bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural and Functional Basis of GusR‐Mediated Regulation of ß‐Glucuronidase Expression in Enterobacteriaceae Gastrointestinal Pathobionts
Authors: Little Michael Steven , Pellock Samuel J. , Walton William W. , Redinbo Matthew R. 
Published: November 2023

Gastrointestinal (GI) microbial dysbioses have been linked to the on‐set or exacerbation of mammalian metabolic disorders, inflammatory bowel diseases, and colorectal cancer, particularly with increases in the levels of Proteobacteria. Furthermore, elevated activities of microbial ß‐Glucuronidase (GUS) enzymes correlates to colon carcinogenesis and poor outcomes in cancer treatment. Here, we determine the structural and functional basis for the regulation of GUS expression in Proteobacterial pathobionts of the Enterobacteriaceae family, species Escherichia coli and Salmonella enterica. In the GI microbiota, only the Enterobacteriaceae maintain an operon of GUS and glucuronide transporters under the control of a glucuronide repressor GusR, and presumably this operon can provide a growth advantage in the competitive intestinal milieu. The crystal structures of E. coli GusR and S. enterica GusR in complexes with glucuronidated ligands were determined to 2.0 Å resolution. Isothermal titration calorimetry (ITC) was used to determine E. coli GusR's DNA binding affinity to the GusABC operon operator site and identify an endogenous glucuronide ligand with low micro‐molar affinity. Structural‐guided GusR ligand binding pocket mutants coupled with ITC pinpointed residues essential for glucuronide binding. Finally, we confirm using in vivo cell‐based assay that GusR robustly induces GUS activity in the presence of GusR‐targeted ligands. Taken together, these data provide the first molecular insights into the control of GUS expression by a family of pathobionts that proliferate in the GI during disease.Support or Funding InformationNational Science Foundation Graduate Research Fellowship & National Institute of Health
Targeting the Hendra Virus Fusion Protein Transmembrane Domain to Inhibit Viral Fusion
Authors: Barrett Chelsea , Webb Stacy , Dutch Rebecca 
Published: November 2023

Hendra Virus is a zoonotic virus in the family Paramyxoviridae. It is an enveloped, negative sense single stranded RNA virus that is highly pathogenic, but currently there is no approved antiviral therapeutic or vaccine. Further elucidation of the mechanisms that underlie viral entry into a cell will assist in presenting additional targets for therapeutics. Two surface glycoproteins, the attachment protein (G) and the fusion protein (F), are critical for viral binding and fusion. When a proper signal is received by the Hendra F protein, it undergoes a large, essentially irreversible, conformational change resulting in the fusion of the virus and target cell membranes. Previous research has demonstrated that the Hendra F protein associates as a homo‐trimer, and that the transmembrane domain (TMD), plays a key role in this association, as well as overall protein stability. To further characterize this role of the TMD in the fusion process, exogenous Hendra TM constructs were created containing the cytoplasmic tail, a signal peptide, an HA tag and varying lengths of the heptad repeat B region. When these TM constructs were co‐expressed with the full length Hendra F protein, a significant reduction in the expression and stability of the full length protein was observed. In contrast, when the Hendra TM constructs were co‐expressed with Parainfluenza Virus 5 (PIV5) F protein, another member of Paramyxoviridae, no decrease in F protein expression was observed, suggesting the interactions between transmembrane domains are sequence specific. Further experimentation is ongoing to further characterize the TMD interactions, however specifically targeting the TMD of F proteins may present a unique therapeutic target.Support or Funding InformationUniversity of Kentucky, Department of Molecular and Cellular Biochemistry
Role of the Transmembrane Domain of Mucin 1 in Nuclear Localization
Authors: O'Connor Gerald Joseph 
Published: November 2023

