bluuebunny/crossref_metadata_2025 huggingface dataset:
An isoform of actin is associated with the cancer status of ovarian and breast epithelial cells
Authors: Lanner Carita , Hobbs Hailey , Gauthier Nicole , Peltsch Heather , Cutuli Facundo , Boyle Amanda , Naryzhny Stanislav , Lee Hoyun
Published: June 2021

An isoform of actin associated with non‐malignant ovarian and breast epithelial cells was detected in a study investigating cancer specific forms of PCNA (proliferating cell nuclear antigen) in ovarian and breast cancer materials. An antibody (B1 antibody) generated against ten residues from the interdomain connector loop in PCNA, detected not only PCNA, but a novel protein in non‐malignant cells. Immunoprecipitation was performed using the B1 antibody and proteins were cut from gels. In‐gel trypsin digestion was performed for MALDI‐TOF analysis. The novel protein was identified as a form of actin using Mascot peptide fingerprinting software. Immunoblotting with actin antibodies support the identification of the B1 protein as an isoform of actin. A local alignment between PCNA and beta‐actin was performed and found a ten‐residue area of overlap with 50% homology between PCNA and beta‐actin which corresponded to the antigen for the B1 antibody. This homology is likely the basis of the cross reaction with beta‐actin.

To avoid the cross reaction with PCNA exhibited by the B1 antibody, a second antibody (ACTB358) was generated to 11 residues starting at residue 358 of beta actin The ACTB358 antibody is highly specific for a novel isoform of actin with a mass of 86 kDa and a pI of 5.5, but does not interact with cytoplasmic or other types of actin. We are investigating the sequence and expression of the novel isoform of actin in non‐malignant and malignant ovarian and breast cells.

Support by NOSMFA Research Development Fund
Paraquat‐Induced Early Phase Changes in Protein Expression in Whole Tissue and Mitochondria from Rat Liver
Authors: Venkatakrishnan Priya , Nakayasu Ernesto S , Almeida Igor C , Miller R. Timothy
Published: June 2021

Paraquat (PQ) is a widely used herbicide whose toxicity to plants and humans involves redox‐cycling. PQ is a substrate for polyamine transporters in lung, resulting in accumulation and lung toxicity. However, most human deaths are due to multisystem failure, including hepatotoxicity. Inside cells, PQ redox cycles, consumes NAD(P)H and produces superoxides. In mitochondria (MT), PQ inhibits complex‐1 activity. Proteomic analyses were performed with purified MT and liver homogenate from PQ‐treated (40 mg/kg; ip) rats, using nanospray‐coupled linear ion‐trap mass spectrometry (LTQ‐MS) combined with 18O‐labeling in an effort to understand the early effect of PQ on MT. The proteins that were modulated above 2‐fold were analyzed using GO ontology and Interpro scan. In MT, 29 and 8 proteins were down‐, or up‐regulated, respectively, including down‐regulation of the MT complex‐1 subunits Ndufs5 and Ndufs7. Other MT proteins included oxidoreductases, transferases, hydrolases and transporters. In liver homogenate, 20 and 17 proteins were down‐, or up‐regulated, respectively. These proteins were involved in cell metabolism and binding functions. Ubiquitination and proteolytic pathway proteins as well as oxidoreductases and transferases were also affected. Studies are ongoing to identify the regulators of, and role(s) of, protein responses in liver tissue homogenates and MT following PQ exposure.
Understand liver functions by proteomic profiling of isolated rat liver perfusates
Authors: Zhang Yang , Wang Yan , Sun Wei , Wu Shuzhen , Ma Sucan , Gao Youhe
Published: June 2021

Current proteomic research is mainly focused on tissue, cellular and subcellular levels with disrupted structural and functional integrity. Here we tried to understand liver functions by proteomic profiling of perfusates from isolated rat liver, which preserved intact hepatic structure and functions during the experiments. There are two major concerns in this type of study. The first one is the structural and functional integrity of the liver. Perfusion conditions were carefully controlled and alanine amiotransferase/aspartate aminotransferase levels in the perfusates were monitored as indicators of liver integrity. The second concern was the blood contamination in the perfusates. The amount of immunoglobins identified in the perfusates was monitored as indicator of blood contamination. More than 800 proteins were identified by 2DLC‐MS/MS with false positive rate less than 1%. This data was analyzed to provide interesting insight to liver functions. This is the first time using proteomic strategy to help understand liver functions by studying its perfusates. This strategy is useful for biomarker discovery for liver diseases and applicable to the studies of other organs' functions.
An Efficiency machine learning system prediction coupled with yeast two hybrid confirmations to identify HPV 16 E6 interacting PDZ proteins
Authors: Hu Siqi , Shao Chen , Zhang Ling , Ma Sucan , Gao Youhe
Published: June 2021

Identification of protein interactions by experimental screening is not only labor‐intensive but almost futile in screening low abundance species due to the suppression by high abundance species. A better way is to use high‐throughput computational prediction to screen protein sequence databases to produce manageable potential ligands and to validate experimentally. Here, we used an integrated machine learning system developed in the lab coupled with yeast two‐hybrid (Y2H) conformations to identify Human papillomavirus (HPV) type 16 E6 interacting PDZ proteins. It has been reported that carcinogenic HPV 16 E6 has PDZ interacting C‐ terminal, which non‐carcinogenic ones do not have. Using prediction plus validation strategy, VELI 3 was found to interact with HPV 16 E6 in Y2H system, suggest that VELI3 is a potential cellular target of HPV 16 E6. This strategy can be easily extended to a variety of viral proteins with PDZ binding motif for identification of their targeting cellular PDZ domain proteins.
Effects of ADP‐ribosylarginine Hydrolase (ARH1) on Cell Proliferation and Tumorigenesis
Authors: KATO JIRO , Zhu Jainfeng , Kasamatsu Atsushi , Liu Chengyu , Hoffmann Victoria , Lizak Martin J , Moss Joel
Published: June 2021

