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Role of a 19S Proteasome Subunit- PSMD10(Gankyrin) in Neurogenesis of Human Neuronal Progenitor Cells

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Role of a 19S Proteasome Subunit- PSMD10(Gankyrin) in Neurogenesis of Human Neuronal Progenitor Cells University of Massachusetts Medical School eScholarship@UMMS Open Access Articles Open Access Publications by UMMS Authors 2019-08-31 Role of a 19S Proteasome Subunit- PSMD10(Gankyrin) in Neurogenesis of Human Neuronal Progenitor Cells Indrajit Sahu Technion - Israel Institute of Technology Et al. Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/oapubs Part of the Amino Acids, Peptides, and Proteins Commons, Cancer Biology Commons, Cell Biology Commons, Developmental Biology Commons, Developmental Neuroscience Commons, Enzymes and Coenzymes Commons, and the Nervous System Commons Repository Citation Sahu I, Nanaware PP, Mane M, Mulla SW, Roy S, Venkatraman P. (2019). Role of a 19S Proteasome Subunit- PSMD10(Gankyrin) in Neurogenesis of Human Neuronal Progenitor Cells. Open Access Articles. https://doi.org/10.15283/ijsc19007. Retrieved from https://escholarship.umassmed.edu/oapubs/3950 Creative Commons License This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in Open Access Articles by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. eISSN 2005-5447 International Journal of Stem Cells Published online August 31, 2019 https://doi.org/10.15283/ijsc19007 BRIEF REPORT Role of a 19S Proteasome Subunit- PSMD10Gankyrin in Neurogenesis of Human Neuronal Progenitor Cells Indrajit Sahu1,2,3, Padma Nanaware1,3,4, Minal Mane1, Saim Wasi Mulla1,3, Soumen Roy5, Prasanna Venkatraman1,3 1Advance Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, Kharghar, Navi Mumbai, India 2Faculty of Biology, Technion – Israel Institute of Technology, Haifa, Israel 3Homi Bhabha National Institute, BARC Training School Complex, Mumbai, Maharashtra, India 4Department of Pathology, University of Massachusetts Medical School, Worcester, MA, USA 5Department of Physics, Bose Institute, Kolkata, India PSMD10Gankyrin, a proteasome assembly chaperone, is a widely known oncoprotein which aspects many hall mark proper- ties of cancer. However, except proteasome assembly chaperon function its role in normal cell function remains unknown. To address this issue, we induced PSMD10Gankyrin overexpression in HEK293 cells and the resultant large- scale changes in gene expression profile were analyzed. We constituted networks from microarray data of these differ- entially expressed genes and carried out extensive topological analyses. The overrecurring yet consistent theme that appeared throughout analysis using varied network metrics is that all genes and interactions identified as important would be involved in neurogenesis and neuronal development. Intrigued we tested the possibility that PSMD10Gankyrin may be strongly associated with cell fate decisions that commit neural stem cells to differentiate into neurons. Overexpression of PSMD10Gankyrin in human neuronal progenitor cells facilitated neuronal differentiation via β-catenin Ngn1 pathway. Here for the first time we provide preliminary and yet compelling experimental evidence for the involve- ment of a potential oncoprotein – PSMD10Gankyrin, in neuronal differentiation. Keywords: Gankyrin, Human neuronal progenitor cells, Microarray, Neurogenesis, Proteasome, PSMD10 Introduction Received: January 11, 2019, Revised: June 21, 2019, Accepted: August 6, 2019, Published online: August 31, 2019 PSMD10, also called Gankyrin (PSMD10Gankyrin), is a Correspondence to Prasanna Venkatraman Advance Centre for Treatment, Research and Education in Cancer, well-established proteasome assembly chaperone and an Tata Memorial Centre, Kharghar, Navi Mumbai 410210, India oncoprotein. Initially identified as p28 component (Nas6/ Tel: +91-2227405091, Fax: +91-2227405080 PSMD10) of 19S regulatory subunit of the 26S proteasome E-mail: [email protected] (1), PSMD10 later gained importance for its tumorigenic Co-Correspondence to Soumen Roy Department of Physics, Bose Institute, 93/1 Acharya PC Roy Road, potential in many cancers by regulating multiple signaling Gankyrin Kolkata 700 009, India pathways (2, 3). From a structural perspective PSMD10 Tel: +91-3323031150, Fax: +91-3323506790 belongs to ankyrin repeat family proteins, well-known for E-mail: [email protected] their role as mediators of protein-protein interactions that This is an open-access article distributed under the terms of the Creative regulate numerous physiological processes (4, 5). Most im- Commons Attribution Non-Commercial License (http://creativecommons.org/ Gankyrin licenses/by-nc/4.0/), which permits unrestricted non-commercial use, dis- portantly PSMD10 acts as a dual-negative