Investigating the role of SUMOylation of eukaryotic translation initiation factors

Dysregulation of translation has a direct effect on growth control in mammalian cells. The initiation step of translation is an important point of control of gene expression and a rate-limiting step for protein synthesis. Initiation requires initiation factors (eIFs) that are important for the activation of both mRNA and the recruitment of ribosomal subunits. Previously, post-translational modifications (PTMs), such as phosphorylation, have been shown to be an important part in fine-tuning translation. SUMOylation is another PTM that affects a number of fundamental cellular processes, including the response to DNA damage, metabolic regulation and protein trafficking. Many eIFs have been identified in proteomic screens as SUMOylated targets, but to date most of these modifications have not been confirmed. Preliminary work from the Watts lab indicated that some eIFs co-purified with the S. pombe SUMO protease Ulp2. The aim of my project was to determine whether components of the eIF4F complex are SUMOylated and to initiate studies to investigate the role of this modification. The first results chapter investigates eIF4G, eIF3h and Sla1 (the La protein homologue) and demonstrates that eIF4G and Sla1, but not eIF3h, are SUMOylated in S. pombe. These experiments were then extended to mammalian cells. The effects of stress conditions on protein synthesis and SUMOylation in a range of cell lines were first analysed. SUMO localisation was altered in response to sodium arsenite (AR) and ionising radiation (IR). In most cell types tested, for example, after IR treatment, SUMO1 went to nuclear foci in HeLa cells, but was more abundant in the cytoplasm following exposure to AR. Next, in vivo and in vitro SUMOylation assays were used to demonstrate that mammalian eIF4G and eIF4A are both SUMOylated. Mass spectrometric analysis identified the SUMOylation sites in eIF4G, as K1386 and K1588. Those of eIF4AI and eIF4AII are K225 and K226, respectively. Mutated eIF4AII was introduced into cells to investigate the role of SUMOylation of this factor. Colocalisation of eIF4A/eIF4G and SUMO1 shows that, in AR-treated cells, SUMO1 colocalises with eIF4A and eIF4G in the cytoplasmic stress granules, especially at their edges. In contrast, in IR-treated cells, the colocalisation of eIF4G/eIF4A with SUMO1 is much more in the nucleus, compared to that in untreated cells, suggesting that eIF4G/eIF4A and SUMO1 may have a cellular role in some aspects in response to AR and IR

Sumoylation of eIF4A2 affects stress granule formation

Regulation of protein synthesis is crucial for cells to maintain viability and to prevent unscheduled proliferation that could lead to tumorigenesis. Exposure to stress results in stalling of translation, with many translation initiation factors, ribosomal subunits and mRNAs being sequestered into stress granules or P bodies. This allows the re-programming of the translation machinery. Many aspects of translation are regulated by post-translational modification. Several proteomic screens have identified translation initiation factors as targets for sumoylation, although in many cases the role of this modification has not been determined. We show here that eIF4A2 is modified by SUMO, with sumoylation occurring on a single residue (K226). We demonstrate that sumoylation of eIF4A2 is modestly increased in response to arsenite and ionising radiation, but decreases in response to heat shock or hippuristanol. In arsenite-treated cells, but not in hippuristanol-treated cells, eIF4A2 is recruited to stress granules, suggesting sumoylation of eIF4A2 correlates with its recruitment to stress granules. Furthermore, we demonstrate that the inability to sumoylate eIF4A2 results in impaired stress granule formation, indicating a new role for sumoylation in the stress response

The S. pombe translation initiation factor eIF4G is sumoylated and associates with the SUMO protease Ulp2

SUMO is a small post-translational modifier, that is attached to lysine residues in target proteins. It acts by altering proteinprotein interactions, protein localisation and protein activity. SUMO chains can also act as substrates for ubiquitination, resulting in proteasome-mediated degradation of the target protein. SUMO is removed from target proteins by one of a number of specific proteases. The processes of sumoylation and desumoylation have well documented roles in DNA metabolism and in the maintenance of chromatin structure. To further analyse the role of this modification, we have purified protein complexes containing the S. pombe SUMO protease, Ulp2. These complexes contain proteins required for ribosome biogenesis, RNA stability and protein synthesis. Here we have focussed on two translation initiation factors that we identified as co-purifying with Ulp2, eIF4G and eIF3h. We demonstrate that eIF4G, but not eIF3h, is sumoylated. This modification is increased under conditions that produce cytoplasmic stress granules. Consistent with this we observe partial co-localisation of eIF4G and SUMO in stressed cells. Using HeLa cells, we demonstrate that human eIF4GI is also sumoylated; in vitro studies indicate that human eIF4GI is modified on K1368 and K1588, that are located in the C-terminal eIF4A- and Mnk-binding sites respectively

Multifunctional core‐shell electrospun nanofibrous fabrics of poly(vinyl alcohol)/silk sericin (core) and poly(lactide‐ co ‐glycolide) (shell)

