bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional studies of human polymerase η A264P (736.6)
Authors: Lentscher Anthony , Jiang Xiaohua
Published: June 2021

DNA is the blueprint which contains the information necessary for a cell to maintain normal structure and function. This essential function of DNA means the cell must necessarily preserve the integrity of its blueprint. Harmful mutations must be minimalized where they cannot be completely eliminated. One such method for maintaining the integrity of DNA is through the function of human polymerase η, a replication enzyme which gains its utility in its ability to correct thymine dimerization mutations to DNA as a result of exposure to ultraviolet radiation. Mutations in human polymerase η lead to a xeroderma pigmentosum variant (XPV) which is limited in its ability to repair damage done to DNA. This research will include introducing the A264P mutation to the catalytic core of the enzyme through site‐directed mutagenesis, transformation of the resultant DNA into E. coli cells, eventually to overexpress and purify the protein, and run translesion synthesis assays in order to determine how restricted the function of the A264P mutant is when compared to the wild‐type protein.
Lysosomal interaction of Akt with Phafin2: a critical step in the induction of autophagy (737.1)
Authors: Noguchi Masayuki , Matsuda‐Lennikov Mami , Hirata Noroyuki , Hashimoto Manabu , Kimura Kohki , Edamura Tatsuma , Suizu Futoshi
Published: June 2021

Autophagy is an evolutionarily conserved mechanism for the gross disposal of intracellular proteins in mammalian cells and dysfunction in this pathway has been associated with human disease. Although the serine threonine kinase Akt is suggested to play a role in this process, little is known about the molecular mechanisms by which Akt induces autophagy. Using a yeast two‐hybrid screen, Phafin2, a lysosomal protein with a unique structure of N‐terminal PH (pleckstrin homology) domain and C‐terminal FYVE (Fab 1, YOTB, Vac 1, and EEA1) domain was found to interact with Akt. A sucrose gradient fractionation experiment revealed that both Akt and Phafin2 co‐existed in the same lysosome enriched fraction after autophagy induction. Confocal microscopic analysis and BiFC analysis demonstrated that both Akt and Phafin2 accumulate in the lysosome after induction of autophagy. BiFC analysis using PtdIns (3)P interaction defective mutant of Phafin2 demonstrated that lysosomal accumulation of the Akt‐Phafin2 complex and subsequent induction of autophagy were lysosomal PtdIns (3)P dependent events. Furthermore, in murine macrophages, both Akt and Phafin2 were required for digestion of fluorescent bacteria and/or LPS‐induced autophagy. Taken together, these findings establish that lysosomal accumulation of Akt and Phafin2 is a critical step in the induction of autophagy via an interaction with PtdIns (3)P.
Four conserved cysteines of VKORC1L1 function in concert in the vitamin K cycle (739.1)
Authors: Tie Jian‐Ke , Jin Da‐Yun , Stafford Darrel
Published: June 2021

VKORC1 reduces vitamin K epoxide in the vitamin K cycle for the posttranslational modification of proteins that are involved in a variety of biological functions. VKORC1 has been extensively studied, but few studies of VKORC1L1, a paralogous enzyme sharing over 50% protein identity with VKORC1, have been reported. Here we show via cell‐based assay that VKORC1L1 reduces vitamin K epoxide to support vitamin K‐dependent carboxylation as efficiently as does VKORC1. However, unlike VKORC1, VKORC1L1 is a four‐transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1 function. Results from intermediate disulfide trapping allow us to hypothesize the role of VKORC1L1’s four conserved loop cysteines function in concert: first the catalytic C58 attacks the active site disulfide, forming an intermediate disulfide with C139, and the resolving C50 then attacks the intermediate disulfide, which results in active site reduction. VKORC1L1’s and VKORC1’s different membrane topologies and reaction mechanisms suggest that these two enzymes might have different physiological functions.
Role of protein prenyltransferases in modifying effectors from infectious bacterium (739.7)
Authors: Wright Elia , Fierke Carol
Published: June 2021

Protein prenylation is an important post‐translational modification. Many G‐proteins, such as Ras and Rho, are prenylated in order to anchor them to the cell membrane. Protein farnesyltransferase (FTase) and geranylgeranyltransferase (GGTase) add 15‐carbon farnesyl groups and 20‐carbon geranylgeranyl groups to proteins, respectively. Substrate specificity for FTase and GGTase is minimally described by the Cza2X paradigm where C is a cysteine residue four amino acids from the C‐terminus, followed by two amino acids (z is variable; a2 is aliphatic) and an X group that specifies which prenyltransferase is preferred, though there is significant substrate overlap. Recent experiments have demonstrated that infectious bacterium Legionella pneumophila requires host cell farnesylation machinery to induce pulmonary disease. Ankyrin b, an essential Legionella effector protein, is farnesylated by the host FTase, which is important for evasion of the endocytic pathway and protein function. Prenylation of effector proteins may be a general method used by pathogenic bacteria to anchor proteins in hosts. To investigate the prevalence of prenylation of bacterial proteins, we have measured the reactivity of mammalian FTase and GGTase‐I both in vivo and in vitro with peptide sequences derived from proteins expressed in pathogenic bacteria. We have shown that FTase and GGTase are capable of prenylating bacterial Cza2X motifs in vitro and in vivo through peptide assays and in vivo cellular localization of GFP fusion proteins. We have also tested a series of prenyl donor analogs synthesized in the Gibbs laboratory with FTase and GGTase to identify analogs with similar substrate specificity to in vivo prenyl donors to use as tools for defining the prenylome. These studies will further our understanding of the many ways protein prenyltransferases regulate cellular processes and how inhibition of these enzymes may be incorporated into treatment of bacterial infections and other diseases.