regulator tribution, and reproduction in any medium, provided the original work is of both pRb and P53 tumor suppressor pathways, by mod- properly cited. ulating their ubiquitin mediated proteasomal degradation Copyright ⓒ 2019 by the Korean Society for Stem Cell Research 1 2 International Journal of Stem Cells Published online August 31, 2019 via specific protein-protein interactions (6, 7). ATG) replacing the RFP gene from the vector. PAX2 and The well-established association of PSMD10Gankyrin with pMD2G packaging vectors were used for virus production. various cancers overshadows its role in normal cells. As a 19S assembly chaperone, PSMD10Gankyrin is central to the Cell culture, generation of stable cells and transduction building of the base complex and aids in its association HEK293 and HEK293-FT cells were grown in DMEM Gankyrin with the 20S core particles (8). Ability of PSMD10 with 10%FBS supplement, 5% CO2 at 37℃. Stable cells to retain NF-κB in the cytoplasm (9) and regulate its for PSMD10Gankyrin overexpression were generated by transcriptional activity at the basal level, may be a critical transfecting HEK293 cells with pCMV10-3XFlag- checkpoint in normal cells. PSMD10Gankyrin enhances the PSMD10Gankyrin construct and selected with (800 μg/mL) transcriptional activity of β-catenin/TCF3 complex (10) G418 (Sigma). For PSMD10Gankyrin silencing HEK293 cells that drives Wnt signaling, a major player in cellular dif- were transfected with siRNA using Lipofectamine ferentiation process. Our recent study reported number of RNAiMax (Invitrogen) and incubated for 48 hrs. Human proteins that interact with endogenous PSMD10Gankyrin in Neural progenitor cells (ReNcell VM, purchased from mammalian cells (11). PSMD10Gankyrin interactions with Millipore) were grown on laminin (10 μg/mL: Sigma) coat- Hsp70 and Hsp90 chaperones that help in the turnover ed plates or cover slips in Neural Stem Cells maintenance of proteins may be critical during heat shock response or media (Millipore) supplemented with EGF and FGF (20 ER stress. These limited and yet important interactions ng/mL each: Sigma). hNPCs grow rapidly as a monolayer bring forth the role of PSMD10Gankyrin in cellular homeo- on laminin with a doubling time of 20∼30 hours (Supple- stasis. mentary Fig. S1). For differentiation studies cells were To obtain deeper insights on the biological role of differentiated into astrocytes, oligodendrocytes and neu- PSMD10Gankyrin, we generated microarray gene expression rons (Supplementary Fig. S1B, S1C, S1D, S1E) in 10∼14 profiles by overexpressing PSMD10Gankyrin in HEK293 cells days of incubation period by withdrawing EGF and FGF used as a model system in our previous studies. The data as per the manufacturers protocol (Millipore). hNPCs were were analyzed using a combination of topological inves- transduced with high titer lentiviral particles along with tigation of networks, gene-set enrichment and pathway polybrene for 3XFlag-PSMD10Gankyrin overexpression analysis. This strategy led us to a remarkable prediction, (More details in Supplementary methods). namely – the potential involvement of PSMD10Gankyrin in the biogenesis of neurons and neuronal functions. Al- Microarray and data analysis though such networks can have wider biological implicati- RNA was isolated from ∼1×106 HEK293 stable clone ons, this analysis focused our attention on a human neuro- cells overexpressing PSMD10Gankyrin and the Vector Con- nal progenitor cells (hNPCs) model system for differen- trol HEK293 clone by the TRiZol (Invitrogen) method fol- tiation. Here we provide preliminary and yet compelling lowing manufacturers protocol. The isolated RNA was evidence for the role of PSMD10Gankyrin in neurogenesis. outsourced to Genotypic Technology (Bangalore, India). We show that both the mRNA and protein levels of After their quality control report, cDNA was prepared in- PSMD10Gankyrin increase in differentiated cells compared house and was later analyzed by microarray using the the to the hNPCs. This increase in PSMD10Gankyrin parallel the Agilent platform. The data were analyzed using Agilent time-dependent increase in β-tubulin, a marker of the expression reference guide. Network reconstruction and differentiated neurons. Overexpression of PSMD10Gankyrin extensive topological analysis of networks were carried out in hNPCs cell line induces the expression of Ngn1 and using various bioinformatics tools (More details in Sup- alters the ratio of neurons to astrocytes favoring the proc- plementary methods). ess of neurogenesis. Semi quantitative and real-time PCR Materials and Methods mRNA from PSMD10Gankyrin stable cells, control HEK293 cells, hNPCs (UD) and Differentiated (DF) cells Plasmids and cloning were isolated by TRiZol (Invitrogen)
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