29 results on '"Jovcevski B"'
Search Results
2. Polyphenol characterisation and diverse bioactivities of native Australian lilly pilly (Syzygium paniculatum) extract
- Author
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Pachulicz, R. J., Yu, L., Jovcevski, B., Bulone, Vincent, Pukala, T. L., Pachulicz, R. J., Yu, L., Jovcevski, B., Bulone, Vincent, and Pukala, T. L.
- Abstract
Magenta lilly pilly (Syzygium paniculatum) is an Australian native tree that produces berry fruits that are rich in phytochemicals reportedly beneficial to human health. Here we explored the biological activities of polyphenol-enriched extracts from the magenta lilly pilly fruit, benchmarking it against traditional sources including purple sweet potato and blackberry. We show that the extracts exert potent antioxidant and neuroprotective properties as well as antimicrobial activity against Staphylococcus aureus. The phenolic composition of lilly pilly was investigated using liquid chromatography coupled to mass spectrometry (HPLC-DAD-MS), revealing anthocyanins to be the primary component in high abundance compared to traditional anthocyanin-containing plants. Three anthocyanins from lilly pilly, along with their glycosylation patterns and stability, were characterised. Altogether, our results demonstrate the potential to exploit magenta lilly pilly fruits as a high-yielding source of phenolics with beneficial biological properties of potential interest for multiple downstream applications., QC 20230522
- Published
- 2022
- Full Text
- View/download PDF
3. Crystal structure of arginine deiminase from group A streptococcus
- Author
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Henningham, A., primary, Ericsson, D.J., additional, Langer, K., additional, Casey, L., additional, Jovcevski, B., additional, Chhatwal, G.S., additional, Aquilina, J.A., additional, Batzloff, M.R., additional, Kobe, B., additional, and Walker, M.J., additional
- Published
- 2013
- Full Text
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4. Profiling and optimized extraction of bioactive polyphenolic compounds from young, red-fleshed apple using eco-friendly deep eutectic solvents.
- Author
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Yu L, Jovcevski B, Pukala TL, and Bulone V
- Subjects
- Chromatography, High Pressure Liquid, Plant Extracts chemistry, Choline chemistry, Glycerol chemistry, Neuroprotective Agents isolation & purification, Neuroprotective Agents chemistry, Mass Spectrometry, Malus chemistry, Polyphenols analysis, Polyphenols isolation & purification, Antioxidants analysis, Antioxidants chemistry, Fruit chemistry, Deep Eutectic Solvents chemistry
- Abstract
Red-fleshed apple cultivars with an enhanced content of polyphenolic compounds have attracted increasing interest due to their promising health benefits. Here, we have analysed the polyphenolic content of young, red-fleshed apples (RFA) and optimised extraction conditions of phenolics by utilising natural deep eutectic solvents (NDES). We also compare the antioxidant, neuroprotective and antimicrobial activities of NDES- and methanol-extracted phenolics from young RFA. High-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS) was used for phenolics identification and quantification. Besides young RFA, ripe red-fleshed, young and ripe white-fleshed apples were analysed, revealing that young RFA possess the highest phenolic content (2078.4 ± 4.0 mg gallic acid equivalent/100 g), and that ripe white-fleshed apples contain the least amount of phenolics (545.0 ± 32.0 mg gallic acid equivalent/100 g). The NDES choline chloride-glycerol containing 40 % w/w H
2 O gave similar yields at 40 °C as methanol. In addition, the polyphenolics profile, and bioactivities of the NDES extract from young RFA were comparable that of methanol extracts. Altogether, our data show that NDES extracts of young RFA are a promising source of bioactive polyphenolics with potential applications in diverse sectors, e.g., for functional food production, smart material engineering and natural therapies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)- Published
- 2024
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5. Biochemical and structural characterization of meningococcal methylenetetrahydrofolate reductase.
- Author
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Pantong W, Pederick JL, Maenpuen S, Tinikul R, Jayapalan JJ, Jovcevski B, Wegener KL, Bruning JB, and Salaemae W
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- Humans, NAD chemistry, NADP, Models, Molecular, Folic Acid chemistry, Folic Acid metabolism, Adenine, Methylenetetrahydrofolate Reductase (NADPH2) genetics, Methylenetetrahydrofolate Reductase (NADPH2) chemistry, Methylenetetrahydrofolate Reductase (NADPH2) metabolism, Neisseria meningitidis metabolism
- Abstract
Methylenetetrahydrofolate reductase (MTHFR) is a key metabolic enzyme in colonization and virulence of Neisseria meningitidis, a causative agent of meningococcal diseases. Here, the biochemical and structural properties of MTHFR from a virulent strain of N. meningitidis serogroup B (NmMTHFR) were characterized. Unlike other orthologs, NmMTHFR functions as a unique homohexamer, composed of three homo-dimerization partners, as shown in our 2.7 Å resolution crystal structure. Six active sites were formed solely within monomers and located away from the oligomerization interfaces. Flavin adenine dinucleotide cofactor formed hydrogen bonds with conserved sidechains, positioning its isoalloxazine ring adjacent to the overlapping binding sites of nicotinamide adenine dinucleotide (NADH) coenzyme and CH
2 -H4 folate substrate. NmMTHFR utilized NADH (Km = 44 μM) as an electron donor in the NAD(P)H-CH2 -H4 folate oxidoreductase assay, but not nicotinamide adenine dinucleotide phosphate (NADPH) which is the donor required in human MTHFR. In silico analysis and mutagenesis studies highlighted the significant difference in orientation of helix α7A (Phe215-Thr225) with that in the human enzyme. The extended sidechain of Met221 on helix α7A plays a role in stabilizing the folded structure of NADH in the hydrophobic box. This supports the NADH specificity by restricting the phosphate group of NADPH that causes steric clashes with Glu26. The movement of Met221 sidechain allows the CH2 -H4 folate substrate to bind. The unique topology of its NADH and CH2 -H4 folate binding pockets makes NmMTHFR a promising drug target for the development of new antimicrobial agents that may possess reduced off-target side effects., (© 2023 The Protein Society.)- Published
- 2023
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6. PPARγ Corepression Involves Alternate Ligand Conformation and Inflation of H12 Ensembles.