Mono‐ADP‐ribosylation, a post‐translational modification involving the transfer of ADP‐ribose from NAD to an acceptor protein is regulated by enzymes that catalyze opposing arms of ADP‐ribosylation cycles. ADP‐ribosyltransferases (ARTs) catalyze the modification of ADP‐ribose acceptors, and ADP‐ribosyl‐acceptor hydrolases (ARHs) cleave the ADP‐ribose‐acceptor bond. ARH1 catalyzes for the hydrolysis of α‐ADP‐ribose‐(arginine)protein bonds formed by NAD:arginine ARTs. ARH1−/− cells grown from embryonic tissues of an ARH1 knockout mouse, and ARH1−/− cells transformed with an inactive, double‐mutant ARH1 gene (ARH1−/−+dm) had higher proliferation rates than wild‐type ARH1+/+ cells and ARH1−/− cells expressing the wild‐type ARH1 gene (ARH1−/−+wt). Like ARH1−/− cells, ARH1−/−+dm cells formed more colonies in soft agar than did ARH1+/+ cells or ARH1−/−+wt cells. Similarly, ARH1−/− and ARH1−/−+dm cells, but not ARH1+/+ and ARH1−/−+wt cells, produced tumors in nude mice. ARH1−/− mice also developed diverse tumors spontaneously more frequently than did their wild‐type littermates. As expected based on the function proposed for ARH1 in an ADP‐ribosylation cycle(s), basal ADP‐ribose‐arginine content of ARH1−/− cells and ARH1−/− mouse tissues was higher than those of wild‐type cells and tissues. Thus, tumorigenesis appears to be related to mono‐ADP‐ribosylation of arginine residues in proteins.
Structural Properties of Human Urine‐Sourced Neutrophil Gelatinase Associated Lipocalin (huNGAL)
Authors: Rae Tracey D , Bonn Ryan , Workman Ryan , Ali Salman , Syed Hina , Rupprecht Kevin , Tu Bailin , Fishpaugh Jeffrey , Grenier Frank
Published: June 2021

Elevated levels of Neutrophil Gelatinase Associated Lipocalin (NGAL) in the urine have been established as a promising diagnostic indicator for acute kidney injury (AKI). Development of a quantitative diagnostic immunoassay for NGAL in urine prompts an understanding of the native protein analyte and its structural similarity to the selected recombinant protein used to calibrate the assay. Here we present findings from various analyses on the native human NGAL protein in urine (huNGAL). Chromatographic size‐fractionation of individual human urine specimens indicate that the predominant form of huNGAL is monomeric, although oligomeric forms are detected in some specimens. When observed, NGAL‐containing oligomers are shown to occur through inter‐protein disulfide linkage. An enriched huNGAL protein sample from a pool of urine specimens was analyzed by two‐dimensional electrophoresis (2DE) with Western Blotting to elucidate size and charge distribution of NGAL‐active isoforms. Although all identified huNGAL isoforms displayed molecular weights near the predicted intact polypeptide sequence, the charge distribution of the isoforms was surprisingly wide (pI 5.9‐9.1) given a theoretical pI of 9.0 for the native polypeptide. Structural properties of the enriched huNGAL are compared to those of the immunoassay calibrator protein as well as NGAL protein from other recombinant and native sources.
Polyoxygenated cholesteryl ester hydroperoxides are proatherogenic components of minimally oxidized low density lipoprotein
Authors: Harkewicz Richard , Miller Yury , Almazan Felicidad , Witztum Joseph , Dennis Edward
Published: June 2021

The oxidation of low‐density lipoprotein (LDL) occurs in vivo and significantly contributes to the development of atherosclerosis. An important mechanism of LDL oxidation in vivo is its modification by 12/15‐lipoxygenase (LO) and we are currently working to determine the identities of bioactive components produced through the action of 12/15‐LO and that play a central role in atherogenesis. We have developed a model of minimally oxidized LDL (mmLDL) in which native LDL is modified by cells overexpressing 12/15‐LO. This mmLDL activates macrophages inducing cell spreading, activation of extracellular signal‐regulated kinase 1/2 (ERK1/2) and (protein kinase B) Akt signaling, and secretion of proinflammatory cytokines. Employing both normal and reversed phase liquid chromatography coupled to mass spectrometry demonstrates the presence of polyoxygenated hydroperoxide cholesteryl ester species in mmLDL that are not observed in native LDL. Remarkably, many of these same polyoxygenated cholesteryl ester hydroperoxide species were observed in the extracts of atherosclerotic lesions isolated from hyperlipidemic apoE−/− mice.

This work is supported by the LIPID MAPS Large Scale Collaborative Grant number GM069338 from the U.S. National Institutes of Health.
Investigation of HIV‐1 Protein‐Lipid Interactions During Assembly at the Plasma Membrane
Authors: Nemer David M , Stahelin Robert V
Published: June 2021

Human immunodeficiency virus (HIV‐1) is an enveloped retrovirus that infects T‐helper lymphocytes and leads to acquired immune deficiency syndrome (AIDS) through destruction of this crucial arm of the human immune system. HIV‐1 replicates through virion assembly and budding at the plasma membrane of T‐helper lymphocytes, leading to virus proliferation and lymphocyte depletion. Several virus‐encoded proteins direct this process, including the p17 matrix domain (MA). This 17‐kDa protein binds to the plasma membrane and is essential for membrane localization, assembly, and budding of nascent virions. Recent structural, computational, and cell studies have provided insight into how HIV‐1 MA is targeted to the plasma membrane. This study supplements previous research by providing biophysical data on the membrane binding properties of MA. We demonstrate the lipid affinity, specificity, and membrane penetration of MA in addition to showing how phosphorylation events may mediate membrane targeting. Together, these data help elucidate the mechanism of MA targeting to the plasma membrane and the role matrix‐lipid interactions play in the assembly and budding of HIV‐1 virions. Further examination of matrix‐membrane interactions will provide a greater understanding of the role of lipids in the viral life cycle and of their potential as targets for exciting and novel therapeutic strategies.
UVB Irradiation on Melanoma Cells Regulates α4β1 Integrin (VLA‐4) Avidity for Its Ligand VCAM‐1
Authors: Wang Lei , Burdick Monica , Wu Shiyong
Published: June 2021

