bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of a high school enrichment opportunity in the anatomical sciences (534.3)
Authors: Taylor Melissa , Bareither Mary Lou
Published: June 2021

High school students, especially students from underrepresented minority groups, may not have much exposure to anatomy in high school classrooms. These students may later be at a disadvantage when they take science college courses. This study examines the implementation of an afterschool anatomy enrichment program to a select group of high school students. The program was two semesters long and introduced students to various anatomy topics such as bones, skin, muscles, etc. They also had the opportunity to view cadavers and do some dissections. Under the guidance of undergraduate mentors from the University of Illinois‐Chicago, the high school students completed their own research projects on an anatomy topic of interest using literature and web resources and presented the projects at the spring undergraduate research forum. Students’ interests in science were surveyed after completing the enrichment program. The results of the questionnaires showed that the majority of the students enjoyed this program and wanted to pursue a science or health major in college. The results also showed that the students would recommend that future high school students partake in the program.

Grant Funding Source: None
Complementary roles for Rock1 and myosin light chain kinase in actin coat compression on fused secretory vesicles (538.4)
Authors: Frick Manfred , Ehinger Konstantin , Miklavc Pika
Published: June 2021

Alveolar type II epithelial cells secrete pulmonary surfactant via exocytosis of lamellar bodies (LBs). Surfactant is a poorly‐soluble lipo‐protein substance that does not readily diffuse out of fused LBs following opening of the fusion pore. LBs are coated with actin following fusion with the plasma membrane (PM) and actin coating and subsequent actin coat compression is essential to drive surfactant secretion. We have previously demonstrated that myosin II is involved in actin coat compression. However detailed mechanisms of myosin II activation and coat compression were still missing. Here we report that actin coat compression is a multiphasic process. High‐resolution life cell imaging and immunocytochemistry revealed that Rock1 and myosin light chain kinase 1 (MLCK) are recruited to LBs following fusion with the PM. Recruitment of Rock1 was preceded by transient recruitment and activation of Rho GTPases to the actin coat. Inhibition of Rock1 did not affect initial compression of the actin coat but significantly impaired full compression of fused vesicles. Inhibition of MLCK did not have a significant effect on actin coat compression at all. Combined inhibition of Rock1 and MLCK, however, completely blocks vesicle compression. Our data suggest that Rock1 and MLCK act via complementary pathways to regulate actin coat compression likely regulating activation of regulatory myosin II light chain.

Grant Funding Source: Deutsche Forschungsgemeinschaft (DFG), BiU
Npr2 regulates autophagy via Gtr1‐Gtr2‐dependent target of rapamycin complex 1 signaling (539.11)
Authors: Noda Takeshi , Kira Shintaro , Shirahama‐Noda Kanae
Published: June 2021

Autophagy is an intracellular degradation process that delivers cytosolic material to lysosomes and vacuoles. To understand the mechanisms that regulate autophagy, we performed a genome‐wide screen using a yeast deletion mutant collection, and identified Npr2 and Npr3. Mutation of Npr2 and Npr3 disrupted autophagy upstream of Target of Rapamycin Complex 1 (TORC1). Additionally, we found that the Npr2‐Npr3 complexes function upstream of Gtr1‐Gtr2, a GTPase complex that is homologous to Rag. A constitutively active GTP‐bound Gtr1 mutant suppressed autophagy by activating TORC1, whereas a constitutively GDP‐bound Gtr1 mutant induced autophagy. The autophagic defects observed in ∆npr2 cells and cells expressing the GTP‐bound Gtr1 mutant were quite comparable, but different from Gtr1‐GDP expressing cell. We hypothesized that the autophagic defect in the ∆npr2 cells resulted from a defect in the switching of the nucleotide‐binding state of Gtr1. Thus, Npr2‐Npr3 appears to regulate GTP hydrolysis in Gtr1, which affects TORC1 activity and autophagy.
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
Ectopic expression studies of the J‐protein Swa2 and its potential role in yeast prion propagation (567.2)
Authors: Rockman Michael , Troisi Elizabeth , Hines Justin
Published: June 2021