Grant Funding Source: Supported by NIH F31 Diversity Fellowship
pH‐Dependent structural ensembles of haptoglobin suppress β2‐microglobulin amyloidosis: role in lysosomal localization and extracellular protein quality control (752.7)
Authors: Sultan Abdullah , Bakthisaran Raman , Rao C , Tangirala Ramakrishna
Published: June 2021

Several diseases involve extracellular deposition of misfolded proteins, which is associated with inflammation and local acidosis. We have investigated the chaperone‐like activity of haptoglobin towards the amyloid aggregation of β2‐microglobulin (β2m) and of α‐synuclein and its role in clearing the amyloid deposits from the extracellular space. At neutral pH, haptoglobin prevents the formation of β2m amyloid fibrils but not that of α‐synuclein. Under conditions of local acidosis, pH 5.5‐6.5, it prevents β2m fibril formation with relatively decreased efficiency. We have investigated the structural and stability aspects of haptoglobin as a function of pH. Between pH 5.5‐8.0, it exists in a continuum of conformational states with subtle perturbation in tertiary structure, moderately exposed hydrophobic surfaces and distinct latent heat of unfolding. Below pH 5.0 it exists in a molten‐globule‐like conformation with no chaperone activity. Live cell imaging and flow cytometry studies show that the species of β2m formed in the presence of haptoglobin are not cytotoxic and readily undergo lysosomal degradation. Analytical ultracentrifugation and dot blot studies reveal that haptoglobin interacts with prefibrillar oligomeric species formed during β2m fibril formation. Thus, haptoglobin is an extracellular chaperone with promising therapeutic implications in extracellular amyloid diseases.
Minotoring adenine nucleotides levels and their relevance to biochemical and cellular metabolic function using bioluminescence‐based strategies (766.5)
Authors: Goueli Said , Hsiao Kevin
Published: June 2021

Adenine nucleotides are major determinants of the energy status of the cell and thus any modulation of their cellular concentration has significant consequences to cellular metabolism, cellular growth and cell death. ATP generating enzymes are usually involved in anabolic processes while ATP consuming enzymes are involved in catabolite processes. We have developed biochemical and cell based assays that monitor the concentrations of ATP, ADP and AMP in biochemical reactions and in cells. These universal assays can measure the activity of various enzymes with no modification of the native substrate and the ability to use diverse substrates such as proteins, peptides, lipids, sugars, etc. The AMP generating reactions such as ubiquitin ligases, aa aminoacyl t‐RNA synthetases, DNA ligases, cAMP‐dependent phosphodiesterases, etc. can be also monitored with high sensitivity and reproducibility. The ADP‐generating (kinases and ATPases, and ATP‐consuming reactions (kinases and ATPases) can be monitored as well using this luminescent approach. We will also show data using luminescent technology to monitor the cellular levels of these adenine nucleotides under various stress and physiological conditions.
Mechanisms of atypical protein kinase C activation in insulin signaling (767.9)
Authors: Tobias Irene , King Charles , Newton Alexandra
Published: June 2021

Atypical protein kinase C (aPKC) is a key modulator of insulin signaling that regulates glucose transport. Dysfunction of aPKC correlates with insulin‐resistant states in both mice and humans. Despite the engaged interest in this enzyme’s importance to type 2 diabetes, much less is known about the molecular mechanisms that govern its cellular functions than for the conventional and novel classes of PKCs. Here we examine the regulation of aPKC by phosphorylation and lipid binding in order to better define these mechanisms of activation in the context of insulin signaling pathways. Specifically, we show that key drivers of aPKC activity are 1) constitutive phosphorylation at the activation loop and turn motif phosphorylation sites and 2) that phospholipids regulate activity and susceptibility to dephosphorylation. Methods include in vitro kinase activity assays performed on recombinant aPKC purified from insect cells, using phospho‐resistant or phospho‐mimetic mutations placed at key phosphorylation sites. Additional biochemical activity studies were performed following dephosphorylation and in the presence of phospholipids. Our data are consistent with aPKCs sharing similar constitutive phosphorylation as conventional and novel PKCs, with dephosphorylation and activity accelerated by the presence of lipids.

Grant Funding Source: This work was supported by P01 DK054441 to ACN and T32 GM007752 to IT
Group III phospholipase A2: collective unity amidst structural and functional diversity (769.1)
Authors: Hariprasad Gururao , Singh Reema
Published: June 2021

Secretory phospholipase A2 (PLA2) catalyses the hydrolysis of the sn‐2 position of glycerophospholipids to liberate arachidonic acid, a precursor of eicosanoids, that mediate inflammation. The group III PLA2 enzymes are present in a wide array of organisms across many species with completely different functions. For a systematic classification of the group, fifty protein sequences from different species of the group were taken for sequence, structural and phylogenetic studies. In addition to the conservation of calcium binding motif and the catalytic histidine, the sequences exhibit specific ‘amino acid signatures’. Structural modeling reveals that these enzymes have a conserved globular structure with species specific variations seen at the active site, calcium binding loop, hydrophobic channel, the C‐terminal domain and the quaternary conformational state. Phylogenetic analyses are in accordance with the structural features. The results provide a convincing platform for the classification the group III PLA2s into‐ 1A: venomous insects; IB: non‐venomous insects; II: mammals; IIIA: gila monsters; IIIB: reptiles, amphibians, fishes, sea anemones, liver fluke; IV: scorpions. This classification helps to understand structure‐function relationship, substrate specificity, designing of potent inhibitors against the drug target isoforms and understand evolutionary perspective of constituents.

