Student Number : 0213014A - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / Cytosolic glutathione transferases (GSTs) are obligate stable homo- and heterodimers comprising two GST subunits. Interactions across the subunit interface play an important role in stabilising the subunit tertiary structure and maintain the dimeric structure required for activity. The crystal structure of a rat Mu class GST consisting of two type one subunits (rGST M1-1) reveals a lock-and-key motif and a mixedcharge cluster at the subunit interface. Previous investigations revealed the lock-andkey motif was not essential for dimerisation. It was therefore postulated that the mixed-charge cluster at the dimer interface is primarily responsible for subunit association. Statistical analyses of individual rGST M1-1 chains did not predict the presence of any charge clusters. This suggests that the mixed-charge cluster forms only upon dimerisation and reinforces the probability that quaternary structure stabilisation is a major role of the mixed-charge cluster. Arginine 81 (Arg-81), a structurally conserved residue in the GST family involved in the mixed-charge cluster, was mutated to alanine. Phenylalanine 56 (Phe-56), the ‘key’ residue in the lock-and-key motif, was mutated to serine. These changes were engineered to disrupt the mixed-charge cluster and the lock-and-key motif situated at the dimer interface of rGST M1-1. Sizing by gel filtration chromatography of the mutant GST identified that these engineered amino acids resulted in a stable monomeric protein (F56S/R81A rGST M1). The F56S/R81A rGST M1 displayed almost no catalytic activity, suggesting perturbations of the active site or substrate binding sites. Structural investigations of the monomer by far- and near-UV circular dichroism revealed a similar secondary structural content to the wild-type. However, the tryptophan fluorescence properties suggested the tryptophans were situated in more hydrophilic environments than in the wild-type. ANS binding studies indicated a large increase in the accessible hydrophobic surface area of the monomer. Ureainduced equilibrium unfolding of F56S/R81A rGST M1 follows a cooperative twostate unfolding model. The unfolding data indicates decreased conformational stability and a large increase in the solvent exposed surface area of the monomer. In conclusion, the mixed-charge cluster at the dimer interface of rGST M1-1 is essential for monomeric association, which subsequently contributes to catalytic activity of the dimer and the stabilities of individual rGST M1-1 subunits.

O excesso de tecido cicatricial (quelóides e cicatrizes hipertróficas) é um defeito do processo de cicatrização das feridas, caracterizado por um aumento na produção da matriz extracelular. Neste estudo, fibroblastos dérmicos humanos tratados com 50 <font face=\"symbol\">mM verapamil apresentaram discreta modificação na distribuição dos microfilamentos e alteraram sua morfologia de fusiformes para estrelados/arredondados. Estes efeitos poderiam estar associados a baixos níveis de cálcio citosólico. Esta hipótese foi confirmada através marcação de fibroblastos tratados com calcium green. Observamos também, que o verapamil inibiu a proliferação celular em 64,4%, aumentou a secreção de MMP1 e diminuiu o colágeno sintetizado pelos fibroblastos, sem aparentes efeitos citotóxicos. O metabolismo celular do cálcio está aparentemente relacionado a produção da matriz extracelular e portanto as patologias hipertróficas da cicatrização (quelóides e cicatrizes hipertróficas) podem responder ao tratamento com bloqueadores do canal de cálcio (verapamil). / Excessive scar tissue (keloids and hypertrophic scars) is a defective wound healing process characterized by overproduction of extracellular matrix. In the present study human dermal fibroblasts treated with 50 <font face=\"symbol\">mM verapamil changed their normal spindle-shaped morphology to stellate/rounded and showed discrete reorganization of microfilaments We hypothesized that these effects would be associated to lower levels of cytosolic Ca2+. Indeed, short time loading with calcium green confirmed that verapamil-treated fibroblasts exhibited lower intracellular calcium levels. We also observed that verapamil decrease cellular proliferation by 64.4%, increase the secretion of MMP1 and decrease synthesis of collagen in cultured fibroblasts. This alterations induced by verapamil are not associated with cytotoxic effects. The cellular calcium metabolism appears to regulate extracellular matrix production and so those hypertrophic disorders of wound healing (keloids and hypertrophic scars) may respond to therapy with calcium antagonist drugs (verapamil).

