bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel adenosine/uridine‐rich element‐binding proteins recognize interleukin‐3 ARE in leukemia T cells (749.4)
Authors: Martinez Marina , Gonzalez Jose , Lopez Armando , Gonzalez Carlos 
Published: June 2021

Human Interleukin‐3 (hIL‐3) is a cytokine that promotes myelopoiesis, differentiation of macrophages and granulocytes. Aberrant expression of this lymphokine has been associated with several hematological cancers. IL‐3 3’‐UTR harbors Adenosine/Uridine‐Rich Elements (AREs) involved in its post‐transcriptional control. These regulatory sequences are recognized by specific ARE‐Binding Protein (ARE‐BP) complexes. Previous results from our laboratory estimated five ARE‐BP complexes from 34 to 88 kDa binding to the hIL‐3 ARE using UV‐crosslinking assay. Our goal is to identify novel ARE‐BPs that mediate the post‐transcriptional regulation of hIL‐3. To achieve this goal, RNA affinity purification coupled with MS/MS analysis was performed. Also, an immunoblot analysis to confirm the presence of specific proteins was carried out. These results identified ~40 proteins between 12 to 82kD interacting with the hIL‐3 ARE in acute lymphoblastic leukemia cells. MS/MS results in accordance with previous results from our laboratory showed that HuR and p42hnRNP C1/C2 are components of the ARE‐BPs that recognize the hIL‐3 ARE. In addition, our results identified KSRP and SAM68 as novel hIL‐3 ARE‐BPs. Besides, we found that p45AUF‐1 protein interacts with hIL‐3 ARE. Taken together, these data imply that novel ARE‐BP complexes can play an important role in the IL‐3 post‐transcriptional regulation. Ultimately, elucidating the role of these ARE‐BPs in IL‐3 expression can provide new insights about the ARE‐mediated post‐transcriptional regulatory pathway and blood cancer.Grant Funding Source: MBRS‐RISE Program R25GM061838, UPR‐PES Intitutional funding
Small RNA regulation of a multidrug efflux pump (750.1)
Authors: Parker Ashley , Gottesman Susan 
Published: June 2021

Multidrug efflux pumps are a major mechanism used by bacteria to export drugs and toxic compounds from the cell. One of the most important efflux pumps in E. coli is the AcrAB‐TolC system. In E. coli and among other enteric organisms, acrAB and tolC deletion mutants are known to be hypersensitive to antibiotics, and very little has been done to investigate regulation at the post‐transcriptional level of these efflux genes. Small ribonucleic acids (sRNAs) are known to be major post‐transcriptional regulators in bacteria, they usually enhance or repress translation by binding to the 5’ untranslated region (UTR) of mRNA targets with the help of a small chaperone protein, Hfq. In this study we hypothesized that Hfq‐dependent sRNAs might regulate acrA, acrB, and/or tolC. Translational fusions of the 5’ UTR and start of the open reading frame for each gene with lacZ were constructed and transformed with a sRNA plasmid library to test for sRNA regulation, measured by beta‐galactosidase assays. RyeB, a sRNA expressed during stationary phase, was found to repress the expression of tolC, which encodes the outer membrane protein of many multidrug resistant efflux pumps. Compensatory mutants were used to validate that the repression was by direct base pairing, upstream from the ribosomal binding site. We have confirmed by Western blot analysis that RyeB regulation of tolC at the mRNA level leads to a decrease in TolC protein levels. Our results suggest that the decreased amounts of TolC in stationary phase is due to the increase in expression of RyeB, and this may limit efflux of some compounds under these conditions.Grant Funding Source: National Institutes of Health, Intramural Research
microRNA miR‐3185 mediated alternative translation initiation of peroxiredoxin 5 mRNA in human breast cancer cells (750.3)
Authors: Misra Smita , Mittal Mukul , Chaudhuri Gautam 
Published: June 2021

