bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased protein synthesis of IRES containing mRNAs, that induce cell survival and proliferation, by the daidzein metabolite equol may contribute to breast cancer malignancy.
Authors: de la Parra Columba , Dharmawardhane Suranganie
Published: June 2021

The effects of soy isoflavones on established breast cancer remain to be understood. We recently reported that dietary daidzein increased primary mammary tumor growth and metastasis, and upregulated eukaryotic protein synthesis initiation factors (eIF) eIF4G and eIF4E. The increased eIF expression in tumor extracts of mice after daidzein diets was associated with protein expression of mRNAs with internal ribosome entry sites (IRES). Results with metastatic cancer cell lines show that the effects of daidzein in vivo can be recapitulated by its metabolite equol. Equol increased metastatic cancer cell viability and up‐regulated gene and protein expression of the transcription factor c‐Myc. In vitro, equol, but not daidzein, up‐regulated eIF4G without affecting eIF4E or its regulator, 4E‐BP, levels. This elevated eIF4G was not associated with eIF4E or 4E‐BP in cap affinity chromatography assays or co‐immunoprecipitations. In dual luciferase assays, IRES‐dependent protein synthesis was increased by equol. As shown by western blotting and polysomal associations, equol specifically increased the expression of IRES‐containing mRNAs of cancer promoting molecules. Therefore, upregulation of eIF4G by equol may result in increased translation of pro‐cancer mRNAs with IRESs and, thus, promote cancer malignancy. Supported by US Army/BCRP W81XWH‐11–1‐0199 to CD and NIH/NIGMS SC3GM084824 to SD.
Binding of Eukaryotic Initiation Factor (eIF) 4F to Barley Yellow Dwarf Virus (BYDV) 3′ UTR Correlates Well With Protein Synthesis Efficiency
Authors: Banerjee Bidisha , Goss Dixie J.
Published: June 2021

BYDV belonging to the luteoviridae family is an economically important plant virus lacking a 5′ cap and 3′poly (A) tail and is translated by cap‐independent translation (CIT) mechanism. The BYDV 3′ Untranslated Region (UTR) translation enhancer element (BTE) stimulates CIT initiation at the 5′upstream AUG via base‐pairing (kissing stem loop interaction) to a complementary loop, 5′UTR SL‐D. 3′ BTE recruits eIFs and transfers them to the 5′ end of the viral RNA where translation initiates.

Wild type BTE and BTE mutants with in‐vitro translation efficiencies from 5–164 % were studied. eIF4F binding is the first committed step in cap‐dependent protein synthesis. The role of eIF4F in viral protein synthesis is not fully understood.eIF4F binding to BTE and BTE mutants gave equilibrium binding constant values (Kds) which are a measure of relative complex stability. We show: 1) translation efficiencies correlated well with eIF4F binding with one exception; 2) Kinetics, higher activation energy and interaction of other eIFs contribute; 3) BTE and BTE mutants‐eIF4F interaction is both enthalpically (52–90%) and entropically favorable with more enthalpic contribution to ΔG° at 25°C. The stability measurements and kinetics determine the nature of interactions and provides unique information on the sequential assembly of the initiation complex and it's binding to viral mRNA.

Grant Support: NSF MCB 1157632
Mechanism of Activator (eIF4F) and Suppressor (IRP) Binding to Iron Responsive Element mRNA
Authors: Ma Jia , Khan Mateen , Merrick William C , Theil Elizabeth C. , Goss Dixie J.
Published: June 2021

Cellular iron metabolism is regulated by expression of iron responsive element (IRE) containing‐mRNAs to control iron related protein levels (e.g. ferritin). The cytoplasmic iron regulatory proteins, IRPs, repress IRE‐mRNA translation by binding to the iron response element (IRE). Our previous study (Ma et al. (2012) PNAS 109: 8417–8422) showed that Fe2+ decreased IRP (suppressor)/IRE binding affinity and increased eIF4F (activator)/IRE binding affinity. Fluorescence studies showed that Fe2+can bind to the IRE‐RNA structure and induce conformational changes. These conformational changes affect IRP/IRE interacting bases and consequently affect protein binding. Mn2+(O2resistant Fe2+surrogate) both decrease the association rate (kon) for IRP and increases the dissociation rate (koff), lowering the binding affinity of IRP for IRE‐RNA. In contrast to IRP/IRE binding, Fe2+increases eIF4F/IRE binding and eIF4F:IRP competitively bind to the IRE. Kinetic and thermodynamic data suggest a model for IRE‐RNA riboregulator function, illustrating the potential of specific metal ion interactions with folded untranslated mRNA structures for gene regulation.

Grant support: NSF MCB 1157632 NIH DK20251
Enhancement of ubiquitin conjugating activity promotes the clearance of aggregation‐prone mutant proteins in living cells
Authors: Shang Fu , Liu Zhenzhen , Taylor Allen , Zhang Xinyu , Wu Mingxin , Liu Yizhi , Liang Jack J.N.
Published: June 2021

Intracellular accumulation and aggregation of damaged proteins is a common cause of many age‐related degenerative diseases. In this study, we investigated whether enhancement of the ubiquitin‐proteasome pathway (UPP) promotes clearance of damaged proteins and prevents their intracellular accumulation and aggregation. A conformational‐defect T5P mutant γC‐crystallin (T5P‐γC ) was used as a model substrate. Similar to other damaged proteins, T5P‐γC was degraded by the UPP more rapidly than wild type γC‐crystallin (wt‐γC). Supplementation of C‐terminus of Hsp70 interacting protein (CHIP), or a ubiquitin conjugating enzyme (Ubc5) accelerates the degradation T5P‐γC. When expressed in living lens or HeLa cells, T5P‐γC formed intracellular aggregates whereas wt‐γC were evenly distributed. Co‐expression of CHIP or Ubc5 reduced the aggregation of T5P‐γC in cells. Cyclohexmide chase assay showed that CHIP and Ubc5 promoted the degradation of T5P‐γC, but not wt‐γC. Mutant CHIP with either a defective TPR‐domain or U‐box domain failed to reduce the protein level of T5P‐γC, suggesting molecular chaperones are involved in targeting the damaged proteins for degradation. Together, the data suggest that enhancing UPP activity can be an effective strategy to eliminate aggregation‐prone proteins.
Interaction of TAG1 and CASPR2, Proteins Associated to Autism Disorder?
Authors: Rubio‐Marrero Eva N. , Falivelli Giulia , Kim Hyuck , Comoletti Davide
Published: June 2021

