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A Hypomorphic Stip1 Allele Reveals the Requirement for Chaperone Networks in Mouse

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A Hypomorphic Stip1 Allele Reveals the Requirement for Chaperone Networks in Mouse bioRxiv preprint doi: https://doi.org/10.1101/258673; this version posted February 1, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 A hypomorphic Stip1 allele reveals the requirement for chaperone networks in mouse 2 development and aging 3 Rachel E. Lackie1,2, Marilene H. Lopes1,3, Sali M.K. Farhan4, Abdul Razzaq1,2, Gilli Moshitzky5, 4 Mariana Brandao Prado3, Flavio H. Beraldo1,7, Andrzej Maciejewski1,6, Robert Gros1,7,8, Jue 5 Fan1, Wing-Yiu Choy6, David S. Greenberg5, Vilma R. Martins11, Martin L. Duennwald9,10, 6 Hermona Soreq5, Vania F. Prado1,2,7,10*, Marco. A.M. Prado1,2,7,10 * 7 1Molecular Medicine, Robarts Research Institute, 2Program in Neuroscience, University of 8 Western Ontario, 3Laboratory of Neurobiology and Stem cells, Department of Cell and 9 Developmental Biology; Institute of Biomedical Sciences, University of Sao Paulo,4Analytic and 10 Translational Genetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, 11 Harvard Medical School, and The Stanley Center for Psychiatric Research, Broad Institute of 12 MIT and Harvard, Boston, MA, USA, 5The Edmond and Lily Safra Center for Brain Sciences, 13 Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The 14 Hebrew University of Jerusalem, 6Department of Biochemistry, 7 Department of Physiology and 15 Pharmacology, 8Department of Medicine, 9Department of Pathology and Laboratory Medicine, 16 10 Department of Anatomy & Cell Biology, Schulich School of Medicine and Dentistry, 17 University of Western Ontario, London Ontario, Canada. 11International Research Center, 18 A.C.Camargo Cancer Center, São Paulo, Brazil. 19 Correspondence: 20 Dr. Marco A.M. Prado 21 [email protected] 22 Dr. Vania F Prado 23 [email protected] 24 25 26 Robarts Research Institute 27 1151 Richmond St. N, N6A 5B7 28 The University of Western Ontario 29 London, Ontario, Canada 30 Tel: 519-9315777 Ext. 24888 or 24889 31 32 Short title: STI1 in mammalian proteostasis 33 Keywords: Aging / Hsp70 / Hsp90 / Proteostasis / STI1/ stem cells/STIP1/HOP/ 34 Neurodegeneration 35 1 bioRxiv preprint doi: https://doi.org/10.1101/258673; this version posted February 1, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 36 ABSTRACT 37 The chaperone machinery is well conserved from yeast to mammals, however our knowledge of 38 their impact on mammalian physiology is lagging. Stress-inducible phosphoprotein-1 (STI1; 39 STIP1; Hop) is a co-chaperone that simultaneously interacts with Hsp70 and Hsp90 via three 40 tetratricopeptide repeat (TPR) domains, of which TPR1 and TPR2B may be redundant in yeast. 41 In-depth analysis of human datasets indicated that STI1 belongs to a set of co-chaperones that is 42 essential in humans and that the TPR1 domain is evolutionarily conserved, suggesting that in 43 mammals it may be required for optimal STI1 activity in vivo. We generated mice with a 44 hypomorphic Stip1 allele lacking the TPR1 domain. While these mice are viable, they presented 45 decreased levels of Hsp90 client proteins and co-chaperones, suggesting profound dysregulation 46 of chaperone networks. We used this hypomorphic STI1 mutant mouse line to investigate the 47 requirement of STI1-mediated regulation of chaperone networks in mouse physiology. 48 Embryonic cell pluripotency was severely affected by decreased STI1 activity, contributing to 49 the abnormal development in these mice. Moreover, adult TPR1-deprived STI1 mice presented 50 age-related hippocampal neurodegeneration, resulting in compromised memory recall. Our 51 findings reveal a requirement for optimal regulation of chaperone networks and their clients 52 during development and strict dependence on full STI1 activity for healthy neuronal aging. 53 These experiments demonstrate the unique experimental power of using hypomorphic alleles to 54 reveal how chaperone networks regulate mammalian physiology. 55 56 2 bioRxiv preprint doi: https://doi.org/10.1101/258673; this version posted February 1, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 57 INTRODUCTION 58 Regulation of proteostasis by the Heat-shock protein 70 (Hsp70) and 90 (Hsp90) is thought to be 59 particularly important during development [1,2], aging [3] and in neurodegenerative diseases [4- 60 6]. The heat shock response, mainly controlled by the transcription factor Hsf1, protects 61 organisms from different types of insults and thus modulates resilience in adulthood [7]. Also, 62 Hsp90 and Hsp70 help to buffer deleterious changes due to genetic mutations and both 63 chaperones seem to contribute to phenotypic variability [8,9]. However, despite our extensive 64 knowledge of the roles of chaperone networks in yeast and worms, how chaperones and co- 65 chaperones regulate physiological processes in vivo in mammals is not fully appreciated. 