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Item Biopolymer-polyphenol conjugates: Novel multifunctional materials for active packaging(Elsevier B V, 2024-11) Sahraeian S; Abdollahi B; Rashidinejad AThe development of natural active packaging materials and coatings presents a promising alternative to petroleum-based packaging solutions. These materials are engineered by incorporating functional ingredients with preservative capabilities. Concurrently, research has highlighted the diverse physicochemical, functional, and health-promoting properties of protein-polyphenol, polysaccharide-polyphenol, and protein-polysaccharide-polyphenol conjugates within various food formulations. However, a critical gap exists regarding the exploration of these biopolymers as active packaging materials. In contrast to conventional approaches for developing active packaging materials, this review presents a novel perspective by focusing on biopolymer-polyphenol conjugates. In this work, we delve into the realm of active packaging materials and coatings constructed from these conjugates, highlighting their potential as multifunctional active components in food packaging and preservation. This review comprehensively investigates the physicochemical properties, functionalities, and health-promoting activities associated with biopolymer-polyphenol conjugates. Their emulsification, antioxidant, and antimicrobial activities, coupled with enhancements in mechanical strength and permeability properties, contribute to their multifunctional nature. Furthermore, we explore the potential advantages and limitations of utilizing these conjugates in active packaging applications. Finally, the review concludes by proposing crucial research avenues for further exploration of biopolymer-polyphenol conjugates within the domain of active food packaging.Item Structure-guided inhibition of the cancer DNA-mutating enzyme APOBEC3A.(Springer Nature Limited, 2023-10-11) Harjes S; Kurup HM; Rieffer AE; Bayarjargal M; Filitcheva J; Su Y; Hale TK; Filichev VV; Harjes E; Harris RS; Jameson GBThe normally antiviral enzyme APOBEC3A is an endogenous mutagen in human cancer. Its single-stranded DNA C-to-U editing activity results in multiple mutagenic outcomes including signature single-base substitution mutations (isolated and clustered), DNA breakage, and larger-scale chromosomal aberrations. APOBEC3A inhibitors may therefore comprise a unique class of anti-cancer agents that work by blocking mutagenesis, slowing tumor evolvability, and preventing detrimental outcomes such as drug resistance and metastasis. Here we reveal the structural basis of competitive inhibition of wildtype APOBEC3A by hairpin DNA bearing 2'-deoxy-5-fluorozebularine in place of the cytidine in the TC substrate motif that is part of a 3-nucleotide loop. In addition, the structural basis of APOBEC3A's preference for YTCD motifs (Y = T, C; D = A, G, T) is explained. The nuclease-resistant phosphorothioated derivatives of these inhibitors have nanomolar potency in vitro and block APOBEC3A activity in human cells. These inhibitors may be useful probes for studying APOBEC3A activity in cellular systems and leading toward, potentially as conjuvants, next-generation, combinatorial anti-mutator and anti-cancer therapies.Item Seven-membered ring nucleobases as inhibitors of human cytidine deaminase and APOBEC3A.(Royal Society of Chemistry, 2023-06-21) Kurup HM; Kvach MV; Harjes S; Jameson GB; Harjes E; Filichev VVThe APOBEC3 (APOBEC3A-H) enzyme family as a part of the human innate immune system deaminates cytosine to uracil in single-stranded DNA (ssDNA) and thereby prevents the spread of pathogenic genetic information. However, APOBEC3-induced mutagenesis promotes viral and cancer evolution, thus enabling the progression of diseases and development of drug resistance. Therefore, APOBEC3 inhibition offers a possibility to complement existing antiviral and anticancer therapies and prevent the emergence of drug resistance, thus making such therapies effective for longer periods of time. Here, we synthesised nucleosides containing seven-membered nucleobases based on azepinone and compared their inhibitory potential against human cytidine deaminase (hCDA) and APOBEC3A with previously described 2'-deoxyzebularine (dZ) and 5-fluoro-2'-deoxyzebularine (FdZ). The nanomolar inhibitor of wild-type APOBEC3A was obtained by the incorporation of 1,3,4,7-tetrahydro-2H-1,3-diazepin-2-one in the TTC loop of a DNA hairpin instead of the target 2'-deoxycytidine providing a Ki of 290 ± 40 nM, which is only slightly weaker than the Ki of the FdZ-containing inhibitor (117 ± 15 nM). A less potent but notably different inhibition of human cytidine deaminase (CDA) and engineered C-terminal domain of APOBEC3B was observed for 2'-deoxyribosides of the S and R isomers of hexahydro-5-hydroxy-azepin-2-one: the S-isomer was more active than the R-isomer. The S-isomer shows resemblance in the position of the OH-group observed recently for the hydrated dZ and FdZ in the crystal structures with APOBEC3G and APOBEC3A, respectively. This shows that 7-membered ring analogues of pyrimidine nucleosides can serve as a platform for further development of modified ssDNAs as powerful A3 inhibitors.Item DL-PPI: a method on prediction of sequenced protein-protein interaction based on deep learning(BioMed Central Ltd, 2023-12) Wu J; Liu B; Zhang J; Wang Z; Li JPURPOSE: Sequenced Protein-Protein Interaction (PPI) prediction represents a pivotal area of study in biology, playing a crucial role in elucidating the mechanistic underpinnings of diseases and facilitating the design of novel therapeutic interventions. Conventional methods for extracting features through experimental processes have proven to be both costly and exceedingly complex. In light of these challenges, the scientific community has turned to computational approaches, particularly those grounded in deep learning methodologies. Despite the progress achieved by current deep learning technologies, their effectiveness diminishes when applied to larger, unfamiliar datasets. RESULTS: In this study, the paper introduces a novel deep learning framework, termed DL-PPI, for predicting PPIs based on sequence data. The proposed framework comprises two key components aimed at improving the accuracy of feature extraction from individual protein sequences and capturing relationships between proteins in unfamiliar datasets. 1. Protein Node Feature Extraction Module: To enhance the accuracy of feature extraction from individual protein sequences and facilitate the understanding of relationships between proteins in unknown datasets, the paper devised a novel protein node feature extraction module utilizing the Inception method. This module efficiently captures relevant patterns and representations within protein sequences, enabling more informative feature extraction. 2. Feature-Relational Reasoning Network (FRN): In the Global Feature Extraction module of our model, the paper developed a novel FRN that leveraged Graph Neural Networks to determine interactions between pairs of input proteins. The FRN effectively captures the underlying relational information between proteins, contributing to improved PPI predictions. DL-PPI framework demonstrates state-of-the-art performance in the realm of sequence-based PPI prediction.Item Enhanced properties of non-starch polysaccharide and protein hydrocolloids through plasma treatment: A review(Elsevier B V, 2023-09-30) Sahraeian S; Rashidinejad A; Niakousari MHydrocolloids are important ingredients in food formulations and their modification can lead to novel ingredients with unique functionalities beyond their nutritional value. Cold plasma is a promising technology for the modification of food biopolymers due to its non-toxic and eco-friendly nature. This review discusses the recent published studies on the effects of cold plasma treatment on non-starch hydrocolloids and their derivatives. It covers the common phenomena that occur during plasma treatment, including ionization, etching effect, surface modification, and ashing effect, and how they contribute to various changes in food biopolymers. The effects of plasma treatment on important properties such as color, crystallinity, chemical structure, rheological behavior, and thermal properties of non-starch hydrocolloids and their derivatives are also discussed. In addition, this review highlights the potential of cold plasma treatment to enhance the functionality of food biopolymers and improve the quality of food products. The mechanisms underlying the effects of plasma treatment on food biopolymers, which can be useful for future research in this area, are also discussed. Overall, this review paper presents a comprehensive overview of the current knowledge in the field of cold plasma treatment of non-starch hydrocolloids and their derivatives and highlights the areas that require further investigation.Item Small-Angle X-ray Scattering (SAXS) Measurements of APOBEC3G Provide Structural Basis for Binding of Single-Stranded DNA and Processivity(MDPI (Basel, Switzerland), 2022-09-06) Barzak FM; Ryan TM; Mohammadzadeh N; Harjes S; Kvach MV; Kurup HM; Krause KL; Chelico L; Filichev VV; Harjes E; Jameson GB; De la Torre JC; Andrei GAPOBEC3 enzymes are polynucleotide deaminases, converting cytosine to uracil on single-stranded DNA (ssDNA) and RNA as part of the innate immune response against