Mucin 1 (MUC1) is a highly glycosylated, single pass membrane protein that provides the mucous surfaces of epithelial cells and protects against pathogens. It consists of two parts, MUC1‐N, which includes the extracellular domain, and MUC1‐C, which includes the cytosolic and transmembrane domains. MUC1 overexpression has been observed in 80 to 90% of human solid tissue cancers. Studies have shown that this overexpression may lead to malignant unregulated gene expression when MUC1‐C enters the nucleus. The transport of MUC1‐C, from the plasma membrane to the nucleus, appears to be the result of MUC1‐C homodimerization. Our lab has utilized ToxR Assays to elucidate the role of the transmembrane domain in MUC1‐C dimerization. Using this assay we have seen that an Ala1180Leu mutation decreases the propensity of MUC1‐C to dimerize, implying it may be of importance to the dimer interface. Because this mutation is shown to decrease the ability of MUC1‐C to form dimers, the hypothesis is that it will also decrease the transport of MUC1‐C to the nucleus. Currently, we are working with mammalian (HEK) cells to determine if the Ala1180Leu mutation also affects nuclear localization of MUC1. Understanding of the mechanisms of MUC1‐C dimerization and nuclear localization is important in designing effective therapeutics.Support or Funding InformationSaint Joseph's University Department of Biology Saint Joseph's University Summer Scholars Program Edwin Li, Ph.D.
Ligand induced shift in FRET spectrum of MDA5/LGP2 signaling complex
Authors: Corby Josie , Stoneman Michael , Biener Gabriel , Raicu Valerica , Frick David 
Published: November 2023

The innate immune system is an intracellular first line of defense against invading organisms. It consists of a group of enzymes called pattern recognition receptors (PRRs), which identify conserved features of bacteria and viruses known as pathogen associated molecular patterns (PAMPs). One important group of PRRs is the RIG‐I like receptors (RLRs) which recognize nucleic acid PAMPs. The three known RLRs (RIG‐I, MDA5, and LGP2) are ATP‐fueled helicases that assemble as spiral‐like oligomers on mitochondria upon ligand recognition. Previously, these filaments have only been visualized using electron microscopy and crystallography.Most current models, based on cellular fractionation experiments, suggest that RIG‐I, MDA5 and LGP2 reside in the cytoplasm in the absence of PAMPS, but when PAMPs are present, they migrate to the mitochondria and assemble into filaments seeded on the mitochondrial antiviral signaling protein (MAVS). To study the interaction of LGP2, RIG‐I, and MDA5 in live cells, we used fluorescent fusion proteins and Förster resonant energy transfer (FRET) spectrometry. Plasmids encoding donor (mCFP) and acceptor (mYFP) fused to MDA5, RIG‐I, and LGP2 respectively were generated. HEK293T cells were co‐transfected so they express various combinations of fluorescently labeled LGP2, RIG‐I and/or MDA5. After which the transfected cells were treated with either poly I:C/Turobofect® or Turobofect® alone. After four hours, the live cells were stained with Mito‐tracker red® and imaged using an optical micro‐spectroscope (Optimis) which consists of a two photo excitation scanning optical microscope and high spectral resolution.The fluorescently labeled LGP2, RIG‐I and MDA5 were visualized in live HEK293T cells. The mitochondrial regions were selected and the donor concentration, acceptor concentration and FRET efficiency were calculated for each mitochondrion selected. Most of RLRs appeared at the mitochondria, even in the absence of poly I:C. FRET was noted in the mitochondrial regions for all combinations, but in most cases only small FRET differences were seen when the PAMP was added. Remarkably, however, when labeled MDA5 and LGP2 were expressed together, a 4‐fold increase in average FRET was only observed when poly I:C was added, indicating a dramatic change in oligomeric structure.
Allosteric Landscape of a Stress‐inducible Human Hsp70 Molecular Chaperone
Authors: Meng Wenli , Clerico Eugenia M , McArthur Natalie , Gierasch Lila M 
Published: November 2023