- Author
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Frkic RL, Pederick JL, Horsfall AJ, Jovcevski B, Crame EE, Kowalczyk W, Pukala TL, Chang MR, Zheng J, Blayo AL, Abell AD, Kamenecka TM, Harbort JS, Harmer JR, Griffin PR, and Bruning JB
- Subjects
- Co-Repressor Proteins metabolism, Drug Inverse Agonism, Ligands, Protein Conformation, Insulins, PPAR gamma metabolism
- Abstract
Inverse agonists of peroxisome proliferator activated receptor γ (PPARγ) have emerged as safer alternatives to full agonists for their reduced side effects while still maintaining impressive insulin-sensitizing properties. To shed light on their molecular mechanism, we characterized the interaction of the PPARγ ligand binding domain with SR10221. X-ray crystallography revealed a novel binding mode of SR10221 in the presence of a transcriptionally repressing corepressor peptide, resulting in much greater destabilization of the activation helix, H12, than without corepressor peptide. Electron paramagnetic resonance provided in-solution complementary protein dynamic data, which revealed that for SR10221-bound PPARγ, H12 adopts a plethora of conformations in the presence of corepressor peptide. Together, this provides the first direct evidence for corepressor-driven ligand conformation for PPARγ and will allow the development of safer and more effective insulin sensitizers suitable for clinical use.
- Published
- 2023
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7. Structural Study of Potent Triazole-Based Inhibitors of Staphylococcus aureus Biotin Protein Ligase.
- Author
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Stachura DL, Nguyen S, Polyak SW, Jovcevski B, Bruning JB, and Abell AD
- Abstract
The rise of multidrug-resistant bacteria, such as Staphylococcus aureus , has highlighted global urgency for new classes of antibiotics. Biotin protein ligase (BPL), a critical metabolic regulatory enzyme, is an important target that shows significant promise in this context. Here we report the in silico docking, synthesis, and biological assay of a new series of N1 -diphenylmethyl-1,2,3-triazole-based S. aureus BPL ( Sa BPL) inhibitors ( 8 - 19 ) designed to probe the adenine binding site and define whole-cell activity for this important class of inhibitor. Triazoles 13 and 14 with N1 -propylamine and -butanamide substituents, respectively, were particularly potent with K
i values of 10 ± 2 and 30 ± 6 nM, respectively, against Sa BPL. A strong correlation was apparent between the Ki values for 8 - 19 and the in silico docking, with hydrogen bonding to amino acid residues S128 and N212 of Sa BPL likely contributing to potent inhibition., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)- Published
- 2023
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8. Escherichia coli YgiC and YjfC Possess Peptide─Spermidine Ligase Activity.
- Author
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Pederick JL, Klose J, Jovcevski B, Pukala TL, and Bruning JB
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- Catalytic Domain, Ligases metabolism, Polyamines metabolism, Escherichia coli Proteins metabolism, Escherichia coli metabolism, Spermidine
- Abstract
Polyamines and polyamine-containing metabolites are involved in many cellular processes related to bacterial cell growth and survival. In Escherichia coli , the bifunctional enzyme glutathionylspermidine synthetase/amidase (GspSA) controls the production of glutathionylspermidine, which has a protective role against oxidative stress. E. coli also encodes two enzymes with homology to the synthetase domain of GspSA, YgiC, and YjfC; however, these do not catalyze the formation of glutathionylspermidine, and their catalytic function remained unknown. Here, we detail the structural and functional characterization of YgiC and YjfC. Using X-ray crystallography, the high-resolution crystal structures of YgiC and YjfC were obtained. This revealed that YgiC and YjfC possess multiple substitutions in key residues required for binding of glutathione in GspSA. Despite this difference, these enzymes share a similar active site structure to GspSA, suggesting that they catalyze the formation of an alternate peptide─spermidine conjugate. As the physiological substrates of YgiC and YjfC are unknown, this was probed using the peptide triglycine as a model substrate. A combination of enzyme activity assays and mass spectrometry revealed that YgiC and YjfC can function as peptide-spermidine ligases, forming a triglycine-spermidine conjugate. For both enzymes, conjugate formation was only observed in the presence of spermidine, but not other common polyamines, supporting that spermidine or a spermidine derivative is the physiological substrate. Importantly, since YgiC and YjfC are widely distributed in Gram-negative bacterial species, this suggests that these enzymes function in a conserved cellular process, representing a currently unknown aspect of bacterial polyamine metabolism.
- Published
- 2023
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9. Structural Analysis and Identity Confirmation of Anthocyanins in Brassica oleracea Extracts by Direct Injection Ion Mobility-Mass Spectrometry.
- Author
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Pachulicz RJ, Yu L, Jovcevski B, Bulone V, and Pukala TL
- Abstract
Anthocyanins are a subclass of plant-derived flavonoids that demonstrate immense structural heterogeneity which is challenging to capture in complex extracts by traditional liquid chromatography-mass spectrometry (MS)-based approaches. Here, we investigate direct injection ion mobility-MS as a rapid analytical tool to characterize anthocyanin structural features in red cabbage ( Brassica oleracea ) extracts. Within a 1.5 min sample run time, we observe localization of structurally similar anthocyanins and their isobars into discrete drift time regions based upon their degree of chemical modifications. Furthermore, drift time-aligned fragmentation enables simultaneous collection of MS, MS/MS, and collisional cross-section data for individual anthocyanin species down to a low picomole scale to generate structural identifiers for rapid identity confirmation. We finally identify anthocyanins in three other Brassica oleracea extracts based on red cabbage anthocyanin identifiers to demonstrate our high-throughput approach. Direct injection ion mobility-MS therefore provides wholistic structural information on structurally similar, and even isobaric, anthocyanins in complex plant extracts, which can inform the nutritional value of a plant and bolster drug discovery pipelines., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)
- Published
- 2023
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10. A New 1,2,3-Triazole Scaffold with Improved Potency against Staphylococcus aureus Biotin Protein Ligase.
- Author
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Stachura DL, Nguyen S, Polyak SW, Jovcevski B, Bruning JB, and Abell AD
- Subjects
- Biotin, Staphylococcus aureus drug effects, Staphylococcus aureus enzymology, Triazoles chemistry, Triazoles pharmacology, Anti-Bacterial Agents chemistry, Anti-Bacterial Agents pharmacology, Carbon-Nitrogen Lyases antagonists & inhibitors, Bacterial Proteins antagonists & inhibitors
- Abstract
Staphylococcus aureus , a key ESKAPE bacteria, is responsible for most blood-based infections and, as a result, is a major economic healthcare burden requiring urgent attention. Here, we report in silico docking, synthesis, and assay of N1 -diphenylmethyl triazole-based analogues ( 7 - 13 ) designed to interact with the entire binding site of S. aureus biotin protein ligase ( Sa BPL), an enzyme critical for the regulation of gluconeogenesis and fatty acid biosynthesis. The second aryl ring of these compounds enhances both Sa BPL potency and whole cell activity against S. aureus relative to previously reported mono-benzyl triazoles. Analogues 12 and 13 , with added substituents to better interact with the adenine binding site, are particularly potent, with K
i values of 6.01 ± 1.01 and 8.43 ± 0.73 nM, respectively. These analogues are the most active triazole-based inhibitors reported to date and, importantly, inhibit the growth of a clinical isolate strain of S. aureus ATCC 49775, with minimum inhibitory concentrations of 1 and 8 μg/mL, respectively.- Published
- 2022
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11. Discovery of an ʟ-amino acid ligase implicated in Staphylococcal sulfur amino acid metabolism.