Yeast prions require a core set of chaperone proteins (Sis1, Ssa1, and Hsp104) for stable propagation in yeast, and yet some prions exhibit requirements for additional chaperone activities, indicating that prion‐chaperone requirements are heterogeneous. Preliminary data indicate an additional uncharacterized role for the J‐protein Swa2 in yeast prion biology. Swa2 is the sole yeast homolog of mammalian protein auxilin, which functions in vesicle‐mediated endocytosis by disassembling the structural lattice formed by the protein clathrin. Likewise, Swa2 is required for clathrin‐vesicle uncoating in yeast, presumably by recruiting Hsp70 and stabilizing the Hsp70•clathrin complex. Gene dissection analysis by method of plasmid shuffling and tetracycline repression of Swa2 protein expression are the two primary methods utilized to explore the potential role of Swa2 in yeast prion propagation. Given Swa2’s known role in clathrin uncoating, we expect that mechanisms identified with further research may involve the cellular vesicle trafficking, providing a direct connection between these systems and yeast prion inheritance.
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.
Fatty acid specificity of the P450 decarboxylase OleT(JE) (580.5)
Authors: Mitchell Megan , Grant Job , Hsieh Chun , Makris Thomas
Published: June 2021

The metal catalyzed oxidative cleavage of inert carbon‐carbon bonds is central to a plethora of industrially and biochemically important transformations. Recently, a P450 peroxygenase (OleTJE) has been isolated that efficiently decarboxylates fatty acids, yielding carbon dioxide and alkene co‐products. The reactivity of OleTJE, which closely resembles P450 fatty acid peroxygenases that are known to normally afford monooxygenation products, is unprecedented for a P450 cytochrome. In order to explore its viability to synthesize short chain alkenes, we have used optical absorption spectroscopy to explore the chain length specificity of OleTJE. The binding of long chain fatty acids, C18 and above, results in a significant conversion of the heme‐iron from low‐ to high‐spin, which has been used to determine equilibrium binding constants and establish binding kinetics using transient kinetic techniques. Using a reverse binding approach, the displacement of bound substrate by short chain‐length fatty acids (C16 and below), which do not result in significant spin state conversions, has been similarly explored. Together, these results indicate a dramatic chain length selectivity of P450 OleTJE for fatty acid substrates, which may be critical for utilizing this enzyme for the synthesis of short alkene products in the future.
Rational design of a functional NOR in Mb to understand reaction mechanism (581.1)
Authors: Reed Julian , Chakraborty Saumen , Ross Matthew , Nilges Mark , Schulz Charles , Lu Yi
Published: June 2021

Nitric oxide reductases (NORs) catalyze the two electron reduction of nitric oxide to nitrous oxide (2NO + 2H+ + 2e‐ N2O + H2O). Nitric oxide is a fundamental cellular signaling molecule, while nitrous oxide is both a potent greenhouse gas (310x GWP of CO2) as well as a powerful ozone depleting substance. Thus, understanding this process is both chemically and environmentally significant. The active site structure in NORs has been elucidated, but the mechanism is still unknown. Due to the inherent difficultly in studying these complex enzymes, rational design of simpler protein‐based model systems is desired. To this end, a structural and functional biosynthetic model of NOR was engineered through redesign of sperm whale myoglobin (swMb), and two generations (FeBMb1 and FeBMb2) have been successfully developed. Heme replacement with isostructural Zn protoporphyrin IX into FeBMb1 has allowed us to directly probe the interaction of FeB with NO. Utilizing numerous spectroscopic techniques, the mechanism of NO reduction by these biosynthetic models has been elucidated and is shown to follow the trans mechanism. Additionally, enzymatic assays have begun which seek to determine catalytic rate, and preliminary results are comparable to some native systems.
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
Baculovirus display for discovery of low‐affinity extracellular receptor‐ligand interactions using protein microarrays (595.2)
Authors: Tom Irene , Estevez Alberto , Bowman Krista , Gonzalez Lino
Published: June 2021

