Browsing by Author "Sturrock, Edward"
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- ItemOpen AccessCharacterisation of dysregulated proteins in macrophages infected with Mycobacterium smegmatis focusing on matrix metalloproteases and their effectors(2019) Seele, Palesa Pamela; Sturrock, Edward; Blackburn, JonathanCavitation is a key facilitator in transmission of Mycobacterium tuberculosis (M. tb). Upregulation of matrix metalloproteases (MMPs) has been documented in patients with tuberculosis (TB), while their tissue inhibitor (TIMPs) levels remained the same. Animals which can develop cavities have well-conserved MMP-1 orthologs suggesting a pivotal role of MMP-1 in cavitation. The migration of immune cells to the site of infection and maturation of the granuloma is associated with MMP-9 expression. Our understanding of the phenotypic changes induced by Mycobacterium smegmatis (M. smeg) in THP-1 macrophages is central to understanding its avirulent nature especially its effects on MMPs. The aim of this study was to evaluate the role of MMP-1 and MMP-9, and their effectors in macrophages infected with M. smeg. Differentiated THP-1 monocytes were incubated in serum-free media with or without bacilli. Thereafter, the secretome and lysate were harvested at different time points. The activity of MMPs was analysed by zymography. The activity of MMP-1 and MMP-9 were specifically determined using an MMP-1 fluorogenic assay and a non-fluorogenic MMP-9 substrate monitored using the HPLC. Discovery proteomics was performed for the 18-hour time point with the use of mass spectrometry. The generated data was used to evaluate dysregulated proteins and those that act as upstream and downstream effectors of MMPs. The phenotypic changes induced by M. smeg were also analysed. In addition to that, the hosts’ response to lipoarabinomannan H37Rv (LAM) treatment was assessed by discovery proteomics and zymography. There was an increase in gelatinase activity of secreted MMP-9 which was maintained between the 1 and 18-hour time points. The fold difference in activity between uninfected and infected declined at 24 hours, and at the 72-hour time point the uninfected was slightly higher versus the infected. The data also suggests a switch in the proteolytic repertoire of the macrophages between the 6- and 18-hour time points to one that potentially generates the same degradation products as the uninfected macrophages. The intracellular gelatinase activity of MMP-9 (82 kDa) was not significantly altered by the M. smeg infection, in fact the activity was slightly higher in the uninfected in the 18 and 24-hour time points. In contrast to MMP-9, MMP-1 was secreted in the later time points and was significantly decreased by the infection. This supports the postulation that upregulation of MMP-1 is specific for M. tb infection. The proteomics data depict significant upregulation of MMP-9 in the lysate and secretome, while TIMP-1 was exclusively expressed and secreted by infected macrophages, validating the non-destructive ECM phenotype induced by M. smeg. The dysregulation of IL-1β and COX pathways were implicated in the overexpression of MMP-9, as well as tRNA aminoacylation in alternative splicing of MMPs. The GO enrichment of exosomes is postulated to play a role in the recycling of MMP-9. Intercellular communication is hypothesised to be delivered to neighbouring cells through exosomes carrying DNA, RNA, proteins and DNA/RNA binding proteins, and via signalling scaffolds formed by the 14-3-3 proteins amongst others. LAM treatment did not induce dysregulation in the activity of expressed and secreted MMP-9, however, TIMP-1 was upregulated explaining the lack of differential gelatinase activity between treated and non-treated macrophages. The C-type mannose receptor 2 (MRC2) and C-type lectin domain family 11 (CLEC11A) were only expressed by the LAM-treated macrophages and may partake in the recognition and uptake of M. tb. Interestingly, the data indicates the presence of chromatin in the secretome which may be responsible for the formation of extracellular traps (NETs) and facilitating the transport of LAM across the glomerular basal membrane (GBM) through exosomes. Inhibition of MMP activity by TIMPs could result in decreased aggregation of NETS (aggNETS) that trap the LAM from being transported by binding to the chromatin. This decreases the concentration of LAM in urine and means MMP inhibitors that chelate the active-site zinc could decrease the sensitivity of urine-LAM detection kits.
