Biochemistry Commons^™

Enhancing Translation: A Need To Leverage Complex Preclinical Models Of Addictive Drugs To Accelerate Substance Use Treatment Options, Christa Corley, Ashley Craig, Safiyah M. Sadek, Julie A. Marusich, Samar N. Chehimi, Ashley M. White, Lexi J. Holdiness, Benjamin C. Reiner, Cassandra D. Gipson

Markey Cancer Center Faculty Publications

Preclinical models of addictive drugs have been developed for decades to model aspects of the clinical experience in substance use disorders (SUDs). These include passive exposure as well as volitional intake models across addictive drugs and have been utilized to also measure withdrawal symptomatology and potential neuro- behavioral mechanisms underlying relapse to drug seeking or taking. There are a number of Food and Drug Administration (FDA)-approved medications for SUDs, however, many demonstrate low clinical efficacy as well as potential sex differences, and we also note gaps in the continuum of care for certain aspects of clinical ex- periences in individuals …

Biliverdin Reductase-A Integrates Insulin Signaling With Mitochondrial Metabolism Through Phosphorylation Of Gsk3Β, Chiara Lanzillotta, Antonella Tramutola, Simona Lanzillotta, Viviana Greco, Sara Pagnotta, Caterina Sanchini, Silvia Di Angelantonio, Elena Forte, Serena Rinaldo, Alessio Paone, Francesca Cutruzzolà, Flavia Agata Cimini, Ilaria Barchetta, Maria Gisella Cavallo, Andrea Urbani, D. Allan Butterfield, Fabio Di Domenico, Bindu D. Paul, Marzia Perluigi, Joao M.N. Duarte

Markey Cancer Center Faculty Publications

Brain insulin resistance links the failure of energy metabolism with cognitive decline in both type 2 Diabetes Mellitus (T2D) and Alzheimer’s disease (AD), although the molecular changes preceding overt brain insulin resistance remain unexplored. Abnormal biliverdin reductase-A (BVR-A) levels were observed in both T2D and AD and were associated with insulin resistance. Here, we demonstrate that reduced BVR-A levels alter insulin signaling and mitochondrial bioenergetics in the brain. Loss of BVR-A leads to IRS1 hyper-activation but dys- regulates Akt-GSK3β complex in response to insulin, hindering the accumulation of pGSK3βS9 into the mito- chondria. This event impairs oxidative phosphorylation and fosters …

Therapeutic Potential Of Berberine In Attenuating Cholestatic Liver Injury: Insights From A Psc Mouse Model, Yanyan Wang, Derrick Zhao, Lianyong Su, Yun-Ling Tai, Grayson W. Way, Jing Zeng, Qianhua Yan, Ying Xu, Xuan Wang, Emily C. Gurley, Xi-Qiao Zhou, Jinze Liu, Jinpeng Liu, Weidong Chen, Philip B. Hylemon, Huiping Zhou

Markey Cancer Center Faculty Publications

Background and aims

Primary sclerosing cholangitis (PSC) is a chronic liver disease characterized by progressive biliary inflammation and bile duct injury. Berberine (BBR) is a bioactive isoquinoline alkaloid found in various herbs and has multiple beneficial effects on metabolic and inflammatory diseases, including liver diseases. This study aimed to examine the therapeutic effect of BBR on cholestatic liver injury in a PSC mouse model (Mdr2 −/− mice) and eluci‑ date the underlying mechanisms.

