+2, Mg+2, GTP and GDP); none of these conditions enhanced or inhibited a-tocopherol binding to SPF. This work suggests that only aTTP serves as the physiological mediator of a-tocopherol, yet structural homology between proteins allows common recognition of similar ligand features. In addition, several photo-affmity analogs of a-tocopherol were evaluated for their potential utility in further elucidation of a-TTP function or identification of novel tocopherol binding proteins.

Amputation and heat induced protein synthesis in the regenerating forelimb of Notophthalmus viridescens

This thesis compares the responses of regenerating forelimb tissues of the newt Notophthalmu..f vlridescens to the stresses of hyperthermia and ID.echanical injury of amputation. In particular, both quantitative and qualitative changes in the synthesis of soluble proteins in stump tissues, including those of the heat shock protein family (HSP70-1ike) were examined. Results from SDS-PAGEfluorography indicate that the trauma of amputation mimics the heat shock response both quantitatively and temporally in its transient repression of the synthesis of most normal cellular proteins, and qualitatively. in the locaJized expression of two unique proteins (hsp30 and hsp70). Fluorography of proteins separated by twodimensional gets revealed that thelCl4:alizedt amputation induced 70kDa protein (amp70) was distinct from the more basic newt hsp/hsc70 isoforms. Although limb amputation resulted in an increase in the synthesis of HSP70 mRNA analogous to that induced by heat 3.b.OCKf amp70 did not cross-react with murine monoclonal antibodies directed against both the inducible and cognate HSP70 proteins of the human. Thus, the possible relationship of amp70 to other members of the HSP70-1ike protein family remains unclear. Western analyses indicated that the levels of the constitutive form of HSP70 (hsc70) were found to be regulated in a stage-dependent manner in the distal stump tissues of the regen,erating forelimb of the newt. The highest levels were found in the mid-late bud stage, a period during which rapidly dividing blastema cells begin to redifferentiate in a proximodistal direction. Immediately after amputation) hsc70 synthesis and accumulation was depressed below steady-state levels measured in the unamputated limb~ The results are discussed in light of a possible role for HSPs and amputatio~ induced proteins in the epimorphic regeneration of the amphibian limb.

Fimbriae of coprinus cinereus, schizophyllum commune and ustilago violacea = structural aspects and a role in conjugation

Surface fibrils (fimbriae) have been observed on fungi from every major group. Fimbriae are thought to be involved in the following cell to cell interactions: conjugation, flocculation and adhesion. Several higher fungi exibit two other types of interactions: hyphal fusion (anastomosis) and clamp connection formation. As a prelude to examining the role of fimbriae in these processes, the fimbriae of two fungi that undergo these fusion events were examined. Electron microscopy studies revealed that Coprinus cinereus and Schizophyllum commune are fimbriated. C. cinereus fimbriae were 5 nm in diameter and 0.5 to 20 11m in length. Fimbriae of C. cinereus oidia were more numerous and longer than those of the hyphal stage. S. commune fimbriae were also 5 nm in diameter, but were only 0.5 to 2 11m in length. There was an unequal distribution of fimbriae on the hyphal surfaces of S. commune . Fimbriae were sparsely distributed over the entire hyphal surface, with higher densities of fibrils present on the side growths of the hyphae found in the older sections of the mycelium. Antiserum raised against Ustilago violacea fimbrial protein (AU) crossreacted strongly with 37 and 39 kd C. cinereus mycelial proteins. In contrast, AU bound very weakly to 89 and 92 kd S. commune mycelial proteins. Since AU cross-reacted poorly with S. commune fimbrial proteins, it was impossible to further characterize the fimbriae of this specIes. The 37 and 39 kd C. cinereus proteins, were isolated by electroelution and were shown to be able to form fibrils the same diameter as oidial fimbriae. The 37 kd protein was shown to be composed of several proteins with isoelectric points ranging from pH 6.1 to 7.63. Furthermore, the 37 kd protein was found to be multimeric, while the 39 kd protein was not. These results strongly suggested that the 37 kd protein is the structural fimbrial protein of C. cine reus . Finally, a series of experiments were designed to determine whether fimbriae are required for conjugation in U. violacea Conjugation was inhibited significantly with AU, but not with pre-immune serum or AU preincubated with purified fimbrial protein. Thus, it was concluded that fimbriae play a central role in mating in this organism.

