Molecular Biology
Kylie Kibler, kylie_kibler1@baylor.edu
Baylor University, with Dr. Jonathan Kelber
Combinatorial Mechanisms of DHPS and SLC3A2 Function During Metastasis in Solid Tumors
Triple-negative breast cancer (TNBC) remains the most aggressive and lethal subtype of breast cancer and is characterized by high recurrence rates, poor prognosis, and limited therapeutic options. The patients diagnosed with metastatic disease have a dire median survival of 8-10 months. Thus, there is an urgent need to identify targetable mechanisms to improve patient outcomes. TNBC is characterized by significant intratumoral heterogeneity, which contributes to metastasis and therapy resistance. Previous studies from our group demonstrated that the hypusination pathway is involved in TGFB signaling and solid tumor progression and recently showed a prognostic and cooperative role for deoxyhypusine synthase (DHPS) and solute carrier 3A2 (SLC3A2) in TNBC. Both DHPS and SLC3A2 are implicated in cancer progression, and pharmacological inhibitors for these targets already exist, underscoring the importance of understanding the molecular basis of their cooperativity. Based on these findings, we hypothesize that DHPS and SLC3A2 cooperate to potentiate TNBC metastasis. Our objective is to investigate the functional roles of DHPS and SLC3A2 in TNBC progression by pharmacological inhibition, gene expression analysis, and 3D organoid models. We observed that inhibition of both SLC3A2 and DHPS reduced total and hypusinated eIF5A1/2 levels most effectively. Also, we show that blockade of DHPS, SLC3A2 or both reduced BC spheroid growth. Taken together, these data identify an uncharacterized mechanism through which DHPS/SLC3A2 signaling axis controls TNBC cells growth, providing a prognostic utility for targeting DHPS/SLC3A2 to improve breast cancer treatment responses.
Hannah Skene, hannahjskene@gmail.com
Earlham College. with Dr. Lexie Kuzmishin Nagy
How Does theR. Palustris Proxp-X Enzyme Recognize its Substrate?
ProXp-x is a trans-editing enzyme in Rhodopseudomonas palustris bacteria that ensures protein synthesis accuracy. ProXp-x removes incorrect amino acids from tRNAs, but the mechanism by which ProXp-x finds and separates incorrectly formed aminoacyl-tRNAs is unknown. Aminoacyl-tRNAs are important in protein synthesis to deliver the correct amino acids to the ribosome. Cells can survive without ProXp-x, but they are more likely to be susceptible to environmental stressors, like D-amino acids. We hypothesize that ProXp-x prevents D-amino acid toxicity in Rhizobia (soil-dwelling bacteria). This study will aminoacylate tRNA molecules with D-amino acids and L-amino acids to measure ProXp-x’s binding affinity for these molecules and the kinetics of removing amino acids from tRNA. To investigate, ProXp-x will be purified using fast protein liquid chromatography with a nickel affinity column followed by size exclusion chromatography (SEC). tRNA will be synthesized via in vitro transcription and purified via gel electrophoresis and ethanol precipitation. The resulting tRNA and ProXp-x enzyme will be used in electrophoretic mobility shift assays to assess how ProXp-x finds tRNAs and differentiates between tRNAs charged with D-amino acids and L-amino acids. During SEC, ProXp-x did not elute due to an unknown contaminant interacting with ProXp-x and interfering with the purification. However, gel electrophoresis confirms the presence of protein and tRNA. Future directions include reattempting SEC and performing electrophoretic mobility shift assays to further assess how ProXp-x functions.
Armeen Nasir, axn0675@mavs.uta.edu
University of Texas Arlington, with Dr. Byung Ran So and Ayesha Ali Khan
Snipping with Precision: A U1C Isoform’s role in Spliceosome Assembly
U1 small nuclear ribonucleoproteins (snRNPs) are essential components of the spliceosome, responsible for recognizing the 5' splice site during pre-mRNA splicing. One of these U1 subunits includes U1C. Recent studies suggest that alternative isoforms of U1C may contribute not only to spliceosome function but also to pathological conditions such as cancer and Alzheimer's disease. Our research focuses on U1C 159, a truncated isoform composed of 159 amino acids. We hypothesized that this shorter isoform may regulate U1 snRNP biogenesis and post-transcriptional splicing events. To investigate this, we cloned and transfected plasmids with U1C isoforms called U1C-shortest (118 aa), U1C 159, and U1C-longest (180 aa) to study their expression and impact on snRNP assembly. This system can help generate stable cell lines with HeLa cells expressing each isoform and essentially distinguish their function in U1 snRNP formation and regulation. Future studies can elucidate the underlying mechanisms of spliceosome dysregulation involving U1C isoforms in disease contexts.