Abstract
Defects in the activity of the proteasome or its regulators are linked to several pathologies, including neurodegenerative diseases. We hypothesize that proteasome heterogeneity and its selective partners vary across brain regions and have a significant impact on proteasomal catalytic activities. Using neuronal cell cultures and brain tissues obtained from mice, we compared proteasomal activities from two distinct brain regions affected in neurodegenerative diseases, the striatum and the hippocampus. The results indicated that proteasome activities and their responses to proteasome inhibitors are determined by their subcellular localizations and their brain regions. Using an iodixanol gradient fractionation method, proteasome complexes were isolated, followed by proteomic analysis for proteasomal interaction partners. Proteomic results revealed brain region-specific non-proteasomal partners, including gamma-enolase (ENO2). ENO2 showed more association to proteasome complexes purified from the striatum than to those from the hippocampus. These results highlight a potential key role for non-proteasomal partners of proteasomes regarding the diverse activities of the proteasome complex recorded in several brain regions.
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Data Availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
References
Abdullah A, Sane S, Freeling JL et al (2015) Nucleocytoplasmic translocation of UBXN2A is required for apoptosis during DNA damage stresses in colon cancer cells. J Cancer 6:1066–1078. https://doi.org/10.7150/jca.12134
Asano S, Fukuda Y, Beck F et al (2015) A molecular census of 26S proteasomes in intact neurons. Science 347:439–442. https://doi.org/10.1126/science.1261197
Bellavista E, Martucci M, Vasuri F et al (2014) Lifelong maintenance of composition, function and cellular/subcellular distribution of proteasomes in human liver. Mech Ageing Dev 141–142:26–34. https://doi.org/10.1016/j.mad.2014.09.003
Bose S, Stratford FLL, Broadfoot KI et al (2004) Phosphorylation of 20S proteasome alpha subunit C8 (α7) stabilizes the 26S proteasome and plays a role in the regulation of proteasome complexes by γ-interferon. Biochem J 378:177–184. https://doi.org/10.1042/BJ20031122
Bousquet-Dubouch MP, Baudelet E, Guérin F et al (2009) Affinity purification strategy to capture human endogenous proteasome complexes diversity and to identify proteasome-interacting proteins. Mol Cell Proteomics 8:1150–1164. https://doi.org/10.1074/mcp.M800193-MCP200
Giannini C, Kloß A, Gohlke S et al (2013) Poly-Ub-substrated-degradative activity of 26S proteasome is not impaired in the aging rat brain. PLoS ONE 8:64042. https://doi.org/10.1371/journal.pone.0064042
Coux O, Tanaka K, Goldberg AL (1996) Structure and functions of the 20S and 26S proteasomes. Annu Rev Biochem 65:801–847. https://doi.org/10.1146/annurev.bi.65.070196.004101
Dahlmann B (2016) Mammalian proteasome subtypes: their diversity in structure and function. Arch Biochem Biophys 591:132–140. https://doi.org/10.1016/j.abb.2015.12.012
Dang FW, Chen L, Madura K (2016) Catalytically active proteasomes function predominantly in the cytosol. J Biol Chem 291:18765–18777. https://doi.org/10.1074/jbc.M115.712406
Ding Q, Keller JN (2001) Proteasomes and proteasome inhibition in the central nervous system. Free Radic Biol Med 31:574–584. https://doi.org/10.1016/S0891-5849(01)00635-9
Drews O, Wildgruber R, Zong C et al (2007) Mammalian proteasome subpopulations with distinct molecular compositions and proteolytic activities. Mol Cell Proteomics. https://doi.org/10.1074/mcp.M700187-MCP200
Fabre B, Lambour T, Delobel J et al (2013) Subcellular distribution and dynamics of active proteasome complexes unraveled by a workflow combining in vivo complex cross-linking and quantitative proteomics. Mol Cell Proteomics 12:687–699. https://doi.org/10.1074/mcp.M112.023317
