Biology subjects

Walker, S. E.

Publications and source records attributed to Walker, S. E..

8 recordsLinked to original sources

bioRxiv2025

Translation Inhibition by Rocaglamide A Enhances Susceptibility of Yeasts to Caspofungin

Fks1 catalyzes the synthesis of {beta}-1,3-glucans, a major structural component of the fungal cell wall, and is the primary target of the antifungal drug caspofungin. Mutations in Fks1 confer caspofungin resistance by disrupting its interaction with the drug, thereby reducing inhibition of Fks1 enzymatic activity. Previous studies demonstrated that translation of the FKS1 mRNA was highly dependent on the translation initiation helicases eIF4A and Ded1 (1). Therefore, we investigated whether treatment of Saccharomyces cerevisiae cells with the eIF4A inhibitor Rocaglamide A (RocA) or mutation of Ded1 affects translation of FKS1 and susceptibility to caspofungin. Using WT and temperature-sensitive ded1-ts strains, we demonstrated that RocA enhanced caspofungin-mediated growth inhibition and translation repression. Sensitivity to both drugs was further enhanced in ded1-ts strains, suggesting specifically targeting Ded1 in fungi could be an effective mechanism to prevent caspofungin resistance. We extended the analysis to Candida glabrata, a related fungal pathogen, and found similar results. Importantly, combining RocA with caspofungin was fungicidal in both species, suggesting the combination could decrease development of caspofungin resistance in pathogenic yeasts. Together these findings highlight the potential of targeting translation initiation helicases for effective combination antifungal treatments.

molecular biology↗

bioRxiv2025

Analysis of Divergent Gene Expression between HPV+ and HPV- Head and Neck Squamous Cell Carcinoma Patients

Human Papillomavirus (HPV) is a non-enveloped virus with a circular double-stranded DNA genome. It is one of the most common sexually transmitted infections, with high-risk types such as HPV-16 and HPV-18 linked to anogenital and head and neck squamous cell carcinomas (HNSCC). HNSCC includes cancers of the oral cavity, pharynx, larynx, and related regions, caused by carcinogens or persistent viral infections. HPV-positive (HPV+) HNSCC cases are more prevalent in Western countries and exhibit better prognosis and treatment response compared to HPV-negative (HPV-) cases. These differences suggest distinct fundamental differences between each subtype. This study analyzed RNA-seq data from the PanCancer Atlas 2018 dataset to investigate molecular distinctions between HPV+ and HPV-HNSCC. Using dimensionality reduction techniques such as Principal Component Analysis (PCA) and Uniform Manifold Approximation and Projection (UMAP), a clear clustering of HPV+ cases was observed, suggesting a unique gene expression profile. HPV+ tumors exhibited upregulation of genes involved in nucleic acid processing and downregulation of genes associated with apoptosis and epidermis development. These findings underscore the biological differences between HPV+ and HPV-HNSCC, offering insights into HPV-driven oncogenesis. Understanding these distinctions may improve patient stratification and inform targeted therapeutic strategies for HNSCC.

cancer biology↗

bioRxiv2024

Protein Arginine Methylation of the Translation Initiation Factor eIF1A Increases Usage of a Near-cognate Start Codon

Protein arginine methylation has emerged as a key post-translational modification responsible for many facets of eukaryotic gene expression. To better understand the extent of this modification in cellular pathways, we carried out bioorthogonal methylation profiling in Saccharomyces cerevisiae to comprehensively identify the in vivo substrates of the major yeast protein arginine methyltransferase Hmt1. Gene ontology analysis of candidate substrates revealed an enrichment of proteins involved in the process of translation. We verified one such factor, eIF1A, by in vitro methylation. Three sites on eIF1A were found to be responsible for its methylation: R13, R14, and R62, with varied capacity by which each site contributed to the overall methylation capacity in vitro. To determine the role of methylation in eIF1A function, we used a battery of arginine-to-alanine substitution mutants to evaluate translation fidelity in these mutants. Our data show that substitution mutants at R13 and R14 in the N-terminal tail improved the fidelity of start codon recognition in an initiation fidelity assay. Overall, our data suggest that Hmt1-mediated methylation of eIF1A fine-tunes the fidelity of start codon recognition for proper translation initiation.