The major aspect contributing to the mortality of melanoma is its ability to spread, or metastasize. Ultraviolet light B (UVB) is considered an indirect cause of melanoma formation. However, little is known about the potential effects of UVB to melanoma metastasis. Integrins, a large family of cell adhesion molecules expressed on the melanoma cell surface, are important for cell signaling, growth, and migration during metastasis. Most critically, tumor cell tissue invasion is dependent on the initial interaction of tumor cells with vascular endothelium at the target organ, and there is increasing evidence for a prominent role of melanoma VLA‐4 integrin binding to its endothelial ligand vascular cell adhesion molecule‐1 (VCAM‐1) in this process. This research focuses on the quantitative modulation of VLA‐4 integrin expression and function on melanoma cells after UVB irradiation. The present data show that 3 hours post‐UVB irradiation, VLA‐4 expression was unchanged relative to untreated cells, but adhesion to VCAM‐1 decreased ~70%. At 12 hours post‐treatment, VLA‐4 expression was reduced to ~50% of untreated cells, yet adhesion to VCAM‐1 was similar to the 3‐hour time point. Further studies imply that the spatial organization of VLA‐4 in the melanoma membrane contributes the changes in avidity for VCAM‐1. Understanding of the molecular mechanism underlying melanoma‐endothelia interactions upon UVB irradiation can guide the development of clinical advances for melanoma.
Posttranslational Regulation of the Tumor‐Suppressor Ikaros by Vasoactive Intestinal Peptide Receptor 1 in Human HuT 78 Cells
Authors: Van der Steen Travis , Hermann Rebecca J , Meinhardt Steven , Dovat Sinisa , Dorsam Glenn Paul
Published: June 2021

Ikaros (IK) is a krupple‐like zinc finger and master regulator of lymphopoesis. Mutations to the IK DNA binding domain result in T cell leukemia. The phosphorylated posttranslational modifications of IK have been shown to be regulated by casein kinase II (CKII) throughout the primary sequence. Putative consensus sequences for additional kinases such as GSK3, Cdk, CaMKII, and PKA have been identified. Hyperphosphorylation of IK reduces its DNA binding affinity and mediates G1/S transition. Vasoactive intestinal peptide/pituitary adenylyl cyclase activating peptide receptor 1 (VPAC1) is a G protein coupled receptor that negatively regulates T cell receptor (TCR) activation through a cAMP/PKA signaling dependent pathway when bound by its ligand, vasoactive intestinal peptide (VIP). Our hypothesis is that VIP/VPAC1 signaling suppresses CKII phosphorylation of IK in a PKA dependent mechanism. Upon T cell activation, a new immunoreactive pool of IK was detected, and VIP (10‐8M) ablated the detection of this hyperphosphorylated species. We conclude that VIP/VPAC1 signaling may block the immunoreactive pool of a certain hyperphosphorylated IK species thereby negatively regulating T cell activation. Research supported by NIH‐K01 1K01DK064828 and COBRE 2P20RR05566.
G‐protein coupled receptor‐54 is required for embryonic kidney branching morphogenesis and glomerular development through regulating Bmp7 expression
Authors: Yi Tingfang , Liu Mingyao
Published: June 2021

Kidney branching morphogenesis and glomerular development are two key processes in embryonic kidney formation. Bmp7 has been identified to be essential for these processes. However, the molecular mechanism of regulating Bmp7 expression in kidneys has not been fully understood. Here we report that G‐protein coupled receptor 54 (Gpr54) regulates Bmp7 expression and is required for mouse embryonic kidney development. Gpr54 deletion leads to severely retarded kidney branching morphogenesis, S‐shaped body formation, glomerular development, and nephron underdosing in embryonic kidneys in vivo and in explanted kidneys in vitro. Deletion of Gpr54 significantly decreases Bmp7 expression in developing kidneys. Using CHIP and luciferase assays, we demonstrate that Gpr54 regulates NFAT2, but not NFAT1, NFAT3 or NFAT4, mediated Bmp7 transcription. Furthermore, we show that Gpr54 regulates Sp1, but not phosphorylated Sp1, mediated Bmp7 transcription. Together, these data suggest that Gpr54 signaling pathway regulate Bmp7 expression and play important roles in embryonic kidney branching morphogenesis and development. Gpr54 could be a new therapeutic target for kidney diseases.
Focal Adhesion Signaling Down‐Regulation Increases Myocyte Apoptosis During Acute Stress of Volume Overload: Role of Beta1‐Adrenergic Receptor Stimulation
Authors: seqqat rachid , Rafiq Khadija , Hanscom Marie , Margulies Kenneth , Houser Steven , Dell'Italia Louis , Sabri Abdelkarim
Published: June 2021

We examined the role of ß1‐adrenergic receptors (ß1‐ARs) in focal adhesion (FA) signaling alteration and myocyte apoptosis in acute stress of volume overload (VO). Rats subjected to aorto‐caval fistula (ACF) developed cardiac dilatation associated with a decrease in interstitial collagen deposition along with a decrease in tyrosine phosphorylation of key FA signaling molecules FAK, Pyk2, and paxillin. This impared FA signaling induced by ACF was associated with an increase in myocyte apoptosis. ß1‐AR blockade (extended‐release metoprolol succinate, 100 mg QD) prevented collagen loss, FA signaling down‐regulation, and myocyte apoptosis induced by ACF at 2 days, but failed to reduce cardiac dilatation and wall stress. The phosphatase PTEN activation was increased in ACF animals and this increase was prevented with ß1‐AR blockade. Chronic stimulation of cultured neonatal rat cardiomyocytes with ß1‐AR agonist or expression of ß1‐ARs induced PTEN activation, FA signaling down‐regulation, and myocyte apoptosis. Moreover, expression of a catalytically inactive PTEN mutant or wild‐type FAK prevented ß1‐AR‐induced myocyte apoptosis. These results show that PTEN activation and FA signaling alteration are critical downstream signals for myocyte apoptosis following ß1‐AR stimulation in‐vitro and affects myocyte‐extracellular matrix scaffolding necessary for cardiac remodeling during acute VO.
Structural analysis of GPS autoproteolysis of LNB‐TM7 receptors‐Characterization of the interaction between the extracellular α‐subunit and the 7TM β‐subunit
Authors: Huang Yi‐Shun , Lin Hsi‐Hsien
Published: June 2021

