1,720,967 research outputs found
Investigating the role of eukaryotic initiation factor 5B (eIF5B) in oral squamous cell carcinoma
No full textOral squamous cell carcinoma (OSCC) is a common malignancy of the mucosal epithelium affecting ~600,000 patients a year. Patient prognosis remains poor despite improvements in the therapeutic regime. Therefore, new therapeutic targets must be identified that improve the current standard of care. Regulation of mRNA translation plays a critical role in oncogenesis and cancer progression. Particularly, the IRES-mediated non-canonical translation of distinct mRNAs has been implicated in tumorigenesis. Eukaryotic initiation factor 5B (eIF5B) is a key factor that drives IRES-mediated translation of distinct anti-apoptotic proteins and is implicated in the pathophysiology of several malignancies. Single-cell RNAseq data analysis demonstrated that EIF5B is predominantly expressed in cancer cells compared to other cells in the tumor microenvironment. Further bioinformatic analyses revealed that higher EIF5B mRNA is correlated with poor patient prognosis for OSCC patients. Therefore, we aimed to establish the pre-clinical rationale for eIF5B as a therapeutic target for OSCC. Cell viability data suggested that RNAi-mediated eIF5B depletion significantly increased OSCC cell death under TRAIL treatment. eIF5B depletion also resulted in decreased levels of multiple antiapoptotic proteins. Bromodeoxyuridine (BrdU) incorporation, invasion, and wound healing assays suggested eIF5B depletion hinders proliferation, invasion and migration phenotypes, respectively. Western blot analysis revealed that proteins involved in ERK and NF-κΒ signalling, VEGF and HIF-1α, decreased upon eIF5B depletion. eIF5B depletion also resulted in the decrease of angiogenic biomarkers and endothelial tube formation, suggesting a role of eIF5B depletion in decreasing the angiogenic capability of OSCC cells. Stable eIF5B depletion was achieved with the use of shRNA. Under these conditions, eIF5B depletion increased cell death in the presence of cisplatin. Decreased invasion phenotypes were also observed using shRNA-mediated knockdown, setting up the pipeline to transition experiments into preclinical mouse models. Thus, my work has a strong potential to establish eIF5B as a therapeutic target for OSCC treatment
Establishing and optimizing a pipeline for using a combined multi-omic approach to assessing the role of eIF5B in glioblastoma cells
Get PDFTranslation is an important and highly regulated process. Specifically, translation initiation is of interest when considering the ways in which cells respond to treatments and environmental changes. Previously, we demonstrated that eIF5B is responsible for the translation of a subset of mRNAs encoding anti-apoptotic proteins which regulated the sensitivity of U343’s resistance to TRAIL. Through this project, I have established an important multi-omics pipeline for investigating the impact of eIF5B on glioblastoma cells. By utilizing this pipeline, I have demonstrated that both TRAIL and eIF5B individually affected the glioblastoma cell metabolome, and in combination revealed further changes. Specifically, these treatment conditions produced robust shifts in metabolites such as myo-inositol. I have successfully prepared sequencing-ready RNA libraries for Ribo-seq with corresponding RNA for RNA-seq. This research will provide our lab the opportunity to expand my research on eIF5B through this pipeline as I have demonstrated that eIF5B-modulated metabolites are very cancer-relevant
Regulation of polyhydroxyalkanoic acid synthase by riboswitch in Cupriavidus necator
Get PDFRiboswitches provide an opportunity for inducible gene expression at the transcriptional and translational levels. The riboswitches utilized in this thesis function by creating secondary mRNA structures that sequesters the ribosome binding site (RBS), preventing translation. This secondary structure can bind to theophylline, causing the mRNA to refold, exposing the RBS and allowing translation to occur. Riboswitches are currently underutilized, tested in only a few model organisms and primarily explored through reporter genes for proof-of-concept. This thesis demonstrates the functionality of theophylline riboswitches in the industrially relevant bacteria Cupriavidus necator, and for inducible control of the industrially relevant enzyme polyhydroxyalkanoic acid synthase (phaC), both of which have previously not been demonstrated. This thesis tests the applicability of the riboswitch in industry-relevant bacteria and addresses a significant gap in the knowledge about riboswitch functionality
Elucidating the role of eukaryotic initiation factor 5B (eIF5B) in non-canonical translation initiation
