bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification of MBP‐ and Strep‐tag II proteins
Authors: Heijbel Anna , Carlsson Marianne , Karlsson Anneli
Published: June 2021

Recombinant tagged proteins can be purified to high purity in one step using affinity chromatography. The tag can either be a peptide or a protein, and the choice of tag depends on target protein characteristics and the aim for purifying the protein. Two new chromatography media are presented, StrepTactinTM SepharoseTM High Performance that utilizes that the small 8 amino acid peptide, Strep(II)‐tag, has high affinity for the Strep‐tactin ligand. Dextrin Sepharose High Performance, on the other hand is based on the protein tag, Maltose binding protein (MBP, Mr: ~42 000), which binds specific to carbohydrates (e.g. dextrin). The specificity of the two media towards their respective tag gives high purity of the target proteins.

In this study, simple protein purifications on ÄKTAdesignTM chromatography systems using MBPTrapTM HP (prepacked with Dextrin Sepharose High Performance) and StrepTrapTM HP, (prepacked with StrepTactin Sepharose High Performance) are presented. Purifications were also scaled‐up giving equivalent results. Robustness was tested by running six repeated purifications of a Strep(II)‐tagged protein expressed in E. coli on the same StrepTrap HP column including regeneration with 0.5 M NaOH between each run. No tendencies in reduction of final purity and yield of the target protein were observed, demonstrating the high reproducibility during repeated usage of StrepTrap HP.

In summary, the results show the ease of using prepacked StrepTrap HP and MBPTrap HP columns together with chromatography systems. Equivalent results are obtained when scaling up showing the simplicity to purify larger quantities of pure target protein. Results also show that StrepTrap HP can be efficiently regenerated repeated times with 0.5 M NaOH without losing binding capacity.
Midbody targeting of the ESCRT machinery by a noncanonical coiled coil in CEP55
Authors: Lee Hyung Ho , Elia Natalie , Ghirlando Rodolfo , Lippincott‐Schwartz Jennifer , Hurley James H.
Published: June 2021

The ESCRT (endosomal sorting complex required for transport) machinery is required for the scission of membrane necks in processes including the budding of HIV‐1 and cytokinesis. An essential step in cytokinesis is recruitment of the ESCRT‐I complex and the ESCRT‐associated protein ALIX to the midbody (the structure that tethers two daughter cells) by the protein CEP55. Biochemical experiments show that peptides from ALIX and the ESCRT‐I subunit TSG101 compete for binding to the ESCRT and ALIX‐binding region (EABR) of CEP55. We solved the crystal structure of EABR bound to an ALIX peptide at a resolution of 2.0 angstroms. The structure shows that EABR forms an aberrant dimeric parallel coiled coil. Bulky and charged residues at the interface of the two central heptad repeats create asymmetry and a single binding site for an ALIX or TSG101 peptide. Both ALIX and ESCRT‐I are required for cytokinesis, which suggests that multiple CEP55 dimers are required for function.
SNARE‐mediated homotypic fusion in Golgi apparatus ribbon formation
Authors: Ritter Alex Taylor , Linstedt Adam , Sengupta Debrup
Published: June 2021

The ribbon‐like structure of the Golgi apparatus is thought to be the end result of homotypic fusion of cisternal membranes of ministacks, which are pre‐Golgi intermediate structures. Organization of ministack compartments for fusion is facilitated in part by the protein GRASP65 that links homotypic compartments of the ministacks and brings them into close contact. This process is called tethering. However, the Grasp65 protein alone is not sufficient to facilitate membrane fusion. Once the membranous compartments of the ministacks are in close proximity, a different set of proteins is believed to fuse the membranes into one continuous, ribbon‐like structure. The identity of these proteins is currently unknown. Soluble NSF attachment receptor (SNARE) proteins mediate membrane fusion in eukaryotic cells. A number of different SNARE proteins have been identified in Golgi membranes including Syntaxin 5, hBet1, Sec22, and Membrin. We believe that these four SNARE proteins along with GRASP 65 are sufficient to drive homotypic membrane fusion, possibly bringing about the fusion step that is required for Golgi ribbon formation. To test this hypothesis, I utilized a non‐Golgi membranous structure to model the activity of the proteins. Mitochondrial outer membranes provide an accessible model membrane to use in this case. I constructed plasmids that target the suspected Golgi SNAREs to the mitochondrial outer membrane. I then expressed these plasmids in HeLa cells along with GRASP 65 and assayed for membrane fusion using Fluorescence Recovery After Photobleaching (FRAP). As a result of my efforts, we are well on our way to determining the role of Golgi SNAREs in membrane fusion. This work was supported by the National Science Foundation's Research Opportunities for Undergraduates Program and the Mellon Institute of Carnegie Mellon University.
Cidea and FSP27: novel proteins associated with triglyceride storage in adipocytes
Authors: Puri Vishwajeet , Ranjit Srijana , Perugini Richard A , Czech Michael P
Published: June 2021

Lipid droplets are now been characterized as organelles existing in most types of cells and tissues of higher eukaryotes. Storage of energy as triglyceride in large adipose‐specific lipid droplets is a fundamental need in all mammals. Recently we found that the Cide domain‐ containing proteins, Cidea and FSP27 (Cidec), are associated with lipid droplets in adipocytes. Expression of Cidea and FSP27 greatly enhance lipid droplet size and neutral lipids when expressed ectopically in COS cells and 3T3‐L1 fibroblasts. Their expression in adipocytes is differentiation dependent, controlled by PPARγ and their depletion causes fragmentation of lipid droplets accompanied by markedly elevated lipolysis. Our results indicate an important role of Cidea and FSP27 in lipid droplet dynamics. Strikingly, in omental and subcutaneous white adipose tissue (WAT) from BMI‐matched obese humans, expression of Cidea, Cidec and well known lipid droplet protein, perilipin, correlates positively with insulin sensitivity. Thus, Cidea, Cidec and Perilipin define a novel, highly regulated pathway of triglyceride deposition in human WAT. The data support a model whereby failure of this pathway results in ectopic lipid accumulation, insulin resistance and its associated co‐morbidities in humans.
Lysosomal sorting of ABC transporter TAP‐like (ABCB9)
Authors: Fujimoto Yasuyuki , Maeda Masatomo
Published: June 2021

TAP‐like (TAPL, ABCB9) is an ATP‐binding cassette (ABC) transporter with similarity to TAP, a transporter associated with antigen processing. Both are half‐type transporters of ABCB subfamily and they localize distinctly in lysosome and endoplasmic reticulum, respectively. Here, we identified a region necessary for lysosomal targeting of TAPL.

