bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural insights into aspartoacylase deficiency in Canavan disease (769.4)
Authors: Wijayasinghe Yasanandana , Pavlovsky Alexander , Viola Ronald
Published: June 2021

Canavan disease (CD) is a fatal neurodegenerative disorder with impaired N‐acetyl‐L‐aspartic acid (NAA) metabolism. This disease is caused by mutations in the ASPA gene, leading to a deficiency in the aspartoacylase (ASPA) enzyme that hydrolyzes NAA in the brain. The majority of the defects are missense mutations found remote from the active site, resulting in decreased stability and activity. To study the possible structural perturbations triggered by these mutations, ASPA mutants associated with different disease severities have been cloned, purified, and crystallized. E285A, one of the most common mutants shows that loss of the hydrogen bond interaction with Asp 285 disturbs the active site architecture leading to altered substrate binding. The loss of a hydrophobic interaction in the F295S mutant leads to a local collapse of the structure, contributing to low protein stability. But K213E, a mutant associated with a mild disease phenotype, has minimal structural differences compare to the native enzyme. Our improved understanding of the structural changes that occur in ASPA mutants will allow the design of small molecule chaperones that could lead to recovery of the activity of this defective enzyme and therefor a treatment therapy for Canavan disease.

Grant Funding Source: Supported by Canavan Research Foundation, and Jacob's Cure Organization
Regulation of endothelial nitric oxide synthase phosphorylated at Thr495 by interaction with calmodulin (769.8)
Authors: Piazza Michael , Guillemette J. Guy , Dieckmann Thorsten
Published: June 2021

Nitric oxide synthase (NOS) plays a major role in a number of key physiological and pathological processes and it is important to understand how this enzyme is regulated. Calmodulin (CaM), a small acidic calcium binding protein, is required to fully activate the enzyme. The exact mechanism of how CaM activates NOS is not fully understood at this time. Studies have shown CaM to act like a switch that causes a conformational change in NOS to allow for the electron transfer between the reductase and oxygenase domains, through a process that is thought to be highly dynamic and at least in part controlled by several possible phosphorylation sites. We have determined the solution structure of CaM bound to a peptide of eNOS that contains a phosphorylated threonine corresponding to Thr495 in order to investigate the structural and functional effects that the phosphorylation of this residue may have on nitric oxide production. Our biophysical studies show that phosphorylation of Thr495 introduces electrostatic repulsions between the target sequence and CaM as well as a diminished propensity for the peptide to form an α‐helix. The calcium affinity of the CaM‐target peptide complex is reduced due to phosphorylation and this leads to weaker binding under low physiological calcium concentrations. This study provides an explanation for the reduced NO production by eNOS carrying a phosphorylated Thr495 residue.

Grant Funding Source: National Science and Engineering Research Council (NSERC)
Thermal denaturation of Mycobacterium tuberculosis protein Rv0045c monitored by intrinsic tryptophan fluorescence (770.1)
Authors: Drake Lindsey , Hoops Geoffrey
Published: June 2021

Rv0045c is a novel esterase of Mycobacterium tuberculosis, the causative bacterium of the still prevalent and deadly disease tuberculosis. The six tryptophan residues in this enzyme were of interest to monitor the denaturation process by intrinsic fluorescence. To simplify the signals, the six tryptophans (W78, W98, W122, W246, W248, W263) were mutated to phenylalanine to preserve the relative size of the residue but remove the fluorescence capability; the goal being to create a quadruple mutant with only one pair of tryptophans interacting. Mutated proteins were synthesized through site directed mutagenesis, overexpressed in E.coli hosts, and purified using a Ni2+ affinity resin. Differential scanning fluorimetry and kinetic assays were performed to test the initial stability of the mutants. W78F and W98F were not as stable as the wild type and therefor were not used in further double and triple mutations. Significant changes in the thermal stability of Rv0045c triple and quadruple mutants will be analyzed via intrinsic fluorescence.

Grant Funding Source: NSF TUES
Identification and biochemical characterization of a Kunitz‐type serine protease inhibitor from an insect, Manduca sexta (772.1)
Authors: Watkins Jamilah , Takahashi Daisuke , Hiromasa Yasuaki , Kanost Michael
Published: June 2021

Hemolymph (blood) of insects contains serine proteases and serine protease inhibitors that function in innate immune responses. The objective for this research was to investigate low molecular weight protease inhibitors from hemolymph of a lepidopteran insect, Manduca sexta. We identified serine protease inhibitor activity against trypsin, chymotrypsin, and elastase in plasma from naïve larvae and in plasma from larvae that were previously injected with Micrococcus luteus. We separated hemolymph plasma proteins using size exclusion chromatography, and the fractions with protease inhibitor activity were analyzed by SDS‐PAGE and liquid chromatography‐electrospray ionization tandem mass spectrometry. A serine protease inhibitor belonging to the Kunitz family was identified, and using primers based on the sequence encoding this protein found in the M. sexta genome sequence, we cloned its cDNA from larval fat body RNA. The cDNA encodes a protein with a secretion signal sequence followed by the mature secreted inhibitor protein of 56 amino acid residues. This sequence is highly similar to a Kunitz inhibitor previously purified from M. sexta (Ramesh, N., Sugumaran, M., Mole, J.E. 1988. J Biol Chem. 263:11523‐7). Using a synthesized and codon‐optimized DNA encoding the protease inhibitor, we are currently producing the recombinant inhibitor in E. coli for further biochemical characterization. We will study its inhibitory activity as a potential regulator of proteases in hemolymph that function in the innate immune system of M. sexta. The genome of M. sexta contains nine genes for single‐domain Kunitz proteins closely spaced on a single scaffold. The ultimate goal of this project is to understand the biological functions of these Kunitz protease inhibitors in innate immunity.