The novel class B2 GPCRs (LNB‐TM7) have been found to undergo post‐translational autoproteolysis at the G protein‐coupled receptor proteolytic site (GPS) in the ER. Therefore, the GPS autoproteolysis of LNB‐TM7 receptors produces constitutively two subunits, a large extracellular domain (α‐subunit) resembling cell‐adhesion protein and a TM7 domain (β‐subunit) that is a typical TM7 moiety of GPCR. Intriguingly, the cleaved α‐subunit was shown to remain firmly on the cell membrane. It has long been believed that the α‐subunit interacts non‐covalently with the β‐subunit to form a heterodimeric structure on the cell membrane. However, based on our data and others, it is shown that the α‐subunit and β‐subunit do not normally interact on the cell membrane and behave rather as two separate proteins. First, we find that PFO can dissociate the α‐subunit of cellular form but not soluble form. Second, we find that the α‐subunit does not associate with soluble CD4 fragment secreted into conditioned medium and does not associate with the CD4 fragment moved to the lipid raft region. Third, we find that the α‐subunit is retained in the ER by non‐covalent interaction with the β‐subunit. Hence, it is hoped that the results from our investigation will provide some important clues to decipher the cellular functions and human diseases associated with the LNB‐TM7 receptors. NSC96‐2320‐B‐182‐005
Cross‐Linking of a DOPA‐Containing Peptide Ligand into its G Protein‐Coupled Receptor
Authors: Umanah George E. , Son Cagdas D , Ding FaXiang , Naider Fred , Becker Jeffrey M
Published: June 2021

The interaction between a 3,4‐dihydroxylphenylalanine (DOPA) labeled analog of the tridecapeptide α‐factor and Ste2p, a Saccharomyces cerevisiae model G protein‐coupled receptor (GPCR), has been analyzed by periodate‐mediated cross‐linking. Chemically synthesized α‐factor with DOPA substituting for tyrosine at position 13 and biotin tagged onto lysine7 [Lys7(Biotin),Nle12,DOPA13]α ‐factor (Bio‐DOPA‐α ‐factor) was used for cross‐linking into Ste2p. The biological activity of Bio‐DOPA‐α ‐factor was about one‐third that of native α‐factor and exhibited about a ten‐fold lower binding affinity. Bio‐DOPA‐α ‐factor cross‐linked into Ste2p, and cross‐linking was inhibited by native α‐factor. The Ste2p‐ligand complex was purified using a metal ion affinity column, and after cyanogen bromide treatment, avidin affinity purification was used to capture Bio‐DOPA‐α ‐factor‐Ste2p cross‐linked peptides. Mass‐spectrometric analyses of the cross‐linked fragments showed that Bio‐DOPA‐α ‐factor reacted with the Phe55‐Met69 region of Ste2p. Cross‐linking of Bio‐DOPA‐α ‐factor was reduced by 80% using a cysteine‐less Ste2p (Cys59Ser). These results suggest an interaction between position thirteen of α‐factor and residue Cys59 of Ste2p. This study is the first to report DOPA cross‐linking of a peptide hormone to a GPCR and the first to identify a residue‐to‐residue cross‐link between Ste2p and α‐factor.

Supported by NIH GM‐22086 and GM‐22087 1
Structural basis for EGFR ligand sequestration by Argos
Authors: Klein Daryl E. , Stayrook Steven E. , Shi Fumin , Narayan Kartik , Lemmon Mark A.
Published: June 2021

Members of the epidermal growth factor receptor (EGFR) or ErbB/HER family and their activating ligands are essential regulators of diverse developmental processes. Inappropriate activation of these receptors is a key feature of many human cancers, and its reversal is an important clinical goal. A natural secreted antagonist of EGFR signaling, called Argos, was identified in Drosophila. Argos functions by directly binding to (and sequestering) growth factor ligands that activate EGFR. Here we describe the 1.6Å‐resolution crystal structure of Argos bound to Spitz, an EGFR ligand. Contrary to expectations and previous reports, Argos itself does not contain an EGF‐like domain. Instead, a trio of closely related domains (resembling a three‐finger toxin fold) form a clamp‐like structure around the bound EGF ligand. Although structurally unrelated to the receptor, Argos mimics EGFR by using a bipartite binding surface to entrap EGF domain of Spitz. The three‐domain clamp of Argos resembles the human urokinase‐type plasminogen activator (uPA) receptor, which uses a similar mechanism to engulf the EGF‐like module of uPA. Our results indicate that undiscovered mammalian counterparts of Argos may exist among other poorly characterized structural homologues.
ErbB2/HER2/Neu resembles an autoinhibited invertebrate EGF receptor
Authors: Alvarado Diego , Klein Daryl E , Lemmon Mark A
Published: June 2021