- Author
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Pederick JL, Horsfall AJ, Jovcevski B, Klose J, Abell AD, Pukala TL, and Bruning JB
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- Adenosine Triphosphate metabolism, Aspartic Acid chemistry, Aspartic Acid metabolism, Dipeptides biosynthesis, Phylogeny, Methionine chemistry, Methionine metabolism, Staphylococcus aureus enzymology, Bacterial Proteins chemistry, Bacterial Proteins classification, Bacterial Proteins genetics, Peptide Synthases chemistry, Peptide Synthases classification, Peptide Synthases genetics, Cysteine chemistry, Cysteine metabolism
- Abstract
Enzymes involved in Staphylococcus aureus amino acid metabolism have recently gained traction as promising targets for the development of new antibiotics, however, not all aspects of this process are understood. The ATP-grasp superfamily includes enzymes that predominantly catalyze the ATP-dependent ligation of various carboxylate and amine substrates. One subset, ʟ-amino acid ligases (LALs), primarily catalyze the formation of dipeptide products in Gram-positive bacteria, however, their involvement in S. aureus amino acid metabolism has not been investigated. Here, we present the characterization of the putative ATP-grasp enzyme (SAOUHSC_02373) from S. aureus NCTC 8325 and its identification as a novel LAL. First, we interrogated the activity of SAOUHSC_02373 against a panel of ʟ-amino acid substrates. As a result, we identified SAOUHSC_02373 as an LAL with high selectivity for ʟ-aspartate and ʟ-methionine substrates, specifically forming an ʟ-aspartyl-ʟ-methionine dipeptide. Thus, we propose that SAOUHSC_02373 be assigned as ʟ-aspartate-ʟ-methionine ligase (LdmS). To further understand this unique activity, we investigated the mechanism of LdmS by X-ray crystallography, molecular modeling, and site-directed mutagenesis. Our results suggest that LdmS shares a similar mechanism to other ATP-grasp enzymes but possesses a distinctive active site architecture that confers selectivity for the ʟ-Asp and ʟ-Met substrates. Phylogenetic analysis revealed LdmS homologs are highly conserved in Staphylococcus and closely related Gram-positive Firmicutes. Subsequent genetic analysis upstream of the ldmS operon revealed several trans-acting regulatory elements associated with control of Met and Cys metabolism. Together, these findings support a role for LdmS in Staphylococcal sulfur amino acid metabolism., Competing Interests: Conflict of interest J. L. P. and A. J. H. are recipients of an Australian Government Research Training Program stipend scholarship. The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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12. Designer D-peptides targeting the N-terminal region of α-synuclein to prevent parkinsonian-associated fibrilization and cytotoxicity.
- Author
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Horsley JR, Jovcevski B, Pukala TL, and Abell AD
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- Brain metabolism, Caco-2 Cells, Humans, Peptides pharmacology, Parkinson Disease drug therapy, alpha-Synuclein chemistry
- Abstract
The deposition of α-synuclein (αS) aggregates in the gut and the brain is ever present in cases of Parkinson's disease. While the central non-amyloidogenic-component (NAC) region of αS plays a critical role in fibrilization, recent studies have identified a specific sequence from within the N-terminal region (NTR, residues 36-42) as a key modulator of αS fibrilization. Due to the lack of effective therapeutics which specifically target αS aggregates, we have developed a strategy to prevent the aggregation and subsequent toxicity attributed to αS fibrilization utilizing NTR targeting peptides. In this study, L- and D-isoforms of a hexa- (VAQKTV-Aib, 77-82 NAC) and heptapeptide (GVLYVGS-Aib, 36-42 NTR) containing a self-recognition component unique to αS, as well as a C-terminal disruption element, were synthesized to target primary sequence regions of αS that modulate fibrilization. The D-peptide that targets the NTR (NTR-TP-D) was shown by ThT fluorescence assays and TEM to be the most effective at preventing fibril formation and elongation, as well as increasing the abundance of soluble monomeric αS. In addition, NTR-TP-D alters the conformation of destabilised monomers into a less aggregation-prone state and reduces the hydrophobicity of αS fibrils via fibril remodelling. Furthermore, both NTR-TP isoforms alleviate the cytotoxic effects of αS aggregates in both Neuro-2a and Caco-2 cells. Together, this study highlights how targeting the NTR of αS using D-isoform peptide inhibitors may effectively combat the deleterious effects of αS fibrilization and paves the way for future drug design to utilise such an approach to treat Parkinson's disease., (Copyright © 2022 Elsevier B.V. All rights reserved.)
- Published
- 2022
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13. Polyphenol characterisation and diverse bioactivities of native Australian lilly pilly ( Syzygium paniculatum ) extract.
- Author
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Pachulicz RJ, Yu L, Jovcevski B, Bulone V, and Pukala TL
- Subjects
- Anthocyanins chemistry, Antioxidants chemistry, Australia, Chromatography, High Pressure Liquid, Fruit chemistry, Humans, Phenols chemistry, Plant Extracts chemistry, Rosaniline Dyes analysis, Polyphenols analysis, Polyphenols pharmacology, Syzygium chemistry
- Abstract
Magenta lilly pilly ( Syzygium paniculatum ) is an Australian native tree that produces berry fruits that are rich in phytochemicals reportedly beneficial to human health. Here we explored the biological activities of polyphenol-enriched extracts from the magenta lilly pilly fruit, benchmarking it against traditional sources including purple sweet potato and blackberry. We show that the extracts exert potent antioxidant and neuroprotective properties as well as antimicrobial activity against Staphylococcus aureus . The phenolic composition of lilly pilly was investigated using liquid chromatography coupled to mass spectrometry (HPLC-DAD-MS), revealing anthocyanins to be the primary component in high abundance compared to traditional anthocyanin-containing plants. Three anthocyanins from lilly pilly, along with their glycosylation patterns and stability, were characterised. Altogether, our results demonstrate the potential to exploit magenta lilly pilly fruits as a high-yielding source of phenolics with beneficial biological properties of potential interest for multiple downstream applications.