PROCEDURES

Various truncated forms of TAPL tagged with GFP were stably expressed in CHO‐K1 cells. Subcellular distributions of the tagged proteins were analyzed by confocal microscopy.

RESULTS

TAPL has ten transmembrane helices followed by conserved ABC region. Amino‐terminal four helices are unique to TAPL. The remaining six helices and the ABC region form the core structure of ABC transporters. The amino‐terminal four helices were revealed to be sufficient for the lysosomal targeting. A truncated protein lacking this region failed in sorting to lysosomes. The latter construct could dimerize, which is essential for half‐type transporters, while the former could not (BBRC, in press). The results demonstrate that TAPL can be dissected into two parts, each required for subcellular localization and transporter machinery.

Supported by JSPS grants (13142206, 18590072).
Effects of Triglyceride‐Rich Lipoproteins on the Inflammatory Potential of Endothelial Cells
Authors: Wang Ying , Wong Maelene Ling , Raymond Nadine , Passerini Anthony , Simon Scott Irwin
Published: June 2021

Atherosclerosis, a chronic inflammatory arterial disease featured by focal accumulation of fat‐laden deposits, is exacerbated by prolonged circulation of elevated levels of triglyceride‐rich lipoproteins (TGRL). This study aims to examine the effects of TGRL on the inflammatory potential of endothelial cells and the mechanisms for enhanced recruitment of mononuclear cells using vascular mimetics. We have developed an ex vivo atherogenesis model, in which human aortic endothelial cells (HAEC) in culture are perfused with postprandial TGRL freshly isolated from high‐fat diet fed individuals under varied shear rates. The expressions of cell adhesion molecules (CAM) are examined by flow cytometry, while the binding and internalization of TGRL are visualized by immunofluorescence. The results show an enhanced CAM upregulation upon TNF‐stimulation in TGRL perfused HAECs. Moreover, a member of low density lipoprotein receptors, LR11 is found involved in TGRL binding and upregulated in TNF‐α stimulated HAECs. We conclude that excessive exposure of endothelium to TGRL primes them for enhanced inflammatory response to cytokines or pro‐athero shear flow profiles. This elevated inflammatory state further enhances the recruitment of TGRL, thus revealing a positive feedback loop for metabolically driven development of atherosclerosis.

This study is supported by NIH RO1 HL 082689 to S.I.S.
C/EBPbeta knockdown prevents fatty acid induced inflammatory gene progression, macrophage infiltration and insulin resistance in vivo and in vitro
Authors: Rahman Shaikh Mizanoor , Janssen Rachel , de la Houssaye B.A. , Friedman Jed
Published: June 2021

Obesity is associated with chronic, low grade inflammation, abnormal cytokine production, macrophage infiltration into adipose tissue, and insulin resistance. CCAAT/enhancer binding protein‐beta (C/EBPb) is a transcription factor that plays a key role in hepatic steatosis, inflammation, and ER stress. Here we show that global C/EBPb gene deletion prevented high fat diet (16 wks) induced increases in adipose tissue inflammatory genes TNFa, MCP1, and IL6 along with macrophage marker gene CD68. Immunohistochemistry with F4/80 revealed absent macrophage infiltration into adipose tissue of C/EBPb KO mice. Moreover, C/EBPb KO mice had increased mRNA expression of anti‐inflammatory adiponectin and superoxide dismutase (SOD) in adipose tissues. In RAW 264.7 macrophage cells and in 3T3L1 mature adipocytes, palmitate treatment robustly increased C/EBPb and inflammatory genes, while siRNA mediated knock down of C/EBPb prevented palmitate mediated induction of C/EBPb, MCP1, and IL6 and loss of insulin stimulated AKT activation and PI3‐kinase activity. These results suggest that C/EBPb plays a crucial role in dietary fat induced inflammation and insulin resistance in vivo and in vitro. Thus, C/EBPb might be an important therapeutic target for prevention of obesity induced inflammation, insulin resistance and macrophage infiltration. Funded by NIH DK059767 & DERC P&F (University of Colorado Denver).
A Phospholipid Substrate Molecule Residing in the Membrane Surface Me‐diates Opening of the Lid Region in Group IVA Cytosolic Phospholipase A2
Authors: Burke John Edmund , Hsu Yuan‐Hao , Woods Virgil L , Dennis Edward A
Published: June 2021

The Group IVA phospholipase A2 (GIVA PLA2) associates with natural membranes in response to an increase in intracellular Ca2+, or increases in certain lipid mediators. Employing deuterium exchange mass spectrometry (DXMS), we have identified the regions of the protein binding the lipid surface, and conformational changes upon a single substrate binding in the absence of a lipid surface. Lipid binding produced both increase and decreases in exchange. The regions with decreased exchange included the C2 domain, and a charged patch of residues on the catalytic domain. The regions with an increase in exchange are all located near the lid and hinge regions from 403‐457. Using the GIVA PLA2 irreversible inhibitor methyl‐arachidonyl flourophosphonate (MAFP) we were able to isolate structural changes caused only by pseudo‐substrate binding. The changes seen in the catalytic domain are the same with substrate and inhibitor, which implies that most of the changes are due to a substrate mediated, not interface mediated, lid opening which exposes the active site to water. Finally experiments with inhibitor plus phospholipid vesicles showed decreases at the C2 domain as well as charged residues on the putative membrane binding surface of the catalytic domain revealing the binding sites of the enzyme to the lipid surface. This represents a new methodology to study membrane associated proteins.
V‐ATPase/small GTPase/aldolase complex and regulation of endosomal/lysosomal protein degradative pathway
Authors: Merkulova Maria , Hurtado‐Lorenzo Andres , Brown Dennis , Ausiello Dennis , Marshansky Vladimir
Published: June 2021

