Browsing by Subject "Chemical and Systems Biology"
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- ItemOpen AccessCharacterising the mechanism of DCUN1D1 activity in prostate cancer and identifying DCUN1D1 inhibitors for prostate cancer treatment(2020) Vava, Akhona; Zerbini, Luiz FernandoDCUN1D1 is an E3 ligase of the neddylation pathway. It mediates the posttranslational modification of majority of the cullin family of proteins with NEDD8. This activity is known to enhance ubiquitination of the cullin RING E3 ligases, however, the extent of the impact of DCUN1D1's activity is underexplored. Studies performed previously in our lab demonstrated the role of DCUN1D1 in prostate cancer in vitro and in vivo. We also identified potential inhibitors of DCUN1D1 which inhibited the proliferation of prostate cancer cells in a DCUN1D1-specific manner. This study seeks to determine the mechanism of action of DCUN1D1 in prostate cancer and to identify DCUN1D1 inhibitors using a proteomics approach. Immunoprecipitation-coupled mass spectrometry was performed to identify DCUN1D1 binding partners and we identified some known substrates of DCUN1D1 in the form of cullin 3, cullin 4B and cullin 5. We also observed that the DCUN1D1 pulldown products implicated the ubiquitin proteasome pathway, transcription, lipid metabolism and inflammatory pathways. SILAC quantitative proteomics analysis was also performed to determine the proteins that were differentially expressed in DU145 DCUN1D1 knockdown cells relative to DU145 control cells. Interestingly, we did not identify the cullin proteins or classical components of the neddylation pathway but identified the ubiquitin activating enzyme, UBA1. We also found that dysregulation of DCUN1D1 in prostate cancer led to a dysregulation in translation-related and protein processing activities such as dysregulation of eukaryotic protein translation, and protein processing in the endoplasmic reticulum. We also observed the recurrence of the WNT signalling pathway across the proteomics approaches. This culminated in the exploration of the mechanism of action of DCUN1D1 in prostate cancer using changes in protein expression as measured by western blot analysis. Significantly, we determined that DCUN1D1 mediates its mechanism of action in prostate cancer, through the neddylation pathway and preferential neddylation of cullin proteins. We also observed that knockdown of DCUN1D1 in prostate cancer led to the dysregulation of the ubiquitination and WNT/β-catenin pathways. Furthermore, advanced connectivity map analysis was performed to identify potential inhibitors of DCUN1D1 based on a proteomics approach. The drugs found to strongly connect with the DCUN1D1 knockdown signature included kinase inhibitors and anti-inflammatory agents. The above observations could lead to improved understanding of DCUN1D1 and its potential for molecular target based treatment of prostate cancer.
- ItemOpen AccessDevelopment of SNAP-tag based fusion proteins as novel auristatin F-containing immunoconjugates and photoimmunotheranostics in the detection and treatment of triple-negative breast cancer(2022) Mungra, Neelakshi; Barth, StefanBreast cancer represents one of the most common forms of female malignancy of the 21st century. Among the various breast cancer subtypes, triple-negative breast cancer (TNBC) is phenotypic of breast tumors lacking expression of the estrogen receptor (ER), the progesterone receptor (PR) and the human epidermal growth factor receptor 2 (HER2). As an idiosyncratic disease, TNBC displays a conspicuously aggressive and invasive clinical course, with an unexplained partiality towards women of African ancestry. Its acute heterogeneity and complexity behave as mutually reinforcing negative factors, which further complicate prognosis, thereby increasing the burden of breast cancer-related mortality. With the absence of well-defined molecular targets in TNBC, there is a heightened reliance on tri-modality therapy (surgery, radiotherapy and chemotherapy), albeit with an increasing incidence of adverse effects and disease relapse. To this end, there is an urgent need to develop an arsenal of targeted diagnostics and therapeutics, which can be synergized to cover the vast majority of triple-negative breast tumors, paving the way towards the development of personalized regimens suitable for the particular needs and disease of each patient. As such, achieving selective cytotoxicity, with minimal or no collateral damage to healthy tissues, embodies the holy grail of targeted anti-cancer therapies. For instance, the high affinity and specificity of monoclonal antibodies (mAbs) and derivatives thereof, have cemented their application as revolutionary tools in the selective delivery of drugs to malignant cells. These therapeutic proteins, also known as antibody-drug conjugates (ADCs), might exhibit several advantages compared to their small-molecule counterparts, but their widespread clinical use is hampered by various developmental considerations. Traditional conjugation strategies employed to arm mAbs with