Grant Funding Source: Supported by FAST TRACK grant No. SR/FT/18 given by Department of Science and Technology
Exploring enzyme conformational landscape with pressure‐perturbation: allosteric rearrangements in P450 3A4 revealed with FRET and SDSL‐EPR (769.16)
Authors: Davydov Dmitri , Yang Znongyu , Halpert James , Hubbell Wayne
Published: June 2021

We used a combination of pressure‐perturbation techniques to explore conformational rearrangements in cytochrome P450 3A4. Site‐directed incorporation of a FRET donor‐acceptor pair allowed us to discover an equilibrium between open and closed states revealed in pressure‐induced increase in the distance between fluorophores placed in the α‐ and β‐domains. This transition is characterized by a volume change (ΔV°) of ‐33 ± 14 ml/mol and ΔG° of 0.7 ± 0.2 kcal/mol, so that only 20% of the enzyme adopts the open conformation at ambient pressure. Addition of testosterone, an allosteric activator, changes the ΔV° and ΔG° to ‐18 ± 7 ml/mol and 0.2 ± 0.1 kcal/mol respectively, increasing the fraction of the open state to 40%. The studies by site‐directed spin labeling (SDSL) and pressure‐perturbation EPR demonstrated that this transition is also revealed in pressure‐dependent alteration of the mobility of a nitroxide probe placed at positions 85, 166, 374, 409, or 495. We also detected a pressure‐induced displacement of emission maxima of environment‐sensitive fluorophores placed at residues 85 and 409 suggesting their relocation to a more polar environment. Importantly, the parameters of these testosterone‐dependent changes were similar to those found in our FRET studies. Our results elucidate the mechanism of effector‐dependent conformational equilibrium in P450 3A4 and demonstrate a high potential of pressure‐perturbation strategies in the studies of enzyme conformational landscapes.

Grant Funding Source: Supported by NIH GM54995
Time‐course analysis of gene expression in a mouse model simulating aspects of human post‐traumatic stress disorder (776.7)
Authors: Muhie seid , Srinivasan Seshamalini , Gautam Aarti , Chakraborty Nabarun , Hammamieh Rasha , Jett Marti
Published: June 2021

We profiled transcriptome changes in brain regions and blood of socially defeated mouse model of post traumatic stress disorder. Brain regions involved in fear circuitry and blood were collected from 5 days and 10 days aggressor exposed and control mice. Changes in gene expression after 1 day, 10 days and 42 days of rest (post 5 days and 10 days defeat periods) were profiled using microarray and real time PCR. Differentially expressed blood transcripts were validated by assaying the corresponding plasma proteins.

Functional and pathway analyses of differentially regulated genes in trauma response brain regions (Aygdala, hippocampus, medial prefrontal cortex, ventral striatum and nucleus acumbens) of defeated mice were found to be involved in startle response, associative learning, long term memory, inflammation, regulation of circadian rhythm, glucocorticoid receptor activity, dendritic branching, and synaptic depression.

Blood transcripts with increased expression with longer rest period were involved in T‐cell immunity, serotonorgic and dopaminergic synaptic processes and axonal guidance. Transcripts in blood with decreased expression with longer rest period were involved in associative learning, opoid signaling and glutamatergic pathways. Changes at the transcriptome level indicated the pervasive nature of traumatic stress in altering functions and pathways which normally precipitate in phenotypic behavioral disorders and pathologies. With increased rest period, some of the important functions and pathways showed regaining vitality indicating a sense of recovery with time.
Electron paramagnetic resonance characterization of drug‐binding to monoacylglycerol lipase using the dynamic signature of a protein spin‐label (779.3)
Authors: Wong Brian , Budil David , Karaogeorgos Ioannis , Tyukhtenko Sergiy , Makriyannis Alexandros
Published: June 2021

In recent years, monoacylglycerol lipase (MGL) has been an important pharmaceutical target because of its relationship to pathways that have been implicated in cancer proliferation. The purpose of this study is to find an efficient and streamlined way of characterizing drug‐binding to MGL using electron paramagnetic resonance (EPR). The free nitroxide radical methyl thiosulfonyl spin label (MTSL) was used to label the single cysteine mutant S208C on a domain of MGL that is thought to constitute a “lid” covering the substrate binding pocket of MGL. EPR spectra shows reproducible line shape changes reflecting a significant reduction in the mobility of the lid when MGL binds reversible inhibitors. The results are consistent with NMR data that reveal an equilibrium between the putative “open” and “closed” positions of the lid as a function of temperature or pH. These findings establish a useful assay for screening potential cancer drugs that bind in a specific way to MGL that can be carried out more rapidly and with significantly smaller amounts of protein than other assays for the dynamic motion of the drug target.

Grant Funding Source: Supported by grants DA09158, DA00493, DA03801 and DA07312
Expression and activity of single‐chain recombinant eel follicle stimulating hormone in mammalian cells (792.2)
Authors: Park Chae‐Won , Nanjidsuren Tsevelmaa , Kim Min‐Su , Seo Eun‐Bi , Hong Sun‐Mi , Kang Myung‐Hwa , Kim Dae‐Jung , Min Kwan‐Sik
Published: June 2021

