bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interleukin‐17A expression in endometriotic tissue and plasma samples from women with endometriosis (539.2)
Authors: Ahn Soo , Edwards Andrew , Nakamura Diane , Lessey Bruce , Tayade Chadrakant , Reifel Conrad 
Published: June 2021

Cytokines present within the environment of endometriosis are believed to contribute to the pathogenesis of the disease by promoting inflammatory conditions surrounding the implantation of endometriotic lesions. Interleukin‐17A is a member of proinflammatory cytokines implicated in the pathogenesis of various chronic inflammatory diseases. However, its role in the disease progression of endometriosis is poorly understood. The objective of this study was to elucidate the involvement of IL‐17A in the pathogenesis of endometriosis. To investigate this, IL‐17A concentration was measured in plasma, eutopic and ectopic samples from women with endometriosis and in plasma and eutopic samples from women without endometriosis. Immunostaining for IL‐17A was conducted on eutopic and ectopic tissue sections. To elucidate biological function of IL‐17A, WST‐1 proliferation assay, cell cycle analysis with Propidium Iodide (PI) , and supernatant analysis were performed with endometrial epithelial carcinoma cells (EECCs) . Expression of IL‐17A, IL‐17RA, and IL‐17RC mRNA from EECCs and primary EECs was measured using qPCR. Flow cytometry was performed on EECCs to detect IL‐17RA expression. Results show presence of IL‐17A in plasma samples and ectopic tissue samples from women with endometriosis. Imunohistochemistry confirmed the presence of IL‐17A positive cells within both the epithelium and stroma of eutopic and ectopic tissue sections. Even though IL‐17A has shown no proliferative or apoptotic effect on EECCs, its stimulation on EECCs induced increased production of G‐CSF, VEGF, PDGF‐AA, and SDF‐1. Follow up investigation is needed to elucidate the understanding of the relationship between the presence of IL‐17A and the pathogenesis of endometriosis.Grant Funding Source: Supported by CIHR
Structural basis for highly promutagenic replication across O6‐methylguanine by human DNA polymerase β (551.3)
Authors: Lee Seongmin , Koag Myong‐Chul , Min Kyungjin 
Published: June 2021

O6‐methylguanine (O6MeG) is a minor, yet highly genotoxic lesion that promotes misincorporation of T opposite O6MeG by various DNA polymerases in vitro. Human DNA polymerase β (polβ) has been shown to insert T opposite O6MeG ~30‐fold more frequently than C, albeit with a ~1000‐fold reduced insertion efficiency relative to the insertion of C opposite G. Currently, the structural basis for this slow, yet promutagenic phenomenon is unknown. To gain insight into this process, we solved seven X‐ray structures of polβ bound to O6MeG‐containing DNA: a binary structure with a gap opposite O6MeG, two ground state and two pre‐catalytic ternary structures with an incoming dC/T nucleotide paired with O6MeG, and two binary post‐insertion structures. The two ground‐state ternary structures with active site Mg2+ showed an open protein conformation and staggered base pair conformation, rather than the closed protein conformation and co‐planar base pair conformation typically observed for correct insertion. This structural difference suggests that polβ slows nucleotide incorporation opposite O6MeG by inducing an alternate conformation that is suboptimal for nucleotidyl transfer. The two pre‐catalytic ternary structures with active site Mn2+ showed that, remarkably, the T:O6MeG‐Mn2+ structure adopts closed protein conformation and Watson‐Crick‐type base pairing, whereas the C:O6MeG‐Mn2+ structure adopts open protein conformation and staggered base pairing. These striking structural differences between the T:O6MeG‐Mn2+ and the C:O6MeG‐Mn2+ complexes clearly explain preferential T misincorporation over C opposite O6MeG during replication by the enzyme. Our polβ structures featuring metal ion‐dependent conformational reorganization reveal structural basis for the slow, yet highly promutagenic replication across O6MeG by polβ.Grant Funding Source: Supported by the NIH ES23101
Biochemical and mass spectrometric analysis of the Klebsiella aerogenes urease accessory proteins (555.10)
Authors: Farrugia Mark , Carter Eric , Hausinger Robert 
Published: June 2021