When used in conjunction with multivalent baits, protein microarrays offer a robust technology for discovery of low‐affinity extracellular protein‐protein interactions (ePPIs). Baits for receptor‐matching screens generally consist of recombinantly expressed and purified soluble domains. Given that approximately two‐thirds of extracellular proteins are transmembrane‐containing proteins, it would be desirable to develop a system to express and display bait receptors in a native‐like membrane environment. Towards this end, we evaluated the use of baculovirus (BV) display as a platform for generating multivalent baits for protein microarray screens. Virion particles were generated displaying single‐transmembrane receptors BTLA, CD200 or EFNB2, representing a range of affinities for their interacting partners. Virions labeled with Cy5 fluorophore were screened against a microarray containing over 600 extracellular proteins and the results were compared to data derived from soluble Fc‐protein or bait‐coated protein A microbeads. An optimized protocol employing a blocking step with a non‐bait expressing control BV allowed the identification of the expected interactions with signal‐to‐noise similar or higher than obtained with the other formats. Our results demonstrate that BV display is suitable for detection of high‐ and low‐affinity ePPIs on protein microarrays. The platform eliminates the need for direct protein purification and provides a native‐like lipid environment for membrane‐associated receptors.
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
Effect of APOE genotype and Alzheimer pathology formation on synaptic membrane lipids of human brains (598.2)
Authors: Oikawa Naoto , Hatsuta Hiroyuki , Murayama Shigeo , Suzuki Akemi , Yanagisawa Katsuhiko
Published: June 2021

APOE genotype has great impact on the development of Alzheimer’s disease (AD), i.e., ε4 allele accelerates whereas ε2 allele suppresses the development of AD; however, how APOE genotype affects AD development remains unclear. On the basis of the previous finding that the assembly of the amyloid ß‐protein (Aß) into fibrils in the brain, an early and invariable pathological feature of AD, depends on the lipid environment of membrane, we examined whether lipid composition of synaptic plasma membrane, which is an initial site of amyloid deposition, of human brain varies in an APOE genotype dependent manner. Levels of cholesterol and ganglioside in synaptic plasma membranes prepared from human cortices of aged individuals were examined using Amplex‐Red cholesterol assay kit and by LC‐MS analyses, respectively. In the comparison between amyloid‐free ε2/ε3 and ε3/ε3 brains, the levels of cholesterol were significantly lower in the subjects carrying ε2 allele. Alternatively, the presence of AD pathology substantially decreased the levels of cholesterol in the subjects carrying ε3 allele. This study suggests that ε2 allele suppresses AD development by keeping cholesterol in synaptic membranes under a certain level, which is prerequisite for the initiation of Aß assembly into fibrils.
Peritoneal mesothelial cells give rise to myofibroblasts via mesothelial‐mesenchymal transition in peritoneal fibrosis (60.10)
Authors: Lua Ingrid , Asahina Kinji
Published: June 2021

Mesothelial cells (MCs) cover the surface of the internal organs, and the peritoneal cavities as it have a phenotype intermediate between epithelial and mesenchymal cells, expressing markers indicative of both cell types. Peritoneal mesothelium is used as a semipermeable barrier for the exchange of toxic substance in peritoneal dialysis, a renal failure therapy. When peritoneal MCs are exposed to non‐physiological dialysis fluids in long term, tissue fibrosis and failure of treatment occurs. Myofibroblasts (MFs) synthesize excessive extracellular matrix proteins and participate in peritoneal fibrosis. However, the origin of MFs remains obscure. Epithelial‐mesenchymal transition (EMT), typically defined by the loss of E‐cadherin and up regulation of mesenchymal markers, been suggested to involved in this process. However, as MCs do not express E‐cadherin, this mechanism needs to be clarified as we propose mesothelial‐mesenchymal transition (MMT) instead of EMT. In the present study, we examined whether peritoneal MCs undergo MMT and give rise to MFs in mouse peritoneal fibrosis induced by injections of chlorhexidine gluconate (CG) solution. Peritoneal MCs expressed glycoprotein M6A (GPM6A), vimentin, cytokeratin 19, and Wilms tumor 1 (WT1). We isolated peritoneal MCs from mice using antibodies against GPM6A and magnetic beads. In culture, MCs formed epithelial colonies and changed shape to myofibroblastic cells in the presence of TGF‐β. To trace lineages of peritoneal MCs, we used Wt1‐CreERT2 and Rosa26mTmGflox mice. By tamoxifen injection, we specifically labeled peritoneal MCs as GFP+ cells. After injections of CG, most of the single MC layer was eliminated and GFP+ MFs accumulated in the fibrotic area of the peritoneum. Interestingly, when we stopped injections of CG, the mesothelial layer re‐formed and some of MCs expressed GFP in the regenerating mesothelium. These results demonstrate that mesothelial cells are the source of myofibroblasts in peritoneal fibrosis.

