bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of the sRNA cosR in the oxidative stress response of Caulobacter crescentus (750.2)
Authors: Ramos Kevin , Ngadiaga Djamila , Laub Michael , Peterson Celeste
Published: June 2021

The ability to survive oxidative stress is crucial for all aerobically growing organisms. Reactive oxygen species (ROS) can disrupt cell membranes, proteins, electron transport chain, and DNA if they are not mitigated. Here we examine the role of a sRNA in the oxidative stress response in the model alpha‐proteobacterium Caulobacter cresentus. Upon oxidative stress, C. crescentus activates the transcription factor OxyR, which in turn directly regulates catalase, alphadyroxyl peroxidase and a sRNA called cosR. We monitor the survival of C. crescentus after exposure to hydrogen peroxide using colony forming unit viability assays and live dead stains. We find that overexpression of the sRNA confers protection to oxidative stress while the deletion of the sRNA causes mild impairment in survival after oxidative stress. Thus, OxyR appears to both directly and indirectly, through the sRNA cosR, activate genes that help the C. crescentus survive oxidative stress.
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
Biochemical and structural insight into the iterative oxidation mechanism of deoxy‐5‐methylcytosine on DNA by a Naegleria gruberi enzyme, 5‐methylpyrimidine dioxygenase 1 (768.16)
Authors: Saleh Lana , Tamanaha Esta , Trottier Karine , Dai Nan , Noren Christopher
Published: June 2021

Reversible methylation of DNA is a powerful and ubiquitous mechanism for dynamic control of gene expression. The recent discovery that deoxy‐5‐methylcytosine (d5mC) in mammalian DNA can be iteratively oxidized by ten eleven translocation (TET) proteins to generate deoxy‐5‐hydroxymethylcytosine (d5hmC), deoxy‐5‐formylcytosine (d5fC), and deoxy‐5‐carboxycytosine (d5caC), marks a breakthrough in the field of epigenetics. These oxidized bases are predicted to be both novel epigenetic markers and part of the d5mC demethylation pathway. Mammalian TETs (mTETs) belong to the FeII/α‐ketoglutarate‐dependent dioxygenase (FeαKGO) family, which is widely distributed across all three kingdoms of life, including the heterolobosean amoeboflagellate, Naegleria gruberi. Little is known about the mechanistic and structural bases for substrate specificity and product profile in members of the FeαKGO TET/JBP subfamily, with oxidative activities on 5‐methylpyrimidines in nucleic acids, due to challenges in in vitro protein production of these enzymes. Herein, we report the first biochemical and structural characterization of the N. gruberi protein, 5‐methylpyrimidine dioxygenase 1 (mYOX1), which bears ~ 39 % similarity to mTETs. Similar to mTETs, mYOX1 iteratively oxidizes d5mC on DNA to d5caC in vitro. Our structure and detailed kinetic studies offer novel insight into the mode of substrate recognition and product release by mYOX1. Furthermore, the ease of production of mYOX1 as a recombinant protein in Escherichia coli renders it an ideal molecular diagnostic tool for the detection and mapping of oxidized d5mC epigenetic markers in various genomic contexts.
Designing oligonucleotide conformational states for quantitative transcription factor detection (768.2)
Authors: Bonham Andrew
Published: June 2021

Nucleic acids undergo dynamic conformational rearrangements that have enabled a variety of bio‐sensors, biologically‐inspired probes with applications from real‐time PCR to the detection of small molecules. Understanding the forces that drive these rearrangements enables more sophisticated and accurate bio‐sensors. Transcription factor beacons (TF beacons) are versatile, nucleic acid‐based optical bio‐sensors that report the concentration of TFs. As many cancers over‐express oncogenic TFs, optimizing tools for early detection of these biomarkers would improve diagnosis of early‐stage cancers. We have investigated how changing the thermodynamic and kinetic stability of TF beacons’ ground state conformations and excited state conformations enable tuning of sensitivity and detection limit. To aid this process, and for wider adoption of TF beacons, we have developed web servers for the automated design of TF beacons against arbitrary protein targets. Ultimately, better understanding of nucleic acid conformational changes and stability enables the development of efficacious bio‐sensors with a range of applications, including detecting TF level changes in patient samples for earlier point‐of‐care diagnosis.
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 & 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
Elucidation of the mechanism by which β‐arrestin‐1 regulates LIM kinase 1 (773.3)
Authors: Lee Kyu , Lin Alice , Awan Rashid , Defea Kathryn
Published: June 2021