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that affects brain development, social and communication skills. Although the causes of the disease are mostly unknown rare variations of the CANTNAP2, the gene that codes for contactin‐associated protein‐like 2 (CASPR2), have been implicated in ASD. We compared the cellular localization and glycosylation processing of wild type CASPR2 to twelve CASPR2 point mutations found in autistic individuals. In contrast to the wild‐type protein that localizes to the cell surface, mutants showed altered cellular disposition with endoplasmic reticulum retention and activation of one signaling pathway of the unfolded protein response. As CASPR2 is associated with a set of neuropsychiatric diseases, it is of interest to study the interaction of CASPR2 with proteins such as the transient axonal glycoprotein‐1 (TAG1). We are currently studying the interaction between different CASPR2 mutants and TAG1 by various biochemical techniques. Preliminary results show that we can measure the interaction between wild‐type TAG1 and wild type CASPR2, but not with one CASPR2 mutants. This data suggest that the interaction is disrupted by the mutation but also that another protein could be mediating this interaction. These studies will help understand better the biology of CASPR2 and its specific role in the development of autism and other neurodevelopmental disorders.
N‐linked glycosylation in the ATP binding cassette class A3 (ABCA3) transporter
Authors: Mulugeta Surafel , Zhao Ming , Hawkins Arie , Wang Ping , Zhang Peggy , Gonzales Linda , Guttentag Susan H. , Beers Michael F.
Published: June 2021

Glycosylation plays a critical role in the proper maintenance of protein structure to promote stability and/or to mediate trafficking and function. The first luminal loop of ABCA3 (A3) contains three putative N‐linked glycosylation sites at residues 53, 124, and 140. We previously reported that alanine mutagenesis of these sites appeared to affect A3 glycosylation. In this study, we used glutamine mutagenesis to generate EGFP‐tagged fusion constructs containing either wild type (A3WT), single motif mutants (A353, A3124, A3140) or a double mutant (A3124+140). Fluorescence analyses of transfected A549 or primary human alveolar type II cells showed while single motif mutants exhibited a vesicular distribution similar to A3WT, the double mutant showed both vesicular and ER distribution. By immunoblot, A353failed to alter either band migration or expression level. In contrast, mutants A3124, A3140, and A3124+140 showed faster band migration rates (suggesting reduced glycosylation) and significantly reduced expression levels compared to A3WT. Treatment of cells with a proteasome inhibitor partially restored the expression levels supporting proteasome dependent degradation of the three mutant isoforms. Together, these studies suggest that disruption of two glycosylation sites of ABCA3 results in the production of an unstable protein that is susceptible to proteasomal degradation.
Study of Self Catalyzed Protein Glycation on Monoclonal Antibody A (MAb A)
Authors: Liu Hong , Yang Yi , Flores Heather , Zhang Boyan , Swartz Trevor , Patapoff Tom
Published: June 2021

Protein glycation is known as a nonenzymatic reaction between reducing sugars, and amine groups. It may lead to functional changes of a protein. Recently, a site specific glycation on lysine Kn of MAb A was reported, with a hypothesis that MAb A self catalysis in reaction with glucoses at specific site. To elucidate the mechanism of the site specific glycation, an in vitro forced glycation study was employed to generate fully glycated MAb A. Subsequently, characterization of the glycated MAb A and its thermal degradation products were performed, through boronate affinity HPLC, ESI‐Mass spectrometry, Ion‐exchange HPLC, and peptide map. Finally, molecular dynamics (MD) simulations were used to map the local electrostatic environment around Kn under glycation conditions. The in vitro study showed that Kn is preferably glycated up to 90%, with four other lysine sites glycated at various low levels. Thermal stress study demonstrated that the main degradation pathway of highly glycated MAb A is hydrolysis of the Schiff base, the reverse reaction of glycation. Glycated Kn was preferably hydrolyzed as compared to the other glycated lysine sites. These results demonstrated the site specific glycation at Kn is self catalyzed. MD analysis suggested the observed self catalysis is caused by a strong electron‐donating environment surrounding Kn with three aspartates potentially involved in Kn reaction with a glucose.
Using Molecular Dynamics (MD) in modeling broadly neutralizing antibodies against influenza viruses and its implications for developing a “universal flu vaccine”
Authors: LaCosta Will , Beckman Sarah , Friend Julia , Krautler Elliabeth , Pacific Luke , Pulapura Sharanya , Shim Peter C , Herman Tim , Wriggers Willy , Xu Huafeng , De Luke
Published: June 2021

Influenza evolves rapidly to evade existing human immunity and seasonal flu vaccines. The recent discovery of broadly neutralizing antibodies against influenza points to the possibility of “universal flu vaccines”, which may protect against viral strains over many seasons. The SMART team from Pingry High School, funded by grants from NIH‐CTSA, is using a 3D‐printer from MSOE to model the interactions between one lineage of such antibodies and influenza hemagglutinin. By targeting the receptor binding pocket of influenza hemagglutinin, these antibodies make it difficult for the virus to develop escape mutations without compromising its ability to bind to sialic acid, a critical requirement for the virus to attach to and infect the host cells. Based on biochemical experiments and molecular dynamics simulations carried out at multiple research institutions including Harvard Medical School and D. E. Shaw Research, the Pingry SMART Team will create 3D models of antibody‐hemagglutinin complexes. These models will be used to investigate how the immune system generates effective antibodies against pathogens while exploring how biochemical data and molecular simulations contribute to development of the next‐generation influenza vaccines.
Rab1a regulates endocytic vesicle processing through interaction with Kifc1
Authors: Quiroz Jose , Mukhopadhyay Aparna , Wolkoff Allan W.
Published: June 2021

Rab1a regulates endocytic vesicle processing by recruiting the microtubule‐based motor Kifc1. The mechanism by which this occurs is unknown and therefore we examined whether Kifc1 and Rab1a interact with each other either directly or through a protein complex.