V‐ATPase‐driven acidification is crucial for regulation of endosomal/lysosomal protein degradative pathway. Recently we demonstrated that the V‐ATPase has a novel function as endosomal pH‐sensor which scaffolds ARNO and Arf6 small GTPases. Using the combination of pull‐down experiments and mass‐spectrometry analysis, here we demonstrated that ARNO's protein‐protein interaction network also includes: i) a1‐, a3‐ and a4‐isoforms of V‐ATPase; ii) enzymes of glycolytic pathway, iii) adaptors; iv) coats and v) cytoskeletal proteins. Immunoprecipitation experiments demonstrated that ARNO specifically interacts with aldolase, but not with other enzymes of glycolytic pathway. The direct interaction of aldolase with ARNO was further confirmed in pull‐down experiments with recombinant proteins. Moreover, additional experiments demonstrated that specific interaction of aldolase with ARNO is taking place via its PH‐domain. Thus using the combination of proteomic approaches we uncover the direct interaction of ARNO with aldolase, the crucial enzyme of glycolytic pathway and modulator of V‐ATPase. Our data indicate on novel regulatory role of ARNO in function of aldolase and potentially in assembly/disassembly of V‐ATPase. The cell biological significance of the interaction between V‐ATPase, ARNO and aldolase in regulation of endosomal/lysosomal protein degradative pathway is further discussed.
Large‐scale Detection of O‐GlcNAc Modification Sites using Lectin Chromatography and Electron Transfer Dissociation Mass Spectrometry
Authors: Chalkley Robert , Thalhammer Agnes , Schoepfer Ralf , Burlingame A L
Published: June 2021

O‐GlcNAc modification is a transient, regulatory post‐translational modification of serine and threonine residues in nuclear and cytoplasmic proteins. It has been shown to have roles in cell regulation in response to nutritional state and cellular stress. The modification is poorly understood, partly due to the difficulty in detecting and identifying sites of modification by proteomic approaches.

Levels of O‐GlcNAc modification are known to be high in the brain. In this study we demonstrate how the combination of lectin weak‐affinity chromatography and subsequent analysis by electron transfer dissociation mass spectrometry has allowed identification of large numbers of O‐GlcNAc modification sites in post‐synaptic density preparations, doubling the number of known sites in the literature. Several of the identified sites of O‐GlcNAc modification have also been observed in a phosphorylated state, further supporting the proposition that these two modifications may regulate each other.

This work is supported by NIH NCRR grant RR001614, the Wellcome Trust and the Biotechnology and Biological Sciences Research Council.
Phosphorylation by CDK1 and JNK1 regulate the DNA binding activity of transcription factor Sp1 during mitosis
Authors: Chuang Jian‐Ying , Hung Jan‐Jong
Published: June 2021

The transcription factor Sp1 is ubiquitously expressed in different cells and thereby regulates the expression of genes involved in many cellular processes. This study reveals that Sp1 was phosphorylated at threonine 739 by JNK and CDK1 and not co‐localized with the chromatin at the beginning of mitotic stage. EMSA, DNA affinity precipitation assay, and ChIP assay were performed and revealed that during mitosis the DNA‐binding ability of phosphorylated Sp1was decrease as well as dephosphorylation of Sp1 by phosphatase restored the DNA binding affinity. In addition, the phosphorylation of Sp1 during mitotic stage also suppressed Sp1 to interact with p300, Brg1, and RNA polII and reduced the transcriptional activity of Sp1 target genes. Moreover, in late mitosis, the Pin1 interacted with the high phosphorylation of Sp1 and recruited the PP2A to dephosphorylate Sp1. In Pin1 knockout MEF cells, there was a delay in the relocalization of Sp1 to the nucleus during telophase. Taken together, our results indicated that Sp1 interacts with JNK and CDK1, followed by phosphorylation of Sp1 at early mitosis. Sp1, then, is disassociated from DNA and loses interaction with co‐activator. In late mitosis, Pin1 and PP2A are recruited by phosphorylation of Sp1, leading to the enhancement of the DNA binding‐ability and protein interactional function of Sp1.
Putative Cross‐Talk Among Posttranslational Modifications (PTMs) of Human IRS‐1
Authors: Ball Lauren Elizabeth , Klein Amanda Leigh , Buse Maria G , Berkaw Mary Neale
Published: June 2021

The purpose of this study is to interrogate the functional relevance of O‐GlcNAc modification of insulin receptor substrate‐1 (IRS‐1) on insulin/IGF‐1 receptor signaling. The interactions of IRS‐1 with binding partners are modulated by many S/T kinases and may be further modulated by O‐GlcNAc glycosylation. To identify the sites of O‐GlcNAc modification and putative cross‐talk among PTMs on this highly phosphorylated adaptor protein, IRS‐1 was expressed in HEK293 cells in the presence of the O‐GlcNAcase inhibitor, PUGNAc (50 μM). The protein was enriched by Ni/NTA chromatography, gel purified, reduced and alkylated with iodoacetamide, and proteolytically digested. Peptides were analyzed by LC‐MS/MS with an LTQ ion‐trap MS equipped with ETD (Thermo). LC‐MS/MS of IRS‐1 yielded 80% sequence coverage of the 131 kDa protein and revealed 3 O‐GlcNAc modified peptides. All of the glycopeptides were S/T phosphorylated, indicating that these modifications are not mutually exclusive. One site of O‐GlcNAc modification occurred adjacent to an SH2 binding motif. Analyses are ongoing to elucidate the temporal dynamics of these modifications and their impact on insulin and IGF‐1 signaling.
GIV is a Non‐Receptor GEF for Gαi with a Unique Motif that Regulates Akt Signaling
Authors: Garcia‐Marcos Mikel , Ghosh Pradipta , Ear Jason , Farquhar Marilyn Gist
Published: June 2021

