eSymposia | Proteomics in Cell Biology and Disease



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KS|QA: Kathryn Lilley, PhDKS|QA: Kathryn Lilley, PhD

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KS|QA: Kathryn Lilley, PhD



KS|QA with Dr. Kathryn Lilley, PhD

In collaboration with EMBO Press

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In this exclusive interview, EMBO editor Maria Polychronidou speaks with meeting co-organizer Dr. Kathryn Lilley, about her vision for the “Proteomics in Cell Biology and Disease” eSymposia virtual meeting, and many of the exciting new advances and directions that will be covered. Hear from Dr. Lilley about:

  • the latest single cell proteomics approaches that are revealing a whole new world of proteomics exploration and insight.
  • how spatial and temporal protein interactions and dynamics elucidate intricacies of cellular processes and disease mechanisms.
  • the virtual meeting format, and how the scientific community is adapting to new platforms and technologies during these times.
  • her recent election to EMBO, and what becoming a member means to her career.

Dr. Kathryn Lilley is the Professor of Cellular Dynamics in the Department of Biochemistry, University of Cambridge, UK, where she directs the Cambridge Center for Proteomics. Her research program creates and applies technologies to measure the dynamics of the transcriptome and proteome in high throughput in space and time during critical cellular processes. Her groups has also contributed many informatics tools to efficiently mine spatiotemporal proteomics data. In 2018 she was awarded the HUPO Distinguished Achievements in Proteomics Award. In 2020 she was elected as a member of EMBO.

Dr. Maria Polychronidou is a Senior Scientific Editor at Molecular Systems Biology (EMBO Press). She received her PhD from the University of Heidelberg, where she studied the role of nuclear membrane proteins in development and aging. During her post-doctoral work, she focused on the analysis of tissue-specific regulatory functions of Hox transcription factors using a combination of computational and genome-wide methods. She joined Molecular Systems Biology as an Editor in 2013.

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Phosphoproteomics revealed excessive phosphate burden perturbed intracellular signaling pathways


Phosphoproteomics revealed excessive phosphate burden perturbed intracellular signaling pathways Phosphoproteomics revealed excessive phosphate burden perturbed intracellular signaling pathways Ping He1, Alexis Janoczkin1, Spencer Kiers1, Erik Beeler1, Belinda B. Willard2, Mohammed S. Razzaque 3 1 Department of Biochemistry, Lake Erie College of Osteopathic Medicine, Erie, PA, USA 2 Proteomics and Metabolomics Core, Cleveland Clinic Lerner Research Institute, Cleveland, Ohio, USA. 3 Department of Pathology, Lake Erie College of Osteopathic Medicine, Erie, PA, USA Phosphate (Pi) is an essential nutrient for the human body that maintains and modulates normal cellular functions. Insufficient intake of dietary Pi leads to skeletal deformities, whereas Pi overload causes cytotoxicity. Preclinical and clinical studies have shown systemic organ damages induced by Pi-mediated cytotoxicity. However, the underlying molecular mechanisms are poorly understood. In our earlier studies, we have shown abnormally high Pi-mediated profound changes in protein expression and phosphorylation in components regulating AKT and mitogen-activated protein kinase (MAPK) signaling cascades, endoplasmic reticulum (ER) stress, and epithelial-mesenchymal transition (EMT). To expand our understanding of Pi-rewired signaling networks, we employed proteomics and phosphoproteomics to systematically analyze Pi-mediated changes in protein abundance and protein phosphorylation. HEK293 cells were treated with 1mM, 10mM (pro-survival) and 40mM (pro-death) Pi for 24 hours in triplicate settings. After trypsinization, the digested peptides were desalted followed by TMT labeling. For global proteomics, the TMT labeled samples were separated using a high pH reversed-phase HPLC method, and the collections were combined into 4 fractions. For phosphoproteomics, the peptides were PO3-enriched followed by TMT labeling. The labeled phosphopeptide samples were combined without fractionation. The peptides were analyzed on a Thermo Scientific Fusion Lumos mass spectrometry system, and the data was analyzed by Sequest programs to search against the human UniProtKB database for peptide/protein identification and quantification. A total of 4704 proteins and 5041 phosphopeptides were identified of which 4249 peptides had quantitative values. We thereafter compared the protein abundance and protein phosphorylation changes between 1mM and 10mM, and 1mM and 40mM Pi treated groups. Compared to 1mM Pi treated groups, 10mM Pi treatment caused one protein (Calmodulin-3) down-regulated over 2 folds and 20 phosphopeptides from 12 proteins down-regulated over 1.5 folds. In contrast to 10mM Pi, 40mM Pi treatment elicited more pronounced changes in both global protein expression and protein phosphorylation. The treatment resulted in over 2-fold change in 18 downregulated proteins and 44 upregulated proteins. It led to over 1.5-fold change in 560 downregulated phosphopeptides from 187 proteins and 19 upregulated phosphopeptides from 16 proteins. Bioinformatic pathway analysis and literature searching revealed that the differentially expressed and phosphorylated proteins were enriched in signal transduction pathways (such as Calcium and AKT signaling) and diverse biological processes (such as Cell cycle/DNA repair, apoptosis, ER stress, and mRNA alternative splicing). Molecular cross-talks were also identified among the Pi-induced pathological pathways. Further biological validation studies are underway to verify the changes of the selected candidates triggered by excessive Pi and to explore their pathological functions in the context of high Pi. Phosphoproteomics-based investigation of Pi-mediated alterations of the global phosphorylation landscape will offer a panoramic view of Pi-associated signaling networks, which will deepen our understanding of molecular mechanisms of phosphate toxicity and likely to provide the potential therapeutic targets to reduce high Pi-related pathologies.