- ItemOpen AccessExploring the Impact of ACE Inhibition in Immunity and Disease(2022-08-04) Oosthuizen, Delia; Sturrock, EdwardAngiotensin-converting enzyme (ACE) is a zinc-dependent dipeptidyl carboxypeptidase and is crucial in the renin-angiotensin aldosterone system (RAAS) but also implicated in immune regulation. Intrinsic ACE has been detected in several immune cell populations, including macrophages and neutrophils, where its overexpression results in enhanced bactericidal and antitumour responses, independent of angiotensin II. With roles in antigen presentation and inflammation, the impact of ACE inhibitors must be explored to understand how ACE inhibition may impact our ability to clear infections or malignancy, particularly in the wake of the coronavirus (SARS-CoV2) pandemic and as antibiotic resistance grows. Patients using ACE inhibitors may be more at risk of postsurgical complications as ACE inhibition in human neutrophils results in decreased ROS and phagocytosis whilst angiotensin receptor blockers (ARBs) have no effect. In contrast, ACE is also elevated in certain autoimmune diseases such as rheumatoid arthritis and lupus, and its inhibition benefits patient outcome where inflammatory immune cells are overactive. Although the ACE autoimmune landscape is changing, some studies have conflicting results and require further input. This review seeks to highlight the need for further research covering ACE inhibitor therapeutics and their potential role in improving autoimmune conditions, cancer, or how they may contribute to immunocompromise during infection and neurodegenerative diseases. Understanding ACE inhibition in immune cells is a developing field that will alter how ACE inhibitors are designed in future and aid in developing therapeutic interventions.
- ItemOpen AccessHeterologous production of recombinant peptidylglycine α-amidating monooxygenase for the production of biosimilar α-amidated peptides(2022) Morrison, David Graham; Sturrock, Edward; Steenkamp L SA biological method for peptide synthesis provides increased production capacity of inexpensive peptide pharmaceuticals with environmentally safe procedures relative to current chemical peptide synthesis. Most precursor peptides are readily produced from yeast and bacterial systems using recombinant DNA technologies but require C-terminal amidation for maximum biological activity. Peptidylglycine α-amidating monooxygenase (PAM) is the only enzyme that catalyses the C-terminal amidation of peptides in vivo through its two catalytic cores, peptidylglycine α-hydroxylating monooxygenase (PHM) and peptidylglycine αamidating lyase (PAL). The cost and limited quantities of the commercial PAM variants available have necessitated research into low cost, scalable quantities of PAM and peptide amidation to enable inexpensive biological peptide production. In the present study, an assay for measuring the product of PAM activity, glyoxylate, was developed based on a 2-aminobenzaldehyde-glycine-glyoxylate (AGG) absorbance assay. The AGG chromophore synthesised was identified with ultra-performance liquid chromatography mass-spectroscopy (UPLC-MS). PAM activity was measurable with glyoxylate between 25 µM and 1600 µM with the AGG assay. Furthermore, the activity of PHM alone was measured by the inclusion of an alkaline hydrolysis step to lyse glyoxylate as a substitute for PAL catalytic activity. Multiple candidate proteins and DNA sequences for PAM were identified by genetic sequence searches and a novel fungal PHM modelled in silico. Fourteen PAM, PHM, PAL and truncated constructs were expressed in the non-conventional yeast host, Yarrowia lipolytica. The novel fungal PHM's nutrient, temperature, and pH conditions were optimised to maximise protein expression. Enzyme purification was optimised with scalable industrial appropriate methods to purify milligram amounts of fungal PHM. The AGG assay was validated with a commercially obtained PAM, demonstrating a simple medium-throughput method to measure PAM activity. The novel fungal PHM was characterised with a pH optimum of 4.0 and maximum enzymatic activity at 45°C. Deglycosylation of fungal PHM enhanced enzyme activity by 1.83 fold, but lowered the temperature optimum to 37°C. The novel PHM and alkaline hydrolysis catalysed the conversion of the peptide pharmaceutical precursor for exenatide into its final bioactive form.
