bluuebunny/crossref_metadata_2025 huggingface dataset:
Predictive Models of Peptide RMS Fluctuations in the Context of HLA‐A*02:01 Through Sequence Alone
Authors: Ayres Cory , Riley Timothy , Corcelli Steven , Baker Brian
Published: November 2023

Much of the current day understanding of T‐Cell receptor (TCR) recognition of peptide major histocompatibility complexes (pMHCs) is based on studies of crystal structures of TCR/pMHC complexes. Though insightful, these studies fail to directly investigate the role of dynamics in these interactions. Accordingly, recent effort has gone into characterizing differences in dynamics of these proteins, and relating them to affinity, stability and immunogenicity. In particular, in a recent study we observed a relationship between differences in RMS fluctuations of wild‐type and tumor derived peptides, and whether the tumor derived peptide was immunogenic. While utilizing differences in peptide dynamics to predict immunogenicity seems promising, the method is limited by computational cost and time required to generate the necessary molecular dynamics simulations. As such, we sought to generate models to predict RMS fluctuations of a peptide through sequence alone. Predictive models based on 53 1μs simulations of HLA‐A*02:01 presenting different nonameric peptides. Through differences in chemical composition, seven models were generated which predict the Cα RMS fluctuations of a theoretical nonameric peptide. These models provide accurate predictions of expected RMS fluctuations, and also highlight how a single point mutation can impact the fluctuations of distal peptide residues.
A high‐throughput approach to annotate the lysine methylome
Authors: Cornett Evan M , Dickson Bradley M , Krajewski Krzysztof , Cowles Martis W , Sun Zu‐Wen , Rothbart Scott B.
Published: November 2023

Lysine methylation is a widespread posttranslational modification (PTM) in eukaryotes whose protein regulatory functions have been illuminated through studies of histone lysine methylation and its key connections to chromatin and epigenetic regulation. Beyond histones, a major gap in knowledge is broadening between the rapidly expanding lysine methylome and the proteins responsible for the addition, removal, and recognition of these PTMs. As several inhibitors of lysine methylation regulators are in preclinical and clinical development for cancer therapy, defining the full target spectrum of these proteins is critical for understanding mechanisms of action for these drugs, to rationalize potential combination therapies, and to identify reliable biomarkers for clinical activity. Here we debut the development of a high‐throughput and unbiased functional proteomics platform that queries the activities of lysine methyltransferases (KMTs) and generates molecular footprints that can be mapped to protein targets. We demonstrate the utility of this platform towards the discovery of novel substrates for KMTs. Full implementation of this platform will provide critical systems level information to enable the construction of lysine methylation signaling networks and will facilitate fundamental understanding of lysine methylation function in human health and disease.

Support or Funding Information

Van Andel Research Institiute, Van Andel Institute Employee Impact Campaign, and EpiCypher
The Importance of a Salt Bridge in FNR Transcription Factor Activity
Authors: Kazmouz Sobhi Gheath , Badang Louis J , Moore Laura J
Published: November 2023

Fumarate Nitrate Reductase transcription factor (FNR) regulates the expression of genes involved in anaerobic respiration within E. coli bacteria. In the absence of oxygen, the FNR protein contains a [4Fe‐4S] cluster. This [4Fe‐4S] cluster causes a change in the dimerization helix of FNR, allowing the protein to dimerize and bind to DNA. In a recent publication, the crystal structure of A. fischeri FNR, which has high homology to E. coli FNR, was reported[1]. The structure suggests that the [4Fe‐4S] cluster promotes dimerization through a salt bridge interaction between residues Asp 130 and Arg 140. This investigation focuses the effect of the salt bridge interaction on the activity of E. coli FNR. Two mutant variants of FNR (Asp130Arg and Arg140Glu) were constructed in a pET11a plasmid containing the FNR gene. In each variant, one of the amino acid residues constituting the salt bridge was replaced with an oppositely charged amino acid side chain. The activity of the FNR mutant proteins was then measured under anaerobic conditions using the narG‐lacZ reporter strain. The results show that the activity of FNR decreases significantly when the salt bridge is broken. In addition, the results of the mutations that swap the amino acids of the salt bridge will be discussed.
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 Information