Methods

Mdr2−/− mice (12–14 weeks old, both sexes) received either BBR (50 mg/kg) or control solution daily for eight weeks via oral gavage. Histological and serum biochemical …

A Novel Protozoa Parasite-Derived Protein Adjuvant Is Effective In Immunization With Cancer Cells To Activate The Cancer-Specific Protective Immunity And Inhibit The Cancer Growth In A Murine Model Of Colorectal Cancer, Rajesh Mani, Chloe G. Martin, Kanal E. Balu, Qingding Wang, Piotr G. Rychahou, Tadahide Izumi, B. Mark Evers, Yasuhiro Suzuki

Markey Cancer Center Faculty Publications

Cancer-specific CD8+ cytotoxic T cells play important roles in preventing cancer growth, and IFN-γ, in addition to IL-12 and type I interferon, is critical for activating CD8+ cytotoxic T cells. We recently identified the capability of the amino-terminus region of dense granule protein 6 (GRA6Nt) of Toxoplasma gondii, an intracellular protozoan parasite, to activate IFN-γ production of microglia, a tissue-resident macrophage population. Therefore, in the present study, we examined whether recombinant GRA6Nt protein (rGRA6Nt) functions as an effective adjuvant to potently activate cancer-specific protective immunity using a murine model of MC38 colorectal cancer (CRC). When mice were immunized with non-replicable …

Structure, Dynamics, And Redox Reactivity Of An All-Purpose Flavodoxin, Sharique Khan, Ahmadullah Ansari, Monica Brachi, Debarati Das, Wassim El Housseini, Shelley Minteer, Anne-Francis Miller

Markey Cancer Center Faculty Publications

The flavodoxin of Rhodopseudomonas palustris CGA009 (Rp9Fld) supplies highly reducing equivalents to crucial en- zymes such as hydrogenase, especially when the organism is iron-restricted. By acquiring those electrons from photodriven electron flow via the bifurcating electron transfer flavoprotein, Rp9Fld provides solar power to vital metabolic processes. To understand Rp9Fld’s ability to work with diverse partners, we solved its crystal structure. We observed the canonical fla- vodoxin (Fld) fold and features common to other long-chain Flds but not all the surface loops thought to recognize part- ner proteins. Moreover, some of the loops display alternative structures and dynamics. To advance studies …

Sedimentation Of Large, Soluble Proteins Up To 140 Kda For 1h-Detected Mas Nmr And 13c Dnp Nmr – Practical Aspects, Dallas Bell, Florian Lindemann, Lisa Gerland, Hanna Aucharova, Alexander Klein, Daniel Friedrich, Matthias Hiller, Kristof Grohe, Tobias Meier, Barth Van Rossum, Anne Diehl, Jon Hughes, Leonard J. Mueller, Rasmus Linser, Anne-Frances Miller, Hartmut Oschkinat

Markey Cancer Center Faculty Publications

Solution NMR is typically applied to biological systems with molecular weights < 40 kDa whereas magic-angle-spinning (MAS) solid-state NMR traditionally targets very large, oligomeric proteins and complexes exceeding 500 kDa in mass, including fibrils and crystalline protein preparations. Here, we propose that the gap between these size regimes can be filled by the approach presented that enables investigation of large, soluble and fully protonated proteins in the range of 40–140 kDa. As a key step, ultracentrifugation produces a highly concentrated, gel-like state, resembling a dense phase in spontaneous liquid-liquid phase separation (LLPS). By means of three examples, a Sulfolobus acidocaldarius bifurcat- ing electron transfer flavoprotein (SaETF), tryptophan synthases from Salmonella typhimurium (StTS) and their dimeric β-subunits from Pyrococcus furiosus (PfTrpB), we show that such samples yield well-resolved proton-detected 2D and 3D NMR spectra at 100 kHz MAS without heterogeneous broadening, similar to diluted liquids. Herein, we provide practical guidance on centrifugation conditions and tools, sample behavior, and line widths expected. We demonstrate that the observed chemical shifts correspond to those obtained from μM/low mM solutions or crystalline samples, indicating structural integrity. Nitrogen line widths as low as 20–30 Hz are observed. The presented approach is advantageous for proteins or nucleic acids that cannot be deuterated due to the expression system used, or where relevant protons cannot be re-incorporated after expression in deuterated medium, and it circumvents crystallization. Importantly, it allows the use of low-glycerol buffers in dynamic nuclear polarization (DNP) NMR of proteins as demonstrated with the cyanobacterial phytochrome Cph1.

