Modest Declines in Proteome Quality Impair Hematopoietic Stem Cell Self-Renewal

Abstract

Low protein synthesis is a feature of somatic stem cells that promotes regeneration in multiple tissues. Modest increases in protein synthesis impair stem cell function, but the mechanisms by which this occurs are largely unknown. We determine that low protein synthesis within hematopoietic stem cells (HSCs) is associated with elevated proteome quality in vivo. HSCs contain less misfolded and unfolded proteins than myeloid progenitors. Increases in protein synthesis cause HSCs to accumulate misfolded and unfolded proteins. To test how proteome quality affects HSCs, we examine Aars^sti/sti mice that harbor a tRNA editing defect that increases amino acid misincorporation. Aars^sti/sti mice exhibit reduced HSC numbers, increased proliferation, and diminished serial reconstituting activity. Misfolded proteins overwhelm the proteasome within Aars^sti/sti HSCs, which is associated with increased c-Myc abundance. Deletion of one Myc allele partially rescues serial reconstitution defects in Aars^sti/sti HSCs. Thus, HSCs are dependent on low protein synthesis to maintain proteostasis, which promotes their self-renewal.

Keywords: HSC; hematopoietic stem cell; protein homeostasis; protein quality control; protein synthesis; proteostasis; self-renewal; stem cell; translation.

Figure 1.. HSCs Depend upon Low Protein Synthesis to Maintain Proteome Quality

(A) Western blot examining ubiquitylated protein in 3 × 10⁴ HSCs/MPPs, CMPs, GMPs, and MEPs (one of >5 blots). (B) Flow cytometry analysis showing ubiquitylated protein content relative to HSCs (n = 11 mice). (C) Representative histograms of ubiquitylated protein content in HSCs, CMPs, GMPs, and MEPs. (D) Cell volume of HSCs, CMPs, GMPs, and MEPs (n ≥ 34 cells/population). (E) Representative gel showing total protein content following SYPRO Ruby staining in HSCs/MPPs, CMPs, GMPs, and MEPs (one of 4 blots). (F) Total protein content relative to HSCs (n = 4 experiments). (G) Ubiquitylated protein relative to total protein content in HSCs, CMPs, GMPs, and MEPs (from B and E). (H) Diagram showing that TMI fluoresces when it binds to free cysteine thiols in unfolded proteins. (I) Relative TMI fluorescence in bone marrow cells after a 4-h incubation at 37°C or 42°C (n = 8 mice). (J) Total protein content in bone marrow cells after a 4-h incubation at 37°C or 42°C (n = 3 mice). (K) TMI fluorescence in bone marrow cells from mice treated 18 h earlier with bortezomib (BZ) or vehicle (DMSO) (n = 6 mice/treatment). (L) Relative TMI fluorescence in HSCs and progenitors (n = 11 mice). (M) OP-Puro incorporation by HSCs, CMPs, GMPs, and MEPs in vivo (n = 4 mice). (N) Diagram representing effects on HSC protein synthesis in wild-type (Pten^fl/fl;Rpl24^+/+), Pten-deficient (Mx1-Cre⁺Pten^fl/fl;Rpl24^+/+), and Mx1-Cre⁺;Pten^fl/fl;Rpl24^Bst/+ mice. (O) Western blot examining ubiquitylated protein in 8.5 × 10³ HSCs/MPPs from Pten^fl/fl;Rpl24^+/+, Mx1-Cre⁺Pten^fl/flRpl24^+/+, and Mx1-Cre⁺;Pten^fl/fl; Rpl24^Bst/+ mice (one of 3 blots). (P) TMI fluorescence in wild-type, Mx1-Cre⁺;Pten^fl/fl;Rpl24^+/+, and Mx1-Cre⁺;Pten^fl/fl;Rpl24^Bst/+ mice (n = 3–4 mice/genotype). Data represent mean ± standard deviation. Statistical significance was assessed using a Student’s t test (I-K) or an ANOVA followed by Dunnett’s multiple comparisons test relative to HSCs (B, D, F, G, L, M, and P). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2.. A tRNA Editing Defect that Reduces Proteome Quality Impairs HSC Self-Renewal