Grant Funding Source: Supported by NIH grant GM41247.
Modulation of eukaryotic elongation factor 2 kinase activity via phosphorylation on Ser‐500 (773.1)
Authors: Tavares Clint , Kaoud Tamer , Ferguson Scarlett , Wellmann Rebecca , Stancu Bogdan , Giles David , Chitjian Catrina , Dalby Kevin
Published: June 2021

Eukaryotic elongation factor 2 kinase (eEF‐2K) is an atypical calcium/calmodulin (Ca2+/CaM)‐dependent kinase known to modulate protein synthesis through the inhibitory phosphorylation of elongation factor 2. Despite its pivotal role in translation regulation and potential role in tumorigenesis, the mechanism of regulation of eEF‐2K activity by phosphorylation is still unclear. Our objective was to analyze the activation of eEF‐2K by phosphorylation at Ser‐500 (phosphorylated by protein kinase A or Ca2+/CaM‐induced autophosphorylation). In vitro analysis indicates that autophosphorylation of eEF‐2K induces Ca2+‐independent activity. Western blotting analysis as well as mutagenesis of Ser‐500 to Ala or Asp suggests that Ser‐500 phosphorylation is responsible for this Ca2+‐independent activity. Interestingly, CaM appears to be essential for this Ca2+‐independent activity of eEF‐2K. Mechanistic studies indicate that Ser‐500 phosphorylation increases eEF‐2K affinity for CaM in the presence or absence of Ca2+. Ser‐500 phosphorylation also increases the rate of Thr‐348 phosphorylation (a key site for the allosteric regulation of eEF‐2K activity). We are currently studying the dynamic nature of Ser‐500 phosphorylation in cells. Phosphorylation of eEF‐2K on Ser‐500 could be a significant mechanism for its regulation, especially upon the influx of Ca2+ during cellular signaling events.

Grant Funding Source: NIH (GM059802 and CA167505) to KND
The crystal structure of SOD5: an unusual copper‐only superoxide dismutase (774.2)
Authors: Waninger‐Saroni Jessica , Gleason Julie , Galaleldeen Ahmad , Hart P. , Culotta Valeria
Published: June 2021

Superoxide dismutases (SODs) are antioxidant enzymes that catalyze the dismutation of superoxide into molecular oxygen and hydrogen peroxide. Candida albicans is a polymorphic fungus that causes the majority of fungal infections in humans. In the immunocompetent population, colonization of C. albicans can result in acute infections of the mucosal surfaces both in the oral and genital cavities, however, immunocompromised individuals encounter life‐threatening systemic infections. Several factors contribute to the pathogenesis of C. albicans including the expression of SOD5; a postulated copper‐zinc SOD. SOD5 enables C. albicans to evade the immune response of the host, namely the oxidative burst. In the absence of SOD5, reactive oxygen species (ROS) accumulate and C. albicans exhibit a severe loss in viability. Our studies sought to examine the crystal structure of SOD5. The protein was overexpressed in E. coli and due to its insolubility; it was denatured, refolded, and further purified. Following protein purification, a sedimentation velocity experiment was performed. The data reveal that SOD5 is a monomeric protein in solution. The protein was then crystallized and the structure of SOD5 was determined confirming that SOD5 is a novel monomeric SOD possessing a copper‐only active site and lacking a zinc binding site. In addition, this protein was devoid of the electrostatic loop that typically characterizes all SOD1s. The SOD5 copper site is solvent accessible, cupped in an electrostatic architecture that could serve in superoxide guidance.
MauG catalysis: a tale of ferryl iron, radicals and long distance hopping (774.8)
Authors: Wilmot Carrie , Yukl Erik , Davidson Victor
Published: June 2021

Methylamine dehydrogenase (MADH) enables some methylotrophic/autotrophic bacteria to grow on methylamine as a sole source of carbon and energy. MADH catalysis depends on the cofactor tryptophan tryptophylquinone (TTQ) that is a posttranslational modification of two Trp residues in the MADH β‐subunit. The maturation of MADH depends on four gene products located in the methylamine utilization (mau) gene cluster. One of these, mauG, encodes a c‐type di‐heme enzyme that completes synthesis of the TTQ cofactor. The potent oxidant is an unusual bis‐Fe(IV) MauG species composed of a ferryl heme (Fe(IV)=O) with an oxidizing equivalent stored as Fe(IV) at the second heme, which has an unusual His, Tyr axial ligation. The bis‐Fe(IV) oxidant is formally Fe(V) and equivalent to Compound I. Completion of TTQ to generate active MADH involves long‐range electron transfer and a radical hopping mechanism to effect catalysis over a 40 Å distance. The MauG catalyzed reaction occurs in three discrete 2‐electron events in a H2O2 or O2 (+ reducing equivalents) dependent process. A crystal structure of MauG in complex with its protein substrate, a precursor form of MADH known as preMADH, has been solved. The crystals are catalytically active. The order of the 2‐electron chemistry catalyzed by MauG was determined through a series of structures from crystals harvested after different amounts of time following crystallization. H2O2 to initiate the reaction was generated by the slow breakdown of polyethylene glycol used in crystallization. These in crystallo data are corroborated by mass spectrometry in solution experiments.