Speaker(s):
  • Ping He, MD, PhD, Lake Erie College of Osteopathic Medicine (LECOM)

A robust preparation method for micro-sample scale discovery proteomics


A robust preparation method for micro-sample scale discovery proteomics Benjamin Cooper1,2,3*, Dalia Ponce1, Frédéric Hollande2,3, Jonathan Mangum1. 1. Department of Pharmacology & Therapeutics, University of Melbourne, Australia. 2. Department of Clinical Pathology, University of Melbourne, Australia 3. University of Melbourne Centre for Cancer Research, Victorian Comprehensive Cancer Centre, Melbourne, Australia. The application of discovery proteomics to scarce samples is restricted by consumptive preparation methodologies. Here, we aimed to develop a sample preparation method which demonstrates robust, scalable, and affordable performance at the middle to low microgram scale. Using a variation of an in-StageTip (iST) protocol1, 50 µg of HEK-293T cell lysates were prepared with varying digestive enzyme concentrations, incubation times, and liquid strong cation exchange resin (SCX) volumes. Each variable condition was sequentially tested on three independent cell lysates with 10 µg of protein being analysed (LC-MS/MS with LFQ) using a 6545XT Advance-Bio ESI-LC-Q/TOF (Agilent Technologies). We observed the greatest number of IDs from protein samples incubated overnight with 1:50 Trypsin-LysC followed by peptide capture and sample clean-up using 10 µL of liquid SCX resin (mean number of IDs ± SD, 595 ± 18). We sought to benchmark this performance of our ‘homemade’ liquid SCX inStage-Tip (HiST) against a commercially available iST sample preparation kit (PreOmics). Our method generated significantly greater numbers of protein IDs (mean number of IDs ± SD, 376 ± 95) and lower levels of intra-replicate variability (CV = HiST 3.08 vs. PreOmics 25.25) when compared with the commercial kit. These data highlight the robustness of our method in middle to low microgram sample preparation contexts and supports its use in future high-throughput proteomic analysis of scarce samples. 1 Kulak, N., Pichler, G., Paron, I. et al. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat Methods 11, 319–324 (2014). https://doi.org/10.1038/nmeth.... This research was supported by Agilent technologies in the provision of MS equipment and PhD stipend for lead Author. This research is also supported by an Australian Government Research Training Program (RTP) Scholarship. *email benc1@student.unimelb.edu.au

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Integrative assessment of apical and molecular respiratory exposure effects in an 18-month systems toxicology study with A/J mice