Human embryonic kidney‐derived HEK293 cell lines expressing super folder green fluorescent protein (SFGFP) or Rab1a‐SFGFP fusion protein were prepared. To test whether Kifc1 interacts with Rab1a, a plasmid encoding Kifc1‐flag was transfected into Rab1a‐SFGFP expressing HEK293 cells. Cell lysates were subjected to immunoprecipitation with GFP antibody. Following SDS‐PAGE, analysis revealed the presence of Kifc1 in immunoprecipitates from Rab1a‐SFGFP expressing cells.

In summary, immunofluorescence microscopy verified stable cell lines expressing Rab1a‐SFGFP in HEK293. Immunoprecipitation of Rab1a‐SFGFP cells transiently transfected with Kifc1‐Flag showed that Rab1a and Kifc1 were in the immunoprecipitate. We conclude that Rab1a and Kifc1 are associated with each other in a protein complex and do not just independently bind to endocytic vesicles. Whether other proteins are in this complex remains to be discovered.
Quantitative redox proteomic analysis of reversible cysteine oxidation in hearts from mice fed a Western diet: implications for metabolic cardiovascular disease
Authors: Behring Jessica B , Kumar Vikas , Siwik Deborah A , Whelan Stephen A , Colucci Wilson S , Cohen Richard A , McComb Mark E , Bachschmid Markus M
Published: June 2021

Global changes in reversible cysteine oxidation of cardiac proteins from mice fed a high fat, high sucrose (HFHS) diet for 8 months were quantified using novel 6‐plex Iodo‐based isobaric labels in a switch assay and high resolution mass spectrometry. 400 reversibly oxidized proteins were identified, 56 with cysteines exhibiting more than 1.5‐fold change in oxidation by HFHS diet. As an improvement on “fold‐change”, our design also defined the percentage of oxidized cysteine at each site, or “site occupancy”. Nearly 50% of the proteins localized to mitochondria, consistent with this organelle being the primary target of metabolic stress resulting in mitochondrial oxidant formation. Multiple proteins in each of the critical metabolic pathways, including: the electron transport chain, tricarboxylic acid cycle, acyl‐carnitine shuttle, beta‐oxidation, and ketolysis, were reversibly oxidized. This suggests that substrate utilization pathways are dysfunctional. The condition further worsens as arterial stiffness increases energy demand of an overworked heart. All of these factors likely contribute to cardiac diastolic dysfunction and hypertrophy that occurs in the mice. Identifying and quantifying reversible oxidative protein changes will elucidate novel signaling mechanisms for switches in metabolism; these modifications could potentially serve as biomarkers for diet‐induced metabolic disease. NHLBI HHSN268201000031C
The role of modulator residues in PDZ domain binding interactions
Authors: Amacher Jeanine , Cushing Patrick R , Brooks Lionel , Boisguerin Prisca , Madden Dean R
Published: June 2021

PDZ domains are the most common interaction domain in the human proteome. PDZ interactions are classified by motif residues, or, preferences distinguished by high throughput techniques. However, these motifs are degenerate, and fail to predict the affinities of each peptide‐binding domain for its unique set of interacting partners, making design of specific therapeutic inhibitors a challenge. Through biochemical, structural, and computational studies, we reveal that targeted inhibitor design requires an understanding of modulatory, or non‐motif affinity determinant, residues in PDZ domains. Our work investigates modulatory preferences in the CFTR Associated Ligand (CAL) protein.

Despite a motif dependent on two residues, we previously reported the engineering of a selective inhibitor of CAL PDZ via a peptide array pipeline. Our approach allowed us to identify multiple residues along the binding cleft that influenced affinity and/or selectivity. We now investigate the stereochemistry and binding affinities of over a dozen co‐complex structures of CAL PDZ bound to high, intermediate, and low affinity peptides, revealing distinct modulator preferences that are hidden from high throughput motif analysis. We extend our analysis to additional PDZ domains, which share cellular targets. Our data suggests that modulator preferences dictate endogenous PDZ networks.
Evaluation of synthetic thiosemicarbazone containing compounds as inhibitors of cathepsin K
Authors: Wen Lisa , Feeley Ariel P , Sexton Buddy K , Smith Samantha K , Pallempati Kirthi , Huang Jenq‐Kuen , McConnell Rose M
Published: June 2021

Cathepsin K is a cysteine protease that is highly expressed in osteoclasts. Because of its strong collagenolytic activity cathepsin K has been described as the major enzyme responsible for the degradation of organic bone matrix, and is believed to play a fundamental role in bone resorption. Cathepsin K has become an established drug target for osteoporosis. Here, we report screening and evaluation of synthetic thiosemicarbazone containing compounds against cathepsin K. The studies were performed using recombinant human cathepsin K and colorimetric enzyme inhibition assay with N‐Carbobenzoxy‐L‐Phenylalanyl‐Arginine‐4‐nitroanilide hydrochloride as the chromogenic substrate. Data analysis of the effectiveness of these inhibitors will be used to foster future development of newer effective inhibitors for the treatment osteoporosis. Funding provided by National Cancer Institute at NIH (Grant No. 3R15CA086933–04 and 3R15CA086933–04A2S1) and Western Illinois University.
Enzyme‐Substrate Interactions in the Helicobacter pylori 5′‐Methylthioadenosine/S‐adenosylhomocysteine Nucleosidase
Authors: Mishra Vidhi , Ronning Donald R.
Published: June 2021