Grant Funding Source: Supported by NIH grants R37GM41574, R01GM66569, F32GM97779
Recognition of metal ions using ProMOL to improve protein function assignation (777.1)
Authors: Kovuri Venkata Aditya , Craig Paul , Bernstein Herbert
Published: June 2021

ProMOL is a plugin for PyMOL that utilizes the 3D visualization and measurement capacities of PyMOL to predict enzyme function by aligning query structures with a collection of annotated active sites found in our motif template library. The focus of this initiative is a response to the >3000 structures in the Protein Data Bank that are labeled as having “unknown function”. ProMOL undergoes constant revision and development from the various team members from RIT and Dowling College. The motif capabilities of ProMOL have been expanded to include metal ions and prosthetic groups that are found in enzyme active sites, as nearly 40% of all the PDB structures contain at least one metal ion. This is biologically significant as metal ions such as Mn, Zn, Ca and Fe, play a vital role in both the structure and function of many different proteins. The addition of metal ion recognition to ProMOL/PyMOL promises to lead us to better function assignments to this collection of protein structures. There are current plans to launch a database with specific information pertaining to the metal ion binding sites and 3D visualization of motifs with metal ions. This work has been supported in part by National Institute of General Medical Sciences 2R15GM078077‐02, 3R15GM078077‐02S1.

Grant Funding Source: Supported by NIGMS 2R15GM078077‐02, 3R15GM078077‐02S1
Characterization of CTCF protein around the ‐224 A/G single nucleotide polymorphism in the neuropeptide receptor Y2 (NPY2R) gene in predisposition to hypertension (777.4)
Authors: Albino‐Rodriguez Elinette , Nunez‐Marrero Angel , Porrata‐Doria Tirtsa , Dutil Julie
Published: June 2021

Hypertension (HTN) is a complex trait resulting from the interactions of multiple genes and environmental factors. Neuropeptide Y receptor 2 (NPY2R) was identified as a candidate gene for HTN in animal models and human populations. Previous work showed that the luciferase activity induced by the NPY2R promoter with the G allele in position ‐224 is reduced by 42% compared to the promoter containing an A in that position. Furthermore, an electrophoretic shift assay (EMSA) showed an allele‐specific binding with the oligonucleotide containing an A nucleotide in ‐224 position. We were also able to detect a super shift using the CTCF antibody. The aim of this study was to characterize the CTCF protein in HEK 293 cells transfected with NPY2R ‐224 A/G variations. Changes in CTCF expression and distribution were analyzed using western blot and immunofluorescence (IF) experiments. Interestingly, the western blot showed higher quantification of CTCF in HEK 293 cells transfected with NPY2R ‐224 A compared to NPY2R ‐224 G (p < 0.001). Analysis of nuclear localization of CTCF by IF revealed that it is highly distributed in HEK 293 cells transfected with NPY2R ‐224 A compare to a lower distribution in NPY2R ‐224 G. CTCF is the only protein identified so far that mediates enhancer‐blocking activity of insulators. These data provides strong evidence of the CTCF role in the transcriptional regulation.

Grant Funding Source: Supported by Grant RR003050/MD007579
Bioinformatics analysis of uman NOTCH4 protein (778.10)
Authors: Bowman Jacob , Tansey John , Bennett Jennifer
Published: June 2021

NOTCH4 is a receptor protein in the highly conserved Notch signaling pathway that plays a critical role in many cellular processes, including differentiation and apoptosis. The structure of NOTCH1 has been determined and several functions elucidated. NOTCH1 is used as the model, in function and structure, for the rest of the Notch proteins. The objective of the current study is to use available bioinformatics data to better characterize functional domains and vital residues of the NOTCH4 protein. Amino acid sequence data of NOTCH4 were aligned against templates of homologous domains‐Ankyrin repeats, epidermal growth factor, and calcium‐binding epidermal growth factor ‐ from standards to find a predicted 3‐D structure of NOTCH4. The threading program RaptorX, has been used to generate a predicted structure of one domain of NOTCH4 and then Cn3D was used for visualization. This study plans to expand to identify important residues in all of the major domains. Data from this predicted structure will be used to inform future experiments and develop a clearer picture of the Notch signaling pathway.
New imaging applications with Amershamœ imager (779.2)
Authors: Johansson Johan , Svensson Erika , Bjerneld Erik , Winkvist Maria , Roos Håkan
Published: June 2021

Electrophoretic based protein‐ and DNA separation are well established techniques that are widely used in Life Sciences. However, the development of highly sensitive detection reagents together with advanced imaging techniques has shifted the obtained results from qualitative towards quantitative. Moreover, the combination of new reagents and imaging solutions opens for new applications. To obtain quantitative results, the whole workflow has to be considered. Here we have focused on the importance of the CCD imager system, suitable applications and important considerations when working quantitatively. Fluorescent detection systems are typically characterized by their high sensitivity and broad linear dynamic range, and are as such well adapted to quantitative Western blotting (WB). The obtained fluorescent signals are stable and directly proportional to the protein amount. Fluorescence enables simultaneous detection of more than one protein on the same blot (multiplexing), even when the proteins have similar size. Here we have utilized the strength of fluorescence and the new Amersham Imager, optimized for quality work in 1D and WB applications. Sensitivity and dynamic range are important not only for protein detection, but also for DNA and RNA applications. These key features will be discussed.
Evaluation of cytotoxicity of CdS/maltodextin nanoparticles bioconjugated with Cry protein in hepatic cells (780.1)
Authors: Gutiérrez Ivonne , Reyes‐Esparza Jorge , Rodriguez‐Fragoso Patricia , Rodriguez‐Fragoso Lourdes
Published: June 2021

