Abalone peptide increases stress resilience and cost‐free longevity via SKN‐1‐governed transcriptional metabolic reprogramming in C. elegans

Abstract A major goal of healthy aging is to prevent declining resilience and increasing frailty, which are associated with many chronic diseases and deterioration of stress response. Here, we propose a loss‐or‐gain survival model, represented by the ratio of cumulative stress span to life span, to quantify stress resilience at organismal level. As a proof of concept, this is demonstrated by reduced survival resilience in Caenorhabditis elegans exposed to exogenous oxidative stress induced by paraquat or with endogenous proteotoxic stress caused by polyglutamine or amyloid‐β aggregation. Based on this, we reveal that a hidden peptide (“cryptide”)—AbaPep#07 (SETYELRK)—derived from abalone hemocyanin not only enhances survival resilience against paraquat‐induced oxidative stress but also rescues proteotoxicity‐mediated behavioral deficits in C. elegans, indicating its capacity against stress and neurodegeneration. Interestingly, AbaPep#07 is also found to increase cost‐free longevity and age‐related physical fitness in nematodes. We then demonstrate that AbaPep#07 can promote nuclear localization of SKN‐1/Nrf, but not DAF‐16/FOXO, transcription factor. In contrast to its effects in wild‐type nematodes, AbaPep#07 cannot increase oxidative stress survival and physical motility in loss‐of‐function skn‐1 mutant, suggesting an SKN‐1/Nrf‐dependent fashion of these effects. Further investigation reveals that AbaPep#07 can induce transcriptional activation of immune defense, lipid metabolism, and metabolic detoxification pathways, including many SKN‐1/Nrf target genes. Together, our findings demonstrate that AbaPep#07 is able to boost stress resilience and reduce behavioral frailty via SKN‐1/Nrf‐governed transcriptional reprogramming, and provide an insight into the health‐promoting potential of antioxidant cryptides as geroprotectors in aging and associated conditions.


List of supplementary data
) is defined as AUC ratio of stress span to life span (S/L), where S/L<1 or >1 indicates a resilience loss or gain, respectively.FIGURE S2 Survival resilience of C. elegans treated with 0-1 mM paraquat.See Table S1 and also Figure 1a.S8.   * Identified against a set of dipeptide and tripeptide antioxidant indicators in the BIOPEP-UWM database, which was used here for in silico analysis of peptide sequences.# The relative change in area under the survival curve of C. elegans with versus without peptide treatment in paraquat survival assay, indicating a relative total survival gain or loss.

FIGURE
FIGURE S1 A loss-or-gain model for quantification of stress survival resilience.FIGURE S2 Survival resilience of C. elegans treated with 0-1 mM paraquat.

FIGURE
FIGURE S3 Effect of selected peptides on C. elegans survival against oxidative stress induced by paraquat.FIGURE S4 Effect of abalone peptide on polyQ aggregation in C. elegans.FIGURE S5 Measurement of motility-related health metrics of C. elegans using worm tracking systems.

FIGURE
FIGURE S1 A loss-or-gain model for quantification of stress survival resilience.The area under the curve (AUC) is used to quantify the cumulative survival capacity of C. elegans against stress.The survival resilience (Equation1) is defined as AUC ratio of stress span to life

FIGURE
FIGURE S3 Effect of selected peptides on C. elegans survival against oxidative stress induced by paraquat.Wild-type L4 nematodes were pretreated with selected abalone (a) and sea cucumber (b) peptides for 24 h and then exposed to 50 mM paraquat.Live and dead nematodes were scored every 12 h or 24 h until all dead.Representative results are presented as Kaplan-Meier survival curves.Information of the tested peptides are shown in (c).

FIGURE
FIGURE S4 Effect of abalone peptide on polyQ aggregation in C. elegans.(a) Representative fluorescence micrographs of AM140 nematodes on day 5 of adulthood showing Q35::YFP aggregates in body wall muscle cells.Scale bar, 100 μm.(b) Quantification of Q35::YFP aggregates in body wall muscle cells of AM140 nematodes treated with or without AbaPep#07 at 20°C from L4 stage.Data are representative of three independent experiments and presented as mean ± SD (n ≥ 30 nematodes for each treatment).* p<0.05; *** p<0.001.

FIGURE
FIGURE S6 Effect of abalone peptide on fitness metrics of C. elegans.Wild-type nematodes were treated with AbaPep#07 from L4 until day 1 or day 10 of adulthood as indicated prior to fitness analysis.Body size and motility were analyzed as described in Figure 3. Representative results are shown as bar and scatter plots with mean ± SD from at least three independent experiments.* p<0.05; ** p<0.01; *** p<0.001.