Elucidation of the mechanism by which β‐arrestin‐1 regulates LIM kinase 1 Kyu Lee, Alice Lin, Rashid Awan, and Kathryn Defea. Department of Biochemistry and Molecular Biology, University of California, Riverside, Riverside, California 92521 β‐ arrestins are multifunctional proteins which can mediate termination of G‐protein signaling and internalization of receptor upon binding to G‐protein‐coupled receptor (GPCR). Also, β‐arrestins can transduce signals to downstream targets in G‐protein independent signaling manner. Protease‐activated receptor‐2 (PAR2), G‐protein couple receptor, can be activated by proteolytic cleavage of N‐terminus, which exposes a tethered ligand (SLIGRL/SLIGKV,human/mouse) leading to Gαq signaling while simultaneously promote activation of Cofilin, actin filament severing protein, through β‐arrestins. Cofilin, which can sever the existing filaments and create many barbed ends for the rapid changes, is necessary for chemotaxis or cell migration. Cofilin can be regulated by LIM kinase (LIMK), inactivating by phosphorylation at Ser 3, and regulated by chronophin (CIN), activating by dephosphorylation at Ser 3. We have previously shown that β‐arrestin can interact with cofilin, LIMK, and CIN and regulate their activities. However, the mechanism how β‐arrestin‐1 (β1) can interact and regulate LIMK and cofilin upon PAR‐2 activation has not been investigated explicitly. Here, using multiple recombinant β1 truncations in sandwich immunoassays, in vitro kinase assays and co‐immunoprecipitation, we demonstrate that the specific regions in β1 scaffold and regulate the kinase activity of LIMK. Thus, this study furthers characterize the interaction between β1 and LIMK.
In pursuit of mammalian polyamine oxidase crystal structure (774.4)
Authors: Zamora Diana , T. Melendez Jose , Galaleldeen Ahmad
Published: June 2021

Mouse Polyamine Oxidase (mPAO) is a mammalian flavoprotein that is necessary for the catabolism of polyamines. mPAO oxidizes the endo carbon nitrogen bonds of N1‐acetylspermine and N1‐acetylspermidine when oxidized to spermine and spermidine respectively. It is known that normal levels of polyamines are important for cell growth, yet their actual function is not well understood. The structure of mammalian PAO has yet to be determined; however structures for maize and yeast PAO (Fms1) exist. Although the identity between mPAO, Fms1, and maize PAO is only 20%, it has been shown, through sequence comparison and mutational analysis, that a conserved histidine residue at position 67 of Fms1 and 64 of mPAO helps to properly position the amine substrate for oxidation. Determining the crystal structure of mPAO will help identify other residues that play a role in substrate binding and catalysis.

mPAO was cloned into pAG8H, a modified pET19d vector that introduces a cleavable his‐tag at the N‐terminus of the protein. mPAO was expressed and purified using a Nickel column followed by an anion exchange column. Following the purification process, analytical ultracentrifugation sedimentation velocity experiments were performed to determine the oligomerization state and homogeneity of the protein.

The sedimentation velocity experiments revealed a homogeneous, monomeric mPAO in solution, in contrast to the dimeric form of Fms1 in solution. Crystallization trials are underway to determine the structure of mPAO.
Crystal structure of MraY, an essential membrane enzyme in bacterial cell wall synthesis (775.1)
Authors: Lee Seok‐Yong , Chung Ben
Published: June 2021

The Phospho‐MurNAc‐pentapeptide translocase (MraY) is a membrane‐spanning enzyme involved in an essential process of bacterial cell wall synthesis: transfer of the peptidoglycan precursor phosphor‐MurNAc‐pentapeptide to the carrier lipid undecaprenyl phosphate. MraY belongs to a subfamily of the polyprenyl‐phosphate N‐acetyl hexosamine 1‐phosphate transferase (PNPT) superfamily whose members are involved in various biological processes including prokaryotic cell envelope polymer synthesis and eukaryotic N‐linked glycosylation. MraY has been a promising target for the development of antibiotics because it is the target of many different classes of natural product antibiotics. Despite the importance, the lack of a structure has hampered our understanding of the mechanism of the enzyme function and inhibition by natural product inhibitors. We report the crystal structure of MraY from Aquifex aeolicus (MraYAA) at 3.3 Å resolution, the first structure of a member of the PNPT superfamily. The crystal structure, together with crystallographic and functional studies, reveals the architecture of MraYAA, the location of Mg2+ at the active site and the putative binding sites of both substrates. Our crystallographic studies provide insights into the mechanism of how MraY attaches a building block of peptidoglycan to a carrier lipid.
Protein function prediction using ProMOL and PyMOL (777.3)
Authors: McKay Talia , Hart Kaitlin , Bernstein Herbert , Tedla‐Boyd Weinishet , Craig Paul
Published: June 2021