To evaluate of cytotoxicity of CdS/maltodextrin nanoparticles (CdS/MD) bioconjugated with Cry protein in hepatic cells. Previous studies have shown that CdS/MD nanoparticles are innocuous in several cell lines at low concentrations. For this study, the CdS/MD nanoparticles and bioconjugate were analyzed by fluorescence microscopy and electron microscopy. HepG2 cells were treated with CdS/MD alone and bioconjugated with Cry protein. We performed a dose‐response curve of CdS/MD and CdS/MD‐Cry protein [0.01, 0.1, 1‐9 μg/L] and cell viability was measured. The type of cell death was identified by fluorescence microscopy. It was found that the bioconjugate was 20 times bigger than the CdS/MD nanoparticles. It was shown that nanoparticles and the bioconjugate emit green fluorescence in cells HepG2. In cells treated with NPs was observed fully fluorescence, while those treated with the bioconjugate only was observed fluorescence in the cell membrane. The cytotoxicity assay showed that the nanoparticles and Cry protein were non‐toxic at low concentrations (<2μg/L). At low concentrations the bioconjugate did not produce toxicity in HepG2 cells (<0.1 μg/L). The Cry protein produced apoptosis while the bioconjugate produced necrosis. In conclusion, at low concentrations the bioconjugate did not produce toxicity in HepG2 cells.

Grant Funding Source: CONACYT
Sphingosine 1‐phosphate signaling regulates exosomal cargo sorting (783.3)
Authors: Kajimoto Taketoshi , Okada Taro , Miya Satoshi , Zhang Lifang , Nakamura Shun‐ichi
Published: June 2021

During late endosome maturation, cargo molecules are sorted into intralumenal vesicles (ILVs) of multivesicular endosomes (MVEs) and are either delivered to lysosomes for degradation or fused with the plasma membranes for exosome release. For delivering to lysosomes, the endosomal sorting complex required for transport (ESCRT) regulates both budding of ILVs and cargo sorting into ILVs. For exosome release, ceramide triggers budding of ILVs of MVEs, but the mechanism underlying cargo sorting into exosomal ILVs is still unclear. Here we show that inhibitory G protein (Gi)‐coupled sphingosine 1‐phosphate (S1P) receptors regulate exosomal cargo sorting. In concrete terms we demonstrated two major parts using fluorescence resonance energy transfer (FRET) technique, fluorescence recovery after photobleaching (FRAP) technique, or newly developed single exosome quantification technique; 1) Gi‐coupled S1P receptors on MVEs are constitutively activated through a constant supply of S1P on MVEs, 2) the continuous activation of Gi‐coupled S1P receptors on MVEs is essential for cargo sorting into ILVs destined for exosome release. Our results provide a new paradigm for a mechanism underlying cargo sorting into ILVs of exosomal MVEs.
Role of N‐acetylglucosaminyltransferase‐V and galectin‐3 binding protein in anoikis stress of cancer cells (788.1)
Authors: Song Kyoungjin , Ko Jeong‐Heon , Kim Yong‐Sam
Published: June 2021

Anoikis is an anchorage‐dependent cell death. Anoikis resistance is known to be a crucial step for cancer metastasis. We have previously identified that N‐Acetylglucosaminyl transfer ase‐V (GnT‐V) is a key protein inducing metastasis in human colon cancer cells. And the secretion of galectin‐3 binding protein (LGALS3BP) was lowered in GnT‐V overexpressing WiDr cells. In this study, we hypothesized that GnT‐V has an inhibitory effect on anoikis of cancer cells, while LGALS3BP stimulates the apoptotic process. To prove this, we established the stable transfectants (WiDr:GnT‐V), the control cells (WiDr:mock). As a result, GnT‐V was observed to mitigate anoikis and promote cell aggregation in soft‐ agar plate system. Among human colon cancer cells tested, HCT116 cells formed aggregates poorly and thus showed low cell viability. Of note, the secretion of LGALS3BP was higher than any other colon cancer cellline, which is in line with the result that anoikis resistance was observed in LGALS3BP‐suppressed WiDr cells. Taken together, these results may suggest that GnT‐V induces anoikis resistance of cancer cells either alone or in cooperation with the target molecules, such as LGALS3BP.
Matrix‐mediated mechanical regulation of tissue morphogenesis (79.1)
Authors: Nelson Celeste
Published: June 2021

Tissues develop into their mature three‐dimensional (3D) forms via the process of morphogenesis. Using lithographic approaches in combination with 3D traction force microscopy, we have elucidated the mechanical forces required for morphogenesis of the airways of the vertebrate lung, as well as the forces exerted by the airway epithelium as it branches into its characteristic tree‐like structure. I will describe how we combine these experimental strategies with computational approaches to define the mechanical regulation of branching morphogenesis. I will also discuss efforts into understanding the evolutionary relationships between mechanical forces and biochemical signals across a variety of branched organs and species.
Rhamnetin and cirsiliol regulate radioresistance and EMT by suppression of Notch‐1 expression in NSCLC (794.1)
Authors: Kang JiHoon , Seo HyunJeong , Park Kang Hyun , Youn BuHyun
Published: June 2021

Despite optimal radiotherapy, chemotherapy and/or surgery, the majority of patients with NSCLC fail treatment. Radioresistance is a major cause of decreasing the efficiency of radiotherapy. To understand its mechanisms, we focused on radiation‐induced Notch‐1 signaling pathway. In this study, we investigated the use of Notch‐1‐regulating flavonoids, rhamnetin and cirsiliol, as novel therapeutic drugs to regulate radiosensitivity in NSCLC. Treatment with two compounds reduced the proliferation of NSCLC cells through the suppression of radiation‐induced Notch‐1 expression. Both compounds increased the expression of miR‐34a in a p53‐dependent manner, leading to inhibition of Notch‐1 expression. Consequently, reduced Notch‐1 expression promoted apoptosis through significant down‐regulation of NF‐κB pathway, resulting in a radiosensitizing effect on NSCLC cells. EMT induced by radiation was also notably attenuated by two compounds. Moreover, an in vivo xenograft mouse model confirmed the radiosensitizing effects of both flavonoids as we observed in vitro. Altogether, our findings provided evidence that rhamnetin and cirsiliol can act as novel radiosensitizers via targeting Notch‐1 expression.