The glycoprotein hormone family consists of luteinizing hormone (LH), follicle‐stimulating hormone (FSH), and thyroid‐stimulating hormone (TSH), which are secreted by the pituitary gland in all mammalian species, and chorionic gonadotropin (CG), which is secreted by placental trophoblast cells in primates and equids. These hormones consists of non‐covalently associated α and β subunits. Within a species, the amino acid sequence of the α subunit is identical across all glycoprotein hormones and is encoded by a single gene. The αβ dimer is the active form of the hormone, and biological specificity is conferred by the β subunit. Also in fish, the duality of gonadotropin hormone (GTH), GTH‐1 (FSH‐like GTH), and GTH‐II (LH‐like GTH) is found in certain teleost. In this study, in order to understand the fundamental mechanisms involved in teleost reproduction and to proved a broader basis for comparative studt of teleost GTHs, we isolated a cDNA encoding the α and β subunit of GHT‐1 from eel and produced it's recombinant protein from E.coli and CHO cells. To obtain tethered eelFSH, the cDNA encoding the full‐length eel FSH β‐subunit (signal sequence of 22 amino acid residues and the mature protein of 105 amino acid residues) was fused with the mature protein (93 amino acids) of the α‐subunit by overlapping PCR mutagenesis. Subsequently, PCR fragement was inserted into the pCR2.1 clon ing vector and sequenced. After then, it was digested with the EcoRI and SalI enzymes and ligated into the eukaryotic expression vector pcDNA3. The gene was completely sequenced to confirm the presence of the Kozak site and, myc‐tag and to role out the possibility of any PCR erros. Eel FSH protein was expressed in the E. coli system using the pREST expression plasmid vector, and the protein was purified by NI‐NTA column. Eel FSH protein was detected at the predicted molecular weight of 24.3 kDa by SDS‐PAGE in E.coli and 32‐45 kDa in CHO cell lines.

Grant Funding Source: This work was supported by NFRDI
Gigaxonin regulates vimentin structure by modulating the Ras pathway through the degradation of galectin‐1 (796.11)
Authors: Martin Kyle , Beard Samuel , Loden Kelsey , Payne R. Mark
Published: June 2021

Giant axonal neuropathy (GAN) is a progressive neurodegenerative disorder characterized by intermediate filament abnormalities. GAN is caused by a loss of gigaxonin (Giga), which functions as an E3 ligase adaptor in the ubiquitin proteasome system. A previous bioinformatics study showed that levels of galectin‐1 (Gal‐1), which stabilizes activated Ras, were significantly increased in GAN cells. We tested the hypothesis that Giga regulates intermediate filament structure via Gal‐1 using GAN fibroblasts. We showed that Giga controls the degradation of Gal‐1, as restoring Giga decreased Gal‐1 protein levels and rescued the vimentin structure in GAN fibroblasts. In addition, directly decreasing the levels of Gal‐1 using siRNA rescued the vimentin structure, which was quantified using the absence of vimentin in the GAN cell periphery, termed vimentin free zones (VFZs). VFZs were observed in 82.5% of control GAN cells, and after siRNA inhibition of Gal‐1, only 20.5% of cells displayed the same phenotype. Furthermore, disruption of downstream Ras signaling rescued the GAN phenotype, as siRNA directed against H‐Ras and MEK1/2 decreased the percentage of cells with VFZs to 37.6% and 43.7% respectively. These results show that accumulation of Gal‐1 from the loss of Giga activates the Ras pathway leading to the abnormal vimentin structure in GAN cells and that inhibition of Ras signaling may be therapeutic option for GAN.
MEK kinase 2 regulates integrin signaling to control paxillin ubiquitylation and focal adhesion stability in breast tumor cells (797.3)
Authors: Cuevas Bruce
Published: June 2021

We have recently shown by gene silencing in invasive breast tumor cells that the kinase MEKK2 is required for xenograft metastasis, and our objective in this work was to determine how MEKK2 influences tumor cell function. We discovered that silencing MEKK2 expression significantly inhibits tumor cell migration, and was associated with a marked increase in the number and size of focal adhesion complexes. We discovered that tumor cell attachment to fibronectin, but not collagen, induces MEKK2 activation and translocalization to focal adhesions. Furthermore, MEKK2 knockdown greatly reduces fibronectin‐induced focal adhesion kinase phosphorylation, suggesting that MEKK2 mediates integrin‐induced signaling. We reveal that MEKK2 associates with the scaffold protein paxillin in an interaction that requires the amino‐terminal leucine‐rich motif (LD1) of paxillin. We demonstrate that the MEKK2/paxillin complex promotes paxillin ubiquitylation and consequent re‐distribution to the cytoplasm, thereby promoting focal adhesion turnover and cell migration. Finally, we show that MEKK2 associates with the ubiquitin ligase RNF5 and promotes paxillin ubiquitylation. Taken together, our results reveal a novel function for MEKK2 as a regulator of ubiquitylation‐dependent paxillin redistribution that promotes focal adhesion turnover and breast tumor cell migration.

Grant Funding Source: NIH CA120881
CRTC2 regulates miRNAs and binds to chromatin remodeling factors to control germinal center B cell fate (798.1)
Authors: Najdi Rani , Zambrano Angara , Sherman Mara , Damore Michael , Teitell Michael
Published: June 2021

When B cells are stimulated by T cell‐dependent antigens during an immune response, they form germinal centers (GCs) to undergo clonal expansion and affinity‐based selection of surface Ig proteins. GC B cells are highly proliferative and misregulation of normal GC processes results in hematologic malignancies. Our lab has shown that inactivation of CRTC2, a CREB transcriptional co‐activator, and the repression of its target genes are required for B cells to exit the GC and differentiate into plasma cells. CRTC2 is inactivated by a signaling pathway triggered by DNA double strand breaks during Ig gene remodeling. This inactivation process ends with the nuclear export of CRTC2, shutting down a gene program to enable GC B cells to differentiate into plasma cells. Despite the significance of this signaling pathway and its CRTC2‐regulated target genes, key details and mechanisms of CRTC2 regulation during B cell differentiation have not been fully explored. Using a bioinformatics‐coupled molecular approach, we identified multiple candidate CRTC2‐target microRNAs that could regulate GC B cell physiology. Our data also reveals that CRTC2 interacts with components of chromatin remodeling complexes at target promoters in GC B cells. Our findings predict that CRTC2, through its control of miRNA expression and interactions with co‐regulatory factors, regulates B cell development and lymphomagenesis.