Heterotrimeric G proteins are molecular switches that control signal transduction. Ligand‐occupied G‐protein coupled receptors serve are the canonical guanine nucleotide exchange factors (GEFs) that activate heterotrimeric G proteins. A few unrelated non‐receptor GEFs have also been described, but little or nothing is known about their structure, mechanism of action or cellular functions in mammals. We have discovered that GIV/Girdin serves as a non‐receptor GEF for Gαi through an evolutionarily conserved motif that forms a unique interface with the Gα‐subunit. Using insights from the 3‐dimensional structure of this interface, we demonstrate that this GEF motif enhances Akt signaling via the G ??‐PI3K pathway and that mutational disruption of the Gαi‐GIV interface inhibits Akt enhancement and cell migration in cancer cells. Recently we also found that GIV is a metastasis‐related protein in carcinomas by virtue of its ability to promote unrestricted Akt activation. Thus, the novel regulatory motif described here provides the structural and biochemical basis for the pro‐metastatic features of GIV, making the functional disruption of the Gαi‐GIV interface a promising target for therapy against cancer metastasis.

Supported by NIH grants CA100768 and DKI7780 to M.G.F, by Basque Government Postdoctoral fellowship BFI06.300 and Susan G. Komen Postdoctoral fellowship KG080079 to MG‐M and by training grant NIH/T32 DK07202 and a Research Scholar Award from American Gastroenterology Association (AGA) to PG.
Nuclear receptor ligands LG268 and rosiglitazone inhibit collagen destruction by chondrocytic cells stimulated with interleukin‐1 beta
Authors: Schmucker Adam , Burrage Peter , Brinckerhoff Constance
Published: June 2021

Matrix metalloproteinase‐1 (MMP‐1) and MMP‐13 mediate cartilage destruction in arthritis. Our objective was to evaluate the inhibition of IL‐1β‐induced MMP‐1 and ‐13 expression in SW1353 chondrocytic cells by retinoid x receptor (RXR) ligand LG100268 (LG268), and peroxisome proliferator‐activated receptor gamma (PPARγ??ligand rosiglitazone (rosi).

We pretreated SW1353 cells with LG268 and/or rosi, then added IL‐1ß (1 ng/ml). MMP‐1 and ‐13 hnRNA and mRNA was measured by realtime RT‐PCR, and cells were embedded in collagen to measure collagenolysis. Chromatin immunoprecipitation (ChIP) detected PPARγ?and acetylated histone H4 (AcH4) at the Activator Protein‐1/PPARγ response element (AP‐1/PPRE) in the MMP‐1 and ‐13 promoters. Modification of PPARγ and RXR by small ubiquitin‐like modifier (SUMO) was measured by IP and Western blot.

LG268 and rosi reduced MMP‐1 and ‐13 mRNA, AcH4 at the AP‐1/PPRE, and collagen destruction, and combined rosi/LG268 treatment had an additive effect. Treatment with rosi or LG268 led to SUMOylation of PPARγ and RXR, respectively, and induced "cross SUMOylation" of the other partner in the PPARγ:RXR heterodimer. Unexpectedly, ChIP indicated increased PPARγ at the AP‐1/PPRE in IL‐1β‐treated vs. untreated cells, while rosi and LG268 had little effect.

LG268 and rosi inhibit IL‐1ß‐induced MMP‐1 and ‐13, and reduce collagen destruction in vitro, likely by multiple mechanisms.

Funding Support

ACS: T32‐AR‐07576; PSB: T32‐AI‐07363; ACS, PSB, CEB: NIH ‐AR‐26599, 2.
MLL2 histone methylase in estrogen dependent trans‐activation of high density lipoprotein receptors
Authors: Mandal Subhrangsu S , Ansari Khairul I
Published: June 2021

The estrogen receptors (ERs) are nuclear hormone receptors that are activated by estrogen and play critical roles in mediating hormonal effects in reproductive tissues, cardiovascular functions and various human diseases. Mixed Lineage Leukemias (MLL) are histone methyl‐transferases (HMTs) that post‐translationally methylate histone proteins and play key roles in gene expression and epigenetic regulation in human. Recently, we demonstrated that histone methylase MLL2, in coordination with ERs, play critical roles in estrogen dependent trans‐activation of high density lipoprotein receptor (HDLR) genes that maintain blood cholesterol level. Knock down of histone methylases MLL2 down regulates the estrogen dependent trans‐activation of HDLR genes. Herein, we will present the mechanism by which MLL2 regulate HDLR genes in an estrogen dependent manner.
Role of Alpha Actinin4 in Transcriptional Regulation
Authors: Khurana Simran , Chakraborty Sharmistha , Liu Yu , Su Yu‐Ting , Lam Minh , Bruggeman Lesilie A , Saleem Moin A , Mathieson Peter W , Kao Hung‐Ying
Published: June 2021