Integrative assessment of apical and molecular respiratory exposure effects in an 18-month systems toxicology study with A/J mice Smoking cessation is the most effective measure for reducing the risk of smoking-related diseases. However, switching to less harmful products (modified risk tobacco products [MRTP]) can be an alternative to help reduce such risk for adult smokers who would otherwise continue to smoke. Standard toxicology endpoints for toxicological assessment of aerosols from less harmful products and cigarette smoke (CS) can lack sensitivity, and systems toxicology can yield deeper insights into toxicological mechanisms. In an 18-month chronic carcinogenicity/toxicity study in A/J mice (OECD Test Guideline 453), we assessed the aerosol of Tobacco Heating System 2.2 (THS 2.2), a candidate MRTP based on the heat-not-burn principle, in comparison with 3R4F CS. To capture toxicity- and disease-relevant mechanisms, we complemented standard toxicology endpoints with multiomics systems toxicology analyses, including proteomics and mRNA/miRNA transcriptomics analyses. Here, we report the integrative assessment of the apical and molecular exposure effects on the respiratory tract (nose, larynx, and lungs). Compared with 3R4F CS, THS 2.2 aerosol exerted far fewer effects on respiratory tract histology, including adaptive tissue changes in nasal and laryngeal epithelia and inflammation and emphysematous changes in the lungs. Omics analyses revealed inflammatory responses following 3R4F CS exposure (e.g., antimicrobial peptide response in the nose) and both shared and distinct oxidative and xenobiotic responses upon 3R4F CS exposure across the respiratory tract. Integrative computational analyses confirmed the substantially lower impact of THS 2.2 aerosol than 3R4F CS on toxicologically and disease-relevant molecular processes such as inflammation, oxidative stress responses, and xenobiotic metabolism. Overall, this work exemplifies how apical and molecular endpoints, including proteomics endpoints, can be combined effectively for toxicological assessment and further supports the findings on the reduced respiratory risks of THS 2.2 aerosol. Reference: Titz et al. Respiratory effects of exposure to aerosol from the candidate modified-risk tobacco product THS 2.2 in an 18-month systems toxicology study with A/J mice. Toxicological Sciences. accepted.

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Genomic and multi-tissue proteomic integration for understanding the biology of disease and other complex traits


Genomic and multi-tissue proteomic integration for understanding the biology of disease and other complex traits Understanding the tissue-specific genetic architecture of protein levels is instrumental to understand the biology of health and disease. We generated a genomic atlas of protein levels in multiple neurologically relevant tissues (380 brain, 835 cerebrospinal fluid (CSF) and 529 plasma), by profiling thousands of proteins (713 CSF, 931 plasma and 1079 brain) in a large and well-characterized cohort. We identified 274, 127 and 32 protein quantitative loci (pQTL) for CSF, plasma and brain respectively. cis-pQTL were more likely to be shared across tissues but trans-pQTL tend to be tissue-specific. Between 44% to 68.2% of the pQTL do not colocalize with expression, splicing, methylation or histone QTLs, indicating that protein levels have a different genetic architecture to those that regulate gene expression. By combining our pQTL with Mendelian Randomization approaches we identified potential novel biomarkers and drug targets for neurodegenerative diseases including Alzheimer disease and frontotemporal dementia. Here we present the first multi-tissue study yielding hundred of novel pQTLs. This data will be instrumental to identify the functional gene from GWAS signals, identify novel biological protein-protein interactions, identify novel potential biomarkers and drug targets for complex traits. Chengran Yang1,2,3, Fabiana G. Farias1,2,3, Laura Ibanez1,2,3, Brooke Sadler4, Maria Victoria Fernandez1,2,3, Fengxian Wang1,2,3, Joseph L. Bradley1,2,3, Brett Eiffert1,2,3, Jorge A. Bahena1,2,3, John P. Budde1,2,3, Zeran Li1,2,3, Umber Dube1,2,3, Yun Ju Sung1,2,3, Kathie A. Mihindukulasuriya1,2,3, John C. Morris3,5,6, Anne Fagan3,5,6, Richard J. Perrin3,5,6, Bruno Benitez1,2,3, Herve Rhinn7, Oscar Harari1,2,3,6† and Carlos Cruchaga1,2,3,6†* 1. Department of Psychiatry, Washington University School of Medicine, St Louis, MO, USA 2. NeuroGenomics and Informatics, Washington University School of Medicine, St Louis, MO, USA 3. Hope Center for Neurological Disorders, Washington University School of Medicine, St Louis, MO, USA 4. Pediatrics Hematology/Oncology, Washington University School of Medicine, St Louis, MO, USA 5. Department of Neurology, Washington University School of Medicine, St Louis, MO, USA 6. The Charles F. and Joanne Knight Alzheimer Disease Research Center, Washington University School of Medicine, St Louis, MO, USA. 7. Department of Bioinformatics. Alector, Inc. 151 Oyster Point Blvd. #300 South San Francisco, CA, USA.