molecular biology↗

bioRxiv2023

Blunted glucocorticoid responsiveness to stress causes behavioral and biological alterations that lead to posttraumatic stress disorder vulnerability

BACKGROUNDUnderstanding why only a subset of trauma-exposed individuals develop posttraumatic stress disorder (PTSD) is critical for advancing clinical strategies. A few behavioral (deficits in fear extinction) and biological (blunted glucocorticoid levels, small hippocampal size, and rapid-eye movement sleep (REMS) disturbances) traits have been identified as potential vulnerability factors. However, whether and to what extent these traits are interrelated and whether one of them could causally engender the others are not known. METHODSIn a genetically selected rat model of reduced corticosterone responsiveness to stress, we explored PTSD-related biobehavioral traits using ex vivo magnetic resonance imaging, cued fear conditioning, and polysomnographic recordings combined with in vivo photometric measurements. RESULTSWe showed that genetic selection for blunted glucocorticoid responsiveness leads to a correlated multitrait response, including impaired fear extinction, small hippocampal volume, and REMS disturbances, supporting their interrelatedness. Fear extinction deficits and concomitant disruptions in REMS could be normalized through postextinction corticosterone administration, causally implicating glucocorticoid deficiency in two core PTSD-related risk factors and manifestations. Furthermore, reduced REMS was accompanied by higher norepinephrine levels in the hippocampal dentate gyrus that were also reverted by postextinction corticosterone treatment. CONCLUSIONSOur results indicate a predominant role for glucocorticoid deficiency over the contribution of reduced hippocampal volume in engendering both REMS alterations and associated deficits in fear extinction consolidation, and causally implicate blunted glucocorticoids in sustaining neurophysiological disturbances leading to fear extinction deficits.

neuroscience↗

bioRxiv2022

Robust eIF4B levels undermine invasive growth and immune evasion mechanisms in murine triple negative breast cancer models.

Dysregulated protein synthesis is seen in many aggressive cancers, including metastatic breast cancer. However, the specific contributions of certain translation initiation factors to in vivo disease remain undefined. This is particularly true of eIF4B, an RNA-binding protein and cofactor of the RNA helicase eIF4A and associated eIF4F cap-binding complex. While eIF4A, eIF4G, and eIF4E are well-known to contribute to the progression of many cancer types including metastatic breast cancers, the role played by eIF4B in breast cancer remains relatively unclear. We therefore explored how naturally divergent and experimentally modulated eIF4B levels impact tumor growth and progression in well-characterized murine triple negative breast cancer (TNBC) models. Surprisingly, we found that higher eIF4B levels in mouse and human breast cancers were associated with less aggressive phenotypes. shRNA-mediated eIF4B knockdown in TNBC lines failed to markedly alter proliferation and global translation in the cells in vitro and only modestly hindered their growth as primary mammary tumors growth in mice. However, eIF4B knockdown significantly enhanced invasive growth in vitro and exacerbated both tumor burden and mortality relative to nontargeting shRNA controls in a model of metastatic disease. Analysis of eIF4B levels and breast cancer patient survival reinforced a link to better outcomes. Interestingly, low eIF4B expression was also associated with more formidable immune evasion in vitro and in vivo, implicating a novel immunomodulatory role for this factor in the malignant setting that suggests a mode of action beyond its historical role as a co-activator of eIF4A/F. Significance StatementMetastasis is the leading cause of cancer-related mortality. Despite many advances in our understanding of this complex process and the molecular and cellular events involved, mechanisms that allow secondary tumors to arise and persist remain incompletely understood. Uncharacterized metastatic determinants active at the level of translational control may be exploitable as novel therapy targets or biomarkers predicting a tumors potential for spread and recurrence. Here we describe previously unrecognized consequences of dysregulated eIF4B levels in murine breast cancer that shed light on how this translation initiation factor contributes to disease outcomes. Our findings suggest that eIF4B levels direct metastatic risk and immune evasion, and further study should establish its value in personalized treatment decisions and development of future therapies.

cancer biology↗

bioRxiv2022

Dynamic Phosphorylation Regulates Eukaryotic Translation Initiation Factor 4A Activity During the Cell Cycle