viruses and retrotransposons. APOBEC3G is a two-domain protein that restricts HIV. Although X-ray single-crystal structures of individual catalytic domains of APOBEC3G with ssDNA as well as full-length APOBEC3G have been solved recently, there is little structural information available about ssDNA interaction with the full-length APOBEC3G or any other two-domain APOBEC3. Here, we investigated the solution-state structures of full-length APOBEC3G with and without a 40-mer modified ssDNA by small-angle X-ray scattering (SAXS), using size-exclusion chromatography (SEC) immediately prior to irradiation to effect partial separation of multi-component mixtures. To prevent cytosine deamination, the target 2'-deoxycytidine embedded in 40-mer ssDNA was replaced by 2'-deoxyzebularine, which is known to inhibit APOBEC3A, APOBEC3B and APOBEC3G when incorporated into short ssDNA oligomers. Full-length APOBEC3G without ssDNA comprised multiple multimeric species, of which tetramer was the most scattering species. The structure of the tetramer was elucidated. Dimeric interfaces significantly occlude the DNA-binding interface, whereas the tetrameric interface does not. This explains why dimers completely disappeared, and monomeric protein species became dominant, when ssDNA was added. Data analysis of the monomeric species revealed a full-length APOBEC3G-ssDNA complex that gives insight into the observed "jumping" behavior revealed in studies of enzyme processivity. This solution-state SAXS study provides the first structural model of ssDNA binding both domains of APOBEC3G and provides data to guide further structural and enzymatic work on APOBEC3-ssDNA complexes.Item Characterising parameters in Gcn1 relevant for Gcn2 activation : this dissertation is submitted for the degree of Doctor of Philosophy, Biochemistry, Massey University, Auckland, New Zealand. EMBARGOED to 29 March 2026.(Massey University, 2022) Gottfried, SusanneThe protein kinase Gcn2 ("General control non-depressible" 2) is present in virtually all eukaryotic cells assessed thus far, from yeast to humans. Gcn2 supports the cells to cope with nutrient deficiency. Upon amino acid starvation, Gcn2 phosphorylates the alpha subunit of eukaryotic translation factor (eIF2α), which subsequently stimulates the translation of the transcription factor Gcn4. Gcn4 then induces the expression of stress-response genes involved in amino acid biosynthesis. At the same time, intracellular protein synthesis is downregulated. Activation of Gcn2 requires the binding of uncharged tRNA to the HisRS-like domain, which depends on the physical interaction between Gcn2 and Gcn1. Formation of the Gcn1-Gcn2 complex involves direct binding of the N-terminal RWD domain in Gcn2 to the RWD binding domain (RWDBD) in Gcn1. Malfunction of Gcn2 is implicated in various diseases such as cancer and Alzheimer's. By hyper-activating Gcn2, cancer cells take advantage of Gcn2 to satisfy their high nutrition demand. Healthy cells do not critically depend on Gcn2, making Gcn2 a promising target for potential drug development. Understanding Gcn2 function in detail may help identify suitable targets that prevent or treat such diseases. The aim of the study was to gain a deeper understanding of properties in Gcn1 determining binding to Gcn2 and test whether targeting the protein-protein interaction between Gcn1-Gcn2 could be a suitable approach to inhibit Gcn2.--Shortened abstractItem Structure-rheology relationships of protein-polysaccharide complexes at oil/water interfaces : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Physics at Biophysics and Soft Matter Group, School of Fundamental Science, Massey University(Massey University, 2021) Ramamirtham, SashikumarThe complexation of proteins with polysaccharides to form bio-complexes is being utilized in a variety of applications including food formulations, microencapsulation, protein separation and bioactive deliveries. Understanding the impact of these biomolecules on each other with discernment will not only improve our existing usages but also aid in devising newer applications. The duo of beta -lactoglobulin (beta -lg), a surface active globular whey protein, and pectin, a plant-derived polysaccharide, is the model protein-polysaccharide system of this study. Beta -lg and pectin have been reported to undergo complexation driven by electrostatic attraction leading to contrasting interfacial rheological properties depending on the fine structures of the polysaccharide. The aim of this thesis is to understand the role of fine structures of the polysaccharide in protein adsorption and the interfacial film formation. Given that beta -lg is the interfacially active molecule in this study, assemblies