Iron sulfur (FeS) clusters are ubiquitous cofactors required for numerous fundamental biochemical processes, including DNA replication and repair, transcription, and translation. In the cell, these metallocofactors require a dedicated protein pathway for assembly. The Cytosolic Iron Sulfur Cluster Assembly (CIA) pathway is conserved across higher-level eukaryotes and is responsible for building and inserting these cofactors into the FeS proteins that need them. A major unsolved problem in the FeS cluster biogenesis field is how so many diverse FeS proteins are identified for cluster insertion. Several studies have identified a multiprotein complex containing Cia1, Cia2, and Met18 as the CIA targeting complex responsible for FeS cluster recognition and target maturation. The CIA targeting complex has been shown to associate with an FeS cluster protein, Nar1. Nar1 is a CIA factor that plays an unknown role in cluster transfer. Little information is known about the structure of the CIA targeting complex its mechanism of FeS cluster protein recognition. In this thesis, I investigate the architecture of the CIA targeting complex as well as the role each subunit plays in identification of apo-proteins and iron-sulfur cluster insertion. Previous proteomic and cell biological studies from the Lill lab propose that the CIA targeting complex exists as a mixture of discrete complexes in vivo. Each of these complexes is responsible for recognizing a distinct subset of targets. Herein, we utilize affinity co-purification and size exclusion chromatography investigate connectivity of the targeting complex, identify stable subcomplexes, and define their roles in recognizing our two model targets Rad3 and Leu1. We determine the CIA targeting complex contains one Met18, two Cia1, and four Cia2 polypepides. This complex is required to recognize Leu1. Our experiments reveal the formation of the stable subcomplexes Cia1-Cia2 and Met18-Cia2, which is sufficient to identify to Rad3. We also interrogate the role of Nar1 in binding to targets and cluster transfer, excluding the model that it acts as an adapter for cluster transfer. Furthermore, using site directed mutagenesis, combined with our co-purification and in vivo assays, we map the key interfaces required to form the targeting complex and investigate how their mutations impacts CIA function in vivo. We identify the binding site of Cia1 on Cia2, as well as the general region in which Cia2 binds to Met18. Through these experiments, we shed light on the role these subunits of CIA targeting complex and Nar1 play in FeS target recognition and FeS cluster transfer.

Rhomboids are unique membrane proteins that use a serine protease hydrolysis mechanism to cleave a transmembrane substrate within the lipid bilayer. This remarkable proteolytic activity is achieved by a core domain comprised of 6 transmembrane segments that form a hydrophilic cavity submerged in the membrane. In addition to this core domain, many rhomboids also possess aqueous domains of varying sizes at the N- and/or C-terminus, the sequences of which tend to be rhomboid-type specific. The functional role of these extramembranous domains is generally not well understood, although it is thought that they may be involved in regulation of rhomboid activity and specificity. While extramembranous domains may be important for rhomboid activity, they are absent in all x-ray crystal structures available. For this reason, we have focused on uncovering the structural and functional relationship between the rhomboid cytoplasmic domain and its catalytic transmembrane core. To investigate the structure and function of the bacterial rhomboid cytoplasmic domain, full-length rhomboids from Escherichia coli and Pseudomonas aeruginosa were studied using solution nuclear magnetic resonance (NMR) spectroscopy, mutation and activity assays. The P. aeruginosa rhomboid was purified in a range of membrane-mimetic media, evaluated for its functional status in vitro and investigated for its NMR spectroscopic properties. Results from this study suggested that an activity-modulating interaction might occur between the catalytic core transmembrane domain and the cytoplasmic domain. Further investigation of this hypothesis with the E. coli rhomboid revealed that protease activity relies on a short but critical sequence N-terminal to the first transmembrane segment. This sequence was found to have a direct impact on the rhomboid active site, and should be included in future structural studies of this catalytic domain. The structure of the cytoplasmic domain from the E. coli rhomboid was also determined by solution NMR. We found that it forms slowly-exchanging dimers through an exchange of secondary structure elements between subunits, commonly known as three-dimensional domain swapping. Beyond this rare example of domain swapping in a membrane protein extramembranous domain, we found that the rate of exchange between monomeric and dimeric states could be accelerated by transient interactions with large detergent micelles with a phosphocholine headgroup, but not by exposure to other weakly denaturing conditions. This novel example of micelle-catalyzed domain swapping interactions raises the possibility that domain swapping interactions might be induced by similar interactions in vivo. Overall, the results of this thesis have identified detergent conditions that preserve the highest level of activity for bacterial rhomboids, defined the minimal functional unit beyond what had been identified in available x-ray crystal structures, and characterized a novel micelle-catalyzed domain-swapping interaction by the cytoplasmic domain.

