Targeted inhibition of the Shroom3–Rho kinase protein–protein interaction circumvents Nogo66 to promote axon outgrowth

BMC Neuroscience

Anne B Vojtek²  Mathew A Young¹  Ashley A Reinke²  Amanda Wilbur²  Heather M Dickson² 

[1] Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI, USA;Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, USA

关键词: Protein–protein interaction inhibitors;    POSH;    PirB;    ROCK;    Shroom3;    NogoA;    Neural regeneration;   

Others  :  1220316
DOI  :  10.1186/s12868-015-0171-5

 received in 2014-10-23, accepted in 2015-06-03,  发布年份 2015

【 摘 要 】

Background

Inhibitory molecules in the adult central nervous system, including NogoA, impede neural repair by blocking axon outgrowth. The actin-myosin regulatory protein Shroom3 directly interacts with Rho kinase and conveys axon outgrowth inhibitory signals from Nogo66, a C-terminal inhibitory domain of NogoA. The purpose of this study was to identify small molecules that block the Shroom3–Rho kinase protein–protein interaction as a means to modulate NogoA signaling and, in the longer term, enhance axon outgrowth during neural repair.

Results

A high throughput screen for inhibitors of the Shroom3–Rho kinase protein–protein interaction identified CCG-17444 (Chem ID: 2816053). CCG-17444 inhibits the Shroom3–Rho kinase interaction in vitro with micromolar potency. This compound acts through an irreversible, covalent mechanism of action, targeting Shroom3 Cys1816 to inhibit the Shroom3–Rho kinase protein–protein interaction. Inhibition of the Shroom3–Rho kinase protein–protein interaction with CCG-17444 counteracts the inhibitory action of Nogo66 and enhances neurite outgrowth.

Conclusions

This study identifies a small molecule inhibitor of the Shroom3–Rho kinase protein–protein interaction that circumvents the inhibitory action of Nogo66 in neurons. Identification of a small molecule compound that blocks the Shroom3–Rho kinase protein–protein interaction provides a first step towards a potential new strategy for enhancing neural repair.

【 授权许可】

   
2015 Dickson et al.

【 图 表 】

Figure1.

Figure2.

Figure3.

Figure4.

Figure5.

Figure6.