The orphan receptor tyrosine kinase ErbB2 (HER2/Neu) is potently transforming upon overexpression, and is an important therapeutic target in human cancer. ErbB2 lacks a key autoinhibitory 'tether' in its extracellular region that is present in other human ErbB receptors (including the EGF receptor), providing a potential explanation for its oncogenic properties and activity. Here, we present structural studies of the tightly ligand‐regulated Drosophila melanogaster EGFR (dEGFR) that challenge the notion that ErbB2 lacks autoinhibition. Inactive dEGFR is stabilized by a set of interdomain interactions distinct from the 'tether' in human EGFR. Surprisingly, all of these autoinhibitory interactions in the unliganded dEGFR structure are maintained (and even extended) in ErbB2. This similarity to dEGFR argues that normal and pathogenic ErbB2 signaling may also be ligand‐regulated. Our findings have important implications for ErbB2 regulation in human cancer, and for developing therapeutic approaches to target novel aspects of this orphan receptor.
Glutamate induces cell surface translocation of annexin A2 in retinal ganglion cells
Authors: Valapala Mallika , Borejdo Julian , Vishwanatha Jamboor K
Published: June 2021

Glutamate‐induced elevation in intracellular Ca2+ has been implicated in the loss of retinal ganglion cells (RGCs) in glaucoma. RGCs respond to excitotoxic injury by activating an extracellular proteolytic cascade involving the components of the plasmin‐plasminogen system. Annexin A2 is a Ca2+ dependent phospholipid binding protein and serves as an extracellular proteolytic center by recruiting tissue plasminogen activator (tPA) and plasminogen and mediating localized generation of plasmin. Here we characterize the expression of annexin A2 in the rat RGCs. Ca2+ imaging and time lapse microscopy demonstrated glutamate‐induced Ca2+ influx. We showed that glutamate mobilized both endogenous and GFP fused annexin A2 to the cell surface in a process dependent on the NMDA receptor activity. By in vitro plasmin generation assay we demonstrated that annexin A2 forms a catalytically active plasmin generating complex on glutamate treatment and this activity is inhibited by blocking the N‐terminus of annexin A2. Phosphorylation of Y23 at the N‐terminus by Src kinase is critical and mutation of Y23 to Y23F inhibits the translocation process. Mutations in the p11 binding site also inhibited annexin A2 cell surface translocation. A hexapeptide against the N‐terminal tPA binding site inhibited annexin A2‐mediated plasmin generation. These results suggest an involvement of annexin A2 in neuronal cell death processes
Adenylyl cyclase type 6 regulates Akt activity via PHLPP2
Authors: Gao Mei Hua , Tang Tong , Guo Tracy , Miyanohara Atsushi , Hammond H. Kirk
Published: June 2021

Adenylyl cyclase (AC) is the enzyme that catalyzes ATP to generate cyclic AMP (cAMP). Cardiac‐directed expression of adenylyl cyclase type 6 (AC6) has pronounced favorable effects on heart function in normal and failing hearts. The mechanisms explaining the favorable effects of AC6 are not entirely known, but may reflect effects on cellular events that are not replicated by cAMP production. Because of the central role of Akt in intracellular signaling, and its importance to cardiac myocyte survival and function, we tested the hypothesis that increased AC6 expression influences Akt signaling. Studies were performed in cultured neonatal cardiac myocytes; AC6 gene transfer was performed using recombinant adenovirus encoding AC6 (Ad.AC6). In the basal (unstimulated) state, AC6 gene transfer increased Akt phosphorylation and activity 5‐fold. However, when these cardiac myocytes were stimulated with a ß‐adrenergic receptor agonist (isoproterenol) or with an adenylyl cyclase activator (forskolin), AC6‐associated Akt phosphorylation rapidly was reduced (<2 min). This reduction in Akt phosphorylation required an Akt‐specific phosphatase, PH domain leucine‐rich phosphatase (PHLPP2), but was cAMP‐independent and reversible. The Mg2+ binding domain in the C1 region of AC6 is required for agonist‐induced activation of PHLPP2. This novel bidirectional regulation of Akt activity may contribute to the unexpected favorable effects of AC6 on the failing heart.
Calcineurin Mediates Natriuretic Peptide Receptor‐A Desensitization in MA‐10 Cells
Authors: Henesy Michelle B , Rich Thomas C
Published: June 2021

Natriuretic peptides (e.g., ANP) activate particulate guanylyl cyclases (e.g. NPR‐A) leading to increased intracellular cGMP. Cellular mechanisms underlying the regulation of NPR‐A are poorly understood. It is known that both homologous and heterologous NPR‐A desensitization result in decreased guanylyl cyclase activity correlating with loss of phosphate. The phosphatase responsible for NPR‐A desensitization has not been identified. Here we identify the phosphatase responsible for both homologous and heterologous NPR‐A desensitization in murine leydig tumor cells. We measured ANP‐induced increases in intracellular cGMP levels in the presence and absence of an array of phosphatase inhibitors and following si‐RNA‐mediated knockdown of calcineurin. Results suggest that calcineurin (PP2B) is primarily responsible for both homologous and heterologous desensitization. In addition, we observed that calcinuerin inhibitors attenuate ANP‐induced loss of phosphate from NPR‐A. Taken together, these data suggest that calcineurin is the phosphatase primarily responsible for dephosphorylation and subsequent desensitization of NPR‐A. Supported by AHA 0715101B and 0335084N, NIH HL74278.
Structure and chemistry of the human p300/CBP and yeast Rtt109 histone acetyltransferase
Authors: Tang Yong , Liu Xin , Wang Ling , Holbert Marc A. , Meeth Katrina , Wurtele Hugo , Rocha Walter , Gharib Marlene , Jiang Eva , Zhao Kehao , Luo Cheng , Thibault Pierre , Verrault Alain , Cole Philip A. , Marmorstein Ronen
Published: June 2021

