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Item Nphos: Database and Predictor of Protein N-phosphorylation.(Oxford University Press, 2024-04-10) Zhao M-X; Ding R-F; Chen Q; Meng J; Li F; Fu S; Huang B; Liu Y; Ji Z-L; Zhao Y; Xue YProtein N-phosphorylation is widely present in nature and participates in various biological processes. However, current knowledge on N-phosphorylation is extremely limited compared to that on O-phosphorylation. In this study, we collected 11,710 experimentally verified N-phosphosites of 7344 proteins from 39 species and subsequently constructed the database Nphos to share up-to-date information on protein N-phosphorylation. Upon these substantial data, we characterized the sequential and structural features of protein N-phosphorylation. Moreover, after comparing hundreds of learning models, we chose and optimized gradient boosting decision tree (GBDT) models to predict three types of human N-phosphorylation, achieving mean area under the receiver operating characteristic curve (AUC) values of 90.56%, 91.24%, and 92.01% for pHis, pLys, and pArg, respectively. Meanwhile, we discovered 488,825 distinct N-phosphosites in the human proteome. The models were also deployed in Nphos for interactive N-phosphosite prediction. In summary, this work provides new insights and points for both flexible and focused investigations of N-phosphorylation. It will also facilitate a deeper and more systematic understanding of protein N-phosphorylation modification by providing a data and technical foundation. Nphos is freely available at http://www.bio-add.org/Nphos/ and http://ppodd.org.cn/Nphos/.Item GCN2 in Viral Defence and the Subversive Tactics Employed by Viruses(Elsevier Ltd, 2024-07-01) Gibbs VJ; Lin YH; Ghuge AA; Anderson RA; Schiemann AH; Conaglen L; Sansom BJM; da Silva RC; Sattlegger E; Freed EOThe recent SARS-CoV-2 pandemic and associated COVID19 disease illustrates the important role of viral defence mechanisms in ensuring survival and recovery of the host or patient. Viruses absolutely depend on the host's protein synthesis machinery to replicate, meaning that impeding translation is a powerful way to counteract viruses. One major approach used by cells to obstruct protein synthesis is to phosphorylate the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α). Mammals possess four different eIF2α-kinases: PKR, HRI, PEK/PERK, and GCN2. While PKR is currently considered the principal eIF2α-kinase involved in viral defence, the other eIF2α-kinases have also been found to play significant roles. Unsurprisingly, viruses have developed mechanisms to counteract the actions of eIF2α-kinases, or even to exploit them to their benefit. While some of these virulence factors are specific to one eIF2α-kinase, such as GCN2, others target all eIF2α-kinases. This review critically evaluates the current knowledge of viral mechanisms targeting the eIF2α-kinase GCN2. A detailed and in-depth understanding of the molecular mechanisms by which viruses evade host defence mechanisms will help to inform the development of powerful anti-viral measures.Item A role for β-catenin in diet-induced skeletal muscle insulin resistance.(Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society, 2023-02-17) Masson SWC; Dissanayake WC; Broome SC; Hedges CP; Peeters WM; Gram M; Rowlands DS; Shepherd PR; Merry TLA central characteristic of insulin resistance is the impaired ability for insulin to stimulate glucose uptake into skeletal muscle. While insulin resistance can occur distal to the canonical insulin receptor-PI3k-Akt signaling pathway, the signaling intermediates involved in the dysfunction are yet to be fully elucidated. β-catenin is an emerging distal regulator of skeletal muscle and adipocyte insulin-stimulated GLUT4 trafficking. Here, we investigate its role in skeletal muscle insulin resistance. Short-term (5-week) high-fat diet (HFD) decreased skeletal muscle β-catenin protein expression 27% (p = 0.03), and perturbed insulin-stimulated β-cateninS552 phosphorylation 21% (p = 0.009) without affecting insulin-stimulated Akt phosphorylation relative to chow-fed controls. Under chow conditions, mice with muscle-specific β-catenin deletion had impaired insulin responsiveness, whereas under HFD, both mice exhibited similar levels of insulin resistance (interaction effect of genotype × diet p < 0.05). Treatment of L6-GLUT4-myc myocytes with palmitate lower β-catenin protein expression by 75% (p = 0.02), and attenuated insulin-stimulated β-catenin phosphorylationS552 and actin remodeling (interaction effect of insulin × palmitate p < 0.05). Finally, β-cateninS552 phosphorylation was 45% lower in muscle biopsies from men with type 2 diabetes while total β-catenin expression was unchanged. These findings suggest that β-catenin dysfunction is associated with the development of insulin resistance.Item Histone H1 phosphorylation during mitosis : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand(Massey