Human peroxiredoxin 5 (PRDX5) gene, encodes a member of the peroxiredoxin family of antioxidant enzymes, which reduces hydrogen peroxide and alkyl hydroperoxides. Apart from its antioxidant function, PRDX5 is also predicted to have a role in the regulation of gene transcription. The predicted PRDX5 protein has a strong mitochondrial localization signal how the protein goes to the nucleus and acts as transcription factor is not known. The PRDX5 mRNA has two in‐frame AUG codons, translation from the first ‘AUG’ results in the full length protein (~24 kDa) and translation from second ‘AUG’ results in a shorter protein (~18 kDa). We found that the shorter form of PRDX5 which lacks the N‐terminal mitochondrial import signal is located in the nucleus and regulates BRCA2 gene expression in human breast cancer cells in a redox dependent manner. Here we report a miRNA mediated mechanism for the regulation of alternative translation initiation of PRDX5 mRNA. The microRNA hsa‐miR 3185 has a strong binding site within the two in‐frame AUG codon of the PRDX5 ORF. Increase in the oxidation state leads to an increased expression of hsa‐miR‐3185. Increase amount of miR‐3185 leads to an increase in short form of PRDX5 translation from the second AUG perhaps by blocking the translation initiation from the first AUG codon. To our knowledge this is the first report of regulation of alternative translation initiation of a human gene by a microRNA.Grant Funding Source: Supported by the DOD‐CDMRP BCRP Grants W81XWH‐06‐1‐0466, W81XWH‐08‐1‐0446, BC086542, and the Susan G
The ribosome‐associated Hsp40 chaperone Zuo1 antagonizes prion formation in yeast (752.11)
Authors: Cameron Dale , Pontoriero David , Castanzo Dominic , Keyser Erich , Selechnik Daniel 
Published: June 2021

Prions are self‐propagating infectious protein conformations. In mammals prions underlie the transmissible spongiform encephalopathies, a set of invariably fatal neurodegenerative disorders associated with misfolding of the mammalian prion protein, PrP. In recent years increasing numbers of fungal proteins capable of infectiously propagating altered conformations have been discovered. Fungal prions are typically not lethal and debate continues as to whether they may have evolutionarily conserved physiological roles rather than representing disease states. To gain insight into the mechanisms by which cells regulate prion formation, propagation and toxicity, we have examined the role of the ribosome‐associated complex (RAC) chaperones in the formation and toxicity of the [PSI+] prion in yeast. The [PSI+] prion results from a self‐propagating aggregated form of the essential translation termination factor Sup35, and is manifested phenotypically as elevated levels of nonsense suppression. We find that yeast cells lacking the Hsp40 chaperone Zuo1 exhibit higher frequencies of spontaneous and induced [PSI+] formation, consistent with a role for the RAC in chaperoning nascent Sup35 to prevent misfolding of the N‐terminal prion domain as it emerges from the ribosome. Notably, formation of [PSI+] in cells lacking Zuo1 is associated with a reduction in fitness. As both ZUO1 deletion and the [PSI+] prion can independently enhance readthrough of stop codons, toxicity may be due to the elevated levels of nonsense suppression in Δzuo1 [PSI+] strains. Studies are currently underway to understand the mechanisms by which deletion of Zuo1 enhances both the formation and toxicity of the [PSI+] prion.Grant Funding Source: Supported by Research Corporation for Science Advancement
Context‐specific action of linezolid and chloramphenicol, the inhibitors of ribosomal peptidyl transferase (752.12)
Authors: Marks James , Orelle Cedric , Kannan Krishna , Klepacki Dorota , Roncase Emily , Vazquez‐Laslop Nora , Mankin Shura 
Published: June 2021

Protein synthesis has been a successful target for antibiotics, with many different inhibitors targeting functional sites of the ribosome. Understanding the mode of antibiotic action is critical for drug development and elucidating the fundamental mechanisms of protein synthesis.Chloramphenicol and linezolid have been shown to bind at the A site in the peptidyl transferase center, and in theory should prevent any peptide bond formation. However, in the cell free translation system, ribosomes in the presence of either antibiotic are not arrested at the initiator codon but instead are able to catalyze the formation of a number of peptide bonds before halting translation. The exact sites of arrest appear to be context dependent, and possibly different for these two antibiotics despite binding to the same ribosomal site. No obvious consensus sequence defining the sites of antibiotic‐induced arrest emerged from analysis of in vitro translation of several selected genes. Therefore, we are expanding these studies using genome‐wide ribosome profiling. This approach should reveal the context of the gene sites at which these inhibitors arrest translation in vivo. We believe our investigation will provide critical insights into mechanism of translation inhibition by chloramphenicol and linezolid and pave the way for future drug design. Importantly, our studies may reveal unknown aspects of catalysis of peptide bond formation by the ribosome.Grant Funding Source: Supported by NIH
Elongation factor G undergoes an extensive structural rearrangement during ribosomal translocation (752.3)
Authors: Ermolenko Dmitri , Salsi Enea , Farah Elie 
Published: June 2021