Non-Transcriptional Irf7 Interacts With Nf-Κb To Inhibit Viral Inflammation, Shumin Fan, Sonam Popli, Sukanya Chakravarty, Ritu Chakravarty, Saurabh Chattopadhyay

Markey Cancer Center Faculty Publications

Interferon (IFN) regulatory factors (IRF) are key transcrip- tion factors in cellular antiviral responses. IRF7, a virus- inducible IRF, expressed primarily in myeloid cells, is required for transcriptional induction of interferon α and antiviral genes. IRF7 is activated by virus-induced phosphory- lation in the cytoplasm, leading to its translocation to the nu- cleus for transcriptional activity. Here, we revealed a nontranscriptional activity of IRF7 contributing to its antiviral functions. IRF7 interacted with the pro-inflammatory tran- scription factor NF-κB-p65 and inhibited the induction of in- flammatory target genes. Using knockdown, knockout, and overexpression strategies, we demonstrated that IRF7 inhibited NF-κB–dependent inflammatory …

Foxs1 Is Increased In Liver Fibrosis And Regulates Tgfβ Responsiveness And Proliferation Pathways In Human Hepatic Stellate Cells, Evelyn A. Bates, Zachary A. Kipp, Wang-Hsin Lee, Genesee J. Martinez, Lauren Weaver, Kathryn N. Becker, Sally Pauss, Justin F. Creeden, Garrett B. Anspach, Robert N. Helsley, Mei Xu, Maria E. C. Bruno, Marlene E. Starr, Terry D. Hinds Jr.

Markey Cancer Center Faculty Publications

Liver fibrosis commences with liver injury stimulating transforming growth factor beta (TGFβ) activation of hepatic stellate cells (HSCs), causing scarring and irreversible damage. TGFβ induces expression of the transcription factor Forkhead box S1 (FOXS1) in hepatocytes and may have a role in the pathogenesis of hepatocellular carcinoma (HCC). To date, no studies have determined how it affects HSCs. We analyzed human livers with cirrhosis, HCC, and a murine fibrosis model and found that FOXS1 expression is significantly higher in fibrotic livers but not in HCC. Next, we treated human LX2 HSC cells with TGFβ to activate fibrotic pathways, and FOXS1 …

Cell Aggregation Activates Small Gtpase Rac1 And Induces Cd44 Cleavage By Maintaining Lipid Raft Integrity, Dong Li, Younhee Park, Hami Hemati, Xia Liu

Markey Cancer Center Faculty Publications

Lipid rafts are highly ordered membrane domains that are enriched in cholesterol and glycosphingolipids and serve as major platforms for signal transduction. Cell detachment from the extracellular matrix (ECM) triggers lipid raft disruption and anoikis, which is a barrier for cancer cells to metastasize. Compared to single circulating tumor cells (CTCs), our recent studies have demonstrated that CD44-mediatd cell aggregation enhances the stemness, survival and metastatic ability of aggregated cells. Here, we investigated whether and how lipid rafts are involved in CD44-mediated cell aggregation. We found that cell detachment, which mimics the condition when tumor cells detach from the ECM …

Cyclin D1 Extensively Reprograms Metabolism To Support Biosynthetic Pathways In Hepatocytes, Heng Wu, Betsy T. Kren, Andrew N. Lane, Teresa A. Cassel, Richard M. Higashi, Teresa Whei-Mei Fan, George S. Scaria, Laurie L. Shekels, Mark A. Klein, Jeffrey H. Albrecht

Markey Cancer Center Faculty Publications

Cell proliferation requires metabolic reprogramming to accommodate biosynthesis of new cell components, and similar alterations occur in cancer cells. However, the mechanisms linking the cell cycle machinery to metabolism are not well defined. Cyclin D1, along with its main partner cyclindependent kinase 4 (Cdk4), is a pivotal cell cycle regulator and driver oncogene that is overexpressed in many cancers. Here, we examine hepatocyte proliferation to define novel effects of cyclin D1 on biosynthetic metabolism. Metabolomic studies reveal that cyclin D1 broadly promotes biosynthetic pathways including glycolysis, the pentose phosphate pathway, and the purine and pyrimidine nucleotide synthesis in hepatocytes. Proteomic …