Rhomboids are unique membrane proteins that use a serine protease hydrolysis mechanism to cleave a transmembrane substrate within the lipid bilayer. This remarkable proteolytic activity is achieved by a core domain comprised of 6 transmembrane segments that form a hydrophilic cavity submerged in the membrane. In addition to this core domain, many rhomboids also possess aqueous domains of varying sizes at the N- and/or C-terminus, the sequences of which tend to be rhomboid-type specific. The functional role of these extramembranous domains is generally not well understood, although it is thought that they may be involved in regulation of rhomboid activity and specificity. While extramembranous domains may be important for rhomboid activity, they are absent in all x-ray crystal structures available. For this reason, we have focused on uncovering the structural and functional relationship between the rhomboid cytoplasmic domain and its catalytic transmembrane core. To investigate the structure and function of the bacterial rhomboid cytoplasmic domain, full-length rhomboids from Escherichia coli and Pseudomonas aeruginosa were studied using solution nuclear magnetic resonance (NMR) spectroscopy, mutation and activity assays. The P. aeruginosa rhomboid was purified in a range of membrane-mimetic media, evaluated for its functional status in vitro and investigated for its NMR spectroscopic properties. Results from this study suggested that an activity-modulating interaction might occur between the catalytic core transmembrane domain and the cytoplasmic domain. Further investigation of this hypothesis with the E. coli rhomboid revealed that protease activity relies on a short but critical sequence N-terminal to the first transmembrane segment. This sequence was found to have a direct impact on the rhomboid active site, and should be included in future structural studies of this catalytic domain. The structure of the cytoplasmic domain from the E. coli rhomboid was also determined by solution NMR. We found that it forms slowly-exchanging dimers through an exchange of secondary structure elements between subunits, commonly known as three-dimensional domain swapping. Beyond this rare example of domain swapping in a membrane protein extramembranous domain, we found that the rate of exchange between monomeric and dimeric states could be accelerated by transient interactions with large detergent micelles with a phosphocholine headgroup, but not by exposure to other weakly denaturing conditions. This novel example of micelle-catalyzed domain swapping interactions raises the possibility that domain swapping interactions might be induced by similar interactions in vivo. Overall, the results of this thesis have identified detergent conditions that preserve the highest level of activity for bacterial rhomboids, defined the minimal functional unit beyond what had been identified in available x-ray crystal structures, and characterized a novel micelle-catalyzed domain-swapping interaction by the cytoplasmic domain.

No description available.