National 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 Information

University 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 Information

Saint 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 Information

Supported 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 Information

National 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 Information

This 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 Information

This 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 Information

The 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 Information

All 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 Information

American 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 Information

This 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).
Drugging the undruggable steroid receptor coactivators
Authors: Wang Jin
Published: November 2023

Protein–protein interactions (PPIs) play a central role in most biological processes, and therefore represent an important class of targets for therapeutic development. However, disrupting PPIs using small‐molecule inhibitors (SMIs) is challenging and often deemed as “undruggable.” We developed a cell‐based functional assay for high throughput screening to identify SMIs for steroid receptor coactivator‐3 (SRC‐3 or AIB1), a large and mostly unstructured nuclear protein. Without any SRC‐3 structural information, we identified SI‐2 as a highly promising SMI for SRC‐3. SI‐2 meets all of the criteria of Lipinski's rule for a drug‐like molecule and has a half‐life of 1 h in a pharmacokinetics study and a reasonable oral availability in mice. As a SRC‐3 SMI, SI‐2 can selectively reduce the transcriptional activities and the protein concentrations of SRC‐3 in cells through direct physical interactions with SRC‐3, and selectively induce breast cancer cell death with IC50 values in the low nanomolar range (3–20 nM), but not affect normal cell viability. Furthermore, SI‐2 can significantly inhibit primary tumor growth and reduce SRC‐3 protein levels in a breast cancer mouse model. In a toxicology study, SI‐2 caused minimal acute cardiotoxicity based on a hERG channel blocking assay and an unappreciable chronic toxicity to major organs based on histological analyses. We believe that this work could significantly improve breast cancer treatment through the development of “first‐in‐class” drugs that target oncogenic coactivators.

Support or Funding Information

This work is supported in part by National Institutes of Health Grants R01GM115622, Cancer Prevention and Research Institute of Texas Grants R1104, Welch Foundation Grant Q‐1798; the Center for Comparative Medicine, the Cytometry and Cell Sorting Core, the Center for Drug Discovery, and the Dan L. Duncan Cancer Center at Baylor College of Medicine; and the Texas Medical Center Digestive Diseases Center.
Total Synthesis, Antileishmanial and Anticancer Activity of the Acetylenic Fatty acids 6‐Hexadecynoic, 10‐Phenyl‐6‐decynoic, and 10‐Cyclohexyl‐6‐decynoic Acids
Authors: Álvarez Ester , Carballeira Néstor , Morales Christian , Delgado Yamixa , Tinoco Arthur , Reguera Rosa , Álvarez Raquel , Balaña Rafael
Published: November 2023

