bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the Ordered Domain of an Epstein Barr Viral Tegument Protein
Authors: Marlowe Megan E. , Gorres Kelly
Published: November 2023

Epstein Barr virus (EBV) is the first known human herpes virus to cause cancer. The virus also causes mononucleosis. Within the viral life cycle, the virus can switch between latent and lytic phases. In the latent phase, very few genes are expressed and new virions are not produced. During the lytic, or active, phase there is an increase in viral gene expression, proteins are produced, and infectious viral particles are released from the cell. The structure of Epstein‐Barr virus, like all herpesviruses, contains a tegument layer, which surrounds the genome‐containing capsid. The proteins in the tegument are delivered to the cell immediately upon infection. These proteins conduct a wide variety of functions in the viral life cycle. This project investigates an EBV tegument protein essential for virus production, but its role is unknown. Bioinformatics analysis identified homologs in other herpesviruses, but no similarity to any protein in any other organism. Amino acid sequences within a protein can exhibit order and disorder, relative to its three dimensional structure. A database called MobiDB was used to predict regions of order versus disorder from the amino acid sequence. The C‐terminal portion of the protein is calculated to be low complexity or disordered, whereas an N‐terminal domain is ordered. To express this structured part of the protein, an early stop codon was inserted into the gene. The protein with a His‐tag was overexpressed in E. coli and purified by nickel‐affinity chromatography. The objective of the experiments conducted is to study the function and characteristics of the structured domain of a tegument protein expressed in the lytic phase of the EBV infection.

Support or Funding Information

This work was supported by the UWL College of Science and Health and a UWL Faculty Research Grant to K.G.
Structure, Dynamics and Folding of an Immunoglobulin‐like Domain of Actin Binding Protein Palladin
Authors: Vattepu Ravi , Beck Moriah R
Published: November 2023

While members of the immunoglobulin (Ig) share a very conserved folding pattern, this family is also the most diverse in terms of function and sequence. The conserved ®–sandwich fold of Ig domains provides a stable scaffold to display a variety of binding site sequences on its surface, however it has been suggested that small variations in the fold may add additional flexibility to fulfill specific functions. Among the few known intracellular proteins containing Ig‐domains, the muscle‐related family is the most common and includes cell adhesion molecules like titin and telokin. Palladin is a recently identified protein from a novel family of actin binding protein that includes myopalladin and myotilin. All of these family members utilize immunoglobulin‐like (Ig) domains to bind and crosslink actin filaments. Palladin isoforms contain three to five immunoglobulin‐like (Ig) domains that have been implicated in a variety of protein‐protein interactions. Our previous results suggest that palladin Ig3 domain is directly involved in binding and crosslinking of actin, promoting actin polymerization, and phospholipid binding. Although the Ig4 domain of palladin does not interact with actin, the tandem Ig3–4 domains display much more efficient actin binding, crosslinking, lipid binding and polymerization than Ig3 alone. Recently we established that palladin dimerizes upon binding to actin, which likely results in a conformational change. Now we have carried out a detailed structural and dynamical characterization of the isolated Ig3 and Ig4 domains of palladin by NMR spectroscopy to provide a basis for understanding the underlying differences between the isolated and tandem domain functions. In the present study, we describe the use of a range of solution‐state NMR techniques and other biophysical methods to monitor dynamics and folding properties of the Ig3 and Ig4 domains of palladin. We have found that the most flexible regions of the Ig3 domain include regions that overlap with actin binding sites, suggesting that dynamics may be critical for function. We also employ NMR backbone relaxation measurements to explore conformational exchange taking place at the hydrophobic core of both Ig domains and compare these results to other closely related proteins.