Core–shell fibres (CSFs) offer a simple route to multifunctional hybrid materials for a wide range of applications. Herein, we report the design of a core–shell electrospun nanofibrous fabric containing a hydrophilic core and hydrophobic shell. CSFs were fabricated for the first time from poly(vinyl alcohol)/silk sericin (from silk cocoons) as the core and poly(lactide-co-glycolide) as the shell. The core serves as a potential carrier for water-soluble bioactive agents, and the shell works as a barrier to prevent premature release of water-soluble agents from the core. The effect of the molecular weight of poly(lactide-co-glycolide) and the loading of silk sericin on the morphology of fibres was studied. The parameters that significantly influence the core–shell electrospinning process were studied to elucidate the most effective conditions to create our multifunctional nanofibrous fabrics with smooth fibre morphology (diameters in the range 800–1300 nm) and low bead formation. Our CSFs were shown to degrade in saline buffer solution (pH 7.4) and were readily rendered with anti-bacterial properties against Staphylococcus aureus and Escherichia coli by the post-spinning deposition of silver nanoparticles (AgNPs, 40 nm diameter) or cinnamon essential oil (CEO). The fibres were non-toxic to normal human dermal fibroblast cell lines, as the cells were shown to attach and proliferate on CSFs, CSF/AgNPs and CSF/CEO with good cell tolerance for 72 h of incubation. These smart multifunctional CSFs show great potential towards smart delivery fabrics/dressings capable of carrying water-soluble bioactive agents surrounded by a protective, but degradable, antibacterial shell to guard the cargo for more effective controlled release

Weighing up the possibilities: controlling translation by ubiquitylation and sumoylation

Regulation of protein synthesis is of fundamental importance to cells. It has a critical role in the control of gene expression, and consequently cell growth and proliferation. The importance of this control is supported by the fact that protein synthesis is frequently upregulated in tumor cells. The major point at which regulation occurs is the initiation stage. Initiation of translation involves the interaction of several proteins to form the eIF4F complex, the recognition of the mRNA by this complex, and the subsequent recruitment of the 40S ribosomal subunit to the mRNA. This results in the formation of the 48S complex that then scans the mRNA for the start codon, engages the methionyl-tRNA and eventually forms the mature 80S ribosome which is elongationcompetent. Formation of the 48S complex is regulated by the availability of individual initiation factors and through specific protein-protein interactions. Both of these events can be regulated by post-translational modification by ubiquitin or Ubls (ubiquitin-like modifiers) such as SUMO or ISG15. We provide here a summary of translation initiation factors that are modified by ubiquitin or Ubls and, where they have been studied in detail, describe the role of these modifications and their effects on regulating protein synthesis

Investigating Rice Blast Resistance Gene Distribution among Landrace Rice Varieties in Lower Northern Thailand for Improving Rice Cultivars

Rice blast disease caused by the fungus Pyricularia oryzae is considered as one of the severe diseases, leading to reduce tremendous rice productivity in its cultivated areas of Thailand. Due to the rapid evolution and high genetic diversity of the pathogen, the innate rice resistance (R) genes associated with defense mechanisms are significantly considered as the most important for rice breeding program to create new rice varieties, resistant to blast disease. This study aimed to investigate the rice blast R genes (Pi9, Pib, and Pi-ta) in 98 landrace rice germplasms collected from three different provinces in lower northern Thailand, Phichit (PCT), Phitsanulok (PLK), and Sukhothai (STI) through PCR assay. The results showed that the Pi-ta gene was presented in 29 different varieties, making it the most widespread, whereas the Pi9 and Pib genes were found in 28 and 25 varieties, respectively. The distribution percentage of studied genes in PLK and STI germplasms is higher than in PCT germplasms. Interestingly, only eight landrace rice varieties (varieties no. 46, 47, 48, 51, 66, 76, 81, and 90) collected from PLK and STI germplasms contain all of these three resistance genes. This finding provided the genetic information and diversity of the R genes across landrace rice varieties in the lower north of Thailand. Moreover, these R genes could be useful as genetic resources for rice improvement with resistance to blast disease through breeding program in the future

Weighing up the possibilities: controlling translation by ubiquitylation and sumoylation

Regulation of protein synthesis is of fundamental importance to cells. It has a critical role in the control of gene expression, and consequently cell growth and proliferation. The importance of this control is supported by the fact that protein synthesis is frequently upregulated in tumor cells. The major point at which regulation occurs is the initiation stage. Initiation of translation involves the interaction of several proteins to form the eIF4F complex, the recognition of the mRNA by this complex, and the subsequent recruitment of the 40S ribosomal subunit to the mRNA. This results in the formation of the 48S complex that then scans the mRNA for the start codon, engages the methionyl-tRNA and eventually forms the mature 80S ribosome which is elongationcompetent. Formation of the 48S complex is regulated by the availability of individual initiation factors and through specific protein-protein interactions. Both of these events can be regulated by post-translational modification by ubiquitin or Ubls (ubiquitin-like modifiers) such as SUMO or ISG15. We provide here a summary of translation initiation factors that are modified by ubiquitin or Ubls and, where they have been studied in detail, describe the role of these modifications and their effects on regulating protein synthesis

