bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterizing inhibitors of the human mobile element L1 endonuclease
Authors: Darthard Loryn , Meyers Jade , Servant Geraldine , Streva Vincent , DeFreece Cecily
Published: June 2021

The Long interspersed element‐1 (L1) an active autonomous retroelement within the human genome. L1 replicates itself through a process known as retrotransposition. While L1 mutagenic L1 insertion events have been associated with diseases like breast and colon cancer, the full effect of L1 endonuclease activity on the stability of the genome is undetermined. We know that L1 endonuclease expression is associated with a DNA double strand break (DSB) and that the DNA DSB must occur during retrotransposition prior to the integration of novel L1 elements. However, not all of the L1‐associated DNA DSBs are repaired by insertion of a novel L1 element, and the breaks must be repaired faithfully or DNA mutations can occur. Thus, we hypothesize that an inhibitor of L1 endonuclease would be beneficial for estimating L1‐associated damage and to potentially minimize L1‐related genetic instability. We have tested novel small molecules for their ability to inhibit L1 endonuclease using in vitro endonuclease cleavage assays and cell based L1 retrotransposition assays. We have thus far identified a subset of small molecules that inhibit the L1 endonuclease. We are still in the process of confirming these inhibitors through additional testing and locating more inhibitors. Once the inhibitor is located we can begin to answer the question of whether DNA damage associated with the activity of the LINE1 endonuclease leads to mutation and disease.
Irreversible Inhibitors of Bovine Aortic Lysyl Oxidase
Authors: Greenaway Frederick , Peng Kai , Severson Elizabeth , Mool Shreya , Jakobsche Charles
Published: June 2021

Lysyl oxidase (LOX) is a copper‐containing amine oxidase with a unique lysyl tyrosyl quinone (LTQ) cofactor formed by post‐translational modification of Tyr‐187 followed by a cross‐linking reaction with the Lys‐152 side chain. The enzyme is irreversibly inhibited by hydrazines, hydrazides and semicarbazides and by β‐aminopropionitrile, βAPN. While hydrazines are known to inhibit by forming an irreversible adduct on the LTQ, the mechanism of inhibition of βAPN is known to be a mechanism‐based inhibitor although the exact site of inhibition has never been determined. We report the results of kinetic and mass spectrometric studies of the reaction of LOX with several irreversible inhibitors, including βAPN. Alkyl and aryl hydrazines were studied and KI and kinact parameters compared to determine how the nature of the organic group affects inhibition with, in general, aryl hydrazines being better inhibitors than alkyl hydrazines. The site of βAPN inhibition was investigated by LC/MS/MS to identify the nucleophilic side chain responsible for the inhibition. The results are interpreted in terms of a computational model structure obtained by molecular modeling. Supported in part by Nuclea Biotechnologies.
Inhibitor Development Against Aspartate‐β‐Semialdehyde Dehydrogenase‐ A Novel Target for Drug Development
Authors: Thangavelu Bharani , Pavlovsky Alexander , Bhansali Pravin , Viola Ronald
Published: June 2021

Aspartate‐β‐semialdehyde dehydrogenase (ASADH) is located at the first branch point in the aspartate metabolic pathway, which leads to the biosynthesis of several essential amino acids and important metabolites. This pathway is present only in plants and microbes but absent in mammals. The Aspartate pathway is crucial for bacteria for various metabolic processes and the microbial enzymes involved in this pathway are attractive targets for new antibiotic and antifungal compounds. The structures of ASADHs have been determined from Gram‐positive bacteria, Gram‐negative bacteria and fungal species. These enzymes share the same substrate binding and active site catalytic groups; however they show different inhibition patterns when screened against low molecular weight fragment libraries. In the present study we describe several approaches, including fragment based drug discovery (FBDD), docking studies, kinetic and crystal structure studies which were combined to characterize selective inhibitors of ASADH. Coupling reactions were employed to expand the initial set of inhibitors of ASADH. Structure‐guided development of these lead compounds has produced moderate inhibitors of our target enzyme, with some selectivity observed between the Gram‐negative and Gram‐positive orthologs of ASADH and greater selectivity for the fungal enzyme form. This initial group of inhibitors is being optimized for better potency and selectivity. This combined approach of inhibitor design, along with kinetic and structural characterization, illustrates the potential for potent drug development against this essential microbial enzyme. This work is supported by funding from the NIH (AI077720).
Bacterial Cytochrome c Peroxidases: Insight into the Structure‐Function Relationship
Authors: Walsh Kelly , Sullivan Kristal , Frato Katherine , Elliott Sean
Published: June 2021

Understanding how a sequence of amino acids serves as a functional, catalytic unit is a fundamental question in enzymology. The Elliott group studies the structure‐function relationship using bacterial cytochrome c peroxidases (bCCPs) as a model system for probing the interplay between structure, redox chemistry, and catalysis. bCCPs are diheme periplasmic enzymes that reduce hydrogen peroxide to water, requiring two c‐type heme cofactors and two electrons from the cytochrome c pool. Peroxide detoxification is an essential bacterial defense mechanism. A hallmark of bCCPs lies in the potentials of their hemes: a low potential peroxidatic heme (FeL) and a high potential electron transfer heme (FeH). Interestingly, the bCCPs from Shewanella oneidensis (SoCCP) and Nitrosomonas europaea (NeCCP) feature a high sequence identity (60%), yet have key catalytic differences: SoCCP requires reductive activation of FeH, where NeCCP does not. Further, the reduction potentials governing respective FeH are very different: where SoCCP (+245 mV vs NHE) is average for bCCPs, and NeCCP (>400 mV vs NHE) is unusually high. Previous experiments in the Elliott group have found that point mutants in one heme environment can globally affect the redox properties. Specifically, mutating a key glutamate to lysine at FeL, which is expected to directly hinder activity, has unexpectedly altered the reduction potentials of FeH as well as FeL. The high sequence and structural similarities, yet differences in electrochemical properties, present a robust model to probe the influence of nearby residues on redox potentials and to understand bacterial defense mechanisms against damaging oxygen species.
A Fluorimetric Readout Reporting the Kinetics of Nucleotide‐induced Human Ribonucleotide Reductase Oligomerization
Authors: Fu Yuan , Lin Hongyu , Wisitpitthaya Somsinee , Blessing William , Aye Yimon
Published: June 2021