Urease, which hydrolyzes urea into carbonic acid and ammonia, requires nickel, GTP, bicarbonate, and several accessory proteins for its activation through metallation. This work focuses on the soluble accessory protein complex containing a maltose binding protein/UreD fusion (MBP‐UreD), UreF, and UreG of Klebsiella aerogenes, and its interaction with the cognate urease. The properties of this complex were examined using gel permeation chromatography along with chemical cross‐linking and mass spectrometry of tryptic peptides, revealing it to be an overall dimeric species. Variants of UreD within the complex were analyzed for their ability to bind urease and facilitate its activation. Select UreD variants lacking the maltose binding protein solubility tag were analyzed for their ability to restore urease activation in a ∆ureD urease gene cluster. Also presented are biochemical studies that give further insight into the processes behind urease activation.Grant Funding Source: DK045686
Characterization of the activity and biological function of human protein arginine methyltransferase 9 (555.6)
Authors: Hadjikyriacou Andrea , Clarke Steven 
Published: June 2021

Protein arginine methyltransferases (PRMTs) make up a family of enzymes that transfer methyl groups onto arginine residues and play important roles in DNA repair, splicing, transcriptional control and signaling. In mammals, a family of nine PRMTs are known. PRMT1,2,3,4,6, and 8 catalyze the formation of asymmetric dimethylarginine (ADMA) and ω‐monomethylarginine (MMA), PRMT5 catalyzes symmetric dimethylarginine (SDMA) and MMA formation, and PRMT7 catalyzes only MMA formation. The last member of this family, PRMT9 (also designated PRMT10), has not previously been characterized. In humans, it is encoded on a gene on chromosome 4 in position 4q31. We have now expressed human PRMT9 both as a GST‐fusion protein in bacterial cells and as a GFP‐fusion protein in human HEK293 cells. For both expression systems, we have also prepared catalytic mutants to serve as controls for possible contamination of other types of PRMTs. Using an in vitro methylation assay, we show that both forms of PRMT9 catalyze the methylation of myelin basic protein and a GST‐fusion protein of the N‐terminal domain of human fibrillarin. Studies are in progress to characterize the type of activity and physiological substrates for PRMT9.Grant Funding Source: Supported by Ruth L. Kirschstein National Research Service Award GM007185
Regulation of ALC1 (amplified in liver cancer) involves interplay between the SNF2 ATPase domain, PAR binding macrodomain and other conserved elements (563.1)
Authors: Trivedi Rushi , Gottschalk Aaron , Conaway Joan , Conaway Ronald 
Published: June 2021

Regulation of ALC1 (Amplified in Liver Cancer) involves interplay between the SNF2 ATPase domain, PAR binding macrodomain and other conserved elements
Rushi D. Trivedi*1,2, Aaron J. Gottschalk1, Joan W. Conaway 1,2, Ronald C. Conaway 1,2
1Stowers Institute for Medical Research, Kansas City, MO; 2Department of Biochemistry, University of Kansas Medical Center, Kansas City, KS.ALC1, also known as CHD1L, was originally identified as a gene present on a human chromosome 1q21 region amplified in ~50% of human hepatocellular carcinomas. ALC1, a member of the SNF2 family of ATPases, has an N‐terminal SNF2‐like ATPase that is most closely related to that of ISWI, and a C‐terminal macrodomain that binds selectively to poly(ADP‐ribose) (PAR). Between the ATPase and the macrodomain is an evolutionarily conserved region with no clear homology to any known domains. This “linker” region can be further divided into three sub‐regions of greatest conservation.We have previously shown that wild type ALC1 possesses DNA‐dependent ATPase and ATP‐dependent nucleosome remodeling activities that are strongly activated by nucleosome bound PARylated PARP1. A point mutation in the ALC1 macrodomain that interferes with PAR binding prevents PARP1‐ and NAD‐dependent ALC1 activation.Our current line of studies are aimed at investigating the interplay between ALC1 ATPase and macrodomain in the context of nucleosome remodeling. Preliminary results suggest that the SNF2 ATPase of ALC1 is inherently inactive but is activated upon interaction with PARylated PARP. Upon performing an extensive mutagenesis study of the conserved regions within the “linker”, we have identified potential regulatory elements and a putative DNA binding domain that could facilitate the communication between the two domains. Furthermore, we are analyzing the interplay between the SNF2 ATPase, macrodomain, and “linker” by characterizing the activities of chimeric ALC1 constructs with macrodomains exchanged from different proteins.Grant Funding Source: Stowers Institute for Medical Research
From Stop to Go: role of the specialized elongation factor eEFSec in recoding UGA for selenocysteine incorporation (566.3)
Authors: Dubey Aditi , Szwed Angelia , Copeland Paul 
Published: June 2021