- Published
- 2022
- Full Text
- View/download PDF
14. A structural model of the human plasminogen and Aspergillus fumigatus enolase complex.
- Author
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Nguyen S, Jovcevski B, Truong JQ, Pukala TL, and Bruning JB
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- Antifungal Agents, Humans, Models, Structural, Phosphoenolpyruvate metabolism, Plasminogen metabolism, Protein Binding, Aspergillus fumigatus, Phosphopyruvate Hydratase genetics, Phosphopyruvate Hydratase metabolism
- Abstract
The metabolic enzyme, enolase, plays a crucial role in the cytoplasm where it maintains cellular energy production within the process of glycolysis. The main role of enolase in glycolysis is to convert 2-phosphoglycerate to phosphoenolpyruvate; however, enolase can fulfill roles that deviate from this function. In pathogenic bacteria and fungi, enolase is also located on the cell surface where it functions as a virulence factor. Surface-expressed enolase is a receptor for human plasma proteins, including plasminogen, and this interaction facilitates nutrient acquisition and tissue invasion. A novel approach to developing antifungal drugs is to inhibit the formation of this complex. To better understand the structure of enolase and the interactions that may govern complex formation, we have solved the first X-ray crystal structure of enolase from Aspergillus fumigatus (2.0 Å) and have shown that it preferentially adopts a dimeric quaternary structure using native mass spectrometry. Two additional X-ray crystal structures of A. fumigatus enolase bound to the endogenous substrate 2-phosphoglycerate and product phosphoenolpyruvate were determined and kinetic characterization was carried out to better understand the details of its canonical function. From these data, we have produced a model of the A. fumigatus enolase and human plasminogen complex to provide structural insights into the mechanisms of virulence and aid future development of small molecules or peptidomimetics for antifungal drug design., (© 2022 Wiley Periodicals LLC.)
- Published
- 2022
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15. Structural insights into the antifungal drug target guanosine monophosphate synthase from Aspergillus fumigatus.
- Author
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Nguyen S, Jovcevski B, Pukala TL, and Bruning JB
- Subjects
- Candida albicans metabolism, Carbon-Nitrogen Ligases, Fungal Proteins metabolism, Guanosine Monophosphate metabolism, Humans, Antifungal Agents metabolism, Antifungal Agents pharmacology, Aspergillus fumigatus genetics, Aspergillus fumigatus metabolism
- Abstract
Purine biosynthesis is a fundamental cellular process that sustains life by maintaining the intracellular pool of purines for DNA/RNA synthesis and signal transduction. As an integral determinant of fungal survival and virulence, the enzymes in this metabolic pathway have been pursued as potential antifungal targets. Guanosine monophosphate (GMP) synthase has been identified as an attractive target as it is essential for virulence in the clinically prominent fungal pathogens Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans. However, a lack of structural information on GMP synthase has hindered drug-design efforts. Here, the first structure of a GMP synthase of fungal origin, that from A. fumigatus (at 2.3 Å resolution), is presented. Structural analysis of GMP synthase shows a distinct absence of the D1 dimerization domain that is present in the human homologue. Interestingly, A. fumigatus GMP synthase adopts a dimeric state, as determined by native mass spectrometry and gel-filtration chromatography, in contrast to the monomeric human homologue. Analysis of the substrate-binding pockets of A. fumigatus GMP synthase reveals key differences in the ATP- and XMP-binding sites that can be exploited for species-specific inhibitor drug design. Furthermore, the inhibitory activities of the glutamine analogues acivicin (IC
50 = 16.6 ± 2.4 µM) and 6-diazo-5-oxo-L-norleucine (IC50 = 29.6 ± 5.6 µM) against A. fumigatus GMP synthase are demonstrated. Together, these data provide crucial structural information required for specifically targeting A. fumigatus GMP synthase for future antifungal drug-discovery endeavours.- Published
- 2022
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16. Structural and mechanistic insights into amyloid-β and α-synuclein fibril formation and polyphenol inhibitor efficacy in phospholipid bilayers.
- Author
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Sanders HM, Jovcevski B, Marty MT, and Pukala TL
- Subjects
- Amyloid drug effects, Amyloid ultrastructure, Amyloid beta-Peptides genetics, Amyloid beta-Peptides ultrastructure, Amyloidogenic Proteins antagonists & inhibitors, Amyloidogenic Proteins genetics, Catechin analogs & derivatives, Catechin pharmacology, Humans, Lipid Bilayers metabolism, Membrane Lipids genetics, Parkinson Disease drug therapy, Parkinson Disease pathology, Phospholipids biosynthesis, Phospholipids genetics, Polyphenols pharmacology, alpha-Synuclein ultrastructure, Amyloid genetics, Amyloid beta-Protein Precursor genetics, Parkinson Disease genetics, alpha-Synuclein genetics
- Abstract
Under certain cellular conditions, functional proteins undergo misfolding, leading to a transition into oligomers which precede the formation of amyloid fibrils. Misfolding proteins are associated with neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. While the importance of lipid membranes in misfolding and disease aetiology is broadly accepted, the influence of lipid membranes during therapeutic design has been largely overlooked. This study utilized a biophysical approach to provide mechanistic insights into the effects of two lipid membrane systems (anionic and zwitterionic) on the inhibition of amyloid-β 40 and α-synuclein amyloid formation at the monomer, oligomer and fibril level. Large unilamellar vesicles (LUVs) were shown to increase fibrillization and largely decrease the effectiveness of two well-known polyphenol fibril inhibitors, (-)-epigallocatechin gallate (EGCG) and resveratrol; however, use of immunoblotting and ion mobility mass spectrometry revealed this occurs through varying mechanisms. Oligomeric populations in particular were differentially affected by LUVs in the presence of resveratrol, an elongation phase inhibitor, compared to EGCG, a nucleation targeted inhibitor. Ion mobility mass spectrometry showed EGCG interacts with or induces more compact forms of monomeric protein typical of off-pathway structures; however, binding is reduced in the presence of LUVs, likely due to partitioning in the membrane environment. Competing effects of the lipids and inhibitor, along with reduced inhibitor binding in the presence of LUVs, provide a mechanistic understanding of decreased inhibitor efficacy in a lipid environment. Together, this study highlights that amyloid inhibitor design may be misguided if effects of lipid membrane composition and architecture are not considered during development., (© 2021 Federation of European Biochemical Societies.)
- Published
- 2022
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17. Nucleoside selectivity of Aspergillus fumigatus nucleoside-diphosphate kinase.