University, 2016) Bond, Sarah DHistone H1 phosphorylation is important for the regulation of high order chromosome organisation during mitosis. One of these phosphorylation sites in the linker histone subtype H1.4 is shown here to be phosphorylated by Aurora B kinase, a master regulator of mitosis. Altered phosphorylation of H1.4 on this phosphorylation site at serine 27 illustrated the significance of the timing of this phosphorylation. When serine 27 of H1.4 is mutated to prevent this phosphorylation chromosome congression to the equatorial plate during metaphase is hindered. In contrast, in the presence of the constitutive H1.4 serine 27 phosphorylation mimic, bridging and lagging chromosomes occurred, leading to a corresponding increase in the proportion of cells with a micronucleus. These phenotypes could be brought about through disruption of the Heterochromatin protein 1 family members bound to the adjacent methylated lysine. Such aberrations during mitosis can lead to genetic instability and ultimately aneuploidy, a hallmark of cancer. With the frequently reported over-expression of Aurora B in cancer this shows another mechanism in which this kinase, via histone H1.4 phosphorylation, can push a cell toward malignancy. Another important mitotic kinase, Cyclin dependent kinase 1 together with cyclin B, is responsible for the hyperphosphorylation of histone H1.4 during mitosis; which is required for condensing the cells genetic information into highly compact metaphase chromosomes. This vital mitotic event ensures the faithful transmission of the duplicated DNA into the dividing daughter cells. The mechanisms through which histone H1 hyperphosphorylation contribute to chromosome condensation are poorly understood. One mechanism through which this may occur is via the recruitment of condensation factors such as the condensins or Topoisomerase II. Here the interaction between the Condensin I subunit, CAPD2, and histone H1.4 is explored. CAPD2 interacts with the two most prominent linker histone subtypes, H1.4 and H1.2, through their C-terminal tails. H1.4 and CAPD2 can interact in vitro whilst each is phosphorylated by cyclin dependent kinase as they are during mitosis, in a manner dependent on RNA. Overall, these results indicate that histone H1.4 is a vital component of higher order chromatin and its phosphorylation is essential for the normal progression through mitosis.Item ATM and p400 : characterisation of a novel interaction between a DNA repair enzyme and a chromatin remodeler : a thesis presented to Massey University in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry(Massey University, 2014) Smith, Rebecca JaneThe ability to maintain genomic integrity prevents unrestricted cell proliferation and the progression of cancer. DNA repair pathways such as the DNA double-strand break (DSB) response are essential in maintaining this integrity. This system requires activation of the serine/threonine kinase ataxia telangiectasia mutated (ATM) through acetylation by TIP60, a histone acetyl transferase, and subsequent ATM autophosphorylation. During DNA repair, activated ATM phosphorylates the histone variant H2AX several kilobases either side of the break site. This phosphorylation acts a signal for additional repair proteins and chromatin remodeling complexes which repairs DNA. In a previous study, H2AX phosphorylation was induced through the over expression of TIP60 or the SWI3-ADA2-N-CoR-TFIIIB (SANT) domain of p400. It was hypothesised that over expressed TIP60 or SANT domain was able to sequester a putative negative regulator from the ATM-TIP60 complex and artificially induce activation. This study aimed to investigate if a single domain of TIP60 or if a single helix from the three helix SANT domain was responsible for the activation of the ATM-TIP60 complex. Here, the ability of the chromo domain and zinc domain of TIP60 individually and the combined zincHat domain of TIP60 to induce H2AX phosphorylation as well as three helix deletion mutants of the SANT domain of p400 was examined. While all constructs were able to be expressed in human cell lines, the induction of H2AX was variable and non-reproducible. ATM belongs to the phosphatidylinositol 3-kinase-related kinase family (PIKK). Members of the PIKK family show domain homology, where the domain of one protein is replaced with the homologous domain of another member and the function of the protein is not altered. As p400 has been previously shown to interact with TIP60 and also Transformation/transcription domain-associated protein (TRRAP), a member of the PIKK family, it was hypothesised that p400 could interact with ATM (which also interacts with TIP60). This study confirms this novel interaction between ATM and p400 through the use of co-immunoprecipitation and protein localisation using confocal microscopy. This study provides a platform to further investigate the involvement of an ATM-p400 complex during DNA repair.