bluuebunny/crossref_metadata_2025_split huggingface dataset:
Host vs Virus: HSV has evolved to evade host antiviral mechanisms by manipulating the host proteostasis machinery
Authors: Weller Sandra , Adlakha Mitali 
Published: November 2023

Cells have evolved elaborate homeostatic mechanisms to recognize and respond to environmental and other forms of stress, including viral infections. Disturbances to protein homoeostasis (proteostasis) can be caused by the expression of aggregation prone proteins in neurodegenerative diseases such as Huntington's or Spinocerebellar ataxia. These disturbances lead to the accumulation of microscopically visible deposits of aggregated proteins (nuclear or cytoplasmic aggresomes). Degradation of aggregated proteins can be achieved through the action of the UPS (ubiquitin proteasome system) or by the induction of autophagy. Interestingly, the UPS and autophagy have both been implicated in clearance of viral pathogens and antigen presentation (Wileman, 2007; Gobeil and Leib, 2012). Specifically, Hsc70 has been implicated in antigen trafficking, MHC class II presentation and chaperone mediated autophagy (Cuervo and Wong, 2014; Deffit and Blum, 2015). These observations suggest that the cellular proteostasis machinery is important for intrinsic antiviral response mechanisms. Viruses, in turn, have adapted to counteract antiviral defenses.HSV‐1 infection causes a drastic spatial reorganization of components of the cellular proteostatic machinery as well as misfolded proteins into Virus Induced Chaperone Enriched (VICE) domains in the nucleus (Burch et al 2004; Livingston et al 2008,2009). In cells infected with WT HSV, Hsc70 is localized predominantly to VICE domains; whereas, in cells infected with a mutant virus lacking the viral immediate early protein ICP22, Hsc70 is not relocalized. When an aggregation prone protein is expressed by transfection, large cytoplasmic aggresomes are observed; however, in the presence of ICP22, cytoplasmic aggresomes are not detected. In these cells, nuclear inclusions are formed which contain ICP22, Hsc70 and misfolded proteins. Thus, ICP22 appears to reorganize Hsc70 and misfolded proteins into nuclear inclusions. The sequestration of Hsc70 into VICE domains may prevent it from carrying out antiviral activities elsewhere in the cell.Support or Funding InformationNIH Grant AI021747.
Sulfide Oxidation and Transfer Catalyzed by a Bacterial Persulfide Dioxygenase Fused to a Rhodanese
Authors: Motl Nicole , Kabil Omer , Skiba Meredith , Smith Janet , Banerjee Ruma 
Published: November 2023

Hydrogen sulfide (H2S) is the third eukaryotic gaseous signaling molecule that evokes a broad range of physiological effects. H2S levels are governed by the rates of both production and clearance. In eukaryotes, sulfide clearance occurs primarily via the mitochondrial sulfide oxidation pathway which consists of the enzymes sulfide: quinone oxidoreductase (SQR), persulfide dioxygenase (PDO also known as ETHE1), rhodanese and sulfite oxidase. Recently, bacterial proteins that are fusions between PDO and rhodanese have been identified. Existence of such a fusion suggests that their mitochondrial counterparts in higher organisms may interact and that characterization of such fusion proteins will facilitate the modeling of interactions between human mitochondrial PDO and rhodanese and aid in elucidating the metabolic intermediates in the sulfide oxidation pathway. Here we have characterized a bacterial PDO‐rhodanese fusion (PRF) protein from Burkholderia phytofirmans which displays 1.8‐fold higher PDO activity and 4.8‐fold lower Km for GSSH as compared to human PDO, and rhodanese activity comparable to other bacterial rhodaneses. Additionally, the rhodanese domain catalyzed the formation of GSSH and sulfite from thiosulfate and glutathione, but no detectable sulfur transfer activity was observed for the reverse reaction. Similar observation were made for the stand‐alone rhodanese domain; however, several fold increases in specific activity and Km values for thiosulfate and glutathione were observed for the various sulfur transfer reactions, suggesting that the PDO domain modulates the activity of the rhodanese domain. Combined, these results suggest that the fusion is poised to produce sulfite as its main product. Bioinformatic analysis suggests that this enzyme may be involved in sulfur assimilation pathways rather than in sulfide oxidation as in mammals. Spectral changes were observed with the ferric form of the enzyme in the presence of Na2S. Further analysis of this interaction displayed that Na2S binds with moderate affinity resulting in polysulfide production and suggesting a potentially novel role for non‐heme iron proteins in polysulfide production. To obtain structural insights into its mechanism, the x‐ray crystal structure of PRF co‐crystallized with thiosulfate was determined at 1.79 Å resolution. Notably, in this structure the persulfidated form of the rhodanese domain active site cysteine, C314, was captured.Support or Funding InformationNational Institutes of Health (GM112455 to RB) and National Institute of General Medical Sciences Training Grant support (T32GM008353 to NM)
Temperature and Pressure Dependence of the Activity of Inteins from Extreme Thermophiles
Authors: Comeau Hannah Y , Long Joshua D. , Pierre Isabelle V , Reitter Julie N , Mills Kenneth V 
Published: November 2023