Grant Funding Source: Supported by NICHD T32 Training Program
Evidence for a role of p130Cas in JAM‐A‐dependent signaling events (60.3)
Authors: Matthews Jason , Weight Caroline , Williams Holly , Leoni Giovanna , Neumann Philipp , Farkas Attila , Nava Porfirio , Nusrat Asma , Parkos Charles
Published: June 2021

Junctional adhesion molecule‐A (JAM‐A) is a tight junction protein that plays a key role in regulation of intestinal mucosal homeostasis. Previous studies have shown that loss of JAM‐A decreases beta‐1 integrin‐mediated cell migration and increases Akt‐dependent proliferation however signaling pathways downstream of JAM‐A remain incompletely defined. Levels of known actin‐dependent regulators of cell migration and proliferation in epithelial cells derived from JAM‐A deficient mice and ex vivo cultures of small intestinal enteroids were thus examined. We observed dramatic changes in p130Cas, a critical signaling component of focal adhesions that is regulated by ABL kinase. Western blots of JAM‐A deficient intestinal epithelial cells (IEC) and enteroids revealed significantly decreased levels of phosphorylated p130Cas. In addition, JAM‐A deficient colonic IEC showed decreased levels of the migration‐regulating integrin beta‐1 in parallel with decreased E‐cadherin, a negative regulator of proliferation. Treatment of cultured IEC with the ABL kinase inhibitor Imatinib resulted in delayed epithelial wound closure, inhibited cell spreading and decreased p130Cas phosphorylation at Y410 similar to that observed with JAM‐A deficiency. These findings suggest that p130Cas may act to transduce JAM‐A‐dependent signals to beta‐1 integrin to regulate migration, and E‐cadherin to inhibit proliferation.

Grant Funding Source: Supported by NIH T32 DK007771‐12, Pathobiology of Mucosal/Epithelial Disease
The mechanism of serine protease‐induced increases in epithelial barrier function in intestinal epithelial cells (60.7)
Authors: Lahey Kelcie , MacNaughton Wallace
Published: June 2021

Apical serine proteases increase barrier function in intestinal epithelial cells (IECs). We investigated the requirement for proteolytic, protein kinase C (PKC)ζ, and epidermal growth factor receptor (EGFR) activities in the prolonged transepithelial resistance (TER) response to trypsin in IECs.

Methods. 1. SCBN epithelial cell monolayers grown on Transwells were treated with trypsin (45BAU/ml) and collected for measurement of phospho‐PKCζ at 2, 5, 10, 15, 30, and 60 min using Western blot. 2. TER across SCBN monolayers was measured in Ussing chambers. Trypsin activity was inhibited by soybean trypsin inhibitor (SBTI) treatment at 10 min (start of plateau phase of TER response) or 30 min (after plateau phase had been established). 3. To investigate the role of EGFR, cells were pretreated with an EGFR inhibitor (PD15305) before addition of trypsin.

Results. 1. Upregulation of phospho‐PKCζ was observed in trypsin‐treated SCBNs for up to 60 min. 2. Apical trypsin caused an increase in TER that reached a sustained plateau by 10 min. SBTI at 30 or 60 μg/ml decreased the trypsin‐induced increase in TER by 65% and 80% (respectively) at 10 min and by 70% and 80% at 30 min. 3. Inhibition of EGFR with PD153035 reduced the trypsin‐induced TER by 55%.

Our data show the requirement for ongoing proteolytic activity, sustained PKCζ activation and EGFR activity in the trypsin‐induced increase in intestinal epithelial barrier function.

