bluuebunny/crossref_metadata_2025_split huggingface dataset:
Developing a Physical Model of HigB Toxin and its Endonuclease Cleavage Mechanism
Authors: Chabbra Alesa , Alzamora Carolina , Ghali Helana , Handal Marina , Kathiria Mahima , Martinek Alexander , Nguyen Jenny , Patel Chandni , Patel Shreja , Pham Antoine , Lavin Emily Schmitt 
Published: June 2021

The CREST (Connecting Researchers, Educators, and STudents) team at Nova Southeastern University created a physical model depicting the mechanism of endonuclease cleavage by the HigB protein as described in the literature. The proteins, toxin HigB and antitoxin HigA, are part of a type II toxin‐antitoxin system that cleaves ribosome‐bound mRNAs in bacteria. Chemical or physical stresses, such as extreme temperatures and lack of nutrients, lead to antitoxin degradation that releases HigB. Temporary inhibition of growth by HigB allows cells to more efficiently utilize current resources. Although HigB is referred to as a toxin, it is not inherently detrimental and can be beneficial to cellular survival. Bacteria that use this mechanism include Escherichia coli, Pseudomonas aeruginosa, and Proteus vulgaris. As such, HigB is of clinical interest due to its contribution in prolonging bacterial infections through antibiotic tolerance (persistence). When cells are not in a stressed state, HigA binds to and inactivates HigB, allowing growth and translation to proceed. Details of HigB in complex with the ribosome as well as residues important for mRNA cleavage were found in the Protein Data Bank file, 4ZSN, and imported into Jmol, a protein visualization software. The 4ZSN file consisted of several nucleic acid and protein bundles; Bundle 1 was examined in more detail. Specific program commands were developed to manipulate the original file into a format that was later 3‐D printed to create an instructional molecular model, which was funded in part by NSF‐DUE 1725940 for the CREST Project. This model highlights the ribosomal proteins, ribosomal RNA, and HigB along with its residues that are essential for mRNA cleavage. The N and C termini of the HigB protein were also indicated. This molecular model can be used to explain how ribosome‐dependent proteins (HigB) target ribosome‐bound mRNAs in bacterial systems. Providing a 3‐D tool allows students to engage actively in understanding the complex process of endonuclease cleavage.Support or Funding InformationThe development of this 3‐D molecular model was funded in part by NSF‐DUE 1725940 for the CREST (Connecting Researchers, Educators, and STudents) Project.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cell Type‐ and Protein‐Specific Induction of LAMP3
Authors: Zhao Ming , Venosa Alessandro , Tomer Yaniv , Beers Michael F. , Mulugeta Surafel 
Published: June 2021

RATIONALEThe family of lysosome‐associated membrane proteins (LAMP) includes LAMPs 1, 2, and 3. While LAMPs 1 and 2 are ubiquitously expressed and account for half of the proteins in the lysosomal membrane, LAMP3 (DC‐LAMP) is only expressed in distinct cell types. LAMP3 expression is associated with poor prognosis of certain cancers, Parkinson's disease, and is believed to contribute to the unfolded protein response by promoting protein degradation and cell survival during proteasome dysfunction. LAMP3 is constitutively expressed in and has been used as a subcellular marker for alveolar type 2 (AT2) cells primarily localizing in the lamellar bodies (the principal storage site of pulmonary surfactant). In this study, the expression of LAMP3 in various cell types and the mechanisms regulating its induction were evaluated.METHODSIn vitro cell line models (including lung epithelial A549 and H441 cells, kidney epithelial HEK292 cells, and cervical cancer cell line HeLa cells) transiently expressing fluorescent‐tagged proteins were used for co‐expression studies with immunostained LAMP3 by confocal microscopy. Additionally, LAMP3 mRNA levels in cells prior to and following transient expression were assessed by real‐time PCR.RESULTSWe discovered that none of the cell lines studied constitutively expresses LAMP3 (mRNA or protein). However, upon stimulation of cells by transient expression of certain proteins, A549 and HeLa cells strongly and weakly induced LAMP3 expression, respectively, while no LAMP3 induction was observed in either H441 or HEK293 cells. In A549 cells, transient expression of the AT2 cell specific proteins (ABCA3 and SP‐C) and the huntingtin poly‐Q repeat peptides (Mtt‐25 and Mtt‐72) induced LAMP3 expression. Media‐transfer assays also showed that LAMP3 induction takes place via paracrine signaling. In contrast, LAMP3 protein expression was absent in cells expressing empty fluorescence vector constructs including GFPC1, DsRedC1, and pcDNA3, as well as fluorescent‐tagged proteins including mCherry/LC3, RFP/Rab7, and GFP/NEDD4‐2. LAMP3 mRNA levels in these cells were comparable to those that induced protein expression, but the proteins appeared to be rapidly degraded by the cell's quality control system as demonstrated by the appearance of LAMP3 protein expression following autophagy and proteasome inhibition assays.CONCLUSIONThese results confirm, in vitro, the specific nature of LAMP3 expression in certain cells types and demonstrate its dependency on stimulation by distinct proteins, paracrine signaling, and disruption of cell quality control.Support or Funding InformationNIH HL 129150 (SM), NIH HL 119436 (MFB), VA Merit Award BX001176 (MFB)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanism and stability of type II bacterial toxin‐antitoxin complexes and the role of regulated antitoxin proteolysis that releases toxin
Authors: Pavelich Ian , Dunham Christine M. 
Published: June 2021