Früh K, Gossen M, Wang K et al (1994) Displacement of housekeeping proteasome subunits by MHC-encoded LMPs: a newly discovered mechanism for modulating the multicatalytic proteinase complex. EMBO J 13:3236–3244. https://doi.org/10.1002/j.1460-2075.1994.tb06625.x
Grabbe C, Husnjak K, Dikic I (2011) The spatial and temporal organization of ubiquitin networks. Nat Rev Mol Cell Biol 12:295–307
Graham JM (2002) Fractionation of Golgi, endoplasmic reticulum, and plasma membrane from cultured cells in a preformed continuous iodixanol gradient. Sci World J 2:1435–1439. https://doi.org/10.1100/tsw.2002.286
Grimm S, Höhn A, Grune T (2012) Oxidative protein damage and the proteasome. Amino acids. Springer, Berlin, pp 23–38
Grumati P, Dikic I (2018) Ubiquitin signaling and autophagy. J Biol Chem 293:5404–5413. https://doi.org/10.1074/jbc.TM117.000117
Guerrero C, Milenković T, Pržulj N et al (2008) Characterization of the proteasome interaction network using a QTAX-based tag-team strategy and protein interaction network analysis. Proc Natl Acad Sci USA 105:13333–13338. https://doi.org/10.1073/pnas.0801870105
Jenkins EC, Shah N, Gomez M et al (2020) Proteasome mapping reveals sexual dimorphism in tissue-specific sensitivity to protein aggregations. EMBO Rep. https://doi.org/10.15252/embr.201948978
Ji WU, Eunju I, Joongkyu P et al (2010) ASK1 negatively regulates the 26 S proteasome. J Biol Chem 285:36434–36446. https://doi.org/10.1074/jbc.M110.133777
Jung T, Catalgol B, Grune T (2009) The proteasomal system. Mol Aspects Med 30:191–296
Kabashi E, Agar JN, Strong MJ, Durham HD (2012) Impaired proteasome function in sporadic amyotrophic lateral sclerosis. Amyotroph Lateral Scler 13:367–371. https://doi.org/10.3109/17482968.2012.686511
Keller JN, Hanni KB, Markesbery WR (2000a) Possible involvement of proteasome inhibition in aging: Implications for oxidative stress. Mech Ageing Dev 113:61–70. https://doi.org/10.1016/S0047-6374(99)00101-3
Keller JN, Hanni KB, Markesbery WR (2000b) Impaired proteasome function in Alzheimer’s disease. J Neurochem 75:436–439. https://doi.org/10.1046/j.1471-4159.2000.0750436.x
Keller JN, Huang FF, Markesbery WR (2000c) Decreased levels of proteasome activity and proteasome expression in aging spinal cord. Neuroscience 98:149–156. https://doi.org/10.1016/S0306-4522(00)00067-1
Keller JN, Huang FF, Zhu H et al (2000d) Oxidative stress-associated impairment of proteasome activity during ischemia-reperfusion injury. J Cereb Blood Flow Metab 20:1467–1473. https://doi.org/10.1097/00004647-200010000-00008
Kelmer Sacramento E, Kirkpatrick JM, Mazzetto M et al (2020) Reduced proteasome activity in the aging brain results in ribosome stoichiometry loss and aggregation. Mol Syst Biol. https://doi.org/10.15252/msb.20209596
Kim HT, Goldberg AL (2018) UBL domain of Usp14 and other proteins stimulates proteasome activities and protein degradation in cells. Proc Natl Acad Sci USA 115:E11642–E11650. https://doi.org/10.1073/pnas.1808731115
Kors S, Geijtenbeek K, Reits E, Schipper-Krom S (2019) Regulation of proteasome activity by (post-)transcriptional mechanisms. Front Mol Biosci 6:48. https://doi.org/10.3389/fmolb.2019.00048
Lee D, Takayama S, Goldberg AL (2018) ZFAND5/ZNF216 is an activator of the 26S proteasome that stimulates overall protein degradation. Proc Natl Acad Sci USA 115:E9550–E9559. https://doi.org/10.1073/pnas.1809934115
Lehtonen Š, Sonninen T-M, Wojciechowski S et al (2019) Dysfunction of cellular proteostasis in Parkinson’s disease. Front Neurosci. https://doi.org/10.3389/fnins.2019.00457
Liu K, Jones S, Minis A et al (2019) PI31 is an adaptor protein for proteasome transport in axons and required for synaptic development. Dev Cell 50:509-524.e10. https://doi.org/10.1016/j.devcel.2019.06.009
Liu X, Xiao W, Zhang Y et al (2020) Reversible phosphorylation of Rpn1 regulates 26S proteasome assembly and function. Proc Natl Acad Sci USA 117:328–336. https://doi.org/10.1073/pnas.1912531117