Alpha actinins (ACTNs) are known for their ability to modulate cytoskeletal organization and cell motility by crosslinking actin filaments. However, recently it has been shown that the ACTNs can also play an important role in transcriptional regulation. ACTN4 mutations including K228E, T232I and S235P are linked to familial focal segmental glomerulosclerosis (FSGS), a kidney disease characterized by proteinuria. The mechanisms by which these mutations in ACTN4 are associated with FSGS are not well defined. We have demonstrated here that ACTN4 potently activated transcription by nuclear receptors including the retinoid acid receptor (RAR), the vitamin D3 receptor (VDR) and peroxisome proliferator‐activated receptor (PPARγ). ACTN4 physically interacted with nuclear receptors in a hormone dependent manner and mutation in the nuclear receptor interacting motif disrupted its ability to stimulate transcription. Furthermore, FSGS‐linked ACTN4 mutants localized aberrantly and failed to potentiate transcriptional activation by nuclear hormone receptors. Our data demonstrate that the inability of FSGS to activate transcription by nuclear receptors correlates with their aberrant cytoplasmic localization and their inability to interact with nuclear receptors.
Identification of activated receptor tyrosine kinases that are important for motility, invasion, colony formation and proliferation in human osteosarcoma
Authors: Rettew Ashley , Getty Patrick J , Greenfield Edward M
Published: June 2021

The prognosis for osteosarcoma (OS) remains poor, with 20‐30% of patients succumbing to pulmonary metastasis. Therefore, novel therapeutic agents are needed. In this study, we first screened for activated receptor tyrosine kinases (RTKs) in highly metastatic LM7 and 143B human osteosarcoma cell lines using a phospho‐RTK antibody array. In the LM7 cells, 12 of the 42 RTKs on the array were phosphorylated. Nine of the 12 RTKs were also phosphorylated in the 143B cells. siRNAs targeting the 12 RTKs found to be activated in the arrays were screened to identify the RTKs that are important for motility, invasion, colony formation and/or proliferation in OS cells. In 143B cells, Eph kinase B2 (EphB2) knockdown resulted in >85% inhibition of motility, invasion, colony formation and proliferation compared to cells treated with Luciferase control siRNA. Colony formation was also reduced by 70% in LM7 cells treated with EphB2 siRNA. Knockdown of EphA4 inhibited all 4 assays by >39% in the 143B cells. In the LM7 cell line, IGF‐1R silencing inhibited motility by 85%, colony formation by 58% and proliferation by 32%. Overall, EphB2, EphA4 and IG‐1R were activated in the metastatic cell lines and knockdown of these tyrosine kinases had the greatest effect on motility, invasion, colony formation and proliferation. These results indicate that EphB2, EphA4 and IGF‐1R may be valuable targets for the treatment of OS.
Regulation of the epidermal growth factor receptor intracellular domain
Authors: Choi Sung Hee , Lemmon Mark A
Published: June 2021

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase comprised of extracellular, transmembrane, and intracellular domains. The intracellular domain contains a juxtamembrane region (JM), a tyrosine kinase domain (TKD), and a C‐terminal tail. Binding of activating ligands to the EGFR extracellular region induces receptor dimerization, leading to TKD activation through formation of an asymmetric intracellular dimer. We have investigated the role of the JM region and the C‐terminal tail on TKD activation. We expressed the EGFR intracellular domain (E1ICD) harboring various point or deletion mutations in the JM region and the C‐tail using a baculovirus system. The effect of mutations on dimerization and in vitro enzyme activity were investigated using analytical ultracentrifugation and multiple kinase assays. The results point to a role for the JM region in allosteric TKD activation, and for the C‐tail in promoting receptor‐receptor interactions. Using small angle X‐ray scattering (SAXS) and X‐ray crystallography, we have also studied the structural relationship between the JM, TKD and C‐tail regions of the EGFR intracellular domain, and how it may be altered upon EGFR activation. This study was funded by a pre‐doctoral fellowship from the Great Rivers affiliate of the American Heart Association (Award 0715334U).
Structural Basis of Protein Lysine Methylation
Authors: Trievel Raymond
Published: June 2021

Lysine methylation is a prominent covalent modification of histones, transcription factors, and other nuclear proteins. Site‐specific lysine methylation has been implicated in regulating a myriad of genomic processes, including transcription, DNA damage response, X‐chromosome inactivation, and genome stability. In addition to the site of methylation, the lysine epsilon‐amine group can undergo mono‐, di‐, or trimethylation, imparting a hierarchical level of specificity in methyllysine signaling. Both the site and degree of methylation are dynamically regulated through the concerted activities of lysine methyltransferases and demethylases, permitting the fine‐tuning of lysine methylation status in vivo. Structural and biochemical studies of the SET domain family of lysine methyltransferases and the JmjC class of lysine demethylases have furnished insights into the substrate specificities of these enzymes and have yielded a framework for understanding the molecular mechanisms underlying the dynamics of lysine methylation. This research is supported by grants from the National Institutes of Health (GM073839), the University of Michigan's Office of the Vice President for Research, and the Canadian Institutes of Health Research.
Histone and factor covalent modifications in genome regulation
Authors: Berger Shelley L.
Published: June 2021

Genomic structure and function is regulated in part through covalent post‐translational modifications (PTMs) of factors and histones, including acetylation (ac), methylation (me), phosphorylation (ph), ubiquitylation (ub), and sumoylation (su). There are an enormous number of factor and histone PTMs. To make sense of this bewildering complexity, we focus on patterns, temporal sequences, and cross‐talk between PTMs in various models, including in yeast S. cerevisiae and in mammalian cells.

In yeast we focus on dynamic changes in histone PTMs during transcription and other processes. We are trying to unravel the molecular outcomes of these PTMs, as well as their role in complex physiological processes, such as during gametogenesis and during replicative cell aging. We use the genetic approaches available in yeast to determine whether altered function of chromatin may have a direct role in these epigenetic pathways.