Get PDFCap-dependent translation drives the global synthesis of proteins. Under stress conditions global protein production is attenuated, yet the translation of ATF4 is upregulated through uORF- mediated translation initiation. eIF5B has been shown to deliver initiator-tRNA during non-canonical translation initiation. As such, we defined the role of eIF5B in the non-canonical translation of ATF4, and p27. Through polysome profiling and luciferase reporter assays, we confirmed that eIF5B facilitates uORF2-mediated repression of ATF4 translation. We determined that eIF5B has transcriptome-wide effects on signaling pathways, verifying activation of the JNK arm of the MAPK pathway and upregulation of dyskerin. I investigated the role eIF5B has in regulation of p27, and suggest that the mechanism is IRES-dependent. This study furthers the understanding into mechanisms of alternative translation initiation, which is critical to gene expression regulation
Regulation of mRNA translation by eIF5B in head and neck squamous cell carcinoma (HNSCC)
Get PDFStudies have demonstrated that eIF5B promotes cap-independent translation of the anti-apoptotic factor, XIAP, under stress conditions. Also, eIF5B depletion results in the decreased levels of several pro-survival proteins, thus sensitizing glioblastoma cells to TRAIL-induced apoptosis. Here, we have shown high EIF5B mRNA expression in HNSCC was associated with poor patients’ survival. Further, to assess the impact of eIF5B on HNSCC biology, we depleted eIF5B in three HNSCC cell lines. We also show that eIF5B depletion enhanced the sensitivity of Cal-33 and UM-SCC-29 cells to TRAIL-induced apoptosis. Further, we show the levels of anti-apoptotic proteins Bcl-xL, cIAP1, cFLIPs, and XAIP were decreased upon eIF5B depletion in Cal-33 suggesting the role of eIF5B in the translation of these proteins which was further supported by our polysome profiling data. Altogether our findings suggest that eIF5B may play an important role in the translation of mRNAs encoding key anti-apoptotic proteins that evade apoptosis
Characterizing the interaction between PDCD4 and eIF3 with respect to translation regulation
Get PDFProgrammed cell death protein 4 (PDCD4) inhibits IRES-mediated translation of anti-apoptotic proteins such as XIAP. PDCD4 was shown to directly interact with the XIAP IRES element and inhibit translation initiation. Additionally, our lab reported that a eukaryotic initiation factor, eIF3 interacts with the XIAP IRES to facilitate ribosome recruitment. Interestingly, the activity of PDCD4 and eIF3 are regulated by common regulatory kinases called S6K1 and 2. Therefore, to investigate the possibility of interaction between PDCD4 and eIF3 as well as their co-regulation by S6K1 and 2, I have performed co-immunoprecipitation assays in glioblastoma and S6K double knockout MEFs. The results of in cellulo assays demonstrate RNA-independent PDCD4-eIF3 interactions. In addition, eIF3F, one of the 13 eIF3 subunits has been demonstrated to interact directly with PDCD4. This study suggests that the interaction of PDCD4 with eIF3F may have a role in regulating global and/or transcript-specific translation.SG
Prevalence and antimicrobial susceptibility of Mycoplasma bovis and Pasteurella multocida isolated from Albertan feedlot cattle
No full textBovine respiratory disease (BRD) is a significant health problem for the Canadian feedlot industry. While often polymicrobial in nature, Mycoplasma bovis and Pasteurella multocida are considered important respiratory pathogens for BRD. This study aimed to evaluate the prevalence and antimicrobial resistance of M. bovis and P. multocida isolated from Albertan feedlot cattle that were sampled 8 years apart. In the first study, nasopharyngeal swabs from cattle sampled at feedlot entry and after 60 days on feed were collected in 2008-2009 (Cohort 1). In a second study conducted in 2015-2016 (Cohort 2), nasopharyngeal swabs were collected from cattle diagnosed with BRD and matching healthy controls. Trans-tracheal samples were also collected from Cohort 2 cattle for M. bovis evaluation. For Cohort 1, the prevalence of M. bovis was lower in cattle at entry compared to when the same individuals were sampled ≥60 days later (P<0.05). For Cohort 2, the prevalence of M. bovis was greater in both nasopharyngeal and tracheal samples from cattle diagnosed with BRD, compared to controls (P<0.05). Similarly, P. multocida was more frequently isolated from the nasopharynx of BRD cases. Antimicrobial-resistant patterns changed broadly over the 8-year time period with resistance being lower (P<0.05) in Cohort 1 bacteria for florfenicol and tulathromycin. When evaluated for resistance genes by PCR, the majority (98%) of oxytetracycline-resistant isolates carried tet(H) while only 16 (15%) out of 106 tulathromycin-resistant isolates from Cohort 2 carried a known macrolide resistance gene. The genomes of nine tulathromycin-resistant isolates were sequenced, leading to the identification of a conserved gene cluster that was present in all isolates with tulathromycin-resistance but unknown macrolide resistance genes. One of the genes was a novel putative methylase, which doubled the minimum inhibitory concentration against tulathromycin when cloned into Escherichia coli. This study showed that macrolide resistance in M. bovis and P. multocida increased over an 8-year span, coinciding with the approval and adoption of tulathromycin to prevent BRD in Canadian cattle. Additionally, a novel putative macrolide resistance gene was identified and shown to be widespread in P. multocida from the feedlots enrolled in this study. The rapid development of resistance to a newly used antimicrobial indicates the need to reserve essential antimicrobials for the treatment of cattle, to maintain their efficacy