Md_Harmonize: A Python Package For Atom-Level Harmonization Of Public Metabolic Databases, Huan Jin, Hunter N. B. Moseley

Markey Cancer Center Faculty Publications

A major challenge to integrating public metabolic resources is the use of different nomenclatures by individual databases. This paper presents md_harmonize, an open-source Python package for harmonizing compounds and metabolic reactions across various metabolic databases. The md_harmonize package utilizes a neighborhood-specific graph coloring method for generating a unique identifier for each compound via atom identifiers based on a compound’s chemical structure. The resulting harmonized compounds and reactions can be used for various downstream analyses, including the construction of atom-resolved metabolic networks and models for metabolic flux analysis. Parts of the md_harmonize package have been optimized using a variety of computational …

Design And Characterization Of A Novel Eef2k Degrader With Potent Therapeutic Efficacy Against Triple-Negative Breast Cancer, Changxin Zhong, Rongfeng Zhu, Ting Jiang, Sheng Tian, Xiaobao Zhao, Xiaoya Wan, Shilong Jiang, Zonglin Chen, Rong Gong, Linhao He, Jin-Ming Yang, Na Ye, Yan Cheng

Markey Cancer Center Faculty Publications

Dysregulated eEF2K expression is implicated in the pathogenesis of many human cancers, including triple-negative breast cancer (TNBC), making it a plausible therapeutic target. However, specific eEF2K inhibitors with potent anti-cancer activity have not been available so far. Targeted protein degradation has emerged as a new strategy for drug discovery. In this study, a novel small molecule chemical is designed and synthesized, named as compound C1, which shows potent activity in degrading eEF2K. C1 selectively binds to F8, L10, R144, C146, E229, and Y236 of the eEF2K protein and promotes its proteasomal degradation by increasing the interaction between eEF2K and the …

Benchmark Dataset For Training Machine Learning Models To Predict The Pathway Involvement Of Metabolites, Erik D. Huckvale, Christian D. Powell, Huan Jin, Hunter N. B. Moseley

Markey Cancer Center Faculty Publications

Metabolic pathways are a human-defined grouping of life sustaining biochemical reactions, metabolites being both the reactants and products of these reactions. But many public datasets include identified metabolites whose pathway involvement is unknown, hindering metabolic interpretation. To address these shortcomings, various machine learning models, including those trained on data from the Kyoto Encyclopedia of Genes and Genomes (KEGG), have been developed to predict the pathway involvement of metabolites based on their chemical descriptions; however, these prior models are based on old metabolite KEGG-based datasets, including one benchmark dataset that is invalid due to the presence of over 1500 duplicate entries. …

Human Schwann Cells In Vitro I. Nerve Tissue Processing, Pre-Degeneration, Isolation, And Culturing Of Primary Cells, Gabriela I. Aparicio, Paula V. Monje

Markey Cancer Center Faculty Publications

This paper presents versatile protocols to prepare primary human Schwann cell (hSC) cultures from mature peripheral nervous system tissues, including fascicles from long spinal nerves, nerve roots, and ganglia. This protocol starts with a description of nerve tissue procurement, handling, and dissection to obtain tissue sections suitable for hSC isolation and culturing. A description follows on how to disintegrate the nerve tissue by delayed enzymatic dissociation, plate the initial cell suspensions on a two-dimensional substrate, and culture the primary hSCs. Each section contains detailed procedures, technical notes, and background information to aid investigators in understanding and managing all steps. Some …

Copy Number Variation That Influences The Ionizing Radiation Sensitivity Of Oral Squamous Cell Carcinoma, Tadahide Izumi, Piotr G. Rychahou, Li Chen, Molly H. Smith, Joseph Valentino