In mammals we study PTMs of DNA‐bound transcription factors, using the tumor suppressor and transcription factor p53 as a model. We currently focus on methylation and phosphorylation of specific residues in the N and C termini of p53. We are investigating cross‐talk between these PTMs on p53, and mechanistically‐interrelated PTMs on histones. Such PTM cascades may play a central role in signaling to chromatin.
Stabilization of Reaction Intermediates in the Catalytic Cycle of Nitric Oxide Synthases
Authors: Tejero Jesus , Biswas Ashis , Wang Zhi‐Qiang , Page Richard C. , Haque Mohammed Mahfuzul , Hemann Craig , Zweier Jay L. , Misra Saurav , Stuehr Dennis J.
Published: June 2021

Nitric oxide synthases (NOS) catalyze the synthesis of nitric oxide from L‐Arginine. NOS enzymes have a tryptophan residue that forms a hydrogen bond with the heme‐thiolate and a stacking interaction with the heme. To assess the importance of this residue we replaced it by histidine in inducible NOS. The W188H mutation had relatively small effects on L‐Arg binding and on enzyme heme‐CO and heme‐NO absorbance spectra, but increased the heme midpoint potential by 88 mV relative to wild‐type iNOSoxy. The crystal structure of the mutant showed that the His188 imidazole still stacked with the heme and was in position to form a hydrogen bond with the heme thiolate. Analysis of a single turnover L‐Arg hydroxylation reaction revealed that a new heme species formed during the reaction. Formation of this species required the presence of tetrahydrobiopterin (H4B) and its buildup appears to be coupled with the formation of the bound H4B radical. The decay of the intermediate species correlates with L‐Arg hydroxylation and formation of ferric enzyme.

This indicates that the W188H mutation stabilizes an intermediate heme‐oxy species whose reactivity becomes rate‐limiting for L‐Arg hydroxylation. The conserved Trp residue in NOS may facilitate NO synthesis by tuning heme‐thiolate electronegativity. Additional research to identify the intermediate will be discussed.
Biphasic regulation of cell cycle by nitric oxide donors in promyelocytic HL‐60 cell line
Authors: Kumar Sachin , Barthwal Manoj Kumar , Dikshit Madhu
Published: June 2021

Nitric oxide (NO) regulates a wide array of cell functions, proliferation, cytostasis and apoptosis. Role of NO in cell proliferation is however is less explored, which has been suggested to be dependent on the NO concentration, cell type and intracellular redox status. Present study explores the effect of NO donors [DETA‐NONOate (DETA‐NO) and SNAP] on the promyelocytic HL‐60 cells. NO induced proliferation of HL‐60 cell at lower concentration (1‐100 µM), which was evaluated by Thymidine incorporation, BrdU labelling and cell cycle analysis regardless of any apoptosis, while higher concentration (250µM‐1mM) promoted apoptosis as evident by change in mitochondrial potential, Caspase activation, nick and PI labelling. To investigate further the mechanism involved, expression profile of various CDK/ cyclins including CDK2, CDK6, Cyclin A, Cyclin B, Cyclin D1, p15, p27 was monitored, Expression of Cyclin B, CDK2 was increased at 10‐50 µM by DETA‐NO, while most of these cell cycle regulators were reduced at 1 mM DETA‐NO. This proliferative effect seems to be redox sensitive as effect was abolished by pretreatment of cells with DTT (2mM). NO (10‐50µM) also augmented nitrosylation of various proteins suggesting nitrosylation of important targets. The results obtained thus suggest that NO in a concentration dependent manner modulated cell cycle in the HL‐60 cells.

The work was supported by CSIR and DBT India.
Regulation of catecholamine biosynthetic enzymes by nitric oxide
Authors: Ansell Dominique R. , Crispo J. , Wharton A. , Tai T.C.
Published: June 2021

The neurotransmitters/neurohormones catecholamines (CA) are involved in the sympathetic control of arterial blood pressure and cardiac function. Recent studies show that nitric oxide (NO) can regulate the release of CA from the adrenal medulla. The in vitro PC12 cell line was used to examine the effect of NO on the regulation of the CA biosynthetic enzymes: tyrosine hydroxylase (TH), dopamine‐β‐hydroxylase (DBH) and phenylethanolamine N‐methyltransferase (PNMT). Results obtained from drug treatments with the NO donor, sodium nitroprusside (SNP) constitute an assessment of its effects in PC12 cells. Treatment of cells with SNP for 6 hours significantly increased TH and PNMT mRNA levels. Combination drug studies with SNP and intracellular kinase activators (forskolin, PMA, 8‐Br‐cGMP) and inhibitors for PKA (H‐89), cGMP (6‐anilinoquinone) PKG (DT‐2) and PKC (GF109203X) were also conducted. Increases in transcript levels for TH and PNMT were obtained under combination drug treatments of SNP and activators of PKA and PKG (p<0.05). mRNA transcript levels of TH, DBH and PNMT showed significant decreases when cells were pre‐treated with PKA, PKC and PKG inhibitors. Furthermore, preliminary transfections showed activation of the PNMT promoter by SNP treatment. Results from this study suggest that NO is capable of regulating CA biosynthetic enzymes, TH and PNMT via activation of the PKA and PKG pathways.
The Differential Role of Nitric Oxide Synthases in Ultraviolet Light B‐induced Apoptosis
Authors: liu wei , Wu Shiyong
Published: June 2021

Ultraviolet light (UV) activates nitric oxide synthase(s) (NOSs), which produces nitric oxide (NO). However the involvement of each NOS in UV‐induced signaling is not clear. The role of NO in UV‐induced apoptosis remains controversial. In this study, we analyzed expression and activation of NOSs in UVB‐irradiated keratinocytes HaCaT. We also determined the roles of NOSs in UV‐induced apoptosis. Our data showed that the expression of nNOS was increased and then decreased at 12 h and 24 h post‐UV respectively. While eNOS expression was not changed by UV, a decreased amount of coupled eNOS with an increased amount of uncoupled eNOS was observed within half‐hour of UV‐treatment. There was no increased level of iNOS was detected after UV‐irradiation. These results suggested that UV‐induced NO elevation is mainly regulated by cNOS.