Rhomboids are unique membrane proteins that use a serine protease hydrolysis mechanism to cleave a transmembrane substrate within the lipid bilayer. This remarkable proteolytic activity is achieved by a core domain comprised of 6 transmembrane segments that form a hydrophilic cavity submerged in the membrane. In addition to this core domain, many rhomboids also possess aqueous domains of varying sizes at the N- and/or C-terminus, the sequences of which tend to be rhomboid-type specific. The functional role of these extramembranous domains is generally not well understood, although it is thought that they may be involved in regulation of rhomboid activity and specificity. While extramembranous domains may be important for rhomboid activity, they are absent in all x-ray crystal structures available. For this reason, we have focused on uncovering the structural and functional relationship between the rhomboid cytoplasmic domain and its catalytic transmembrane core. To investigate the structure and function of the bacterial rhomboid cytoplasmic domain, full-length rhomboids from Escherichia coli and Pseudomonas aeruginosa were studied using solution nuclear magnetic resonance (NMR) spectroscopy, mutation and activity assays. The P. aeruginosa rhomboid was purified in a range of membrane-mimetic media, evaluated for its functional status in vitro and investigated for its NMR spectroscopic properties. Results from this study suggested that an activity-modulating interaction might occur between the catalytic core transmembrane domain and the cytoplasmic domain. Further investigation of this hypothesis with the E. coli rhomboid revealed that protease activity relies on a short but critical sequence N-terminal to the first transmembrane segment. This sequence was found to have a direct impact on the rhomboid active site, and should be included in future structural studies of this catalytic domain. The structure of the cytoplasmic domain from the E. coli rhomboid was also determined by solution NMR. We found that it forms slowly-exchanging dimers through an exchange of secondary structure elements between subunits, commonly known as three-dimensional domain swapping. Beyond this rare example of domain swapping in a membrane protein extramembranous domain, we found that the rate of exchange between monomeric and dimeric states could be accelerated by transient interactions with large detergent micelles with a phosphocholine headgroup, but not by exposure to other weakly denaturing conditions. This novel example of micelle-catalyzed domain swapping interactions raises the possibility that domain swapping interactions might be induced by similar interactions in vivo. Overall, the results of this thesis have identified detergent conditions that preserve the highest level of activity for bacterial rhomboids, defined the minimal functional unit beyond what had been identified in available x-ray crystal structures, and characterized a novel micelle-catalyzed domain-swapping interaction by the cytoplasmic domain.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2005. / Previous studies indicated that cytosolic aldolase (ALDc) could be a rate limiting step in glycolysis and thus play a role in the regulation of carbon partitioning in sink tissues. In this study the role of ALDc in sugarcane was studied. Expression patterns of both ALDc transcript and protein were examined. In contrast to the leaves where ALDc expression is very low, the enzyme (transcript and protein) levels were high in all internodal tissues at all stages of maturity. In the leaves the plastidic isoform was prevalent as found previously in other C4 plants. The similar pattern of expression in transcript and protein abundance illustrate that there are no activators or inhibitors of ALDc activity present in sugarcane. The control on ALDc activity in sugarcane is therefore regulation of gene expression. To investigate the possibility that ALDc could be regulating carbon partitioning in sugarcane a series of transgenic sugarcane plants in the varieties NCo310 and N19 were produced. The presence and expression of the transgene and resultant effect on ALDc levels were determined for all the transgenic lines. The degree of ALDc reduction varied, with the biggest suppression of aldolase being 90% of that of the control plants. Alteration of ALDc activity caused no obvious phenotype. In both the varieties large decreases in ALDc tended to to lead to higher sucrose levels than that of the the control plants. 14C radiolabelling studies were conducted to investigate the effect of reduced ALDc levels on respiration and carbon partitioning. No differences in carbon metabolism could be found between the transgenic and control plants. Even in the line exhibiting a more than 90% decrease, the residual ALDc was sufficient for plants to grow normally under favourable glasshouse conditions. This would suggest that ALDc does not play a role in the regulation of flux through glycolysis, carbon partitioning and sucrose accumulation.