5′‐methylthioadenosine/S‐adenosylhomocysteine nucleosidase (MTAN) is a multi‐functional bacterial enzyme that catalyzes the hydrolysis of the N‐glycosidic bond of many adenosine‐based metabolites. This hydrolytic activity places MTAN at the hub of at least seven different metabolic pathways, which includes the purine salvage pathway, the methionine salvage pathway, various S‐adenosylmethionine (SAM)‐dependent methylation pathways, polyamine biosynthesis, the production of quorum sensing molecules and menaquinone biosynthesis. The bacterial species such as Campylobacter and Helicobacter are dependent on MTAN for menaquinone synthesis, an essential metabolite for bacterial viability. As MTAN is absent in humans but essential for bacteria like Helicobacter pylori, it is an excellent target for the development of new treatments for Helicobacter infections. To structurally characterize the interactions between MTAN and its various substrates, complexes of an inactive mutant of the Helicobacter pylori MTAN with two known substrates, 5′‐methylthioadenosine (MTA) and S‐adenosylhomocysteine (SAH) were formed and crystallized. The crystal structures of mutant MTAN with SAH and MTA bound in its active site were solved to 1.2 and 1.6 Å, respectively. The MTAN‐SAH co‐crystal structure represents the first visualization of interactions between the homocysteine moiety of SAH and the 5′‐alkylthiol‐binding sub‐site of the MTAN active site. The co‐crystal structure of wild‐type MTAN with products, adenine and S‐ribosylhomocysteine, was also determined to 1.54 Å resolution. The similarities and differences in these three structures highlight features that can be exploited to design H. pylori specific drugs.
Suramin Distinguishes Between the Calmodulin Binding Sequences of Inducible and Neuronal Nitric Oxide Synthase
Authors: Sweda Nicholas , McNamara Alyssa , Stevens‐Truss Regina
Published: June 2021

Nitric oxide synthase (NOS) catalyzes the production of nitric oxide (•NO) from L‐arginine in several tissues in the body. Nitric oxide is believed to play a critical role in cardiovascular disease, stroke, and cancer. For this reason, selective inhibition of NOS isoforms is currently under investigation for their potential as targets in drug design. All known isoforms of NOS require binding of calmodulin (CaM), a calcium binding protein ubiquitously expressed in eukaryotes, for activity. The interactions among the NOS isoforms and CaM vary, and therefore pose a good place to study in differentiating these enzymes. We have previously demonstrated that suramin, a polysulfonated naphthylurea, can distinguish between the CaM binding region of the inducible NOS and the neuronal NOS. When bound to NOS, suramin inhibits its activity by preventing CaM binding and activation. Suramin's inhibition was found not to be time dependent, and IC50 values were found to be ~8 μM and ~120 μM for the inducible NOS and neuronal NOS, respectively. SDS‐PAGE and Western analyses demonstrated that suramin is able to inhibit the interaction between NOS and CaM. AutoDoc analysis further demonstrated that suramin most likely interacts with a portion of the NOS molecule believed to be crucial to CaM's interaction. These studies implicate suramin as a potential lead compound.
POLYISOPRENYLATED METHYLATED PROTEIN METHYL ESTERASE AS A PUTATIVE BIOMARKER AND DRUG TARGET FOR PROSTATE CANCER
Authors: POKU ROSEMARY ANIWAA , AMISSAH FELIX , DUVERNA RANDOLPH , AGUILAR BYRON J. , LAMANGO NAZARIUS S.
Published: June 2021

Prostate cancer is the most frequently diagnosed cancer among men in the U.S., with an estimated 241,740 new cases and 28,170 deaths in 2012. Mutation and overexpression‐induced abnormal activities of polyisoprenylated proteins have been implicated in various neoplasms including prostate cancer. We determined whether polyisoprenylated methylated protein methyl esterase (PMPMEase) is overexpressed and hyperactive in prostate cancer, possibly contributing to progression and metastasis. PMPMEase specific activities were 4‐and 6‐fold higher in androgen‐dependent (22Rv1 and LNCaP) cells, 2‐ and 17‐fold higher in androgen‐independent (PC‐ 3 and DU 145) cells respectively, compared to normal WPE1‐NA22 prostate cells. Treatment of the cells with L‐28, a specific PMPMEase inhibitor induced apoptosis (EC50 of 1.8 to 4.6 μM). PMPMEase activity of the different cell lines followed a similar profile (IC50 of 2.3 to 130 μM). Analysis of prostate cancer tissue microarray for PMPMEase expression revealed intermediate, strong and very strong staining in 94.5% of the 92 adenocarcinoma cases compared to only trace staining in the normal tissue controls. The results suggest that PMPMEase is overexpressed, is hyperactive and is involved in prostate cancer cell progression. Its activity, immunoreactivity and inhibition could serve in the diagnosis and targeted therapeutic management of the disease.
Resistin expression study used to develop a simplified mouse model of adipokine expression
Authors: Quijada Elizabeth , Dean Diane , Marcucci Melissa
Published: June 2021

