Eukaryotic translation initiates upon recruitment of the EIF2‐GTP·Met‐tRNAi ternary complex (TC) to the ribosomes. EIF2 (α, β, γ subunits) is a GTPase. The GDP to GTP exchange within EIF2 is facilitated by the guanine nucleotide exchange factor EIF2B (α‐ε subunits). During stress‐induced conditions, phosphorylation of the α‐subunit of EIF2 turns EIF2 into an inhibitor of EIF2B. In turn, inhibition of EIF2B decreases TC formation and triggers the internal stress response (ISR), which determines the cell fate. Deregulated ISR has been linked to neurodegenerative disorders and cancer, positioning EIF2B as a promising therapeutic target. Hence, a better understanding of the mechanisms/factors that regulate EIF2B activity is required. Here, combining transcript and protein level analyses, we describe an intronically polyadenylated (IPA) transcript of EIF2B's γ‐subunit. We show that the IPA mRNA isoform is translated into a C‐terminus truncated protein. Using structural modeling, we predict that the truncated EIF2Bγ protein has unfavorable interactions with EIF2γ, leading to a potential decrease in the stability of the nonproductive EIF2:EIF2B complex. While we discovered and confirmed the IPA mRNA isoform in breast cancer cells, the expression of this isoform is not cancer‐specific and is widely present in normal tissues. Overall, our data show that a truncated EIF2Bγ protein co‐exists with the canonical protein and is an additional player to regulate the equilibrium between productive and nonproductive states of the EIF2:EIF2B complex. These results may have implications in stress‐induced translation control in normal and disease states. Our combinatorial approach demonstrates the need to study noncanonical mRNA and protein isoforms to understand protein interactions and intricate molecular mechanisms.
Epigenetic deregulation is a critical theme which needs further investigation in bladder cancer research. One of the most highly mutated genes in bladder cancer is KDM6A, which functions as an H3K27 demethylase and is one of the MLL3/4 complexes. To decipher the role of KDM6A in normal versus tumor settings, we identified the genomic landscape of KDM6A in normal, immortalized, and cancerous bladder cells. Our results showed differential KDM6A occupancy in the genes involved in cell differentiation, chromatin organization, and Notch signaling depending on the cell type and the mutation status of KDM6A. Transcription factor motif analysis revealed HES1 to be enriched at KDM6A peaks identified in the T24 bladder cancer cell line; moreover, it has a truncating mutation in KDM6A and lacks a demethylase domain. Our co-immunoprecipitation experiments revealed TLE co-repressors and HES1 as potential truncated and wild-type KDM6A interactors. With the aid of structural modeling, we explored how truncated KDM6A could interact with TLE and HES1, as well as RUNX and HHEX transcription factors. These structures provide a solid means of studying the functions of KDM6A independently of its demethylase activity. Collectively, our work provides important contributions to the understanding of KDM6A malfunction in bladder cancer.
Cyclin-dependent kinases (CDKs) are commonly known by their role in cell cycle regulation which affects cancer mechanism. In many cancer types, CDKs show extreme activity or CDK inhibiting proteins are dysfunctional. Specifically, CDK2 plays an indispensable role in cell division especially in the G1/S phase and DNA damage repair. Therefore, it is important to find new potential CDK2 inhibitors. In this study, ligand-based drug design is used to design new potential CDK2 inhibitors. Y8 L ligand is obtained from the X-ray crystal structure of human CDK2 (PDB ID: 2XNB) (www.pdb.org) and used as a structure model. By adding hydrophilic and hydrophobic groups to the structure, a training set of 36 molecules is generated. Each molecule examined with Spartan’14 and optimized structures are used for docking to CDK2 structure by AutoDock and AutoDock Vina programs. Ligand-amino acid interactions are analysed with Discovery Studio Visualizer. Van der Waals, Pi-Pi T-shaped, alkyl, pi-alkyl, conventional hydrogen bond and carbon-hydrogen bond interactions are observed. By docking results and viewed interactions, some molecules are identified and discussed as potential CDK2 inhibitors. Additionally, 8 different QSAR descriptors obtained from Spartan’14, Preadmet and ALOGPS 2.1 programs are investigated with multiple linear regulation (MLR) analysis with SPSS program for their impact on affinity value.
Epigenetic deregulation is a critical theme which needs further investigation for bladder cancer research. One of the highly mutated genes in bladder cancer is KDM6A, functioning as a H3K27 demethylase and is part of the MLL3/4 complexes. To decipher the role of KDM6A in normal versus tumor setting, we identified the genomic landscape of KDM6A in normal, immortalized and cancer bladder cells. Our results showed differential KDM6A occupancy at the genes involved in cell differentiation, chromatin organization and Notch signaling depending on the cell type and the mutation status of KDM6A. Transcription factor motif analysis revealed HES1 to be enriched at KDM6A peaks identified for T24 bladder cancer cell line, which has a truncating mutation in KDM6A, lacking demethylase domain. Our co-immunoprecipitation experiments reveal TLE co-repressors and HES1 as potential truncated-KDM6A interactors. With the aid of structural modeling, we explored how the truncated KDM6A could interact with TLE, HES1, as well RUNX, HHEX transcription factors. These structures provide a solid mean to study the functions of KDM6A independent of its demethylase activity. Collectively, our work provides important contributions to the understanding of KDM6A malfunction in bladder cancer.
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