Naturally occurring acetylenic fatty acids have been recognized for their biological activity towards cancer and protozoal parasites. Lung cancer is one of the deadliest types of cancer and the second most incidental cancer in both men and women. Previous research has shown that these acids display cytotoxicity against the human lung carcinoma cell line A549. On the other hand, leishmaniasis is a neglected disease caused by the protozoan parasite Leishmania donovani(infantum) that affects mostly underdeveloped countries and can be fatal in the most severe cases (visceral leishmaniasis). Our laboratory has shown that the Δ6 alkynoic fatty acids display antiprotozoal activity and inhibit the Leishmania donovani topoisomerase IB enzyme (LdTopIB). In this work, the total syntheses of the naturally occurring 6‐hexadecynoic acid and the synthetic 10‐phenyl‐6‐decynoic and 10‐cyclohexyl‐6‐decynoic acids were completed in 5 steps (67–77% overall yields). The 6‐hexadecynoic acid displayed good inhibition of LdTopIB (EC‐‐50=61 μM) and cytotoxicity towards L. infantum iRFP70 promastigotes (IC50=100 μM). In addition, the 6‐hexadecynoic acid also displayed cytotoxicity towards the human lung A549 cell line (EC‐50= 65 μM). On the other hand, the 10‐phenyl‐6‐decynoic acid did not inhibit LdTopIB (EC‐‐50>200 μM) and displayed no cytotoxicity towards L. infantum iRFP70 promastigotes (IC50>100 μM) as well as no cytotoxicity towards A549 (EC50>100 μM). The 10‐cyclohexyl‐6‐decynoic acid also showed no inhibition of LdTopIB, no cytotoxicity towards L. infantum promastigotes, but displayed cytotoxicity against the A549 cell line (EC‐50= 87 μM). Our results show that the 6‐hexadecynoic acid was the best of the series due to its inhibitory activity towards LdTopIB and toxicity towards L. infantum promastigotes and the A549 cell line.

Support or Funding Information

NIH‐MARC Program undergraduate fellowship (5T34GM00781‐37) and the NIH‐RISE Program graduate fellowship (5R25GM061151‐15)
In vivo drug discovery for progressive supranuclear palsy using a novel zebrafish model
Authors: Burton Edward A , Bai Qing
Published: November 2023

Progressive supranuclear palsy (PSP) is a common neurodegenerative disorder causing progressive motor, oculomotor, bulbar, and cognitive deficits. Current treatments do not arrest disease progression and the prognosis for functional independence and survival is correspondingly poor. Pathologically, PSP is characterized by accumulation of insoluble hyperphosphorylated forms of the 4‐repeat isoform of the microtubule associated protein tau (4R‐Tau) and subsequent loss of CNS neurons. Convergent lines of evidence suggest that 4R‐Tau is mechanistically involved in the neurodegeneration of PSP. Clinical and pathological PSP phenocopies can result from splice site mutations in the MAPT gene causing over‐expression of 4R‐Tau. Furthermore, PSP is strongly associated with the H1 MAPT haplotype that gives rise to relative over‐production of 4R‐Tau. Consequently, compounds that prevent neurodegeneration caused by 4R‐Tau in vivo may be effective therapeutic agents in PSP.

The long‐term goal of our work is to develop effective treatments for progressive supranuclear palsy (PSP). Development of therapies for PSP and other tauopathies has been limited by lack of animal models that allow discovery‐based approaches, for example screening candidate chemical modifiers against disease‐relevant phenotypes in vivo. The zebrafish is a genetically and chemically tractable vertebrate model that is particularly suited to drug discovery and comparative efficacy testing and is being used increasingly for studying human neurological diseases. The objective of this study was to develop a zebrafish model of PSP and to carry out a phenotype based chemical modifier screen in vivo.

We generated transgenic zebrafish that express human 4R‐Tau in CNS neurons. Tau transgenic zebrafish replicated many clinic‐pathological features of PSP, including: hypokinesia; oculomotor deficits; somato‐dendritic accumulation of hyperphosphorylated tau; neuronal apoptosis; and impaired survival. Importantly, these phenotypes were quantitative, replicable and occured rapidly allowing their use as endpoints in a phenotype‐driven screen. We tested over 200 bioactive compounds to identify chemicals that rescued motor function in the 4R‐Tau zebrafish, using a custom automated video tracking and motion analysis method. We identified several ‘hits’ that rescued motor function and showed activity in subsequent verification assays.

Our completion of the first unbiased phenotype‐based chemical modifier screen in a vertebrate tauopathy model provides proof‐of‐concept that drug discovery can be carried out in vivo by assaying disease‐relevant neurological deficits. Importantly our screen has so far identified several candidates for further development as possible tau therapeutics.