High fat diets can impact the expression of bioactive peptide signaling molecules known as adipokines. Adipocyte cells synthesize adipokines and these molecules regulate energy metabolism, neuroendocrine function and immune function. One particular adipokine is the 12 kDa protein, resistin. Resistin was originally identified in a study that concluded that mice heterozygous for a null PPARγ allele exhibit adipocyte hypertrophy on a high fat diet and have an increased sensitivity to insulin. Additional research has shown resistin was linked to increased insulin resistance; however, the specific mechanism responsible for the change in insulin sensitivity has not yet been established. The goal of this research project is to provide a simplified and comprehensive model system for understanding adipokine expression by analyzing the expression of resistin using western blot analysis. Developing a simplified model framework will allow for subsequent investigations into adipokine expression that exhibit a clear understanding of the impact of excess adipose tissue on adipokine expression. The mice have been fed a high fat diet over a period of three weeks and the epididymal fat pads have been removed. western blot analysis will be utilized to compare resistin expression in the fat obtained from the low and high fat fed mice.
The role of stearoyl‐CoA desaturase‐3 in lipid metabolism
Authors: Bond Laura , Strable Maggie , Ade Lacmbouh , Ntambi James
Published: June 2021

Stearoyl‐CoA desaturase (SCD) is a rate‐limiting enzyme in de novo lipogenesis that catalyzes the synthesis of monounsaturated fatty acids from saturated fatty acids. Four SCD isoforms have been identified in mouse (SCD1–4); SCD isoform 3 exhibits unique tissue expression and substrate specificity. While SCD1, 2, and 4 convert palmitate and stearate to palmitoleate and oleate, respectively, SCD3 preferentially desaturates palmitate. SCD3 expression is restricted to skin, harderian glands, and preputial glands. Despite the limited tissue expression of this isoform, SCD3−/− mice exhibit a global phenotype when fed a lipogenic low‐fat high‐carbohydrate diet. While deletion of SCD1 protects mice from diet‐induced obesity and hepatic steatosis, SCD3 deletion predisposes mice to increased lipogenesis in white adipose tissue and hepatic triglyceride accumulation. Gene expression studies in the harderian gland reveal that deletion of SCD1 causes a dramatic decrease in Scd3, while deletion of SCD3 results in an increase in Scd1. We propose that the observed increase in lipogenesis upon the deletion of this lipogenic enzyme stems from a regulatory mechanism between SCD isoforms. Funded by NIH.
Expression of an endocytic‐defective VE‐cadherin mutant cannot restore cell‐cell‐adhesion in the absence p120 in the endothelium
Authors: Garrett Joshua P , Lowery Anthony M , Kowalczyk Andrew P , Vincent Peter A
Published: June 2021

Vascular endothelial cadherin (VE‐cad) is an Adherens Junction (AJ) transmembrane protein found at the cell‐cell junction in endothelial cells (EC) which participates in cell‐cell adhesion. The juxtamembrane domain of VE‐cad binds to p120 to regulate the levels of VE‐cad. Previously, we found that depletion of p120 using shRNA results in a decrease in the level of VE‐cad and a decrease in monolayer integrity as assessed by trans‐endothelial electrical resistance (TEER). Re‐expression of p120 can restore both the decrease in TEER and VE‐cad levels. In contrast, re‐expression of VE‐cad to normal levels in the absence of p120 will not rescue TEER even though VE‐cad is found at cell‐cell junctions. We hypothesize that re‐expression of VE‐cad does not rescue TEER in the absence of p120 due to an increase in VE‐cad endocytosis. To test this hypothesis, we expressed a VE‐cad mutant (DEE‐VE‐cad) that has limited endocytosis even in absence of p120 binding. Expression of DEE‐VE‐cad, following p120 depletion, was unable to rescue TEER even though the levels of DEE‐VE‐cad were equal to or above levels of endogenous VE‐cad and was localized at the cell‐cell junction. These results indicate that prevention of VE‐cad endocytosis is not the only mechanism by which p120 regulates the formation of a mature AJ in EC monolayers.
DEPTOR: A Novel Cell‐Intrinsic Molecule That Plays A Critical Role In The Regulation Of Endothelial Activation, Proinflammatory And Angiogenic Responses
Authors: Bruneau Sarah , Woda Craig Bryan , Briscoe David Michael
Published: June 2021

Cytokine‐mediated activation of endothelial cells (EC) is characteristic of immune inflammation, and therapeutics that inhibit EC activation have broad implications for the treatment of chronic diseases. Here, by qPCR and Western blot analyses, we find that DEPTOR, an intracellular inhibitor of mTOR signaling, is expressed in vascular EC. Using a siRNA knockdown approach and phosphokinase arrays, we found that DEPTOR functions to inhibit the mTOR, ERK1/2 and STAT1 signaling pathways in EC. Moreover, using pharmacological inhibitors of mTOR (Rapamycin, Torin1) and ERK1/2 (U0126), we found that DEPTOR regulates each of these pathways through independent mechanisms. Importantly, we also found that knockdown of DEPTOR in EC results in marked activation responses, including up to an ~1000 fold increase in the mRNA expression of T cell chemokines (CXCL9, CXCL10, CXCL11, CX3CL1 and CCL5) and adhesion molecules (VCAM‐1 and ICAM‐1). Functionally, we also found that DEPTOR siRNA transfected EC, a) bind increased numbers of PBMC and CD3+ T cells (P<0.005) in adhesion assays and b) have increased migration and angiogenic responses (P<0.01) in the wound healing and spheroid sprouting assays. Collectively, these findings identify DEPTOR as a novel regulatory molecule in EC, and suggest that its relative level of expression is of great importance in EC‐dependent mechanisms of inflammation and in immune angiogenesis.
A role for endomucin‐1 in maintaining a non‐inflammatory endothelial surface and in the regulation of leukocyte‐endothelial cell interactions
Authors: Yang Jinling , Zahr Alisar , Alcaide Pilar , Jones Alexander , Gregory‐Ksander Meredith , Luscinskas Francis W. , Ksander Bruce , Argueso Pablo , D'Amore Patricia A
Published: June 2021