ProMOL, a plugin for the molecular graphics system PyMOL, was designed to characterize protein structures that lack functional annotation; there are currently over 3700 PDB entries with “unknown” functions. ProMOL was created, and is constantly being revised, by members of the RIT and Dowling College communities. ProMOL compares query protein structures against a library of active site motif templates, calculates Levenshtein distances and RMSD values, and generates a 3D visual alignment between query and motif template. The best results are then submitted to BLAST, Dali and Pfam for analysis. If the data produced by these four bioinformatics programs lead to a preliminary conclusion concerning the function of an uncharacterized structure, the behavior of the protein is then tested in vitro. To date, we have found >90 significant “hits” for uncharacterized protein structures using ProMOL’s motif library. These structures are currently being analyzed via BLAST, Dali, and Pfam. To date the most promising alignments include PDB entries 2HRZ (proposed as isomerase/NAD‐dependent epimerase), 4EZI (confirmed as an esterase; see the related poster by Kaitlin Hart), 3L1W (proposed as a nuclease), and 2O14 (proposed as a hydrolase/aminopeptidase). This work has been supported in part by National Institute of General Medical Sciences 2R15GM078077‐02, 3R15GM078077‐02S1.

Grant Funding Source: Supported by NIGMS 2R15GM078077‐02, 3R15GM078077‐02S1
Metallomics and proteins expression of diabetic rats (778.2)
Authors: Braga Camila , Vieira José , Moraes Paula , Cavecci Bruna , Fernandes Ana Angélica , Okoshi Marina , Padilha Pedro
Published: June 2021

The aim of this study was to analyze changes in the expression of proteins and compare metal ions (Cu, Zn, Se= using atomic absorption spectrometry with flame and Mg= using atomic absorption spectrometry with oven graphite) in plasma gels (two‐dimensional electrophoresis, pool, eight replicates/group, strips with pH 4‐7, polyacrylamide gels at 10% w/v) of normal (G1), diabetic (G2) and diabetic treated with insulin (G3) rats. In the context, 251 spots were found in G1, 300 in G2 and 227 in G3 (percentual matching of 77, 84 and 85%. The differential analysis indicated difference in expression (p<0.05‐ ANOVA) of 13 (G1xG2), 10 (G1xG3) and 18 (G2xG3) spots when groups were compared. The qualitative determination of metal ions of interest indicated to G1, G2 and G3 that 87 (48.60%), 154 (75.49%), 48 (23.36%) contained Cu; 33 (18.43%), 47 (23.03%), 141 (71.57%) Mg; 148 (82.68%), 108 (52.94%), 155 (78.68%) Se and 19 (10.61%), 7 (3.43%), 69 (35.02%) Zn. Observed difference in the presence of ions in the same protein in relation to different groups, and the next is to understand how these ions are coordinated to protein.

Grant Funding Source: Financial Support: Fapesp 2011/21672‐1
Growth of Escherichia coli DL41(DE3) in enhanced chemically defined media in the presence of L‐telluromethionine (778.7)
Authors: Cooper Brent , Taylor Savannah , Broderick Kathleen , Boles Jeffrey
Published: June 2021

In analyzing the three‐dimensional structure of a protein, the x‐ray crystallography endeavor can be improved by incorporating unnatural amino acids into that protein. Most naturally occurring atoms do not have sufficient mass to diffract electrons. Using unnatural amino acids containing a metalloid provides a solution to this problem. Bioincorporation of L‐Telluromethionine can provide residues with sufficient mass for electron scattering when incorporated serendipitously. However telluromethionine has cytotoxic effects which reduce cell growth and uptake. A proteomic analysis of telluromethionyl Escherichia coli was carried out using the methionine auxotroph E. collie DL41(DE3) (pCock) expression system. Both telluromethionyl and methionyl exposed cultures were analyzed with the objective of identifying differentially expressed proteins. The biological roles of differentially expressed proteins was elucidated and have been applied to improving methods for the bioincorporation of telluromethionine via the supplementation of chemically defined media with metabolites that were negatively affected due to the exposure to L‐Telluromethionine.
Development of stabilizers for live cell preservation at room temperature (779.10)
Authors: Diaz Paul , Muller Rolf , Desharnais Joel , Brewer Kai , Whitney Scott
Published: June 2021