Histone actyltransferase (HAT) enzymes form a superfamily of proteins that transfer an acetyl group from the acetyl‐coenzyme A (Ac‐CoA) cofactor to the epsilon amino group of histone or sometimes non‐histone proteins to promote gene activation. Paradoxally, despite the similar chemistry carried out by these enzymes, and their structurally related core regions, they fall into subfamilies with very limited to no sequence homology, structurally divergent core flanking regions and they use divergent mechanisms for catalysis. Four well studied HAT families include Gcn5/PCAF, MYST, p300/CBP and Rtt109. Although the structure and chemistry of the evolutionarily conserved Gcn5/PCAF and MYST HATs have been previously characterized, the metazoan‐specific p300/CBP and fungal‐specific Rtt109 have only recently been characterized at the structural and chemical levels. These recent studies provide new insights into the ancestral relationship between HATs and their functions and point to a common HAT ancestor that has evolved around a common structural framework to form HATs with divergent catalytic and substrate binding properties. These studies also point to the importance of regulatory loops within HATs and autoacetylation in HAT function. Implications for future studies will also be discussed.
Surface Residue Glu762 of the FMN Subdomain Impacts Ferrous Heme‐NO reactivity in Neuronal Nitric Oxide Synthase
Authors: HAQUE MOHAMMAD MAHFUZUL , Wang Zhi‐Qiang , Fadlalla Mohammed , Stuehr Dennis J.
Published: June 2021

Nitric Oxide Synthases (NOSs) are calmodulin (CaM)‐dependent flavoheme enzymes that oxidize L‐arginine to nitric oxide (NO) and L‐citrulline. Their catalytic behaviors are determined by their rates of heme reduction (kr), ferric heme‐NO dissociation (kd), and ferrous heme‐NO oxidation (kox). We found that point mutation (Glu762N) of a conserved residue on the enzyme's FMN module doubled the NO synthesis activity compared to wild type (WT) nNOS. However, in the absence of Arg, NADPH oxidation rates suggested that electron flux through the heme was slower in E762N nNOS, and this correlated with the mutant having a 60% slower kr. During NO synthesis, little heme‐NO complex accumulated in the mutant, compared to approximately 50‐70% accumulation in WT nNOS. We concluded that the E762N mutation is hyperactive because it minimizes buildup of an inactive ferrous‐NO complex during the steady state. Indeed, we found that kox was 2 times faster in the E762N mutant than in WT. Computer simulation and experimental measures both indicated that the slower kr coupled with the faster kox lowers the mutant's apparent KmO2 for NO synthesis, which in turn enables its hyperactivity in steady‐state NO synthesis assays. This represents a novel and unexpected way for the FMN module to impact the activity of NOS enzymes.This work was supported by NIH Grant CA53914 to Dennis Stuehr.
The 3D structures of Ca2+‐S100B bound to small molecules within the target cleft of S100B as determined by x‐ray crystallography
Authors: Charpentier Thomas H , Wilder Paul T , Liriano Melissa A , Varney Kristen M , Pozharski Edwin , MacKerell Alexander D , Coop Andrew , Toth Eric A , Weber David J
Published: June 2021

Structural studies are part of a rational drug design program underway to inhibit the S100B‐p53 interaction and restore p53 function in malignant melanoma. Three small molecules were found to bind S100B and structures of these compounnds SBi132, SBi1279 and SBi523 were determined by X‐ray crystallography at 2.10Å, 1.98Å and 1.90 Åresolution, respectively. These small molecules were seen to bind in separate locations in the hydrophobic pocket of Ca2+‐S100B and were compared. SBi523 interacts with residues in loop2 that is found below the hydrophobic cleft. SBi132 binds in the hydrophobic pocket of Ca2+‐S100B and is nearby residues in loop2 and helix 4. SBi279 overlaps both sites of SBi132 and SBi523 and makes contacts with both with loop2 and helix 4. There are very minor changes in S100B when comparing the structures of S100B in complex with each SBi molecule. The orientations of SBi compounds 132, 279 and 523, in their respective binding sites, were consistent with NMR data including saturation transfer difference and 15N backbone and 13C sidechain chemical shift perturbations. These small molecules in close proximity to each other will be useful for developing novel inhibitors of protein‐protein interactions of S100B. This work was supported by grants from the National Institutes of Health [GM58888 and CA107331], the American Cancer Society [CDD107745], and the University of Maryland Drug Design Center.
FIZZ1 binds to B7‐H3 on fibroblasts and activates myofibroblast differentiation
Authors: Hu Biao , Wu Zhe , Phan Sem H.
Published: June 2021

Found in inflammatory zone 1(FIZZ1) is a secreted protein involved in regulation of a wide range of biological processes, such as angiogenesis, apoptosis, cell proliferation and cell differentiation. However, its cognate receptor on target cells, such as the fibroblast, remains to be identified. To address this issue and seek out the putative receptor of FIZZ1, a FIZZ1‐AD fusion protein was constructed and used as the "bait" to screen against the mouse cDNA library fused with the BD domain in a yeast two‐hybrid system. This yielded several clones that interacted positively with the FIZZ1 bait, and which were found to contain B7‐H3, a new member of the B7 family. This FIZZ1‐B7‐H3 interaction was confirmed by co‐immunoprecipitation and FRET assays. B7‐H3 expression by fibroblasts was confirmed by immunostaining and real time PCR. Further studies indicated that FIZZ1 induced the phosphorylation of B7‐H3. In contrast to wild type fibroblasts, B7‐H3 deficient cells from knockout mice failed to undergo myofibroblast differentiation in response to FIZZ1 treatment. Taken together these data suggest that B7‐H3 may serve as the cognate receptor for FIZZ1 on fibroblasts, which mediates the downstream signaling associated with myofibroblast differentiation.