Markey Cancer Center Faculty Publications

Genome instability in cancer cells causes not only point mutations but also structural variations of the genome, including copy number variations (CNVs). It has recently been proposed that CNVs arise in cancer to adapt to a given microenvironment to survive. However, how CNV influences cellular resistance against ionizing radiation remains unknown. PRMT5 (protein arginine methyltransferase 5) and APE1 (apurinic/apyrimidinic endonuclease 1), which enhance repair of DNA double-strand breaks and oxidative DNA damage, are closely localized in the chromosome 14 of the human genome. In this study, the genomics data for the PRMT5 and APE1 genes, including their expression, CNVs, and …

Distinct Camp Signaling Microdomains Differentially Regulate Melanosomal Ph And Pigmentation, Maftuna Yusupova, Dalee Zhou, Jaewon You, Jeydi Gonzalez-Guzman, Megha B. Ghanta, Hong Pu, Zalfa Abdel-Malek, Qiuying Chen, Steven S. Gross, John August D'Orazio, Shosuke Ito, Kazumasa Wakamatsu, Melissa L. Harris, Jonathan H. Zippin

Markey Cancer Center Faculty Publications

cAMP signaling is a well-established regulator of melanin synthesis. Two distinct cAMP signaling pathways—the transmembrane adenylyl cyclase pathway, activated primarily by the MC1R, and the soluble adenylyl cyclase (sAC) pathway—affect melanin synthesis. The sAC pathway affects melanin synthesis by regulating melanosomal pH, and the MC1R pathway affects melanin synthesis by regulating gene expression and post-translational modifications. However, whether MC1R genotype affects melanosomal pH is poorly understood. We now report that loss of function MC1R does not affect melanosomal pH. Thus, sAC signaling appears to be the only cAMP signaling pathway that regulates melanosomal pH. We also addressed whether MC1R genotype …

Phosphatidic Acid Binding To Patched Contributes To The Inhibition Of Smoothened And Hedgehog Signaling In Drosophila Wing Development, Jie Zhang, Yajuan Liu, Chi Wang, Craig W. Vander Kooi, Jianhang Jia

Markey Cancer Center Faculty Publications

Hedgehog (Hh) signaling controls growth and patterning during embryonic development and homeostasis in adult tissues. Hh binding to the receptor Patched (Ptc) elicits intracellular signaling by relieving Ptc-mediated inhibition of the transmembrane protein Smoothened (Smo). We uncovered a role for the lipid phosphatidic acid (PA) in the regulation of the Hh pathway in Drosophila melanogaster. Deleting the Ptc C-terminal tail or mutating the predicted PA-binding sites within it prevented Ptc from inhibiting Smo in wing discs and in cultured cells. The C-terminal tail of Ptc directly interacted with PA in vitro, an association that was reduced by Hh, and increased …

Regulated Translocation Of Neutral Sphingomyelinase-2 To The Plasma Membrane Drives Insulin Resistance In Steatotic Hepatocytes, Salim S. El-Amouri, Alexander A. Karakashian, Erhard Bieberich, Mariana N. Nikolova‑Karakashian

Markey Cancer Center Faculty Publications

Abstract Obesity-associated diabetes is linked to the accumulation of ceramide in various organs, including the liver. The exact mechanisms by which ceramide contributes to diabetic pathology are unclear, but one proposed scenario is that ceramide accumulation may inhibit insulin signaling pathways. It is unknown however whether the excess ceramide is generated proximal to the insulin receptor, that is, at the plasma membrane (PM), where it could affect the insulin signaling pathway directly, or the onset of insulin resistance is due to ceramide- induced mitochondrial dysfunction and/or lipotoxicity. Using hepatic cell lines and primary cultures, gain- and loss- of function approach, …

Bacteroides Fragilis In The Gut Microbiomes Of Alzheimer’S Disease Activates Microglia And Triggers Pathogenesis In Neuronal C/Ebpβ Transgenic Mice, Yiyuan Xia, Yifan Xiao, Zi-Hao Wang, Ashfaqul M. Alam, John P. Haran, Beth A. Mccormick, Xiji Shu, Xiaochuan Wang, Keqiang Ye