FIGURE
FIGURE S7 Effect of abalone peptide on survival of pmk-1 mutant C. elegans against oxidative stress.The pmk-1(km25) mutant nematodes (strain KU25) were treated from L4 stage with the peptide AbaPep#07 at the indicated concentrations for 24 h and then exposed to 50 mM paraquat.Live and dead nematodes were scored every 12 h until all dead.Representative results from three independent experiments are shown as Kaplan-Meier survival curves.SeeTable S6 for survival statistics.

FIGURE
FIGURE S8 Consensus SKN-1-binding motifs.Two consensus sequences of predicted SKN-1 binding sites in C. elegans.RSAT and WebLogo are used to identify binding motifs and generate graphs, respectively.See also TableS8.

FIGURE
FIGURE S9 Effect of abalone peptide on expression of lipogenesis genes and composition of fatty acids in C. elegans.(a) Schematic of lipogenesis pathway in C. elegans.(b) Effect of peptide AbaPep#07 on expression of fat synthesis genes.Except fat-7, most lipogenesis genes were not significantly regulated by AbaPep#07.Data derived from RNA-seq are shown as a heat map.(c) Profiling of individual fatty acid fraction relative to the total fatty acid pool in C.elegans.Wild-type nematodes were incubated with or without AbaPep#07 from L4 to day 10 of adulthood and harvested for fatty acid analysis by GC-MS.

TABLE S1 Survival
resilience of C. elegans to exogenous oxidative stress induced by paraquat.

TABLE S2 Statistics
for C. elegans survival assays under paraquat-induced oxidative stress.

TABLE S3 Peptides
identified in abalone protein hydrolysate and tested for antioxidant capacity.

TABLE S4 Survival
resilience of C. elegans to endogenous proteotoxic stress.

TABLE S5
Statistics for C. elegans life span assays.

TABLE S6
Statistics for survival assays of mutant C. elegans under paraquat-induced oxidative stress.

TABLE S7
List of differentially expressed genes (separate Excel file).

TABLE S9
List of primers used for quantitative real-time PCR analysis.

TABLE S1
Survival resilience of C. elegans to exogenous oxidative stress induced by paraquat.Stress span is expressed as the area under the curve (AUC) of lifelong survival curves of wild-type C. elegans populations exposed to the indicated concentration of paraquat.Life span is the AUC of lifelong survival curves of wild-type C. elegans populations under normal conditions in the references corresponding to the stress span.Survival resilience (S/L) is defined as the AUC ratio of stress span to life span.Related to Figure 1a.

TABLE S2
Statistics for C. elegans survival assays under paraquat-induced oxidative stress.The survival time is the period that the wild-type nematodes pretreated with or without protein hydrolysate/AbaPep#07 were still alive after exposure to 50 mM paraquat.Related to Figure

TABLE S3
Peptides identified in abalone protein hydrolysate and tested for antioxidant capacity.The hydrolysate was subjected to activity-guided fractionation by ultrafiltration and gel filtration, and the peptide sequences in antioxidant fractions were identified by RP-nano-LC-MS/MS.After in silico analysis of the sequences for antioxidant potential using BIOPEP-UWM database, the peptides of interest were synthesized and tested for antioxidant activity using paraquat survival assay in C. elegans.

TABLE S4
Survival resilience of C. elegans to endogenous proteotoxic stress.Stress span is expressed as the area under the curve (AUC) of population survival curves of transgenic C. elegans expressing polyQ or Aβ in body wall muscle cells.Life span is the AUC of population survival curves of wild-type or transgenic control C. elegans (without polyQ/Aβ expression) in the references corresponding to the stress span.Survival resilience (S/L) is defined as the AUC ratio of stress span to life span.

TABLE S5
Statistics for C. elegans life span assays.Life span is the days of adulthood of wild-type nematodes treated with the indicated concentrations of AbaPep#07 from L4 stage.

TABLE S6
Statistics for survival assays of mutant C. elegans under paraquat-induced oxidative stress.The survival time is the period that the nematodes pretreated with or without AbaPep#07 were still alive after exposure to 50 mM paraquat.The data for skn-1(zu67) (strain

TABLE S7
List of differentially expressed genes.Differentially expressed genes (DEGs) regulated by AbaPep#07 in day 10 wild-type C. elegans.Related to Figure 5 and Figure 6.See separate Excel file for details.

TABLE S8
References for SKN-1-targeted genes.Related to Figure 6b.Number Reference TABLE S9 List of primers used for quantitative real-time PCR analysis.