Support or Funding Information

US Department of Veterans' Affairs (BX003168‐01)

NINDS (NS080881)

CurePSP (441‐05, 468‐08)

Pittsburgh Foundation (AD201577642)
The Antimicrobial Property of the Acetone Extract of Cola Acuminate
Authors: Thomas Brianna J , Telles Caroline M
Published: November 2023

The increase in drug‐resistant bacteria during the recent years has sparked an interest in antimicrobial properties of medicinal plants. Thus, the mechanism by which medicinal plants kill bacteria is certainly an important topic that merits further research. Cola acuminate, also known as Obi or Bizzy nut to the Ettu people of Jamaica, is a “cure‐all” herbal medicine. Cola acuminate (Bizzy Nut), possesses a variety of bioactive compounds which exhibit important inhibitory activities against the growth of certain mammalian cell lines, bacteria and fungi. However, the specific chemicals responsible for this bioactivity have not been identified. Thus, we hypothesized that the solid‐liquid extraction of Bizzy Nut, followed by antimicrobial screening would lead to the identification of active antimicrobial agents present in Bizzy Nut.

As an initial step in testing our hypothesis, finely ground samples of Bizzy nut were sequentially extracted in a Soxhlet apparatus using 100% hexane, ether, acetone, methane, or water to produce five independent extracts of unique polarity. The antimicrobial activity of the acetone extract was carried out using Staphylococcus aureus and Pseudomonas aeruginosa as test organisms. Our results show that the acetone extract (Biz‐3) possesses antimicrobial activity in Staphylococcus aureus at concentrations as low as 40ppm. There was a dose‐dependent increase in the zone of inhibition of Biz‐3 from 8.5±0.30mm at 40ppm to 11.7±0.1 mm at 120ppm. To purify this antimicrobial activity, we have used SPE to generate three unique fractions of the acetone extract. The antimicrobial screening (Minimum Inhibitory Concentration, MIC) of each of the SPE fraction will be discussed. Our conclusion from this work is that the acetone extract of Cola acuminate (Bizzy Nut), contains putative antimicrobial bioactivity compounds that are effective again gram positive bacteria.
An investigation of the oxidatively damaged transcriptome in the human neuronal cells
Authors: Kharel Prakash , Gadepalli Venkat , Chattopadhyay Anshuman , McDonough Jennifer , Basu Soumitra
Published: November 2023

Reactive oxygen species (ROS) are produced intracellularly and can cause oxidative damage to the biomolecules. Under normal physiological conditions ROS are used in cellular homeostasis and cell signaling but during stress condition they are often overproduced. The overproduction of the ROS is associated with age and neurological disorders, such as, Alzheimer's disease, Parkinson's disease and multiple sclerosis. The weakened antioxidant defense mechanism in the brain of the patients with the neurological disorders makes the neurons susceptible to oxidative damage. The ROS can damage every major class of biomolecules including DNA, RNA and proteins. In the past the focus was mainly on studies of DNA and protein damages, while the detrimental effects of RNA damages only began to be highlighted in recent years. 8‐Hydroxyguanosine (8‐OHG) is the most common oxidative modification found in the RNA. By identifying the modified mRNA molecules during oxidative stress in the neuronal cells (SH‐SY5Y), we aim to delineate the relationships between specific mRNA oxidation and neurodegeneration. We have isolated the modified mRNA from the stressed SH‐SY5Y cells by immunoprecipitation with anti 8‐OHG antibody. Using RT‐qPCR assay we have shown the differential mRNA expression during the oxidative stress, for example, high expression of superoxide dismutase 1 and carbonyl reductase mRNAs. Additionally, we have identified the selectively modified mRNA molecules by using iLumina® RNA sequencing platform. This study will shed light on the yet to be understood link between mRNA oxidation and neurodegeneration.