Grant Funding Source: Supported by R01CA90571, R01CA156674, P01GM081621 (all to M.A.T.) and T32‐CA009056 (to R.N.)
Regulation of the epigenetic signature in leukemia by Ikaros (800.5)
Authors: Payne Jonathon , Casiano Carlos , Dovat Elanora , Song Chunhua , Payne Kimberly , Payne Marvin , Dovat Sinisa
Published: June 2021

Ikaros encodes a DNA binding protein that regulates gene expression via chromatin remodeling and epigenetic mechanisms. The loss of Ikaros activity due to genetic or functional inactivation results in the development of high‐risk leukemia in humans. Our goal is to determine the mechanisms by which Ikaros regulates transcription in human leukemia. Previous studies showed that Ikaros function in leukemia is controlled through its direct phosphorylation by Casein Kinase II (CK2). Treatment of leukemia cells with CK2 inhibitors results in enhanced Ikaros activity, which leads to cessation of cell growth. We have studied the mechanism by which inhibition of CK2 regulates Ikaros‐induced epigenetic changes in leukemia. The human Nalm6 pre‐B cell leukemia was treated with CK2 inhibitor, and the epigenetic signature of the histone modifications H3K4me3 and H3K36me3 were determined using chromatin immunoprecipitation coupled with next generation sequencing (ChIP‐SEQ). Both H4K4me3 and H3K36me3 histone modifications are associated with positive regulation of gene expression. The enrichment of a particular histone modification was confirmed by quantitative chromatin immunoprecipitation (qChIP). Our data demonstrate that the inhibition of CK2 activity in leukemia results in a marked alteration in the epigenetic signature of H3K4me3 and H3K36me3 as compared to untreated cells. This is directly related to altered Ikaros binding to its target genes following CK2 inhibition. Current bioinformatics analysis is directed toward establishing a link between epigenetic modifications and Ikaros binding in leukemia. These results suggest that CK2 and Ikaros regulate gene transcription in leukemia via histone modifications.

Grant Funding Source: Supported by NIH R01 HL095120 (SD); NIHR21CA162259 (KP), St. Baldrick's Foundation Awards (SD,KP,CC)
The EGF receptor is activated during mitosis and its cell signaling is mediated differently than in interphase (802.13)
Authors: Wee Ping , Shi Huaiping , Jiang Jennifer , Chen Xinmei , Wang Zhixiang
Published: June 2021

The Epidermal Growth Factor Receptor (EGFR)‐mediated signaling cascades have been intensely studied, especially as to how they relate to many cancers. It has been proposed that the G0 to S interval is the only portion of the cell cycle regulated by the EGF, and that the activation of the EGFR is inhibited during mitosis (M‐phase) so to preserve energy needs required for mitotic structural changes. Here we show that all the major tyrosine residues in the EGFR C‐terminus that are phosphorylated in interphase are also phosphorylated in nocodazole‐induced M‐phase in response to EGF, indicating full activation of EGFR. We further studied EGF‐induced activation of downstream signaling pathways in M‐phase in comparison to interphase. Some signaling proteins such as PLC‐γ1 and CBL are strongly phosphorylated in M‐phase. While two isoforms of AKT were phosphorylated in interphase, only the AKT2 isoform was phosphorylated in M‐phase. p38 MAPK was constitutively phosphorylated in M‐phase regardless of EGF stimulation. Interestingly, ERK1/2 was not phosphorylated in M‐phase. We further showed that the decoupling of ERK1/2 activation from EGFR activation was due to the incomplete activation of RAF‐1. In conclusion, our results demonstrate that in M‐phase, EGF stimulated strong EGFR activation. The activated EGFR is able to selectively activate certain downstream signaling pathways.
Mitotic kinase CDK1 cooperates with polo kinase to ensure the temporal regulation of kinetochore microtubule dynamics and spindle checkpoint (802.14)
Authors: Yu Huijuan , Zhuang Xiaoxuan , Mo Fei , Fang Guowei , 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, at which mitotic kinase circuitry synergized to ensure the quality control of cell division. Mitotic master kinase Cdk1 controls many aspects of mitotic chromosome behavior and spindle microtubule dynamics to ensure accurate chromosome segregation. Polo kinase 1 (PLK1) is a highly conserved mitotic kinase and activation of PLK1 requires phosphorylation of the T‐loop. Here we show that phosphorylation of PLK1 T‐loop by CDK1 in conserved Thr214 is essential for temporal control of kinetochore microtubule dynamics and spindle checkpoint signaling. Phosphorylates PLK1 at Thr214 suppresses microtubule dynamics and promotes the stabilization of initial kinetochore‐microtubule attachments in prometaphase. Using fluorescence resonance energy transfer‐based reporter of PLK1 activity at the kinetochore and quantitative analysis of native PLK1 substrate phosphorylation, we show that constitutively phosphorylation of Thr214 reduces inter‐kinetochore tension and turnover of kinetochore microtubules, resulting in increased microtubule attachment errors and a checkpoint dependent mitotic arrest. Therefore, we reason that phosphorylation of PLK1 by CDK1 is essential to ensure the temporal regulation of kinetochore microtubule dynamics and spindle checkpoint control.