Translocation of mRNA and tRNAs through the ribosome is catalyzed by the universally conserved elongation factor (EF‐G in prokaryotes and EF‐2 in eukaryotes). It has been hypothesized that EF‐G undergoes structural rearrangements during translocation. Here, we test this hypothesis by following the movement of EF‐G relative to the small ribosomal subunit using single‐molecule Förster resonance energy transfer (smFRET). We show that ribosome‐bound EF‐G adopts distinct conformations corresponding to the pre‐ and post‐translocation states of the ribosome. Our results suggest that upon ribosomal translocation, domain IV of EF‐G, which is essential for the catalysis of translocation, moves toward the A site of the small ribosomal subunit and promotes translocation of peptidyl‐tRNA.Grant Funding Source: These studies were supported by grant from the US National Institute of Health no. GM‐099719
p53 Aggregation and prionoid effect and the modulation of this process by PRIMA‐1 and its active metabolite, 2‐methylene‐3‐quinuclidinone hydrate (754.4)
Authors: Rangel Luciana , da Costa Caroline , da Costa Danielly , da Silva Jerson 
Published: June 2021

Among other functions, p53 acts as a transcription factor and is involved in cell cycle control, leading to apoptosis or to the arrest of the cell cycle for DNA damage repair. It is mutated in around 50% of all tumors. In general, mutations occur on the DNA‐binding domain, leading to its loss of function as a transcription factor. The mutation in a single allele leads to the complete loss of function due to a phenomenon called negative dominance. It has been postulated that mutant p53 can form aggregates that are related to loss‐of‐function and cancer development. Also, new data have contributed to the spread the application of the prion concept on different amyloid disesases. In this work, we show that the effect of mutant p53 on wild type p53 aggregation occurs in a prionoid manner. We have also observed that PRIMA‐1, a classical drug described to stabilize mutant p53 structure and function, exerts its effect on aggregated mutant p53. Also, 2‐methylene‐3‐quinuclidinone hydrate (MQ) has been shown to inhibit WT and mutant recombinant p53 central core domain (p53C) aggregation at 37oC. The WT form has been protected in a lower degree. MQ has also been shown to inhibit the seeding promoted by mutant p53 cellular extracts fractions enriched in oligomers on WTp53C. The same seeding inhibition effect was observed for extracts from cells treated with PRIMA‐1. These results might lead to a different understanding on the controversial mechanism of action of PRIMA‐1 and to further understanding of the seeding and aggregation process of p53, as well as its modulation.Grant Funding Source: Supported by FAPERJ and CNPq
Quantifying effects of glycerol and low molecular weight polyethylene glycols on Cro repressor folding and dimerization (755.3)
Authors: Sternke Matthew , Knowles D. Benjamin , Phan Noel , Lingeman Emily , Record M. Thomas 
Published: June 2021

Small solutes affect noncovalent protein processes by their competitive‐with‐water preferential interactions with the chemical functional groups on the protein surface that is buried or exposed during the process. Solute effects on protein processes are exploited by in vitro biochemists to drive folding or binding, but many of these effects are not understood. Here we determine the effects of glycerol and low molecular weight polyethylene glycols (PEGs) on equilibrium folding/unfolding and dimerization of a variant Cro repressor dimer and equilibrium folding/unfolding of a variant Cro repressor monomer (generously provided by Prof. Michael Mossing of University of Mississippi Department of Chemistry and Biochemistry) and dissect the effects of these solutes on each Cro repressor specie into effects from the terminal (‐CH¬2OH) and interior (‐CH2OCH2‐) groups. We find that glycerol and diEG are protein‐stabilizing solutes, triEG has a neutral effect on protein stability, and tetraEG and above are protein‐destabilizing solutes. From this we conclude that the exterior ‐CH2OH groups are stabilizing and the interior ‐CH2OCH2‐ groups are destabilizing. We also find that the effects of each solute scale in an approximate 2:1 ratio between the variant dimer and the variant monomer, showing that the effects of the solutes scale linearly with surface area buried or exposed in the process.Grant Funding Source: Supported by NIH Grant 47022
Mitochondrial protease Oma1 plays important roles in stress management and chronological lifespan control in yeast (756.3)
Authors: Bohovych Iryna , Donaldson Garrett , Christianson Sara , Zahayko Nataliya , Khalimonchuk Oleh 
Published: June 2021