Cyropreservation at ultra‐low temperatures utilizing freezing mediums containing DMSO is the current best practice for long term storage of mammalian cells. Such conditions can impart devastating losses caused by common failures of the cold storage systems as well as the sensitivity of valuable cell types both primary and engineered to the freezing and thawing process. Hence recovery of viable cells from the frozen state is challenging and as is typical for many cell types only a fraction of the cells survive with the fully desired phenotype. Based on the studies of extremophiles such as tardigrades, rotifers or brine shrimp that can survive for many years in the dry state we hypothesized that techniques and protocols could be developed that mimic this molecular phenomena in eukaryotic cell cultures. Many previous candidate approaches or the last 15 years have failed to preserve cells under these conditions over the short term and no method to date has been successful at preserving cells under such conditions for more than a few hours. We applied a combinatorial screening approach to identify formulations containing both biostabilizers that preserve air‐dried eukaryotic cells at ambient temperature. The use specific biostabilizers in specific formulations protect cells from the stress of dehydration, dry‐storage, and rehydration while preventing the execution of apoptosis programs. We report here that we are able to achieve statistically significant improvements in cell survival rates and preservation time as compared to conventionally published dry down methods. These results provide a roadmap for achieving room temperature storage of mammalian cells in a dry state. The technology can be further developed for products that extend cell survival at ambient temperatures. This technology could eventually be applied to ambient storage of eukaryotic cell stocks for biomedical research, toxicology screens, cell‐based therapeutics and blood supply logistics.
A novel dynamic delivery system enabling high efficiency transfection of cells (779.4)
Authors: Ludtke James , Lauer Anthony , Storck Austin , Rossi Nicholas , Neder Karen , Pinchuk Anatoly , Juckem Laura
Published: June 2021

Transfection is a technique to study expression and knockdown of gene expression. Over the past decade, many researchers have utilized liposomal systems to deliver nucleic acids; however, this method has many caveats including low transfection efficiency and high cellular toxicity. To address these shortcomings, Mirus Bio has developed a novel non‐liposomal technology called the TransIT‐X2™ Dynamic Delivery System. This unique polymeric delivery system is able to condense negatively charged nucleic acids (plasmid DNA and/or siRNA), enter cells and break out of the endosomal compartment, a critical barrier for transfection. The plasmid and siRNA can then be delivered to their respective functional locations, the nucleus and the cytoplasm.

In this study, we demonstrate functional transfection of labeled plasmid DNA co‐delivered with labeled siRNA illustrating the dual abilities of the system. Additionally, extensive head‐to‐head comparisons with Lipofectamine® 2000 were performed using a luciferase expression assay in 30 cell types; TransIT‐X2 demonstrated superior performance in 20 cell types with 11 showing greater than 2‐fold higher expression compared to Lipofectamine 2000. Transfection efficiency of green fluorescent protein (GFP) was measured using flow cytometry and visualized by microscopy; most cell types had greater than 50% GFP efficiency. Lastly, gene knockdown after delivery of siRNA or miRNA was assessed. Two endogenous genes, GAPDH and AHA1, were targeted for siRNA knockdown and mRNA levels were assessed by qRT‐PCR. miRNA function was assessed by the decrease in the miR‐1 target mRNA PTK9. TransIT‐X2 shows effective knockdown via siRNA or miRNA.

The TransIT‐X2 Dynamic Delivery System offers researchers exceptional broad‐spectrum transfection of plasmid DNA and siRNA. This cutting edge, non‐liposomal polymeric delivery method enhances endocytic release of the transfection complexes allowing researchers to obtain very high transfection efficiencies.
Cytosolic calcium regulates nanoparticle egress from alveolar epithelial cells (780.11)
Authors: Sipos Arnold , Chow Robert , Borok Zea , Kim Kwang‐Jin , Crandall Edward
Published: June 2021