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Dengue virus non-structural protein 1 (NS1) protein-protein interactions in mosquito cells reveal participation in gene expression regulation


Dengue virus non-structural protein 1 (NS1) protein-protein interactions in mosquito cells reveal participation in gene expression regulation Abstract: The dengue virus (DENV) NS1 protein is a multifunctional protein essential for viral replication and also acts as an immunomodulator. Yet, increasing evidence indicates that some properties of NS1 differ between vertebrate and mosquito cells. In order to gain insight of DENV NS1 functions in mosquito cells, the protein-protein interactions (PPIs) of DENV NS1 in Aedes albopictus C6/36 cells was obtained using a proximity biotinylation system (BioID). This system is based on a plasmid that expresses the DENV NS1 protein fused to a promiscuous biotin-ligase (BirA) enzyme from E. coli, which will add biotin to proteins that potentially interacts with NS1. The biotinylated proteins were purified using streptavidin beads and identified by mass spectrometry. A total of 817 proteins possibly interacting with DENV NS1 were identified. Of these, near 10% matched with previously reported ontology groups for NS1 PPIs in vertebrate cells; including proteins of the oligosaccharide transferase complex (OST), the chaperonin containing TCP-1 (CCT), nuclear import and export, vesicle localization and ribosomal proteins. Interestingly, other protein pathways such as epigenetic regulation, RNA silencing and apoptosis, not previously reported in vertebrate cells, were also found as part of the NS1 PPIs in mosquito cells. The interactions between NS1 and 4 selected proteins (Dido1, RPL26, Sec61A and GRP78) were validated in DENV infected C6/36 cells by colocalization and by proximity ligation assays. Due to the strong and novel interaction observed between Dido1 (Death Inducer-Obliterator 1) and NS1, we further explore the role of Dido 1 in viral infection. Dido1 knockdown in C6/36 and Aag2 (Aedes aegypti) cells results in a significant (one log) reduction in DENV and ZIKV progeny, suggesting that Dido 1 is a host factor necessary for flavivirus replication in the mosquito vector. Transcription analysis of C6/36 cells where compared in conditions of normal or knockdown expression of Dido1 and infection with DENV2 or not. Results reveal variations in gene expression of multiple genes consistent with the molecular role of Dido1, which has been related to transcription and epigenetic regulation. Ontology of differentially expressed genes include pathways previously associated with DENV infection such as RNA surveillance, IMD and Toll pathways. These results could help to understand the heterogenicity of NS1 as a multifunctional protein essential for Flavivirus infection in mosquito cells. Autors: Caraballo-Hernández Gerson I.1, Rosales-Ramírez Romel1, Siyuan Ding2, Harry-B. Greenberg2; Ludert Juan E.1. 1 Department of Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies (CINVESTAV-IPN), Mexico City, Mexico. Emails: gerson.caraballo@cinvestav.mx; jludert@cinvestav.mx 2 Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA.

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Proteograph™, a multi-nanoparticle platform, enables plasma proteomics profiling at scale and speed, significantly improving coverage and scalability versus traditional deep fractionation methods