- ItemOpen AccessInvestigation of ACE-overexpression in Myeloid Cell Lines through Whole-Proteome Analysis(2023) Batteson, Delia; Sturrock, EdwardAbstract Angiotensin converting enzyme (ACE) plays an important role in blood pressure regulation and is a key component of the renin-angiotensin aldosterone system (RAAS). A dipeptidyl carboxypeptidase, ACE also hydrolyses many different substrates across its N- and C-domain. This substrate variability has uncovered novel ACE function in other biological systems and disease. In the immune system, ACE and angiotensin II influence inflammation and wound repair. Independently of angiotensin II, ACE overexpression in murine macrophages (ACE 10/10) and neutrophils (NeuACE) has shown remarkable enhanced immune phenotypes with the ability to improve murine survival against B16 melanoma, methicillin-resistant Staphylococcus aureus (MRSA) and Listeria monocytogenes. Current literature points to the C-domain as the main proponent. The myeloid cells have enhanced reactive oxygen species (ROS) generation, pro-inflammatory cytokine production and phagocytosis with increased ATP and TCA cycle intermediate production. ACE overexpressing macrophages also improve cognitive ability in murine Alzheimer's models by amyloid-β (1-42) protein cleavage and degradation. Despite lengthy characterization of ACE overexpression and the enhanced immune phenotype of ACE 10/10 macrophages and NeuACE neutrophils, the mechanism, and substrate(s) by which it occurs is unknown. Understanding the biological processes influenced by ACE overexpression may provide alternative therapies where standard medicine is no longer effective including resistant bacterial infections and tumours. Importantly, ACE inhibition in human and murine neutrophils, and ACE 10/10 macrophages has shown reduced extracellular and intracellular microbicidal function. This and the immune benefit associated with ACE overexpression has prompted interest in the mechanism responsible. This project aimed to identify differentially expressed and significantly dysregulated biological pathways and proteins in ACE overexpressing murine (ACE 10/10 PTM) and human macrophages (ACE +/+ THP-1) whilst also analysing global proteomic changes with respects to ACE inhibition using discovery mass spectrometry (MS). ACE overexpressing murine and human macrophage whole cell protein lysates underwent label-free discovery MS to identify proteomic shifts in comparison to control macrophages. Using data-independent methodology, 270 and 442 differentially expressed proteins were identified in murine and human ACE overexpressing macrophages, respectively. Functional enrichment for several immune processes including phagocytosis, ROS generation, and antigen processing and presentation were identified whilst metabolic enrichment for TCA, fatty acid oxidation, electron transport chain (ETC) and glucose was present in murine ACE 10/10 macrophages. Human ACE +/+ THP-1 macrophages saw similar ETC, ATP synthase and glucose protein up-regulation and cytokine signalling, antigen processing and presentation functional enrichment. Unique to ACE +/+ human macrophages was neutrophil degranulation whereupon ACE C-domain inhibition by Lis-Trp dysregulated these proteins. Both murine and human ACE overexpression identified KEGG peroxisome proliferator-activated receptor (PPAR) signalling as significantly enriched, providing a possible target pathway for future mechanistic validation studies. ACE C-domain inhibition following Lis-Trp treatment led to a general downregulation of the functionally enriched ETC, TCA and ATP synthase components identified as up-regulated in the literature and our own murine MS results. Murine phosphoproteomic analysis identified ERK2/MAPK1 and PKA kinase-substrate enrichment with ACE overexpression. Using the human cell line, THP-1, Lis-Trp uptake was quantified through MS and ACE enzymatic activity assays over two hours. Minimal internalization took place for both 10 µM and 100 µM with < 1% intracellular Lis-Trp detected. However, partial ACE inhibition was achieved for both treatments despite low intracellular concentrations. To study the impact of this partial inhibition, the role of ACE in phagocytosis was explored. Using elastomeric micropattern contraction as a proxy for phagocytic uptake, ACE C- and N-domain inhibition both led to significant reduction in micropattern contraction in comparison to uninhibited control THP-1 macrophages. ACE +/+ THP-1 macrophages showed enhanced phagocytosis as observed in murine ACE 10/10 macrophage literature by means of increased micropattern contraction. Following ACE domain inhibition by 10 µM Lis-Trp (C-domain) and 10 µM RXP407 (N-domain) a statistically significant decrease in contraction measurements was observed, implying that both the N- and C-domain of ACE play a role in phagocytosis. Partial ACE inhibition may therefore be sufficient in reducing macrophage microbicidal function as observed in NeuACE and human neutrophils, increasing the potential risk of dangerous infection in immunocompromised patients. This work demonstrated an altered proteomic profile in ACE overexpressing murine and human macrophages, and confirmed findings on ACE 10/10 murine macrophages, whilst providing a novel and deeper understanding of ACE +/+ human macrophages. Furthermore, ACE C-domain inhibition likely negatively impacts macrophage function including phagocytosis whilst increasing neutrophil degranulation, but further biochemical characterization is required. Importantly, novel lipid metabolism and PPAR signalling were functionally enriched in both species, providing an exciting path for future studies in ACE overexpression and enhanced immunity.