Human ribonucleotide reductase (hRNR) is one of the primary targets of nucleotide cancer drugs in clinical use. The nucleotide‐induced oligomeric regulation of hRNR subunit a is increasingly recognized as an innate as well as a drug‐relevant mechanism to enzyme activity modulations. In the presence of negative feedback inhibitor dATP and leukemia drug clofarabine nucleotides [ClFD(T)P], hRNR‐a assembles into catalytically inert hexameric complexes, whereas nucleotide effectors that govern substrate specificity typically trigger a dimerization. To date, both knowledge about and tools to interrogate the oligomeric assembly pathway of RNR in any species in real time are lacking. To fill this void, we developed a fluorimetric assay that can reliably report on the oligomeric state changes of a with high sensitivity. The oligomerization‐directed fluorescence quenching of hRNR‐a covalently labeled with two different fluorophores enables the direct readout of hRNR dimeric and hexameric states. Proof of concept was demonstrated with all three known hexamerization inducers: dATP and ClFD(T)P.We then applied the newly developed platform to reveal the timescales of a self‐assembly pathway driven by the feedback regulator dATP. Dimerization of hRNR‐awas found to be the rate‐limiting step along the oligomerization pathway. (Dimerization rate constant: 6×104 M‐1s‐1) Kinetics of the subsequent steps were estimated to be an order of magnitude faster. The study sheds new light on kinetic understanding of hRNR‐a oligomeric regulation in response to feedback inhibitors.
Incorporating Unnatural Amino Acids into the Protein Coat of Adeno‐Associated Virus as New Tools for Virology and Theraputics
Authors: Kelemen Rachel , Chatterjee Abhishek
Published: June 2021

The co‐translational incorporation of genetically encoded unnatural amino acids has emerged as a valuable tool for studying and engineering protein structure and function, but this technique has typically been restricted to the study of individual proteins. We have successfully incorporated unnatural amino acids into the protein capsid of adeno‐associated virus (AAV), facilitating the study and engineering of fully assembled viral particles. AAV is a promising vector for gene therapy and a simple model of parvovirus assembly, infection, and genome release. Using our unique chemical handles for subsequent modification, we have site‐specifically attached a variety of chemical groups including fluorophores and small molecules for viral vector re‐targeting. This method provides a unique and versatile toolset for both studying viral processes and developing vectors which target cells of theraputic interest, and we anticipate that the incorporation of other amino acids will open up new applications.
Casein Kinase 1 Alpha (CK1a) Promotes Nuclear Entry of PERIOD and Represses CLOCK transcriptional activity in the Drosophila melanogaster Circadian Clock
Authors: Lam Vu , Li Ying , Murphy Katherine , Kwok Rosanna , Chiu Joanna
Published: June 2021

Circadian clocks endow organisms with the ability to temporally coordinate and synchronize their physiology with the surrounding environment, thereby conferring an adaptive advantage. Although the specific clock genes are not highly conserved, the basic design and regulation of the circadian oscillator is remarkably similar across species. In Drosophila melanogaster, the central oscillator exists as interconnected transcriptional translational feedback loops that drive and sustain rhythm. Essential to this regulatory network are the transcriptional activator and repressor: CLOCK (CLK) and PERIOD (PER), respectively, as mutations in these proteins result in arrhythmicity. Functional PER and CLK, however, undergo daily cycle of phosphorylation that regulates both the abundance and the timing for which they act as regulators of the circadian transcriptome. To comprehensively identify proteins responsible for these phosphorylation events, we performed proteomics studies using affinity purification (AP) and label‐free quantitative mass spectrometry (MS) using Drosophila S2 cells. We annotate our result by incorporating published PPIs, knowledge about common AP‐MS contaminants, as well as protein coevolution data. We identified Casein Kinase 1 alpha (CK1α) as a clock kinase that phosphorylates CLK to promote CLK clearance at the per promoter and thereby terminating CLK transcriptional activity. Furthermore, CK1α regulates the timing of PER nuclear entry in the cytoplasm, whereas in the nucleus, CK1α phosphorylates and destabilizes PER.
A Newly Uncovered PPDK Regulatory Protein Isoform in Maize Catalyzes Regulatory Phosphorylation but not Dephosphorylation of PPDK
Authors: Chastain Chris
Published: June 2021

