Student Number : 0002482E - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / Proteins in the glutathione transferase family share a common fold. The close packing of secondary structures in the thioredoxin fold in domain 1 forms a compact hydrophobic core. This fold has a bababba topology and most proteins/domains with this fold have a topologically conserved isoleucine residue at the N-terminus of a-helix 3. Class Alpha glutathione transferases are one of 12 classes within the glutathione transferase family. To investigate the role of the conserved isoleucine residue in the structure, function and stability of glutathione transferases, homodimeric human glutathione transferase A1-1 (hGST A1-1) was used as a representative of the GST family. Ile71 was replaced with valine and the properties of I71V hGST A1-1 were compared with those of wildtype hGST A1-1. The spectral properties monitored using far-UV CD and tryptophan fluorescence indicated little change in secondary or tertiary structure confirming the absence of any gross structural changes in hGST A1-1 due to the incorporation of the mutation. Both wildtype and mutant dimeric proteins were determined to have a monomeric molecular mass of 26 kDa. The specific activity of I71V hGST A1-1 (130 mmol/min/mg) was three times that of wildtype hGST A1-1 (48 mmol/min/mg). I71V hGST A1-1 showed increased kinetic parameters compared to wildtype with a 10-fold increase in kcat/Km for CDNB. The increase in Km of I71V hGST A1-1 suggests the mutation had a negative effect on substrate binding. The DDG for transition state stabilisation was –5.82 kJ/mol which suggest the I71V mutation helps stabilise the transition state of the SNAR reaction involving the conjugation of reduced glutathione (GSH) to 1-chloro-2,4-dinitrobenzene (CDNB). A 2-fold increase in the IC50 value for I71V hGST A1-1 (11.3 mM) compared to wildtype (5.4 mM) suggests that the most noticeable change due to the mutation occurs at the H-site of the active site. Conformational stability studies were performed to determine the contribution of Ile71 to protein stability. The non-superimposability of I71V hGST A1-1 unfolding curves and the decreased m-value suggest the formation of an intermediate state. The conformational stability of I71V hGST A1-1 (16.5 kcal/mol) was reduced when compared to that of the wildtype (23 kcal/mol). ITC was used to dissect the binding energetics of Shexylglutathione to wildtype and I71V hGSTA1-1. The ligand binds 5-fold more tightly to wildtype hGST A1-1 (0.07 mM) than I71V hGST A1-1 (0.37 mM). The I71V mutant displays a larger negative DCp than wildtype hGST A1-1 (DDCp = -0.41 kJ/mol/K). This indicates that a larger solvent-exposed hydrophobic surface area is buried for I71V hGST A1-1 than for wildtype hGST A1-1 upon the binding of S-hexylglutathione. Overall the results suggest that Ile71 conservation is for the stability of the protein as well as playing a pivotal indirect role in catalysis and substrate binding.

There are several diseases well known that are due to aberrant protein folding. These types of diseases can be divided into three main categories: Loss-of-function diseases Gain-of-toxic-function diseases Infectious misfolding diseases   Most loss-of-function diseases are caused by aberrant folding of important proteins. These proteins often misfold due to inherited mutations. The rare disease marble brain disease (MBD) also known as carbonic anhydrase II deficiency syndrome (CADS) can manifest in carriers of point mutations in the human carbonic anhydrase II (HCA II) gene. We have over the past 10-15 years studied the folding, misfolding and aggregation of the enzyme human carbonic anhydrase II. In summary our HCA II folding studies have shown that the protein folds via an intermediate of molten-globule type, which lacks enzyme activity and the molten globule state of HCA II is prone to aggregation. One mutation associated with MBD entails the His107Tyr (H107Y) substitution. We have demonstrated that the H107Y mutation is a remarkably destabilizing mutation influencing the folding behavior of HCA II. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II towards populating ensembles of intermediates of molten globule type under physiological conditions. The enormously destabilizing effects of the H107Y mutation is not due to loss of specific interactions of H107 with residue E117, instead it is caused by long range sterical destabilizing effects of the bulky tyrosine residue. We also showed that the folding equilibrium can be shifted towards the native state by binding of the small-molecule drug acetazolamide, and we present a small molecule inhibitor assessment with select sulfonamide inhibitors of varying potency to investigate the effectiveness of these molecules to inhibit the misfolding of HCA II H107Y. We also demonstrate that high concentration of the activator compound L-His increases the enzyme activity of the mutant but without stabilizing the folded protein.   The infectious misfolding diseases is the smallest group of misfolding diseases. The only protein known to have the ability to be infectious is the prion protein. The human prion diseases Kuru, Gerstmann-Sträussler-Scheinker disease (GSS) and variant Creutzfeldt-Jakob are characterized by depositions of amyloid plaque from misfolded prion protein (HuPrP) in various regions of the brain depending on disease. Amyloidogenesis of HuPrP is hence strongly correlated with prion disease. Our results show that amyloid formation of recHuPrP90-231 can be achieved starting from the native protein under gentle conditions without addition of denaturant or altered pH. The process is efficiently catalyzed by addition of preformed recHuPrP90-231 amyloid seeds. It is plausible that amyloid seeding reflect the mechanism of transmissibility of prion diseases. Elucidating the mechanism of PrP amyloidogenesis is therefore of interest for strategic prevention of prion infection.

