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The Chaperone Protein Cct5 is Essential for Hematopoietic Stem Cell Maintenance

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Abstract

Proper proteostasis is indispensable for the long-term maintenance of hematopoietic stem and progenitor cells (HSPCs). The TRiC/CCT (chaperonin-containing TCP-1) complex, a key constituent of cellular machinery facilitating accurate protein folding, has remained enigmatic in its specific function within HSPCs. Here we show that conditional knockout (KO) of Cct5 significantly impairs the maintenance of murine HSPCs. Primary and secondary transplantation experiments unequivocally demonstrate the incapacity of Cct5 KO HSPCs to reconstitute both myeloid and lymphoid lineage cells in recipient mice, highlighting the pivotal role of the TRiC/CCT complex in governing these cellular lineages. Furthermore, leveraging an integrated approach that merges a Protein–Protein Interaction (PPI) database with RNA sequencing (RNA-seq) data of HSPCs, our analysis reveals intricate interactions between Cct5 and vital transcription factors crucial for HSC homeostasis. Notably, Cct5 engages with MYC, PIAS1, TP53, ESR1, HOXA1, and JUN, intricately regulating the transcriptional landscape governing autophagy, myeloid differentiation, and cytoskeleton organization within HSPCs. Our study unveils the profound significance of TRiC/CCT complex-mediated proteostasis in orchestrating the maintenance and functionality of HSPCs.

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Raw data will be provided upon reasonable request.

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Funding

This study was supported by National Key Research and Development Project (2019YFA0111800), Major Research Plan of National Natural Science Foundation of China (92268116, 91957107), General Program of National Natural Science Foundation of China (81970095) to BG, and by Innovative Research Team of High-Level Local Universities in Shanghai (SHSMU-ZDCX20212000). We thank the support from Shanghai Frontiers Science Center of Cellular Homeostasis and Human Diseases, and thank Flow Cytometry and Animal Core Facilities of Shanghai Jiao Tong University School of Medicine for the technical support.

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Author notes

  1. Linxi Li, Jie Huang and Suping Zhang contributed equally to this work.

Authors and Affiliations

  1. Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, 280, Chong-Qing South Road, West Bldg #2, Rm 804, Shanghai, 200025, China

    Linxi Li, Suping Zhang, Chunxu Yao, Yandan Chen & Bin Guo

  2. Children’s Hospital, Fudan University, Minhang, Shanghai, China

    Jie Huang

  3. Department of Hematology, Xin Hua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, 200090, China

    Yandan Chen & Bin Guo

  4. Thoracic Surgery Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA

    Haitao Wang

Authors
  1. Linxi Li
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  2. Jie Huang
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  3. Suping Zhang
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  4. Chunxu Yao
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Contributions

BG, HW and YC conceived the research, designed and supervised the experiments, interpreted data and wrote the manuscript. LL, JH and SZ performed most of the experiments, analyzed data and wrote the manuscript. CY prepared samples for immune-staining analysis.

Corresponding authors

Correspondence to Yandan Chen, Haitao Wang or Bin Guo.

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Ethics Approval (Include Appropriate Approvals or Waivers)

All animal experiments followed protocols approved by The Institutional Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine.

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The authors are alone responsible for the content and writing of the paper. All authors reviewed and approved the final version of the manuscript.

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The authors declare no competing interests.

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Supplementary Information

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12015_2023_10670_MOESM1_ESM.pdf

Supplementary file1 Supplemental Figure 1. The CRUs was significantly decreased in the BM of Cct5-/- mice. (A) CRUs in the BM of Cct5fl/fl and Cct5-/- mice, as determined by LDA. (B) The summary chart showing the percentage of recipient mice with positive engraftment (>1% CD45.2 cell chimerism), as well as the CRUs and Range calculated using ELDA software. (C) The bar graph showing the CRUs in 106 BM cells of Cct5fl/fl and Cct5-/- mice. Poisson statistical analysis, ***p < 0.001. (PDF 143 kb)

12015_2023_10670_MOESM2_ESM.pdf

Supplementary file2 Supplemental Figure 2. Knockdown of CCT5 impairs the engraftment of human cord blood HSPCs. (A) Experimental schematics for the transplantation experiments of Control shRNA and CCT5 shRNA transduced cord blood CD34+ cells. (B) Representative FACS plot showing human CD45 chimerism in the BM of recipient immune-deficient mice transplanted with Control shRNA and CCT5 shRNA infected CD34+ cells. (C) Quantification data showing the percentage of human CD45 chimerism in the BM of recipient mice transplanted with Control shRNA and CCT5 shRNA infected CD34+ cells. ***p < 0.001. (D-E) Quantification data showing the percentage of human myeloid and B cell chimerism in the BM of recipient mice transplanted with Control shRNA and CCT5 shRNA infected CD34+ cells. ***p < 0.001. (PDF 388 kb)

Supplemental Table 1.

Protein-protein interaction (PPI) analysis for CCT5/Cct5 using data sourced from the BioGRID and IntAct databases (XLSX 280 kb)

Supplemental Table 2.

Identification of Cct5-interacting TFs using the TFDB database and their downstream target genes (XLSX 2.93 mb)

Supplemental Table 3.

The shared functions between Cct5-TF targets (PPI) and downregulated genes in Cct5 knockout HSPCs (RNA-seq) (XLSX 416 kb)

Supplemental Table 4.

Gene set enrichment analysis (GSEA) showing the down-regulation of genes involved in autophagy, myeloid cell development and actin filament organization in Cct5 knockout HSPCs (XLSX 6.90 mb)

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Li, L., Huang, J., Zhang, S. et al. The Chaperone Protein Cct5 is Essential for Hematopoietic Stem Cell Maintenance. Stem Cell Rev and Rep 20, 769–778 (2024). https://doi.org/10.1007/s12015-023-10670-7

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