Rhomboids are unique membrane proteins that use a serine protease hydrolysis mechanism to cleave a transmembrane substrate within the lipid bilayer. This remarkable proteolytic activity is achieved by a core domain comprised of 6 transmembrane segments that form a hydrophilic cavity submerged in the membrane. In addition to this core domain, many rhomboids also possess aqueous domains of varying sizes at the N- and/or C-terminus, the sequences of which tend to be rhomboid-type specific. The functional role of these extramembranous domains is generally not well understood, although it is thought that they may be involved in regulation of rhomboid activity and specificity. While extramembranous domains may be important for rhomboid activity, they are absent in all x-ray crystal structures available. For this reason, we have focused on uncovering the structural and functional relationship between the rhomboid cytoplasmic domain and its catalytic transmembrane core. To investigate the structure and function of the bacterial rhomboid cytoplasmic domain, full-length rhomboids from Escherichia coli and Pseudomonas aeruginosa were studied using solution nuclear magnetic resonance (NMR) spectroscopy, mutation and activity assays. The P. aeruginosa rhomboid was purified in a range of membrane-mimetic media, evaluated for its functional status in vitro and investigated for its NMR spectroscopic properties. Results from this study suggested that an activity-modulating interaction might occur between the catalytic core transmembrane domain and the cytoplasmic domain. Further investigation of this hypothesis with the E. coli rhomboid revealed that protease activity relies on a short but critical sequence N-terminal to the first transmembrane segment. This sequence was found to have a direct impact on the rhomboid active site, and should be included in future structural studies of this catalytic domain. The structure of the cytoplasmic domain from the E. coli rhomboid was also determined by solution NMR. We found that it forms slowly-exchanging dimers through an exchange of secondary structure elements between subunits, commonly known as three-dimensional domain swapping. Beyond this rare example of domain swapping in a membrane protein extramembranous domain, we found that the rate of exchange between monomeric and dimeric states could be accelerated by transient interactions with large detergent micelles with a phosphocholine headgroup, but not by exposure to other weakly denaturing conditions. This novel example of micelle-catalyzed domain swapping interactions raises the possibility that domain swapping interactions might be induced by similar interactions in vivo. Overall, the results of this thesis have identified detergent conditions that preserve the highest level of activity for bacterial rhomboids, defined the minimal functional unit beyond what had been identified in available x-ray crystal structures, and characterized a novel micelle-catalyzed domain-swapping interaction by the cytoplasmic domain.

O excesso de tecido cicatricial (quelóides e cicatrizes hipertróficas) é um defeito do processo de cicatrização das feridas, caracterizado por um aumento na produção da matriz extracelular. Neste estudo, fibroblastos dérmicos humanos tratados com 50 <font face=\"symbol\">mM verapamil apresentaram discreta modificação na distribuição dos microfilamentos e alteraram sua morfologia de fusiformes para estrelados/arredondados. Estes efeitos poderiam estar associados a baixos níveis de cálcio citosólico. Esta hipótese foi confirmada através marcação de fibroblastos tratados com calcium green. Observamos também, que o verapamil inibiu a proliferação celular em 64,4%, aumentou a secreção de MMP1 e diminuiu o colágeno sintetizado pelos fibroblastos, sem aparentes efeitos citotóxicos. O metabolismo celular do cálcio está aparentemente relacionado a produção da matriz extracelular e portanto as patologias hipertróficas da cicatrização (quelóides e cicatrizes hipertróficas) podem responder ao tratamento com bloqueadores do canal de cálcio (verapamil). / Excessive scar tissue (keloids and hypertrophic scars) is a defective wound healing process characterized by overproduction of extracellular matrix. In the present study human dermal fibroblasts treated with 50 <font face=\"symbol\">mM verapamil changed their normal spindle-shaped morphology to stellate/rounded and showed discrete reorganization of microfilaments We hypothesized that these effects would be associated to lower levels of cytosolic Ca2+. Indeed, short time loading with calcium green confirmed that verapamil-treated fibroblasts exhibited lower intracellular calcium levels. We also observed that verapamil decrease cellular proliferation by 64.4%, increase the secretion of MMP1 and decrease synthesis of collagen in cultured fibroblasts. This alterations induced by verapamil are not associated with cytotoxic effects. The cellular calcium metabolism appears to regulate extracellular matrix production and so those hypertrophic disorders of wound healing (keloids and hypertrophic scars) may respond to therapy with calcium antagonist drugs (verapamil).