N Sierra, E Albekioni, Nalani Coleman, Rachel Khoury, An Hoang, Christal Sohl. R132Q IDH1 sensitivity to reducing agents, Sixteenth Annual Student Research Symposium of the division research and innovation at San Diego State University, San Diego, CA, 2023. N Sierra, E Albekioni, Nalani Coleman, Rachel Khoury, An Hoang, Christal Sohl. R132Q IDH1 sensitivity to reducing agents, ABRCMS (Annual Biomedical research conference for minoritized scientists) at Phoneix, AZ,2023.
The proto-oncogene IDH1, isocitrate dehydrogenase 1, is a gene that provides enzymes the ability to break down fats for energy and protect cells. In the normal wild-type oxidative decarboxylation reaction, isocitrate produces alpha-ketoglutarate, with the reactant NADP+ being converted to NADPH. However, mutant IDH1 can catalyze a neomorphic reduction, the NADPH-dependent reduction of alpha-ketoglutarate to D-2-hydroxyglutarate, D2HG, which can competitively inhibit alpha ketoglutarate-dependent enzymes. We hope to investigate the catalytic efficiency of the enzyme to discover the relationship between kinetics and tumor phenotypes. We have previously shown that the mutation R132Q produces high levels of D2HG while still being able to produce alpha-ketoglutarate, unlike other mutants. Furthermore, we previously solved a crystal structure of R132Q with the mutant bound to isocitrate and NADP+ substrates under reducing conditions to stimulate the cellular environment. This crystal structure led to the discovery of the reducing agent, TCEP, forming an adduct with NADP+. The impact of this adduct on the catalytic activity of the mutant R132Q IDH1 is unknown. We hypothesize that the NADP+-dependent normal reaction will be inhibited due to the unavailability of the NADP+ substrate. We have conducted steady-state kinetic assays on R132Q mutant at varying concentrations of reducing agents to determine the impact of the TCEP-NADP adduct on R132Q catalysis. We show that observed rates of R132Q decrease as reducing agent concentrations increase, with different reducing agents having unique tendencies for inhibition. This project can reveal possible precautions for researchers to be aware of when crystallizing IDH1 and performing catalytic reactions, as well as help us clarify mechanisms of catalysis.
N Sierra, E Albekioni, K Sabo, Christal Sohl. Exploring the mechanisms of IDH1 pH sensitivity, Fifteenth Annual Student Research Symposium of the division research and innovation at San Diego State University, San Diego, CA, 2022.
N Sierra, E Albekioni, K Sabo, Christal Sohl. Exploring the mechanisms of IDH1 pH sensitivity, SACNAS Society for Advancement of Chicanos/Hispanics and Native Americans in Science at San Juan, Puerto Rico, 2022.
Globally, cancer patients are significantly impacted by the proto-oncogene isocitrate dehydrogenase 1 (IDH1). While many cancers are driven by mutations in IDH1, resulting in the catalysis of a new reaction that has been shown to drive tumor formation, wild-type (WT) IDH1 has also been shown to drive many cancer types. WT IDH1 drives the oxidative decarboxylation reaction of isocitrate to alpha-ketoglutarate, with concomitant conversion of NADP+ to NADPH. This reaction can support tumor growth by synthesizing alpha keto-glutarate. We know from previous structural informatics and pKa calculations that the residue D273 in IDH1 is sensitive to changes in pH, allowing IDH1 to serve as a pH sensor. By affecting the activity of pH sensors, cellular pH can regulate protein-protein and protein-ligand interactions, including the stability and activity of a protein. D273 is located in the first third of the α10 helix, an important regulatory domain. To determine the role of D273 in pH sensitivity, experimental mutants were designed to have minimal disruption in the overall structure but still destroy the ability of the residue to ionize. The mutants produced very drastic decreases in catalytic efficiency for the forward reaction as compared with WT IDH1. Here we describe the pH sensitivity of a new ionizable mutant, D273E IDH1. Using site-directed mutagenesis, the formation of the D273E mutant was made and the enzyme was heterogeneously expressed and purified. We predict this mutant will retain pH sensitivity when measuring the rates of αKG production since it can change its ionization state.