Grant Funding Source: Supported by the National R&D Program for Cancer Control, Ministry for Health and Welfare(1320100)
Mutational analysis of calcineurin B homologous protein isoform 2 binding to the Na+ ‐ H+ exchanger isoform 1 (NHE1): NHE1 mutations H523I and H523G (796.7)
Authors: Berthelsen Nicholas , Novak Alexander , Cottle Wayne , Hasle Daniel , Provost Joseph , Wallert Mark
Published: June 2021

The Na+‐H+ exchanger isoform 1 (NHE1) is a transmembrane protein that regulates a spectrum of function in cells from solid tumors including cell proliferation, adhesion, migration and invasion. The calcineurin B homologous proteins (CHP1 and CHP2) appear to be essential cofactors for NHE1 function. Both CHP1 and CHP 2 bind to the same domain on NHE1, amino acids 515 to 530. CHP2 is expressed primarily in tumor cells where it binds to NHE1 with a 5‐10 fold higher affinity than CHP1. CHP2 expression in tumor cells activation of NHE1, leading to enhances tumorigenic behavior. We will investigate the ability of mutations to a key amino acid in the binding domain (H523) to alter CHP2 binding to NHE1 in vivo. Two distinct site directed mutations to NHE1, H523I and H523G, have been constructed along with the accompanying stable cell lines expressing NHE1 with these mutations. We will present data evaluating CHP2 binding to NHE1 in these cells using a GFP‐CHP construct as well as how changes in CHP2 binding alters adhesion, proliferation, and migration in cells expressing the NHE1 mutations relative to cells expressing wild‐type NHE1.
Levels of DNA topoisomerases induced by interleukin‐2 in HuT 78 cells (798.2)
Authors: Foglesong Paul , Nair Rajeev , Gupta Nikita
Published: June 2021

The activities of DNA topoisomerase I, DNA topoisomerase IIα, and DNA topoisomerase IIβ are induced in three transient peaks in HuT 78 cells, a human T cell lymphoma cell line, within 12 hrs after treatment of the cells with 1,000 units of interleukin‐2 (IL‐2)/ml. The identities of these DNA topoisomerases were determined by immunoprecipitations of the enzymes from nuclear extracts of IL‐2‐treated HuT 78 cells using specific polyclonal antibodies. These results have been confirmed by ELISAs and western blots using specific monoclonal antibodies. The levels of each of these three DNA topoisomerases were determined by ELISAs, and the results were compared to the specific activities of the enzymes. There is no direct correspondence of the levels of any of these enzymes with the profile of its specific activity in the 12 hours following IL‐2 treatment of the cells. These results suggest that the biochemical basis for the observed activation of these three enzymes is primarily posttranslational modification (such as phosphorylation and/or SUMOylation which have been described for each) rather than increased synthesis/degradation of these proteins. This work was supported by grants from the William & Ella Owens Medical Research Foundation and University of the Incarnate Word.
Mitotic kinesin CENP‐E cooperates with microtubule‐bundling protein PRC1 in temporal control of central spindle assembly (801.2)
Authors: Liu Xing , Yao Phil , Wang Wenwen , Qin Bo , Dou Zhen , Ding Xia , Yao Xuebiao
Published: June 2021

Chromosome movements during mitosis are orchestrated primarily by the interaction of spindle microtubules with the kinetochore, the site for attachment of spindle microtubules to the centromere. Kinetochore‐associated mitotic kinesin CENP‐E plays key roles in chromosome congression and spindle checkpoint satisfaction. We have recently identified and characterized syntelin, a novel selective CENP‐E inhibitor (Ding et al., 2010. Cell Res. 20, 1386‐1390). Cells treated with syntelin progress through interphase, enter mitosis normally with a bipolar spindle and lagging chromosomes around the poles. Syntelin is an allosteric inhibitor which tightens CENP‐E‐microtubule interaction by slowing inorganic phosphate release. To delineate the role of CENP‐E in reorganization of interpolar microtubules into an organized central spindle, metaphase synchronized cells were exposed to syntelin and other mitotic motor inhibitors. Syntelin does not perturb interpolar microtubule assembly but abrogates the anti‐parallel microtubule bundle formation. Real‐time image shows that CENP‐E inhibited cells undergo central spindle splitting and exhibits chromosome instability phenotypes. Interestingly, inhibition of CENP‐E did not alter the interaction between CENP‐E and PRC1 but perturbed temporal assembly of PRC1 to the midzone. Surprisingly, inhibition of CENP‐E perturbs the temporal control of PRC1 dephosphorylation which led to a persistent phosphorylation of PRC1 and an inhibition of central spindle assembly. These findings reveal a previously uncharacterized role of CENP‐E motor in temporal control of central spindle assembly.