【 参考文献 】

• [1]Mironova YA, Giger RJ: Where no synapses go: gatekeepers of circuit remodeling and synaptic strength. Trends Neurosci 2013, 36(6):363-373.
• [2]Overman JJ, Carmichael ST: Plasticity in the injured brain: more than molecules matter. Neuroscientist 2014, 20(1):15-28.
• [3]Schwab ME, Strittmatter SM: Nogo limits neural plasticity and recovery from injury. Curr Opin Neurobiol 2014, 27C:53-60.
• [4]Lee JK, Kim JE, Sivula M, Strittmatter SM: Nogo receptor antagonism promotes stroke recovery by enhancing axonal plasticity. J Neurosci 2004, 24(27):6209-6217.
• [5]Markus TM, Tsai SY, Bollnow MR, Farrer RG, O’Brien TE, Kindler-Baumann DR, et al.: Recovery and brain reorganization after stroke in adult and aged rats. Ann Neurol 2005, 58(6):950-953.
• [6]Papadopoulos CM, Tsai SY, Alsbiei T, O’Brien TE, Schwab ME, Kartje GL: Functional recovery and neuroanatomical plasticity following middle cerebral artery occlusion and IN-1 antibody treatment in the adult rat. Ann Neurol 2002, 51(4):433-441.
• [7]Seymour AB, Andrews EM, Tsai SY, Markus TM, Bollnow MR, Brenneman MM, et al.: Delayed treatment with monoclonal antibody IN-1 1 week after stroke results in recovery of function and corticorubral plasticity in adult rats. J Cereb Blood Flow Metab 2005, 25(10):1366-1375.
• [8]Adelson JD, Barreto GE, Xu L, Kim T, Brott BK, Ouyang YB, et al.: Neuroprotection from stroke in the absence of MHCI or PirB. Neuron 2012, 73(6):1100-1107.
• [9]Atwal JK, Pinkston-Gosse J, Syken J, Stawicki S, Wu Y, Shatz C, et al.: PirB is a functional receptor for myelin inhibitors of axonal regeneration. Science 2008, 322(5903):967-970.
• [10]Fournier AE, GrandPre T, Strittmatter SM: Identification of a receptor mediating Nogo-66 inhibition of axonal regeneration. Nature 2001, 409(6818):341-346.
• [11]Mi S, Lee X, Shao Z, Thill G, Ji B, Relton J, et al.: LINGO-1 is a component of the Nogo-66 receptor/p75 signaling complex. Nat Neurosci 2004, 7(3):221-228.
• [12]Shao Z, Browning JL, Lee X, Scott ML, Shulga-Morskaya S, Allaire N, et al.: TAJ/TROY, an orphan TNF receptor family member, binds Nogo-66 receptor 1 and regulates axonal regeneration. Neuron 2005, 45(3):353-359.
• [13]Kempf A, Schwab ME: Nogo-A represses anatomical and synaptic plasticity in the central nervous system. Physiology (Bethesda) 2013, 28(3):151-163.
• [14]Dickson HM, Zurawski J, Zhang H, Turner DL, Vojtek AB: POSH is an intracellular signal transducer for the axon outgrowth inhibitor Nogo66. J Neurosci 2010, 30(40):13319-13325.
• [15]Kim T, Vidal GS, Djurisic M, William CM, Birnbaum ME, Garcia KC, et al.: Human LilrB2 is a beta-amyloid receptor and its murine homolog PirB regulates synaptic plasticity in an Alzheimer’s model. Science 2013, 341(6152):1399-1404.
• [16]Taylor J, Chung KH, Figueroa C, Zurawski J, Dickson HM, Brace EJ, et al.: The scaffold protein POSH regulates axon outgrowth. Mol Biol Cell 2008, 19(12):5181-5192.
• [17]Mohan S, Das D, Bauer RJ, Heroux A, Zalewski JK, Heber S, et al.: Structure of a highly conserved domain of Rock1 required for Shroom-mediated regulation of cell morphology. PLoS One 2013, 8(12):e81075.
• [18]Mohan S, Rizaldy R, Das D, Bauer RJ, Heroux A, Trakselis MA, et al.: Structure of the Shroom Domain 2 reveals a three-segmented coiled-coil required for dimerization, rock binding, and apical constriction. Mol Biol Cell 2012, 23(11):2131-2142.
• [19]Nishimura T, Takeichi M: mediated recruitment of Rho kinases to the apical cell junctions regulates epithelial and neuroepithelial planar remodeling. Development 2008, 135(8):1493-1502.
• [20]Haigo SL, Hildebrand JD, Harland RM, Wallingford JB: Shroom induces apical constriction and is required for hingepoint formation during neural tube closure. Curr Biol 2003, 13(24):2125-2137.
• [21]Farah MH, Olson JM, Sucic HB, Hume RI, Tapscott SJ, Turner DL: Generation of neurons by transient expression of neural bHLH proteins in mammalian cells. Development 2000, 127(4):693-702.
• [22]Vojtek AB, Taylor J, DeRuiter SL, Yu JY, Figueroa C, Kwok RP, et al.: Akt regulates basic helix-loop-helix transcription factor-coactivator complex formation and activity during neuronal differentiation. Mol Cell Biol 2003, 23(13):4417-4427.
• [23]Alabed YZ, Grados-Munro E, Ferraro GB, Hsieh SH, Fournier AE: Neuronal responses to myelin are mediated by rho kinase. J Neurochem 2006, 96(6):1616-1625.