Grant Funding Source: DK56292; CA164133
Investigating proximity‐mediated catalysis by a protein kinase: how docking affects MAPK specificity and processivity (802.16)
Authors: Kaoud Tamer , Dalby Kevin
Published: June 2021

To understand how MAP kinases phosphorylate their substrates we previously investigated the mechanism of Ets‐1 phosphorylation by ERK2. Using pre‐steady state kinetic approaches we showed that the interaction between ERK2 and Ets‐1 is transient and that turnover is limited by both phosphorylation and ADP dissociation. Here, we investigate the mechanism of phosphorylation of ATF2 by JNK2 using pre‐steady state kinetics and mutagenesis. We find that JNK2 catalysis is limited by dissociation of ATF2 from JNK2 and that this difference in mechanism has significant implications for both the site‐specificity and processivity of the MAPK. We discuss these results in terms of a general mechanism for substrate turnover by protein kinases.

Grant Funding Source: Supported by NIH GM59802 and Welch Foundation (F‐1390)
Thr‐412 of coronin‐1 is phosphorylated by protein kinase C α (802.23)
Authors: Oku Teruaki , Kaneko Yutaka , Ishii Rie , Toyoshima Satoshi , Tsuji Tsutomu
Published: June 2021

Coronin‐1, a hematopoietic cell‐specific actin‐binding protein, is thought to be involved in phagocytic process through its interaction with actin filaments. The dissociation of coronin‐1 from phagosomes after its transient accumulation on the phagosome surface is associated with lysosomal fusion. We previously reported that 1) coronin‐1 is phosphorylated by protein kinase C (PKC), 2) coronin‐1 has two phosphorylation sites, Ser‐2 and Thr‐412, 3) Thr‐412 of coronin‐1 is phosphorylated during phagocytosis. In this study, we examined which PKC isoforms have influence on phosphorylation of coronin‐1 at Thr‐412 using isotype‐specific PKC inhibitors and short interfering RNAs (siRNAs). The phosphorylation of coronin‐1 at Thr‐412 was blocked by Gö6976, an inhibitor of PKCα/β. This phosphorylation was attenuated by PKCα siRNA, but not by PKCβ siRNA. We next analyzed the intracellular distribution of coronin‐1 in Gö6976‐treated HL60 cells during phagocytosis. The confocal fluorescence microscopic observation showed that coronin‐1 was not dissociated from phagosomes. These results indicate that phosphorylation of coronin‐1 at Thr‐412 by PKCα regulates its interaction with actin filaments and intracellular distribution during phagocytosis.
RNA interference of MRCK‐1 in Caenorhabditis elegans to explore oxidative stress responses (802.26)
Authors: Robben Michael , Erickson Patti
Published: June 2021

The Caenorhabditis elegans myotonic dystrophy kinase‐related, Cdc42 binding kinase (MRCK‐1) is activated by the Rho GTPase CDC‐42 (cell division control protein 42), which contains a conserved redox‐sensitive sequence motif on the phosphoryl binding loop. If reactive oxygen species (ROS) can activate CDC‐42, MRCK‐1 may be required to elicit a downstream response to oxidative stress. To test this, mrck‐1 expression was targeted using two RNA interference (RNAi) constructs in transgenic lines of C. elegans that produce green fluorescent protein (GFP) driven by redox‐sensitive promoters. After heat‐shock and chemically‐induced oxidative stress, GFP expression levels were quantified using epifluorescent microscopy. Preliminary data from heat‐shock protein reporter strain CL2070 (hsp‐16.2:GFP) indicate no significant GFP expression changes in the RNAi‐treated worms before or after heat stress, but further studies with different oxidative stressors and reporter strains are underway. If reduction of mrck‐1 is found to affect oxidative stress responses, a new mechanism for prevention of degenerative diseases and aging could be suggested.

Grant Funding Source: Supported by the Henson Undergraduate Research fund.
Identification of Gα12‐specific binding determinants in AKAP‐Lbc (802.8)
Authors: Cavagnini Kyle , Martin Joseph , Brawley Douglas , Meigs Thomas
Published: June 2021

The Rho family of small GTP‐binding proteins are key regulators of cytoskeletal dynamics and cell growth. Rho activation is triggered by guanine nucleotide exchange factors (RhoGEFs), and a specific subset of these proteins, including leukemia‐associated RhoGEF (LARG), are stimulated by trimeric G protein α subunits of the G12/13 class. This interaction is mediated by a regulator of G protein signaling (RGS) homology domain common to these RhoGEFs; however, a RhoGEF lacking an RGS homology domain, termed AKAP‐Lbc, is also activated by the G12/13 class in a signaling pathway required in cardiac development. To identify AKAP‐Lbc regions involved in Gα12 binding, we compared AKAP‐Lbc to other Gα12 targets and found a C‐terminal region with weak homology to axin. A GST‐fusion of this region co‐precipitated Gα12 in constitutively active and inactive forms, in contrast to the activation‐dependent interaction of Gα12 with LARG. In addition, binding experiments utilizing Gα12 and Gα13 harboring a common epitope tag revealed this AKAP‐Lbc domain as preferential for Gα12 interaction, whereas the RGS homology domain of LARG bound Gα13 with greater affinity than Gα12. These findings reveal a novel, Gα12‐specific mechanism for engagement of a non‐RGS RhoGEF by the G12/13 class of trimeric G proteins.

Grant Funding Source: Supported by the GlaxoSmithKline Foundation and Lineberger Comprehensive Cancer Center
An adapted education program to increase adherence to the dietary guidelines for Americans: a feasibility study (807.1)
Authors: McGee Bernestine , Johnson Glenda
Published: June 2021

The National Institutes of Health (NIH) We Can! (Ways to Enhance Children's Activity & Nutrition) was culturally tailored in order to increase adherence to the Dietary Guidelines for Americans, as the vehicle for preventing childhood overweight and obesity in Louisiana families.

