LCAT deficiency is a rare autosomal recessive disease characterized by low levels of plasma HDL and an inability of the enzyme lecithin:cholesterol acyltransferase (LCAT) to esterify cholesterol. An understanding of the structure and function of the LCAT protein will add significantly to the understanding of reverse cholesterol transport. This understanding can be gained, in part, by studying different mutations within the LCAT gene and their resultant phenotypes. Recombinant DNA technology has been used to determine the nature of a mutation in an LCAT gene of a previously described homozygote with this disorder. Southern blot analysis determined there were no major rearrangements in the genomic DNA at the LCAT locus. An attempt was made to follow segregation of the mutant alleles in three generations of a large pedigree by linkage analysis. There are known polymorphisms at the haptoglobin (Hp) locus, which is linked to LCAT on the long arm of chromosome 16, and in the adenosine phosphoribotransferase (APRT) and choesterol ester transfer protein (CETP) loci which are also on the long arm of chromosome 16, but have not been shown linked to LCAT. The information gained was uninformative in this pedigree. An extensive restriction fragment length polymorphism (RFLP) search in the immediate vicinity of the LCAT gene did not reveal any polymorphic sites. 2.4 kb of the ⋋ phage clone SF1020, obtained from one of the homozygotes, containing exons 1-5 plus 0.5 kb of DNA 5¹ to the LCAT gene, but not exon 6, was subcloned into M13 and sequenced. A cytosine to thymidine (C->T) transition was discovered in exon 4. This would result in a substitution of tryptophan for arginine at amino acid 135. The amino acid arginine is positively charged and resides in one of the most highly charged segments along the amino acid chain of the LCAT protein indicating that this region is likely involved in protein folding. Tryptophan, on the other hand is the most hydrophobic of the amino acids and would, therefore, severely disrupt the interaction of charged amino acids in that region, preventing normal folding of the LCAT protein. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Les récepteurs tyrosine kinase (RTKs) CSF-1R et KIT sont médiateurs importants de la signalisation cellulaire. Leur fonction basale est altérée par des mutations associées à divers types de cancer. Ces mutations modifient également leur sensibilité à l’imatinib, utilisé en clinique dans le traitement des cancers. Dans cette thèse, nos objectifs sont (i) étudier les effets structuraux et dynamiques induits par la mutation D802V chez CSF-1R; (ii) caractériser l’affinité de l’imatinib aux formes sauvages (WT) et mutés de KIT (V560G, S628N et D816V) et CSF-1R (D802V). Par simulations de Dynamique Moléculaire (DM), nous avons montré que la mutation D802V interrompt la communication allostérique entre la boucle d’activation et le domaine auto-inhibiteur juxtamembranaire (JMR). Néanmoins, cette rupture n’est pas suffisante pour induire le départ du JMR. L’effet subtil de la mutation chez CSF-1R a été attribué aux différences de séquence primaire entre KIT et CSF-1R dans la région du JMR. L’affinité de l’imatinib aux différentes cibles a été calculée par simulations de docking, DM et calculs d’énergie de liaison. Les interactions électrostatiques constituent la force motrice de la résistance, les mutations D802/816V étant les plus délétères en termes d’énergie. Comme conclusion générale, nous avons établi que la mutation D802V chez CSF-1R n’entraine pas les mêmes effets structuraux provoqués par la mutation D816V chez KIT. En outre, l’étude des deux récepteurs dans leurs formes WT et mutés complexés avec l’imatinib indiquent que le changement structural induit par les mutations associé aux interactions électrostatiques avec le ligand expliqueraient le phénomène de résistance. / The receptors tyrosine kinase (RTKs) CSF-1R and KIT are important mediators of signal transduction. Their normal function is altered by gain-of-function mutations associated with cancer diseases. A secondary effect of the mutations is the alteration of receptors’ sensitivity imatinib, employed in cancer treatment. Our goals in this thesis consist of (i) study the structural and dynamical effects induced by the D802V mutation in CSF-1R; (ii) characterize imatinib’s affinity to the wild-type (WT) and mutant forms of KIT (V560G, S628N and D816V) and CSF-1R (D802V). By means of molecular dynamics (MD) simulations, we have shown that the D802V mutation disrupts the allosteric communication between the activation loop and the auto-inhibitory juxtamembrane (JMR) domain. However, this rupture is not sufficient to induce the JMR’s departure. The subtle effect of this mutation in CSF-1R was associated with differences in the primary sequence between CSF-1R and KIT in the JMR region. The affinity of imatinib to the different targets was estimated by docking, DM and binding energy calculations. The electrostatic interactions showed to be the main force driving the resistance, with mutations D802/816V being the most deleterious in energy contribution. As a general conclusion, we have established that the D802V mutation in CSF-1R does not provoke the same structural effects as its equivalent in KIT. In addition, the study of both receptors in their WT and mutant forms complexed with imatinib indicate that the conformational changes induced by the mutations allied to the electrostatic interactions with the ligand could explain the resistance phenomena.