We previously reported that polystyrene nanoparticles (PNP) translocate across alveolar epithelium. In this study, we investigated PNP exit from alveolar epithelial cells (AEC) using confocal live‐cell imaging. Primary type I cell‐like rat AEC monolayers on Anopore filters were apically exposed to 80 μg/mL PNP (20 nm, carboxyl‐modified, near‐infrared labeled) for 12 h, followed by washing and measuring cytoplasmic [PNP] as a function of time, in the presence/absence of agents that elevate (e.g., ATP) or chelate (e.g., BAPTA‐AM) intracellular [Ca2+] ([Ca2+]i), as confirmed with fluo‐4, and/or nocodazole (Noc), an inhibitor of microtubule polymerization. Results show that cytoplasmic PNP both co‐localize with lysotracker‐positive endosomes and exit cells in two stages: initial rapid egress for ~2 h, followed by slower linear egress. Overall t0.5 for egress is 1.5 h, much faster than that for apical PNP uptake. ATP increases [Ca2+]i and egress rate (to t0.5 ~0.5 h), while BAPTA prevents increased [Ca2+]i and egress rate after ATP exposure. Noc, with or without ATP, inhibits PNP exit via the rapid initial egress process. These data suggest that: (1) PNP egress rate is dependent on [Ca2+]i, (2) PNP uptake is rate‐limiting for transepithelial PNP translocation, (3) PNP egress occurs via both rapid lysosomal exocytosis and slow ‘diffusion’‐like process(es), and (4) kinetics of egress of PNP from AEC can be modulated.

Grant Funding Source: NIH; Hastings & Whittier Foundations
Functionalizing silica‐coated iron oxide nanoparticles for imaging and targeted cancer therapeutics (780.3)
Authors: Nagalla Raji , Liao Zi Wei , Webb Andrew , Flynn Nolan , Kolodny Nancy
Published: June 2021

This project focuses on the surface modification of silica‐coated superparamagnetic iron oxide NPs to achieve tissue specific drug delivery to pancreatic tumors. The treatment of pancreatic cancer remains a challenge in the biomedical community. Over half of cases are diagnosed after the cancer has spread to other tissues, and in these cases the 5‐year survival rate is 2%. The non‐specific nature of currently available chemotherapies has spurred interest in developing vehicles to deliver potent therapies specifically to tumors. Nanoparticles (NPs) present an ideal vehicle due to their high surface area, small size, and low toxicity. Recent work explored the conjugation of NPs to a targeting moiety: monoclonal antibody CHO 31.1, which recognizes gpA33, a membrane glycoprotein overexpressed in 50% of pancreatic cancers and 95% of colorectal cancers. Building on existing methods, the antibody was attached to the silica surface of the NPs via a polyethylene glycol (PEG) linker. In vitro techniques were also developed to quantify NP detection using magnetic resonance imaging (MRI). Attachment of multiple moieties was explored using orthogonal surface chemistries. Cellular internalization was determined using confocal microscopy. Moving forward these novel nanoparticles will be further characterized for morphology, size and zeta potential, and for their utility as contrast agents for tracking in vivo.

Grant Funding Source: Supported by NIH grant #1R15CA156393‐01A1
In vitro reconstitution of lipid‐dependent membrane protein topological switching (785.1)
Authors: Vitrac Heidi , Bogdanov Mikhail , Dowhan William
Published: June 2021

The mechanism by which membrane proteins exhibit structural and functional duality in the same membrane or different membranes is unknown. Systematic alteration of lipid membrane composition uncovered a role for lipid‐protein interactions in determining dynamic post‐assembly changes in and initial topogenesis of membrane proteins. Using an in vitro proteoliposome system in which lipid composition can be systematically controlled not only before (liposomes) but also after (fliposomes) reconstitution, we have determined the minimum and sufficient requirements for lactose permease (LacY) to flip between topologically distinct states. Changes in phosphatidylethanolamine levels after final protein folding resulted in re‐orientation of the original conformer mixture, establishing a dynamic reversibility in topology without molecular chaperone or translocon involvement. Using Förster resonance energy transfer approaches, we further investigated the kinetics of lipid exchange and LacY flipping. The rates of protein flipping in both directions were on a second scale and occurred almost instantaneously as the lipid composition of the proteoliposomes changed. These observations demonstrate a potential thermodynamically driven lipid‐dependent biological switch for generating dynamic structural and functional heterogeneity for a protein within cells independent of other cellular factors.