The PPDK regulatory protein (PDRP) catalyzes the reversible phosphorylation of a Thr residue located within the central catalytic active‐site of pyruvate phosphate dikinase (PPDK). In higher plants, PPDK is an integral enzyme of the C4 photosynthetic pathway and therefore its concomitant up‐/down phospho‐regulation by PDRP is believed to be critical for optimizing photosynthetic efficiency. PDRP is a member of the bacterial and plant DUF‐299 family of proteins now recognized from 1° structure as either PDRP or PEP synthetase regulatory protein (PSRP). The catalytic properties of PDRP (and PSRP) are singularly unique among protein phosphorylating/dephosphorylating enzymes. Among these are: (i) bifunctionality, catalyzing both PPDK phosphorylation and dephosphorylation, (ii) use of ADP versus ATP as a phosphoryl donor, and (iii) utilization of an Pi‐dependent, PPi‐forming phosphorolytic dephosphorylation mechanism. Notably, the PDRP 1° structure is devoid of any canonical subdomains or motifs that unifies all known eukaryotic and prokaryotic Ser/Thr protein kinases into one of three superfamilies. Recently, we identified a second PDRP isoform in maize (PDRP2) that lacks the phospho‐PPDK dephosphorylating activity of its bifunctional counterpart, PDRP1. Alignment of the highly similar PDRP1 and PDRP2 primary sequence does not reveal any discernable residue differences that could account for the lack of dephosphorylating activity of PDRP2 vs. PDRP1. We conclude that (3‐D) structural features not evident in the primary structures of PDRP1 and PDRP2 likely accounts for the stark contrast in catalytic properties of two isoforms.
Investigating the Use of Targeted Therapies for Triple‐Negative Breast Cancer
Authors: Bindeman Wendy , Redwood Abena , Lukose Benjamin , Piwnica‐Worms Helen
Published: June 2021

Triple‐negative breast cancer (TNBC) accounts for 15‐20% of all diagnosed breast cancers. It is characterized by lack of expression of estrogen receptor and progesterone receptor and an absence of HER2 amplification. TNBC is aggressive, highly metastatic, and resistant to current chemotherapies. TNBC frequently harbors mutations in TP53, suggesting that therapies that exploit this vulnerability may be an effective treatment option. In particular, p53‐deficient cells are hypothesized to be highly sensitive to inhibitors of checkpoint kinase 1 (Chk1) due to checkpoint abrogation. We have evaluated the use of mammosphere cultures as an in vitro system for testing the sensitivity of TNBC tumor cells to chemotherapy and targeted therapy. We hypothesized that mammospheres would display higher levels of chemo‐resistance than adherent cells, due to their ability to enrich for tumor initiating cells (TICs), which have been shown to display chemo‐resistance. However, we reasoned that molecularly targeted therapies should maintain their effect in TICs, since the pathways that are targeted would remain vulnerable. To test these hypotheses, we evaluated the effect of cisplatin (DNA damaging agent, chemotherapy) and LY2606368 (inhibitor of Chk1, targeted therapy) on the viability of TNBC cells. Our data suggest that the TIC population of a tumor may retain its sensitivity to targeted therapy despite resistance to cytotoxic chemotherapy. Further studies are necessary to determine if targeted therapy can impair TIC self‐renewal capacity, to evaluate combination therapies, and to verify these results in vivo.

Funding: Komen Foundation; Cancer Prevention & Research Institute of Texas
Evaluating a Dichloroacetate and Metformin Combination in a Mouse Model of Metastatic Cancer
Authors: Ward Nathan , Poff Angela , Van Putten Ashley , Seyfried Thomas , D'Agostino Dominic
Published: June 2021

The increased focus on targeting the glycolytic dependency of cancer cells has yielded a number of new therapeutic options with varying promise. Among the therapies currently being investigated thoroughly are dichloroacetate (DCA) and metformin. These agents have been repurposed from other metabolic disorders because of their activities in altering glucose metabolism. Both have exhibited toxicity against a number of cancers without damaging normal tissue, and are also shown to enhance the efficacy of various chemotherapeutics. DCA, an inhibitor of pyruvate dehydrogenase kinase, promotes glucose oxidation through the activation of pyruvate dehydrogenase and subsequent flux of pyruvate through the TCA cycle. Metformin is shown to indirectly activate AMPK through inhibition of complex I of the mitochondrial respiratory chain. We hypothesize that combining these agents will severely alter energy metabolism in tumor cells, resulting in mitochondrial instability and cancer‐specific cell death. We tested the therapeutic efficacy of DCA and metformin in the VM‐M3 model of metastatic cancer. Dietary administration of 500 mg/kg doses of DCA and metformin significantly increased mean survival time (69%, p<.01 and 58%, p<.05 respectively) over untreated control animals. Tumor volume and metastatic spread were reduced in treated animals compared to controls. In vitro incubation with DCA and metformin resulted in a synergistic reduction of VM‐M3 proliferation and viability (p<.05). These data support the further study of a DCA and metformin combination as a potential metabolic therapy for cancer.
Metabolic actions of metformin in cancer cells and a distinction of metformin from the effects of rotenone, a mitochondrial complex I inhibitor
Authors: Parakhia Rahul , Parras Aldrin , Gadhia Sanket , Barile Frank , Ochs Raymond
Published: June 2021

Metformin, the most widely prescribed anti‐diabetes drug, is correlated to a decreased cancer incidence in diabetes patients, and inhibits cancer cell growth in vitro. We examined metabolic actions of metformin in two cell lines: a pluripotent line from an embryonic carcinoma and a metastatic protstate line. Results with both lines were similar. Metformin increased glucose uptake and lactate formation in these cells, while an inhibitor of phosphofructokinase (acting at PFK‐2) inhibited these processes. Metformin stimulated glucose uptake and lactate formation in the presence of the PFK inhibitor. While metformin is known to stimulate fatty acid oxidation in normal cells, it inhibited this process in the cancer cells. This was traced to a metformin induced decrease in cell viability in the cancer cells in the presence of fatty acids. Decreased cell viability was also observed with rotenone, a known mitochondrial complex I inhibitor. We compared the ability of metformin and rotenone to influence the action of rapamycin on cell viability. Unlike metformin, rotenone further decreased the viability of cells in the presence of rapamycin, suggesting the action of rotenone is not limited to inhibition of mTOR. Finally, we found that Compound C, a known inhibitor of AMPK phosphorylation, suppressed the effects of metformin on glucose uptake. We conclude that metformin exerts anti‐cancer effects apart from Complex I, but involving AMPK and mTOR signaling.
Investigation of the Interaction and Role of CHP1 and CHP2 with NHE1 in Lung Fibroblasts
Authors: Kiefer Lea , Wallert Mark , Provost Joseph
Published: June 2021