- Author
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Nguyen S, Jovcevski B, Pukala TL, and Bruning JB
- Subjects
- Aspergillosis genetics, Aspergillosis microbiology, Aspergillosis pathology, Aspergillus fumigatus pathogenicity, Aspergillus fumigatus ultrastructure, Escherichia coli genetics, Humans, Kinetics, Nucleoside-Diphosphate Kinase chemistry, Nucleoside-Diphosphate Kinase ultrastructure, Nucleosides biosynthesis, Phosphorylation genetics, Substrate Specificity, Aspergillus fumigatus genetics, Nucleoside-Diphosphate Kinase genetics, Nucleosides genetics, Protein Conformation
- Abstract
Aspergillus fumigatus infections are rising at a disconcerting rate in tandem with antifungal resistance rates. Efforts to develop novel antifungals have been hindered by the limited knowledge of fundamental biological and structural mechanisms of A. fumigatus propagation. Biosynthesis of NTPs, the building blocks of DNA and RNA, is catalysed by NDK. An essential enzyme in A. fumigatus, NDK poses as an attractive target for novel antifungals. NDK exhibits broad substrate specificity across species, using both purines and pyrimidines, but the selectivity of such nucleosides in A. fumigatus NDK is unknown, impeding structure-guided inhibitor design. Structures of NDK in unbound- and NDP-bound states were solved, and NDK activity was assessed in the presence of various NTP substrates. We present the first instance of a unique substrate binding mode adopted by CDP and TDP specific to A. fumigatus NDK that illuminates the structural determinants of selectivity. Analysis of the oligomeric state reveals that A. fumigatus NDK adopts a hexameric assembly in both unbound- and NDP-bound states, contrary to previous reports suggesting it is tetrameric. Kinetic analysis revealed that ATP exhibited the greatest turnover rate (321 ± 33.0 s
-1 ), specificity constant (626 ± 110.0 mm-1 ·s-1 ) and binding free energy change (-37.0 ± 3.5 kcal·mol-1 ). Comparatively, cytidine nucleosides displayed the slowest turnover rate (53.1 ± 3.7 s-1 ) and lowest specificity constant (40.2 ± 4.4 mm-1 ·s-1 ). We conclude that NDK exhibits nucleoside selectivity whereby adenine nucleosides are used preferentially compared to cytidine nucleosides, and these insights can be exploited to guide drug design. ENZYMES: Nucleoside-diphosphate kinase (EC 2.7.4.6). DATABASE: Structural data are available in the PDB database under the accession numbers: Unbound-NDK (6XP4), ADP-NDK (6XP7), GDP-NDK (6XPS), IDP-NDK (6XPU), UDP-NDK (6XPT), CDP-NDK (6XPW), TDP-NDK (6XPV)., (© 2020 Federation of European Biochemical Societies.)- Published
- 2021
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18. Polyphenol Honokiol and Flavone 2',3',4'-Trihydroxyflavone Differentially Interact with α-Synuclein at Distinct Phases of Aggregation.
- Author
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Jovcevski B, Das S, Smid S, and Pukala TL
- Subjects
- Amyloid, Biphenyl Compounds, Polyphenols pharmacology, alpha-Synuclein, Flavones pharmacology, Lignans pharmacology
- Abstract
The association between protein aggregation and neurodegenerative diseases such as Parkinson's disease continues to be well interrogated but poorly elucidated at a mechanistic level. Nevertheless, the formation of amyloid fibrils from the destabilization and misfolding of native proteins is a molecular hallmark of disease. Consequently, there is ongoing demand for the identification and development of small molecules which prevent fibril formation. This study comprehensively assesses the inhibitory properties of two small molecules, the lignan polyphenol honokiol and the flavonoid 2',3',4'-trihydroxyflavone, in preventing α-synuclein fibrilization. The data shows that honokiol does not prevent α-synuclein fibril elongation, while 2',3',4'-trihydroxyflavone is effective at inhibiting fibril elongation and induces oligomer formation (for both wild-type α-synuclein and the disease-associated A53T mutation). Moreover, the exposed hydrophobicity of α-synuclein fibrils is reduced in the presence of 2',3',4'-trihydroxyflavone, whereas the addition of honokiol did not reduce the hydrophobicity of fibrils. In addition, ion mobility-mass spectrometry revealed that the conformation of α-synuclein wild-type and A53T monomers after disassembly is restored to a nonaggregation-prone state upon 2',3',4'-trihydroxyflavone treatment. Collectively, this study shows that the mechanisms by which these polyphenols and flavonoids prevent fibril formation are distinct by their interactions at various phases of the fibril-forming pathway. Furthermore, this study highlights how thorough biophysical interrogation of the interaction is required for understanding the ability of inhibitors to prevent protein aggregation associated with disease.
- Published
- 2020
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19. Short Photoswitchable Antibacterial Peptides.
- Author
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Yeoh YQ, Horsley JR, Yu J, Polyak SW, Jovcevski B, and Abell AD
- Subjects
- Anti-Bacterial Agents chemical synthesis, Anti-Bacterial Agents chemistry, Azo Compounds chemistry, Dose-Response Relationship, Drug, Microbial Sensitivity Tests, Molecular Structure, Peptides chemical synthesis, Peptides chemistry, Structure-Activity Relationship, Anti-Bacterial Agents pharmacology, Azo Compounds pharmacology, Peptides pharmacology, Staphylococcus aureus drug effects
- Abstract
Three photoswitchable tetrapeptides, based on a known synthetic antibacterial, were designed and synthesized to determine activity against Staphylococcus aureus. Each peptide contains an azobenzene photoswitch incorporated into either the N-terminal side chain (1), C-terminal side chain (2), or the C-terminus (3) to allow reversible switching between cis- and trans-enriched photostationary states. Biological assays revealed that the C-terminus azobenzene (3) possessed the most potent antibacterial activity, with an MIC of 1 μg/mL. In this study, net positive charge, hydrophobicity, position of the azobenzene, secondary structure, and amphiphilicity were all found to contribute to antibacterial activity, with each of these factors likely facilitating the peptide to disrupt the negatively charged bacterial lipid membrane. Hence, these short photoswitchable antibacterial tetrapeptides provide insights for the future design and synthesis of antibiotics targeting S. aureus., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2020
- Full Text
- View/download PDF
20. Rationally designed peptide-based inhibitor of Aβ42 fibril formation and toxicity: a potential therapeutic strategy for Alzheimer's disease.