Grant Funding Source: Supported by the Canadian Institutes of Health Research
Regulation of ALIX ubiquitination and function in GPCR lysosomal sorting by the alpha‐arrestin ARRDC3 (604.3)
Authors: Lin Huilan , Dores Michael , Trejo JoAnn
Published: June 2021

We recently discovered that protease‐activated receptor‐1 (PAR1), a GPCR for thrombin, is sorted to lysosomes through an ALIX‐mediated pathway independent of receptor ubiquitination. The mechanisms responsible for regulation of ALIX‐mediated ubiquitin‐independent sorting of GPCRs are not known. Here, we show that the arrestin‐domain containing protein‐3 (ARRDC3) is a key regulator of ALIX‐dependent lysosomal sorting of PAR1. We found that activated PAR1 internalized and colocalized with ALIX and ARRDC3 on late endosomes/lysosomes in HeLa cells. ARRDCs, also known as α‐arrestins, contain PPXY motifs within the C‐terminus that mediate interaction with E3 ubiquitin ligases. Co‐expression of an ARRDC3 C‐terminal truncation mutant lacking the PPXY motifs or mutant in which the PPXY motif was mutated to AAXA resulted in mislocalization of ARRDC3 and disruption of agonist‐induced PAR1 lysosomal sorting. Depletion of ARRDC3 by siRNA also inhibited activated PAR1‐induced ALIX ubiquitination. Thus, ARRDC3 may regulate ALIX activity by controlling the ubiquitination status of ALIX through the recruitment of E3 ubiquitin ligases and consequently regulate activated PAR1 sorting into intraluminal vesicles of late endosomes/multivesicular bodies

Grant Funding Source: Supported by GM090689
DNA topoisomerase II inhibitors induce human cholesteryl ester transfer protein expression via an LXR‐dependent pathway (605.17)
Authors: Liu Mengyang , Chen Yuanli , Duan Yajun , Han Jihong
Published: June 2021

DNA topoisomerase II inhibitors induce human cholesteryl ester transfer protein expression via an LXR‐dependent pathway
Mengyang Liu, Yuanli Chen, Yajun Duan, Jihong Han State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin 300071, China The human cholesteryl ester transfer protein (CETP) transfers cholesteryl esters from high‐density lipoproteins (HDL) to the triglyceride‐rich lipoproteins. CETP is mainly expressed and secreted from the liver. It is transcriptionally regulated by liver X receptor (LXR). Etoposide and teniposide are DNA topoisomerase II (Topo II) inhibitors and used as chemotherapeutic medications to treat patients with various cancers. We previously reported that etoposide and teniposide activated macrophage ABCA1 expression, a LXR‐targeted gene. Herein we show that treatment of HepG2 cells with etoposide and teniposide was also able to increase CETP protein expression and secretion which was associated with increased CETP mRNA expression and promoter activity in an LXR‐dependent manner. Knockdown of LXRα/β expression by siRNA reduced the inductive effect of etoposide and teniposide on CETP expression. Moreover, etoposide and teniposide increased the nuclear translocation of LXR while down‐regulated LXR co‐repressor gene expression, thereby resulting in activation of LXR. Taken together, our study demonstrates that etoposide and teniposide induce hepatic CETP expression in an LXR‐dependent mechanism. Our study also reveals another role of Topo II inhibitors in regulation of cholesterol homeostasis gene expression other than their anti‐tumorigenesis function.
Sphingolipid biosynthesis and inflammatory signaling in asthma (605.21)
Authors: Ramachandran Subramaniam , Engel Alex , Barton Greg , Thorner Jeremy
Published: June 2021