Mathematical modeling of eIF5B-mediated non-canonical translation initiation as a chemotherapeutic target
Get PDFGlioblastoma is an extremely aggressive brain cancer that has a median survival time of 15 months, and a 95% mortality rate within 5 years. Standard-of-care therapy has not changed in over 15 years, and has unfortunately been limited in success. Glioblastomas tend to grow rapidly, creating hypoxic conditions within their cells. Under these conditions, the alpha subunit of eIF2 is phosphorylated, resulting in its inability to deliver the initiator tRNA to the ribosome during canonical translation initiation. In healthy cells, if the stress persists and is not alleviated, this may trigger a form of programmed cell death known as apoptosis. However, cancer cells exploit a non-canonical translation initiation pathway that replaces eIF2 with eIF5B to deliver the tRNAi to the pre-initiation complex. Some anti-apoptotic proteins are translated using this pathway, such as X-linked inhibitor of apoptosis (XIAP). The XIAP mRNA contains an IRES element which allows it to be translated using this non-canonical pathway. XIAP is up-regulated in glioblastoma cells, and therefore the eIF5B-mediated non-canonical translation initiation pathway is a promising therapeutic target for those suffering from this deadly disease. In this thesis, ordinary differential equation (ODE) and delay-differential equation (DDE) models are assembled to analyze the canonical and non-canonical translation initiation pathways. Four inhibitor classes are proposed and examined for both pathways. Results are presented in the forms of sensitivity analyses, 3D surface plots and contour plots which allow us to determine several potentially therapeutically effective combinations of inhibitor concentrations and KD values for each mode of inhibition. The results indicate that a direct eIF5B inhibitor or non-canonical ternary complex inhibitor are the most promising therapeutic targets.New Frontiers in Research Fun
Elucidating the role of human obg like ATPase1 (hOLA1) in apoptosis
Get PDFhOLA1 is a purine nucleotide binding protein that belongs to the P-loop GTPase family. hOLA1 suppresses protein synthesis by limiting ternary complex formation thus promoting the integrated stress response (ISR). ISR determines cell fate by activating or inhibiting pro- and anti-apoptotic proteins depending on the type of the stress condition. ISR facilitates cell survival in response to mild stress and promotes apoptosis in response to chronic stress. Depletion of hOLA1 leads to increased cell survival and diminished ISR. This study investigates the role of hOLA1 in apoptosis. My hypothesis was that diminished ISR in hOLA1-depleted cells leads to upregulation of anti-apoptotic proteins thus inhibiting apoptosis. Indeed, this study shows that hOLA1 depletion upregulates anti-apoptotic proteins such as cIAP1, cIAP2, and Bcl-xL, thus inhibiting apoptosis. The inhibition of apoptosis was studied by assessing caspase activation, cleaved poly (ADP-ribose) polymerase (PARP) level, and measuring apoptosis by propidium iodide (PI) staining and flow cytometry.NSERC
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Combined experimental and computational studies of the interactions between small molecule ligands and eukaryotic translation initiation factors eIF5B and eIF4E
Get PDFeIF5B and eIF4E are aberrantly expressed in several cancers, and there is a growing body of literature that recognises their importance in cancer survival. This thesis attempts to study the interactions of small molecule inhibitors with eIF5B and eIF4E by combining cell, molecular, and computational biology techniques. Through western blotting, we identified that ribavirin does not affect the function of eIF5B in BT48 and U343 cells. These results were corroborated by MD simulations, which suggested that the charge on nucleobase of RTP prevents interaction with eIF5B. Using homology modeling and protein validation, the first human eIF5B structure was generated that facilitated the identification of the LWW31 binding site. In addition, MD simulations provided insights into the stability of m7GTP and the binding mode of RBV. Overall, this thesis provides insight into small molecule interactions with eIF5B and eIF4E, which might be beneficial for the future drug design of antineoplastic drugs
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