Proteograph™, a multi-nanoparticle platform, enables plasma proteomics profiling at scale and speed, significantly improving coverage and scalability versus traditional deep fractionation methods Shadi Ferdosi1, Taher Elgierari1, Patrick A. Everley1, Matthew McLean1, Martin Goldberg1, Juan Cruz Cuevas1, John E. Blume1, Omid C. Farokhzad1 and Daniel Hornburg1 1 Seer Inc. 3200 Bridge Parkway - Suite 102 Redwood CIty - CA 94065 U.S.A. Blood plasma, which comprises signals from most tissues, is a rich source of protein biomarkers for disease detection, but its large dynamic range of protein concentrations necessitates complex workflow trade-offs between throughput, scalability, coverage, and precision. We developed a deep, high-throughput quantitative proteome profiling platform, Proteograph, which leverages the selective protein-nanosurface interactions of nanoparticles engineered with distinct physicochemical properties to provide broad coverage of the plasma proteome at scale. In a pilot study we compared the Proteograph to common deep plasma proteome workflows in terms of depth, dynamic range, coverage, throughput, and precision and demonstrated reproducible quantification of approximately 2,000 proteins (Blume et al., Nat Commun 11, 3662 (2020)). We also performed a head-to-head comparison of the Proteograph to typical workflows, including combined abundant protein-immunodepletion and high-pH peptide fractionation, using a pooled common EDTA plasma sample. Samples processed with each method were analyzed by micro LC-MS/MS interfaced to a Sciex 6600+ mass spectrometer (MS) operating in data-independent mode. MS data were processed using Spectronaut (1% peptide and protein false discovery rate). Coverage, depth, precision, and workflow efficiency were evaluated, with all statistical analyses performed in R. Proteograph identified more than 1,600 protein groups in this common sample, spanning nearly the full range of protein abundance in plasma. Compared to standard fractionation workflows, Proteograph captured proteins on average at 10-times lower abundance, outperforming the identification rate of high-pH fractionation fivefold for the two lowest orders of magnitude. In terms of time and resources, the Proteograph workflow upstream of the MS requires only 7 hours with only 30 minutes of hands-on time and has better precision compared to depletion and fractionation methods, which require multiple days. Proteograph demonstrates superior performance on the measures evaluated, distinguishing it as a robust and efficient platform for unbiased, deep, rapid, large-scale proteomics to quantify thousands of proteins across large numbers of samples.

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Dissect RNA-Protein Interactome in Living Cells Using CRISPR-Assisted Proximity Labeling


Dissect RNA-Protein Interactome in Living Cells Using CRISPR-Assisted Proximity Labeling Delineating the protein network associated with long non-coding RNAs (lncRNAs) is fundamental to understanding the functional mechanisms of lncRNAs. Current methods to identify lncRNA binding proteins either rely on crosslinking mediated complex co-precipitation or require extensive molecular engineering, leading to drawbacks such as loss of cellular context and low capture efficiency. We developed CRISPR-Assisted RNA-Protein Interaction Detection (CARPID), which leverages CRISPR/CasRx-based RNA targeting and proximity labeling to identify binding proteins of specific lncRNAs in native cellular context. Applying to the nuclear lncRNA XIST, CARPID reliably captured a list of its known interacting proteins. In addition, we also discovered multiple novel XIST-binding proteins, including a transcription initiation factor TAF15 and an ISWI chromatin remodeler SNF2L, which displayed distinct activities in X-inactivation. Furthermore, we generalized CARPID to explore binding proteins of a tumor-associated lncRNA DANCR and interestingly revealed an enrichment of proteins associated with extracellular exosomes, suggesting a pathway for its presence in circulation. Notably, CARPID can be harnessed to recover the labeled proteins from exosomes. The DANCR-dependent labeling of exosomal proteins were significantly reduced by downregulation of DANCR with active CasRx. We conclude that CARPID is a reliable and robust method to dissect the binding proteins of lncRNA inside living cells. Jingyu Li1,2, Wenkai Yi2, Xiaoxuan Zhu2, Ligang Fan2, Kui Ming Chan1,2, Jian Yan2, Liang Zhang1,2 1. Key Laboratory of Biochip Technology, Biotech and Health Centre, Shenzhen Research Institute of City University of Hong Kong, Shenzhen, China 2. Department of Biomedical Sciences, City University of Hong Kong, Hong Kong SAR, China

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Phospho-proteomic Characterization of IL-2 and IL-15 Signaling in Natural Killer Cells