Leukocyte‐endothelial cell (EC) interactions, including leukocyte rolling, adhesion and transmigration, are central to vascular inflammation. Endomucin‐1 (EMCN‐1) is a transmembrane glycoprotein specifically expressed by venous and capillary endothelium. This study was aimed at determining the role of EMCN‐1 in regulating leukocyte‐EC interaction in inflammation. Whereas leukocytes do not adhere to the surface of cultured human umbilical vein endothelial cells (HUVECs), transfection with siRNA against EMCN‐1 led to dramatic increase in leukocyte‐EC interaction compared to scramble siRNA control by flow adhesion assay. HUVECs were also treated with TNF‐alpha (0.1 – 25 ng/ml), a prominent inflammatory cytokine. TNF‐alpha led to a dramatic reduction of in EMCN‐1 in cell surface protein, total protein and mRNA levels assayed by biotinylation, Western blot and quantitative real‐time PCR. Overexpression of EMCN‐1 by adenovirus (Ad‐EMCN‐1) in HUVECs led to a significant reduction in TNF‐alpha‐induced leukocyte‐EC interaction compared to control cells transduced with GFP‐expressing adenovirus (Ad‐GFP) determined by flow adhesion assay. For analysis of the role of EMCN‐1 in vivo, C57/B6 mice were injected intravitreally with Ad‐EMCN‐1 or Ad‐GFP seven days prior to invtravitreal injection of 10 ng TNF‐alpha or saline. Ciliary bodies, a primary site of leukocyte infiltration, were harvested 48 hr later. Administration of TNF‐alpha led to a reduction in EMCN‐1 protein and to an increase in CD45+ infiltrating inflammatory cells measured by flow cytometry, which was blocked by Ad‐EMCN‐1. These results suggest a critical role for EMCN‐1 in maintaining non‐inflammatory state of ECs at quiescent conditions and regulating leukocyte‐EC interaction during inflammation.

This research was supported by the Department of Defense (PAD), a grant for new collaborative research projects from Schepens Eye Research Institute (PaA and PAD), HL‐097406 (PA) and HL‐36028 (FWL).
A targeted proteomics approach for profiling nuclear proteins in insulin resistant adipocytes
Authors: Ota Asuka , Kovary Kyle , Ahrends Robert , Shen Wen‐Jun , Kraemer Fredric B. , Teruel Mary N.
Published: June 2021

Insulin resistance (IR) predisposes to type 2 diabetes and cardiovascular diseases. Adipose tissue regulates glucose and lipid metabolism, and adipocyte dysfunction is linked to the development of IR. Transcription factors are the major regulators of adipocyte function, but the profiling of nuclear proteins in IR adipocytes has never been done. Here, we used selective reaction monitoring mass spectrometry to quantitatively measure the expression levels of nuclear proteins relevant to adipocyte function in IR adipocytes and pre‐adipocytes. We used 1) OP9 pre‐ and mature adipocytes treated with or without tumor necrosis factor α to induce IR, and 2) primary adipocytes isolated from IR obese (ob/ob) and C57 control mice. Our results show that IR pre‐and mature adipocytes have unique protein expression profiles, and the profiles obtained from IR primary adipocytes are a mix of these signatures, indicating defects in adipogenesis contribute to the development of IR in vivo. This is also indicated by decreased expression of pro‐adipogenic proteins such as CEBPα, PPARγ, and FAPB4 and increased expression of FLNA which is a negative regulator of PPARγ. Thus, we established a quantitative method to profile nuclear proteins in adipocytes. This approach may help diagnostics or drug screening for IR to prevent the onset of a more severe metabolic disease.
Amino‐functionalized nanoparticles inhibit mTOR and induce cell cycle arrest and apoptosis in leukemia cells
Authors: Loos Cornelia , Lunov Oleg , Syrovets Tatiana , Nienhaus G. Ulrich , Mailänder Volker , Landfester Katharina , Simmet Thomas
Published: June 2021

Activation of the mammalian target of rapamycin (mTOR) has been implicated in cancer development, anticancer drug resistance, and aging. Therefore, mTOR is representing a promising therapeutic target. Here we show that amino‐functionalized nanoparticles (PS‐NH2) inhibit, whereas carboxy‐functionalized particles (PS‐COOH) activate mTOR in leukemia cells. Accordingly, PS‐NH2 inhibited proliferation and induced G2 cellcycle arrest in three myeloid leukemia cell lines, whereas PS‐COOH did not. Further, PS‐NH2 inhibited proliferation, angiogenesis, and induced apoptosis in leukemia xenografts in vivo. Consistent with mTOR inhibition, PS‐NH2 inhibit activation of its downstream targets, Akt and p70 ribosomal S6 kinase 1, followed by overexpression of the cell‐cycle regulator p21Cip1/Waf1 and degradation of cyclin B1. Initially, both particles induce autophagy in leukemia cells. However, in leukemia cells treated with PS‐NH2, autophagy finally switched to activation of caspase 3 and induction of apoptosis. In contrast, primary macrophages do not exhibit activated mTOR signaling and are relatively resistant to the PS‐NH2‐induced toxicity. These data indicate that nanoparticles with different surface functionalization might be used as a tool to control activation of mTOR signaling pathway and to influence proliferation, viability, and growth of tumor cells. Supported by the DFG, SPP1313.
Identification of ALDOA as a new Lung adeonocarcinoma predict gene involve cancer metabolism and tumor metastasis
Authors: Chang Yu‐chan , Jan Yi‐Hua , Chan Yung‐Chieh , Yang Yi‐Fang , Su Chia‐Yi , Lai Tsung‐Ching , Liu Yu‐Peng , Hsiao Michael
Published: June 2021