The incorporation of the 21st amino acid selenocysteine (Sec) into proteins requires the recoding of an in‐frame UGA, usually a termination codon, to a Sec codon. While it is known that Sec incorporation utilizes a dedicated set of cis and trans acting factors to synthesize these selenoproteins, how this recoding event takes place at the elongating ribosome is unknown. eEFSec, the selenocystyl‐specific elongation factor, is a specialized elongation factor that bears structural resemblance to the canonical elongation factor eEF1A. However, eEFSec recognizes only one species of tRNA, the Sec‐tRNASec, and delivers it to the ribosome only in response to an in‐frame UGA in the appropriate context. To elucidate eEFSec’s role in recoding UGA to Sec, we have conducted a systematic analysis of all four eEFSec domains using chimeric eEFSec/eEF1A proteins in our recently developed eEFSec‐dependent assay. In addition, through immunoprecipitation in various cell and tissue extracts, we have identified eEFSec‐specific interacting proteins that may act as regulatory elements of this specialized elongation cycle. Altogether, our results shed light on the molecular and cellular mechanism of recoding a Stop codon to selenocysteine in eukaryotes.Grant Funding Source: Supported by NIH Grant GM077073 to PRC
High fat diet induces an increase in the direct phosphorylation of glycerol which can contribute for the increase in white adipose tissue mass (575.2)
Authors: Buzelle Samyra , Ferreira‐Sodré Graziella , Valentim Rafael , Przygodda Franciele , Garófalo Maria Antonieta , Chaves Valéria , Navegantes Luiz Carlos , Kettelhut Isis do 
Published: June 2021

Triacylglycerol (TAG) synthesis in mammalian tissues requires a continuous glycerol‐3‐phosphate (G3P) generation for fatty acids esterification and TAG synthesis. The pathways of G3P generation were examined in retroperitoneal (RWAT) and epididymal (EWAT) adipose tissues from mice fed a high‐fat (HF) diet for 8 wk. After this period we evaluate the in vitro rates of glucose uptake and incorporation of 1‐[14C]pyruvate or U‐[14C]glycerol into TAG‐glycerol, and also the glycerol kinase (GyK) and P‐enolpyruvate carboxykinase (PEPCK‐C) activities in both tissues. Protein content was evaluated by western blot. The plasma glucose and insulin were measured by commercial kits. HF diet induced marked increase in body fat mass and in the plasma levels of glucose and insulin. HF diet feeding induced a marked increase in activity (3,4X and 2,5X) and content (42% and 87%) of GyK in RWAT and EWAT, accompanied by a higher TAG‐glycerol synthesis from glycerol in EWAT (47%), but not in RWAT. In contrast, there was a decrease in G3P generation via glycolysis in RWAT (71%) and EWAT (39%) and also a reduction in TAG‐glycerol synthesis from pyruvate (72 and 75%, respectively), without changes in the PEPCK activity and content in both tissues. HF diet induces loss of reciprocal changes in glyceroneogenesis and glycolysis for G3P generation, and seems to favour the direct phosphorylation of glycerol for TAG synthesis and increase in fat mass.Grant Funding Source: Supported by CAPES, FAPESP and CNPQ
Frataxin or a mutant Fe‐S cluster scaffold protein with frataxin‐bypassing ability directly stimulates mitochondrial cysteine desulfurase by exposing substrate‐binding sites (578.4)
Authors: Pandey Alok , Pain Jayashree , Yoon Heeyong , Dancis Andrew , Pain Debkumar 
Published: June 2021