We Can! Louisiana is an educational program aimed at promoting adherence to the healthful food and physical activity recommendations set forth in the U.S. Dietary Guidelines for Americans and the resulting effect on reducing weight gain and the risk factors for obesity‐related chronic disease in parents and children in rural Louisiana communities.

The education program designed with input from representatives of the target population including time and location of classes and selection of the healthy lifestyle promoter from the community, included topics gathered from the focus groups that were historical, social, cultural, and environmental in nature that influence health behavior of the target population. The We Can! Louisiana weekly parent sessions included food preparation instructions, physical activity and a period for participants to discuss progress and challenges in following recommended child feeding principles, and developing self‐efficacy and social support in meeting the U.S. Dietary Guidelines for Americans recommendations. A gardening component was included to encourage adoption of the U.S Dietary Guidelines for Americans. The biweekly sessions were held with the children to reinforce the parental instructions. Parents and children demonstrated sustained interest in weekly nutrition and physical activities and offered suggestions for making the program interesting.

Grant Funding Source: USDA/ARS project #58‐6251‐8‐041
Chefs Move to Schools: a pilot study of the influence of outside chefs on school lunchroom behavior (808.11)
Authors: Hanks Andrew , Just David , Wansink Brian
Published: June 2021

Objective: Since the announcement of the Chefs Move to School program, little research has been done to document its impact on student behaviors. The objective of this study was to examine how a professional chef’s dish affects student behaviors in school lunchrooms.

Methods: A high school in Western New York invited a local chef to use school cafeteria ingredients to create an entrée compliant with school lunch regulations. On a pre‐determined Thursday, the chef agreed to prepare three types of pizzas that would be served with the other standard entrées. In this school, pizza was Thursday’s main entrée. Lunch sales and consumption data were collected for 370 students on days when the standard pizza was served and on the one day when the chef‐created pizza was served. Before‐after analyses were utilized to document differences in lunch sales and consumption.

Results: High school students were 18% more likely to select the pizza prepared by the chef (p=0.022). Interestingly, among students who purchased lunch at least once during the study days, there was a 20% increase in the chance these students purchased a school lunch (p<0.001), suggesting an increased interest among these students. In terms of consumption, 68% of students ate their whole entrée and these students ate nearly 17% more vegetables (p=0.02) when the chef’s entrées were served. Moreover, these students also ate 13% more of their snacks (p=0.011) when the ne entrées were offered.

Conclusions and Implications: This study provides evidence that guest chefs can increase participation among high school students in the school lunch program. In addition, students may eat a healthier meal, though particular attention should be paid to potentially less nutritious items students enjoy eating with the new dish.
Linking infant iron status, brain function, and looking behavior during a memory task (810.36)
Authors: Hammons Julie , Canfield Richard , Robertson Steven , Haas Jere
Published: June 2021

No definition of iron deficiency (ID) exists for infants under 12 mo, but young infants (4‐6 mo) are at risk of anemia and depleted iron stores. Nutritional insults during peak brain development (late gestation thru early infancy) may affect long‐term memory. Previous human studies used global cognitive measures, and cannot link ID to impaired memory via specific brain abnormalities. This study examined the links between iron status (hemoglobin (Hb), ferritin, transferrin receptor and body iron), memory function, and brain activity in 40 infants, aged 4‐6 mo. Memory performance was assessed via a Paired Comparison (PC) task and brain function via a similar task with synchronous electroencephalography (EEG). Mixed and logistic regression models were used to estimate the effect (OR) of iron status on 1) amplitude and latency of EEG peaks related to memory; 2) novelty preference and look duration in the PC. Covariates include quality of mother‐infant interactions and infant age, gender, and SES. Nearly 10% of infants were anemic (Hb <110 g/L) and over 70% had mild anemia (Hb 110‐120 g/L). Over 60% of infants showed novelty preference in the PC. This work will inform a definition of ID in infants <12 mo.

Grant Funding Source: Supported by the USDA/NIFA (Hatch# 2012‐13‐228) and NSF Graduate Research Fellowship (JEH)
Quality evaluation of noodles produced from wheat, pigeon pea (Cajanus cajan) and carrot flour blend (813.8)
Authors: Banjoko Immaculata , Oyelola Taiwo , Kujore Fowotade , Banjoko Oludotun , Adebowale Folakemi , Ajiboshin Isaac
Published: June 2021

Noodles are becoming an increasingly popular meal among the young and old in Nigeria and other developing countries of the world. There is therefore a need to utilize some less known crops native to the environment in the production.

The noodles were produced from a blend of pigeon pea flour, carrot flour, mixed in different proportion with a constant amount of wheat flour as sample A (100% wheat) as control, sample B (60% wheat, 20% pigeon pea, 20% carrot flour) Sample C (60% wheat, 30% pigeon pea, 10% carrot flour) sample D (60% wheat, 10% pigeon pea, 30% carrot flour) were subjected to the following analysis: proximate composition, functional analysis, cooking qualities and sensory evaluation. The result showed that sample A (control) contained: carbohydrate (80.42%) protein (2.18%) crude fiber (2.0%) ash (2.4%) moisture (8.2%), water absorption capacity (0.9%), swelling capacity (1.01%) and bulk density (0.7%) in comparison with other sample result shows that sample D has the highest amount of carbohydrate (80.23%) close to that of sample A and the least amount of protein (4.27%) but slightly higher than that of sample A, it also has least amount of ash content (0.4%), crude fiber (2.6) and moisture content (9.0%) sample C has the highest amount of protein (13.13%) least amount carbohydrate (51.37%) highest amount of fat (16.6%) much higher than that of sample A highest amount of ash and also the highest amount of moisture. Sample B has the highest amount of crude fiber (2.8%) high amount of protein (11.81%) which is next to sample C high amount of fat (13.4%) moisture (11.6%) carbohydrate (57.59%) and ash (2.8%) next to sample C in value. Sample A is most acceptable of all the samples followed by sample C while Sample D is the least acceptable of the samples which is due to high proportion of carrot flour contained responsible for the colour of the overall acceptance.
Nutriose®FM as a dietary fiber source in dog diets (814.1)
Authors: Panasevich Matthew , Rossoni Serao Mariana , de Godoy Maria , Swanson Kelly , Guérin‐Deremaux Laetitia , Lynch Gary , Fahey George , Dilger Ryan
Published: June 2021