Objective‐ SNPs located within human chromosomal region 17q21 are strongly associated with the incidence of inherited asthma and have been correlated by others to elevated transcript levels of nearby genes, including ORMDL3. Studies in yeast have identified the yeast ORMDL3 orthologs, as negative regulators of the serine palmitoyl‐CoA transferase (SPT) enzyme complex. This study investigates the role of mammalian ORMDL3. Methods and Results‐ To further understand the function ORMDL3, a stable doxycycline‐inducible human embryonic kidney (HEK) cell line was developed that overproduces ORMDL3 up to 40‐fold compared to control cells. SPT activity in ORMDL3 overexpressing cells was reduced to 40‐60% compared to that of control cells. Sphingolipidomic analysis revealed that all sphingolipid species, were reduced in these cells, consistent with significant inhibition of SPT activity. Conversely, combined RNAi‐mediated knockdown of all ORMDL isoforms resulted in higher SPT activity and increased sphingolipids. However, knockdown of any single ORMDL isoform did not alter SPT activity, suggesting that they serve a redundant function in inhibiting SPT. Additionally, individual knockdown of the two, different, small non‐catalytic subunits of SPT_ SPTSSA and SPTSSB _did not alter ORMDL3‐mediated inhibition of SPT. Furthermore, to attempt to determine the consequence of increased ORMDL3 expression in asthma, LPS‐induced TLR4 endocytosis was measured in RAW macrophages overproducing ORMDL3. There was a very modest increase in the fraction of cells displaying cell surface TLR4 in the ORMDL3 overexpressing macrophages as compared to control cells. This result may due to inefficient transfection of the cell population. Conclusions‐ Taken together, these findings suggest that, although Ormdl3 demonstrably impacts sphingolipid biosynthesis at the level of SPT, overproduction ORMDL3 does not dramatically affect at least one arm of the innate immune response.
One E3 ligase targets two key control points in cholesterol synthesis (605.5)
Authors: Brown Andrew , Sharpe Laura , Loregger Anke , Cook Emma , Phan Lisa , Stevenson Julian , Zelcer Noam
Published: June 2021

The mevalonate pathway is used by cells to produce sterol and non‐sterol metabolites and is subject to tight metabolic regulation. We recently reported that squalene monooxygenase (SM), an enzyme controlling a rate‐limiting step in cholesterol biosynthesis, is subject to cholesterol‐dependent proteasomal degradation. However, the E3‐ubiquitin ligase (E3) mediating this effect was not established. Using a candidate approach, we identify the E3 membrane‐associated RING finger 6 (MARCH6, also known as TEB4) as the ligase controlling degradation of SM. We find that MARCH6 and SM physically interact, and consistent with MARCH6 acting as an E3, its over‐expression reduces SM abundance in a RING‐dependent manner. Reciprocally, knockdown of MARCH6 increases the level of SM protein and prevents its cholesterol‐regulated degradation. Additionally, this increases cell‐associated SM activity, but is unexpectedly accompanied by elevated levels of squalene, SM’s substrate. Prompted by this observation, we found that MARCH6 also controls the level of endogenous and ectopically expressed HMGCR in hepatocytes and model cell‐lines. 25‐hydroxycholesterol induces rapid degradation of HMGCR in the presence of MARCH6, and absence of the ligase blunted sterol regulation. In conclusion, degradation of both HMGCR and SM by MARCH6 establishes it as a major regulator of flux through the cholesterol synthesis pathway.
Manipulation of phospholipid composition alters protein binding to lipid droplets (606.3)
Authors: Hains Anastasia , Listenberger Laura
Published: June 2021

Lipid droplets are cytoplasmic organelles that store excess lipid. Each lipid droplet consists of a core of neutral lipid surrounded by a phospholipid monolayer. Proteins interact with the phospholipid surface and regulate lipid droplet synthesis and degradation. We have shown that the phospholipid composition of synthetic lipid droplets can influence protein binding in an in vitro assay. We aimed to determine if protein binding to lipid droplets is influenced by the lipid droplet phospholipid composition in cells. We altered the phospholipid composition of lipid droplets in NIH3T3 cells with hexadecylphosphocholine (HePC), an inhibitor of phosphatidylcholine (PC) synthesis. Isolation of lipid droplets from inhibitor treated and control cells and separation of phospholipids by thin layer chromatography, demonstrated a decrease in PC relative to phosphatidylethanolamine in HePC‐treated cells. Furthermore, HePC treatment prevented the lipid droplet localization of CTP:phosphocholine cytidylyltransferase, the enzyme that catalyzes the rate‐limiting step in PC synthesis. Other changes in protein localization to lipid droplets were observed by SDS‐PAGE and silver staining of lipid droplet proteins in HePC‐treated and control cells. Current and future experiments continue to examine how changes in phospholipid composition alter the protein composition of this important storage compartment.

Grant Funding Source: Supported by an award from Research Corporation for Science Advancement