McKinnon C, Tabrizi SJ (2014) The ubiquitin-proteasome system in neurodegeneration. Antioxid Redox Signal 21:2302–2321. https://doi.org/10.1089/ars.2013.5802
McNaught KSP, Belizaire R, Jenner P et al (2002) Selective loss of 20S proteasome α-subunits in the substantia nigra pars compacta in Parkinson’s disease. Neurosci Lett 326:155–158. https://doi.org/10.1016/S0304-3940(02)00296-3
McNaught KSP, Jenner P (2001) Proteasomal function is impaired in substantia nigra in Parkinson’s disease. Neurosci Lett 297:191–194. https://doi.org/10.1016/S0304-3940(00)01701-8
Minis A, Rodriguez JA, Levin A et al (2019) The proteasome regulator PI31 is required for protein homeostasis, synapse maintenance, and neuronal survival in mice. Proc Natl Acad Sci USA 116:24639–24650. https://doi.org/10.1073/pnas.1911921116
Moodley KK, Chan D (2014) The hippocampus in neurodegenerative disease. The hippocampus in clinical neuroscience. S. Karger AG, Basel, pp 95–108
Morozov AV, Karpov VL (2019) Proteasomes and several aspects of their heterogeneity relevant to cancer. Front Oncol 9:1–21. https://doi.org/10.3389/fonc.2019.00761
Murata S, Takahama Y, Kasahara M, Tanaka K (2018) The immunoproteasome and thymoproteasome: functions, evolution and human disease. Nat Immunol 19:923–931. https://doi.org/10.1038/s41590-018-0186-z
Muratani M, Tansey WP (2003) How the ubiquitin-proteasome system controls transcription. Nat Rev Mol Cell Biol 4:192–201
Ng W, Sergeyenko T, Zeng N et al (2007) Characterization of the proteasome interaction with the Sec61 channel in the endoplasmic reticulum. J Cell Sci 120:682–691. https://doi.org/10.1242/jcs.03351
Noda C, Tanahashi N, Shimbara N et al (2000) Tissue distribution of constitutive proteasomes, immunoproteasomes, and PA28 in rats. Biophys Biochem Res Commun. https://doi.org/10.1006/bbrc.2000.3676
Nopoulos PC (2016) Huntington disease: a single-gene degenerative disorder of the striatum. Dialog Clin Neurosci 18:91–98. https://doi.org/10.31887/dcns.2016.18.1/pnopoulos
Onódi Z, Pelyhe C, Terézia Nagy C et al (2018) Isolation of high-purity extracellular vesicles by the combination of iodixanol density gradient ultracentrifugation and bind-elute chromatography from blood plasma. Front Physiol 9:1479. https://doi.org/10.3389/fphys.2018.01479
Pedrazzini E, Villa A, Longhi R et al (2000) Mechanism of residence of cytochrome b(5), a tail-anchored protein, in the endoplasmic reticulum. J Cell Biol 148:899–913. https://doi.org/10.1083/jcb.148.5.899
Persaud-Sawin DA, Lightcap S, Harry GJ (2009) Isolation of rafts from mouse brain tissue by a detergent-free method. J Lipid Res 50:759–767. https://doi.org/10.1194/jlr.D800037-JLR200
Peters JM, Cejka Z, Harris JR et al (1993) Structural features of the 26 S proteasome complex. J Mol Biol 234:932–937
Rezvani K, Baalman K, Teng Y et al (2012) Proteasomal degradation of the metabotropic glutamate receptor 1α is mediated by Homer-3 via the proteasomal S8 ATPase: signal transduction and synaptic transmission. J Neurochem 122:24–37. https://doi.org/10.1111/j.1471-4159.2012.07752.x
Salon ML, Morelli L, Castaño EM et al (2000) Defective ubiquitination of cerebral proteins in Alzheimer’s disease. J Neurosci Res 62:302–310. https://doi.org/10.1002/1097-4547(20001015)62:2%3c302::AID-JNR15%3e3.0.CO;2-L
Sbardella D, Tundo GR, Coletta A et al (2018) The insulin-degrading enzyme is an allosteric modulator of the 20S proteasome and a potential competitor of the 19S. Cell Mol Life Sci 75:3441–3456. https://doi.org/10.1007/s00018-018-2807-y
Shimada T, Fournier AE, Yamagata K (2013) Neuroprotective function of 14–3-3 proteins in neurodegeneration. Biomed Res Int. https://doi.org/10.1155/2013/564534
Shintani T, Suzuki K, Kamada Y et al (2001) Apg2p functions in autophagosome formation on the perivacuolar structure. J Biol Chem 276:30452–30460. https://doi.org/10.1074/jbc.M102346200