Green fabrication route of robust, biodegradable silk sericin and poly(vinyl alcohol) nanofibrous scaffolds

Silk sericin (SS) has been extensively used to fabricate scaffolds for tissue engineering. However, due to its inferior mechanical properties, it has been found to be a poor choice of material when being electrospun into nanofibrous scaffolds. Here, SS has been combined with poly(vinyl alcohol) (PVA) and electrospun to create scaffolds with enhanced physical properties. Crucially, these SS/PVA nanofibrous scaffolds were created using only distilled water as a solvent with no added crosslinker in an environmentally friendly process. Temperature has been shown to have a marked effect on the formation of the SS sol–gel transition and thus influence the final formation of fibers. Heating the spinning solutions to 70 °C delivered nanofibers with enhanced morphology, water stability and mechanical properties. This is due to the transition of SS from β-sheets into random coils that enables enhanced molecular interactions between SS and PVA. The most applicable SS/PVA weight ratios for the formation of nanofibers with the desired properties were found to be 7.5/1.5 and 10.0/1.5. The fibers had diameters ranging from 60 to 500 nm, where higher PVA and SS concentrations promoted larger diameters. The crystallinity within the fibers could be controlled by manipulation of the balance between PVA and SS loadings. In vitro degradation (in phosphate buffer solution, pH 7.4 at 37 °C) was 30–50% within 42 days and fibers were shown to be nontoxic to skin fibroblast cells. This work demonstrates a new green route for incorporating SS into nanofibrous fabrics, with potential use in biomedical applications

Hemocompatibility Evaluation of Thai Bombyx mori Silk Fibroin and Its Improvement with Low Molecular Weight Heparin Immobilization

Bombyx mori silk fibroin (SF), from Nangnoi Srisaket 1 Thai strain, has shown potential for various biomedical applications such as wound dressing, a vascular patch, bone substitutes, and controlled release systems. The hemocompatibility of this SF is one of the important characteristics that have impacts on such applications. In this study, the hemocompatibility of Thai SF was investigated and its improvement by low molecular weight heparin (LMWH) immobilization was demonstrated. Endothelial cell proliferation on the SF and LMWH immobilized SF (Hep/SF) samples with or without fibroblast growth factor-2 (FGF-2) was also evaluated. According to hemocompatibility evaluation, Thai SF did not accelerate clotting time, excess stimulate complement and leukocyte activation, and was considered a non-hemolysis material compared to the negative control PTFE sheet. Platelet adhesion of SF film was comparable to that of the PTFE sheet. For hemocompatibility enhancement, LMWH was immobilized successfully and could improve the surface hydrophilicity of SF films. The Hep/SF films demonstrated prolonged clotting time and slightly lower complement and leukocyte activation. However, the Hep/SF films could not suppress platelet adhesion. The Hep/SF films demonstrated endothelial cell proliferation enhancement, particularly with FGF-2 addition. This study provides fundamental information for the further development of Thai SF as a hemocompatible biomaterial

Hydroquinine Inhibits the Growth of Multidrug-Resistant <i>Pseudomonas aeruginosa</i> via the Suppression of the Arginine Deiminase Pathway Genes

Hydroquinine has antimicrobial potential with demonstrated activity against several bacteria, including multidrug-resistant (MDR) P. aeruginosa reference strains. Despite this, there is limited evidence confirming the antibacterial activity of hydroquinine against clinical isolates and the underlying mechanism of action. Here, we aimed to investigate the antibacterial effect of hydroquinine in clinical P. aeruginosa strains using phenotypic antimicrobial susceptibility testing and synergistic testing. In addition, we examined the potential inhibitory mechanisms against MDR P. aeruginosa isolates using informatic-driven molecular docking analysis in combination with RT-qPCR. We uncovered that hydroquinine inhibits and kills clinical P. aeruginosa at 2.50 mg/mL (MIC) and 5.00 mg/mL (MBC), respectively. Hydroquinine also showed partial synergistic effects with ceftazidime against clinical MDR P. aeruginosa strains. Using SwissDock, we identified potential interactions between arginine deiminase (ADI)-pathway-related proteins and hydroquinine. Furthermore, using RT-qPCR, we found that hydroquinine directly affects the mRNA expression of arc operon. We demonstrated that the ADI-related genes, including the arginine/ornithine antiporter (arcD) and the three enzymes (arginine deiminase (arcA), ornithine transcarbamylase (arcB), and carbamate kinase (arcC)), were significantly downregulated at a half MIC of hydroquinine. This study is the first report that the ADI-related proteins are potential molecular targets for the inhibitory effect of hydroquinine against clinically isolated MDR P. aeruginosa strains