Intracellular pH (pHi) homeostasis is crucial for cell survival. The membrane protein Sodium‐Hydrogen exchanger 1 (NHE1) is one of the major contributors to a stable pHi. Additionally, NHE1 serves as an anchor for intracellular proteins. NHE1 also plays an essential role in Non‐small cell lung cancer (NSCLC) metastasis as a side effect of the transporter's increased activity to compensate the Warburg effect. Several phosphorylation sites and protein cofactors affect NHE1's activity. The two isoforms calcineurin homologous proteins CHP1 and CHP2 each bind to the C‐terminus of NHE1 and regulate its function. CHP1 is ubiquitous and is key for basel level NHE1 activity while CHP2 is primarily expressed in gut cells, detected in most cancer cells and increases proton transport. To better understand the role of NHE1 and CHP1/CHP2 we generated RFP‐CHP1 and GFP‐CHP2 and transfected each into lung fibroblasts expressing tagged NHE1 (PSN cells) and into lung fibroblasts lacking NHE1 (PS120 cells). Cellular distribution of each CHP was determined and showed significant translocation of CHP1 and CHP2 to the plasma membrane in PSN cells while no localization was observed in PS120 cells. The specific amino acid required for CHP‐NHE1 interaction is determined in cells expressing one of eight different NHE1 amino acid substitutions at the putative CHP‐NHE binding domain of NHE1. To investigate the role of CHP2 binding to NHE we expressed GFP‐CHP2 in PSN and PS120 cells and conducted wound‐healing assays. In addition to cell migration rates, we tracked the migration path of individual cells to assess the impact of each NHE1 mutation on CHP2‐influenced motility.
Pro‐tumorigenic Effects of Hydrogen Sulfide (H2S) on Normal Colonic Fibroblasts (NCF) and Colorectal (CRC) Cancer‐Associated Fibroblasts (CAF)
Authors: Chao Celia , Dikmen Aycan , Coletta Ciro , Pang Lan , Szabo Csaba , Hellmich Mark
Published: June 2021

Introduction

Bidirectional paracrine signaling between cancer cells and fibroblasts in the stroma facilitates tumor progression. We have shown that CRC cells upregulate cystathionine β synthase (CBS), resulting in H2S production, when compared to normal colonic epithelium, and that CRC‐derived H2S increases CRC growth, migration, and tumor angiogenesis. The aims of this study are to determine whether H2S can promote CAF proliferation and migration, and stimulate NCF to develop into a CAF‐like phenotype.

Methods

Human CAF from CRC and NCF were collected under an IRB approved protocol and established in culture. CAF were grown for up to 120h in the presence of vehicle or a slow‐release H2S donor, GYY4137. CAFs were allowed to migrate through transwell chambers toward conditioned media (CM) from HCT116 or normal colon mucosal cells (NCM356). Western blots for NCF and CAF were performed to determine CBS expression levels.

Results

H2S donor GYY4137 (30‐300μM) significantly increased proliferation of CAF, but not NCF. CAF migrate toward HCT116 CM, but not NCM356 CM. CAF migrate significantly less toward CM from HCT116 cells with shRNA‐mediated silencing of CBS or CBS inhibitor aminooxyacetic acid. NCF cells express less basal CBS protein compared to CAF cells. NCF cells can be induced to express CBS enzyme after exposure to HCT116 CM for 48h.

Conclusions

H2S enhances CRC CAF cell proliferation and migration. Furthermore, when NCF cells are exposed to CRC CM, the NCF cells induce expression of CBS enzyme. The results support the hypothesis that the gasotransmitter H2S promotes pro‐tumorigenic activities in the CRC microenvironment.
Hypoxia‐induced carbonic anhydrase IX facilitates lactate transport in human breast cancer cells by non‐catalytic interaction
Authors: Becker Holger , Jamali Somayeh , Klier Michael , Barros Luis , McKenna Robert , Deitmer Joachim
Published: June 2021

The most aggressive and invasive tumor cells, which often reside in hypoxic environments, rely on extensive glycolysis to meet their large demand for energy and biosynthetic precursors. Thereby they release vast amounts of lactate and protons via monocarboxylate transporters (MCTs), which exacerbates extracellular acidification and supports the formation of a hostile environment. We have studied the mechanisms that regulate lactate transport in MCF‐7 human breast cancer cells under normoxia and hypoxia. Under hypoxia, expression of MCT1 and MCT4 remained unchanged, while expression of carbonic anhydrase IX (CAIX) was greatly enhanced. Measurements of intracellular pH and lactate concentration with the FRET‐based lactate nanosensor laconic show that CAIX augments lactate flux via MCT1 by a non‐catalytic interaction. Mutation studies in Xenopus oocytes indicate that CAIX, via its intramolecular H+‐shuttle His200, functions as a “proton‐collecting/distributing antenna” to facilitate rapid lactate flux via MCT1. Knockdown of CAIX significantly reduced proliferation of MCF‐7 cancer cells, suggesting that rapid efflux of lactate and H+, as enhanced by CAIX, contributes to cancer cell survival under hypoxic conditions.