Sparks A, Dayal S, Das J et al (2014) The degradation of p53 and its major E3 ligase Mdm2 is differentially dependent on the proteasomal ubiquitin receptor S5a. Oncogene 33:4685–4696. https://doi.org/10.1038/onc.2013.413
Stanhill A, Haynes CM, Zhang Y et al (2006) An arsenite-inducible 19S regulatory particle-associated protein adapts proteasomes to proteotoxicity. Mol Cell 23:875–885. https://doi.org/10.1016/j.molcel.2006.07.023
Stohwasser R, Giesebrecht J, Kraft R et al (2000) Biochemical analysis of proteasomes from mouse microglia: Induction of immunoproteasomes by interferon-γ and lipopolysaccharide. Glia 29:355–365. https://doi.org/10.1002/(SICI)1098-1136(20000215)29:4%3c355::AID-GLIA6%3e3.0.CO;2-4
Tanahashi N, Murakami Y, Minami Y et al (2000) Hybrid proteasomes. Induction by interferon-γ and contribution to ATP- dependent proteolysis. J Biol Chem 275:14336–14345. https://doi.org/10.1074/jbc.275.19.14336
Trettel F, Rigamonti D, Hilditch-Maguire P et al (2000) Dominant phenotypes produced by the HD mutation in STHdh(Q111) striatal cells. Hum Mol Genet 9:2799–2809. https://doi.org/10.1093/hmg/9.19.2799
Verma R, Chen S, Feldman R et al (2000) Proteasomal proteomics: Identification of nucleotide-sensitive proteasome-interacting proteins by mass spectrometric analysis of affinity-purified proteasomes. Mol Biol Cell 11:3425–3439. https://doi.org/10.1091/mbc.11.10.3425
VerPlank JJS, Goldberg AL (2017) Regulating protein breakdown through proteasome phosphorylation. Biochem J 474:3355–3371. https://doi.org/10.1042/BCJ20160809
VerPlank JJS, Lokireddy S, Zhao J, Goldberg AL (2019) 26S Proteasomes are rapidly activated by diverse hormones and physiological states that raise cAMP and cause Rpn6 phosphorylation. Proc Natl Acad Sci USA 116:4228–4237. https://doi.org/10.1073/pnas.1809254116
Wang X, Majumdar T, Kessler P et al (2016) STING requires the adaptor TRIF to trigger innate immune responses to microbial infection. Cell Host Microbe 20:329–341. https://doi.org/10.1016/j.chom.2016.08.002
Wang Y, Lilley KS, Oliver SG (2014) A protocol for the subcellular fractionation of Saccharomyces cerevisiae using nitrogen cavitation and density gradient centrifugation. Yeast 31:127–135. https://doi.org/10.1002/yea.3002
Weinkauf M, Zimmermann Y, Hartmann E et al (2009) 2-D PAGE-based comparison of proteasome inhibitor bortezomib in sensitive and resistant mantle cell lymphoma. Electrophoresis 30:974–986. https://doi.org/10.1002/elps.200800508
Yee MS, Pavitt DV, Tan T et al (2008) Lipoprotein separation in a novel iodixanol density gradient, for composition, density, and phenotype analysis. J Lipid Res 49:1364–1371. https://doi.org/10.1194/jlr.D700044-JLR200
Yoshimura T, Kameyama K, Takagi T et al (1993) Molecular characterization of the ‗26s‘ proteasome complex from rat liver. J Struct Biol 111:200–211
Zeng BY, Medhurst AD, Jackson M et al (2005) Proteasomal activity in brain differs between species and brain regions and changes with age. Mech Ageing Dev 126:760–766. https://doi.org/10.1016/j.mad.2005.01.008
Zhang H, Pan B, Wu P et al (2019) PDE1 inhibition facilitates proteasomal degradation of misfolded proteins and protects against cardiac proteinopathy. Sci Adv 5:5870. https://doi.org/10.1126/sciadv.aaw5870
Zong C, Gomes AV, Drews O et al (2006) Regulation of murine cardiac 20S proteasomes: role of associating partners. Circ Res 99:372–380. https://doi.org/10.1161/01.RES.0000237389.40000.02
Acknowledgements
We would like to acknowledge Dr. X.J. Wang (Basic Biomedical Sciences, University of South Dakota) for the gift of polyclonal β5 (PSMB5) antibody. We would also like to thank Andy Lemrick (Marketing Communications and University Relations, University of South Dakota) for the graphic designs in Figure 1, as well as Bill Conn and Ryan Johnson from USD IT Research Computing for help with the database installation and server operation.