Phospho-proteomic Characterization of IL-2 and IL-15 Signaling in Natural Killer Cells Abstract: Natural Killer (NK) cells are cytotoxic lymphocytes critical to the innate immune system. Despite their importance in immunology, no studies have characterized how NK cells respond to cytokine stimulation. NK cell proliferation is primarily driven by cytokines, particularly interleukin (IL)-2 and IL-15. Therefore, we performed phospho-proteomic analysis of IL-2 and IL-15 signaling in a model system of NK cells (NK-92). In two independent biological replicates, we identified 4,728 high-confidence phospho-peptides. After 30 minutes of stimulation, analysis revealed that both IL-2 and IL-15 induced significant up-regulation of phosphorylation of Y694 on STAT5 (signal transducer and activator of transcription 5), a phosphorylation site known to be essential to immune cell proliferation. Further analysis with PCA revealed differences between the two cytokines including upregulation of SIPA1 pS74 and RETREG3 pS229 following IL-2 stimulation and upregulation of PRPF3 pS619 and TRIP11 p464 following IL-15 stimulation. Kinase set enrichment analysis (KSEA) revealed several kinases which were differentially activated by IL-2 or IL-15 including Casein Kinase II, Aurora A, and Aurora by IL-2 and GSK-3, ERK1, ERK2, CDK5, PKA, and PKC by IL-15. In addition, time course analysis of NK cells stimulated by either IL-2 or IL-15 for 0, 5, 10, 15, or 30 minutes revealed differential clustering of phospho-peptides, suggesting short-term phosphorylation differences that may contribute to different activation networks. Taken together, these results represent the first phospho-proteomic analysis of NK cells and suggest that the cytokines IL-2 and IL-15 promote NK cell proliferation through different mechanisms. Authors: (presenting author) Melanie A. MacMullan, University of Southern California, Los Angeles, CA, USA, macmulla@usc.edu Pin Wang, University of Southern California, Los Angeles, CA, USA, pinwang@usc.edu Nicholas A. Graham, University of Southern California, Los Angeles, CA, USA, nagraham@usc.edu

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NPC Defects in Alzheimer’s Disease: Investigating NUP153, -93, and -214 and Transport Regulators NTF2, CAMK4, and IPO4 Mislocalization in Hippocampal Neurons


NPC Defects in Alzheimer’s Disease: Investigating NUP153, -93, and -214 and Transport Regulators NTF2, CAMK4, and IPO4 Mislocalization in Hippocampal Neurons Authors *M. Tran1, M. GORAS1, D. BROKAW1, J. NOLZ1, E. DELVAUX1, D. MASTROENI1, P. COLEMAN1; Arizona State University-Banner Neurodegenerative Disease Research Center, Tempe, Arizona, USA. Abstract Alzheimer’s disease (AD) is a progressive, neurocognitive disorder characterized by memory dysfunction as well as the presence of neuropathological aberrations, namely amyloid plaques, and neurofibrillary tangles. However, AD neuropathology has been found to be a poor prognostic indicator and clinical trials targeting AD neuropathology have largely been unfruitful, necessitating new research into novel mechanisms of the underlying pathways mediating the cognitive decline. Eukaryotic cells rely on the movement of proteins between the nucleus and cytoplasm and, for cargo larger than a couple nanometers in diameter, transport is facilitated by the nuclear pore complex (NPC). NPC contain approximately 30 distinct nucleoporins (NUPs) which form a central channel with filaments extending into the nucleus and cytoplasm. Mutations in nucleoporin genes have been linked to various human diseases including nephrotic, cardiac, and neurodegenerative diseases. A recent study showed that mislocalization of nucleoporins from the nuclear membrane in AD may be directly interacting with soluble tau-p12 suggesting tau’s role in NPC deterioration and nuclear-cytoplasmic transport defects. However, this study only investigated four nucleoporins and is thus ill-equipped to make generalizable conclusions regarding global changes to the NPC that contribute to AD nuclear dysfunction and pathophysiology. Furthermore, there is a lack of research studying the effects of nuclear-cytoplasmic transporters and their relationship with cell function deterioration in AD. Mislocalization of NTF2, a protein that maintains the RAN gradient, could have widespread effects on a variety of cell processes on top of nuclear-cytoplasmic transport imbalance. In this study, we evaluate global changes to NPC and nuclear-cytoplasmic transporters in AD by analyzing gene expression from homogenate brain tissue and neuronal data. Three significantly differentially expressed NUPs (NUP-214, -93, -153), representing different parts of the NPC (cytoplasmic filaments, inner ring structure, nuclear basket), and NTF2, CAMK4, and IPO4 were selected from the significant results for further laboratory validation via immunohistochemistry and immunoblotting on human postmortem brain tissue. This laboratory research represents one of the first attempts to categorize differential changes throughout the NPC and transport regulators in AD. Bioinformatic analysis revealed widespread differential NUP and nuclear cytoplasmic transport gene expression across multiple brain regions in AD. These results were reflected in the immunohistochemistry and immunoblotting, which revealed quantity and localization changes of the selected NUPs and transporters in AD. Future studies will explore the hierarchical relationship between neuropathological hallmarks of AD and NPC and transport aberrations to better understand the etiology of impaired nucleocytoplasmic transport in neurodegeneration.

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