Grant Funding Source: DK56292; CA164133
Regulation global sumoylation by a MAP kinase Hog1 and its potential role in osmo‐tolerance in yeast (802.17)
Authors: Li Yang , Irqeba Ameair , Panahi Mahmoud , Zhu Ming , Wang Yuqi
Published: June 2021

Sumoylation, a post‐translational protein modification by small ubiquitin‐like modifier (SUMO), has been implicated in many stress responses. Here we analyzed the potential role of sumoylation in osmo‐response in yeast. We find that osmotic stress induces rapid accumulation of sumoylated species in normal cells. Interestingly, disruption of MAP kinase Hog1 leads to a much higher level of accumulation of sumoylated conjugates that are independent of new protein synthesis. We also find that the accumulation of sumoylated species is dependent on a SUMO ligase Siz1. Notably, overexpression of SIZ1 in Hog1‐disruptive mutants (hog1[[Unsupported Character ‐ Symbol Font □]]) but not in wild type cells leads to a markedly increased and prolonged accumulation of sumoylated species. Examination of osmo‐tolerance of mutants that display either an increase or a decrease in the global sumoylation level revealed an inverse relationship between accumulation of sumoylated conjugates and somo‐tolerance. Further investigation has shown that many of the sumoylated species induced by hyperosmotic stress are actually poly‐sumoylated. Together, these findings indicated that abnormal accumulation of poly‐sumoylated conjugates is harmful for osmo‐tolerance, and suggest that Hog1 promotes adaptation to hyperosmotic stress partially via regulation of global sumoylation level.
PHLPP phosphatases are master regulators of cellular receptor tyrosine kinase levels (802.19)
Authors: Cohen Katsenelson Ksenya , Reyes Gloria , Niederst Matt , Chen Muhan , Nowak Dawid , Kunkel Maya , Brognard John , Trotman Lloyd , Newton Alexandra
Published: June 2021

Growth factor receptor levels are aberrantly high in diverse cancers, driving the proliferation and survival of tumor cells. Understanding the molecular basis for this aberrant elevation has profound clinical applications. Here we show that the PH domain leucine‐rich repeat protein phosphatase (PHLPP) suppresses the steady‐state levels of receptor tyrosine kinases (RTKs) by a novel mechanism unrelated to its previously described function as the hydrophobic motif phosphatase for AKT, protein kinase C and S6 kinase. Specifically, genetic depletion and pharmacological studies reveal that PHLPP1 and PHLPP2 promote histone deacetylase (HDAC)‐dependent repression of the transcription of diverse growth factor receptors, including the EGF receptor. These data uncover a novel, and much broader, role for PHLPP in regulation of growth factor signaling beyond its direct inactivation of AKT: by suppressing RTK levels, PHLPP dampens the downstream signaling output of two major oncogenic pathways, the PI3 kinase/AKT and the RAS/ERK pathways. Our data are consistent with a model in which PHLPP is a master regulator of the RTK signaling output of cells.
Membrane associated RGC‐32 promoted macrophage phagocytosis via direct and specific activation of novel PKC pathway (802.24)
Authors: Tang rui , Chen shiyou
Published: June 2021

Phagocytosis of bacterial pathogens by macrophages requires the activation of protein kinase C (PKC), a family of protein serine/threonine kinases. Previous publications have demonstrated that both classical and novel PKCs were required for macrophage phagocytosis. However recruitment of classical PKCs or novel PKCs or both is macrophage linage specific, which prompt us to hyposize that there exist PKC isozyme specific regulators in macrophage. Here we found that response gene to complement 32 (RGC32) is a direct and specific activator for novel PKC pathway in macrophage phagocytosis. RGC32 was induced and specifically expressed in macrophage lineages in differentiation. While bone marrow progenitor cells from RGC32 knockout mice did not have significant defects in macrophage differentiation, both peritoneal macrophages (PMs) and bone marrow derived macrophages (BMDMs) presented dramatic defects in their phagocytosis activities. Moreover, macrophages over expressed RGC32 showed promoted phagocytosis activity. During phagocytosis, RGC32 was recruited to cell membrane and co‐localized with F‐actin to forming phagocytic cups and macrophages from RGC32 knockout mice showed impaired F‐actin assembling and phagocytic cup formation. Mechanistically, RGC32 participated in phagocytosis through activation of PKC induced alanine‐rich C‐kinase substrate (MARCKS) phosphorylation. Recombinant RGC32 increased PKC activity in vitro. Knocking down RGC32 in macrophage suppressed MARCKS phosphorylation while over expression RGC32 increased MARCKS phosphorylation. Interestingly, RGC32 directly interacted with novel but not classical PKC isozymes and RGC32 induced MARCKS can only be suppressed by novel PKC inhibitor. Finally, RGC32 knockout mice showed defects in pathogens clearance to peritoneal bacterial infection. In summary, for the first time, we identified RGC32 as an membrane associated novel PKC activator during macrophage phagocytosis.
Molecular mechanism of Raf kinase inhibitory protein regulation by cAMP‐dependent protein kinase (802.25)
Authors: Ong Colin , Masterson Larry , Kim Jonggul , Gomes Suzana , Veglia Gianluigi , Rosner Marsha
Published: June 2021

Raf Kinase Inhibitory Protein (RKIP), a 21 kDa protein and member of phosphatidylethanolamine binding protein (PEBP) family, regulates GPCR, MAPK, and NFκB pathways and is a metastasis suppressor. Although, kinases such as ERK2 and PKCα have been shown to phosphorylate RKIP, structural data on the protein kinase‐RKIP interaction are still lacking. Here we report that another kinase, PKA, phosphorylates RKIP at residue 51, triggering a feedback loop, and we determine the structural model of RKIP‐PKA catalytic subunit. Using different biophysical approaches including Nuclear Magnetic Resonance (NMR), Paramagnetic Relaxation Enhancements (PREs) and molecular modeling, we identify the binding interface between RKIP and PKA catalytic subunit. Chemical shift mapping and site directed mutagenesis studies revealed residues within RKIP that are critical to the catalysis and binding mediated by PKA catalytic subunit. As the first description of PKA binding to a full‐length physiological substrate, these results have wide applicability to the mechanism of PKA catalysis