Grant Funding Source: Supported by NIH grant GM R37 20478
Oligomerization of Hsp70 upon insertion into lipid membranes (785.2)
Authors: Armijo Gabrielle , Cauvi David , Okerblom Jonathan , Lopez Victor , Gonzales Derek , Arispe Nelson , De Maio Antonio
Published: June 2021

Heat shock proteins (hsp) are well recognized for their protein folding activity. Additionally, hsp expression is enhanced during stress conditions to preserve cellular homeostasis. Hsp are also detected outside cells, released by an active mechanism independent of cell death. Extracellular hsp appear to act as signaling molecules as part of a systemic response to stress. Extracellular hsp do not contain a consensus signal for their secretion via the classical ER‐Golgi compartment. Therefore, they are likely exported by an alternative mechanism requiring translocation across the plasma membrane. We propose that membrane interaction is the first step in the export process of Hsp70, the major inducible hsp, since this protein has been detected on the surface of stressed cells. The question that emerges is how does this highly charged cytosolic protein interact with lipid membranes? Using a liposome insertion assay, we showed that Hsp70 selectively interacted with negatively charged phospholipids, particularly phosphatidyl serine (PS), within liposomes, which was followed by insertion into the lipid bilayer, forming high molecular weight oligomers. Hsp70 displayed a preference for less fluid lipid environments, and the region embedded into the lipid membrane was mapped to the C‐terminus end of the molecule. The results from our studies provide evidence of an unexpected ability of a large, charged protein to become inserted across a lipid membrane. This observation provides a new paradigm for the interaction of proteins with lipid environments. In addition, it may explain the export mechanism of an increasing number of proteins that lack the consensus secretory signals.

Grant Funding Source: Supported by NIH R01 GM098455
Unraveling mTOR signaling: from two complexes to nuclear dynamics (786.1)
Authors: Sarbassov Dos
Published: June 2021

The protein kinase mTOR is a central component of the essential and highly conserved nutrient‐sensing pathway. Our biochemical characterization of mTOR led to discovery of a novel nuclear envelope mTOR complex that is distinct from the known complexes of mTOR (mTORC1 and mTORC2) by size and also by the sub‐cellular localization. We found that mTOR in association with the nuclear pore component forms the nuclear envelope complex that regulates the nutrient‐dependent nuclear import of ribosomal proteins. We found that similar to the amino acid deprivation, the mTOR kinase inhibitors caused a substantial decrease in abundance of ribosomal proteins in the nuclear but not cytosolic fraction. The mTOR‐dependent nuclear import of ribosomal proteins is also supported by analysis of the GFP‐tagged ribosomal protein in the cell based imaging assays. The nuclear abundance of ribosomal proteins was not affected by inhibition of mTORC1 by rapamycin or loss of mTORC2. Thus, our study identified a novel role of mTOR in regulation of the nuclear import of ribosomal proteins that might play a role in ribosomal biogenesis and contribute to the nutrient‐dependent regulation of cell growth.

Grant Funding Source: Supported by Texas State (CPRIT)
Structure and catalytic mechanism of D‐glucuronyl C5 epimerase, and the role in neuritogenesis regulation in PC12 cell (787.1)
Authors: Qin Yi , Li Jie , Xu Eric , Ding Kan
Published: June 2021

Neurodevelopment is orchestrated by a series of growth factor‐heparan sulfate (HS) interactions which were involved in neuritogenesis. HS is a glycosaminoglycan which is present on the cell surface and in the extracellular matrix interacting with diverse signal molecules and is essential for many physiological functions. D‐glucuronic acid epimerase (Glce) is a critical enzyme involving in HS synthesis, which converts D‐glucuronic acid to L‐Iduronic acid. Here we determined the crystal structures of Glce dimer in apo form and in complex with heparin hexasaccharide. The oligosaccharide bound at a cleft contained within a middle α‐helical domain. We identified that each Glce molecule had one catalytic domain, which was a cleft encircled by eight α‐helixes and enriched with tyrosine and arginine residues. Through mutagenesis and the complex structure, we proposed a catalytic mechanism of sugar epimerization by a proton abstraction and readdition through Y546 and a functional water molecule between Y468 and Y528. Our results also showed that Glce depletion caused PC12 cell growth arrested and promoted the cell neuritogenesis and differentiation induced by nerve growth factor (NGF). PC12 cell growth was boosted by overexpression of Glce, however the neuritogenesis was impaired when Glce‐depletion was rescued. We further showed that Glce‐depletion blocked Smad1/5/8 phosphorylation, however this signaling could be restored by Glce. In addition, the downstream effector of Smad1/5/8, Id3 was induced by Glce. Id3 silence inhibited PC12 cell growth and induced the cell neuritogenesis and differentiation. The results suggest that Glce plays a key role in PC12 cells growth and neuritogenesis through Smad/Id3 signaling and improve our understanding the HS synthesis reaction and aids the drug discovery for nervous system disease treatment.