- Author
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Horsley JR, Jovcevski B, Wegener KL, Yu J, Pukala TL, and Abell AD
- Subjects
- Humans, Ion Mobility Spectrometry, Protein Conformation, beta-Strand, Alzheimer Disease therapy, Amyloid beta-Peptides chemistry, Peptide Fragments chemistry, Protein Aggregation, Pathological
- Abstract
Amyloid beta peptide (Aβ42) aggregation in the brain is thought to be responsible for the onset of Alzheimer's disease, an insidious condition without an effective treatment or cure. Hence, a strategy to prevent aggregation and subsequent toxicity is crucial. Bio-inspired peptide-based molecules are ideal candidates for the inhibition of Aβ42 aggregation, and are currently deemed to be a promising option for drug design. In this study, a hexapeptide containing a self-recognition component unique to Aβ42 was designed to mimic the β-strand hydrophobic core region of the Aβ peptide. The peptide is comprised exclusively of D-amino acids to enhance specificity towards Aβ42, in conjunction with a C-terminal disruption element to block the recruitment of Aβ42 monomers on to fibrils. The peptide was rationally designed to exploit the synergy between the recognition and disruption components, and incorporates features such as hydrophobicity, β-sheet propensity, and charge, that all play a critical role in the aggregation process. Fluorescence assays, native ion-mobility mass spectrometry (IM-MS) and cell viability assays were used to demonstrate that the peptide interacts with Aβ42 monomers and oligomers with high specificity, leading to almost complete inhibition of fibril formation, with essentially no cytotoxic effects. These data define the peptide-based inhibitor as a potentially potent anti-amyloid drug candidate for this hitherto incurable disease., (© 2020 The Author(s).)
- Published
- 2020
- Full Text
- View/download PDF
21. Interrogating the higher order structures of snake venom proteins using an integrated mass spectrometric approach.
- Author
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Wang CR, Bubner ER, Jovcevski B, Mittal P, and Pukala TL
- Subjects
- Animals, Elapidae, Mass Spectrometry, Proteins, Proteomics, Snake Venoms
- Abstract
Snake venoms contain complex mixtures of proteins vital for the survival of venomous snakes. Aligned with their diverse pharmacological activities, the protein compositions of snake venoms are highly variable, and efforts to characterise the primary structures of such proteins are ongoing. Additionally, a significant knowledge gap exists in terms of the higher-order protein structures which modulate venom potency, posing a challenge for successful therapeutic applications. Here we use a multifaceted mass spectrometry approach to characterise proteins from venoms of Collett's snake Pseudechis colletti and the puff adder Bitis arietans. Following chromatographic fractionation and bottom-up proteomics analysis, native mass spectrometry identified, among other components, a non-covalent l-amino acid oxidase dimer in the P. colletti venom and a C-type lectin tetramer in the B. arietans venom. Furthermore, a covalently-linked phospholipase A
2 (PLA2 ) dimer was identified in P. colletti venom, from which the PLA2 species were shown to adopt compact geometries using ion mobility measurements. Interestingly, we show that the dimeric PLA2 possesses greater bioactivity than the monomeric PLA2 s. This work contributes to ongoing efforts cataloguing components of snake venoms, and notably, emphasises the importance of understanding higher-order venom protein interactions and the utility of a combined mass spectrometric approach for this task. SIGNIFICANCE: The protein constituents of snake venoms represent a sophisticated cocktail of biologically active molecules ideally suited for further exploration in drug design and development. Despite ongoing efforts to characterise the diverse protein components of such venoms there is still much work required in this area, particularly in moving from simply describing the protein primary sequence to providing an understanding of quaternary structure. The combined proteomic and native mass spectrometry workflow utilised here gives new insights into higher order protein structures in selected snake venoms, and can underpin further investigation into the protein interactions which govern snake venom specificity and potency., (Copyright © 2020 Elsevier B.V. All rights reserved.)- Published
- 2020
- Full Text
- View/download PDF
22. The molecular chaperone β-casein prevents amorphous and fibrillar aggregation of α-lactalbumin by stabilisation of dynamic disorder.
- Author
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Sanders HM, Jovcevski B, Carver JA, and Pukala TL
- Subjects
- Amyloid metabolism, Mass Spectrometry, Protein Folding, Proteostasis physiology, Caseins chemistry, Caseins metabolism, Lactalbumin antagonists & inhibitors, Lactalbumin chemistry, Lactalbumin metabolism, Molecular Chaperones chemistry, Molecular Chaperones metabolism, Protein Aggregates physiology
- Abstract
Deficits in protein homeostasis (proteostasis) are typified by the partial unfolding or misfolding of native proteins leading to amorphous or fibrillar aggregation, events that have been closely associated with diseases including Alzheimer's and Parkinson's diseases. Molecular chaperones are intimately involved in maintaining proteostasis, and their mechanisms of action are in part dependent on the morphology of aggregation-prone proteins. This study utilised native ion mobility-mass spectrometry to provide molecular insights into the conformational properties and dynamics of a model protein, α-lactalbumin (α-LA), which aggregates in an amorphous or amyloid fibrillar manner controlled by appropriate selection of experimental conditions. The molecular chaperone β-casein (β-CN) is effective at inhibiting amorphous and fibrillar aggregation of α-LA at sub-stoichiometric ratios, with greater efficiency against fibril formation. Analytical size-exclusion chromatography demonstrates the interaction between β-CN and amorphously aggregating α-LA is stable, forming a soluble high molecular weight complex, whilst with fibril-forming α-LA the interaction is transient. Moreover, ion mobility-mass spectrometry (IM-MS) coupled with collision-induced unfolding (CIU) revealed that α-LA monomers undergo distinct conformational transitions during the initial stages of amorphous (order to disorder) and fibrillar (disorder to order) aggregation. The structural heterogeneity of monomeric α-LA during fibrillation is reduced in the presence of β-CN along with an enhancement in stability, which provides a potential means for preventing fibril formation. Together, this study demonstrates how IM-MS and CIU can investigate the unfolding of proteins as well as examine transient and dynamic protein-chaperone interactions, and thereby provides detailed insight into the mechanism of chaperone action and proteostasis mechanisms., (© 2020 The Author(s).)
- Published
- 2020
- Full Text
- View/download PDF
23. Fabrication of Piperazine Functionalized Polymeric Monolithic Tip for Rapid Enrichment of Glycopeptides/Glycans.
- Author
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Sajid MS, Jovcevski B, Pukala TL, Jabeen F, and Najam-Ul-Haq M
- Subjects
- Animals, Cattle, Glycopeptides metabolism, Horseradish Peroxidase chemistry, Horseradish Peroxidase metabolism, Humans, Polysaccharides metabolism, Serum Albumin, Bovine chemistry, Serum Albumin, Bovine metabolism, Glycopeptides blood, Polymers chemistry, Polysaccharides blood
- Abstract
Enrichment strategies are designed for the pretreatment of low-abundance glycans and glycopeptides prior to mass spectrometric (MS) analysis. Here, a tip-based strategy is being reported for the enrichment of glycopeptides and glycans using a piperazine modified polymeric monolithic tip. The tip is fabricated using the free radical polymerization. Fast separation (2 min) is achieved under optimized conditions with 20 cycles per step of loading, incubation, washing, and elution followed by MALDI-MS analysis. A total of 25, 22, and 34 glycopeptides covering all glycosylation sites are enriched by the modified tips from tryptic digests of horse radish peroxidase, chicken avidin, and human immunoglobulin G, respectively. Piperazine exhibits high selectivity 1:400 horse radish peroxidase/bovine serum albumin, sensitivity to 100 attomoles, recovery 89.51%, and batch to batch reproducibility (RSD > 1) in glycopeptides enrichment. Piperazine tips also enrich glycans from ovalbumin and human immunoglobulin G. High selectivity (1:1200, ovalbumin/BSA) and detection limit of 100 attomole is attained for glycans and furthermore 58 glycans are enriched from human serum. Thus, piperazine tips can be used as an enrichment tool for swift, cost-effective routine analysis of biological samples for separation of glycopeptides and glycans.