It has been shown previously in our lab that mutations in the pyrimidine pathway reduced the ability of Pseudomonas aeruginosa to produce virulence factors. Knockout mutations in pyrB, pyrC and pyrD genes of the pyrimidine pathway showed that virulence factor production was decreased. Pyoverdin, pyocyanin, hemolysin, iron chelation, motility, and adherence are all considered virulence factors. Here I further investigate the effects of mutations in the pyrimidine pathway by studying a pyrE mutant. I studied the effect of the pyrE mutation on the production of the above virulence factors. Just like the effect of pyrB, pyrC and pyrD mutations,the pyrE mutation also showed that the bacteria were deficient in producing virulence factors when compared to the wild type. The broader impact of this research would be the possibility of finding drugs that could treat patients infected with P. aeruginosa and possibly extend the lives of chronically infected patients with cystic fibrosis.

A subset of germline mutations that arise de novo each generation are deleterious and may cause severe genetic diseases. Predicting where in the genome and how often we expect to see deleterious mutations requires an understanding both of the distribution of mutation rates and the distribution of fitness effects in the genome. Both aspects are addressed in turn in the two projects described in this thesis. The distribution of mutations in the genome is poorly understood because germline mutations occur very rarely. In Chapter 1 of this work, we investigated the sources of mutations by using the spectrum of low-frequency variants in 13,860 human X chromosomes and autosomes as a proxy for the spectrum of germline de novo mutations. By comparing the mutation spectrum in multiple genomic compartments on the autosomes and between the X and autosomes that have unique biochemical and sex-specific properties, we ascribed specific mutation patterns to replication timing and recombination and identified differences in the types of mutations that accrue in males and females. Understanding mutational mechanisms provides a basis for modeling mutation rate variation in the genome, which is ultimately needed to infer the fitness effects of mutations. In Chapter 2, we used patterns of human genetic variation at methylated CpGsites, known to experience mutations at very high rates, to directly learn about the fitness effects of mutations at these sites. In whole exome sequences now available for 390,000 humans, 99% of putatively-neutral, synonymous CpG sites have experienced a C>T mutation; at current sample sizes, not seeing a C>T mutation at these sites indicates strong selection against that mutation. We leveraged the saturation of neutral C>T mutations and the similarity of mutation rates at methylated CpG sites across annotations to identify the subset of sites in a given functional annotation of interest that are likely to be under strong selection. One implication of this work is that for the vast majority of sites in the genome, there will be little information about strong selection even in samples that are many times larger than at present; the distribution of fitness effects at highly mutable CpG sites may then serve as an anchor for what to expect for other types of sites. Through the two specific cases described, this work illustrates the potential of large contemporary repositories of human genetic variation to inform human genetics and evolution, as well as their limitations in the absence of suitable models of mutation, selection, and other aspects of the evolutionary process.