Support or Funding Information

This project was supported by an Academic Research Enhancement Award (AREA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number R15 GM120670 and start‐up funds from Wichita State University.
Salt‐Dependent Protein Splicing in Extreme Halophiles
Authors: Janton Christopher J. , Reitter Julie N. , Mills Kenneth V.
Published: November 2023

Protein splicing is an autocatalytic, post‐translational process by which an intervening polypeptide, or intein, can excise itself and ligate the flanking polypeptides, or exteins. Inteins have been found in a species of extreme halophilic archaea, Halobacterium salinarum (Hsa). Previous work in the lab demonstrated that splicing of the Hsa DNA Polymerase II (Polll) intein is salt‐dependent. We compared the in vitro activity of the Hsa Polll intein to that of the only other Hsa intein, which interrupts the Cell Division Control protein (Cdc21a). The Hsa Cdc21a intein does not splice in E. coli, but can be induced to splice at very high salt concentrations under reducing conditions in vitro. Additionally, we investigated the salt dependence of isolated C‐terminal cleavage of the Hsa Polll splicing mechanism. We plan to test the splicing activity of the Hsa Cdc21a intein in vivo.

Support or Funding Information

This work was supported by the National Science Foundation (grants MCB‐1244089 and MCB‐1517138 to KVM) and the Dreyfus Foundation (KVM).
Modeling Dynamics in the D‐Amino Acid Oxidase Protein
Authors: Kueffer Lauren , Beyers Wyatt
Published: November 2023

In human brains the enzyme D‐amino acid oxidase (hDAAO) plays a key role in modulating the degradation pathway of the important signaling molecule D‐serine. The hDAAO enzyme is active as a homodimer and is stereospecific for the oxidative deamination of D‐amino acids to give ammonia and an α‐keto acid, coupled to the reduction of the cofactor FAD. The reoxidation of FAD results in hydrogen peroxide. The deregulation of D‐serine signaling has been linked to schizophrenia susceptibility and certain mutations in the hDAAO enzyme itself are associated with familial amyotrophic lateral sclerosis (ALS). Mutations in the hDAAO enzyme are known to impact enzyme function and stability.

We were interested in comparing apo‐enzyme simulations to FAD bound simulations of the wild‐type hDAAO enzyme, a wild‐type pig kidney D‐amino acid oxidase enzyme (pkDAAO), a mutant R279A hDAAO enzyme, and a mutant D31H hDAAO enzyme. Even though there is 85% sequence identity between the two species, hDAAO binds FAD weaker and shows a slower rate of flavin reduction compared to pkDAAO. The R279A mutation exchanges an Arginine for an Alanine at position 279 on both chains of the homodimer. This mutation is close to the FAD binding site and has shown to increase the enzyme's binding affinity for FAD. The D31H mutation exchanges an Aspartate for a Histidine at position 31 on both chains of the homodimer. Even though residue 31 is located far from the FAD binding site, this mutation leads to a higher binding affinity for FAD compared to the human wild‐type. We use molecular dynamic simulations to study how the protein structure changes over time and how the protein dynamics are altered by mutations in the amino acid sequence. Investigating the changes we see at the atomic level in protein structure can help to determine how each mutation can have different effects on enzyme stability or activity.
Dynamics Underlying Cytochrome P450cam Regioselectivity via 2D IR Spectroscopy
Authors: Thielges Megan , Basom Edward
Published: November 2023

Conformational heterogeneity and dynamics likely contribute to the remarkable activity of enzymes, but are challenging to characterize experimentally. We applied linear and 2D IR spectroscopy to compare the conformational heterogeneity and dynamics of wild‐type and Y96F cytochrome P450cam‐CO bound to substrates that are hydroxylated with varying regioselectively. Our results suggest that the binding of different substrates to P450cam variably stabilizes the active site into two distinct states associated with different levels of regioselectivity. P450cam regioselectivity appears to be unrelated to the average electrostatic environment in the protein‐substrate complexes; instead, regioselectivity is correlated with conformational heterogeneity and dynamics within the populated conformations, supporting that fast dynamics contribute to the activity of this enzyme. We are extending the studies to measure conformations and dynamics throughout this and other proteins via the selective incorporations of IR‐active probe groups with spectrally resolved frequencies as an approach to the measurement of dynamics of proteins with high temporal and spatial resolution.
Propagation of tau prions from Alzheimer's disease and chronic traumatic encephalopathy patients in cultured cells
Authors: Woerman Amanda L , Aoyagi Atsushi , Patel Smita , Kazmi Sabeen A , Lobach Iryna , Grinberg Lea T , McKee Ann C , Seeley William W , Olson Steven H , Prusiner Stanley B
Published: November 2023