- ItemOpen AccessRarity of kidney stones in South Africa's black population : studies of urinary macromolecules, crystal matrix extract containing osteopontin, and bone turnover markers in urine and serum from black and white subjects as a key to understanding this paradox(2007) Deppa, Ntsapokazi; Rodgers, Allen; ; Sturrock, EdwardThe work described in this thesis was undertaken to investigate physicochemical, biochemical and physiological factors contributing towards the rarity of kidney stone disease in the South African black population. Healthy, age-matched male subjects from the black and white population groups were recruited for this purpose. In several of the studies, subjects followed a standardized diet and were required to provide 24 hour urine collections. These were analyzed for sodium, potassium, calcium, oxalate, uric acid, citrate, chloride, magnesium, phosphate, sulphate and creatinine using standard laboratory techniques. Urine composition values were used as input data for the calculation of relative supersaturation (RS) values for calcium oxalate (CaOx), calcium phosphate (CaP, or brushite) and uric acid (U A) using the computer programme EQUIL and for the calculation of the Tiselius Risk Index (TRI). CaOx crystallization experiments were performed. These included CaOx metastable limit (MSL) and BONN Risk Index (BRI) determinations, particle formation kinetics, 14 C-oxalate cry stal deposition kinetics and CaOx crystal aggregation and nucleation inhibition. Crystallizati on experiments were also supplemented with scanning electron microscopy (SEM) and zeta potential measurements. Urine compositions, crystallization data and physicochemical risk indices were analyzed statisitically using ANOV A. Several different investigations were undertaken. These included crystallization experiments involving urinary macromolecules from both race groups, crystal matrix extract isolation (with osteopon tin as its major component) from both race groups and its testing for inhibitory capacity in ultrafiltered urine from both race groups. Similar crystallization experiments were conducted with commercially available osteopontin. In addition, a comprehensive trial was conducted in which the ingestion of three sodium salts (sodium chloride, sodium bicarbonate and sodium citrate) was investigated for their effects on urinary risk factors of CaOx stone formation and for their effects on bone turnover markers in urine and in serum. For the biochemical isolation of crystal matrix extract, COD-CME was precipitated in urine from both black and white subjects. The proteins included in COD-CME were detected using sodium dodecyl sulfate polyacryalamide gel electrophoresis (SDS-PAGE). Western Blotting was used for semi-quantitative analysis of OPN v For the trial involving different sodium salts, four experimental protocols were investigated. The four protocols included low NaCl (3 g/day), high NaCl (12 g/day), sodium bicarbonate (6 g/day) and sodium citrate in the form of Citro-Soda (16 g/day). A Latin Square Design was followed for the random assignment of participants to sequences of protocols. The studies on macromolecules showed that those in the urine of black subjects were more potent inhibitors of CaOx crystal deposition and aggregation than those in the urine of white subjects. Isolation and characterization of the crystal matrix extract in COD crystals confirmed that osteopontin is the main intracrystalline protein in the extract. The crystallization experiments performed on the crystal matrix extract isolated from the urine of both race groups demonstrated that the extract isolated from the urine of black subjects was a superior inhibitor of CaOx deposition, growth and aggregation. Crystallization studies performed on commercially available osteopontin in urine from black and white subjects showed that this protein is a more effective inhibitor of CaOx crystal deposition, growth and nucleation in the urine from the former group compared to that from the latter. The studies on supplemental sodium salts demonstrated that the two race groups respond differently to lithogenic and anti-lithogenic dietary challenges. High NaCl protocol resulted in a favourable and counter-intuitive significant decrease in free unbound calcium in samples from black subjects whereas no such change was observed in white subjects. Supplemental sodium bicarbonate and sodium citrate induced favourable decreases in urinary total calcium, urinary ionized calcium, BRI, RS of CaOx, RS of uric acid and RS of brushite, and a favourable increase in urinary citrate and pH in both groups. Interestingly and more importantly, these factors were more prominent in samples from black subjects than those from white subjects. Bone turnover measurements showed that urinary deoxypyridinoline (DPD) levels were lower while serum osteocalcin (OC) levels were higher in blacks than in whites at baseline, but these differences were not statistically significant. Smaller increases in urinary DPD levels after high aCl and sodium bicarbonate and corresponding bigger increases in serum osteocalcin (OC) levels after these protocols (and sodium citrate) in black subjects than in white subjects indicate less bone resorption and higher bone formation, respectively, in the former group. Vl The results presented in this thesis have provided convincing evidence that in the context of CaOx kidney stone formation, several physicochemical, biochemical and physiological factors are different in black and white South African subjects and that these factors are more effective in the former group with respect to providing protective mechanisms against CaOx kidney stone formation