Protein splicing is a post‐translational modification by which intervening polypeptides, or inteins, catalyze their own excision from two flanking polypeptides, or exteins, concomitant with the ligation of the exteins. We studied the activity of inteins that interrupt the DNA Polymerase II (PolII) from the deep‐sea thermophiles Thermococcus kodakarensis (Tko) and Thermococcus barophilus (Tba). Previous work with Pyrococcus abyssi (Pab) and Pyrococcus horikoshii (Pho) PolII inteins, which both possess flexible loop regions, showed temperature‐dependent splicing activity. The Tba and Tko inteins we studied, on the other hand, contain a homing endonuclease region in place of this flexible loop. We found that, unlike the Pab and Pho PolII inteins, the Tba and Tko PolII inteins splice poorly in E. coli, but can be induced to splice in vitro at higher temperatures. Mutagenesis studies suggest that changing the Tba PolII intein's native final reside (Asn to Gln) slowed splicing significantly, as did changing the native penultimate Ser to His. Mutating the Tko PolII intein's native final residue (Gln to Asn) accelerated activity such that it proceeded in vivo, while mutating the penultimate native His to Ser slowed splicing activity. We are currently studying the influence of high pressure on the activity of the inteins.Support or Funding InformationThis material is based upon work supported by the National Science Foundation under grants MCB‐0950245, MCB‐1244089 and MCB‐1517138 and by a Henry Dreyfus Teacher‐Scholar Award (KVM).
The Predicted Human PANK4 Lacks Key Catalytic Residues for Pantothenate Kinase Function
Authors: Yao Jiangwei , Rock Charles O. , Jackowski Suzanne 
Published: November 2023

The human genome is predicted to encode four pantothenate kinase proteins. The PANK1, PANK2, and PANK3 proteins are established functional pantothenate kinases, but the function of the PANK4 protein is disputed. The PANK4 is predicted to encode a fusion of a pantothenate kinase domain at the N‐terminus and a phosphatase domain at the C‐terminus. This putative bifunctional PANK4 is found in the fungi, plant, and animal kingdoms. The plant PANK4 homologue is a functional pantothenate kinase based on its biochemical and genetic characterization. Structural analysis identifies a conserved catalytic glutamate (Glu137 in the human PANK3 protein) as the essential catalytic base for the kinase reaction, while a conserved asparagine (Arg207) forms essential interactions with the pantothenate substrate. While the kinase domain of the human PANK4 has extensive sequence alignment with the human PANK1‐3, the equivalent Glu137 residue in PANK4 is a valine while the equivalent Arg207 residue is a tryptophan. The absence of these two essential catalytic residues suggests that the human PANK4 has lost the pantothenate kinase function. In contrast, the plant PANK4 homologue encodes for the Glu137 and Arg207 residues, consistent with the plant PANK4 homologue indeed functioning as a pantothenate kinase. Bioinformatic analysis showed that the disabling E137V and R207W mutations in the PANK4 protein are conserved in reptiles, birds, and humans (amniotes) while other species in the animal kingdom including amphibians, fish, and insects encode a functional PANK4 protein. These results show that PANK4 experienced conserved, disabling mutations to key catalytic residues during the evolution of amniote. Biochemical experiments will be presented that critically test these bioinformatic predictions concerning the function of the N‐terminal domain of PANK4. The analysis of human PANK4 highlights the importance of confirming bioinformatic predictions with detailed biochemical and structural biology analyses.Support or Funding InformationThis work was supported in whole or in part by National Institutes of Health Grants GM034496 (to C. O. R.), GM062896 (to S. J.), and Cancer Center Support Grant CA21765 and the American Lebanese Syrian Associated Charities.
Redox Control of PRMT1 Substrate Specificity
Authors: Caceres Tamar Betsabe , Price Owen , Morales Yalemi , Hevel Joan 
Published: November 2023

