Translocon Declogger Ste24 Protects against IAPP Oligomer-Induced Proteotoxicity

Abstract

Aggregates of human islet amyloid polypeptide (IAPP) in the pancreas of patients with type 2 diabetes (T2D) are thought to contribute to β cell dysfunction and death. To understand how IAPP harms cells and how this might be overcome, we created a yeast model of IAPP toxicity. Ste24, an evolutionarily conserved protease that was recently reported to degrade peptides stuck within the translocon between the cytoplasm and the endoplasmic reticulum, was the strongest suppressor of IAPP toxicity. By testing variants of the human homolog, ZMPSTE24, with varying activity levels, the rescue of IAPP toxicity proved to be directly proportional to the declogging efficiency. Clinically relevant ZMPSTE24 variants identified in the largest database of exomes sequences derived from T2D patients were characterized using the yeast model, revealing 14 partial loss-of-function variants, which were enriched among diabetes patients over 2-fold. Thus, clogging of the translocon by IAPP oligomers may contribute to β cell failure.

Keywords: IAPP; ZMPSTE24; aggregation; amylin; diabetes; protein folding; proteotoxicity; yeast.

Figure 1. Growth defects and ER stress in yeast expressing 6xIAPP

A) Yeast harboring empty vector or 6xIAPP were induced with 100 nM estradiol and grown on agar plates for 24 hrs. B) Western blot of cells expressing 6xIAPP. C) Cells expressing 6xIAPP-msfGFP fused to a Kar2 signal peptide (Kar2_SS) (panel 1), msfGFP fused to Kar2_SS (panel 2), and msfGFP fused to Kar2_SS and an ER retention peptide (HDEL) (Panel 3). Scale bar = 5 μm. D) RNAseq data from 2 biological replicates of strains harboring a single copy of 6xIAPP. In red are genes upregulated in both the IAPP strains and in the unfolded protein response. The Venn diagram shows the genes typically upregulated in the UPR (blue) and those upregulated >4 fold when 6xIAPP was expressed (pink). The overlap between the two sets is shown in dark pink, along with the p-value calculated using Fisher’s exact test. E) Mean GFP intensity comparison across all fluorescent reporter strains for cells expressing 6xIAPP and controls. The solid line represents equal GFP expression. Filled circles are reporter strains with large intensity changes upon 6xIAPP expression. The most highly perturbed strains (Kar2_ss-GFP-HDEL, Scj1-mNG, Kar2-mNG-HDEL) were measured independently and are shown on the right. Scale bar = 5 μm. See also Figure S1.

Figure 2. Genome-wide screens for modifiers of IAPP toxicity

A) The suppressors and enhancers recovered from the overexpression screen, highlighting the strongest suppressors. A positive score indicates a suppressor of toxicity, while a negative score indicates an enhancer of toxicity. B) Spotting assays demonstrating independent verification of the Ste24 suppressor on both the 6xIAPP and C) 1xIAPP yeast models. 6xIAPP strains and uninduced 1xIAPP strains were grown for 48 hours. The induced 1xIAPP strains were grown for 72 hours. The bar chart is a quantification of the 1xIAPP dilution assay (dashed rectangle) with cells co-overexpressing control vector represented by the black bar (n=6) and cells co-overexpressing STE24 represented by the gray bar (n=9). Statistical analysis was performed using Student’s t-test. D) The enhancers of toxicity recovered from the genome wide deletion of non-essential genes (circles) and temperature-sensitive allele screen of essential genes (squares). A more negative score indicates a stronger enhancer of toxicity. The enrichment for autophagy genes (orange circles) and proteasome genes (turquoise squares) among the enhancers is highlighted. See also Figures S2, S3, and Tables S1-S3.