of beta -lg at dodecane/water interfaces at pHs 3 and 4, and at different conditions of ionic strength, salt type and temperature were studied. These parameters were tuned to vary the relative amounts of two native species, namely, monomer and its smallest aggregate, the dimer, while the interface was monitored using rheology and tensiometry. Unfolding of beta -lg dimers at the interface triggers the formation of disulfide linkages between the free thiol groups located at cys121 of the monomers. In this way, it is demonstrated here for the first time that beta -lg dimers are the smallest elastic network building unit of the protein. A higher concentration of dimers increases the final interfacial elastic strength of the network. The lack of the elastic film forming ability of beta -lg monomer is attributed to the absence of multiple free thiol groups. Moreover, beta -lg monomer exhibited minimal reduction in interfacial tension akin to a pure buffer solution. This fundamental relation between the quaternary structure of beta -lg and its subsequent interfacial network suggests a possible interfacial role in its biological function. Besides, these results will also be used as control for assessing the behaviors of beta -lg/polysaccharide complexes. In the next phase of this study, transient interfacial rheology of pre-mixed solutions of beta -lg and polysaccharides with different lengths and charge densities at pHs 3 and 4 are presented. It was found that, while the interfacial activity of beta -lg/pectin complexes is dictated by the amount of charge on the polysaccharide, the kinetics of the complexed beta -lg’s adsorption and its subsequent interfacial film formation is largely controlled by the contiguity of the charges on the polysaccharide molecule. Using subphase injection techniques, it is further shown that the structure of the beta -lg in the protein/polysaccharide complex prior to adsorption is the major contributor to the lag time duration before the onset of an elastic film formation. This is exemplified by the contrasting behaviors of beta -lg/pectin complexes with high polysaccharide charge density as compared to beta -lg/pectin complexes with low polysaccharide charge density, where the latter can be used as a one shot delivery system to obtain reinforced oil/water interfaces. It is further proposed that the mechanism by which a polysaccharide molecule reinforces beta -lg interfacial film is by concatenating multiple protein units and establishing cross-links in the aqueous subphase. The final phase of this study presents microrheology measurements of oil/water interfaces laden with beta -lg and beta -lg/polysaccharide complexes. Microrheology further ascertains the viscous nature of beta -lg monomer laden interfaces and the elastic nature of the interfaces with beta -lg dimers. In addition, the presence of heterogeneity in the entangled films made of beta -lg dimers in the form of confinements was also observed. A sharp transition was exhibited from an inelastic to elastic interface occurring around a surface dimer concentration of 56 ng/m2 at pH 3, 15 mM NaCl. Further, a slightly denser interface was observed for almost all the beta -lg/polysaccharide complexes at pH 4. The heterogeneity that was observed at dimeric interfaces was not seen for interfaces with beta -lg/polysaccharide complexes indicating the presence of the polysaccharide molecules beneath the interfacial film. On the whole, this thesis demonstrates the advantages of using of interfacial rheological techniques to tease out the structure-rheology relationships of biomolecules such as proteins and protein/polysaccharide complexes and thereby provide valuable insights about molecular manipulations.Item Modular functionalization of engineered polyhydroxyalkanoate scaffolds : a thesis presented in partial fulfilment of the requirements for degree Doctor of Philosophy in Microbiology at Massey University, New Zealand(Massey University, 2019) Wong, Jin XiangMicrobial polyhydroxyalkanoates (PHAs) are spherical polyesters that are naturally synthesized in vivo by a variety of microorganisms as carbon and energy reserves under imbalanced nutrition environments. Notably, PHA particles can be functionalized by the genetic modification of surface-exposed PHA-associated proteins, e.g. PHA synthase (PhaC), and this approach has led to multiple successful proof-of-concept demonstrations for bio-technology applications. However, current recombinant methods to functionalize PHAs require a certain biological complexity, such as simultaneous polyester and protein synthesis within a single cell. The less defined nature of this technology means limited control over particle morphology and surface