Supported by NIH grants HL28737, HL31963 and HL52285.
CMO2 and Human Monocyte Differentiation: Potential mechanisms of lycopene in the prevention of atherosclerosis
Authors: Wiener Doris , Gong Xiaoming , Afeld Jamiee , Marisiddaiah Raju , Rubin Lewis
Published: June 2021

Monocyte‐derived macrophages are precursors of the lipid‐laden foam cells in atherosclerotic fatty‐streak lesions. Depending on diet, circulating monocytes are exposed to lipoprotein packaged carotenoids. Low plasma carotenoid levels have been associated with increased risk of cardiovascular disease. We report that lycopene may alter monocyte‐macrophage phenotype and function through regulating β‐carotene 9',10′‐monooxygenase (CMO2) expression. We demonstrated that the CMO2 gene is differentially expressed in undifferentiated monocytes and monocyte‐derived macrophages. CMO2 is expressed in human primary monocytes and monocytic THP‐1 cells. Reducing CMO2 expression accompanied phorbol 12‐myristate 13‐acetate (PMA) induced differentiation of THP‐1 cells to the macrophage phenotype. Lycopene treatment of THP‐1 cells significantly increased CMO2 mRNA and protein levels. Induced overexpression of CMO2 in THP‐1 cells suppressed NFκB activity but increased PPAR reporter activity, suggesting that elevated CMO2 induces PPAR function. These studies provide the first evidence for the biological role of CMO2 in regulating monocyte differentiation via modulation of inflammation related signaling pathways. Targeting CMO2 with carotenoids may thus serve as a nutritional and therapeutic approach to attenuate vascular wall inflammation and slow the progression of atherosclerosis. (NIH HD42174, Muma Family Endowment)
Synergistic effects of Curcumin and Garcinol on human pancreatic cancer cells
Authors: Parasramka Mansi A. , Wrubel Jennifer Ann , Gupta Smiti V.
Published: June 2021

Pancreatic cancer (PaCa) is the fourth leading cause of cancer related death worldwide with a low five year survival rate of <1% due to its aggressive local invasion and early metastases. Thus early detection and evaluation of novel therapeutic agents is crucial for a favorable prognosis. Curcumin and Garcinol from the plants, Curcuma longa and Garcinia indica respectively have proven anti‐inflammatory, antioxidant, and anticarcinogenic activity in several cancer types. The objective of this study was to investigate the synergistic effect of these two chemopreventive dietary agents in PaCa cells. For this, PaCa cells (BxPC‐3 and Panc‐1: K‐ras wild and mutant) were treated with Curcumin (0‐50µM) and Garcinol (0 ‐ 40µM) and in combination for 24, 48 and 72hrs to examine viability, antiproliferative and apoptotic effects using MTT, Trypan Blue, cytoplasmic histone‐DNA fragment quantification and DAPI staining. The molecular mechanism was investigated by Western immunobloting, targeting different apoptotic signaling moieties. Synergistic effect of these compounds was tested using Isobologram analysis. Cell cycle phase analysis was done by Flow Cytometry. Both compounds individually and in combination significantly enhanced the anti‐proliferative and apoptotic effect in these cells relative to untreated control cells. This is first report on therapeutic effect of Garcinol on PaCa cells.

Grant Funding Source

Graduate Teaching Assistantship
Red wine polyphenolics have anti‐cancer effects in colon‐cancer and target oncogenic microRNAs as potential underlying mechanisms
Authors: Del Follo Armando , Mertens‐Talcott Susanne
Published: June 2021

Red wine has been identified as a food with anti‐cancer properties. The aim of this study was to investigate the anti‐cancer effect of a Texas red wine from Black Spanish grapes (Vitis labrusca) in colorectal adenocarcinoma cells (HT‐29). A polyphenolic extract prepared from the wine was tested (20‐300 mg/L) regarding anti‐cancer effects and underlying mechanisms related to apoptosis and angiogenesis. Reactive oxygen species (ROS) increased up to 6.5‐fold at 300mg/L and mRNA of p53 was increased up to 1.9‐fold compared to the control and caspase‐3 activity was increased up to 1.8 fold within the same concentration‐range, indicating pro‐apoptotic activity of the wine extract. The oncogenic specificity protein Sp1 was significantly decreased, where the zinc‐finger protein ZBTB10 a transcription factor which has been found to inhibit Sp1 was increased up to 1.3‐fold with the wine extract. Vascular endothelial growth factor (VEGF) mRNA a target of Sp1 was decreased up to 3.7‐fold, indicating anti‐angiogenic effects. Additionally, the pro‐oncogenic microRNAs 21 and 27a were significantly decreased within an extract concentration range of 37‐300 mg/L, and 75‐300mg/L, respectively. In conclusion, since ZBTB10 is a relevant target for miRNA 27a, the reduction of miRNA27a may be responsible for a ZBTB10‐involved reduction of the oncogenic Sp1 causing decrease in VEGF and inducing apoptosis as underlying mechanism.

Grant Funding Source

Texas A&M University
Phenethyl isothiocyanate inhibits the migration and invasion of DU145 human prostate cancer cells
Authors: Hong Ji Eun , Park Jung Han Yoon
Published: June 2021

Anti‐carcinogenic effect of cruciferous vegetables is attributed to organic isothiocyanates that occur naturally as thioglucoside conjugates (glucosinolates) in a variety of edible cruciferous vegetables such as broccoli, watercress, and cabbage. Phenethyl isothiocyanate (PITC) is a naturally occurring isothiocyanate compound that has attracted a great deal of attention due to its remarkable cancer chemopreventive properties. Previously, PITC has been reported to suppress the proliferation of human prostate cancer cells by inducing apoptosis and cell cycle arrest. In the present study, we investigated the effects of PITC on the migration and invasion characteristics of DU145 human prostate cancer cells. DU145 cells were cultured in the presence of 0 ‐ 6 μmol/L PITC. PITC inhibited migration and invasion in dose‐dependent manners. Western blot analysis revealed that PITC decreased the secretion of tissue inhibitor of metalloproteinase (TIMP)‐1, vascular endothelial growth factor and urokinase‐type plasminogen activator and increased the levels of TIMP‐2 in DU145 cells. We have demonstrated that PITC inhibits the metastasis potential of DU145 human prostate cancer cells.
Effects of dietary supplementations of tomato extract vs. lycopene on nonalcoholic steatohepatitis related hepatocarcinogenesis in rats
Authors: Wang Yan , Ausman Lynne M , Greenberg Andrew S , Russell Robert M , Wang Xiang‐dong
Published: June 2021