cytotoxic warheads, usually give rise to heterogeneous mixtures of ADC species, bearing non-uniform drug-to-antibody ratios (DARs), pharmacologic characteristics and safety profiles. Fortunately, the implementation of self-labeling tags (such as SNAP-tag, CLIP-tag and Halo-tag) are providing renewed impetus to homogeneous recombinant immunotherapeutics development. More precisely, SNAP-tag is an engineered mutant of the human O(6)-alkylguanine-DNA alkyltransferase, endowed with the ability to specifically and irreversibly react with benzylguanine (BG) derivatives, forming a stable product. Based on the above premises, this research aims to use SNAP-tag technology as a cutting-edge site-specific conjugation method to: (1) develop a comprehensive antibody platform, consisting of single-chain antibody fragments (scFvs) genetically fused to SNAP-tag, to specifically screen and evaluate their predictive potential for chondroitin sulfate proteoglycan 4 (CSPG4), CD44 and aspartate (aspartyl/asparaginyl) β-hydroxylase (ASPH)-positive TNBC cells, (2) generate functional recombinant ADC formulations as robust delivery systems carrying the antimitotic drug monomethyl auristatin F (MMAF/AURIF), concurrently overcoming production constraints to yield therapeutically viable and homogeneous combination products, and (3) provide a fail-safe system that overcomes the lack of specificity of photodynamic therapy (PDT), by coupling scFv-SNAP-tag to the potent near-infrared (NIR) photosensitizer (PS) IRDye700DX (IR700) and to demonstrate its selective dose-dependent cytotoxic activities in vitro. Following in silico design of the open reading frames (ORFs) coding for each construct, standardized molecular cloning techniques were implemented to generate recombinant mammalian expression plasmids, encompassing Ig-Kappa leader as an efficient protein secretory system. After confirmation of the DNA integrity, protein expression was achieved through transient transfection into HEK293T cells. Thereafter, the resulting histidine-tagged fusion proteins (αCSPG4(scFv)-SNAP, αCD44(scFv)-SNAPf and αASPH(scFv)-SNAP) were harvested from the cell culture supernatant and subjected to immobilized metal affinity chromatography (IMAC). In order to evaluate the outcome of this protein expression and purification step, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis were used to confirm the presence of full-length recombinant SNAP-tag based fusion proteins based on their molecular weights. Integration of the fluorescent dye Alexa Fluor 488 into the fusion proteins was carried out to investigate the self-labeling activity of the SNAP-tag moiety, as well as to provide qualitative and quantitative insights into the binding potential of the antibody fragments towards their cognate antigens. Subsequently, the AURIF and IR700-based immunoconjugates were generated by conjugating scFv-SNAP with their respective BG-modified substrates, in a defined 1:1 stoichiometric reaction. The specific and dose-dependent biological activities of the resulting bifunctional therapeutic proteins were then assessed on TNBC cells. In this study, pCB-αCD44(scFv)-SNAPf was successfully cloned and all 3 fusion proteins were effectively expressed, although with low yields and purity, yet adequate for downstream in vitro characterization. After showcasing the self-labeling potential of the SNAP-tag component, surface binding of the fluorescently labeled product was demonstrated on antigen-positive TNBC cell lines through confocal microscopy and flow cytometry. The cell killing ability of the novel AURIF-based recombinant ADCs and IR700 photoimmunoconjugates, was illustrated by the induction of a 50% reduction in cell viability (IC50 value) at nanomolar to micromolar concentrations on target cell lines. This observable selective cytotoxicity revealed that conjugation of BG derivatives to SNAP-tag, did not affect the binding potential of the antibody fragment, nor abrogated the cytocidal activity of the payload. As a proof of concept, this research builds on existing work that promulgates the use of SNAP-tag as a state-of-the-art conjugation strategy that can circumvent the challenges associated with the use of antibodies as effective delivery systems for therapeutic molecules. By harnessing the applicability of SNAP-tag in the unambiguous generation of homogeneous and pharmaceutically acceptable immunoconjugates, the results herein presented, also highlight the prospects of such agents in disease-specific tumor suppression. While various architectural modifications could further improve cytotoxic activities of future combination products, this research underscores the duality of SNAP-tag in the development of immunodiagnostics and therapeutics, that could potentially be instrumental in instilling a shift towards a personalized medicine stratagem. In conclusion, the combination of such immunoconjugates with a robust companion diagnostic panel provided by SNAP-tag, represents a first step towards the effective management of TNBC, with potential impact on the economic, social and clinical settings.