The metabolic alterations in cancer progression remain unknown. In this study, we identified Aldolase A (ALDOA), a glycolytic enzyme that regulates glycolysis process, as the most significant marker in lung cancer by comparative microarray and prognosis database analysis. We observed overexpression of ALDOA mRNA was significantly associated with poor overall survival rate with the hazard ratio of (HR=3.0, P<0.01). Microarray datasets also showed high ALDOA expression in tumor compared with corresponding normal tissue in 27 lung cancer patient (p<0.01). Strong expression of ALDOA protein by IHC was further detected in 94.3% cases (99/105) and the expression level was higher compared to non‐tumor lung parenchyma (p<0.01). High ALDOA expression was found associated with poor survival (p<0.01) of lung cancer patients. ALDOA overexpression promoted invasion and migration in vitro. These complementary function expression consequences were also validated in metastasis colony forming assay in vivo and in‐situ invasion assay. Furthermore, overexpression of ALDOA induces upregulation of GAP1, and MMP2. Knockdown of GAP or MMP2 inhibited ALDOA induced invasion and migration in vitro, suggesting that ALDOA may promote lung cancer cell matastasis by the activation of GAP and MMP2. Our finding may implicate the possibility of targeting ALDOA as a novel therapeutic strategy to inhibit lung cancer metastasis.
Biochemical Characterization of L‐idonate Dehydrogenase from Vitis vinifera
Authors: Steiner Stephen A. , Selby Anna , Moore Kristen , McKinley Katie
Published: June 2021

The enzyme L‐idonate dehydrogenase (L‐IdDH) is an important enzyme in the pathway for the biosynthesis of tartaric acid from ascorbic acid in grapes. IdDH is an NAD+/NADH‐dependent enzyme which catalyzes the conversion of L‐idonate to 5‐keto‐D‐gluconic acid. For this study, the enzyme was produced in BL21 cells which have been transformed by the pET14b plasmid containing the IdDH gene produced from a Vitis vinifera cDNA library. The active enzyme was isolated by TALON affinity chromatography. Initial characterization of the enzyme in a reaction using 5‐keto‐D‐gluconate and NADH as substrates showed a pH optimum of 6.0. The reaction proceeding the opposite direction, with the conversion of L‐idonate to 5‐keto‐D‐gluconate using NAD+ as a cosubstrate, has optimal activity at a significantly higher pH, near 8.0. Kinetics studies have indicated that the Km for 5‐keto‐D‐gluconate is 4 mM in the presence of a saturating concentration of NADH. This work is supported by a grant from the Indiana Academy of Science.
Mitochondrial Targeting of AKAP350C
Authors: Mason Twila A , Kolobova Elena , Goldenring James R
Published: June 2021

Mitochondria are crucial organelles in cell function and survival. Fission and fusion are very important for maintenance of mitochondrial structure and function, and the dynamics of this remain poorly understood. AKAPs (A‐Kinase Anchoring Proteins) were discovered as anchors that provide localized cAMP/PKA signaling, but they play many other roles as well. Rab32, an AKAP, localizes to mitochondria‐associated membranes of the ER (MAM) and plays a role in mitochondrial dynamics by associating with Drp1, a fission protein. AKAP149 localizes to both the ER and mitochondria and plays a role in stress response in some cell types. We are specifically interested in AKAP350 (also AKAP450/AKAP9/CG‐NAP). We have completed cloning of a new splice variant, AKAP350C, which is ubiquitously expressed and contains a unique 55 amino acid carboxyl terminal sequence. Endogenous AKAP350C associates with the mitochondria. Overexpression of AKAP350C causes the collapse of mitochondria towards the nucleus and localizes near the ER and the collapsed mitochondria. The unique 55 amino acid region contains an amphipathic alpha helix that is required for AKAP350C targeting. Mutations disrupting or deleting this alpha helix abolish targeting to the mitochondria. We hypothesize that AKAP350C anchors PKA and other proteins to mitochondria for regulation of mitochondrial dynamics. Research support is provided by NIH grant RO1DK43405.
Effect of X‐rays on mitochondrial DNA in human lymphoblasts with and without elevated levels of the 4977‐bp Common Deletion
Authors: Pogozelski Wendy Knapp , Ribaudo Alexandra , Sinha Shadman , O'Donnell Robert
Published: June 2021

Human mitochondrial DNA (mtDNA) is subject to large‐scale deletions. A deletion of 4977 bp called the “common deletion” makes cells viable but energetically compromised. We compared lymphoblast cell lines in which the deletion was either undetectable or at levels of ~7%. These cells were treated with X‐rays with doses up to 5Gy. Levels of total mtDNA and mtDNA bearing the Common Deletion were measured by real‐time PCR.
The role of FabH in Listeria monocytogenes fatty acid composition
Authors: Saunders Lauren P , Sen Suranjana , Wilkinson Brian J , Gatto Craig
Published: June 2021

Listeria monocytogenes grows at low temperatures by altering its bacterial membrane composition to increase membrane fluidity, primarily by decreasing the length of fatty acid (FA) chains and increasing the anteiso to iso FA ratio. FabH, the initiator of fatty acid synthesis, was identified as the primary determinant of membrane FA composition; the extent of this effect has not been quantified. In this study, previously determined FabH steady‐state parameters and substrate concentrations were used to calculate expected fatty acid compositions at 30 and 10 C. FabH substrates 2‐methylbutyryl‐CoA, isobutyryl‐CoA, and isovaleryl‐CoA produce the primary FA in L. monocytogenes, i.e., anteiso, even chain length iso, and odd chain length iso fatty acids, respectively. The substrate in vivo concentrations were not all resolved and were calculated from overall FA composition and FabH steady‐state parameters. Concentrations were confirmed by supplementing media with precursors of FabH substrates and comparing the FA compositions to those expected from FabH activity. The same relative substrate concentrations were used to calculate the expected FA compositions at 10 °C and our model predicted a greater change in FA composition than was observed, indicative of an additional control step guiding fatty acid composition at low temperatures. This research supported by NIH Grants GM061583 to CG and AI099977 to BJW.
Transmembrane Domain Length is a Determinant of Plasma Membrane Localization via Association with Membrane Rafts
Authors: Levental Ilya , Simons Kai
Published: June 2021