Grant Funding Source: Supported by NIH grant GM087630 to Marsha Rich Rosner, NIH grant GM100310 to Gianluigi Veglia
The small GTPase Rab5 controls cell morphology, sexual development, ion‐stress response and vacuolar formation in fission yeast (802.29)
Authors: Miyamoto Masaaki , Tsukamoto Yuta , Katayama Chisako , Shinohara Miki , Shinohara Akira , Maekawa Shohei
Published: June 2021

Vesicle transport is important for cell functions. Rab family GTPases play a critical role in inner‐membrane transport. In mammalian cells, Rab5 is reported to be involved in the regulation of endosome formation, phagocytosis and chromosome alignment. In order to examine the role of the fission yeast Rab5 homologue Ypt5, we used a point mutant allele and analyzed. Mutant cells displayed abnormal phenotypes in cell morphology, mating, sporulation, endocytosis, vacuole fusion and responses to ion stress. Our data suggest that fission yeast Rab5 is involved in the regulation of various types of cellular functions and regulate cell morphology via actin cytoskeletons.

Grant Funding Source: Supported by Kobe Univ. and Osaka Univ.
Analysis of class‐distinctive Gα13 mutants to identify regions critical for serum response element‐mediated transcriptional activation (802.9)
Authors: Tagliatela Alicia , Hamilton Tyler , Jones Alan , Meigs Thomas
Published: June 2021

Guanine‐nucleotide binding proteins (G proteins) are involved in transmitting signals from cell surface receptors to downstream pathways. The G12/13 class of G proteins includes Gα12 and Gα13, which diverged from a single ancestral G12/13 protein during evolution. These proteins drive proliferation and migration in certain cell types, and also regulate cytoskeleton rearrangements and other cellular events. In order to identify key signaling determinants that evolved in the G12/13 class, we engineered mutants in which class‐distinctive residues in Gα13 were substituted for residues conserved at the same structural position in the non‐G12/13 classes of Gα proteins. These mutants were transfected into human kidney cells, and serum response element (SRE) activation assays identified several substitutions in Gα12 that hindered SRE activation. Surprisingly, the corresponding mutations in Gα13 showed no significant reduction in this serum response. Furthermore, we engineered chimeras in which Gα12 and Gα13 incorporated regions from the fruit fly G12/13 homolog, Concertina, and found a C‐terminal region for which substitution of Concertina sequence abolished Gα12 signaling through SRE yet was fully tolerated in Gα13. These findings suggest Gα12 and Gα13 utilize distinct structural mechanisms for engaging the pathway leading to SRE‐mediated transcription.

Grant Funding Source: Supported by the GlaxoSmithKline Foundation and Lineberger Comprehensive Cancer Center
Relationship between apparent in vivo occupancy at the dopamine transporter and cocaine‐like discriminative stimulus effects of N‐substituted benztropine analogs in mice (803.4)
Authors: Wilkinson Derek , Cohen Marika , Newman Amy , Katz Jonathan
Published: June 2021

Standard inhibitors of the dopamine transporter (DAT) have cocaine‐like subjective effects; however, several atypical DAT inhibitors that bind the DAT with high affinity, such as benztropine (BZT) analogs, do not fully substitute for cocaine in animals trained to discriminate cocaine injections.

The lack of cocaine‐like effects may be related to a slow rate of association to the DAT. Several N‐substituted BZT analogs (R‐2‐amino‐3‐methyl‐n‐butyl; 2‐aminoethyl; 2‐ethyl‐3‐indole; and methylcyclopropyl) were reported to have a relatively fast onset of behavioral effects, but it is unclear if these effects were related to DAT activity. Therefore, the present study examined the in vivo binding to DAT of the above BZT analogs and their cocaine‐like discriminative stimulus effects in mice. None of the BZT analogs fully substituted for cocaine discriminative effects despite the testing of doses that produced in vivo DAT occupancy greater than the ~13% occupancy produced by 10 mg/kg training dose of cocaine. The BZT analogs produced displacement of [I125]RTI‐121 from striatum at maximal rates from 0.86 to 1.28 %/min compared to 1.23%/min for cocaine, indicating a relatively fast apparent rate of DAT association. The results of the present study suggest that DAT occupancy, or rate of association, alone cannot account for the lack of cocaine‐like discriminative effects found with these N‐substituted BZT analogs.