- ItemOpen AccessDifferential Lipidomic and Proteomic Responses Induced by Sub-lethal Drug Challenge in Susceptible and Drug Resistant Mycobacterium smegmatis(2021) Giddey, Alexander D; Blackburn, JonathanTuberculosis remains a major global health challenge and the increasing strength and prevalence of drug resistance threaten to undo much of the good progress made. As one of the primary, frontline anti-tuberculous drugs, growing resistance to rifampicin in particular is concerning. Sub-lethal drug exposure and the development of adaptive phenotypic drug resistance, represent an important avenue by which genetic resistance and treatment failure or relapse may occur. Proteins and general metabolites are molecular classes that are highly dynamic, responsive and essential to understanding the state of an organism, while mass spectrometry-based proteomics and metabolomics are powerful tools by which these can be examined. For mycobacteria in particular, the lipidome and cell wall are compartments of major importance with respect to virulence, adaptation, host-pathogen interactions and persistence. As such, we sought to determine the effect of sub-lethal rifampicin exposure upon the model organism Mycobacterium smegmatis over time and determine what phenotypic adaptations might be observed and explained by alterations in the proteome and lipidome, with special focus on the cell wall sub-proteome. From these data we formed several new hypotheses with respect to virulence and mechanisms of both drug resistance and sensing, which were investigated further. Finally, we examined the effect of sub-lethal rifampicin exposure, and consequent proteomic alterations, upon the M. smegmatis lipidome and propose a model by which mycobacteria respond to sub-lethal challenge with rifampicin: Upon initial insult, drug-susceptible mycobacterial growth slows and stress response networks, including the SOS response, are temporarily activated. For both susceptible and resistant bacteria, cell wall remodelling begins early through dysregulation of cell wall and lipid synthesis enzymes — such as MtrAB, Mur proteins and PimB — resulting in ultimate accumulation of lipids with composition such as to impede passive diffusion of rifampicin into the cell. Some of this lipid accumulation, namely with PIMs, may take place rapidly and so ultimately reveal extremely large increases in abundance, which possibly necessitates downregulation of enzymes such as PimB by ~4 hours post treatment. In concert with ongoing lipid dysregulation, the cell wall proteome is altered as ABC transporter proteins are generally downregulated as an additional mechanism by which to control cell wall permeability through altered cell wall composition — through removal of cell wall penetrating transport proteins — and by limiting controlled influx of exogenous compounds. Bacterial efforts to resume normal growth and adapt to rifamipicin stress involves the dysregulation of numerous virulence factors, such as PknG, which results in impaired virulence. Transcriptional and translational machinery are also gradually upregulated so as to compensate for intracellular rifampicin's inhibition of RpoB, with transcriptional activity regulated separately to that of translational machinery. Ultimately, the combination of increased transcription, translation, and cell wall impermeability allows mycobacteria to overcome rifampicin challenge and resume normal growth. In M. smegmatis specifically, all this is accompanied by the gradual upregulation of the chromosomal resistance factor Arr which, at a later timepoint, modifies extracellular rifampicin to alleviate drug pressure.
