STRC E1659A QC Rate-Limiting Step — Lit Gap-Map
Distilled lit-mining task surfaced by Tafamidis Kinetic-Stabilization Paradigm DR1-distillation: the H01 mech-4 proof is thermodynamic (Phase 5k anion preference), but does not name which step on the E1659A QC reaction coordinate the K1141 binding intercepts. Tafamidis-style mechanistic claim requires identifying the rate-limiting QC step — both for paper §3 and for τRAMD design (per Pharmacochaperone Residence Time Criterion residence-time floor calibration).
This note is a structured gap-map, not a derivation of the answer. Stereocilin ERAD timing is not characterised in any primary literature held in ~/STRC/sources/lit/ as of 2026-04-26. The cheapest path forward is the F508del CFTR proxy, with the canonical ERAD-timing literature retrieved via the sci-hub / annas-archive skills.
What is unknown for stereocilin specifically
- STRC ERAD half-life. No published pulse-chase or cycloheximide-chase data on WT stereocilin or any DFNB16 missense allele.
- STRC calnexin / calreticulin substrate status. Stereocilin has multiple predicted N-glycosylation sites (signal peptide cleaved at position 22; predicted N-glyc at ~5 sites in the central FN3-like and ZP-like domains via NetNGlyc). Whether stereocilin is a calnexin client is not measured; it is inferred by analogy to other ZP-like-domain glycoproteins (TECTA, TECTB, OTOG, ZP1-4 oocyte ZP proteins).
- EDEM1/2/3 substrate status. Mannose-trimming-driven ERAD targeting requires EDEM-class lectin recognition. Not measured for stereocilin.
- DFNB16 missense allele ERAD selectivity. E1659A in particular has not been characterised for ER retention / degradation kinetics in any cell line or animal model the team is aware of.
Closest published analog: F508del CFTR
CFTR F508del is the gold-standard ECM-folding-defect missense. Despite different protein class (multispan membrane channel vs ECM-anchored matrix protein), the QC machinery overlaps:
- Ward & Kopito 1994 J Biol Chem 269:25710 — F508del CFTR core-glycosylated form has ERAD half-life of ~30 min in epithelial cell lines vs. ~12 h for WT.
- Lukacs et al 1994 EMBO J 13:6076 — kinetic dissection of F508del ER retention; calnexin association required.
- Pind, Riordan & Williams 1994 J Biol Chem 269:12784 — calnexin binds CFTR co-translationally; F508del extends calnexin association.
These numbers are the default starting point for any τRAMD residence-time floor on H01 until stereocilin-specific data exists: a lead with τ < 30 min cannot rescue an ERAD half-life of 30 min — too fast to be present during the ERAD timer. Operative τ floor: ≥ 60 min (2× the F508del proxy) for confident rescue.
Canonical ERAD-timing primary literature — retrieval queue
Each entry is a real published paper relevant to defining the rate-limiting step on STRC E1659A’s QC pathway. Retrieve via sci-hub (DOIs) or annas-archive (titles); ingest into ~/STRC/sources/lit/ as standard type: lit notes per Lit Audit Status 2026-04-25.
A. ER QC machinery — calnexin / calreticulin cycle
| Reference | DOI / PMID | What it gives H01 |
|---|---|---|
| Hebert, Foellmer & Helenius 1995 Cell 81:425 | doi:10.1016/0092-8674(95)90395-x | Calnexin substrate retention timer, single-glycoprotein resolution |
| Molinari & Helenius 1999 Nature 402:90 | doi:10.1038/47062 | Glycoprotein folding cycles; quantitative timer |
| Caramelo, Castro, Sigaut & Parodi 2003 PNAS 100:86 | doi:10.1073/pnas.262661199 | UDP-Glc:GT folding-sensor; defines QC reaction-coordinate maximum |
| Hebert & Molinari 2007 Physiol Rev 87:1377 | doi:10.1152/physrev.00050.2006 | Field-defining review; defines the four QC checkpoints |
B. ERAD substrate selection and timing
| Reference | DOI / PMID | What it gives H01 |
|---|---|---|
| Olzmann, Kopito & Christianson 2013 CSH Perspect Biol 5:a013185 | doi:10.1101/cshperspect.a013185 | Modern ERAD review; substrate-selection logic |
| Hosokawa et al 2001 EMBO Rep 2:415 | doi:10.1093/embo-reports/kve084 | EDEM1 discovery; mannose-trimming clock |
| Christianson, Shaler, Tyler & Kopito 2008 Nat Cell Biol 10:272 | doi:10.1038/ncb1689 | Hrd1 / Sel1L ERAD complex composition |
| Bhamidipati, Denic, Quan & Weissman 2005 Mol Cell 19:741 | doi:10.1016/j.molcel.2005.07.027 | Yos9/OS-9 lectin step in ERAD |
C. F508del CFTR — proxy for ECM-class missense ERAD timing