(A) Western blot examining ubiquitylated protein in 2.5 × 10⁴ wild-type (+/+) and Aars^sti/sti (sti/sti) HSCs/MPPs, CMPs, GMPs, and MEPs (one of 2 blots). (B) Flow cytometry analysis showing ubiquitylated protein content in Aars^sti/sti (sti/sti) relative to wild-type (+/+) HSCs (n = 4 mice/genotype). (C) OP-Puro incorporation by wild-type (+/+) and Aars^sti/sti (sti/sti) HSCs and progenitor cells in vivo (n = 3 mice/genotype). (D) Bone marrow cellularity (BM; 1 femur and 1 tibia) in wild-type (+/+) and Aars^sti/sti (sti/sti) mice (n = 6 mice/genotype). (E) Frequency of HSCs in wild-type (+/+) and Aars^sti/sti (sti/sti) bone marrow (n = 6 mice/genotype). (F and G) Absolute number of HSCs in the bone marrow (1 femur and 1 tibia; F) and spleen (G) of wild-type (+/+) and Aars^sti/sti (sti/sti) mice (n = 6 mice/genotype). (H-J) Frequency of CMPs (H), GMPs (I), and MEPs (J) in wild-type (+/+) and Aars^sti/sti (sti/sti) bone marrow (n = 6 mice/genotype). (K) Frequency of colony-forming unit (CFU) progenitors in wild-type (+/+) and Aars^sti/sti (sti/sti) bone marrow (n = 4 mice/genotype). (L) Diagram showing HSC transplantation strategy. (M) Donor cell engraftment when 10 HSCs from wild-type (+/+) and Aars^sti/sti (sti/sti) mice were transplanted with 2 × 10⁵ recipient-type bone marrow cells into irradiated mice. Total hematopoietic cell, B cell, T cell, and myeloid cell engraftment is shown (n = 4 donors and 14–21 total recipients/genotype). (N) Donor cell engraftment in secondary recipients (n = 2 donors and 8–9 total recipients/genotype). (O) Frequency of secondary recipients in (N) that exhibited long-term (16-week) multilineage reconstitution (≥0.5% donor derived peripheral blood B, T, and myeloid cells). Data represent mean ± standard deviation (B-K) or standard error of the mean (M and N). Statistical significance was assessed using a Student’s t test or a Fisher’s exact test (O). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3.. Accumulation of Ubiquitylated Protein Overwhelms the Proteasome in Aars^sti/sti HSCs, Leading to c-Myc Stabilization

(A) Heatmap showing 94 differentially expressed transcripts (≥ 2-fold change; padj < 0.1) up- (red) or downregulated (blue) between wild-type (+/+) and Aars^sti/sti (sti/sti) HSCs (n = 3 experiments/genotype). (B) Gene set enrichment analysis demonstrating no significant activation of the UPR^er in Aars^sti/sti HSCs. (C) Western blot examining phosphorylated (Ser51)-Eif2α in 2 × 10⁴ wild-type (+/+) and Aars^sti/sti (sti/sti) HSCs/MPPs. (D) Frequency of Annexin V⁺ HSCs in wild-type (+/+) and Aars^sti/sti (sti/sti) (n = 3 mice/genotype). (E) Diagram of the proteostasis network. (F) Proteasome activity in 5 × 10³ HSCs/MPPs, CMPs, GMPs, and MEPs (n = 5–9 replicates in 4 experiments). Data are shown in relative luminescence units (RLUs). (G) Representative histogram showing GFP expression in ub^G76V-GFP HSCs/MPPs in vitro treated for 18 h with (gray) or without (black) BZ. (H) Frequency of HSCs that are GFP⁺ in Ub^G76V-GFP (+/+) and Ub^G76V-GFP;Aars^sti/sti (sti/sti) bone marrow (n = 4–5 mice/genotype). (I) Relative proteasome activity in wild-type (+/+) and Aars^sti/sti (sti/sti) HSCs/MPPs (n = 9 replicates in 3 experiments). (J) Number of HSCs in the bone marrow (1 femur and 1 tibia) in mice treated with BZ or vehicle (DMSO) (n = 5–6 mice/treatment). (K) Frequency of wild-type (+/+) and Aars^sti/sti (sti/sti) HSCs that incorporated BrdU after a 72-h pulse in vivo (n = 6–8 mice/genotype). (L and M) Western blot examining c-Myc protein in 10⁴ HSCs/MPPs (L) or 1.8 × 10⁴ CMPs, GMPs and MEPs (M) isolated from wild-type (+/+) and Aars^sti/sti (sti/sti) mice (one of 2 (L) or 3 (M) blots). (N) Myc expression normalized to β-Actin in wild-type (+/+) and Aars^sti/sti (sti/sti) HSCs (n = 3). (O) Fbxw7 expression in wild-type (+/+) and Aars^sti/sti (sti/sti) HSCs (n = 3/genotype). Data represent mean ± standard deviation. Statistical significance was assessed using a Student’s t test (D, H, I, J, K, N, and O) or an ANOVA followed by Dunnett’s test relative to HSCs/MPPs (F). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4.. Reducing c-Myc Expression Largely Rescues Serial Reconstituting Activity of Aars^sti/sti HSCs

(A) Western blot examining c-Myc in 10⁴ HSCs/MPPs isolated from wild-type (+/+), Mx1-Cre⁺;Aars^sti/sti;Myc^fl/+ (sti/sti;Myc+/−) and Aars^sti/sti (sti/sti;Myc+/+) mice. (B) Diagram showing bone marrow transplantation strategy. (C) Donor cell engraftment 16 weeks after 5 × 10⁵ bone marrow cells from wild-type (+/+), Aars^sti/sti (sti/sti;Myc+/+) and Mx1-Cre⁺;Aars^sti/sti;Myc^fl/+ (sti/sti;Myc+/−) mice were transplanted with 5 × 10⁵ recipient-type bone marrow cells into irradiated mice. Total hematopoietic cells, B cell, T cell, and myeloid cell engraftment is shown (n = 4–8 donors and 26–44 total recipients/genotype). (D) Donor cell engraftment in secondary recipients (n = 4–10 donors and 17–44 recipients/genotype). (E) Frequency of secondary recipients in (D) that exhibited long-term multilineage reconstitution. Data represent mean ± standard error of the mean. Statistical significance was assessed using a Student’s t test (C and D) or Fisher’s exact test (E). *p < 0.05; **p < 0.01; ***p < 0.001.