Antibiotic resistance is the most prominent threat facing modern medicine and is unlikely to be resolved by novel antibiotics or reduction of improper antibiotic use. New strategies to combat antibiotic resistance by targeting novel bacterial pathways are necessary. One emerging approach focuses on understanding how bacteria adapt to diverse stresses by the inhibition of growth and transition to a dormant state. These bacteria, termed ‘persisters’, tolerate antibiotics without genetic mutations. This phenotype is triggered by the stringent response whereby transcriptional reprogramming and translational inhibition occurs. A class of ubiquitous bacterial gene pairs called toxin‐antitoxin complexes (TAs) undergo changes during the stringent response that activate the toxin component. Normally antitoxins bind and sequester their cognate toxins to function as autorepressors. In stress conditions, antitoxins are degraded by cellular proteases which frees toxins to inhibit growth until stress passes. We and others demonstrated that proteases recognize C termini of antitoxins. Many studies have focused on identifying toxin targets, yet the stability of TAs and how this influences proteolysis is unknown. Given the structural diversity of antitoxins, it is also unclear how proteases recognize antitoxins or if a common mechanism exists to describe all antitoxin proteolysis. This is an important question as proteolysis is the main step in the release of toxin and the initiation of bacterial persistence. Here, we examine the regulation of E. colitype II TAs DinJ‐YafQ, RelB‐RelE, and YefM‐YoeB. The structures of these pairs are known and demonstrate differences in how DinJ and RelB antitoxins interact with their toxins as compared to YefM. Each pair also forms higher ordered complexes that are differentially regulated. We designed C‐terminal truncations and measured their impact on toxin release and antitoxin stability. We find that DinJ and RelB share common structural motifs at their C termini that are important for toxin sequestration. YefM lacks this motif and its C‐terminal plays no role in toxin sequestration. Further, we explore the role of proteases, these motifs, and effects of diverse stress in targeted antitoxin proteolysis. This work identifies features of antitoxins that may recruit proteases and how proteolysis is a major underlying component of stress allowing bacteria to transition to antibiotic persistent states.Support or Funding InformationThe Dunham laboratory thanks the following sources for funding: NSF CAREER MCB 0953714, NIH NIGMS R01GM072528. Dr. Dunham is a Burroughs Welcome Investigator in the Pathogenesis of Infectious Diseases. Ian Pavelich is an ARCS awardee.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PAM forms an atypical SCF ubiquitin ligase complex that ubiquitinates and degrades NMNAT2
Authors: Desbois Muriel , Crawley Oliver , Evans Paul R. , Baker Scott T. , Masuho Ikuo , Yasuda Ryohei , Grill Brock 
Published: June 2021

PHR (PAM/Highwire/RPM‐1) proteins are conserved RING E3 ubiquitin ligases that function in developmental processes, such as axon termination and synapse formation, as well as axon degeneration. At present, our understanding of how PHR proteins form ubiquitin ligase complexes remains incomplete. Although genetic studies indicate NMNAT2 is an important mediator of PHR protein function in axon degeneration, it remains unknown how PHR proteins inhibit NMNAT2. Here, we decipher the biochemical basis for how the human PHR protein PAM, also called MYCBP2, forms a noncanonical Skp/Cullin/F box (SCF) complex that contains the F‐box protein FBXO45 and SKP1 but lacks CUL1. We show FBXO45 does not simply function in substrate recognition but is important for assembly of the PAM/FBXO45/SKP1 complex. Interestingly, we demonstrate a novel role for SKP1 as an auxiliary component of the target recognition module that enhances binding of FBXO45 to NMNAT2. Finally, we provide biochemical evidence that PAM polyubiquitinates NMNAT2 and regulates NMNAT2 protein stability and degradation by the proteasome.Support or Funding InformationNational Institutes of Health Grant R01 NS072129This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification and Interaction Between Centrosomal Protein Centriolin and the E3 Ubiquitin Ligase, HECTD1
Authors: Salas Jesus , Garcia Alexander , Zora Vancy , Gromley Adam , Gromley Zeynep 
Published: June 2021