The 70‐kDa heat shock proteins, Hsp70s, are molecular chaperones that perform a wide range of cellular functions. They assist in folding of newly synthesized proteins, facilitate assembly of specific protein complexes, and prevent protein misfolding and aggregation. Hsp70s perform these functions by a conserved mechanism that relies on allosteric cycles of nucleotide‐modulated binding and release of client proteins, with the participation of co‐chaperones, including J‐proteins and nucleotide exchange factors. Much of our knowledge about Hsp70 allostery has come from extensive study of the bacterial Hsp70, DnaK. Mechanistic understanding of eukaryotic Hsp70s is of high importance, however, because they have been proposed to be potential drug targets in many diseases including cancer. By using NMR spectroscopy, we find that the allosteric behavior of the eukaryotic inducible cytoplasmic Hsp70, HspA1, varies substantially from that of DnaK. HspA1 favors the state in which the nucleotide‐binding domain (NBD) and substrate‐binding domain (SBD) are intimately docked significantly more than DnaK does. In DnaK, formation of this state requires binding of ATP. By contrast, even in the ADP‐bound state of HspA1, the state with the NBD and SBD docked is the predominant one. We have analyzed the interfaces that govern the allosteric landscape of these Hsp70s and identified sites that contribute to the altered allosteric landscape between HspA1 and DnaK. The impacts of the natural variations are being tested by mutation of residues on the key allosteric interfaces‐‐–the NBD‐SBD interface and the helical lid‐βSBD interface‐–and determining the resulting population shifts between the docked and undocked conformations. These results point to the tunability of Hsp70 allosteric landscapes by modulation of allosteric interfaces through evolutionary diversification and suggest how small molecule modulators could influence Hsp70 functions.Support or Funding InformationSupported by NIH grants GM027616 and GM118161.
Characterization of Sup35, Rnq1, and Ure2 Cotranslational Prion Aggregation in Saccharomyces cerevisiae
Authors: Allwein Benjamin Thomas 
Published: November 2023

Prions are a unique class of self‐propagating proteins that can adopt self‐propagating misfolded conformations and which can spread through templated self‐conversion. In mammals, prions are unerringly deadly elements of disease, underlying the fatal transmissible spongiform encephalopathies. In yeast, however, prions may actually serve to benefit the cell under various stress conditions, and have been shown to confer a fitness advantage in diverse extracellular environments. Prions are thought to originate de novo from a single misfolding event that could potentially occur either cotranslationally on the ribosome or post‐translationally in the cytosol. While post‐translational prion formation is known to occur, cotranslational prion formation has not yet been conclusively demonstrated. To test whether prions can form cotranslationally, we have constructed plasmids encoding Sup35, Rnq1, and Ure2 prion proteins containing an internal hemagglutinin epitope tag and confirmed their expression in yeast. Using these tools, we tested for the association of prion‐forming proteins with ribosomes and assessed their aggregation state on the ribosome. Previous work from our laboratory has demonstrated that the ribosome‐associated complex (RAC), which protects nascent chains from misfolding, antagonizes prion formation. Thus, the potential exists for regulation of co‐translational prion formation in response to environmental stress conditions.Support or Funding InformationNational Institute of General Medical Sciences of the National Institutes of Health Award Number R15GM119081
Investigation of the Third Step of Protein Splicing in Two Similar Cyanobacterial Inteins
Authors: Ramsoomair Christian Kieran , Yakely Anthony E , Reitter Julie , Mills Kenneth 
Published: November 2023

Protein splicing is a post‐translational process facilitated by an intervening polypeptide, or intein. The intein catalyzes its excision from the flanking polypeptides, or exteins, as well as ligation of the exteins. We studied inteins that interrupt the DNA Polymerase II from the cyanobacteria Synechococcus sp. PCC 7002 (Ssp) and Trichodesmium erythraeum (Tery). The two inteins differ mostly in the residues that promote the third step of splicing, side chain cyclization of the C‐terminal residue coupled to peptide bond cleavage. While the Ssp intein has a native C‐terminal Asn, the Tery intein has a native Gln. We hypothesized that the replacement of the native Asn with Gln might slow splicing, whereas replacement of the native Gln with Asn may accelerate splicing, as cyclization of Asn residues is usually considerably faster. Results suggest that our hypothesis is correct for isolated side chain cyclization, but that the overall splicing reaction is less affected by the identity of the C‐terminal residue. This suggests that conformational changes after step one, an amide to thioester rearrangement, might affect the rate of side‐chain cyclization.Support or Funding InformationThis material is based upon work supported by the National Science Foundation under grants MCB‐1244089 and MCB‐1517138, a Henry Dreyfus Teacher‐Scholar Award (KVM), and Mrs. Jeremiah W. O'Connor, Jr.
Conditional protein splicing of inteins from extremophiles
Authors: Gomez Alvin , Mills Kenneth V 
Published: November 2023