Tau prions aggregate in the central nervous system, giving rise to neurodegeneration. Among the tauopathies, Alzheimer's disease (AD) is the most common, whereas chronic traumatic encephalopathy (CTE), Pick's disease (PiD), and progressive supranuclear palsy (PSP) are less prevalent. Brain extracts from deceased individuals with PiD, a neurodegenerative disorder characterized by three‐repeat (3R) tau prions, were used to infect HEK293T cells expressing 3R tau fused to yellow fluorescent protein (YFP). Extracts from argyrophilic grain disease (AGD), corticobasal degeneration (CBD), and PSP patient samples, which contain four‐repeat (4R) tau prions, were transmitted to HEK293 cells expressing 4R tau fused to YFP. These studies demonstrate that prion propagation in HEK cells requires isoform pairing between the infecting prion and the recipient substrate. Interestingly, tau aggregates in AD and CTE, which contain both the 3R and 4R tau isoforms, were unable to robustly infect either 3R‐ or 4R‐expressing cells. However, AD and CTE prions were able to replicate in HEK293T cells expressing both 3R and 4R tau. Unexpectedly, increasing the level of 4R isoform expression alone supported the propagation of both AD and CTE prions. The finding that tau prion strains are isoform specific may have important implications for developing anti‐tau therapeutics and disease‐specific PET probes.

Support or Funding Information

This work was supported by NIH Grants AG002132 and AG031220, Daiichi Sankyo, Dana Foundation, Glenn Foundation, Sherman Fairchild Foundation, and a gift from the Rainwater Charitable Foundation. Control, AD, AGD, CBD, PiD, and PSP tissue samples were provided by the University of California, San Francisco, Neurodegenerative Disease Brain Bank, which is supported by NIH Grants AG023501 and AG19724 (to W.W.S.), Tau Consortium, and Consortium for Frontotemporal Dementia Research. CTE tissue samples were provided by the Boston University Alzheimer's Disease Center Chronic Traumatic Encephalopathy Center, which is supported by the Department of Veterans Affairs, National Institute of Neurological Disorders and Stroke, National Institute of Biomedical Imaging and Bioengineering (NS086559), National Institute on Aging, Boston University Alzheimer's Disease Center (AG13846; Supplement 0572063345–5), Concussion Legacy Foundation, Andlinger Foundation, and World Wrestling Entertainment, Inc.
Protein Aggregation Small Molecule Inhibitor Discovery and Mechanisms
Authors: Xu Bin
Published: November 2023

Epidemiological and clinical studies showed significant association between type 2 diabetes (T2D) and the risk for Alzheimer's disease. Pancreatic hormone amylin is a highly amyloidogenic protein and amylin amyloid deposition in the pancreas are hallmark features of T2D. Recent clinical studies showed that amylin plaques were deposited in the brain of diabetic patients, but not in healthy controls. We performed cell‐based studies, demonstrating that amylin amyloid is highly toxic to pancreatic β‐cells INS1 and neuronal cells SH‐SY5Y and Neuro2A. From a collection of natural compounds used in alternative medicine, we identified multiple potent inhibitors, including rosmarinic acid (RA) and baicalein (200 nM and 1 μM respectively in apparent IC50). These lead compounds disaggregate amylin fibrils from transmission electron microscopic observations and significantly reduce amylin‐induced cytotoxicity. Dissecting the functional groups of these compounds, we demonstrated, for the first time to our knowledge, that the vicinal hydroxyl groups of the catechol groups played key functional roles in amyloid inhibition in more than a dozen catechol‐containing compounds, including physiological neurotransmitters epinephrine, norepinephrine, and dopamine. Compounds with multiple catechol groups, such as rosmarinic acid, exhibited additive/synergistic effects. We provided further mass spectrometric evidence that incubating several of these catechol‐containing inhibitors with amylin leads to covalent adducts consistent with Schiff base conjugation as a mechanism for blocking toxic amyloid formation. The inhibition effects by these compounds were also demonstrated in molecular simulation analyses, providing additional non‐covalent inhibition mechanisms. To expand protein aggregation inhibitor discovery, we applied the concept of drug repurposing. We developed a 384‐well plate based screening platform and screened a NIH Clinical Collection small molecule library that are currently in phased trials. We were able to rapidly identify 10–30 each of “hits” against four different amyloidogenic proteins: amylin, Aβ, tau, and α‐synuclein. Excitingly, we were able to identify compounds that can inhibit all four amyloids but also “protein‐specific” inhibitors. Mechanisms for “general” protein aggregation inhibitors and for “protein‐specific” aggregation inhibitors will be elucidated.