The immune system produces antibodies to recognize and provide protection against infection. The immunoglobulin G (IgG) antibody isotype is present at high serum concentrations and has a longer half-life than other isotypes due to the interaction between its fragment crystallizable (Fc) region with the neonatal Fc receptor (FcRn). This Fc-FcRn interaction, which takes place in many cell types throughout the cardiovascular system, mediates pH-dependent formation of the IgG-FcRn complex and leads to the rescue of IgG from eventual degradation via transport from the low-pH early endosome back to the cell surface for release into serum at pH 7.4. Because this process is the primary determinant of IgG antibody half-life, and because the Fc region is common to all antibodies of the same subtype, the Fc-FcRn system has been a target of numerous antibody design and engineering studies. Indeed, several engineered Fcs have been reported with extended serum half-lives. These novel Fc variants, however, have generally been the result of extensive experimental screening and combinations of individual Fc mutations with known biophysical properties; there are few reports of predominantly structure-based rational Fc design. Notably, simply increasing Fc binding affinity for FcRn at low pH does not appear to be sufficient to achieve the largest increases in half-life (and in some cases, very high affinity results in reduced serum half-life). Most of these engineered Fcs have increased affinity not only at low pH (~6.0), but also at pH 7.4. The longest-lived Fc variant known to date, however, with mutations L309D/Q311H/N434S (“DHS”), has only a modest 5-fold increase in binding affinity compared to wt Fc at low pH, but also exhibits negligible binding to the receptor at pH 7.4 (Lee et al., 2019). This is consistent with previous reports that identify efficient release at physiologic serum pH to be critical to FcRn-mediated half-life extension. Thus, while engineering for affinity at low pH, it is also important to optimize the pH dependence of binding for optimal release at serum pH. The rational design process requires a detailed understanding of the structural and functional details of the interaction, which for a pH-dependent complex like Fc-FcRn must also include an accurate model of the pH-sensing mechanism. Unfortunately, the only publicly available crystal structure of a human Fc-FcRn complex is of the M252Y/S254T/T256E (“YTE”) variant, and was determined only to a relatively low 3.8 Å resolution, leaving the atomic positions of many sidechains, and even regions of the protein backbone, subject to substantial uncertainty. Furthermore, the widely accepted conventional mechanism of pH sensing, involving protonation of key histidine residues on Fc at low pH due to the assumed histidine pKa of 6.5 being within the range of interest (pH 6.0-7.4), is thermodynamically impossible. In this thesis I present an extensive analysis of the Fc-FcRn system, including the generation of all-atom models of human wild-type (wt) and variant complexes and the rat wt complex, and assignment of dominant protonation states at pH 6.0, at which most binding experiments are performed. I validate these models using retrospective molecular dynamics (MD)-based free-energy perturbation (FEP) calculations to compare to a large dataset of wt and mutant binding affinities. During this validation process I identify a residue on FcRn, glutamic acid 133, which adopts a highly unusual configuration in the complex and, due to quantum mechanical electronic polarization effects, is not described well by the fixed-charge molecular mechanics force field used by the FEP calculations, resulting in systematic errors for mutations that affect its hydrogen bonding network. I also identify a new variant, with a V308P mutation in a YTE background (“YTEP”), which induces a previously unreported conformational change that accounts for its high binding affinity compared to YTE and wt. To address the problem of the pH-sensing mechanism, I describe a general method for calculating the pH-sensing free energy of binding for any complex, based on a study of the pH dependence of protein unfolding free energies (Yang and Honig, 1993). The key observation underlying this method is that pH-dependent complex formation must be accompanied by a change in the pKa of one or more titratable groups between the unbound and bound states. Furthermore, the change in binding energy between two pHs can be directly calculated based on those pKas alone. As there are no experimental pKa measurements available for the Fc-FcRn interface residues, I perform these pH-sensing free energy calculations using FEP-based calculated pKas to quantitatively assess which residues at the interface are involved in sensing pH over the physiologically relevant pH range, and present a residue-level model for pH sensing in the Fc-FcRn system. Finally, I present some preliminary work toward the rational design of modified Fc regions with both increased affinity at low pH, and increased pH dependence of binding, using FEP calculations to guide experiment. This type of approach, of computational screening of a large number of different variants, followed by more limited experimental testing of promising leads, has the potential to streamline Fc design efforts and provide further insight into the structural basis of function for the Fc-FcRn system.