A network of conserved proteases known as intramitochondrial quality control (IMQC) is critical for normal mitochondrial activities. IMQC proteases were implicated in establishment of signaling cues for interorganellar communication, but understanding of these events is limited.We report that conserved ATP‐independent metalloprotease Oma1 is important for adaptive responses and preservation of normal mitochondrial function under damage‐eliciting conditions. Our data indicate one or more interorganellar communication lines may be compromised in oma1Δ cells. Oxidative stress‐induced nuclear translocation of the transcription factor Yap1 controlling the expression of antioxidant enzymes is impeded in oma1Δ strain. The defect is consistent with attenuation of catalase expression and activity in peroxide‐stressed oma1Δ cells. Oma1‐deficient cells display marked resistance to the TORC1 signaling pathway‐inactivating drug rapamycin, consistent with significant increase in chronological life span in the absence of stress stimuli. Deletion of TORC1 downstream target kinase Sch9 in oma1Δ background results in a positive genetic effect: the mutants demonstrate synergistic increase in rapamycin resistance and improved tolerance to homeostatic insults.We propose that Oma1 is important for integrating IMQC‐mediated mitochondrial stress sensing into pan‐cellular stress response mechanisms.Grant Funding Source: Supported by NIH‐NIGMS P30 GM103335 and University of Nebraska Layman Award
High‐throughput in vivo quantification of apoptotic induction in yeast liquid culture (762.5)
Authors: Luo Yi , Schwotzer Erica , Chirigos Allison , Whitworth Gregg 
Published: June 2021

We have developed a novel technique that allows for high‐throughput quantification of apoptotic induction in yeast cells growing in batch liquid culture. Our protocol combines cell permeable fluorescent dyes which probe molecular events associated with apoptosis (ROS species release from the mitochondria; nuclear DNA fragmentation) with 96‐well format to allow for the continuous measurement of both growth rate and apoptotic induction. As expected, background fluorescence intensity is much higher than in optimized microscopy‐based procedures. However, we see a significant shift in fluorescence intensity in cultures in which apoptosis has been induced by H2O2 or cation toxicity, compared to control un‐induced cultures. Importantly, this intensity shift is lost in mutants that fail to induce apoptosis under these conditions (ex. aif1Δ). We will describe the quantitative relationship between measurements made in batch culture and apoptotic rate measured by visualizing apoptosis‐specific stains using scanning confocal microscopy. We are currently leveraging the high‐throughput nature of this new assay to assemble a comprehensive dataset describing the quantitative response of yeast to dozens of apoptotic stress conditions across hundreds of mutant strains.Grant Funding Source: EJS, AJC, YL & GW supported by the HHMI Fellows Program & Grant Initiative; GW by Lenfest Grants.
Anti‐hyperlipidemic actions of synthetic nordihydroguaiaretic acid analogs (767.1)
Authors: Singh Madhurima , Tabassum Juveria , Li Yihang , Bittner Stefanie , Shen Wen‐Jun , Inayathullah Mohammed , Rajadas Jayakumar , Kraemer Fredric , Azhar Salman 
Published: June 2021

Previous studies have shown that creosote bush‐derived NDGA exerts beneficial actions on the key components of the Metabolic Syndrome including dyslipidemia, and insulin resistance (IR) in several relevant rodent models. Here we screened several synthetic analogs of NDGA with selective modification of the aromatic groups of NDGA and tested their efficacy against hepatic lipid metabolism in a high‐fructose fed (HFrD) Sprague‐Dawley (SD) rat model of insulin resistance, dyslipidemia and hypertension. Oral gavaging of HFrD‐fed SD rats for 4 days with NDGA analogs 1 and 2 (100 mg/kg, q.d.) suppressed the hepatic TG content, whereas treatment of rats with NDGA analogs 2, 3 and 4, like NDGA, decreased the plasma TG levels by 70‐75%. QRT‐PCR measurements demonstrated that among the NDGA analogs 1, 2 and 4, analog 4 was most effective in inhibiting the mRNA levels of genes and transcription factors involved in lipogenesis, while all three analogs almost equally inhibited the genes involved in TG synthesis. Unlike NDGA, none of these three NDGA analogs affected the genes of hepatic fatty acid oxidation or transport. Our data suggest that these NDGA analogs possess more potent anti‐hyperlipidemic properties than NDGA and have therapeutic potential to ameliorate dyslipidemia and associated IR.Grant Funding Source: Supported by Office of Research and Development, Medical Service, Dpt. of Veterans Affairs
You are what you eat: from high‐fat diet‐fed rodents to human diabesity (767.3)
Authors: Lai MeiChun , Chandrasekera P. Charukeshi , Barnard Neal 
Published: June 2021

