bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of Genetic Interactions Associated with Splicing Factor Prp5
Authors: Merkhofer Evan C. , Moresco Philip , Copeland Jacqueline 
Published: June 2021

Regulation of gene expression occurs at multiple levels, including the removal of non‐coding sequences from protein‐coding pre‐messenger RNA transcripts, commonly known as pre‐mRNA splicing. Correct gene expression requires high fidelity of pre‐mRNA splicing, as up to 15% of mutations leading to disease may be a result of aberrant pre‐mRNA splicing, including cystic fibrosis, spinal muscular atrophy (SMA) and some leukemias. Pre‐mRNA splicing is a multistep process that requires several proteins and RNAs for ATP hydrolysis, including multiple essential DEAD‐box proteins such as Prp5. Here we investigate the role of the DEAD‐box protein Prp5 in splicing using the yeast Saccharomyces cerevisiae as a model. Prp5 has previously been shown to be required for ATP‐dependent RNA remodeling in splicing, facilitating binding of the U2 snRNA to the branchpoint sequence in the pre‐mRNA intron. In addition, unpublished research has revealed genetic interactions between Prp5 and non‐splicing genes such as Gcn5 and Spt8. In this study, additional potential genetic interactions involving Prp5 were investigated. To identify these interactions, a transposon mutagenesis screen was employed. Approximately 20,000 mutations were screened, 61 of which showed putative genetic suppression of the temperature sensitive prp5‐1 mutation. Subsequent growth assays confirmed that many of these mutations exhibited genetic suppression, indicating important genetic interactions between these genes and Prp5. hiTAIL PCR was utilized to identify a number of these genetic interactions, including an interaction between Prp5 and the MMO1 gene, a verified protein in S. cerevisiae with unknown function. Additional experiments to characterize this interaction with MMO1 are currently being performed. Furthermore, we have used high‐throughput sequencing to identify an additional subset of the interactions from the transposon screen. This study will help shed light upon the role of the DEAD‐box protein Prp5 in pre‐mRNA splicing.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Searching for the Function of Ecm14, a Pseudopeptidase, Using a Synthetic Lethal Assay
Authors: McDonald R Christian , Lyons Peter J 
Published: June 2021

Many enzyme families contain both catalytically active and inactive members. Although these inactive enzyme‐homologs (pseudoenzymes) share significant similarity with their active counterparts, key active site modifications seem to suggest a loss of traditional catalytic activity. The human M14 metallocarboxypeptidase (MCP) family is one such enzyme family that contains several active and inactive members. The baker's yeast Saccharomyces cerevisiae contains only one M14 MCP member, Ecm14, which is produced by a non‐essential gene in the yeast genome. We show through a phylogenetic analysis that this gene is present throughout the ascomycete lineage of fungi. Although Ecm14 is very similar to human CPA1 (a catalytically active member of the M14 MCP family), multiple key modifications in Ecm14 seem to indicate a loss of traditional peptidase‐like activity. A key technique for elucidating the function of non‐essential genes, including those producing pseudoenzyme products, is the genome‐wide synthetic lethal screen. Biochemical pathways typically involve multiple gene components and many of these gene players provide redundancy in the event that other genes experience deleterious mutations. The synthetic lethal screen is used to identify novel mutant yeast strains whose survival is dependent on a particular gene of interest. Colonies that contain a putative synthetically lethal gene pair are kept alive and identified by harboring a color‐reporting plasmid that complements the deletion of the endogenous gene of interest. Here we demonstrate the insertion of the wild‐type ECM14 gene into this integral pSLS1 plasmid and the determination of gene insert orientation using restriction endonucleases. Additionally, we demonstrate the preparation of knockout yeast strains using drug resistant deletion cassettes, the transformation of these yeast with the above plasmid, and finally, genome‐wide mutagenesis attempts using the chemical mutagen ethyl methanesulfonate. Identification of synthetic lethal genes will be presented, as a step toward a better understanding of the biological role of Ecm14.Support or Funding InformationGraduate Student Grants in Aid of Research to RCM and Faculty Research Grants to PJL from the Office of Research & Creative Scholarship, Andrews University.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Unveiling the Impact of the Core Circadian Clock Protein FREQUENCY on Output in Neurospora crassa
Authors: Mosier Alexander Eric , Hurley Jennifer Marie 
Published: June 2021