Protein splicing is a post‐translational process that results in the removal of an intervening polypeptide, the intein, and the ligation of the flanking C‐ and N‐terminal polypeptides, the exteins, to form a mature protein. Although protein splicing occurs without the need for any cofactor, chaperone or energy source, the proper conformation of the active site may be influenced by environmental cues. Undergraduates in our lab have carried out experiments to study the structure and mechanism of inteins from halophiles and deep‐sea thermophiles. We have shown that an intein from Halobacterium salinarum (Hsa) can promote protein splicing, but only on incubation at high salt. Students have found that splicing of related inteins from Hsa and from Haloquadratum walsbyi is differentially influenced by salt, and have begun structural studies and experiments to test the physiological relevance of this conditional splicing. They have also studied the temperature dependence of inteins from a wide‐range of deep‐sea thermophiles, and have begun to examine the influence of high pressure. This work provides insight into the mechanism of splicing and the structure of enzymes in unusual conditions, but also provides a context for a training program for young STEM scholars, including first year students participating in a program designed to increase discipline identity and persistence for under‐represented students.Support or Funding InformationThis work was supported by the National Science Foundation (grants MCB‐1244089 and MCB‐1517138) and the Dreyfus Foundation.
Investigating Metallocofactor Assembly and Enzymatic Capability in the Novel Mn/Fe Lipid‐Binding Oxidases
Authors: Miller Effie K , Trivelas Nicholas E , Maugeri Pearson T , Shafaat Hannah S 
Published: November 2023

Recently, the discovery of a novel Mn/Fe protein in a number of pathogens and extremophiles has led to the establishment of a new group of proteins, dubbed R2lox (R2‐like ligand‐binding oxidase). Originally flagged for being highly upregulated in the virulent strain of Mycobacterium tuberculosis, R2lox was initially identified as a sequence homolog for the R2 subunit in class 1 ribonucleotide reductase (RNR); however, a lack of RNR activity combined with x‐ray crystallography data indicate significant differences between R2lox and the typical RNRs. Despite conventional inorganic wisdom and the presence of identical ligands, R2lox has been shown in vitro to bind MnII in the presence of FeII and spontaneously self‐assembles with a site‐differentiated Mn/Fe cofactor. In the absence of MnII, a canonical Fe/Fe cofactor assembles. More interestingly, upon O2 exposure, both cofactors oxidize a nearby valine residue, forming an unprecedented tyrosine‐valine crosslink via C‐H bond activation. However, work in our lab has indicated the mechanisms by which these two cofactors perform this chemistry is thoroughly distinct. It is our goal to utilize the R2lox system as a model to further explore factors governing metallocofactor formation, as well as investigate the potential of these cofactors to perform challenging substrate oxidation reactions using divergent pathways. Utilizing aerobic UV/vis, stopped‐flow, and EPR spectroscopies, the kinetics of metal binding, cofactor assembly, and characterization of discrete intermediates were studied in both Mn/Fe and Fe/Fe R2lox. Through this work, a number of distinctions between the mechanisms for Mn/Fe and Fe/Fe cofactor maturation were identified, and models for the assembly processes have been developed. The implications of these findings are further discussed in the context of potential relevance of R2lox towards therapeutic development and biocatalysis.Support or Funding InformationThe Ohio State University, Pelotonia Fellowship Program, and NIH Cellular, Molecular, and Biochemical Sciences Training Grant
Novel thymidine hypermodifications in viruses encoding a 5‐hydroxymethyl‐5′‐deoxyuridine DNA kinase.
Authors: Weigele Peter R. , Müller Stephanie , Lee Yan‐Jiun , Walsh Shannon , Guan Chudi , Dai Nan , Correa Ivan 
Published: November 2023