66 Stress-inducible phosphoprotein 1 (STI1, STIP1 or Hop for Hsp organizing protein in humans) is 67 a co-chaperone that regulates the transfer of client proteins between Hsp70 and Hsp90 [10-13]. 68 STI1 is a modular protein containing three tetratricopeptide repeat domains (TPR1, TPR2A and 69 TPR2B), which interact with Hsp70 (TPR1 and TPR2B) and Hsp90 (TPR2A), respectively [11]. 70 STI1 and its TPR domains are conserved from yeast to humans. The TPR2A and TPR2B 71 domains are required for activation of client proteins [11,13,14]. In contrast, the TPR1 domain is 72 absent in C. elegans [15], and it may be dispensable in yeast, as mutant STI1 protein lacking the 73 TPR1 domain is functional, albeit with decreased efficiency in forming ternary complex with 74 Hsp70 and Hsp90 [13]. Hence, STI1 has a unique role in the chaperone cycle, but the function of 75 its TPR1 domain remains poorly understood. 76 Although deletion of STI1 in yeast is not lethal, it causes growth impairments in challenging 77 conditions [16]. Also, yeast STI1 interacts genetically with Hsp90, and deletion of STI1 78 sensitizes yeast to Hsp90 suppression [17-20], illustrating the complex relationship between 3 bioRxiv preprint doi: https://doi.org/10.1101/258673; this version posted February 1, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 79 these proteins. Elimination of STI1 in C. elegans is not lethal, but it causes deficits in 80 reproduction, longevity and resilience to thermal stressors [15]. In mice, deletion of STI1 affects 81 the rate of blastocyst survival and escaping embryos died early in development (E10.5), 82 indicating that STI1 is essential in mammals [2]. 83 We surveyed human and mouse databases and found important differences between mammalian 84 and yeast dependence on co-chaperones for survival. To further unveil the impact of chaperones 85 in mammalian physiology, we engineered a mouse line with a STI1 hypomorphic allele coding 86 for a protein lacking the TPR1 domain. Our results reveal that optimal STI1 activity in vivo is 87 required to maintain the functionality of chaperone networks with impact in pluripotency during 88 development and in neuronal aging. This new STI1 hypomorphic mouse line opens new avenues 89 to understand the role of chaperone networks in maintaining client protein levels in different 90 tissues in response to physiological and pathological challenges. 91 4 bioRxiv preprint doi: https://doi.org/10.1101/258673; this version posted February 1, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 92 RESULTS 93 STIP1 loss-of-function mutations in humans are rare 94 Loss of STI1 is tolerated in yeast [16] and C. elegans [15,21], but knockout of Stip1 in mice 95 causes embryonic lethality [2]. To determine whether STIP1 in humans may also be essential, we 96 determined the frequency of variation in STIP1 in relatively healthy individuals using public 97 databases, aggregating genetic information from thousands of exomes and genomes such as 98 ExAC (60,706 individuals) and gnomAD (138,632 individuals) [22]. Protein truncation variants 99 (PTVs) are a class of mutations that typically lead to loss of protein function and can reveal the 100 biological consequences of gene deficiency in humans. After filtering for false PTVs, we have 101 observed 1 and 4 heterozygous STIP1 PTV carriers in ExAC and gnomAD, respectively, at a 102 frequency of <<0.001% (Supplementary Table 1). In comparison, HSP90AA1 presented 8 and 22 103 PTVs in ExAC and gnomAD, respectively, at a 10-fold higher frequency of <0.01% 104 (Supplementary Table 1). While we applied strict false positive PTV filtering criteria (see 105 methods), we are unable to confirm whether these PTVs are real or are the result of sequencing 106 or alignment errors. However, the STIP1 pLI score, which reflects the probability that a given 107 gene is intolerant to loss-of-function variation (haploinsufficient) was 1. This suggested that loss- 108 of-function variations in STIP1 are most likely not tolerated in humans or may result in a disease 109 phenotype [22]. In comparison, the pLI score of HSP90AA1, the stress inducible Hsp90 allele, 110 was 0.68, suggesting that PTVs may be tolerated in HSP90AA1. This is likely due to 111 compensation by the highly redundant HSP90AB1, the constitutive Hsp90 isoform. Interestingly, 112 the HSP90AB1 pLI score is 1. These analyses mirrored the survival of STI1, Hsp90α and Hsp90β 113 knockout mice [2,23,24]. Whereas Hsp90α knockout mice survive to adulthood [23], both STI1 114 and Hsp90β gene ablation causes embryonic lethality [2,24]. We also surveyed the following 5 bioRxiv preprint doi: https://doi.org/10.1101/258673; this version posted February 1, 2018.
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