Markey Cancer Center Faculty Publications

Gut dysbiosis contributes to Alzheimer’s disease (AD) pathogenesis, and Bacteroides strains are selectively elevated in AD gut microbiota. However, it remains unknown which Bacteroides species and how their metabolites trigger AD pathologies. Here we show that Bacteroides fragilis and their metabolites 12-hydroxy-heptadecatrienoic acid (12-HHTrE) and Prostaglandin E2 (PGE2) activate microglia and induce AD pathogenesis in neuronal C/EBPβ transgenic mice. Recolonization of antibiotics cocktail-pretreated Thy1-C/EBPβ transgenic mice with AD patient fecal samples elicits AD pathologies, associated with C/EBPβ/Asparaginyl endopeptidase (AEP) pathway upregulation, microglia activation, and cognitive disorders compared to mice receiving healthy donors’ fecal microbiota transplantation (FMT). Microbial 16S rRNA sequencing …

Unleashing The Potential Of 1,3-Diketone Analogues As Selective Lh2 Inhibitors, Juhoon Lee, Hou-Fu Guo, Shike Wang, Yazdan Maghsoud, Erik Antonio Vázquez-Montelongo, Zhifeng Jing, Rae M. Sammons, Eun Jeong Cho, Pengyu Ren, G. Andrés Cisneros, Jonathan M. Kurie, Kevin N. Dalby

Markey Cancer Center Faculty Publications

Lysyl hydroxylase 2 (LH2) catalyzes the formation of highly stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs), thus promoting lung cancer metastasis through its capacity to modulate specific types of collagen cross-links within the tumor stroma. Using 1 and 2 from our previous high-throughput screening (HTS) as lead probes, we prepared a series of 1,3-diketone analogues, 1−18, and identified 12 and 13 that inhibit LH2 with IC50’s of approximately 300 and 500 nM, respectively. Compounds 12 and 13 demonstrate selectivity for LH2 over LH1 and LH3. Quantum mechanics/molecular mechanics (QM/MM) modeling indicates that the selectivity of 12 and 13 may stem from …

Co-Targeting Nucleus Accumbens Associate 1 And Nf-Κb Signaling Synergistically Inhibits Melanoma Growth, Lixiang Gu, Xingcong Ren, Chrispus Ngule, Xiaofang Xiong, Jianxun Song, Zhiguo Li, Jin-Ming Yang

Markey Cancer Center Faculty Publications

Nucleus-accumbens-associated protein-1 (NAC1) is a cancer-related transcriptional factor encoded by the NACC1 gene, which is amplified and overexpressed in various human cancers and has been appreciated as one of the top potential cancer driver genes. NAC1 has therefore been explored as a potential therapeutic target for managing malignant tumors. Here, we show that NAC1 is a negative regulator of NF-κB signaling, and NAC1 depletion enhances the level of the nuclear NF-κB in human melanoma. Furthermore, the inhibition of NF-κB signaling significantly potentiates the antineoplastic activity of the NAC1 inhibition in both the cultured melanoma cells and xenograft tumors. This study …

Metabolic Disruption Induced By Mtor Signaling Pathway Inhibition In Regulatory T-Cell Expansion For Clinical Application, Roberto Gedaly, Gabriel Orozco, Alexandre P. Ancheta, Mackenzie Donoho, Siddharth Desai, Fanny Chapelin, Aman Khurana, Lillie J. Lewis, Cuiping Zhang, Francesc Marti

Markey Cancer Center Faculty Publications

Background: Regulatory T cell (Treg) therapy is considered an alternative approach to induce tolerance in transplantation. If successful, this therapy may have implications on immunosuppression minimization/withdrawal to reduce drug-induced toxicity in patients. The aim of this study was to assess the efficacy of the mTORC1/C2 inhibitor, AZD8055, in the manufacturing of clinically competent Treg cells and compare the effects with those induced by rapamycin (RAPA), another mTOR inhibitor commonly used in Treg expansion protocols.