Protein arginine methylation is an important post‐translational modification involved in many biological processes. Some examples are: transcription, RNA splicing, signal transduction, DNA repair, and viral replication. Arginine methylation is catalyzed by the Protein Arginine Methyltransferase (PRMT) family of enzymes. These enzymes utilize S‐adenosylmethionine (AdoMet) as a methyl donor to generate one of three distinct species of methylarginine on substrate proteins. In recent years, the significance of PRMTs in human disease has been increasingly studied, especially in cardiovascular disease and cancer. Although the importance of this enzyme is recognized, our understanding of the fundamental biochemistry and regulation of PRMT activity is limited. Adding to the complexity of the PRMT field different methylation states of the same substrate can lead to distinct biological outputs. Therefore, characterizing the kinetic mechanism of the PRMTs and deciphering how the final methylation state of the substrate arginine is determined is crucial in order to fully understand how this family of proteins functions and how it can be controlled or inhibited. Our aim is to determine which intrinsic and extrinsic factors determine how the final methylation state of the substrate arginine is achieved. Recently we documented that the catalytic rate of PRMT1, the major type I PRMT in humans, is significantly and reversibly hindered by oxidation. The results of our study indicate that in addition to altering the catalytic rate of PRMT1 activity, the redox state of the enzyme also appears to alter the product specificity with reduced enzyme generating primarily monomethylated products and oxidized enzyme generating approximately equal amounts of monomethylated and asymmetrically dimethylated products as determined by our methyltransferase assay and HPLC product analysis. Our hypothesis is that this change in product formation involves one or more cysteine residues being reversibly oxidized to sulfenic acid (Cys‐SOH). A thorough understanding of how oxidation affects the product specificity of PRMT1 could have profound implications on our knowledge of processes mediated by PRMT1 such as cell signaling and epigenetic regulation, particularly in disease states correlated with oxidative stress such as cancer and cardiovascular disease.Support or Funding InformationNational Science Foundation/Dominican Republic Government
Physiological significance of persulfide generation by the transsulfuration pathway
Authors: Yadav Pramod Kumar , Martinov Michael , Vitvitsky Victor , Seravalli Javier , Wedmann Rudolf , Filipovic Milos R , Banerjee Ruma 
Published: November 2023

In mammalian cells, cystathionine β‐synthase (CBS), cystathionine γ‐lyase (CSE) and merceptopyruvate sulfurtransferase (MST) catalyze H2S synthesis. Of these three enzymes, CBS is regulated by an unusual heme which is responsive to the gaseous signaling molecules, CO and NO. CBS also catalyzes the condensation of homocysteine and serine to form cystathionine, which is further utilized by CSE to generate cysteine. H2S plays an important role in various physiological process, acting as a neuromodulator, vasorelaxant, cardioprotective agent, and has anti‐inflammatory effects. H2S signals by posttranslational modification of reactive cysteine thiols to persulfides. Recently it has been shown that CBS and CSE can synthesize cysteine persulfide (Cys‐SSH) using cystine as a substrate. In fact, it was reported that Cys‐SSH rather than H2S is the major product of CBS and CSE. However, the intracellular milieu is reducing and the availability of cystine is limiting. In the present study, we examined the kinetics of Cys‐SSH and homocysteine persulfide formation and of Cys‐SSH decay. We also evaluated the effect of increased cellular cystine on persulfide and polysulfide levels and simulated the relative rates of H2S and Cys‐SSH biogenesis at physiologically relevant substrate concentrations.Support or Funding InformationThis work was supported by a grant from the National Institutes of Health (HL58984 to RB) and the American Heart Association (14POST18760003 to PKY).
Designing highly specific protein‐based small molecule biosensors
Authors: Raman Srivatsan 
Published: November 2023