The eukaryotic translation initiation factor 4A (eIF4A) resolves mRNA structures to support protein synthesis, yet little is known about its regulation. Here we analyzed eIF4A phosphorylation during alternate stages of the cell cycle, and found three residues near the DEAD box motif (T73, T146, and S177) underwent substantial phosphorylation changes. Phosphomimetic mutations T73D and T146D led to G2/M phase arrest, and abolished eIF4A interaction with RNA, suggesting eIF4A activity is needed for completion of cell division. In addition to these repressive events, we found that S177, a site immediately adjacent to the DEAD-box, showed diametrically opposed phosphorylation, with only phosphorylated S177 present during G1/S arrest and dephosphorylated S177 peptides during G2/M arrest. Phosphomimetic S177D eIF4A increased polysome levels and enhanced normally reduced eIF4A-eIF4G-interaction during G2/M, while phosphodeficient S177A decreased polysome levels and reduced growth, suggesting phosphorylation of S177 enhances eIF4A-mediated translation during G1/S. Together these results suggest that dynamic phosphorylation of eIF4A S177 serves to stimulate translation during G1/S, while inhibitory phosphorylation of additional sites holds the potential to rapidly transition eIF4A to an inactive state and turn off translation. These results also suggest an important role for eIF4A in coupling translation to cell cycle stages.

molecular biology↗

bioRxiv2020

Early metazoan origin and multiple losses of a novel clade of RIM pre-synaptic calcium channel scaffolding protein homologues

The precise localization of CaV2 voltage-gated calcium channels at the synapse active zone requires various interacting proteins, of which, Rab3 interacting molecule or RIM is considered particularly important. In vertebrates, RIM interacts with CaV2 channels in vitro via a PDZ domain that binds to the extreme C-termini of the channels at acidic ligand motifs of D/E-D/E/H-WC-COOH, and knockout of RIM in vertebrates and invertebrates disrupts CaV2 channel synaptic localization and synapse function. Here, we describe a previously uncharacterized clade of RIM proteins bearing homologous domain architectures as known RIM homologues, but some notable differences including key amino acids associated with PDZ domain ligand specificity. This novel RIM emerged near the stem lineage of metazoans and underwent extensive losses, but is retained in select animals including the early-diverging placozoan Trichoplax adhaerens, and molluscs. RNA expression and localization studies in Trichoplax and the mollusc snail Lymnaea stagnalis indicate differential regional/tissue type expression, but overlapping expression in single isolated neurons from Lymnaea. Ctenophores, the most early-diverging animals with synapses, are unique among animals with nervous systems in that they lack the canonical RIM, bearing only the newly identified homologue. Through phylogenetic analysis, we find that CaV2 channel D/E-D/E/H-WC-COOH like PDZ ligand motifs were present in the common ancestor of cnidarians and bilaterians, and delineate some deeply conserved C-terminal structures that distinguish CaV1 from CaV2 channels, and CaV1/CaV2 from CaV3 channels.

evolutionary biology↗

bioRxiv2019

Binding to the Ribosome by Eukaryotic Initiation Factor 4B Drives Yeast Translational Control in Response to Urea

The yeast eukaryotic initiation factor 4B binds the 40S subunit in translation preinitiation complexes (PICs), promoting mRNA binding. Recent evidence suggests mRNAs have variable dependence on eIF4B, suggesting this factor could promote changes in mRNA selection for translation, in order to adapt to stressors. However, the importance of eIF4B and its constituent domains for mRNA selection under diverse cellular and environmental conditions remain undefined. Here we compared the effects of disrupting eIF4B RNA- and ribosome-binding motifs under ~1400 growth conditions. The RNA-Recognition Motif (RRM) was dispensable for stress responses, but the 40S-binding N-terminal Domain (NTD) promoted growth in response to various stressors. In particular, the NTD conferred a strong growth advantage in the presence of urea. Ribosome profiling revealed that the NTD promoted translation of mRNAs with long and highly structured 5-prime untranslated regions, both with and without urea exposure. Our results suggest eIF4B controls mRNA loading and scanning as a part of the PIC, rather than by activating mRNPs prior to ribosome binding. Furthermore, our data indicate the yeast response to urea includes a translational component, driven by production of proteins associated with the cellular periphery. Together our analyses suggest general eIFs can promote diverse cellular responses.

molecular biology↗