This work was supported by the Research Initiative Membrane Biology, the Stiftung Rheinland‐Pfalz für Innovation, the Graduiertenfördungskommission Rheinland‐Pfalz, and the DFG.
Hexabromocyclododecane and Tetrabromobisphenol A Alter Secretion of Interferon Gamma (IFNγ) from Human Immune Cells
Authors: Almughamsi Haifa , Whalen Margaret
Published: June 2021

Hexabromocyclododecane (HBCD) and Tetrabromobisphenol A (TBBPA) are brominated flame retardant compounds. HBCD is used in polystyrene insulation, accumulates in living organisms and is highly toxic to aquatic organisms. TBBPA is mainly used in plastic, epoxy resin printed circuit board, and other electronic equipment. Interferon gamma (IFNγ) is an inflammatory cytokine that is produced by activated T cells and NK cells and regulates immune responsiveness. HBCD and TBBPA have been found in human blood and previous studies have shown that they alter the ability of human natural killer (NK) lymphocytes to destroy tumor cells. This study examines whether HBCD and TBBPA affect the secretion of IFNγ from human purified NK cells, monocyte‐depleted (MD) human peripheral blood mononuclear cells (PBMCs), and PBMCs. IFNγ secretion was measured by enzyme linked immunosorbent assay (ELISA). Results indicate that exposures of NK cells, MD‐PBMCs and PBMCs to different concentrations of HBCD and TBBPA (ranging from 5‐0.05 µM) for 24h, 48h, and 6 days influence the ability of immune cells to secrete IFNγ. Certain concentrations of HBCD caused increases in IFNγ secretion after 24 and 48 h exposures (all cell preparations). However, TBBPA decreases secretion of IFNγ from NK cells and MD‐PBMCs but at certain concentrations is able to increase secretion of IFNγ from PBMCs. Thus, exposure to these compounds may potentially disrupt the immune regulation mediated by IFNγ. Additional studies were carried out addressing the signaling pathways that may be involved in HBCD‐induced increases in IFNγ secretion.
The APP Interactome: Identifying Targets in Alzheimer's Disease
Authors: Glucksman Marc , Philibert Keith , Marr Robert , Norstrom Eric
Published: June 2021

Alzheimer's disease (AD) is a horrific neurodegenerative disorder affecting the elderly with progressive mental decline and profound effects on memory. Accumulating evidence indicates that the failure to clear the 40‐42 a.a. Aß peptides from the brain is a primary step in the pathology of sporadic AD. We utilized proteomic approaches involving isolation of physiologically relevant macromolecular complexes in vivo of proteins interacting with Amyloid Precursor Protein (APP) or the carboxy terminally located Amyloid IntraCellular Domain (AICD). After in vivo tandem affinity purification in cell culture models, downstream biophysical characterization and mass spectrometry identified individual interactors currently being validated. A new peptidase activity exhibited by metalloendopeptidase, EC 3.4.24.15 (EP24.15) was identified with the ability to degrade Aß at 3 sites and is different from identified activities of neprilysin 1/2 and insulysin. Transthyretin, a carrier protein transporting thyroid hormones was identified and interactions with Aß were quantified in the µM range with ramifications to inhibit Aß multimer formation. Validation has commenced with the target NEEP21, a neuronally expressed single‐pass transmembrane protein was expressed in lentiviral transduced cells and animals to examine the effects on Aß production. Taken together this data suggests that explicating interactions with APP and AICD can have effects on neurotoxic products and may aid in developing effective pharmacological approaches for people afflicted by AD.

Support: RFUMS/DePaul Pilot program and NIH OD010662 (MJG) to the Midwest Proteome Center.
Investigation of the Cellular and Molecular Mechanisms of Facial Branchiomotor Neuron Migration
Authors: Burkard Alexander , Walsh Gregory
Published: June 2021

A fundamental feature of brain development is the migration of neurons from their place of birth to the location where they will assemble and integrate into a neural circuit. We have recently reported that one facial branchiomotor neuron (FBMN) can influence the migration of another FBMN, defining this cell movement as a collective migration. As with any collective migration, cell‐to‐cell contact is required for one cell's influence on another. To determine if this was the case, we imaged the migration of membrane‐mCherry‐expressing FBMNs in live zebrafish embryos. We find that some neurons display persistent, sustained migration in one direction (posterior), whereas other FBMNs stall for short periods of time. We noticed that FBMNs stalled in migration shortly after cell‐to‐cell contact with neuron in their migratory path. This is reminiscent of contact inhibition of locomotion (CIL) observed for many other cell types including neural crest cells. We therefore, quantified the speed and direction of neuron movement before and after neuron‐to‐neuron cell contacts. Consistent with this, centrosomes, which often re‐orient in the direction of migration, were often found on the anterior side of migrating FBMNs. Previous work has shown that planar cell polarity (PCP) genes are essential for FBMN migration. Here we show that PCP mutant neurons migrate in random directions suggesting that PCP proteins are not required for migration per se, but are required for persistent directed movements. Taken together these data suggest the possibility that PCP proteins control contact‐dependent cell polarization in FBMN migration.
Role of Negative Charge on C2 Insert of Nonmuscle Myosin II‐C: Intermittent Structural Oscillation and Aggregation in Neuritogenesis
Authors: Saha Shekhar , Chattoraj Shyamtanu , Halder Debdatta , Goswami Swagata , Bhattacharyya Kankan , Jana Siddhartha
Published: June 2021