Uncontrolled cellular division is a central hallmark of cancer. Thus, studying the molecular functions and interactions of the proteins that regulate the complex processes of cell division is crucial. Two proteins are at the focal point of this study. One is centriolin, a 270 kD protein that has been demonstrated as essential for cytokinesis. The second is a 289 kD E3 ubiquitin ligase called HECTD1. Prior experiments utilizing a yeast two hybrid screen concluded that there is an interaction between centriolin and HECTD1. Furthermore, results of immunoprecipation and western blot from synchronized cells showed that centriolin was diminished in G0, whereas HECTD1 levels were elevated compared to other phases of the cell cycle. In contrast, centriolin concentrations were much more robust in S phase, G1, and M phases, compared to that of G0. In separate experiments, we have used CRISPR to eliminate either centriolin or HECTD1 in HeLa cells to observe the repercussions of the loss of these proteins and their global effects on the cell cycle. Interestingly, we observed significant cell death when we eliminated the HECTD1 gene. Our further experiments revealed that the cell death is due to robust induction of apoptosis. Currently we are investigating whether or not the established interaction between centriolin and HECTD1 plays a role in the cell death that we observed.Support or Funding InformationThis research was supported by Lincoln Memorial University‐DeBusk College of Osteopathic Medicine Intramural Grant.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating proteolytic activity of engineered trypsin variants using naturally occurring macromolecular substrates
Authors: Villa Rodolfo 
Published: June 2021

Serine proteases constitute numerous physiological roles that include but are not limited to the breakdown of food, the participation in immune responses, and with the triggering of cell apoptosis Engineered serine proteases as therapeutic agents constitute a new class of drugs for potential clinical applications. However, one main complication faced when using serine proteases as therapeutic agents is their relatively short half‐life due to the abundance of tight binding inhibitors in the human body that target serine proteases to regulate proteolytic activity and rapidly remove them from circulation. For a protease to be effective it must display resistance towards inhibitors. Previously engineered trypsin variants K60A, K60V, Y39A, Y39F have shown to diminish prime side interactions, affecting micromolecular inhibition through the disruption of hydrogen bond networks occurring at residue positions 39 and 60. These variants displayed resistance towards certain naturally occurring inhibitors, bovine pancreatic trypsin inhibitor and M84R ecotin. Kinetic data was performed using commercially available tripeptide Z‐GPR‐pNA, also indicating no change of specificity. In order to ascertain the viability of these engineered variants as potential therapeutics we must study the interactions between naturally available macro molecular substrates. Experimental methods required the design and utilization of a novel spectroscopicassay tomonitor the proteolytic activity of trypsin variants using chymotrypsinogen (Cg) as our naturally occurring macromolecular substrate. Within the body, trypsin cleaves triggering the activation of zymogen Cg into its active form. Fluorescence will be measured using chymotrypsin specific 4‐methylumbelliferyl p‐tri‐methylammoniocinnamate chloride (MUTMAC). Preliminary data has shown that a 10:1 concentration ratio (100nM Trypsin to 10μM Cg) proved most efficient for activation. However, only ~22% of chymotrypsinogen has been activated using wild‐type trypsin, suggesting that further optimization of this assay is still required. Future work will aim to elucidate the proteolytic rates of these trypsin variant K60A, K60V, Y39A, Y39F where we hope to obtain a better understanding of the interactions between these variants and naturally occurring substrates.Support or Funding InformationNIH MS/PhD Bridge: R25‐GM048972This 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 Allosteric Regulation of BAM2 in Arabidopsis thaliana
Authors: Chandrasekharan Nithesh , Roy Ian R , Monroe Jonathan D , Berndsen Christopher 
Published: June 2021

Starch is an important component of the human diet as it is broken down into glucose to provide energy for metabolic reactions in the body. The amount and sources of starch that humans consume greatly varies around the world depending on the plants that are indigenous to each diet. In plants, starch is hydrolyzed into the disaccharide maltose by β‐amylase enzymes, a process that is important night time metabolism and the response to stress in plants. By understanding starch regulation by β‐amylases, we could potentially improve the quality of food crops which will benefit those who suffer from food insecurity and nutritional deficiencies. Arabidopsis thaliana contains nine β‐amylases (BAMs) that show distinct catalytic activities and functional regulation. Starch is located in the chloroplast and there are 4 chloroplastic BAMs. We are interested in understanding the functional and regulatory differences in these enzymes. The focus of our study is BAM2, which was recently found to be a tetramer and contains a secondary starch binding site which regulates BAM2 activity. Since there are no structures of BAMs from Arabidopsis, we aimed to characterize the structural mechanism of BAM2 regulation by starch binding to the secondary binding site. We first modeled BAM2 as a tetramer using previous experimental information and then performed simulations of this model in several substrate and starch bound states to understand the role of the secondary starch binding site on the active structure. We found that starch binding influences protein dynamics near the active site, which we suggest may be the basis for allosteric regulation. We further identified an amino acid which appears to regulate the T to R transition of the enzyme. We supported these computational studies with small angle X‐ray scattering studies of BAM2 structure and with biochemical assays of amylase activity. These latter studies were aided by the development of a novel fluorescent biosensor assay of maltose production that allows for comparison of diverse substrates from small polysaccharides to soluble starch within a single assay method. These comparisons indicate that BAM2 strongly prefers starch‐like substrates over shorter polysaccharides, likely due to allosteric regulation by the secondary starch binding site. Current studies are focused on understanding the effects of this molecular mechanism on starch structure with the goal of elucidating the functional role of BAM2 in plants.Support or Funding InformationNSF‐RUI MCB‐1616467NSF REU CHE‐17578744‐VA OrganizationJeffrey E. Tickle ScholarshipThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PGRMC1 stabilizes CYP1A2 and other cytochromes P450
Authors: McGuire Meredith R. 
Published: June 2021