Protein stability plays an important role in a wide variety of settings ranging from industrial processes where proteins are used as biocatalysts to medical settings where misfolded proteins are implicated in disease. Understanding protein stability will allow design of improved bioprocess and pharmaceutical formulations as well as aid in the development of therapies for protein-based diseases. The effects of dissolved salts on protein kinetic stability are studied here. We find that ion-solvent interactions, characterized by the Jones-Dole B-viscosity coefficient, are strong indicators of salt effects on protein deactivation. This finding is used to develop a model for predicting protein deactivation in salt solutions in terms of two competing processes. Since protein unfolding and aggregation can lead to a number of protein misfolding diseases, we test the applicability of our model for describing salt effects on transthyretin aggregation. As the factors contributing to protein stability become more understood, the use of enzymes as biocatalyst for industrial process will increase, and the need for enzymes active in a wide range of reaction media will increase. We have developed a process using an enzyme in combination with organic-aqueous tunable solvents (OATS) which allows for monophasic reaction of the enzyme with hypdrophobic substrates. The reaction mixture can be separated into two phases by the addition of carbon dioxide pressure. This separation allows for both convenient recovery of the hydrophobic reaction product from the organic phase as well as recycle of the enzyme in the aqueous phase. Overall reaction conversions of 80% and little enzyme activity loss are observed after six reaction cycles.

The experiments which are required to directly assess the operational stability of thermostable biocatalysts can be time-consuming, troublesome, and, in the context of industry, expensive. In the present work, we develop and validate two methods for quickly estimating the total turnover number (a useful indicator of lifetime productivity) of a biocatalyst for any desired operating temperature. The first method is a heuristic approach, built upon a complete mathematical derivation from first principles, in which the total turnover number can be calculated from two simple biochemical measurements. The second method relies on a single non-isothermal, continuous-mode experiment in conjunction with mathematical modeling to determine the intrinsic deactivation parameters of the biocatalyst. Both methods provide estimates of the total turnover number which are well within one order of magnitude of the values measured directly via isothermal aging tests and therefore are extremely valuable tools in terms of the amount of experimental time eliminated.

The transactivator of transcription protein (Tat) of human immunodeficiency virus (HIV) plays an important role in both viral replication and AIDS pathogenesis. Tat is responsible for enhancing HIV-specific transcription, without which production of infectious virus is severely limited. Ongoing research, however, has identified a plethora of additional functions attributable to Tat including regulating additional HIV processes, altering the homeostasis of both infected and uninfected cells, and being directly involved in AIDS-related neuropathology and cancer. Tat thus plays a diverse role in both viral replication and pathogenesis. What is the relative importance of Tat’s additional functions compared to its principle function of transactivation? This question is explored in Chapter 2 in which tat clones from an epidemiologicallylinked transmission cohort were functionally tested. tat sequences were isolated and cloned from the cohort members at various time points and assessed for transactivation potential. The data revealed that transmission of HIV resulted in the selection of mutations in tat that were host specific and that impacted on the Tat clones’ abilities to transactivate. No correlation, however, was observed between the transactivation potentials of the Tat clones and the AIDS progression statuses of the hosts. The host-specific tat mutations may instead reflect the selection of variants fitter in one or more of Tat’s other functions. This therefore leads to the hypothesis that Tat functions in addition to transactivation are influential in AIDS pathogenesis and are subject to selective pressures during HIV transmission. Tat’s flexibility and diversity of functions are mediated by defined functional domains. One of the most important of these is the basic domain, a highly conserved region encompassing a unique nuclear localisation signal (NLS). The NLS allows Tat to localise to the cell nucleus to participate in transactivation. However, many of Tat’s additional functions require Tat to be outside of the nucleus or even outside of the cell. It is currently unknown how the NLS is modulated to enable Tat nuclear egress, but it may involve post-translational modifications of NLS residues. In Chapter 3, protein arginine methyltransferase 6 (PRMT6) is demonstrated to alter the localisation of Tat within the nucleus and increase the half-life of Tat within the cell. Both of these effects require the methylation activity of PRMT6 and two NLS residues, arginines 52 and 53, previously described to be substrates for PRMT6 methylation. The results suggest that PRMT6 primes Tat for functions outside of the nucleus by altering its subcellular localisation and increasing its stability. The critical role of the basic domain in Tat function is highlighted in Chapter 4 in which mutation of the basic domain led to dysregulation of HIV replication. The mutant, referred to as Nullbasic, has characteristics similar to transdominant Tat mutants, such as suppressing transactivation and localising to the cell cytoplasm. Surprisingly, Nullbasic potently inhibits HIV infectivity by strongly downregulating the expression of envelope, the key molecule involved in cell attachment and entry. Part of this downregulation is attributable to Nullbasic interfering with the nuclear export of envelope-encoding mRNA as a result of altering the subcellular localisation of Rev. However, an additional mechanism of interference is required to fully explain the strong suppression of envelope protein observed in cells. The data allude to a role for Tat in promoting envelope mRNA translation, a role subverted by mutating the basic domain. Taken together, these studies highlight the importance of a diversity of Tat functions to HIV replication and AIDS pathogenesis. Further, they emphasise the critical role of the basic domain to Tat function. Modifications of the basic domain not only influence function, but also regulate Tat spatially and temporally. Dysregulation of the basic domain by mutation not only subverts Tat function, but also elicits antiviral activity at multiple steps of the HIV lifecycle. The study of Tat and its basic domain is crucial in order to understand its role in HIV/AIDS and to recognise Tat as a pathogenic agent in its own right.