Previous studies reported that inclusion of 41 aa (33 aa in human) insert in loop 2 of mouse nonmuscle myosin (NM) II‐C heavy chain makes the molecule to become independent of its regulatory light chain (RLC) phosphorylation in MgATPase activity and in vitro motility, and to localize in the neurites as puncta form. However, the molecular mechanism behind these unique properties among myosin II family is not known. Here, we show that deletion of the N‐terminal 12 aa, but not the C‐terminal 8 aa or the middle 21 aa of C2 insert, diminishes the fluorescence oscillation property of GFP tagged‐NM II‐C2 molecule in the neurites of neuro‐2a cells as determined by fluorescence lifetime imaging microscopy analysis. Also, we demonstrate that inclusion of 41 aa insert increases the population of extended monomers whereas deletion of N‐terminal 12 aa, middle 21 aa or C‐terminal 8 aa of 41 aa insert increases the folded autoinhibited NM II‐C2 monomers as revealed by fluorescence correlation spectroscopy (FCS) analysis in neuronal cells. Furthermore, FCS analysis reveals that NM II‐Δ12C2‐GFP significantly diffuses very slowly compared with NM II‐Δ8C2‐ or NM II‐Δ21C2‐GFP in the neurites, suggesting that radius (rH) of NM II‐Δ12 C2‐GFP is higher than that of wildtype or mutants Δ21 and Δ8C2‐GFP. These studies suggest that N‐terminal 12 aa (contributor of net charge of C2) plays a significant role in determining the oscillation and aggregation properties of the molecule.

Research Support: Department of Science and Technology, India
Inflammatory Lipid Sphingosine‐1‐phosphate Upregulates c‐reactive Protein via C/EBPßß and Potentiates Breast Cancer Progression
Authors: Moon Aree , Kim Eun‐Sook , Cha Yujin , Ham Mina , Bae Mijeong , Lee Hyemin , Jung Joohee , Kim Sang Geon , Hwang Sejin , Kleemann Robert
Published: June 2021

The present study aimed to elucidate the molecular link between S1P and CRP during the invasive process of breast epithelial cells. This is the first report showing that transcription of CRP was markedly activated by S1P in breast cells. Our data suggest that not only S1P treatment but also the endogenously produced S1P may upregulate CRP in breast carcinoma cells. Transcription factors CCAAT/enhancer‐binding protein beta (C/EBPß) and c‐fos were required for S1P‐induced CRP expression. Coupling of S1P3 to heterotrimeric Gαq triggered the expression of CRP, utilizing signaling pathways involving reactive oxygen species (ROS), Ca2+, and ERKs. S1P‐induced CRP expression was crucial for the transcriptional activation of matrix metalloproteinase (MMP)‐9 through ERKs, ROS and c‐fos, leading to breast cell invasion. Using a xenograft mice tumor model, we demonstrated that S1P induced CRP expression both in vitro and in vivo. Taken together, our findings have revealed a molecular basis for S1P‐induced transcriptional activation of CRP and its functional significance in the acquisition of the invasive phenotype of human breast epithelial cells under inflammatory conditions. Our findings may provide useful information on the identification of useful therapeutic targets for inflammatory breast cancer.
Cell type‐specific expression of the human RAP1A and RAP1B genes
Authors: Cruise Jennifer , Niemela Amy , Augustin Ryan , Painter Mark , Hindt Maria
Published: June 2021

Despite their important roles in cell adhesion, motility, immune function, and other processes, little is known about the expression control of the Ras‐family small GTPases RAP1A and 1B, located on human chromosomes 1 and 12, respectively. Although altered expression has been noted in a number of cancers and other disease states, no characterization of these genes' basal promoters, or of their proximal or distal regulatory elements has been reported. We examined native expression of both genes in a set of eight human cell lines, using qRT‐PCR. Human umbilical vein endothelial cells (HUVEC) strongly expressed RAP1A, and Saos‐2 osteosarcoma cells were high expressers of RAP1B. MCF‐10A cells expressed both genes at a moderately high level. Promoter‐less GFP reporter constructs and qRT‐PCR were used to identify functional core promoters for each gene. RAP1A constructs identified a sequence upstream of the TSS containing a negative regulatory element that is functional in more than one cell type. While driving GFP expression in other cell types, RAP1B proximal promoter constructs did not function well in Saos‐2 cells, suggesting that the high native expression observed in that line is under the control of distal elements. Three putative distal regulators have been cloned and are under examination.
EC 3.4.24.15 is a Putative Regulator of Kisspeptin Signaling
Authors: Woitowich Nicole , Philibert Keith , DeJoseph Mary , Urban Janice , Glucksman Marc
Published: June 2021

Inadequate kisspeptin (Kiss) secretion or signaling results in delayed puberty and infertility, indicating the importance of this peptide in controlling reproduction. Regulating Kiss, or peptide concentrations in general, are modulated by enzyme hydrolysis altering their downstream signal. The metalloendopeptidase EC 3.4.24.15 (EP24.15) has been demonstrated to play the key role in the cleavage and subsequent regulation of the reproductive hormone Gonadotropin Releasing Hormone (GnRH) in vitro and in vivo that is downstream of Kiss. The goal of these studies borne from structural analyses and in silico molecular modeling was to determine if Kiss is a substrate of EP24.15 by first identifying the cleavage site and enzyme kinetics, and then if EP24.15 and Kiss are co‐expressed in brain regions relevant to reproductive function. EP24.15 and Kiss were co‐incubated and subjected to matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry to confirm that EP24.15 can cleave Kiss in vitro and enzyme kinetics performed. Double‐label immunohistochemistry demonstrated Kiss and EP24.15 immunoreactivity within the hypothalamus and specifically EP24.15 and Kiss immunoreactivity was co‐expressed within neurons and fibers of the arcuate nucleus. Taken together this data suggests that EP24.15 may act to cleave Kiss in vivo and represents an additional facet of the control mechanism of mammalian reproduction. Understanding the regulation of Kiss by EP24.15 may provide insight into the development of novel therapeutic targets for the treatment of infertility.