No description available.

Thesis (PhD)--University of Stellenbosch, 2004. / 426 Leaves printed single pages, preliminary pages i-xxiv and i-xxvii and 399 numberd pages. Includes bibliography. List of figures, List of tables, List of abbreviations. / ENGLISH ABSTRACT: Hypertrophic cardiomyopathy (HCM) is an autosomal dominantly inherited primary cardiac disease. The primary features of HCM are left ventricular hypertrophy, myocardial disarray, fibrosis and an increased risk of sudden cardiac death. To date, more than 264 HCM-causing mutations, occurring in thirteen genes, have been identified. As the vast majority of HCM-causing mutations occur in components of the cardiac sarcomere, HCM has been considered a disease of the cardiac sarcomere. Functional analyses of HCM-causing mutations in sarcomeric protein-encoding genes revealed that HCM-causing mutations have a vast array of effects on contractile function. The discovery of HCMcausing mutations in the gamma two subunit of adenosine monophosphate activated protein kinase highlighted the fact that mutations in non-sarcomeric proteins can also cause HCM and supports a hypothesis that HCM-causing mutations may result in energy wastage leading to energy depletion. Mutations in the cardiac myosin binding protein C (cMyBPC) gene (MYBPC3) are the second most prevalent cause of HCM. cMyBPC is a modular protein that forms an integral part of the sarcomeric thick filament, where it acts as a regulator of thick filament structure and cardiac contractility. Although cMyBPC has been studied extensively, the mechanisms through which it fulfill these functions have remained elusive, largely due to a lack of a comprehensive understanding of its interactions with other sarcomeric components and its quaternary structure. The aims of the present study were, firstly, to screen MYBPC3 for HCM-causing mutations in a panel of HCM-affected individuals and, secondly, to identify the ligands of domains of cMyBPC in which HCM-causing mutations were found.A panel of deoxyribonucleic acid (DNA) samples obtained from unrelated HCM-affected individuals was screened for HCM-causing mutations in MYBPC3, using polymerase chain reaction (PCR)- based single-strand conformation polymorphism method, as well as restriction enzyme digestion, DNA sequencing and reverse transcription PCR techniques. In order to identify the ligands of domains in which HCM-causing mutations were found, yeast two-hybrid (Y2H) candidate-ligandand library-assays were performed. Three novel and two previously described putative HCM-causing mutations were identified in MYBPC3. Data generated in this and other studies, however, suggest that two of these “mutations” are likely to be either polymorphisms, or disease-modifying factors, rather than main-locus HCMcausing mutations. Recent findings showed a specific interaction between domains C5 and C8 of cMyBPC. This finding identified domains C6 or C10 as candidate ligands of domain C7. Y2H-assays revealed a specific C7:C10 interaction. Additional Y2H assays also identified C-zone titin as a ligand of domain C7 and domain C10 as a ligand of domain C3. Several other Y2H assays, however, yielded no known sarcomeric ligands of the N-terminal region of cMyBPC. Identification of the ligands of specific domains of cMyBPC led to the development of detailed models of cMyBPC quaternary structure when cMyBPC is both unphosphorylated and fully phosphorylated. The integration of these models into an existing model of thick filament quaternary structure allows new insights into the functioning of cMyBPC as a regulator of both thick filament structure and cardiac contractility, as well as the pathophysiology of cMyBPC-associated HCM. / AFRIKAANSE OPSOMMING: Hipertrofiese kardiomiopatie (HKM) is ‘n outsosomaal dominante primêre hartsiekte. Die primêre