Support or Funding Information

This work is in part supported by Virginia Tech new faculty start‐up funds, Commonwealth Health Research Board (CHRB), Alzheimer's and Related Diseases Research Award Fund (ARDRAF) from Virginia Center on Aging, Diabetes Action Research and Education Foundation (DAREF), and Virginia Tech Center for Drug Discovery (VTCDD).
Characterization of Anti‐SOD1 Antibodies and Detection of Intermediary SOD1 Oligomers
Authors: Atlasi Ryan Saleh , Malik Ravinder , Corrales Christian , Tzeplaeff Laura , Cashman Neil , Bitan Gal
Published: November 2023

Mutations in the gene encoding the enzyme copper‐zinc superoxide dismutase (SOD1) are linked to 10–20% of familial amyotrophic lateral sclerosis (fALS) cases. The mutations lead to misfolding and self‐assembly of SOD1 into toxic oligomers and aggregates, resulting in motor neuron degeneration. However, the molecular mechanisms underlying SOD1 aggregation and toxicity are unclear. Characterization and detection of SOD1 oligomers is particularly challenging due to their metastable nature. Antibodies against oligomeric and misfolded SOD1 forms are useful tools for this purpose, provided their specificity and selectivity are well characterized, which is not always the case. In this study, we set out to characterize three novel anti‐misfolded SOD1 antibodies and compare them with two commercial anti‐misfolded SOD1 antibodies raised against G93A SOD1. As controls, we compared the reactivity of these antibodies to two polyclonal anti‐SOD1 antibodies that were expected to be insensitive to misfolding. We asked whether the antibodies could distinguish among native and misfolded conformations of SOD1 and whether any of them were specific for the elusive oligomeric forms of the protein. To follow SOD1 conformational change and aggregation kinetics, wild‐type and G93A SOD1 were incubated under reducing conditions and the reactions were followed using thioflavin T (ThT) fluorescence, electron microscopy (EM), and dot blots with each antibody. Control reactions were followed under non‐reducing conditions. The three new anti‐misfolded SOD1 and one of the commercial antibodies (B8H10) showed maximum affinity at the time oligomers were expected to dominate and lost affinity with fibril formation. Unexpectedly, the two polyclonal antibodies also showed preference for oligomers, especially of G93A SOD1. Surprisingly, the other commercial anti‐misfolded SOD1 antibody, C4F6, showed specificity for G93A SOD1 regardless of misfolding. Two of the new antibodies showed preference for early or late oligomers, suggesting that they could expand currently detectable temporal resolution. In conclusion, our data indicate that certain antibodies are highly sensitive to the misfolded form of SOD1, whereas others are insensitive to either mutation or aggregation.

Support or Funding Information

RGK Foundation
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 Information

NIH 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 Information

National 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 Information

This 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 Information

This 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 Information

National 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 Information

This work was supported by a grant from the National Institutes of Health (HL58984 to RB) and the American Heart Association (14POST18760003 to PKY).
Resurrection of Caspase‐6 Ancestral States
Authors: Grinshpon Robert D , Clark A Clay
Published: November 2023