Supported by RFUMS Pilot Grant Program (JHU, MJG); NIH OD010662 (MJG).
MED28 Modulates Cell Cycle Progression in Human Breast Cancer Cells
Authors: Li Chun‐I , Hsieh Nien‐Tsu , Huang Chun‐Yin , Chang Hsueh‐Chen , Lee Ming‐Fen
Published: June 2021

Mediator subunit MED28 is found highly expressed in several types of tumor including breast cancer. Previously we have reported that MED28 regulates cellular migration and functions as an upstream regulator of the NFkB/MMP9 axis upon EGF stimulationin human breast cancer cells. In the current study we investigated the effect of MED28 on cell growth in human breast cancer cells. Suppression of MED28 expression inhibited cell growth accompanied by lowering the expression of p‐RelA/p65, cyclin D1, and p‐pRb as well as the transcriptional activity of NFkB, but increasing the expression of FOXO3a and p27. Overexpression of MED28 promoted G1 to S transition and, thereby, stimulated cell growth. Our data indicate that MED28 modulates cell growth through FOXO3a and NFkB in human breast cancer cells. Not only is MED28 involved in cellular migration and invasion but also cell cycle progression in human breast cancer cells, reiterating the significance of this multifaceted protein in breast cancer.

(This work was supported by the grants NSC101‐2320‐B‐309‐001 and NSC102‐2320‐B‐309‐001‐MY3 to M‐F Lee.)
Identification of Fyn‐Induced PKA Binding Partners Using Quantitative Proteomics
Authors: Weir Marion , Mann Jacqueline , Ballif Bryan , Deming Paula
Published: June 2021

Protein Kinase A (PKA) is a serine/threonine kinase involved in many cellular processes including migration and proliferation. PKA is known to phosphorylate and activate members of the Src family of tyrosine kinases including Fyn, and new data from our laboratory indicate that Fyn can reciprocally regulate PKA. We hypothesized that co‐expression of Fyn with PKA would induce PKA to bind to and phosphorylate a distinct set of substrates such as scaffolding and signaling molecules. To test this, we used Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC) and mass spectrometry to identify PKA binding partners that are dependent on the expression of Fyn. Several Fyn‐induced binding partners were identified. Further biochemical analyses have been performed for validation and characterization of these distinct signaling complexes.
A Novel Mechanism for 2‐Deoxyglucose Toxicity is Mediated by Glucose Transporter Endocytosis Regulated by Arrestins and AMP‐Activated Protein Kinase
Authors: Schmidt Martin , O'Donnell Allyson
Published: June 2021

Both cancer cells and yeast cells are inhibited by the presence of low concentrations of 2‐deoxyglucose (2DG) although its modes of action have not been fully elucidated. Snf1 kinase, the AMP‐activated protein kinase of yeast, is required for 2DG resistance in cells growing on glucose. Mutations in the SNF1 gene that reduce kinase activity render cells hypersensitive to 2DG, while an activating mutation in SNF1 confers 2DG resistance. Overexpression of either Hxt1 or Hxt3, low‐affinity high‐capacity glucose transporters, suppresses the 2DG hypersensitivity of snf1Δ cells. Addition of 2DG or loss of Snf1 reduce HXT1 and HXT3 expression and stimulate transporter endocytosis and degradation in the vacuole. 2DG‐stimulated trafficking of Hxt1 and Hxt3 requires Rod1 and Rog3, two members of the α‐arrestin family of trafficking adaptors. Mutations in ROD1 and ROG3 that block binding to Rsp5, the yeast NEDD4 ubiquitin ligase, eliminate Rod1‐ and Rog3‐mediated trafficking of Hxt1 and Hxt3. Genetic analysis suggests that Snf1 negatively regulates both Rod1 and Rog3. We propose a novel mechanism for 2DG‐induced toxicity whereby 2DG stimulates modifications of α‐arrestins, which promote glucose transporter internalization and degradation, resulting in glucose starvation, even when cells are in a glucose‐rich environment. Moreover, given recent evidence that endocytosis of a mammalian glucose transporter is under the control of an α‐arrestin and AMPK, our observations suggest that 2DG toxicity in cancer cells may be due to alterations in the endocytosis of glucose transporters.
An Optogenetic Approach to Delineate Spatial Regulation of Plexin Signaling
Authors: Deb Roy Abhijit , Yin Taofei , Wu Yi
Published: June 2021

Plexins are primary receptors of Semaphorins, and transduce guidance signals during embryonic development and tissue remodeling. Previous studies using soluble Semaphorins provide a limited understanding of how intracellular signaling events downstream of Plexins are spatially regulated. Here we developed an optogenetic approach, namely optoPlexin, based on the plant cryptochrome‐2 to specifically activate Plexins at precise subcellular locations with light. Using Plexin‐B1 as an example, we examined the spatiotemporal dynamics of Semaphorin‐4D signaling in osteoblastic MC3T3‐E1 cells. We found that activation of optoPlexin in lamellipodial protrusions led to rapid, local recruitment of PDZ‐containing RhoGEFs, including PDZ‐RhoGEF and LARG (Leukemia associated RhoGEF), subsequent accumulation of myosin and finally, collapse of these protrusions. Biosensors of Rho GTPases identified the specific induction of RhoA activity upon optoPlexin activation. Activation of RhoA pathway required both the presence of a PDZ binding motif in optoPlexin and the interaction of optoPlexin with Rnd1 or Rac1 on the membrane, preserving the specificity of Plexin‐B1 signaling. Activation of optoPlexin also induced redistribution of β‐Pix from sites of illumination to distal regions and concomitant production of new protrusions. We observed activation of Cdc42, rather than Rac1, in these distal protrusions. The optoPlexin‐induced coordination among Rho GTPases was dependent on ROCK kinase activity and myosin‐dependent contractility. These results revealed spatially distinct effects of Plexin‐B1 signaling in regulating Rho GTPases and cell migration, and highlighted the utility of optogenetic approaches in studying spatiotemporal regulation of cellular signaling.
Maternal High Fat Diet‐induced Il‐12b Overexpression in Male Offspring rats is Associated with DNA Hypomethylation
Authors: Hernández‐Saavedra Diego , Zhou Dan , Pan Yuan‐Xiang
Published: June 2021