M Peters, N Sierra, S Jorge. Impacts of Wildfires on Coastal Discharges in the Amazon River Basin, Fourteenth Annual Student Research Symposium of the division research and innovation at San Diego State University, San Diego, CA, 2021.
The vulnerability of coastal areas to terrestrial watershed disturbances, such as wildfires, remains unknown and unquantified. In 2020 alone, there was an unprecedented amount of wildfires, causing damage and disruption throughout the world, with Australia experiencing its largest bushfire in history and the Amazon continuing to battle unusually high numbers of fires. As many coastal waterways are downstream of terrestrial areas that are susceptible to wildfires, it is critical to evaluate the impacts of wildfires on vulnerable marine environments. This study sets out to identify and quantify the impacts of wildfires on coastal regions. Large spatial datasets are utilized to interlink spatial and temporal dynamics of terrestrial processes and disruptions from wildfires with coastal runoff and ecosystem shifts in the Amazon River Basin. This work will focus specifically on the area within the Brazilian state of Roraima where a large number of fires have been observed in recent years upstream of the coast. The extent and magnitude of previous wildfires were characterized using satellite-based products, Enhanced Vegetation Index (EVI), Normalized differential vegetation index (NDVI), and differenced normalized burn ratio (dNBR), to compare pre-and post-fire biomass and estimate the burn severity of plant material and soil. Discharge flows and water quality data were obtained from multiple data repositories available through the Brazilian government, with the majority of the river gauge stations being actively managed by the Geological Survey of Brazil (GSB). This data was compiled for fires that occurred between 2010 to 2020 to evaluate post-fire water quality response. A subset of fires concentrated along the Branco River and Rio Negro, two large tributaries to the Amazon River, was examined in further detail to identify trends in water-quality response. Assembling this extensive dataset provided the unique opportunity to determine the most common post-fire water quality changes in the Branco River, Rio Negro, and Amazon River. Results from this study will further be used to identify shifts in water quality and impacts of the coastal discharge of the Amazon River post-fires.
The proto-oncogene IDH1, isocitrate dehydrogenase 1, is a gene that provides enzymes the ability to break down fats for energy and protect cells. In the normal wild-type oxidative decarboxylation reaction, isocitrate produces alpha-ketoglutarate, with the reactant NADP+ being converted to NADPH. However, mutant IDH1 can catalyze a neomorphic reduction, the NADPH-dependent reduction of alpha-ketoglutarate to D-2-hydroxyglutarate, D2HG, which can competitively inhibit alpha ketoglutarate-dependent enzymes. We hope to investigate the catalytic efficiency of the enzyme to discover the relationship between kinetics and tumor phenotypes. We have previously shown that the mutation R132Q produces high levels of D2HG while still being able to produce alpha-ketoglutarate, unlike other mutants. Furthermore, we previously solved a crystal structure of R132Q with the mutant bound to isocitrate and NADP+ substrates under reducing conditions to stimulate the cellular environment. This crystal structure led to the discovery of the reducing agent, TCEP, forming an adduct with NADP+. The impact of this adduct on the catalytic activity of the mutant R132Q IDH1 is unknown. We hypothesize that the NADP+-dependent normal reaction will be inhibited due to the unavailability of the NADP+ substrate. We have conducted steady-state kinetic assays on R132Q mutant at varying concentrations of reducing agents to determine the impact of the TCEP-NADP adduct on R132Q catalysis. We show that observed rates of R132Q decrease as reducing agent concentrations increase, with different reducing agents having unique tendencies for inhibition. This project can reveal possible precautions for researchers to be aware of when crystallizing IDH1 and performing catalytic reactions, as well as help us clarify mechanisms of catalysis.