- 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.
- ItemOpen AccessUncovering the hidden mechanisms governing the transcriptional regulation of inflammation(2020) Fok, Ezio T; Mhlanga, Musa; Fanucchi, StephanieInflammation provides broad immunological protection that is essential for our survival. This cellular response is characterised by a biphasic cycle consisting of an initial acute pro-inflammatory phase and a subsequent resolving anti-inflammatory phase. Underlying each of these phases are changes in the expression of hundreds of immune genes, which encode for inflammatory mediators called cytokines. Importantly, the biphasic nature of inflammation requires cytokine expression to be highly regulated and coordinated to different timescales during each phase of inflammation. For the initial proinflammatory response, cytokine expression needs to be rapid and robust to efficiently initiate host defence mechanisms and provide effective immunological protection. In contrast, the expression of anti-inflammatory cytokines is temporally delayed to ensure that anti-inflammation always follows pro-inflammation. In order to choreograph the expression of these cytokines during inflammation, numerous mechanisms within the cell serve to regulate and coordinate cytokine transcription. Within the eukaryotic nucleus, multiple modes of transcriptional regulation function cooperatively to provide the regulatory capacity that is required for complex transcription patterns to emerge. These include the organisation of the genome, which confine cognate chromosomal contacts that are causal to transcription, and long-non coding RNAs (lncRNAs) that function to discretely fine tune transcriptional activity. Although many of the mechanisms that regulate transcription have been well described, their role in cytokine expression during inflammation remains largely unknown. In particular, the mechanisms that facilitate rapid and robust cytokine expression during proinflammation and the regulatory networks that coordinate the biphasic regulation of inflammation are unresolved. In this work, two novel lncRNAs were discovered to transcriptionally regulate these key features of cytokine expression during inflammation. The first, UMLILO (Upstream Master LncRNA of the Inflammatory chemokine LOcus), was found to emanate from the ELR+ CXCL chemokine TAD and regulate the transcriptional activation of the pro-inflammatory ELR+ CXCL chemokines (IL-8, CXCL1, CXCL2 and CXCL3). By exploiting the pre-formed local 3D topology, UMLILO is able to epigenetically prime the chemokines for transcriptional activation. This involves the discrete deposition of H3K4me3 onto the promoters of the chemokines, which allows for the pre-loading of transcriptional machinery prior to their signal-dependent activation. This reveals a fundamental mechanism for the epigenetic priming and rapid activation of pro-inflammatory cytokine genes. The second lncRNA, called AMANZI (A MAster Non-coding RNA antagoniZing Inflammation), was found to coordinate the transcription of two functionally opposed cytokines: the master pro-inflammatory IL-1β and the broad antiinflammatory IL-37. AMANZI is encoded in the promoter of IL-1β, which results in its concomitant expression when IL-1β is transcriptionally active. Functionally, AMANZI mediates the formation of a dynamic chromosomal contact between IL-1β and IL-37. This leads to the delayed transcriptional activation of IL-37 ensuring that the pro-inflammatory function of IL-1β precedes IL-37 mediated anti-inflammation. This revealed a novel biphasic circuit that coordinated the expression of IL-1β and IL-37, through the activity of AMANZI, to regulate the two functionally opposed states of inflammation. Clinical observations in healthy individuals revealed that a polymorphism occurring in AMANZI (rs16944) was able to augment the state of this genetic circuit and shift the relative levels of IL-1β and IL-37 to influence an individual's inflammatory capacity. This affected the establishment of innate immunological memory, which is involved in the progression of many inflammatory conditions and the efficacy of certain vaccines. The work described here uncovers novel mechanisms that transcriptionally regulate key features of the inflammatory response. Importantly, this work implicates the role of two novel lncRNAs in inflammation, essentially contributing to the functional annotation to the genome and providing novel targets for the modulation of pathogenic inflammation.