Support or Funding Information

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DC STAMP Domain: Intercompatibility Between SPE‐42 and Other Proteins
Authors: Okeke Ikenna Simon
Published: November 2023

C. elegans is a microscopic, free living nematode. It has a completely sequenced genome, a short generation period, produces large broods, and it is transparent. All of these features makes C. elegans a good model for the research we were conducting. C. elegans exists as self‐fertile hermaphrodites and males that can fertilize hermaphrodites with their sperm. C. elegans hermaphrodites with spe‐42 or let‐479 mutations are self‐sterile because their sperm are incapable of fertilizing oocytes despite being indistinguishable from wild type sperm. When the mutatant hermaphrodite is crossed with a male, the male's sperm are able to rescue the oocyte and fertilize it, showing that the mutant phenotype is a sperm cell‐specific error. This also opens up several questions about the role of spe‐42 in the fertilization of the oocytes. spe‐42 and let‐479 are not specific to C. elegans. Homologs of these genes have been found in all species that use sperm–egg fertilization techniques. In this work, we analyzed the amount of progeny produced when the DC‐STAMP domains, which are required for protein function, were swapped from other proteins into SPE‐42 or LET‐479. By using C. elegans as a model for fertilization help us become more knowledgeable about the reproductive systems of other organisms.
Deciphering the Gene Expression Pattern from Human Blood Samples Collected in Multiple Collection Tubes
Authors: Hoke Allison , Gautam Aarti , Donahue Duncan , Miller Stacy Ann , Srinivasan Seshamalini , Detwiler Leanne , Lynch Jesse , Levangie Michael , Sowe Bintu , Hammamieh Rasha , Jett Marti
Published: November 2023

Theobjective of this study was to investigate how blood storage, extraction methodologies, and the actual blood component itself may influence what genes are expressed in downstream applications. To discover potential biases, blood was collected in triplicate from five donors into RNAgard Blood Tubes, PAXgene Blood RNA Tubes, EDTA Tubes, Sodium Citrate Cell Preparation Tubes (CPT), and Blood Collection Tubes with Acid Citrate Dextrose Solution A (ACD‐A). The RNA was isolated from whole blood and peripheral blood mononuclear cells (PBMCs) using ten different combinations of experimental conditions combined with several widelyutilized RNA isolation methods. After extraction, the RNA was quantified using a spectrophotometer and RNA quality was assessed by the RNA Integrity Number (RIN) value. The RNA was then profiled using Agilent Human Gene Expression micro arrays to observe the expression levels for the different experimental conditions. When the RNA quality was assessed by the RIN value, we found that all PBMC procedures had the highest RIN values as they were processed and stabilized in TRIzol Reagent prior to RNA extraction. Initial data analysis showed that human blood stored at 4°C overnight performed equally well when compared to frozen stabilized blood. We compared within and across donor and method replicates, which indicated signal to noise patterns that were not captured by RIN value alone. Our results provide some new insights into RNA isolation from blood samples and how it is influenced by the choice of storage, handling, and methodologies, and that careful consideration is necessary to avoid bias in downstream processing. Better characterization of collection method idiosyncrasies will facilitate further research in to understanding the effect of gene expression on variability in human sample types.

DISCLAIMERS: Research was conducted in compliance with all Federal Requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army.
Dissecting JADE1 pathway in the Regulation of Cytokinesis
Authors: Panchenko Maria V , Shao Bo
Published: November 2023

Cell cycle regulation is important for normal proliferation of epithelial cells and epithelial tissue homeostasis. The dis‐regulation of the cell cycle progression is linked to genetic instability, aneuploidy, and apoptosis. Cytokinesis, the final stage of cell division, is responsible for the proper partitioning of duplicated chromatin and other cell content between two distinct daughter cells. We previously identified that JADE1 protein associates with chromatin and promotes acetylation of bulk histone H4 by functioning as an adapter and scaffold in the HBO1 complex, which is histone acetyl transferase (HAT) responsible for bulk histone H4 acetylation in vitro and in cultured cells. Our finding of cell cycle mediated JADE1S chromatin shuttling and phosphorylation suggested JADE1 function in cell cycle progression, specifically during G2/M to G1 progression.