Fe‐S cluster biogenesis in mitochondria is essential for cell survival. During the process, the cysteine desulfurase Nfs1 binds the substrate cysteine, removing sulfur from the substrate and forming a persulfide on the active site cysteine in a process requiring the accessory protein Isd11. The persulfide sulfur is then transferred to the Isu scaffold, where it combines with iron to form the Fe‐S cluster intermediate. Frataxin is implicated in the process, although it is unclear where and how. We find that yeast frataxin (Yfh1) directly and specifically stimulates cysteine binding to Nfs1 by exposing substrate‐binding sites. This novel function of frataxin does not require iron, Isu1 or Isd11. The subsequent persulfide formation is independent of frataxin but is strictly dependent on Isd11. Importantly, a point mutation in Isu1 is able to rescue Fe‐S cluster synthesis in yeast mitochondria lacking Yfh1. In agreement with this finding, the Isu1 suppressor is found to mimic the frataxin effects on Nfs1, explaining the bypassing activity. We propose that Nfs1 undergoes at least two conformational changes for optimum activity. One change is mediated by frataxin/Yfh1 interaction and promotes substrate binding. A second change is mediated by Isd11 and induces persulfide formation.Friedreich's ataxia is an incurable neurodegenerative disease associated with frataxin deficiency. A small molecule or an Isu1 suppressor mimic capable of exposing the “buried” cysteine‐binding sites of the cysteine desulfurase perhaps could be developed into a drug for treating the disease.Grant Funding Source: Supported by NIH grants RO1AG030504 to D.P. and R37DK053953 to A.D.
Discovery and characterization of potent and selective inhibitors to human epithelial 15‐lipoxygenase‐2 (582.5)
Authors: Jameson J Brian , Kantz Auric , Jadhav Ajit , Simeonov Anton , Maloney David , Holman Ted 
Published: June 2021

Lipoxygenase (LOX) enzymes catalyzes the peroxidation of arachidonic acid and other polyunsaturated fatty acids to hydroxyeicosatetraenoic acids with varying positional specificity to yield important biological signaling molecules. 15‐LOX‐2 is a highly specific LOX isozyme that is expressed in epithelial tissue and whose activity has been correlated with suppression of tumor growth in prostrate and other epithelial derived cancers. Despite the potential utility of an inhibitor to understand the specific role of this LOX variant in tumor progression, no 15‐LOX‐2 specific inhibitors have currently been reported. The current study employs a high throughput screening method to identify two novel, specific 15‐LOX‐2 inhibitors with < 3uM potency.Grant Funding Source: Supported by the NIH
Study of complex and macro‐molecular systems using small angle x‐ray scattering (593.1)
Authors: Rathore Yogendra , Arif Ehtesham , Nihalani Deepak , Ashish FNU 
Published: June 2021

Understanding the molecular organization of the glomerular filtration assembly is highly desirable that will aid in designing strategies for recovery of this structure in the event of a glomerular injury. Small angle X‐ray scattering (SAXS) is emerging as a powerful tool to understand the complex and multimeric macro‐molecular assemblies in their dynamic environment. We have successfully employed this technique in the recent years to understand structural details of the cytoplasmic domain of Neph1 (a component of the kidney’s filtration system) and its interaction with partner proteins. In the present study, we extended the SAXS based experimental and computational approach to understand the function of cytoplasmic domain of a slit diaphragm protein Nephrin which plays a critical role in maintaining filtration function of the glomerulus. Interestingly, the SAXS analysis suggests that the cytoplasmic domain of Nephrin is structurally similar to the cytoplasmic domain of its closest family member, Neph1. Importantly, the two proteins have been shown to perform similar functions. Additionally, we are the first to determine the solution‐based structure of the motor protein Myo1c and are in a unique position to model the interacting regions of Nephrin and Myo1c using SAXS and molecular modeling. These results will significantly impact the understanding of the molecular organization of the glomerular filtration assembly.Grant Funding Source: NIH Research Grant (R01)
A computational evaluation of the steric and electronic contributions to the stability of the structures of α− and β−D‐glucopyanose Part 3: energy versus geometry for α− and β−D‐glucopyanose in the gas phase (593.4)
Authors: Abdulsalam Maryam , Baello Rofe , Balilli Rahih , Brutofsky Christian , Kasner Marc 
Published: June 2021