The lipid raft hypothesis emphasizes the functional interplay of lipids and proteins as an important mechanism for the regulation of membrane structure and function; however, experimental limitations in defining raft composition and properties have prevented unequivocal demonstration of their functional relevance. Here, we establish a quantitative, functional relationship between raft association and cargo sorting to the plasma membrane. Systematic mutation of a model transmembrane protein showed that both raft phase association and plasma membrane localization were reduced by shortening the transmembrane domain (TMD). Raft partitioning of a variety of TMD and palmitoylation mutants was significantly correlated with plasma membrane (PM) localization, suggesting that, in the absence of other sorting signals, raft association is a determinant of PM sorting for a subset of TM proteins. These findings implicate lipid‐driven phase separation as a mechanism for protein sorting at the latter stages of the secretory pathway.
Unsaturated glycoceramides as molecular carriers for mucosal drug delivery of GLP‐1 analogues
Authors: te Welscher Yvonne Maria , Kaoutzani Lydia , Lencer Wayne I.
Published: June 2021

The goal is to develop technology using glycosphingolipids for oral or nasal administration of therapeutic peptides. We recently discovered that the structure of the ceramide domain of the ganglioside GM1 dictates intracellular trafficking in epithelial cells. GM1‐ceramides with short or unsaturated fatty acids enter the recycling endosome where they are sorted to multiple destinations including to the basolateral cell surface by transcytosis. GM1‐ceramides with saturated fatty acid chains do not enter these pathways. Here, we test whether GM1 with short‐ or unsaturated chain ceramides can be harnessed as vehicles for transepithelial delivery of bioactive peptides. We linked GM1 to stable versions of the therapeutic hormone glucagon‐like peptide‐1 (GLP‐1). The function of the peptide remained intact in the fusion molecules. The GLP1‐GM1 fusions with short or unsaturated fatty acids were incorporated into apical cell membranes, trafficked into the recycling endosome, and reached the basolateral membrane of polarized epithelial cells. The GLP1‐GM1 fusions with long saturated fatty acid were not sorted into these pathways. Thus, ceramide‐based intra‐cellular trafficking may have clinical utility for delivery of therapeutic cargo by transcytosis across epithelial barriers at mucosal surface. The source of research support is from the NIH/NIDDK R21.
Mutation of SIMPLE in Charcot‐Marie‐Tooth 1C Alters Production of Exosomes
Authors: Zhu Hong , Guariglia Sara , Li Wenjing , Chow Chi‐Wing
Published: June 2021

Charcot‐Marie‐Tooth (CMT) disease is an inherited neurological disorder. Mutations in protein SIMPLE accounts for the rare autosomal dominant demyelination in type 1C CMT patients (CMT1C). Understanding the molecular basis of CMT1C pathogenesis is impeded, in part, by the perplexity for the role of SIMPLE, an evolutionarily conserved protein expressed in multiple cell types. Here, we show that SIMPLE plays a role in the production of exosomes, which are nanovesicles secreted extracellularly. We find that SIMPLE resides within the intraluminal vesicles of multivesicular bodies and inside exosomes. Expression of SIMPLE increases the production of exosomes. Conversely, endogenous SIMPLE carrying CMT1C mutation in primary mouse embryonic fibroblasts shows reduced secretion of exosomes, in part, due to improper formation of MVBs. CMT1C patient B cells also show similar defects in exosomes and MVBs. Together, these data indicate that SIMPLE regulates the production of exosomes via modulating the formation of MVBs. Dysregulated endosomal trafficking and changes in the landscape of exosome‐mediated intercellular communications may place an overwhelming burden on the nervous systems and, account for the CMT1C molecular pathogenesis.
Hypo‐glycosylated hFSH binds and activates FSH receptors more rapidly than fully‐glycosylated hFSH
Authors: Bousfield George Russell , Butnev Vladimir Y , May Jeffrey V , Harvey David J , Jiang Chao , Davis John S
Published: June 2021

Follitropin (FSH) heterodimers possess two potential N‐glycosylation sites on both common α and hormone‐specific β subunits. Hypo‐glycosylated hFSH isolated from lutropin preparations possessed a partially glycosylated FSHβ subunit decorated with one glycan, while there were two in the hFSHα subunit. Mass spectrometry revealed the presence of oligomannose glycans, which are normally rare in hFSH preparations. Hypo‐glycosylated hFSH was 5‐ to 26‐fold more active than fully‐glycosylated hFSH in several FSH radioligand assays. In rat testis homogenate, 125I‐hypo‐hFSH bound receptors immediately, without the 45–60 min lag observed for 125I‐fully‐glycosylated hFSH. In porcine granulosa cells, short‐term incubations revealed an earlier onset of cAMP accumulation, protein kinase A (PKA) activity and CREB phosphorylation. Hypo‐glycosylated hFSH increased intracellular cAMP accumulation within one min and PKA‐dependent CREB phosphorylation within 5 min; whereas fully‐glycosylated hFSH induced CREB phosphorylation after a 10–15 min lag. Additionally, CREB phosphorylation was sustained for greater than 60 min in response to hypo‐glycosylated hFSH, but returned to basal within 60 min in response to fully‐glycosylated hFSH. Thus, partial glycosylation of hFSH results in faster binding to its cognate receptor and this is attended by a more rapid cellular response. Supported by NIH grant 5P01 AG‐029531.