Funding
Support for this work was provided by the University of South Dakota Division of Basic Biomedical Sciences (READ award) and CBBRe (Center for Brain and Behavior Research, University of South Dakota). The Proteomics Core facility at the University of South Dakota was supported by NIH Grant Number 2P20 GM103443-19 from the INBRE Program of the IDeA National General Medical Sciences, NIH.
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MN and RA carried out the experiment. NE wrote the manuscript with support from EC and with inputs from all authors. EC performed the proteomic experiments. KR supervised the project. JSP participated in experimental design and contributed to writing and editing the final manuscript. Current Address, NE: Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main Street West, Hamilton ON L8S 4L8, Canada. Current Address, MN: Department of Biostatistics, 170 Rosenau Hall, University of North Carolina—Chapel Hill, Chapel Hill, NC 27599, USA.
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Figure S1:
The NE-PER reagents efficiently separate cytoplasmic and nuclear proteins with minimal cross-contamination. WB results indicate the presence of β-tubulin cytoplasmic protein in the cytoplasmic fractions and not the nuclear fractions in the striatum cell line, confirming the purity of both the cytoplasmic and nuclear compartments used for fractionation experiments. Supplementary file3 (EPS 1528 kb)
Figure S2:
Experimental design of proteasome study. A: Graphs show the linear correlation between fluorescence intensity and the amount of substrate-AMC-hydrolyzing activity in reaction, plotted as arbitrary fluorescence units (AFU). The fluorescence intensity was measured at 380 nm excitation and 460 nm minus background fluorescence. Statistical analyses performed with GraphPad Prism revealed R-squared (R2) for recorded chymotrypsin-like activities in the cytoplasm (R2=0.83, panel A) and nucleus (R2=0.87, panel B) in 4 separate timelines (1, 3, 5, 10 hours). All assays were conducted in the presence of 2 mM ATP to preserve the integrity of the 26S and 30S proteasome complexes. Proteasomal activities recorded at 5 hours were used in the main figures. B: Chymotrypsin-, trypsin-, and caspase-like proteasome activities measured in twenty fractions and collected following iodixanol gradient fractionations of two individual sets of striatum-derived cytoplasmic cell lysates (experiments 1 and 2). The similar distribution of 26S and 30S proteasome complexes in these two sets of experiments confirmed the repeatability of the method. Supplementary file4 (EPS 3905 kb)
Figure S3
: Iodixanol gradient fractionation of proteasome complexes and quality control tests in a hippocampus-derived cell line. A-B: Sedimentation of arylesterase (endoplasmic reticulum markers) and leucine aminopeptidases (Golgi apparatus markers) in the cytoplasm segment of a rat hippocampus cell line. C-E: Cytoplasmic and F-H: nuclear lysates of hippocampus-derived cell were subjected to iodixanol gradient ultra-centrifugation. Subsequently, samples were analyzed for proteasomal catalytic activities with and without the proteasome inhibitors MG132, TLCK, and bortezomib. Arrowheads represent potential non-specific proteasomal activities, while arrows show peaks of three proteasomal activities recorded in collected fractions. I-J: An equal volume of each fraction (cytoplasm and nucleus) was subjected to SDS-PAGE followed by WB using anti-pan alpha (20S proteasome) and anti-RPT6 (19S subunit, S8 ATPase) antibodies to illustrate the distribution of proteasome complexes in the collected fractions. Supplementary file5 (EPS 8500 kb)
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Esfahanian, N., Nelson, M., Autenried, R. et al. Comprehensive Analysis of Proteasomal Complexes in Mouse Brain Regions Detects ENO2 as a Potential Partner of the Proteasome in the Striatum. Cell Mol Neurobiol 42, 2305–2319 (2022). https://doi.org/10.1007/s10571-021-01106-2
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DOI: https://doi.org/10.1007/s10571-021-01106-2