Dietary fermentable fiber is known to benefit intestinal health of companion animals. Nutriose®FM (NU), a soluble corn fiber dextran, was evaluated for in vitro fermentablity, in vivo nutrient digestibility, and fecal fermentation end‐products. Nutriose®FM was subjected to in vitro fermentation using dog fecal inoculum, with fermentative outcomes measured at 0, 3, 6, 9, and 12 h. In the in vivo experiment, ten female dogs (6.36 ± 0.17 yr; 22 ± 2.1 kg) received 5 diets with graded concentrations of NU [0, 0.5, 0.75, 1.0, or 1.25% (as‐is basis)] replacing cellulose in a replicated 5 x 5 Latin square design. Fresh fecal samples were collected to measure pH and fermentation end‐products. In the in vitro fermentation experiment, NU elicited increased (P < 0.05) concentrations of short‐chain fatty acids through 12 h, with acetate, propionate, and butyrate reaching peak concentrations of 1,803, 926, and 112 µmol/g DM, respectively. In comparison to cellulose and pectin, fermentability of NU was higher (P < 0.05) than cellulose, but lower (P < 0.05) than pectin. Few changes in nutrient digestibility, or fecal fermentation end‐products and consistency were observed in the in vivo experiment, suggesting no appreciable alteration of the colonic microbiota. Overall, NU appears to be fermentable in vitro; however, more research is needed to better understand in vivo fermentability of this substrate.
Gene‐specific changes in DNA methylation in response to chronic folic acid supplementation in normal weight and obese women of child‐bearing age (817.3)
Authors: Park Hea Jin , Shade Deanna , Hausman Dorothy , Hohos Natalie , Meagher Richard , Kauwell Gail , Smith Alicia , Bailey Lynn
Published: June 2021

Folate plays an important role in one‐carbon metabolism including DNA methylation. We reported a lower acute pharmacokinetic response to folic acid (FA) in obesity. Both maternal folate deficiency and obesity are associated with increased risk for neural tube defects (NTDs). However, the impact of folate or obesity on DNA methylation of NTD‐related genes remains unclear. We provided 800 ug/d of FA to healthy normal weight (NW; BMI 18.5‐24.9 kg/m2;n = 12) and obese (BMI > 30 kg/m2; n = 6) women (18‐35y) for 8‐wks and determined changes in DNA methylation in CD 16+ cells, isolated from blood using magnetic bead technology. DNA methylation was examined across 2107 CpG sites in 91 NTD‐ and folate pathway‐ related genes using the Infinium Human Methylation450 Bead Chip. For each CpG site, the proportion of DNA methylation that changed over the supplementation was evaluated by fitting a separate linear model. The analysis indicated changes in 57 vs. 101 CpG sites (36 vs. 47 genes) following the supplementation in NW and obese women, respectively. Among them, 68% vs. 76% of the CpG sites (26 vs 37 genes) decreased in methylation. These data suggest that DNA methylation in NTD‐ and folate pathway‐related genes changed in response to FA supplementation and that the changes were different in NW and obese women. Specific pathways influenced by folate status and exhibiting a differential response in obesity are being explored.

Grant Funding Source: Funded in part by HATCH #GEO00706, #GEO00707 and the Obesity Initiative at the University of Georgia
Differential regulation of mammalian tRNA synthetase genes by amino acid availability (818.7)
Authors: Shan Jixiu , Tang Tiffany , Han Jaeseok , Kaufman Randal , Kilberg Michael
Published: June 2021

A low protein diet or one deficient in an essential amino acid (AA) triggers wide‐ranging changes in feeding behavior and gene expression. Mammalian cells have evolved capabilities to cope with this nutritional stress. Activation of either GCN2 (AA limitation) or PERK (ER stress) eIF2 kinases leads to a suppression of global protein synthesis but a paradoxical increase in the translation of selected mRNA species, among them is activating transcription factor 4 (ATF4). Elevated expression of ATF4 protein increases the transcription from a large number of genes involved in a wide spectrum of cellular functions. During ER stress, gene expression profiling showed that ATF4 interacts with another bZIP transcription factor, CHOP, to induce genes associated with protein synthesis, among them were selected tRNA synthetases. In this study, we have quantified the differential expression of all of the genes for the cytoplasmic and mitochondrial localized tRNA synthetases in both wild type and ATF4 KO MEFs in response to AA deprivation. About one‐half of the genes for the cytoplasmic synthetases were up‐regulated and most required ATF4, but only a few of the mitochondrial synthetases were modestly increased and those were largely independent of ATF4. Genomic analysis of the ATF4‐dependent genes revealed the contribution of a C/EBP‐ATF response element (CARE) sequence.

Grant Funding Source: Supported by NIH: DK092062, DK094729 (MSK); HL057346, DK042394, DK088227, HL052173, DK093074 (RJK)