Allosteric transcription factors (aTF) act as switches that transduce small molecule binding into transcriptional actuation to regulate various cellular processes. This switch‐like behavior makes aTFs a cornerstone in synthetic biology applications. In metabolic engineering, they act as in vivo sensors to report on the level of target metabolites, allowing directed evolution of production pathways by revealing rare, high‐producing cells. aTFs also play a pivotal role as switches to control information flow and feedback regulation in synthetic gene networks. Expanding aTFs beyond naturally‐occurring aTF‐small molecule pairs would greatly increase their utility. For example, new chemical recognition capabilities would facilitate the evolution of new biosynthetic routes for natural products and synthetic chemicals useful as therapeutics, novel materials and fuels. Additional switches with orthogonal chemical specificity would allow the engineering of higher‐order synthetic circuits that function more robustly for applications outside the laboratory. However, redesigning an aTF to bind to a new molecule is challenging. Mutations in the ligand‐binding site often disrupt allosteric communication with the DNA‐binding domain, destroying the switch‐like behaviour.Here, we present a general strategy to engineer an aTF to respond to new inducer molecules using the E. coli LacI protein as a test case. We evaluate thousands of candidate designs, derived from computational design to identify those that are both active as a switch and responsive to a target molecule. We enhance the activity of the initial hits toward greater specificity and stronger induction. We demonstrate the utility of this approach by engineering LacI variants to respond to gentiobiose, fucose, lactitol or sucralose with response comparable or superior to the wild‐type LacI response to its synthetic inducer, IPTG. This technology enables us to build highly specific intracellular biosensors for small molecules toward many applications in synthetic biology and cellular engineering.
Phospholipase A2: a unique paradigm of allosteric regulation by membranes
Authors: Mouchlis Varnavas , McCammon J. Andrew , Dennis Edward A. 
Published: November 2023

Defining the details of the molecular recognition between water‐soluble enzymes and membranes is fundamental to understanding protein‐lipid interactions, molecular complementarity and membrane functioning.1 Phospholipase A2 (PLA2) enzymes, which catalyze the hydrolysis of phospholipid substrates that comprise the membrane bilayer provide the ideal system for studying protein‐lipid binding and interactions.2 Our current study focuses on understanding the catalytic cycle of three human PLA2 enzymes: the cytosolic (cPLA2), calcium‐independent (iPLA2) and lipoprotein‐associated (Lp‐PLA2). cPLA2 is the main arachidonic acid provider for the eicosanoid pathway, while iPLA2 is involved in membrane phospholipid remodeling. Lp‐PLA2 was found to associate with LDL and HDL in human plasma in order to hydrolyze phospholipids containing short‐chain and oxidized fatty acids as well as F2‐isoprostanes esterified at the sn‐2 position. Hydrogen/deuterium exchange mass spectrometry was used to experimentally identify distinct PLA2 peptide regions that interact with phospholipid vesicles. Molecular dynamics simulations, guided by the experimental data, showed that the active sites of these enzymes are allosterically regulated by membranes. Membrane phospholipids bind to allosteric sites located on the interfacial surface of PLA2s shifting their conformation from the “closed” to the “open” state. This process facilitates extraction and binding of a phospholipid molecule in the active site where the hydrolysis occur. This constitutes the first detailed study describing the binding and interaction mechanism of PLA2s with the membrane bilayer as well as how they bind a single phospholipid molecule in the catalytic site. These enzymes are implicated in chronic inflammatory diseases and understanding their association with membranes, mechanism of action and interactions at the molecular level, will allow us to identify potent and selective inhibitors that can be further developed as anti‐inflammatory agents.3, 4Support or Funding InformationThis work was supported by NIH Grant GM20501.
New Roles for Dithiolopyrrolones in Disrupting Bacterial Metal Homeostasis and Inhibiting Metalloenzymes
Authors: Chan Andrew N. , Shiver Anthony L. , Wever Walter J. , Razvi Sayeeda Zeenat A. , Traxler Matthew F. 
Published: November 2023