Obesity and type 2 diabetes (T2D) are rapidly growing worldwide epidemics. It is accepted that interactions between obesity, insulin resistance, and β‐cell dysfunction cause T2D, but the mechanisms regulating the interplay among these impairments remain unclear. Rodent models of high‐fat diet (HFD)‐induced obesity are used widely to study human diabesity. However, even with 5000+ rodent‐based HFD publications on PubMed over the last decade, the mechanistic and therapeutic correlation to human diabesity has remained low. We investigated the reasons for this translational discrepancy by conducting a comprehensive review of HFD models. It appears that HFD models have limited translatable benefit due ‐at least in part‐ to divergent dietary compositions/protocols, species/strain variability, and inconsistent disease penetrance, severity, & duration. Combining HFD with other T2D induction modes exacerbates this species barrier ‐ mechanistic insights from these studies are often confined to rodents, rather than being applicable to humans. Therefore, future research dedicated to acquiring transnationally relevant data would accelerate our understanding and development of therapeutics for human diabesity.Grant Funding Source: Supported by Physicians Committee for Responsible Medicine
ACP modification to understand protein‐protein interactions in fatty acid biosynthesis (768.6)
Authors: Burkart Michael 
Published: June 2021

In order to investigate protein‐protein interactions, we have developed a suite of tools to modify the ACP with substrate analogs and reactive probes. These ACP conjugates are used to study the structure of the ACP using NMR and x‐ray crystallography. The interaction between E. coli AcpP and its dehydratase FabA, we have captured both the dynamic and static interactions. These highlight the molecular events by which FabA binds and spreads alpha helices II and III of AcpP, releasing the bound substrate to enter the active site of the enzyme. These tools can be generally applied to any carrier protein dependent pathway, including PKS and NRPS systems.Grant Funding Source: NIH‐NIGMS
Pick a ska or reggae beat? Rates of conformational switching in multi‐domain flavoproteins and their relationship to electron flux through the enzymes (768.8)
Authors: Haque Mohammad , Bayachou Mekki , Im Sang‐Choul , Pearl May , Waskell Lucy , Stuehr Dennis 
Published: June 2021

Multi‐domain enzymes rely on conformational motions to function. However, the conformational setpoints, rates of domain motions, and how they might combine to determine catalytic activity, are not well understood. To address this, we measured and compared conformational setpoints and rates of FMN domain motion in four members of the di‐flavin NADPH oxidoreductase family that catalyze important biological electron transfer reactions in mammals: cytochrome P450 reductase (CPR), methionine synthase reductase (MSR), endothelial and neuronal NO synthase (eNOS &amp; nNOS). We used stopped flow spectroscopy, single turnover methods, and a kinetic model that relates conformational setpoint and rates of FMN domain motion to electron flux through these enzymes. Despite their highly similar protein structures and functions, the four flavoproteins displayed a surprisingly broad range of conformational setpoints (20 to 62% open state) and FMN domain motion rates (vary 100‐fold, CPR > nNOS > MSR > eNOS). Each enzyme had a unique combination of the two parameters, which in eNOS and MSR resulted in their having similar electron flux (cytochrome c reductase activity) despite having fundamentally opposite conformational behaviors (eNOS has slow conformational opening, MSR has slow conformational closing). Together, our study provides the first estimates of conformational rate parameters in this family of redox enzymes, reveals that remarkable differences exist within the family, and explains how these conformational parameters combine to determine each enzyme’s electron transfer activities.Grant Funding Source: Supported by NIH GM51491