kenmerke van HKM is linker ventrikulêre hipertrofie, miokardiale wanorde, fibrose en ‘n verhoogde risiko van skielike dood. Tot dusver is 260 HKM-veroorsakende mutasies in 13 gene geïdentifiseer. Aangesien die oorgrote meerderheid van HKM-veroorsakende mutasies in komponente van die kardiale sarkomeer voorkom, is HKM as ‘n siekte van die kardiale sarkomeer beskryf. Funksionele analise van HKM-veroorsakende mutasies in sarkomeriese protein-koderende gene het aan die lig gebring dat hierdie mutasies ‘n wye spektrum van gevolge op kontraktiele funksie het. Die ontdekking van HKM-veroorsakende mutasies in die gamma-twee subeenheid van adenosien monofosfaat-geaktiveerde proteïen kinase het die feit dat mutasies nie-sarkomeriese proteïene ook HKM kan veroorsaak onderstreep en ondersteun ‘n hipotese dat HKM-veroorsakende mutasies energievermorsing en energie uitputting tot gevolg het. Mutasies in die kardiale miosien-bindingsproteïen C (kMiBPC) geen (MYBPC3) is die tweede mees algemene oorsaak van HKM. kMiBPC is ‘n modulêre protein wat ‘n integrale deel van die sarkomeriese dik filament vorm, waar dit die struktuur van die dik filament en kardiale kontraktiliteit reguleer. Nieteenstaande die feit dat kMiBPC intensief bestudeer is, word die meganismes hoe hierdie funksies vervul word swak verstaan, grotendeels weens die afwesigheid van ‘n in diepte begrip van sy interaksies met ander komponente van die sarkomeer asook sy kwaternêre struktuur. Die doelstellings van hierdie studie was, eerstens, om MYBPC3 vir HKM-veroorsakende mutasies in ‘n paneel van HKM-geaffekteerde individue te deursoek en tweedens, om die ligande van domeine van kMiBPC waarin HKM-veroorsakende mutasies gevind is te identifiseer.‘n Paneel van deoksiribonukleïensuur (DNS) monsters verkry van onverwante HKM-geaffekteerde individue is deursoek vir HKM-veroorsakende mutasies in MYBPC3, deur middel van die polimerase ketting-reaksie (PKR)-gebasseerde enkelstrand konformasie polimorfisme metode, sowel as restriksie ensiem vertering, DNS volgordebepaling en terugtranskripsie PKR tegnieke. Die ligande van domeine van kMiBPC waarin HKM-veroorsakende mutasies gevind is, is geïdentifiseer deur middel van gis twee-hibried (G2H) kandidaat-ligand en biblioteek-siftings eksperimente. Drie onbeskryfde en twee voorheen beskryfde vermeende HKM-veroorsakende mutasies in MYPBC3 is geïdentifiseer. Data gegenereer in hierdie en ander studies dui daarop dat twee van hierdie “mutasies” eerder polimorfismes, of siekte-modifiserende faktore, as hoof-lokus HKMveroorsakende mutasies is. Onlangse bevindings het ‘n spesifieke interaksie tussend die C5 en C8 domeine van kMiBPC getoon. Hierdie bevindings het óf domein C6, óf C10, as kandidaat-ligande van domein C7 geïdentifiseer. G2H eksperimente het ‘n spesifieke interaksie tussen domains C7 en C10 getoon. Addisionele G2H eksperimente het ook C-zone titin as ‘n ligand van domein C7 sowel as domein C10 as ‘n ligand van domein C3 geïdentifiseer. Verdere G2H eksperimente het egter geen sarkomeriese ligande van die N-terminale gedeelte van kMiBPC geïdentifiseer nie. Die identifikasie van ligande van spesifieke domeins van kMiBPC het gelei tot die ontwikkelling van ‘n gedetaileerde model van kMiBPC kwaternêre struktuur wanneer kMiBPC beide ongefosforileerd en ten volle gefosforileerd is. Die intergrasie van hierdie modelle in bestaande modelle van dik filament kwaternêre struktuur werp nuwe lig op die funksionering van kMiBPC as ‘n reguleerder van beide dik filament struktuur en kardiale kontraktiliteit, sowel as die patofisiologie van kMiBPCgeassosieerde HKM.

published_or_final_version / abstract / toc / Biochemistry / Master / Master of Philosophy

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

published_or_final_version / abstract / Microbiology / Doctoral / Doctor of Philosophy