The cyclic structures of α− and β−D‐glucopyranose differ in energy by less than a kcal (~.3 kcal at 298 K in aqueous solution) and both the α− and β− conformers have several relatively stable rotamers. These rotamers are referred to as gg, gt, and tg according to the position of the ‐OH attached to the ‐CH2 at postion C5 on the ring. While the axial versus equatorial orientation of the hydroxyl group at C1 gives rise to the conformational energy, the energies of the gg, gt, and tg rotamers are based on the orientation of the hydroxymethyl group at C5.Relaxed scans are a series of geometry optimizations carried out at 5° intervals as the terminal ‐OH (of the ‐CH2OH group (the HOCC dihedral), the ‐CH2 ‐ (the OCCO dihedral) and the axial/equatorial ‐OH at C1 (the HOCO dihedral) are rotated. Scans of energy as the rotational angles are changed show that only the orientation of hydroxymethyl substituent at C5 makes any significant contribution to the energy. Rotation about the C‐O bond, the HOCO dihedral for the hydroxyl group at C1, shows a low‐level variation in energy (~6 kcal range). Similarly, rotation about the C‐O bond, the HOCC dihedral in the hydroxymethyl group provides energy changes in the same range. However, rotation about the C5‐C6 bond (the OCCO dihedral) show double that range of energies (~12 kcal) and prominent minima indicating the gg, gt, and tg rotamers. Additionally, comparisons of dihedral scans and conformational energies of 2‐hydroxymethyl 6‐hydroxyl‐tetrahydro‐2H‐pyran and 2‐hydroxymethyl‐tetrahydro‐2H‐pyran with 1,5‐anhydro‐D‐glucitol and glucose indicate that the hydroxyl substituents on the pyran ring make little contribution to the stability of the α−, β− conformers and the gg, gt, and tg rotamers of glucose.Three‐dimensional scans of energy versus the two dihedrals in in the hydroxymethyl group (HOCC and OCCO) were done for both α− and β−D‐glucopyranose to develop a potential energy surface to confirm the geometries, energies and number of the low energy structures.
Use of Dendra2 to monitor degradation of the transcription factor Nrf2 in promyelocytic leukemia‐nuclear bodies in living cells (594.1)
Authors: Flores Andrea , Arinze Ifeanyi 
Published: June 2021

Using a cellular fractionation approach, we have shown that the transcription factor Nrf2 can be degraded in the nucleus [Malloy et al. (2013) J. Biol. Chem. 288, 14569‐14583]. The use of photoconvertible proteins, which undergo irreversible change in color upon photoactivation, is a novel approach to monitor real‐time protein movement at the single cell level. In order to use this approach to assess degradation of Nrf2 within the nuclear compartment, we cloned Nrf2 into a plasmid expressing Dendra2, a photoconvertible protein derived from the soft coral species Dendronephthya. When expressed in cells, Dendra2 is a green‐colored fluorescent protein. Upon exposure to a high intensity laser pulse at 405 or 488 nm, its color changes irreversibly from green to red. Photoconversion of the Nrf2‐Dendra2 fusion protein within a region of interest such as within PML‐NB domains facilitates tracking the fate of the fusion protein via time‐lapse imaging. To locate such domains, we also constructed a plasmid expressing the fusion protein Crimson‐PML‐I to identify co‐localization of PML and Nrf2‐Dendra2 within PML‐NB domains. By measuring fluorescence intensity of Nrf2‐Dendra2 in PML‐NBs, we can determine the involvement of these nuclear bodies in the degradation of Nrf2 in the nucleus.Grant Funding Source: Supported by NIH grants SC1CA143985, T32GM007628‐36 and by CTSA award No. UL1TR000445
Rescuing cell polarity with selective small molecule inhibitors of de‐palmitoylation (597.2)
Authors: Martin Brent 
Published: June 2021

Protein palmitoylation is a unique post‐translational modification required for the membrane anchoring and trafficking of numerous regulatory proteins that play key roles in cell growth, polarity, and signaling. In pulse‐chase palmitoylation proteomics studies, we identified the tumor suppressor Scrib as the most dynamic, enzymatically regulated palmitoylated protein in malignant cells. Scrib localizes at the basolateral membrane where it regulates epithelial cell apical‐basolateral polarity, junctional integrity, proliferation, and metastasis. Loss of Scrib at the plasma membrane cooperates with oncogenic Ras or Myc overexpression to drive tumor formation, promote epidermal to mesenchymal transition (EMT), and bypass contact inhibition. Interestingly, overexpression of Snail in polarized cells eliminates Scrib membrane localization. We recently reported a class of in vivo active small selective inhibitors of the acyl protein thioesterases APT1 and APT2. Addition of a small molecule APT2 inhibitor restores Scrib membrane localization and enhances E‐Cadherin expression, overcoming Snail‐mediated repression. These findings suggest that APT2, and not APT1, regulate the palmitoylation of cell polarity proteins, and highlight a strategy to attenuate oncogenic signaling and restore cell polarity pathways in malignant cells.Grant Funding Source: NIH R00CA151460