Circadian rhythms are cellular oscillations that tune an organism's biology to the 24‐hour light/dark cycle on Earth. This cellular time‐keeping mechanism aids in the anticipation of environmental changes and increases organismal survival. These rhythms are maintained by a transcriptional/translational negative feedback loop, called the core circadian clock, that is evolutionarily conserved throughout most branches of life. In this core clock, positive arm proteins act to transcriptionally activate negative arm proteins. The negative arms proteins act back on the positive arm, in a time specific manner, to halt the negative arm's activation before degradation and re‐initiation, thus setting the period of the clock. Beyond this core clock, positive arm proteins also act to transcriptionally activate a variety of other genes that do not participate in the core clock mechanism in a time dependent manor, referred to as clock‐controlled genes (ccgs.) Most research into the mechanisms of circadian regulation on output has focused on transcriptional activation by the positive arm proteins. However, recently published work has demonstrated that there are many oscillating proteins for which the corresponding mRNA is not rhythmic. This has led to the theory that the regulation of circadian output is also accomplished via post‐translational mechanisms, which we hypothesize are imparted by the negative arm proteins.In the model organism Neurospora crassa, the negative arm comprises FREQUENCY (FRQ), Frequency‐Interacting RNA Helicase (FRH), and Casein Kinase 1 (CK‐1). Importantly, FRQ has been computationally and experimentally proven to be an intrinsically disordered protein (IDP). Aside from lacking a single, predetermined three‐dimensional structure, IDPs play key roles in cellular signaling and regulation due, in part, to the transient but highly specific and varied interacting partners they have. These attributes make FRQ a prime candidate to impart post‐translational circadian regulation. The aim of this study is to characterize protein complexes, centered around FRQ, that change in a time specific manner, to determine the impact these interactions could impart post‐translationally on circadian regulatory pathways. To this end, Neurospora samples where crosslinked and purified through co‐immunoprecipitation before protein identification through liquid chromatography mass spectrometry. Initial results suggest that FRQ plays a role in post‐translational regulation on metabolism, the cell cycle, and a variety of other functions.Support or Funding InformationThis work was funded through the support of the NIGMS/NIH R35GM128687 grant and the Rensselaer Polytechnic Institue Start Up Fund.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding the Interacting Mechanism For the Type III Secretion System Through Molecular Dynamics Simulations
Authors: Yang Jing , Gao Meng , Huang Yongqi 
Published: June 2021

The type III secretion system is found in a large number of Gram‐negative bacterial pathogens. They are responsible for the delivery of effector proteins into the cytosol of host cells. It has been found that delivery of effector proteins depends on association with specific chaperones in the bacterial cytosol. The structures of effector/chaperone complex reveal that the chaperone forms a homo/heterodimer which the effector binds to. Upon binding with the chaperone, the intrinsically disordered effector protein forms an extended ordered conformation. Although the overall structures of the chaperones are similar, the structures of the effector proteins and interactions between the effectors and chaperones are rather different. In this work, the effector/chaperone interactions from Pseudomonas aeruginosa, Salmonella typhimurium, and Yersinia pestis were studied to compare their coupled folding‐binding mechanisms through molecular dynamics simulations. Although the sequence similarity among the effectors is very low, the interacting pattern and mechanism are similar. Our results showed that the hairpin structures from both terminuses were critical to stabilize the encounter complex. On the contrary, helices located at the central segment interacted with the chaperone when the binding free energy barrier was crossed. Therefore, preformation of central alpha‐helices has little effect on the association rate. However, interactions between central helices and the chaperone were critical for the complex stability. Our results provided new insights into the binding affinity and kinetics for effector/chaperone interactions.Support or Funding InformationThis work was supported by National Natural Science Foundation of China (Grant number: 21603121) and Hubei University of Technology.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
TIP60 regulation of ΔNp63α is associated with SCC proliferation
Authors: Stacy Andrew J. , Zhang Jin , Craig Michael P. , Kadakia Madhavi P. 
Published: June 2021

The transcription factor ΔNp63α, a p53 family member, is found primarily in the basal stratum of the skin where it functions to promote epithelial morphogenesis, cell proliferation, and cell survival. ΔNp63α is up‐regulated in non‐melanoma skin cancers such as squamous cell carcinoma (SCC), implicating it as a proto‐oncogene. Using cell culture and in vitro techniques, we have identified the acetyltransferase TIP60 as an upstream regulator of ΔNp63α. We observed that overexpression of ΔNp63α with increasing concentrations of TIP60 resulted in a dose‐dependent increase in ΔNp63α protein levels. Also, co‐expression of ΔNp63α with a TIP60 catalytic deficient mutant diminished this increase, thereby suggesting that TIP60 acetyltransferase activity contributed to increased ΔNp63α protein levels. Further, knocking down TIP60 reduced endogenous both ΔNp63α protein and transcript levels. Inhibition of de novo protein synthesis led to increased ΔNp63α protein half‐life in the presence of TIP60. Finally, TIP60 decreased the ubiquitination and proteasomal degradation of ΔNp63α. Cumulatively, these results indicate that ΔNp63α expression is regulated by TIP60 at both the transcriptional and post‐translational levels. We further demonstrated that TIP60 and ΔNp63α can interact with each other in cells as well using recombinant protein and co‐localize within the nucleus of an SCC cell line. We confirmed acetylation of ΔNp63α by TIP60 using an in vitro acetylation assay with recombinant protein and identified acetylated lysines via mass spectrometry. Moreover, site‐directed mutagenesis demonstrates that these lysines are critical to the ability of TIP60 to regulate ΔNp63α protein levels. We also observed that knockdown of both TIP60 and ΔNp63α led to a reduction in SCC proliferation. Using flow cytometry analysis, we found that both TIP60 and ΔNp63α expression promoted G2/M progression. Our results therefore indicate that TIP60 mediates SCC growth in part due to regulation of ΔNp63α. The identification of TIP60 as a novel upstream regulator of ΔNp63α provides researchers with a novel therapeutic target to combat this disease.Support or Funding InformationThe work presented from this project was supported by grant R01 CA154715‐02 (MK) from the National Institute of Health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of the phosphoprotein of human metapneumovirus and its role in formation of viral liquid organelles
Authors: Thompson Rachel , Wilburn Amanda , Cifuentes‐Munoz Nicolas , Creamer Trevor , Dutch Rebecca 
Published: June 2021