Double‐stranded DNA bacterial viruses (bacteriophage) contain the greatest diversity of modified nucleotides in their DNA as compared to any other group of organisms. Some of these modifications consist of sugars, amino acids, or polyamines covalently attached to pyrimidines at the 5‐position, with the ostensible function of blocking the restriction endonuclease defense systems of their bacterial hosts. Phage‐modified thymidines occur through a combination of two processes. First, a suite of phage‐encoded enzymes expressed during infection leads to replacement of 5′‐deoxythymidine triphosphate (dTTP) with 5‐hydroxymethyl‐5′‐deoxyuridine triphosphate (5hmdUTP) at the nucleotide pool level. Then, a phage encoded DNA polymerase synthesizes a precursor DNA containing 5hmdUTP fully substituting for thymidine. Prior to packaging of the phage DNA into the viral capsid, a subset of 5hmdU is further modified by phage‐encoded enzymes resulting in a “hypermodified” base, such as α‐putrescinylthymidine in the Delftia phage ΦW‐14 or α‐glutamylthymidine in the Bacillus phage SP10. These modifications occur on the hydroxymethyl moiety of 5hmdU via a phosphorylated intermediate. Aravind et al. predicted the sequence of a kinase postulated to carry out this step. We have expressed and purified this protein and demonstrate it can transfer a single phosphate to the hydroxymethyl moiety of 5hmdU in a DNA polymer in vitro. Furthermore, we investigated the nucleotide composition of other phages encoding homologs of this kinase and discovered two naturally occurring thymidine modifications: 5‐(2‐aminoethoxy)thymidine and 5‐aminoethyl‐5′‐deoxyuridine. Thus, a single chemical intermediate appears to participate in the formation of three distinct types of DNA modifications; i.e. covalent modification of 5hmdU accomplished through a C‐N, C‐C, and C‐O‐C bond. We will also discuss our current investigation of proteins implicated in the subsequent steps in the biosynthesis of these hypermodifications.Support or Funding InformationAll support is currently funded by New England Biolabs, Inc.
Examining the Mechanism of Egt2 in Ergothioneine Biosynthesis
Authors: Kathuria Karan R. , Irani Seema , Liu Pinghua , Zhang Yan 
Published: November 2023

Ergothioneine (EGT), a unique histidine thiol‐derivative synthesized by certain species of bacteria and fungi, has been recognized as a powerful antioxidant that can have highly beneficial effects on human health. Ergothioneine's limited availability and potential benefits have spurred a great need to develop an efficient industrial scale production, which can best be achieved by engineering the bacterial/fungal production pathway. Interestingly, both of these pathways that produce EGT contain a terminal step in which a pyridoxal 5′‐phosphate (PLP)‐dependent C‐S lyase, termed Egt2, cleaves a carbon‐sulfoxide bond to produce EGT and pyruvate. In this study, we aimed to understand the mechanism of a novel C‐S lyase that uses sulfoxide as its substrate through a structure‐guided approach. In order to overcome a high turn‐over rate and capture the enzyme substrate complex during various stages of the reaction, we employed site‐directed mutagenesis (SDM) to mutate residues interacting with the co‐factor, PLP. Through SDM, we inserted mutations that compromised the enzyme's catalytic rate while preserving structural integrity. The crystallographic analyses revealed the structure of a highly reactive sulfenic acid intermediate that has not previously been visualized in protein chemistry.Support or Funding InformationAmerican Society of Biochemistry and Molecular Biology (ASBMB) & The University of Texas at Austin ‐ Senate of College Councils
Aspartate Cyclization: The “Aspartic Acid Effect” in Pyrococcus abyssi and Deep‐sea Enzyme Pressure Studies
Authors: Minteer Christopher J , Colelli Kathryn M , Reitter Julie N , Mills Kenneth V 
Published: November 2023

Protein splicing is a self‐catalyzed reaction where an intein is excised from precursor flanking polypeptides (exteins) and the exteins are ligated together to produce a mature protein. We are interested in how specific conserved residues catalyze this process and how pressure may affect intein structure and activity.An intein from Pyrococcus abyssi (the Pab PolII intein) can promote splicing in vitro upon incubation at high temperatures. Active site Cys mutations deactivate the intein unless an Asn is substituted for an Asp at the C‐terminus of the N‐terminal extein. N‐terminal cleavage is promoted at low pH, independent of splicing steps. The mechanism of this “Aspartic Acid Effect” occurs via Asp cyclization, as confirmed by anhydride detection via mass spectrometry.We also are interested in how extreme pressure, which would be experienced by deep‐sea thermophiles, may influence protein structure and enzyme activity.Support or Funding InformationThis material is based upon work supported by the National Science Foundation under grants MCB‐1244089 and MCB‐1517138 and by a Henry Dreyfus Teacher‐Scholar Award (KVM).