Figure 3. ZMPSTE24 inhibition unmasked IAPP oligomer toxicity in a pancreatic cell line

6x human IAPP and 6x rat IAPP were introduced into INS-1 823/13 cells by lentiviral infection and induced with doxycycline. Inhibition of ZMPSTE24 with 20 uM lopinavir reduced the survival of cells expressing 6x human IAPP (^***p < 0.001, Student’s t-test) but not cells expressing 6x rat IAPP.

Figure 4. Suppression of toxicity by Ste24 overexpression was not due to reduced IAPP levels and is specific for IAPP

A) Western blot of IAPP expression measured in whole cell lysates prepared after 6 hours of 6xIAPP expression, with and without co-overexpression of STE24. The sum of the upper and lower bands was quantified and normalized to control cells. Overexpression of Ste24 modestly reduced 6xIAPP levels. Pgk1 was the loading control. B) STE24 overexpression rescued 6xIAPP toxicity but not α-synuclein or TDP-43 toxicities. Effects of STE24 overexpression are shown in spotting assays alongside SLA1 (Aβ suppressor), VTS1 (TDP-43 suppressor), and YPT1 (α-synuclein suppressor). 6xIAPP and α-synuclein were induced with 100 nM estradiol. TDP43 was induced with 5 nM estradiol. All strains were grown for 48 hours.

Figure 5. Ste24 overexpression attenuated 6xIAPP-induced changes in Kar2 and Scj1 expression and Kar2_SS-msfGFP-HDEL localization

Representative fluorescence images for cells co-expressing 6xIAPP in combination with either STE24 or a vector control and either Kar2 protein C-terminally fused to mNG at its endogenous locus (Kar2-mNG, top panels); Scj1 protein C-terminally fused to mNG at its endogenous locus (Scj1-mNG, middle panels); or overexpressed msfGFP N-terminally fused to the Kar2_ss and C-terminally fused to the ER-retention sequence HDEL (Kar2_ss-msfGFP-HDEL, bottom panels). The three estradiol doses are for uninduced (0 nM), low toxicity (10 nM), and high toxicity (100 nM) conditions. For Kar2 and Scj1 the mean GFP intensity of the cells was quantified, while the fraction of cells with cytoplasmic, rather than ER-localized GFP was quantified for Kar2_ss-msfGFP-HDEL. At least 100 cells were quantified per replicate and experiments were performed in biological triplicate. Error bars = SD of biological replicates. Scale bar = 5 μm.

Figure 6. Loss of STE24 enhanced IAPP oligomer toxicity

A) Deletion of STE24 produced no growth defect on its own but greatly enhanced 6xIAPP toxicity. This was rescued by reintroducing STE24 on a plasmid under the control of its endogenous promoter. A catalytically inactive E289G Ste24 was much less effective in rescuing the enhanced IAPP toxicity of the Δste24 strain. B) WT human ZMPSTE24 functionally substituted for Ste24 and relieved 6xIAPP toxicity in the Δste24 strain. The effects of substitution with ZMPSTE24 mutants possessing a range of declogging activities are shown in order of decreasing activity (for a measurement of their declogging activity please see (Ast et al., 2016)).

Figure 7. Analysis of the IAPP oligomer toxicity-rescuing ability of 111 ZMPSTE24 missense mutants

ZMPSTE24 mutants identified from the sequencing of diabetes patients and healthy controls were tested for their ability to rescue 6xIAPP toxicity in the Δste24 yeast strain. The growth of 6xIAPP strains expressing ZMPSTE24 variants was quantified as the area under the growth curve at 48 hours. A) Each cell of the heat map is the average growth of four biological replicates, while each column is a technical replicate. B) Retest of poorly growing variants. Variants meeting statistical significance were deemed loss-of-function variants (One-sided ANOVA followed by Dunnett’s test, cutoff: p < 0.01) and are shown in blue bars. Error bars = SD. C) Residues in ZMPSTE24 where amino acid changes produced lower 6xIAPP toxicity rescuing activity are shown on the crystal structure (PDB: 5SYT) in orange, with laminopathy-causing variants shown in red.