Objective

to examine JADE1 role in the regulation of the cell cycle.

Materials and methods

cultured cell lines, cDNA transfection, transduction, RNA interference, FACS, western blot, fluorescent confocal microscopy.

Results

The depletion of JADE1 in synchronized dividing cells increased rates of G2/M to G1 progression as evident from kinetics of G1 cell accumulation. Importantly, JADE1S depletion decreased proportion of cytokinetic cells and increased multinucleates, while JADE1 over‐expression increased cytokinetic proportion. Interestingly, endogenous JADE1 in late telophase was associated with midzone and in cytokinesis with midbody. Inhibition of key mitotic regulator Aurora B kinase with a drug released JADE1S' effects on cytokinesis. These data demonstrate role in cytokinesis and suggest role in abscission delay. Further, we investigated whether HAT HBO1 and unresolved chromatin are involved in JADE1 cytokinesis pathway. According to our results, HBO1 exerts antagonistic effects on JADE1 function by interfering with JADE1‐mediated cytokinesis arrest. Moreover, HBO1 over‐expression compromised midbody associations of JADE1 in early cytokinesis.

Conclusions

JADE1 represents novel cytokinesis factor; HAT HBO1 interferes with JADE1 function in cytokinesis.

Support or Funding Information

NIH RO1 DK087910, Mallory Fund (Boston Medical Center, Pathology)
DETERMINING THE LINK BETWEEN MAMMALIAN p38 STRESS KINASE PATHWAY AND PEROXISOMES
Authors: Singh Rajnish , Chrestensen Carol
Published: November 2023

The inability of the body to fight oxidative stress is the cornerstone of aging and diseases such as inflammation, cancer, diabetes and Alzheimer's. The response of the cell to oxidative stress involves two key players, the p38 stress kinase pathway and the peroxisome, and the interaction between this pathway and peroxisome is relatively unknown. Several pieces of evidence point to a role for protein kinases in phosphorylating peroxisome proteins and regulating important peroxisome functions during stress. Plant kinases and phosphatases have been shown to localize to the peroxisome under stress conditions. Mammalian ATM kinase, a kinase activated by DNA damage, has also been shown to translocate to peroxisomes, during oxidative stress, phosphorylating and activating proteins that regulate peroxisome turnover. Some peroxisome biogenesis factors in yeast require phosphorylation to function.. This project seeks to determine if the p38 activated Mapkap kinase 2 isoform 2 (MK2) translocates to the peroxisome and serves as the link between the p38 stress kinase pathway and the stress alleviating organelle, the peroxisome. Sequence analysis of human MK2 shows the presence of ‐TRL, a putative peroxisome targeting sequence (PTS1) at its C‐terminus. The consensus peroxisome targeting sequence ‐SKL found on many peroxisome proteins, binds to a cytosolic receptor called pex5, which then targets the protein to the peroxisome. In this study, crude peroxisome fractions were isolated from fresh chicken livers using differential centrifugation and probed with isoform specific MK2 antibodies. Preliminary western blotting experiments indicate the presence of both MK2 isoforms in the crude peroxisome fractions. Additionally, experiments were done to determine the physical interaction between the putative peroxisome targeting sequence in mapkap kinase 2 (‐TRL) and pex5, using the yeast two‐hybrid assay. Initial results indicate that MK2, isoform 2, shows a weak interaction with Pex5 suggesting that MK2 isoform 2 maybe localizing to the peroxisome through its putative peroxisome targeting sequence. The link between mapkap kinase 2, a stress activated protein kinase and a cellular organelle that is important in combating cellular stress is novel and has the potential to lead to exciting new mechanisms in stress signaling.

Support or Funding Information

Incentive Funding for Research & Creative Activity, Center for Excellence in Teaching and Learning, Kennesaw State University