Both developmental and postnatal exposure to a high‐fat (HF) diet has been linked to the development and progression of non‐alcoholic fatty liver disease (NAFLD), a condition characterized by an increase in inflammatory cytokine release from hepatocytes and resident macrophages (MΦ). This activates the innate immune system and leads to MΦ polarization from a phagocytic phenotype towards a pro‐inflammatory response (M1), with MΦ interleukin‐12b (Il12b) as the specific marker of the inflammatory hepatic milieu. To determine the dietary regulation of the pro‐inflammatory switch in liver by a maternal and/or postweaning HF diet, female Sprague‐dawley ratswere fed maternally with either a control (C) or high‐fat diet (HF). At weaning, male pups were assigned to either the HF or C diet, which generated four groups with distinct HF dietary exposures: control (C/C), maternal (HF/C), postweaning (C/HF), and lifelong (HF/HF). All HF groups had increased steatosis and activation of the innate immunity in response to a HF. The hepatic mRNA expression of the Il12b gene was increased in all HF groups. This increase was correlated with marked DNA hypomethylation within the coding region flanking a CpG island identified using MeDIP‐seq technology. We conclude that maternal HF exposure regulates hepatic pro‐inflammatory switch of the innate immune system possibly through long lasting changes in DNA methylation in male offspring, and thus contributes to the initiation and progression of NAFLD.
Effect of Zinc & Multiple Micronutrient Supplements on Growth in Tanzanian Children
Authors: Locks Lindsey , Manji Karim , McDonald Christine , Kupka Roland , Kisenge Rodrick , Aboud Said , Wang Molin , Spiegelman Donna , Fawzi Wafaie , Duggan Christopher
Published: June 2021

Background

Poor child growth increases the risk of mortality and impaired development.

Objective

To determine the effect of daily zinc and/or multivitamin (vitamins B, C & E) supplements on the growth of infants in Tanzania.

Methods

In this randomized, 2x2 factorial, double blind clinical trial, 2400 infants were randomized to zinc (Zn), multivitamins (MV), both supplements (Zn + MV), or placebo (P) at age 6 weeks and followed for 18 months.

Results

At 19 months of age, 20%, 6% and 11% of children were stunted, wasted and underweight respectively. Median compliance was 96% of the allocated regimen. Mean change in weight‐for‐age z‐scores (WAZ) in the Zn + MV, Zn, MV and P groups were ‐0.36, ‐0.61, ‐0.52, ‐0.50 respectively (p for interaction <0.001). Mean change in weight‐for‐height (WHZ) in the four groups were ‐0.31, ‐0.57. ‐0.42, ‐0.35 (p for interaction <0.001). There was no significant effect of treatment on change in height‐for‐age (HAZ), nor on rates of stunting, wasting or underweight.

Conclusion

Despite the small effect on mean change in WAZ & WHZ, daily provision of MV and/or Zn supplements to Tanzanian infants beginning in early infancy did not reduce rates of underweight, stunting or wasting over 18 months of follow‐up. Alternative approaches to prevent growth faltering in vulnerable populations should be pursued.
High Sodium Intakes are Associated with High Blood Pressure in South‐Indians
Authors: Bermudez Odiia , Ravi Sripriya , Thanikachalam Mohan , Vanzan Hari , Vasudevan Preethi , Must Aviva , Thanikachalam Sadagopan
Published: June 2021

Objective

To explore the association between sodium intake and hypertension in adults from Tamil Nadu, South India.

Methods

A sample of 8080 men and women (> 20y) recruited for the study was assessed for anthropometry, blood pressure (BP) and key blood parameters. Dietary data were collected using 24‐hr recalls. The association of sodium (Na) intake and BP was analysed using t‐tests. Correlations of Na intake with systolic (SBP) and diastolic (DBP) BP and key variables were estimated.

Results

Compared to recommended intakes <2 g/d of Na, mean NA intakes in the study group was higher at 3g/d, with higher intakes for men than for women (4 vs. 3 g/d, p<0.05), and also for subjects from urban, semi urban and rural locations (3.5, 3.2 and 2.6 g/d, respectively). Those intakes were different (p<0.01) in hypertensive vs. normotensive men (3.7 vs. 3.4 g/d) and women (2.8 vs. 2.7g/d).

Higher Na intakes were associated with increasing waist circumference, BMI, body fat%, higher SES, anxiety and stress levels and lower physical activity levels. With increasing NA intakes there was a trend in increases in prevalence of hypertension (HBP) in both men and women.

Both SBP and DBP increased with increase in quintiles of Na intakes, more in men than women: SBP increased from 123 ± 18 to 128 ± 19 (p<0.01) and DBP increased from 77 ± 11 to 79 ± 12 (p<0.01) in men, while in women SBP increased from 120 ± 20 to 120 ± 20 to (p<0.01) and DBP increased from 75 ±11 to 75 ±11 (p >0.05).

Conclusions

The association between higher Na intakes and HBP is evident. These findings suggest a pressing need for community‐based efforts in the prevention and control of HBP to avert an epidemic of cardiovascular disease.