To determine the role of each cNOS and NO in UV‐induced apoptosis, we analyzed the extent of effect of a broad NOS inhibitor N‐nitro‐L‐arginine methyl ester (L‐NAME) on apoptotic death of HaCaT upon UV‐irradiation. Our data showed that NO was increased almost immediately and peaked at 18 h after UV‐irradiation. A pulse inhibition of NOS in the early phase (0‐0.5 h) partially inhibits UV‐induced apoptosis. However, a prolonged inhibition of NOS (0‐18 h) promotes apoptosis after UV‐treatment. In addition, the caspase 3 activity, but not amount, was changed in the treated cells and was correlated with the PARP cleavage and apoptosis of the HaCaT cells. Base on the results, we propose that in the early stage of UV‐irradiation, eNOS is uncoupled and produces O2‐ and promotes apoptosis. In the late stage of UV‐irradiation, the expression of nNOS is increased and the elevated [NO] produced by nNOS inhibits caspase‐3 activity and apoptosis of the irradiated cells.
Chimeras of Nitric Oxide Synthase Engineered to Have Distinct Kinetic Parameters and Product Profiles
Authors: Binder Katherine Emoke , Wang Zhi‐Qiang , Haque Mohammad Mahfuzul , Stuehr Dennis J.
Published: June 2021

The three Nitric Oxide Synthases (NOSs) play vital roles in biological processes through their synthesis of nitric oxide (NO). According to our catalytic model, three parameters determine the catalytic activity and behavior of any mammalian NOS: heme reduction, ferrous‐NO oxidation and its NO dissociation rate from the ferric heme‐NO complex. These parameters vary significantly among the three NOSs, and cause each NOS to distribute differently between an NO‐releasing cycle and an NO dioxygenase cycle during catalysis. We have investigated if NOS changes in a predictable way when the kinetic parameters vary from their natural ranges. We examined how modulating heme reduction rate and a slow ferric‐NO dissociation rate influences NOS catalytic behavior. To accomplish this, we have constructed four NOS chimeras. Two iNOS‐nNOS chimera proteins (iNOS‐nNOS and iNOS‐nNOS S1412D) were constructed to have non‐native heme reduction rates. These chimeras were found to have a fast rate of heme reduction. In addition, we have generated two other NOS chimeras (V346IiNOS‐nNOS and the V346IiNOS‐nNOSS1412D). These two chimeras were found to have a moderate rate of heme reduction and a slower than normal ferric‐NO dissociation rate. How the measured changes in kinetic parameters correlate with measured NO release and NO dioxygenase activities of each chimera will be discussed. Support by NIH CA53914.
PDE4 inhibitors can induce pro‐inflammatory chemokine expression in human macrophages
Authors: Hertz Angie L , Bender Andrew T , Smith Kimberley C , Gilchrist Mark , Amieux Paul S , Beavo Joseph A
Published: June 2021

Monocytes differentiate into macrophages upon exposure to GM‐CSF. Agonists that increase cAMP are known inhibitors of many inflammatory processes in macrophages activated by lipopolysaccharide and other antigens. We find however, that when cAMP is increased during the differentiation process there is a large increase in the mRNA and protein for several pro‐inflammatory chemokines including CXCL 1, 2, 5, and 7 as well as CCL2, and 13. These pro‐inflammatory effects on chemokine expression are mimicked by stimulation of the Epac pathway. One major phosphodiesterase (PDE) controlling cAMP in macrophages is PDE4. Several PDE4 inhibitors are currently in clinical trials for treatment of inflammatory lung diseases like chronic obstructive pulmonary disorder and asthma. We find that inhibition of PDE4 by rolipram potentiates expression of these pro‐inflammatory chemokines. We also find that under these conditions ATF3, a newly identified negative regulator of NF‐kB activation, is decreased by cAMP and that this also is potentiated by PDE4 inhibitors. These data suggest that co‐administration of chemokine receptor or Epac pathway antagonists should improve the efficacy of PDE4 inhibitor therapy.
Elucidating Compound Selectivity: Integration of Chemoproteomic, Protein Signaling, and Gene Expression Profiles
Authors: Warder Scott Evan , McLoughlin Shaun M , Richardson Paul L , Meuth Joseph L. , Jung Paul M , Hubbard Robert D , Hansen Todd M , Oksenberg Donna , Lamerdin Jane , Liguori Michael J , Wang Jieyi , Bell Randy L , Davidsen Steven K , Westwick John K , Rogers John C. , Blomme Eric A
Published: June 2021

Compound profiling technologies that enable an early assessment of cellular effects related to toxicology and pharmacology can contribute to a reduction of late stage failures in drug development. However, the implementation of ‐omic profiling strategies has been hampered by challenges in analysis and interpretation of these information‐rich data sets. To address this issue, we have rationally combined analytical methods that report compound‐protein interactions (Inhibitor Affinity Capture/IAC; protein arrays) with technologies that monitor down‐stream cellular effects on protein signaling (Protein‐fragment Complementation Assays) and gene expression. IAC is a mass spectrometry‐based technique that discriminates tight from weaker binding proteins using a label‐free affinity binning process. Using these orthogonal approaches and pathway analysis tools, we have: 1) provided additional comprehension of mechanisms driving relevant cellular endpoints for marketed oncology drugs; 2) revealed off‐target profiles for several experimental kinase inhibitors; 3) further elucidated mechanisms of toxicity using a series of fluoroquinolone antibiotics. The multiple lines of evidence generated from this integrated technology scheme provide higher confidence decision‐making and improves the quality of compounds that are progressed towards the clinic.
Influence of Anthocyanins on Carotenoid Bioavailability from Purple Carrots
Authors: Arscott Sara Andrea , Simon Philipp W , Tanumihardjo Sherry A
Published: June 2021