- ItemOpen AccessThe tetrapeptide Ac-SDKP and angiotensin converting enzyme in tuberculous pericarditis and fibrosis(2020) Ramasamy, Vinasha; Sturrock, Edward; Ntsekhe, MpikoTuberculous pericarditis is an extra pulmonary form of tuberculosis (TB) which leads to a lifethreatening form of pericardial fibrosis in up to 25% of patients despite anti tuberculous therapy. The mechanisms leading to the fibrotic phenotype following infection are poorly understood. A proof of concept study revealed decreased levels of the antifibrotic N-acetylseryl-aspartyl-lysyl-proline or Ac-SDKP in tuberculous pericardial fluid as compared to control (non infectious) pericardial fluid. Ac-SDKP is a physiological peptide that is synthesised from its precursor protein thymosin β4 by the sequential action of meprin-α and prolyl oligopeptidase (POP) and is cleaved by angiotensin-1 converting enzyme (ACE). Importantly, a role of ACE and Ac-SDKP in the regulation of inflammation and fibrosis in multiple tissues and organs has been increasingly described in the literature. This has prompted interest in both the mechanisms of and potential for protective benefits of ACE inhibitors and Ac-SDKP analogue administration in fibrotic disease. The aim of this project was to investigate a) the molecular mechanisms of the antifibrotic effects of Ac-SDKP in the development of fibrosis, particularly in TB pericarditis, and b) the potential of ACEi and Ac-SDKP analogues in vitro in fibrosis prevention. Pericardial fluid and blood samples from patients with TB pericarditis or undergoing coronary artery bypass surgery (non-infectious controls) was used to investigate the metabolism of AcSDKP in the tuberculous pericardium. Ac-SDKP levels as measured by ELISA, were significantly decreased (2.3 fold) in TB pericardial fluid as compared to controls. This reduction in Ac-SDKP levels was accompanied by a local 28% increase in the enzymatic activity of ACE, but no change in POP enzyme activity levels, both of which were measured using fluorogenic assays. This suggests that an increase in ACE activity in the pericardium following infection by the mycobacterium leads to a reduction of the levels of the antifibrotic peptide which is likely to contribute to the pathophysiology of fibrosing pericarditis. A mass spectrometric (MS) approach was employed in order to identify proteins whose expression is modulated by the effect of Ac-SDKP in the proteome and secretome of a human lung fibroblast cell line (WI-38). Label free quantitative MS was employed to identify 114 and 44 differentially expressed proteins in Ac-SDKP fibroblast proteome and secretome respectively. Various extracellular matrix components and their related factors such as collagens, cytoskeletal proteins and inflammatory proteins, were identified among the differentially regulated proteins. Reactome pathway analysis confirmed the significant enrichment of Ac-SDKP-related extracellular matrix proteoglycans and extracellular matrix in the differentially expressed proteins of the secretome. Using the same cell line, the antifibrotic effects of Ac-SDKP analogues and ACE inhibitors were investigated through quantitative western blotting for transforming growth factor β (TGF-β) and Smad 3 levels, and using a hydroxyproline assay. Ac-SDKP prevented TGF-β and collagen expression through inhibition of Smad 3 phosphorylation. The Ac-SDψKP analogue (whereby the peptide bond between the aspartate and lysine is reduced) alone prevented TGF-β mediated collagen secretion. The combination of Ac-SDKP and the N domain-selective inhibitor RXP407, but not the non-selective lisinopril had an additive effect on the inhibition of collagen in fibroblasts. However, the antifibrotic effect of Ac-SDψKP was comparable to the combination of Ac-SDKP and RXP407 and was not improved with added ACE inhibition. Finally, the ACE signalling response to Ac-SDKP and the ACE inhibitors RXP407 and lisinopril was investigated using mass spectrometry and quantitative western blotting for phospho JNK and JNK. The ACE inhibitors as well as Ac-SDKP triggered the ACE signalling cascade to induce JNK phosphorylation. This highlights a potential new mechanism for the anti-inflammatory and antifibrotic effects of Ac-SDKP and the inhibitors. This thesis has demonstrated an altered metabolism of Ac-SDKP is associated with increased ACE activity in the tuberculous pericardium. It has also provided a deeper understanding of the antifibrotic action of the tetrapeptide, and in vitro evidence for the use of the analogue AcSDψKP and inhibtion of N domain catalytic activity for decreasing fibrosis. These findings form a solid basis for future in vivo pharmacological studies on the effects of Ac-SDKP analogues and ACE inhibitors in the prevention and management of fibrotic conditions. Importantly, these therapeutic options present an exciting avenue to follow in the prevention of fibrosing pericarditis in TB pericarditis.