- Published
- 2020
- Full Text
- View/download PDF
24. C-Phycocyanin from Spirulina Inhibits α-Synuclein and Amyloid-β Fibril Formation but Not Amorphous Aggregation.
- Author
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Liu Y, Jovcevski B, and Pukala TL
- Subjects
- Amyloid beta-Peptides chemistry, Peptide Fragments chemistry, Protein Aggregates, Amyloid, Amyloid beta-Peptides antagonists & inhibitors, Peptide Fragments antagonists & inhibitors, Phycocyanin pharmacology, Spirulina chemistry, alpha-Synuclein antagonists & inhibitors
- Abstract
Proteinopathies including cataracts and neurodegenerative diseases, such as Alzheimer's and Parkinson's disease, are characterized by a series of aberrant protein folding events, resulting in amorphous aggregate or amyloid fibril formation. In the latter case, research has heavily focused on the development of small-molecule inhibitors with limited success during clinical trials. However, very few studies have focused on utilizing exogenous proteins as potential aggregation inhibitors. C-Phycocyanin, derived from Spirulina sp., has been known to exert anti-inflammatory properties; however, the ability of C-phycocyanin to inhibit protein aggregation has yet to be investigated. We have demonstrated that C-phycocyanin is an effective inhibitor of A53Tα-synuclein at extremely low substoichiometric ratios (200-fold excess of α-synuclein) and Aβ40/42 fibril formation. However, C-phycocyanin is relatively ineffective in inhibiting the reduction-induced amorphous aggregation of ADH and heat-induced aggregation of catalase. In addition, 2D NMR, ion mobility-mass spectrometry, and analytical-SEC demonstrate that the interaction between C-phycocyanin and α-synuclein is through nonstable interactions, indicating that transient interactions are likely to be responsible for preventing fibril formation. Overall, this work highlights how biomolecules from natural sources could be used to aid in the development of therapeutics to combat protein misfolding diseases.
- Published
- 2019
- Full Text
- View/download PDF
25. The influence of the N-terminal region proximal to the core domain on the assembly and chaperone activity of αB-crystallin.
- Author
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Jovcevski B, Andrew Aquilina J, Benesch JLP, and Ecroyd H
- Subjects
- Humans, Mutation, Protein Domains, Protein Multimerization, Protein Structure, Secondary, alpha-Crystallin B Chain genetics, alpha-Crystallin B Chain metabolism, alpha-Crystallin B Chain chemistry
- Abstract
αB-Crystallin (HSPB5) is a small heat-shock protein that is composed of dimers that then assemble into a polydisperse ensemble of oligomers. Oligomerisation is mediated by heterologous interactions between the C-terminal tail of one dimer and the core "α-crystallin" domain of another and stabilised by interactions made by the N-terminal region. Comparatively little is known about the latter contribution, but previous studies have suggested that residues in the region 54-60 form contacts that stabilise the assembly. We have generated mutations in this region (P58A, S59A, S59K, R56S/S59R and an inversion of residues 54-60) to examine their impact on oligomerisation and chaperone activity in vitro. By using native mass spectrometry, we found that all the αB-crystallin mutants were assembly competent, populating similar oligomeric distributions to wild-type, ranging from 16-mers to 30-mers. However, circular dichroism spectroscopy, intrinsic tryptophan and bis-ANS fluorescence studies demonstrated that the secondary structure differs to wild type, the 54-60 inversion mutation having the greatest impact. All the mutants exhibited a dramatic decrease in exposed hydrophobicity. We also found that the mutants in general were equally active as the wild-type protein in inhibiting the amorphous aggregation of insulin and seeded amyloid fibrillation of α-synuclein in vitro, except for the 54-60 inversion mutant, which was significantly less effective at inhibiting insulin aggregation. Our data indicate that alterations in the part of the N-terminal region proximal to the core domain do not drastically affect the oligomerisation of αB-crystallin, reinforcing the robustness of αB-crystallin in functioning as a molecular chaperone.
- Published
- 2018
- Full Text
- View/download PDF
26. Evaluating the Effect of Phosphorylation on the Structure and Dynamics of Hsp27 Dimers by Means of Ion Mobility Mass Spectrometry.
- Author
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Jovcevski B, Kelly MA, Aquilina JA, Benesch JLP, and Ecroyd H
- Subjects
- Fluorescence Resonance Energy Transfer, HSP27 Heat-Shock Proteins metabolism, Heat-Shock Proteins, Humans, Molecular Chaperones, Phosphorylation, Protein Conformation, HSP27 Heat-Shock Proteins chemistry, Ion Mobility Spectrometry, Molecular Dynamics Simulation
- Abstract
The quaternary structure and dynamics of the human small heat-shock protein Hsp27 are linked to its molecular chaperone function and influenced by post-translational modifications, including phosphorylation. Phosphorylation of Hsp27 promotes oligomer dissociation and can enhance chaperone activity. This study explored the impact of phosphorylation on the quaternary structure and dynamics of Hsp27. Using mutations that mimic phosphorylation, and ion mobility mass spectrometry, we show that successive substitutions result in an increase in the conformational heterogeneity of Hsp27 dimers. In contrast, we did not detect any changes in the structure of an Hsp27 12-mer, representative of larger Hsp27 oligomers. Our data suggest that oligomer dissociation and increased flexibility of the dimer contribute to the enhanced chaperone activity of phosphorylated Hsp27. Thus, post-translational modifications such as phosphorylation play a crucial role in modulating both the tertiary and quaternary structure of Hsp27, which is pivotal to its function as a key component of the proteostasis network in cells. Our data demonstrate the utility of ion mobility mass spectrometry for probing the structure and dynamics of heterogeneous proteins.