| Reference | DOI / PMID | What it gives H01 |
|---|---|---|
| Ward & Kopito 1994 J Biol Chem 269:25710 | doi:10.1016/S0021-9258(18)47306-8 | Pulse-chase t½ ER form 30 min; gold-standard timing |
| Lukacs et al 1994 EMBO J 13:6076 | doi:10.1002/j.1460-2075.1994.tb06955.x | Calnexin binding extended on F508del |
| Pind, Riordan & Williams 1994 J Biol Chem 269:12784 | doi:10.1016/S0021-9258(18)99945-6 | Co-translational calnexin association |
| Sun, Mi, Wei, Hong, Hong, Skach 2008 J Cell Biol 182:355 | doi:10.1083/jcb.200712067 | Multi-step folding kinetics; corrector-mode mech logic |
D. Stereocilin-specific (no ERAD data, but interactome / domain context)
| Reference | DOI / PMID | What it gives H01 |
|---|---|---|
| Verpy et al 2001 PNAS 98:9266 | doi:10.1073/pnas.151221698 | STRC discovery; gene/protein characterisation |
| Verpy et al 2008 Nature 456:255 | doi:10.1038/nature07380 | Stereocilin localisation to OHC stereocilia top |
| Avan, Le Gal et al 2019 PNAS 116:25948 | doi:10.1073/pnas.1902781116 | STRC mouse model; horizontal-top-connector phenotype |
| Verpy et al 2011 J Comp Neurol 519:194 | doi:10.1002/cne.22509 | Stereocilin-OTOG/OTOGL co-localisation |
| Kammerer et al 2012 PNAS 109:7160 | doi:10.1073/pnas.1121491109 | CEACAM16 stereocilin interaction (tip-link assembly) |
| Lukashkin et al 2012 PNAS 109:19351 | doi:10.1073/pnas.1210159109 | CEACAM16 KO; tectorial-membrane phenotype |
E. ZP-like domain folding and ECM cross-linking — context for E1659 site
| Reference | DOI / PMID | What it gives H01 |
|---|---|---|
| Bork & Sander 1992 FEBS Lett 300:237 | doi:10.1016/0014-5793(92)80853-9 | ZP-like domain definition |
| Jovine, Qi, Williams & Wassarman 2002 Nat Cell Biol 4:457 | doi:10.1038/ncb802 | ZP polymerisation mechanism |
| Han, Monnard, Wassarman, Williams & Jovine 2010 Cell 143:404 | doi:10.1016/j.cell.2010.09.041 | ZP-N + ZP-C dimer crystal structure |
The ZP-like context matters because E1659 sits in the C-terminal ZP-like domain of stereocilin (per 8 K1141 interface map). ZP-like polymerisation mechanism is field-known (Jovine 2002; Han 2010); whether STRC ZP-like polymerises into a tectorial sub-fibril or simply binds α/β-tectorin is published but not yet ingested into vault.
Operational interpretation for H01 right now
Pending retrieval of the references above:
- Use F508del 30-min ERAD t½ as the working τ-floor for τRAMD design (Phase 5m-equivalent on the K1141 fragment). Lead must show relative residence time ≥ 30 min vs a fast-dissociating reference; geometric mean target τ-floor of 60 min holds.
- Stereocilin is highly likely a calnexin substrate by domain-architecture analogy (multiple N-glycosylation sites + ZP-like + secreted ECM glycoprotein class). H01 paper §3 can defensibly state “stereocilin is predicted to engage the calnexin/calreticulin cycle as a glycoprotein QC client” with the field-knowledge caveat. Direct measurement (anti-calnexin co-IP from iPSC otic-organoid lysate) is the wet-lab closure, not the compute closure.
- “Rate-limiting step on E1659A QC” most likely candidate: extended calnexin retention → eventual EDEM-mediated mannose trimming → Hrd1/Sel1L recognition. The K1141 binding likely intercepts the mannose-trimming-clock step (Hosokawa 2001 / Christianson 2008 paradigm) by lowering the mutant fold’s ΔG enough that it re-enters the calnexin cycle for another folding attempt before the EDEM clock fires. This is a hypothesis, not a measurement.
Companion compute follow-on
Once F508del CFTR analog accepted as proxy:
- Phase 5m-lite (τRAMD on K1141 fragment). ~10-20 replicas × 5-10 ns per ligand on the STRC K1141 Fragment Construct Strategy system once Phase 5l (fragment AF3 + MD ladder) closes. Output: relative residence-time ranking on v5.2 / v5.3 shortlist with explicit τ-floor benchmarking against a known-fast reference (e.g., diflunisal τ_RAMD).
- Phase 5n-lite (calnexin/CRT MSA scan). Project published calnexin-binding consensus motifs onto stereocilin sequence; identify likely glycoprotein-QC-cycle entry points. Pure literature/sequence task; ~1 hr.
Connections
[part-of]h01 hub[refines]Tafamidis Kinetic-Stabilization Paradigm[refines]Pharmacochaperone Residence Time Criterion[depends-on]STRC K1141 Fragment Construct Strategy[supports]STRC Paper Draft Outline 2026-04-25 §3[about]DFNB16 Hearing Loss