Calreticulin and ERp57 as Redox Modulators of IRE1 Oligomerization Determine Tissue‑Specific UPR Imbalance in Aging

Age-dependent loss of the ER luminal chaperones calreticulin (Calr) and ERp57 shifts the ER redox environment, promoting aberrant disulfide‑bonded oligomerization of IRE1α. This altered oligomerization biases IRE1 toward its RNase‑inactive RIDD‑prone state while permitting PERK hyper‑activation, thereby creating the tissue‑specific UPR imbalance observed in aged skeletal muscle (PERK‑dominant) and other tissues (global UPR decline). Restoring Calr/ERp57 redox function should normalize IRE1 oligomerization, re‑balance PERK/IRE1 signaling, and ameliorate age‑related proteostatic decline.

Mechanistic Rationale

1. Calr/ERp57 maintain ER luminal redox homeostasis – both proteins possess thiol‑oxidoreductase activity that buffers luminal glutathione and protein disulfide isomerase (PDI) networks. Their decline with age (1) likely oxidizes the ER lumen, favoring disulfide‑linked IRE1α dimers/oligomers that are competent for RIDD but deficient for XBP1 splicing.
2. IRE1α oligomerization state dictates effector choice – structural studies show that distinct IRE1α oligomers preferentially recruit TRAF2 (activating PERK‑p38/MAPK cross‑talk) or the spliceosome‑like complex for XBP1 mRNA (4). A redox‑shift toward disulfide‑stabilized oligomers would enhance TRAF2 binding, explaining PERK‑driven p38 MAPK hyper‑activation in injured satellite cells (2) while attenuating XBP1s production.
3. Tissue‑specific chaperone expression – skeletal muscle satellite cells retain higher basal ERp57 levels during regeneration, preserving a reducing lumen that favors monomeric IRE1α competent for XBP1 splicing. In contrast, neurons and hepatocytes show a steeper Calr/ERp57 drop with age, leading to a global RIDD‑biased IRE1 phenotype and reduced XBP1s (3).
4. Link to proteostasis failure – Excess RIDD degrades ER‑targeted mRNAs, lowering chaperone synthesis and creating a vicious cycle of further ER stress, protein aggregation, and PERK‑CHOP‑mediated apoptosis (1).

Testable Predictions

• Prediction 1: In aged mouse tissues, biochemical fractionation will show increased disulfide‑linked IRE1α oligomers correlating with reduced Calr/ERp57 levels; young tissues will display predominantly monomeric or non‑disulfide IRE1α.
• Prediction 2: Adenoviral overexpression of Calr or ERp57 in aged skeletal muscle will shift IRE1α oligomers toward a non‑disulfide, XBP1s‑competent state, decreasing p‑eIF2α and CHOP while increasing XBP1s splicing after tunicamycin challenge.
• Prediction 3: Pharmacological reduction of the ER lumen (e.g., with ER‑targeted N‑acetylcysteine) will rescue IRE1α XBP1s activity in aged neuroblastoma cells without affecting PERK phosphorylation, confirming redox specificity.
• Prediction 4: Tissue‑specific knockdown of Calr/ERp57 in young satellite cells will phenocopy the aged PERK‑dominant response to injury (enhanced p38 MAPK, impaired myofiber repair) despite normal IRE1α expression levels.

Experimental Approach

1. Redox‑sensitive IRE1α immunoprecipitation – treat lysates with alkylating agents (N‑ethylmaleimide) under non‑reducing conditions, run SDS‑PAGE, blot for IRE1α; quantify oligomer vs monomer bands across young/old tissues and after Calr/ERp57 manipulation.
2. XBP1 splicing and RIDD assays – RT‑qPCR for spliced XBP1 and degradation of known RIDD substrates (e.g., Blos1, Sec61b) under basal and stress conditions.
3. Functional readouts – measure satellite cell proliferation/differentiation (MyoD, Myogenin) and neuronal viability (caspase‑3 cleavage, aggregate load) following Calr/ERp57 rescue or redox modulation.
4. In vivo validation – administer ER‑targeted redox buffers to aged mice; assess muscle regeneration after cardiotoxin injury and neuronal markers in hippocampus.

Falsifiability

If Calr/ERp57 overexpression or ER lumen reduction fails to alter IRE1α oligomerization state, does not restore XBP1s splicing, and does not improve tissue‑specific outcomes despite correcting chaperone levels, the hypothesis would be refuted. Conversely, observing the predicted shifts would support a redox‑mediated mechanism linking chaperone loss to UPR imbalance in aging.

Implications

This model positions ER luminal redox homeostasis—not merely chaperone abundance—as a proximal regulator of IRE1α signaling bias. It suggests that therapeutic strategies targeting ER redox (e.g., ER‑targeted antioxidants or disulfide‑isomerase mimetics) could rebalance PERK/IRE1 signaling, mitigate age‑related proteotoxic stress, and extend healthspan in a tissue‑specific manner.