- Published
- 2017
- Full Text
- View/download PDF
27. Stability of the octameric structure affects plasminogen-binding capacity of streptococcal enolase.
- Author
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Cork AJ, Ericsson DJ, Law RH, Casey LW, Valkov E, Bertozzi C, Stamp A, Jovcevski B, Aquilina JA, Whisstock JC, Walker MJ, and Kobe B
- Subjects
- Crystallography, X-Ray, Humans, Protein Binding, Protein Conformation, Bacterial Proteins metabolism, Phosphopyruvate Hydratase metabolism, Plasminogen metabolism, Streptococcus pyogenes metabolism
- Abstract
Group A Streptococcus (GAS) is a human pathogen that has the potential to cause invasive disease by binding and activating human plasmin(ogen). Streptococcal surface enolase (SEN) is an octameric α-enolase that is localized at the GAS cell surface. In addition to its glycolytic role inside the cell, SEN functions as a receptor for plasmin(ogen) on the bacterial surface, but the understanding of the molecular basis of plasmin(ogen) binding is limited. In this study, we determined the crystal and solution structures of GAS SEN and characterized the increased plasminogen binding by two SEN mutants. The plasminogen binding ability of SENK312A and SENK362A is ~2- and ~3.4-fold greater than for the wild-type protein. A combination of thermal stability assays, native mass spectrometry and X-ray crystallography approaches shows that increased plasminogen binding ability correlates with decreased stability of the octamer. We propose that decreased stability of the octameric structure facilitates the access of plasmin(ogen) to its binding sites, leading to more efficient plasmin(ogen) binding and activation.
- Published
- 2015
- Full Text
- View/download PDF
28. Phosphomimics destabilize Hsp27 oligomeric assemblies and enhance chaperone activity.
- Author
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Jovcevski B, Kelly MA, Rote AP, Berg T, Gastall HY, Benesch JL, Aquilina JA, and Ecroyd H
- Subjects
- Cell Line, Circular Dichroism, Dimerization, HEK293 Cells, HSP27 Heat-Shock Proteins chemistry, HSP27 Heat-Shock Proteins genetics, Humans, Mass Spectrometry, Mutagenesis, Site-Directed, Phosphorylation, Protein Folding, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Recombinant Proteins genetics, HSP27 Heat-Shock Proteins metabolism, Molecular Chaperones metabolism
- Abstract
Serine phosphorylation of the mammalian small heat-shock protein Hsp27 at residues 15, 78, and 82 is thought to regulate its structure and chaperone function; however, the site-specific impact has not been established. We used mass spectrometry to assess the combinatorial effect of mutations that mimic phosphorylation upon the oligomeric state of Hsp27. Comprehensive dimerization yielded a relatively uncrowded spectrum, composed solely of even-sized oligomers. Modification at one or two serines decreased the average oligomeric size, while the triple mutant was predominantly a dimer. These changes were reflected in a greater propensity for oligomers to dissociate upon increased modification. The ability of Hsp27 to prevent amorphous or fibrillar aggregation of target proteins was enhanced and correlated with the amount of dissociated species present. We propose that, in vivo, phosphorylation promotes oligomer dissociation, thereby enhancing chaperone activity. Our data support a model in which dimers are the chaperone-active component of Hsp27., (Copyright © 2015 Elsevier Ltd. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
29. Structure-informed design of an enzymatically inactive vaccine component for group A Streptococcus.
- Author
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Henningham A, Ericsson DJ, Langer K, Casey LW, Jovcevski B, Chhatwal GS, Aquilina JA, Batzloff MR, Kobe B, and Walker MJ
- Subjects
- Amino Acid Substitution, Animals, Antibodies, Bacterial blood, Antigens, Bacterial genetics, Antigens, Bacterial immunology, Crystallography, X-Ray, Epitope Mapping, Humans, Hydrolases genetics, Hydrolases immunology, Mice, Mice, Inbred BALB C, Models, Molecular, Mutagenesis, Site-Directed, Mutant Proteins chemistry, Mutant Proteins genetics, Mutant Proteins immunology, Mutant Proteins metabolism, Protein Conformation, Protein Multimerization, Streptococcal Vaccines genetics, Streptococcal Vaccines immunology, Streptococcus pyogenes chemistry, Streptococcus pyogenes genetics, Streptococcus pyogenes immunology, Antigens, Bacterial chemistry, Antigens, Bacterial metabolism, Hydrolases chemistry, Hydrolases metabolism, Streptococcal Vaccines chemistry, Streptococcal Vaccines metabolism, Streptococcus pyogenes enzymology
- Abstract
Unlabelled: Streptococcus pyogenes (group A Streptococcus [GAS]) causes ~700 million human infections/year, resulting in >500,000 deaths. There is no commercial GAS vaccine available. The GAS surface protein arginine deiminase (ADI) protects mice against a lethal challenge. ADI is an enzyme that converts arginine to citrulline and ammonia. Administration of a GAS vaccine preparation containing wild-type ADI, a protein with inherent enzymatic activity, may present a safety risk. In an approach intended to maximize the vaccine safety of GAS ADI, X-ray crystallography and structural immunogenic epitope mapping were used to inform vaccine design. This study aimed to knock out ADI enzyme activity without disrupting the three-dimensional structure or the recognition of immunogenic epitopes. We determined the crystal structure of ADI at 2.5 Å resolution and used it to select a number of amino acid residues for mutagenesis to alanine (D166, E220, H275, D277, and C401). Each mutant protein displayed abrogated activity, and three of the mutant proteins (those with the D166A, H275A, and D277A mutations) possessed a secondary structure and oligomerization state equivalent to those of the wild type, produced high-titer antisera, and avoided disruption of B-cell epitopes of ADI. In addition, antisera raised against the D166A and D277A mutant proteins bound to the GAS cell surface. The inactivated D166A and D277A mutant ADIs are ideal for inclusion in a GAS vaccine preparation. There is no human ortholog of ADI, and we confirm that despite limited structural similarity in the active-site region to human peptidyl ADI 4 (PAD4), ADI does not functionally mimic PAD4 and antiserum raised against GAS ADI does not recognize human PAD4., Importance: We present an example of structural biology informing human vaccine design. We previously showed that the administration of the enzyme arginine deiminase (ADI) to mice protected the mice against infection with multiple GAS serotypes. In this study, we determined the structure of GAS ADI and used this information to improve the vaccine safety of GAS ADI. Catalytically inactive mutant forms of ADI retained structure, recognition by antisera, and immunogenic epitopes, rendering them ideal for inclusion in GAS vaccine preparations. This example of structural biology informing vaccine design may underpin the formulation of a safe and efficacious GAS vaccine.
- Published
- 2013
- Full Text
- View/download PDF
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