Purple carrots contain anthocyanins in addition to the provitamin A (pro‐VA) carotenoids contained in typical orange carrots. Simultaneous consumption of these phytochemicals in carrots may affect the bioavailability of the provitamin A carotenoids. We compared the bioavailability of β‐ and α‐carotene in humans from acute feeding of purple and orange carrots. In this randomized, 3 X 3 crossover intervention study, carrot smoothies were served to healthy subjects (n=5, female, aged 21‐26 y) for breakfast after 1 wk on a low carotenoid diet, overnight fasting and a 2‐wk washout period. Carrot smoothies contained one of three different colored carrots and were equalized to 16.5 mg pro‐VA carotenoids per serving (including trans and 9‐cis β‐carotene and α‐carotene): purple carrots [PC treatment; 12.0 mg β‐carotene (4.0% as 9‐cis‐isomer) and 4.6 mg α‐carotene], orange carrots [OC treatment; 10.9 mg β‐carotene (4.0% as 9‐cis‐isomer) and 5.7 mg α‐carotene], or white carrots as a negative control (WC treatment; no β‐carotene or α‐carotene detected). Blood samples were collected at 0, 1, 2, 4, 6, 8, 12, 16, 24, 32, 72, and 144 h following test meal consumption. Carotenoids were measured in the plasma by HPLC. Baseline‐corrected areas under the concentration vs time curves (AUC) will be used as a measure of absorption. (Supported by Standard Process, Inc., Palmyra, WI).
Retention and bioaccessibility of B‐carotene in cassava (Manihot esculanta, Crantz) are affected by style of cooking
Authors: Thakkar Sagar K. , Huo T. , Maziya‐Dixon B. , Failla M. L.
Published: June 2021

We previously demonstrated that the bioaccessibility of β‐carotene (BC) in boiled cassava from various cultivars is highly correlated with BC content. However, cassava is also prepared by fermentation followed by either boiling (fufu) or roasting (gari). The possible impact of such processing on retention and bioaccessibility of BC was investigated. We first compared retention of BC in boiled cassava, gari, and fufu each prepared from the roots of three cultivars. BC content in unprocessed cultivars was 6‐8 µg/g wet weight and cis isomers accounting for approximately one‐third of total BC. Apparent retention of BC was approximately 90% for boiled cassava and fufu. In contrast, roasting fermented cassava at 195°C for 20 min to prepare gari decreased BC content by 90%. Retention was increased to 63% when roasting temperature was reduced to 165°C for 10 min. Processing also was associated with a decline in all trans BC and concomitant increase in 13‐cis BC. The efficiency of micellarization of all trans and cis isomers of BC during simulated digestion was 25‐30% for both boiled cassava and gari and independent of cultivar, but only 12‐15% for fufu. These differences in retention and bioaccessibility of BC from cassava processed in traditional styles suggest that gari and fufu likely provide fewer retinol activity equivalents than an equivalent caloric intake of boiled cassava. Supported by HarvestPlus and OARDC.
Piceatannol inhibits the migration and invasion of DU145 human prostate cancer cells
Authors: Kwon Gyoo Taik , Song Hye Rim , Kim Jin‐Kyung , Park Jung Han Yoon
Published: June 2021

Piceatannol (trans‐3,4,3′,5′‐tetrahydroxystilbene) is a polyphenol found in grapes, red wine, Rheum undulatum and seeds of Euphorbia lagascae. The purpose of the present study was to investigate the effects of piceatannol treatment on the migration and invasion characteristics of DU145 prostate cancer cells. DU145 cells were cultured in the presence of 0 ‐ 5 μmol/L piceatannol with or without 10 μg/L epidermal growth factor (EGF). Piceatannol inhibited the basal and EGF‐induced cell migration and invasion in DU145 cells. Fibrin zymography and Western blot analysis showed that piceatannol reduced the basal and EGF‐induced expression of urokinase‐type plasminogen activator. Gelatin zymography and Western blot analysis revealed that piceatannol reduces the expression of matrix metalloproteinase‐9. Piceatannol decreased the protein levels of vascular endothelial growth factor, tissue inhibitor of metalloproteinase (TIMP)‐1 but increased TIMP‐2 levels in a concentration‐dependent manner. We have demonstrated that piceatannol markedly inhibits the metastasis potential of DU145 prostate cancer cells.
Soy, isoflavones and prostate cancer risk in men: a meta‐analysis
Authors: Yan Lin , Spitznagel Edward L
Published: June 2021

Soy is a major plant source of dietary protein to humans. Epidemiologic studies show that soy consumption may be associated with a reduction in cancer risk in humans. The purpose of this study was to conduct a meta‐analysis on the association between soy and prostate cancer in men. We systematically reviewed studies obtained through a thorough MEDLINE literature search and identified 15 studies on soy consumption and eight on isoflavone intake in relation to prostate cancer risk. We conducted this analysis using a random‐effects model in which studies with smaller standard error of estimate are given greater weight in the summary measure. Our analysis of studies on soy intake yielded an overall risk estimate of 0.74 (95% confidence interval: 0.63, 0.89; P = 0.001). When separately analyzed, studies on non‐fermented soy foods yielded an overall risk estimate of 0.70 (0.56, 0.88; P = 0.002) and those on fermented ones yielded 1.02 (0.73, 1.42; P = 0.922). The analysis of studies on isoflavones yielded an overall risk estimate of 0.88 (0.76, 1.02; P = 0.085). When analyzed on the basis of daily isoflavone intakes, we found an overall risk estimate of 0.63 (0.41, 0.96; P = 0.03) from studies at mg/day and 0.99 (0.85, 1.16; P = 0.909) from those at µg/day, respectively. Results of this analysis suggest that consumption of soy foods is associated with a reduction in prostate cancer risk in men.