Human metapneumovirus (HMPV) is a recently identified RNA virus that frequently causes lower respiratory tract infections in infants, the elderly, and the immunocompromised, yet it has no FDA approved antivirals or vaccines. Understanding how HMPV replicates could help identify targets for development of new therapeutics. A key aspect of the viral replication cycle of HMPV is the formation of membrane‐less, liquid‐like replication and transcription centers termed inclusion bodies (IBs). A major component of IBs is the tetrameric HMPV phosphoprotein (P), which contains N and C terminal domains that lack defined secondary structures, called intrinsically disordered regions (IDRs). IDRs are thought to be involved in formation of liquid organelles through a process called liquid‐liquid phase separation (LLPS). However, the role P plays in IB formation and function is not understood. Our overall hypothesis is that the IDRs and post‐translational modifications of the phosphoprotein allow it to facilitate LLPS for IB formation. Western blots of cell lysate show two distinct bands for HMPV P, both of which contain phosphorylated protein and are detectable with either C terminal or N terminal HA tags. However, a C terminal HA tag disrupts IBs when co‐transfected with N, suggesting interactions through the C terminal domain are critical for IB formation. Purified P can form liquid‐like droplets without the presence of N, indicating it plays an important role in LLPS. Future studies will identify which domains of P are critical for LLPS and how phosphorylation and other modifications of P contribute to IB formation.Support or Funding InformationWe gratefully acknowledge the NIH/NIAID grant R01 AI3200001873 for supporting our research.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Post Translational Modifications in MDH1 May Affect Lung Cancer Survival
Authors: Alexander Sati Amrita , Sohl Christal , Weeks Joi , Hoang An 
Published: June 2021

Cancer cells alter their metabolism by increasing their consumption of glucose and altering pathways to support proliferation. Cytosolic MDH1 is amplified in squamous cell lung carcinomas which corresponds to a 50% reduced rate of progression free survival for patients. The role of malate dehydrogenase (MDH) in normal cells is to catalyze the conversion of oxaloacetate (OAA) and malate using NAD+/NADH. MDH drives the glycolysis cycle by providing NAD+, which we know is crucial in cancer due to the increased use of NAD+ for glucose production. Post translational modifications may affect MDH1 activity in lung cancer. However, the effects of post translational modifications on MDH1 and cancer phenotypes are unclear. Our goal is to understand the mechanism of MDH1 in lung cancer, by mimicking a tumor microenvironment and to understand the consequences of downstream mutations in MDH1, which is sensitive to oxidative stress. We will be using kinetic assays and a cystine C137S post translational modification on MDH1. We hypothesize that the irreversible oxidation of Cystine can impact the folding and stability of a protein, leading to alterations in physiological structure and function. Examining the effect of this post translational modification can show the downstream effect of preventing oxidation and we can observe the degradation and aggregation of proteins linked to cancer. Ultimately, we hope to demonstrate MDH1 to be a future therapeutic target for squamous cell lung cancer.Support or Funding Information1. “Research reported in this [publication/press release] was supported by the National Cancer Institute of the National Institutes of Health under award numbers:U54CA132384 & U54CA132379.2.”The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.”This work was funded by the National Institutes of Health R00 CA187594 (C.D.S.), U54CA132384 (SDSU) & U54CA132379 (UC San Diego), MARC 5T34GM008303 (SDSU), and IMSD 5R25GM058906 (SDSU), as well as the California Metabolic Research Foundation (SDSU) and SDSU startup funds (C.D.S). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.”This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding the Kinetics of Glucan Phosphatases
Authors: Henao Tiffany , Raththagala Madushi Upendrika 
Published: June 2021