Methods: Primary human Treg cells were isolated from leukapheresis product. Cell viability, expansion rates, suppressive function, autophagy, mitochondrial unfolded protein response (mitoUPR), and cell metabolic profile …

The S1p Receptor 1 Antagonist Ponesimod Reduces Tlr4-Induced Neuroinflammation And Increases Aβ Clearance In 5xfad Mice, Zhihui Zhu, Liping Zhang, Ahmed Elsherbini, Simone M. Crivelli, Priyanka Tripathi, Carmen Harper, Zainuddin Quadri, Stefka D. Spassieva, Erhard Bieberich

Markey Cancer Center Faculty Publications

Background Previously, we showed that the sphingosine-1-phosphate (S1P) transporter spinster 2 (Spns2) mediates activation of microglia in response to amyloid β peptide (Aβ). Here, we investigated if Ponesimod, a functional S1P receptor 1 (S1PR1) antagonist, prevents Aβ-induced activation of glial cells and Alzheimer’s disease (AD) pathology.

Methods We used primary cultures of glial cells and the 5XFAD mouse model to determine the effect of Aβ and Ponesimod on glial activation, Aβ phagocytosis, cytokine levels and pro-inflammatory signaling pathways, AD pathology, and cognitive performance.

Findings Aβ42 increased the levels of TLR4 and S1PR1, leading to their complex formation. Ponesimod prevented the …

Enhanced Bypass Of Pd-L1 Translation Reduces The Therapeutic Response To Mtor Kinase Inhibitors, Yanan Cao, Qing Ye, Murong Ma, Qing-Bai She

Markey Cancer Center Faculty Publications

Increased PD-L1 expression in cancer cells is known to enhance immunosuppression, but the mechanism underlying PD-L1 upregulation is incompletely characterized. We show that PD-L1 expression is upregulated through internal ribosomal entry site (IRES)-mediated translation upon mTORC1 inhibition. We identify an IRES element in the PD-L1 50-UTR that permits cap-independent translation and promotes continuous production of PD-L1 protein despite effective inhibition of mTORC1. eIF4A is found to be a key PD-L1 IRES-binding protein that enhances PD-L1 IRES activity and protein production in tumor cells treated with mTOR kinase inhibitors (mTORkis). Notably, treatment with mTORkis in vivo elevates PD-L1 levels and reduces …

Messes: Software For Transforming Messy Research Datasets Into Clean Submissions To Metabolomics Workbench For Public Sharing, P. Travis Thompson, Hunter N. B. Moseley

Markey Cancer Center Faculty Publications

In recent years, the FAIR guiding principles and the broader concept of open science has grown in importance in academic research, especially as funding entities have aggressively promoted public sharing of research products. Key to public research sharing is deposition of datasets into online data repositories, but it can be a chore to transform messy unstructured data into the forms required by these repositories. To help generate Metabolomics Workbench depositions, we have developed the MESSES (Metadata from Experimental SpreadSheets Extraction System) software package, implemented in the Python 3 programming language and supported on Linux, Windows, and Mac operating systems. MESSES …

The Metabolomics Workbench File Status Website: A Metadata Repository Promoting Fair Principles Of Metabolomics Data, Christian D. Powell, Hunter N. B. Moseley

Markey Cancer Center Faculty Publications

Background: An updated version of the mwtab Python package for programmatic access to the Metabolomics Workbench (MetabolomicsWB) data repository was released at the beginning of 2021. Along with updating the package to match the changes to MetabolomicsWB’s ‘mwTab’ file format specification and enhancing the package’s functionality, the included validation facilities were used to detect and catalog file inconsistencies and errors across all publicly available datasets in MetabolomicsWB.