Grant Funding Source: NIH Intramural Research Program
Identification of the attachment site for the cocaine analog [125I]JHC 2‐48 on the dopamine transporter (803.7)
Authors: Dahal Rejwi , Akula Bala Pramod , Sharma Babita , Cha Joo , Cao Jianjing , Boateng Comfort , Lever John , Newman Amy , Foster James , Henry L. , Vaughan Roxanne
Published: June 2021

Cocaine is a highly addictive drug that targets the presynaptic dopamine transporter (DAT) and blocks the reuptake of dopamine resulting in heightened dopaminergic neurotransmission. The DAT is a 12 transmembrane (TM) spanning protein with residues from TMs 1, 3, 6, and 8 comprising the substrate and putative inhibitor binding sites; although recent studies indicate the presence of an inhibitor binding site in the external vestibule located above the substrate‐binding site. Despite decades of research, the binding site of cocaine has yet to be fully characterized. To address this question, we are mapping the attachment sites of irreversible cocaine analogs, [125I]MFZ 2‐24, [125I] RTI 82, and [125I]JHC 2‐48, using insight gained from in silico docking with DAT comparative models. These compounds have a cocaine pharmacophore with analog specific azido group that adducts to the protein. To determine the adduction site of [125I]JHC 2‐48 we are using methionine substitution mutagenesis to generate custom protease sites for cleavage by cyanogen bromide (CNBr). The fragments generated from the CNBr proteolysis of [125I]JHC 2‐48 labeled DAT were visualized via SDS‐PAGE and autoradiography. The preliminary results obtained thus far have determined the [125I]JHC 2‐48 attachment site to be C‐terminal to TM7. Peptide map analyses of other cocaine analogs, [125I]MFZ 2‐24 and [125I] RTI 82, have determined [125I]MFZ 2‐24 adducts to TM1 and [125I]RTI 82 adducts to TM6. Taken together, our data strongly indicate cocaine binds near the substrate binding site.

Grant Funding Source: Supported by: DA027845 and NIDA‐IRP
Unanticipated weight gain during a dietary intervention designed to improve iron status (804.20)
Authors: Chen Vivi , Luna Sarah , Lung'aho Mercy , Gahutu Jean Bosco , Haas Jere
Published: June 2021

Objective: To quantify the effect on weight gain of a 4.5 months feeding trial intended to replace the subjects’ regular meals.

Methods: 145 Rwandan women (18‐26 years) in a double‐blind feeding trial were randomized to receive either iron‐biofortified beans or traditional beans daily for 4.5 months. During the feeding period, the subjects were served 175 g of beans per meal, for lunch and dinner, plus ad libitum consumption of side dishes; they were allowed to consume more beans if desired. Blood biomarkers and anthropometry were measured before and after the feeding period. General linear models were used to evaluate the effect of the intervention on weight gain.

Results: On average, the women consumed 200 g of beans (358 kcals) per meal. After the 4.5 months feeding period, 25% of the women gained 蠅 3 kg in body weight. Both the control and the treatment group consumed the same amount of beans per meal, with the treatment group having a significantly (p>.001) greater increase in iron status. The more beans consumed per meal, the more weight the subjects gained (F= 3.94, p=.05), regardless of iron intake. Women in the upper quartile of the BMI distribution (BMI > 24.2) gained the most weight (F=3.09, p=0.03).

Conclusion: Providing lunch and dinner in a feeding trial to improve micronutrient status resulted in a net addition of calories and subsequent weight gain, especially in women with higher BMI.

Grant Funding Source: Supported by HarvestPlus
Involving children in meal preparation: implications for increasing vegetable intake at home and school (808.9)
Authors: Lakanen Andrew , Reicks Marla
Published: June 2021

The purpose of this study was to test if child involvement in salad preparation increased vegetable intake at school lunch and explore parental barriers and facilitators to child involvement in home meal preparation. Two salad kits (spinach and garden) that children could mix together were offered for school lunch on two occasions in one low‐income elementary school in the Minneapolis metropolitan area. More children took a salad and ate more salad when served as components they could mix together compared to when already mixed, however these increases were not statistically significant. An online survey was developed, pretested, and administered to US adults with children 5‐12 years (n=344) to determine predictors of child involvement in home meal preparation. Multivariate linear regression analysis showed that frequency of child involvement was associated with intentions and parent gender while child age and gender, perceptions of barriers, self‐efficacy and environmental support were not associated. The model explained 33% of the variance in child involvement in home meal preparation (P<0.0001). Parents reported that children were involved with meal preparation several times/week and were generally positive about benefits. Understanding the relationship between parental perceptions of child involvement in meal preparation may enhance effectiveness of programs that promote family meal preparation.
Expressiom of calcium ATPase in different tissues of protein deficient rats supplemented with Moringa olifera leaves (809.6)
Authors: Ajani Emmanuel , Nurain Ismail , Bamisaye Fisayo , Ogunlabi Olugbenga
Published: June 2021

Ca2+‐ATPases are key proteins for the maintenance of calcium homeostasis and essential to the correct functioning of cell machinery. Reports indicates that exposure of cell to oxidative injury will affect Ca2+‐ATPase. In this study, we investigated the effect of Protein energy malnutrition (PEM) on Ca2+‐ATPases and the ameliorating effect of Moringa oliefera leaves. Eighteen Wistar rats randomized into groups A and B were used for the study. Group A (12 rats) were placed on protein deficient diet while group B (6 rats) were placed on protein sufficient diet. They were maintained on respective diets for 6 weeks after which all rats in group B and 6 rats in group A were sacrificed. The remaining rats in group A (relabeled as C) were maintained on protein deficient diet supplemented with Moringa oliefera leaves for another 6 weeks and then sacrificed. The skeletal muscle, brain and liver were removed and homogenized. Ca2+‐ATPase specific activities in the selected tissues and the enzyme’s kinetic constants were then determined and compared. Our result indicates that Ca2+‐ATPases activity (for all the organs) was highest in group C and lowest in the protein deficient rats not supplemented (A). The activity was also highest in the skeletal muscle and lowest in the liver (for all the groups). The Km and Vmax (for all the organs) was lowest in group C and highest in group A. Our result suggest that PEM reduces the activity of Ca2+‐ATPases and that Moringa oliefera is more efficacious than fish in regulating the enzyme activity.