• [24]Darenfed H, Dayanandan B, Zhang T, Hsieh SH, Fournier AE, Mandato CA: Molecular characterization of the effects of Y-27632. Cell Motil Cytoskelet 2007, 64(2):97-109.
• [25]Farber MJ, Rizaldy R, Hildebrand JD: Shroom2 regulates contractility to control endothelial morphogenesis. Mol Biol Cell 2011, 22(6):795-805.
• [26]Hildebrand JD, Soriano P: Shroom, a PDZ domain-containing actin-binding protein, is required for neural tube morphogenesis in mice. Cell 1999, 99(5):485-497.
• [27]Fairbank PD, Lee C, Ellis A, Hildebrand JD, Gross JM, Wallingford JB: Shroom2 (APXL) regulates melanosome biogenesis and localization in the retinal pigment epithelium. Development 2006, 133(20):4109-4118.
• [28]Lee C, Le MP, Cannatella D, Wallingford JB: Changes in localization and expression levels of Shroom2 and spectrin contribute to variation in amphibian egg pigmentation patterns. Dev Genes Evol 2009, 219(6):319-330.
• [29]Armanet N, Metay C, Brisset S, Deschenes G, Pineau D, Petit FM, et al.: Double Xp11.22 deletion including SHROOM4 and CLCN5 associated with severe psychomotor retardation and Dent disease. Mol Cytogenet 2015, 8:8. BioMed Central Full Text
• [30]Hagens O, Dubos A, Abidi F, Barbi G, Van Zutven L, Hoeltzenbein M, et al.: Disruptions of the novel KIAA1202 gene are associated with X-linked mental retardation. Hum Genet 2006, 118(5):578-590.
• [31]Honda S, Hayashi S, Imoto I, Toyama J, Okazawa H, Nakagawa E, et al.: Copy-number variations on the X chromosome in Japanese patients with mental retardation detected by array-based comparative genomic hybridization analysis. J Hum Genet 2010, 55(9):590-599.
• [32]Nizon M, Andrieux J, Rooryck C, de Blois MC, Bourel-Ponchel E, Bourgois B, et al.: Phenotype-genotype correlations in 17 new patients with an Xp11.23p11.22 microduplication and review of the literature. Am J Med Genet Part A 2015, 167A(1):111-122.
• [33]Das D, Zalewski JK, Mohan S, Plageman TF, VanDemark AP, Hildebrand JD: The interaction between Shroom3 and Rho-kinase is required for neural tube morphogenesis in mice. Biol Open 2014, 3(9):850-860.
• [34]Yoder M, Hildebrand JD: Shroom4 (Kiaa1202) is an actin-associated protein implicated in cytoskeletal organization. Cell Motil Cytoskelet 2007, 64(1):49-63.
• [35]Mullard A: Protein–protein interaction inhibitors get into the groove. Nat Rev Drug Discov 2012, 11(3):173-175.
• [36]Wells JA, McClendon CL: Reaching for high-hanging fruit in drug discovery at protein–protein interfaces. Nature 2007, 450(7172):1001-1009.
• [37]Thompson AD, Dugan A, Gestwicki JE, Mapp AK: Fine-tuning multiprotein complexes using small molecules. ACS Chem Biol 2012, 7(8):1311-1320.
• [38]Giger RJ, Hollis ER 2nd, Tuszynski MH: Guidance molecules in axon regeneration. Cold Spring Harb Perspect Biol 2010, 2(7):a001867.
• [39]Overman JJ, Clarkson AN, Wanner IB, Overman WT, Eckstein I, Maguire JL, et al.: A role for ephrin-A5 in axonal sprouting, recovery, and activity-dependent plasticity after stroke. Proc Natl Acad Sci USA 2012, 109(33):E2230-E2239.
• [40]Amano M, Nakayama M, Kaibuchi K: Rho-kinase/ROCK: a key regulator of the cytoskeleton and cell polarity. Cytoskeleton (Hoboken) 2010, 67(9):545-554.
• [41]Riento K, Ridley AJ: Rocks: multifunctional kinases in cell behaviour. Nat Rev Mol Cell Biol 2003, 4(6):446-456.
• [42]Tonges L, Koch JC, Bahr M, Lingor P: ROCKing regeneration: Rho kinase inhibition as molecular target for neurorestoration. Front Mol Neurosci 2011, 4:39.
• [43]Chung KH, Hart CC, Al-Bassam S, Avery A, Taylor J, Patel PD, et al.: Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155. Nucleic Acids Res 2006, 34(7):e53.
• [44]Vojtek AB, Chung KH, Patel PD, Turner DL (2009) MicroRNA-based RNA polymerase II expression vectors for RNA interference in mammalian cells. In: Gaur RK, Rossi JJ (eds) Regulation of gene expression by small RNAs. CRC Press, Boca Raton, pp 301–315
• [45]Robak LA, Venkatesh K, Lee H, Raiker SJ, Duan Y, Lee-Osbourne J, et al.: Molecular basis of the interactions of the Nogo-66 receptor and its homolog NgR2 with myelin-associated glycoprotein: development of NgROMNI-Fc, a novel antagonist of CNS myelin inhibition. J Neurosci 2009, 29(18):5768-5783.
• [46]Venkatesh K, Chivatakarn O, Sheu SS, Giger RJ: Molecular dissection of the myelin-associated glycoprotein receptor complex reveals cell type-specific mechanisms for neurite outgrowth inhibition. J Cell Biol 2007, 177(3):393-399.