N Sierra, E Albekioni, K Sabo, Christal Sohl. Exploring the mechanisms of IDH1 pH sensitivity, Fifteenth Annual Student Research Symposium of the division research and innovation at San Diego State University, San Diego, CA, 2022.
N Sierra, E Albekioni, K Sabo, Christal Sohl. Exploring the mechanisms of IDH1 pH sensitivity, SACNAS Society for Advancement of Chicanos/Hispanics and Native Americans in Science at San Juan, Puerto Rico, 2022.
Globally, cancer patients are significantly impacted by the proto-oncogene isocitrate dehydrogenase 1 (IDH1). While many cancers are driven by mutations in IDH1, resulting in the catalysis of a new reaction that has been shown to drive tumor formation, wild-type (WT) IDH1 has also been shown to drive many cancer types. WT IDH1 drives the oxidative decarboxylation reaction of isocitrate to alpha-ketoglutarate, with concomitant conversion of NADP+ to NADPH. This reaction can support tumor growth by synthesizing alpha keto-glutarate. We know from previous structural informatics and pKa calculations that the residue D273 in IDH1 is sensitive to changes in pH, allowing IDH1 to serve as a pH sensor. By affecting the activity of pH sensors, cellular pH can regulate protein-protein and protein-ligand interactions, including the stability and activity of a protein. D273 is located in the first third of the α10 helix, an important regulatory domain. To determine the role of D273 in pH sensitivity, experimental mutants were designed to have minimal disruption in the overall structure but still destroy the ability of the residue to ionize. The mutants produced very drastic decreases in catalytic efficiency for the forward reaction as compared with WT IDH1. Here we describe the pH sensitivity of a new ionizable mutant, D273E IDH1. Using site-directed mutagenesis, the formation of the D273E mutant was made and the enzyme was heterogeneously expressed and purified. We predict this mutant will retain pH sensitivity when measuring the rates of αKG production since it can change its ionization state.
M Peters, N Sierra, S Jorge. Impacts of Wildfires on Coastal Discharges in the Amazon River Basin, Fourteenth Annual Student Research Symposium of the division research and innovation at San Diego State University, San Diego, CA, 2021.
The vulnerability of coastal areas to terrestrial watershed disturbances, such as wildfires, remains unknown and unquantified. In 2020 alone, there was an unprecedented amount of wildfires, causing damage and disruption throughout the world, with Australia experiencing its largest bushfire in history and the Amazon continuing to battle unusually high numbers of fires. As many coastal waterways are downstream of terrestrial areas that are susceptible to wildfires, it is critical to evaluate the impacts of wildfires on vulnerable marine environments. This study sets out to identify and quantify the impacts of wildfires on coastal regions. Large spatial datasets are utilized to interlink spatial and temporal dynamics of terrestrial processes and disruptions from wildfires with coastal runoff and ecosystem shifts in the Amazon River Basin. This work will focus specifically on the area within the Brazilian state of Roraima where a large number of fires have been observed in recent years upstream of the coast. The extent and magnitude of previous wildfires were characterized using satellite-based products, Enhanced Vegetation Index (EVI), Normalized differential vegetation index (NDVI), and differenced normalized burn ratio (dNBR), to compare pre-and post-fire biomass and estimate the burn severity of plant material and soil. Discharge flows and water quality data were obtained from multiple data repositories available through the Brazilian government, with the majority of the river gauge stations being actively managed by the Geological Survey of Brazil (GSB). This data was compiled for fires that occurred between 2010 to 2020 to evaluate post-fire water quality response. A subset of fires concentrated along the Branco River and Rio Negro, two large tributaries to the Amazon River, was examined in further detail to identify trends in water-quality response. Assembling this extensive dataset provided the unique opportunity to determine the most common post-fire water quality changes in the Branco River, Rio Negro, and Amazon River. Results from this study will further be used to identify shifts in water quality and impacts of the coastal discharge of the Amazon River post-fires.