Caspase proteins predate multicellularity. Caspases regulate cellular homeostasis by balancing cell proliferation with apoptosis. They also participate in crucial non‐apoptotic functions when their activity is regulated below an unknown apoptotic threshold. Aberrant caspase activity causes a multitude of human diseases, ranging from cancer to neurodegenerative disorders. A major obstacle to caspase research is the evolutionarily conserved active‐site structure. Cross‐reactivity of active‐site directed drugs among the family members has prevented meaningful experimentation. Thus, clinical efficacy and an understanding of sub‐threshold behavior remains elusive. Each caspase evolved unique regulatory mechanisms that determine their spatiotemporal activation and niche cellular functions. We hope to learn how these mechanisms evolved in caspases while maintaining the canonical fold and high specificity for aspartate at the P1 cleavage position. This project focuses on a unique regulatory mechanism in caspase‐6 that involves a strand‐to‐helix conformational switch. We collected over 1000 caspase protein sequences ranging from mammals to chondricthys (sharks), and performed an ancestral state reconstruction (ASR). Select ancestral states along the evolutionary path will be resurrected, and characterized in the lab with circular dichroism, activity assays and X‐ray crystallography. Preliminary data suggests that the conformational switch of caspase‐6 arose before mammals diverged, and sometime after mammals diverged from actinopterygii (fish). The mutations along the branch leading up to the neofunctionalization will shed light on how the mechanism evolved. Understanding the various natural allosteric mechanisms among caspase proteins is a crucial step to overcoming the obstacle of drug specificity, and will open up new avenues to meaningful experimentation. Large scale sequence datasets for bioinformatics analysis have recently become available to researchers. This study is a pioneer in exploring the potential of ASR to explain how evolution begets variation in protein family behavior via functional divergence.
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.
Allosteric regulation and enzymatic mechanism of YopJ family of bacterial effectors
Authors: Song Jikui , Zhang Zhimin , Ma Ka‐Wai , Gao Linfeng , Ma Wenbo
Published: November 2023

Bacterial effectors secreted by the type III secretion system manipulate host signaling pathways to promote bacterial pathogenesis. The Yersinia outer protein J (YopJ) family of effectors, found in diverse plant and animal bacterial pathogens, represent a novel class of acetyltransferases that modify cellular proteins to suppress host defense. Distinct from other acetyltransferases, YopJ family of acetyltransferases modifies target proteins through a cysteine protease‐like catalytic core, which is further regulated by inositol hexakisphosphate (IP6), a eukaryote‐specific ligand. However, the substrate recognition and catalytic mechanism of these enzymes have not been well understood. Through structural and biochemical analyses, we demonstrate that both formation of the acetyl‐CoA binding pocket and substrate binding of YopJ effector is allosterically regulated by the IP6‐induced structural rearrangements. Furthermore, we present evidence that YopJ effector employs a “ping‐pong” route of catalysis involving formation of an “acetyl‐cysteine” intermediate. Together, our study sets the structure‐function paradigm for a novel class of acetyltransferases and a conserved regulatory mechanism of YopJ effectors.
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, 4

Support or Funding Information

This 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 Information

This 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).
Electrochemical detection of isatin using flow injection analysis with amperometric detection
Authors: Sanchez Simon Wolfgang , Jarosova Romana , Swain Greg M
Published: November 2023

Isatin is a heterocyclic compound that has been shown to have a wide range of biological activities. Isatin is found endogenously in humans and rodents. In rat models, isatin concentrations have been shown to increase with stress in the heart, brain, blood plasma, and urinary samples. In the rat, highest concentrations are in the vas deferens and seminal vesicles with levels in the heart somewhat higher than in the brain. The highest concentrations in the brain are found in the hippocampus and cerebellum. Isatin is electrochemically active and can be detected with electrochemical techniques. Of these techniques, flow injection analysis is a versatile technique used for the determination of easily oxidizable or reducible analytes. The performance of a nitrogen‐incorporated tetrahedral amorphous carbon electrode and a boron‐doped diamond electrode was evaluated using flow injection analysis with amperometric detection. The boron‐doped diamond electrode is known for its excellent properties such as a low stable background current, weak molecular absorption, and microstructural stability, but has a high deposition temperature required for growth, (600–800 °C). Similarly, the ta‐C:N electrode has been shown to exhibit many of the same attractive properties of the boron‐doped diamond electrode such as a low background current, microstructural stability, and weak molecular absorption with the advantage of a low deposition temperature near room temperature (25–100 °C). The analytical detection figures of merit such as the response precision, sensitivity, linear dynamic range and limit of detection for isatin in a 0.1 M phosphate buffer (pH 7.2) were determined for both electrodes.
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 Information

Aaron Hoskins is a Beckman Young Investigator of the Arnold and Mable Beckman Foundation