Plants comprise two active glucan phosphatases, Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2). These phosphatases belong to the dual phosphatase (DSP) family of the protein tyrosine phosphatase (PTP) superfamily. DSP is a structurally diverse group and carbohydrate is the physiological substrate for glucan phosphatase family of enzymes. The X‐ray crystal structures of SEX4, LSF2 and human glucan phosphatase laforin revealed the primary mechanisms how glucan phosphatases bind carbohydrates and integrate them into the catalytic site. In vitro, enzymatic assays revealed that SEX4 preferentially removes phosphates attached to C6 position of glucose in starch while LSF2 and laforin remove phosphate groups attached to C3 position. However, little is known about the kinetics and regulatory mechanisms of carbohydrate dephosphorylation by SEX4 and LSF2. Here we define the enzymology of SEX4 and LSF2 using the generic substrate para‐nitrophenyl phosphate and physiologically relevant substrates, amylopectin, and glycogen.Support or Funding InformationSkidmore College Start‐up FundThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding oxygen tolerant [Fe‐Fe] hydrogenase
Authors: Silakov Alexey , Corrigan Patrick Sullivan 
Published: June 2021

[Fe‐Fe] hydrogenases are enzymes that perform a reversible heterolytic splitting of hydrogen into two protons and two electrons. The major roadblock in the utilization of these very efficient catalysts in renewable energy devices is the irreversible damage by oxygen and reactive oxygen species. The exact mechanism of the oxygen‐triggered inhibition is not known. However, it has been shown that in the presence of oxygen the active 6Fe metallocenter (H‐cluster) is irreversibly degraded, perhaps via a generation of a reactive oxygen species that react with components of the H‐cluster. In this work, we illustrate that an [Fe‐Fe] hydrogenase from Clostridium beijerinckii shows staggering tolerance to oxygen in atmospheric concentrations. Using activity assays and Fourier Transform infrared (FTIR) spectrometry we illustrate that the H‐cluster forms a stable adduct with oxygen that can be readily reactivated in the presence of hydrogen. Using protein film voltametry (PFV), IR spectroelectromemistry and EPR spectroscopy we provide a detailed description of the oxygen‐reacted state of the active center. Guided by bioinformatics analysis of the [Fe‐Fe] hydrogense family, we performed amino acid substitutions and subsequent biophysical characterization of the variants. The results of this study allow us to delineate the role of protein surrounding of the H‐cluster and of the auxiliary [4Fe‐4S] clusters in facilitating oxygen tolerance and catalytic bias observed in our PFV experiments.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Developing a detailed model of Hexokinase using wREFERASS to implement the King‐Altman method
Authors: Christmas Kevin M 
Published: June 2021

A full kinetic model for enzymatic facilitation of substrate to product should account and provide equations for the binding for all reactants, the multiple enzyme‐reactant states, the inherent ordered or random binding mechanisms for substrates, ions, or protons that modulate the kinetic activity. Kinetic models of this quality are uncommon because the simpler Michaelis‐Menten (MM) representations have been considered adequate. MM approximations are for steady state conditions; they are inaccurate during transients; they cannot accommodate circumstances in which the concentration of reactants are close to the affinity or dissociation constant of the enzyme. The ease in implementing MM has simplified the general understanding of enzymes, biology, and stunted the exploration for better quantitative methods of higher accuracy in enzymology. Here we report the of an enzymatic tool wREFERASS (Garcia‐Sevilla et al., 2010) to develop a complex kinetic model of skeletal muscle hexokinase (EC 2.7.1.1) using the King‐Altman notation: all terms consist of rate constants and reactant concentrations. The wREFERASS tool derives detailed mathematical expressions based on an input file that describes explicitly the kinetic pathways. The expressions provide unitary balance, conserves mass, and require defining and parameterizing the dissociation constants for each binding step. Rate constants and dissociation constants were parameterized using enzymatic data obtained from Lueck and Fromm (1973) on rat skeletal muscle hexokinase II. The kinetic model accounts for all reactants described from the input file and has physical units for all biological elements. The kinetic behavior far surpasses any MM representation due to the consideration of the multiple enzyme states. It accounts for time delay due to binding, shifts in conformational states and product release. After parameterizing each dissociation and rate constant it becomes clear that there is a preferred kinetic pathway for phosphorylation of glucose even though the binding mechanism is referred to as ‘random ordered’. Even with some parameter uncertainty by using detailed kinetic models we can develop targeted hypotheses on the less secure parameters. This approach is also attractive because every element of the pathway is explicit, results in little to no simplification of terms, and allows parameters to better reflect the biology. These models reintroduce time delays that exist in biology and metabolism and increases the accuracy of their predictions. Considerable work goes into developing a kinetic model that is not limited by MM assumptions. This model is archived at www.Physiome.org/Models/providing source code and a model that can be run over the web or downloaded.Support or Funding InformationSupported by NIH grant: U01‐HL122199This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.