Involvement of fimbriae in host-mycoparasite recognition

Extracellular, non-flagellar appendages, termed fimbriae are widespread among fungi. Fungal fimbriae range in diameter from 6-10 nm and exhibit lengths of up to 30 ~m. Fungal fimbriae have been implicated in several functions: adhesion, conjugation and flocculation. A possible role of fimbriae in host-mycoparasite interactions was the focus of this study . Using electron microscopy, fimbriae were observed on the surfaces of Mortiere lla cande labrum, Mortie re lla pusi lla and Phascolomyces articulosus with diameter means of 9.1±0.4 nm, 9.4±0.5 nm and 8.6±0.6 nm, respectively, and lengths of up to 25 ~m. Fimbriae were not observed on the surface of the mycoparasite, Piptocephalis virginiana. Polyclonal antiserum (AU) prepared against the fimbrial protein of Ustilago violacea cross-reacted with 60 and 57 kDa M. candelabrum proteins. In addition, AU cross-reacted with 64 kDa proteins from both M. pusilla and P. articulosus. The proteins that cross-reacted with AU were electroeluted from polyacrylamide gels and were shown to subsequently form fibrils. The diameter means for the electroeluted fibrils were: for M. candelabrum 9.7±0.3 nm, M. pusilla 8.4±0.6 nm and P articulosus 9.2±0.5 nm. Finally, to ascertain the role of fimbriae in host-mycoparasite interactions, AU was incubated with P. virginiana and M. pusilla (mycoparasite/susceptible host) and with P. virginiana and P . articulosus (mycoparasite/ resistant host). It was observed that AU decreased significantly the level of contact between P. virginiana and M. pusilla and between P. virginiana and P. articulosus in comparison to prelmmune serum treatments. Thus, it was proposed that fimbriae might play recognition and attachment roles in early events of mycoparasitism.

The role of skeletal muscle PLIN proteins at rest and following lipolytic stimulation

This thesis investigated the subcellular location of skeletal muscle PLIN proteins (PLIN2, PLIN3, and PLIN5) as well as protein interactions with ATGL and HSL at rest and following lipolytic stimulation. In addition, the serine phosphorylation state of PLIN2, PLIN3, and PLIN5 was determined at rest and following lipolytic stimulation. An isolated whole muscle technique was used to study the effects of contraction and epinephrine-induced lipolysis. This method allowed for the examination of the effects of contraction and epinephrine alone and in combination. Further, the soleus was chosen for investigating the role of PLIN proteins in skeletal muscle lipolysis due to its suitability for isolated incubation, and the fact that it is primarily oxidative in nature (~80% type I fibres). It has also been previously shown to have the greatest reliance on lipid metabolism and for this reason is ideal for investigating the role of PLIN proteins in lipolysis. Immunofluorescence microscopy revealed that skeletal muscle lipid droplets are partially co-localized to both PLIN2 and PLIN5 and that contraction does not affect the amount of colocalization, indicating that PLIN5 is not recruited to lipid droplets with contraction (PLIN2 ~65%; PLIN5 ~56%). Results from the immunoprecipitation studies revealed that with lipolysis in skeletal muscle the interaction between ATGL and CGI-58 is increased (study 2: 128% with contraction, p<0.05; study 3: 50% with contraction, 25% epinephrine, 80% contraction + epinephrine, p>0.05). Further PLIN2, PLIN3, and PLIN5 all interact with ATGL and HSL, while only PLIN3 and PLIN5 interact with CGI-58. Among these interactions, the association between PLIN2 and ATGL decreases with lipolytic stimulation (study 2: 21% with contraction, p<0.05). Finally our results demonstrate that PLIN3 and PLIN5 are serine phosphorylated at rest and that the level of phosphorylation remains unchanged in the face of either contractile or adrenergic stimulation. In summary, the regulation of skeletal muscle lipolysis is a complex process involving multiple proteins and enzymes. The skeletal muscle PLIN proteins likely play a role in skeletal muscle lipid droplet dynamics, and the data from this thesis indicate that these proteins may work together in regulating lipolysis by interaction with both ATGL and HSL.