Higher intake of tomatoes enriched in lycopene (LY) has been associated with lower risk for a variety of cancers. We recently showed that high‐fat diet (HFD) induced nonalcoholic steatohepatitis (NASH) and promoted diethylnitrosamine (DEN)‐initiated hepatocarcinogenesis in a rat model (Wang Y et al., Int. J. Cancer 2009). Using the same model, we investigated the efficacy of equivalent dosage of dietary LY from either tomato extract (TE) or purified LY against NASH related early hepatocarcinogenesis. Six groups of rats (n=8/group) were injected with DEN and then fed either Lieber‐DeCarli control diet or HFD with or without TE or LY for 6 weeks. Results showed similar plasma and hepatic LY concentrations between TE and LY groups, moreover, both TE and LY supplementations in the HFD groups significantly reduced altered hepatic foci multiplicity, lipid peroxidation, expressions of PCNA and cyclinD1, phosphorylated‐ERK, and nuclear NF‐kB p65 subunit in the liver, as compared with the HFD alone. However, TE, but not LY, inhibited HFD‐upregulated inflammatory foci and mRNA levels of proinflammatory cytokines (TNFalpha, IL‐1beta, IL‐12, CD14) in the liver. These data indicate that both TE and LY inhibit NASH related early hepatocarcinogenesis whereas TE provides an additional anti‐inflammatory potency due to a possible synergistic effect of multiple constituents in tomatoes (Supported by NIH grant R01CA104932).

Grant Funding Source

NIH grant R01CA104932
Early Effects of Dietary Soy and Genistein in Rodent Models of Mammary Tumorigenesis
Authors: Simmen Rosalia CM , Su Ying , Pabona John Mark M , Rahal Omar , Simmons Christian Dupre , Hennings Leah
Published: June 2021

The risk of breast cancer is highly modifiable by diet. Breast cancer may have its origins during early mammary development, thus, the increasing popularity of soy food consumption among pregnant and breast‐feeding women and early exposure to soy protein and bioactive components through soy infant formula could have significant implications on adult incidence of this disease. Since soy protein isolate (SPI) and genistein (GEN) diets decreased chemically‐induced tumor incidence in adult female rats, dietary effects on genetic pathways underlying mammary tumorigenesis were evaluated. In rat mammary epithelial cells, SPI and GEN, relative to casein diet increased tumor suppressor PTEN and E‐cadherin expression; these effects were recapitulated in vitro by GEN. Dietary SPI also decreased lipogenic gene expression in rat mammary stromal adipocytes in vivo, which was mimicked by GEN in 3T3‐L1 adipocytes in vitro. Since Wnt signaling perturbation alters the epithelial hierarchy, MMTV‐Wnt1 mice were investigated for dietary SPI and GEN effects on mammary progenitor cell population during disease development. Female mice at weaning were assigned to CAS, SPI‐ or GEN‐based diets and mammary tumor incidence was monitored. Diet‐mediated changes in mammary transcriptional programs and in epithelial subpopulations may underlie protection from developing mammary lesions. USDA‐CRIS‐6251‐5100002‐06S; DOD‐BCRP.
How do we assess the nutritional status in patients with breast cancer?
Authors: Tsutsumi Rie , Katayama Erika
Published: June 2021

Background

The population of breast cancer is increased remarkably and now top of the number in all the cancer in women. Although patients with cancer during treatment usually claim poor appetite, vomit or diarrhea, many women with breast cancer do not and we dietitian have few chance to intervene their nutritional status.

Objective

We evaluated assessment tools to assess and understand their nutritional status and examined what indicator would be appropriate to improvement their QOL and not to have recurrence.

Design

We enrolled 50 patients, performed anthropometric measurements and assessed other factors, BMI, food intake, side effects and incidence of recurrence.

Results

Patients with breast cancer in treatment tended to maintain their body weight and appetite but showed anemia and decreased blood protein. Patients with high BMI had little side effects and problems like decreased white blood cells, but most of them did not improve their meal and kept high fat diet. The patients in normal range of BMI had lower incidence of recurrence and prognosis.

Conclusions

The patients with high BMI achieve the treatment but showed higher incidence of recurrence, especially in patients with high fat diet. We need individualized consultation to improve the diet and BMI. Fat intake and BMI are deeply associated with increased recurrence and deteriorated prognosis.
Associations among Circulating Concentrations of Micronutrients and Risk of Being Diagnosed with Primary Non‐Small Cell Lung Cancer (PNSCLC)
Authors: Rahman Nuzhat , Badiga Suguna , Thomas Dana‐Marie , Kim Young , Cerfolio Robert James , Piyathilake Chandrika
Published: June 2021

Cigarette smoking is the main risk factor for developing PNSCLC. Observational and intervention studies have examined the isolated effects of several micronutrients (folate, ß‐carotene vitamins A, E, or C) which may modify the risk of the disease. We investigated whether circulating concentrations of a panel of micronutrients are associated with the risk of being diagnosed with PNSCLC adjusting for age, gender, ethnicity and histories of smoking and alcohol consumption. Study populations included 218 patients referred to UAB for bronchoscopy based on histological diagnosis of biopsy specimens; 123 diagnosed with PNSCLC (32 women, 91 men), and 95 patients free of lung cancer (48 women, 47 men). Information on demographics and social histories were obtained from medical records. Fasting blood samples were collected for assessment of micronutrients. Logistic regression models tested the associations among micronutrients and risk of being diagnosed with PNSCLC. For all micronutrients concentrations the lowest tertile combined with medium tertile were compared with those of the highest tertile. Patients with plasma folate in the highest tertile demonstrated 58% reduced odds of being diagnosed with PNSCLC (OR = 0.42, 95% CI 0.19 ‐ 0.88, P = 0.02). Patients with ß‐carotene in the highest tertile were found to be at increased risk of PNSCLC (OR = 2.06, 95% CI 1.29 ‐ 3.31, P = 0.002). To our knowledge, this is the first study to demonstrate opposite and independent effects of two micronutrients on PNSCLC. Our results corroborate the concern that higher intakes of ß‐carotene may exert harmful effects while improving folate status may reduce the risk of smoking related LC.