Natural products have been a major source of life‐saving antibiotics. Antibiotic resistance is a growing health threat that has created an urgent need for new antimicrobial therapies. Many “old” classes of natural products, discovered in the “golden age” of antibiotics, have been overlooked in part due to a lack of understanding of their mode of actions. The dithiolopyrrolone class of natural products contains a disulfide bond, which our group has shown to be important for potent antimicrobial activity. As a class, these natural products have shown potent activity against a range of microbes, including Escherichia coli, methicillin‐resistant Staphylococcus aureus, Klebsiella pneumoniae, and Acinetobacter baumannii. The dithiolopyrrolones were previously reported to inhibit various in vivo processes such as RNA synthesis and carbon utilization; however, confounding evidence leaves the mechanism unclear. To rectify the activity of the dithiolopyrrolones, we conducted a chemical genomics screen in Escherichia coli, which revealed an unusual mechanism of action – the redistribution of cellular metals and the disruption of cellular metal homeostasis*. Using holomycin as a representative dithiolopyrrolone, we determined that the reduced form of holomycin binds transition metals with high affinity using spectrometric methods. In exploitation of this metal‐chelation activity, we showed that reduced holomycin inhibits the in vitro activity of E. coli zinc‐dependent aldolase, a unique antimicrobial target, as well as the metallo‐®‐lactamase, NDM‐1. Significantly, the spread of NDM‐1 and other metallo‐β‐lactamases has enabled clinical resistance to last‐resort β‐lactams, the carbapenems. The in vitro inhibition of NDM‐1 by holomycin serves as a starting point for the further development of holomycin and analogs for enhanced activity against these targets in vivo. This work links the chemical reactivity of the dithiolopyrrolones through their disulfide bond to their antimicrobial action. Our work shows that dithiolopyrrolones are a privileged scaffold due to their potent redox‐controlled antimicrobial activity, demonstrates the power of chemical genomics in characterizing antibiotic mode of action, and highlights the potential of metal‐chelating natural products in antimicrobial therapy.Support or Funding InformationThis work was supported by National Institute of Health GM099904‐04 (B.L.). S.Z.A.R is supported by the National Science Foundation Graduate Research Fellowship Program (DGF 1106401).
Characterizing the Use of the RNA Mango Aptamer for RNA Pull‐Downs and Single Molecule Fluorescence
Authors: Poe Hannah M , van der Feltz Clarisse , Chen Xin , Unrau Peter , Hoskins Aaron 
Published: November 2023

Methods for investigating the conformation and interactions of RNA and RNA/protein complexes are currently limited. This in turn has restricted our ability to understand the dynamics and interactions of small nuclear RNAs of the spliceosome. One way to address this challenge is by “tagging” the RNAs with an aptamer so that they can be readily purified and visualized. RNA Mango is an aptamer that has a high affinity (KD<10 nM) and specificity for derivatives of its fluorescent ligand, thiazole orange (TO1). Fluorescence of TO1 is enhanced 103‐fold when it is bound to the parallel‐stranded G‐quandruplex of RNA Mango, making the use of this ligand‐aptamer pair a promising strategy in RNA localization and purification experiments1. What makes this aptamer truly exciting is its ability to enable studies by a variety of biochemical techniques, including single molecule Förster Resonance Energy Transfer (smFRET) experiments and pull‐down assays. The goal of this work was to explore the versatility of RNA Mango incorporation into snRNAs through such experiments.For smFRET experiments, we labeled distinct positions of RNA Mango with Cy5 and Cy3 fluorophores, and determined that the aptamer has a folded FRET state of ~0.7 in monovalent ionic buffer solutions. Additional smFRET experiments suggest that the presence of TO1 derivatives in solution do not affect the folding of RNA Mango, whose properly folded G‐quadruplex is critical for the fluorescence of TO1. Thus, the RNA Mango aptamer itself is stable under a variety of conditions and TO1 binding may only introduce small conformational changes.We created a Saccharomyces cerevisiae strain with RNA Mango incorporated into the U4 snRNA, a component of the spliceosome, such that RNA Mango and TO1 derivatives can be used to isolate U4 and U4 containing splicing complexes. Primer extension assays comparing S. cerevisiae with wildtype and U4‐Mango showed that RNA Mango was successfully incorporated into the genome of our modified strain. We were able to purify snRNP complexes using U4 snRNA:RNA Mango and biotinylated TO1. This work demonstrates that RNA Mango can be integrated into the U4 snRNP to give further insights into the complexities of interactions in the spliceosome.Support or Funding InformationAaron Hoskins is a Beckman Young Investigator of the Arnold and Mable Beckman Foundation