functionalization. Seeking to overcome these limitations, the work presented in this thesis is to introduce the concept of modularity to the PHA particle technology, by merging the PHA particle technology with Tag/Catcher protein ligation systems. The Catcher domain can rapidly form a covalent bond with its pairing short peptide tag in a site-specific manner, without the need of additional reagents nor enzymes at broad ligation conditions. The SpyTag/SpyCatcher pair was merged recombinantly with PHA particle technology, where the resulting SpyCatcher-coated PHA particles were able to immobilize various SpyTagged proteins in vitro in a tunable manner and remained functional. This thesis further demonstrates several functionalization processes to streamline this modular strategy by assessing the possibility of whether non-purified SpyTagged proteins could ligate with the PHA particles in complex environments. The results demonstrated that SpyCatcher-coated PHA particles could be functionalized adequately using two of the proposed methods. To further expand the design space of this generic modular platform towards programmable multi-functionalization, various bimodular PHA particles utilizing alternative Tag/Catcher pairs (e.g. SdyTag/SdyCatcher and SnoopTag/Snoop-Catcher pairs) were designed and studied. One of the constructs resulted in the simultaneous multi-functionalization of plain PHA particles in one-step with two differently tagged proteins in in vivo and ex vivo reaction conditions. This work presents the modular design of PHA scaffolds and several streamlined manufacturing processes to the production of task-specific designer PHAs. Introducing the concept of modularity to the PHA particle technology enabled better control of particle uniformity, reproducibility, and immobilized protein density while remaining functional. These concepts should be broadly applicable to the design and manufacture of advanced functional materials for industrial applications.Item Unravelling the molecular contributions to collagen higher order structure : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Manawatu, New Zealand(Massey University, 2019) Visser, Danielle ReneeAbnormal levels of cross-linking in fibrillar collagen strands have been shown to cause a number of human and animal diseases. Cross-linking is a vital step in fibrillogenesis and contributes greatly to the structural integrity of collagenous tissues. Conversely, defects in cross-link formation can significantly alter fibrillar organisation and lead to pathogenesis. Because collagen cross-links form on collagen-specific hydroxylated lysine residues, an understanding of the link between hydroxylysine and cross-link concentrations is needed to determine whether the level of hydroxylysine, the stereochemistry of these hydroxylysine residues, or other post-translational modifications such as glycosylation affect the level of cross-linking in tissue. While some research has been done to elucidate the connection between the two in different tissue types from the same animal, little has been undertaken to relate hydroxylation and glycosylation of lysine and hydroxylysine to the concentration and types of cross-links in different species. Furthermore, no research has been done to compare the relative distribution of diastereomers of hydroxylysine even within the same species. In order to make a valid comparison, collagen needs to be purified from skin to a high degree and separated into different collagen types and sub- structures as much as possible. To achieve this, the extraction and purification of collagen from the skins of four different mammalian species displaying different skin tensile strengths has been optimised. Different extraction methods were used to prevent the loss of specific features of the collagens that were characterised that may otherwise be lost. Amino acid analysis revealed that while the ratios of the two hydroxylysine diastereomers differed between different animals and extraction methods, the differences were not significant. Mass spectral analysis of cross-links showed that goat skin differed from the other three animals in its cross-link profile. Amino acid analysis combined with mass spectral analysis revealed that on average 70% of proline residues were hydroxylated, a figure much higher than previously thought. Mass spectral analysis also revealed that there are some differences between the glycosylation pattern of different animals, and the ratios of the different types of collagen which are extracted from each animal. While these findings need to be confirmed, they challenge some long held beliefs about the collagen molecule and provide a firm foundation for future work.