Cytochromes P450 are a family of monooxygenase enzymes required for biosynthetic reactions in the body and for the metabolism of pharmaceutical drugs. Compared to our understanding of P450 enzymology, little is known about the post‐translational control of P450 activity. Progesterone Receptor Membrane Component 1 (PGRMC1) is a membrane‐bound hemoprotein that is highly expressed in the liver and binds >20 P450s in vivo. Dap1p/PGRMC1 binds and promotes the activity of CYP51A1 in yeast and cultured cells. To understand the function of PGRMC1 in relation to P450 biology, we generated whole‐body Pgrmc1 KO mice. Proteomic and transcriptomic data in Pgrmc1−/Y mouse livers showed that 81% of detectable P450s are downregulated at the protein level (17/21), while 83% of detectable P450s are unchanged at the transcript level (66/80). The constitutively expressed cytochrome P450 Cyp1A2, which is homologous to human CYP1A2, follows this trend. Consistent with a reduction in protein level, the Vmax of the Cyp1A2‐dependent O‐deethylation of 7‐ethoxycoumarin is 57% lower in Pgrmc1−/Y liver microsomes. In PGRMC1 KO human fibroblasts, PGRMC1 doubles the half‐life of CYP1A2 in a heme‐independent manner. These proteomic, transcriptomic, and turnover data establish PGRMC1 as a novel post‐translational stabilizer of Cyp1A2. Given that our proteomic data identified 17 P450s that are downregulated in Pgrmc1−/Y liver, these data suggest a broad role for PGRMC1 in post‐translational cytochrome P450 stability.Support or Funding InformationNIHF31HL131185 / AHA15PRE25250008 / NIHT32GM007445 / NIHRO1HL077588This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ATP synthase: A potential molecular drug target
Authors: Ahmad Zulfiqar 
Published: June 2021

BackgroundAccording to the Review on Antimicrobial Resistance, antibiotic resistance will result in 10 million additional deaths worldwide per year by 2050 surpassing cancer. Fast‐encroaching antibiotic resistance by microbes in general and E. coli in particular is the main reason for this situation. Thus, finding alternative ways to kill microbes is of paramount importance. ATP synthase is the principal source of cellular energy in almost all organisms. Selective inhibition of microbial ATP synthase may deprive microbes of required energy leading to cell death. A wide variety of inhibitors including phytochemicals and peptides bind and inhibit ATP synthase. These phytochemicals and peptides bind to the explicit binding sites on ATP synthase. Although binding sites are highly conserved, they are flanked by variable amino acids, which participate in selective binding of inhibitors. Our lab is interested in identifying the selective inhibitors of ATP synthase using Escherichia coli as a model organism.MethodWild type, null, and mutant E. coli strains are used to select the potent inhibitors of ATP synthase. Growth of E. coli strains is measured on fermentable and non‐fermentable carbon sources. Membrane bound F1Fo ATP synthase is isolated by harvesting cells in the minimal media, breaking cells by French Press, and separating the enzyme by multiple high‐speed ultracentrifugation. A variety of phytochemical and peptide inhibitors are used to inhibit membrane bound ATP synthase. Inhibitory potency of inhibitors is amplified through addition or subtraction of functional groups.ResultsNatural inhibitors caused variable degree of inhibition. Structural modifications resulted in augmented inhibition. Re‐positioning of −OH, NH2, PO4 groups in phytochemicals or addition of a c‐terminal NH2 group in peptides augmented the inhibitory effects by about 100‐fold. Phytochemicals and peptides also showed synergistic inhibitory effects on ATP synthase.ConclusionsPhytochemicals and peptides potently kill E. coli cells by binding at the F1 sector of ATP synthase. Thus, ATP synthase can be used as a potent molecular drug target to combat antibiotic resistant microbial infections.Support or Funding InformationA.T. Still University Warner/Fermaturo Research Grant, grant 501–605 to ZA.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.