Master of Science / Division of Biology / Nicholas A. Wallace / Cancer is not a single story, but rather numerous often interwoven tales, each with its own characters and progression. In the case of human papillomavirus (HPV) induced cervical cancer (CaCx), the narrative is about the relationship between virus and host, with the consequences of evolution’s shortsightedness driving the plot. Along with the increased proliferative state characteristic of cancer, cells experience frequent, inaccurate replication and replication stresses (ex. DNA damage and nucleotide starvation). To prevent replication fork stall and collapse generated by these stresses, the cell employs translesion synthesis (TLS). Notably, most of the genes in this pathway are upregulated in CaCx; however, the key protein polymerase eta is not. We have observed that upregulation in this pathway is complicated. It occurs at numerous levels, increasing both mRNA and protein abundance. This research further dissects how TLS upregulation occurs. Data shows that in CaCx-derived cell lines, the stability of some TLS proteins is increased, while the stability of other TLS proteins is unchanged. The increased proliferation, typical of these cell lines, cannot account for the enhanced stability. Despite increased TLS protein stability, these cells fail to adequately activate TLS increasing the risk of DNA damage. Genomic instability is a driving factor in HPV genome integration that prevents viral propagation and leads to cell transformation. It also raises mutagenesis rates, likely creating a selective pressure for tolerating failed TLS. The elevated mutation rate known to be associated with failed TLS could also provide a mechanism for acquired resistance to the drugs commonly used to treat CaCx. Changes in protein abundance are routinely used as biomarkers that can lead to the improved outcomes associated with early cancer detection. Elevated TLS protein could be leveraged to ensure cervical cancers are detected during Stage 1, when the 5-year survival rate is 80-90%, rather than at Stage IV, when the rate dips to around 15%.

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Indiana University-Purdue University Indianapolis (IUPUI) / The overexpression of the multi-domain enzyme fatty acid synthase (FASN) has long been associated with poor clinical prognosis and treatment outcome in various cancers. Previous research in the Zhang lab has determined a role for FASN in mediating increases in non-homologous end-joining (NHEJ) DNA double-strand break repair activity allowing for increased cancer cell survival, and this mechanism was found to involve inhibition of NF-kB/p65. The mechanism responsible for the regulation of NF-kB/p65 by FASN in cancer cells, however, remains unknown. To this end, I was able to determine that FASN negatively regulates both the expression and activity of NF-kB/p65 in breast cancer cells, and that this effect was likely mediated by the 16-carbon saturated fatty acid palmitate, the end product of FASN catalytic activity. Specifically, FASN was found to negatively regulate p65 expression by disrupting its protein stability as a result of an increase in poly-ubiquitination of p65 protein and subsequent proteasomal degradation. Further, I found that the phosphorylation site Thr254 of p65 is involved in the regulation of p65 protein stability by FASN, in that mutation of this residue resulted in a disruption in p65 stability. Finally, I was able to determine that FASN likely inhibits the ability of the peptidyl-prolyl cis/trans isomerase Pin1 to assist in maintaining p65 stability, in that both siRNA knockdown and pharmacological inhibition of Pin1 resulted in a reduction of p65 expression in FASN shRNA knockdown cells. The determination of this signaling mechanism serves to expand our understanding of the role of FASN in breast cancer cells and has the potential to assist in uncovering more effective ways to target the oncogenic FASN pathway to kill breast tumor cells and to overcome resistance to drug treatment.

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