Mapping Stress-Responsive Signaling Pathways Induced by Mitochondrial Proteostasis Perturbations

Abstract

Imbalances in mitochondrial proteostasis are associated with pathologic mitochondrial dysfunction implicated in etiologically-diverse diseases. This has led to considerable interest in defining the biological mechanisms responsible for regulating mitochondria in response to mitochondrial stress. Numerous stress responsive signaling pathways have been suggested to regulate mitochondria in response to proteotoxic stress, including the integrated stress response (ISR), the heat shock response (HSR), and the oxidative stress response (OSR). Here, we define the specific stress signaling pathways activated in response to mitochondrial proteostasis stress by monitoring the expression of sets of genes regulated downstream of each of these signaling pathways in published Perturb-seq datasets from K562 cells CRISPRi-depleted of individual mitochondrial proteostasis factors. Interestingly, we find that the ISR is preferentially activated in response to mitochondrial proteostasis stress, with no other pathway showing significant activation. Further expanding this study, we show that broad depletion of mitochondria-localized proteins similarly shows preferential activation of the ISR relative to other stress-responsive signaling pathways. These results both establish our gene set profiling approach as a viable strategy to probe stress responsive signaling pathways induced by perturbations to specific organelles and identify the ISR as the predominant stress-responsive signaling pathway activated in response to mitochondrial proteostasis disruption.

Figure 1.. Target gene sets define stress pathway activation in Perturb-seq datasets.

A. Expression, measured by z-score, of ISR target gene set comprising 13 genes in K562 cells CRISPRi-depleted of the indicated ISR component. The ISR target genes are shown in Table S1. B-F. Expression, measured by z-score, for UPR, ISR, HSR, OSR, and control target gene sets in K562 cells CRISPRi-depleted of ATF4 (B), EIF2B4 (C), EIF2B5 (D), HSPA5 (E), or KEAP1 (F). The target gene sets used in this experiment were defined as in and are shown in Table S1. *p<0.05, **p<0.01, ***p<0.005 for Brown-Forsythe and Welch ANOVA.

Figure 2.. ER proteostasis perturbations selectively activate the unfolded protein response.

A. Heat map showing the expression, measured by z-score, of ISR, UPR, HSR, and OSR target genes in K562 cells CRISPRi-depleted of components of the indicated ER proteostasis pathway, as defined in . B. Pathway activation score vs p-value for UPR, ISR, HSR, and OSR target genes in K562 cells CRISPRi-depleted of individual ER proteostasis factors. Pathway activation score was defined by the average z-score for pathway-specific target genes and p-values were calculated by comparing the expression of pathway specific target gene set to the expression of the control gene sets. C-G. Expression, measured by z-score, for ATF6, IRE1/XBP1s, and control target gene sets in K562 cells CRISPRi-depleted of HSPA5 (C), DDOST1 (D), DAD1 (E), SPR72 (F), or SRP68 (G). ATF6 and IRE1/XBP1s gene sets are defined in Table S1. *p<0.05, **p<0.01, ***p<0.005 for Brown-Forsythe and Welch ANOVA.

Figure 3.. Mitochondrial proteostasis gene perturbation preferentially activates the ISR.

A. Heat map showing the expression, measured by z-score, of ISR, UPR, HSR, and OSR target genes in K562 cells CRISPRi-depleted of components of the indicated mitochondrial proteostasis pathway, as defined in . B. Pathway activation score vs −log p-value for UPR, ISR, HSR, and OSR target genes in K562 cells CRISPRi-depleted of individual mitochondrial proteostasis factors. Pathway activation score was defined by the average z-score for pathway-specific target genes. P-values were calculated by comparing the pathway specific target gene set expression to the expression of the control gene sets. C. Expression, measured by z-score, for UPR, ISR, HSR, OSR, and control target genesets in K562 cells CRISPRi depleted of HSPA9, HSPE1, TIMM23B, or TOMM22, as indicated. *p<0.05, **p<0.01, ***p<0.005 for Brown-Forsythe and Welch ANOVA, as compared to the control geneset. D. Comparison of gene expression, as measured by z-score, in K562 cells CRISPRi-depleted of either HSPA9 or TIMM23B. Only genes increased or decreased |value| > 0.5 in response to either perturbation are shown. E. Comparison of gene expression, measured by z-score, in K562 cells CRISPRi-depleted of HSPA9 or EIF2B3, EIF2B4, or EIF2B5, as indicated. Only genes increased or decreased |value| > 0.5 in response to either perturbation are shown.

Figure 4.. Mitochondrial gene perturbations preferentially activate the ISR.

A. Pathway activation score vs −log p-value for UPR, ISR, HSR, and OSR target genes in K562 cells CRISPRi-depleted of genes encoding mitochondrial proteins, as defined in . Pathway activation score was defined by the average z-score for pathway-specific target genes. P-values were calculated by comparing the pathway specific target gene expression to the expression of the control gene sets. B. Expression, measured by z-score, for UPR, ISR, HSR, OSR, and control target gene sets in K562 cells CRISPRi depleted of SLC25A42, IARS2, or PRELID3B, as indicated. **p<0.01, ***p<0.005 for Brown-Forsythe and Welch ANOVA, as compared to the control geneset. C. Comparison of gene expression, as measured by z-score, in K562 cells CRISPRi-depleted of either SLC25A42 or EIF2B5. Only genes increased or decreased |value| > 0.5 in response to either perturbation are shown.