Results: The MetabolomicsWB File Status website was developed to provide continuous validation of MetabolomicsWB data files and a useful interface to all found inconsistencies and errors. This list of detectable issues/errors include format …

Differential Inhibition Of Anaplerotic Pyruvate Carboxylation And Glutaminolysis-Fueled Anabolism Underlies Distinct Toxicity Of Selenium Agents In Human Lung Cancer, Teresa W-M Fan, Jason Winnike, Ahmad Al-Attar, Alex C. Belshoff, Pawel Lorkiewicz, Jin Lian Tan, Min Wu, Richard M. Higashi, Andrew N. Lane

Markey Cancer Center Faculty Publications

Past chemopreventive human trials on dietary selenium supplements produced controversial outcomes. They largely employed selenomethionine (SeM)-based diets. SeM was less toxic than selenite or methylseleninic acid (MSeA) to lung cancer cells. We thus investigated the toxic action of these Se agents in two non-small cell lung cancer (NSCLC) cell lines and ex vivo organotypic cultures (OTC) of NSCLC patient lung tissues. Stable isotope-resolved metabolomics (SIRM) using 13C6-glucose and 13C5,15N2-glutamine tracers with gene knockdowns were employed to examine metabolic dysregulations associated with cell type- and treatment-dependent phenotypic changes. Inhibition of key anaplerotic processes, pyruvate carboxylation (PyC) and glutaminolysis were elicited by …

Noncovalent Interactions That Tune The Reactivities Of The Flavins In Bifurcating Electron Transferring Flavoprotein, María González-Viegas, Rajiv K. Kar, Anne-Frances Miller, Maria-Andrea Mroginski

Markey Cancer Center Faculty Publications

Bifurcating electron transferring flavoproteins (Bf-ETFs) tune chemically identical flavins to two contrasting roles. To understand how, we used hybrid quantum mechanical molecular mechanical calculations to characterize noncovalent interactions applied to each flavin by the protein. Our computations replicated the differences between the reactivities of the flavins: the electron transferring flavin ( ETflavin) was calculated to stabilize anionic semiquinone (ASQ) as needed to execute its single-electron transfers, whereas the Bf flavin (Bfflavin) was found to disfavor the ASQ state more than does free flavin and to be less susceptible to reduction. The stability of ETflavin ASQ was attributed in part to …

Novel Isolation Method Reveals Sex-Specific Composition And Neurotoxicity Of Small Extracellular Vesicles In A Mouse Model Of Alzheimer’S Disease, Ahmed Elsherbini, Zhihui Zhu, Zainuddin Quadri, Simone M. Crivelli, Xiaojia Ren, Hemendra J. Vekaria, Priyanka Tripathi, Liping Zhang, Wenbo Zhi, Erhard Bieberich

Markey Cancer Center Faculty Publications

We developed a new method to isolate small extracellular vesicles (sEVs) from male and female wild-type and 5xFAD mouse brains to investigate the sex-specific functions of sEVs in Alzheimer’s disease (AD). A mass spectrometric analysis revealed that sEVs contained proteins critical for EV formation and Aβ. ExoView analysis showed that female mice contained more GFAP and Aβ-labeled sEVs, suggesting that a larger proportion of sEVs from the female brain is derived from astrocytes and/or more likely to bind to Aβ. Moreover, sEVs from female brains had more acid sphingomyelinase (ASM) and ceramide, an enzyme and its sphingolipid product important for …

Combination Therapy With Trastuzumab And Niraparib: Quantifying Early Proliferative Alterations In Her2+ Breast Cancer Models, Ameer Mansur, Patrick N. Song, Yun Lu, Andrew C. Burns, Luke Sligh, Eddy S. Yang, Anna G. Sorace

Markey Cancer Center Faculty Publications

HER2–targeted treatments have improved survival rates in HER2+ breast cancer patients, yet poor responsiveness remains a major clinical obstacle. Recently, HER2+ breast cancer cells, both resistant and responsive to HER2–targeted therapies, have demonstrated sensitivity to poly–(ADP– ribose) polymerase (PARP) inhibition, independent of DNA repair deficiencies. This study seeks to describe biological factors that precede cell viability changes in response to the combination of trastuzumab and PARP inhibition. Treatment response was evaluated in HER2+ and HER2– breast cancer cells. Further, we evaluated the utility of 3′–Deoxy–3′–[18F]–fluorothymidine positron emission tomography ([18F]FLT–PET) imaging for early response assessment in a HER2+ patient derived xenograft …