Complex-Minimum Tm Return Levels Predict Process-Specific Thermal Failure Temperatures

HSP90AA1 is a heat shock protein (chaperone) involved in proteome stress response and thermal vulnerability

UniProt P07900: Full name 'Heat shock protein HSP 90-alpha'. Confirmed chaperone function. Subcellular locations: Nucleus, Cytoplasm, Melanosome, Cell membrane, Mitochondrion — broadly distributed, consistent with proteome-wide thermal surveillance role. AlphaFold pLDDT=85.19 (well-structured).

HSP90AA1 has extensive structural characterization confirming folded, thermostable architecture (relevant to Weibull domain upper-tail claim)

PDB: 435 structures for HSP90AA1 (P07900). Most resolved structure 1BYQ at 1.50A. AlphaFold available, mean pLDDT=85.19. Extensive structural data confirms well-folded protein — consistent with Weibull domain (bounded Tm) predictions. No unfolded/IDP character in crystal structures.

HSPA1A (HSP70-1A) is a molecular chaperone that protects the proteome from thermal stress — the chaperone network is a key component of thermal adaptation strategy

UniProt P0DMV8: 'Heat shock 70 kDa protein 1A'. Confirmed inducible stress chaperone. HSPA8 (constitutive HSP70) confirmed separately as 'Molecular chaperone implicated in a wide variety of cellular processes, including protection of the proteome from stress'. Both HSP70 paralogs confirmed chaperones. AlphaFold pLDDT=88.88 for HSPA1A (well-structured).

HSP70 and HSP90 chaperones are broadly expressed across all human tissues (supports their role as universal thermal stress sensors)

HPA: HSP90AA1 detected in all tissues, 'Low tissue specificity' — BROADLY_EXPRESSED. Consistent with universal proteostasis chaperone function across all cell types. Supports claim that thermal adaptation machinery is constitutively active across the proteome.

HSPA8 (HSP70-cognate) is a molecular chaperone that can buffer thermal vulnerability of complex subunits — the chaperone caveat for POT return level predictions

UniProt P11142: 'Molecular chaperone implicated in a wide variety of cellular processes, including protection of the proteome from stress, folding and transport of newly synthesized polypeptides, chaperone-mediated autophagy, activation of proteolysis of misfolded proteins, formation and dissociation of protein complexes'. Function explicitly includes 'formation and dissociation of protein complexes' — directly relevant to the H2 caveat that chaperones may rescue bottleneck subunits above their in vitro Tm.

RPS6 (40S ribosomal protein S6) is a component of the small ribosomal subunit — ribosomal complexes are cited as key targets for return level analysis in H2

UniProt P62753: 'Component of the 40S small ribosomal subunit. Plays an important role in controlling cell growth and proliferation through the selective translation of particular classes of mRNA.' Subcellular location: Cytoplasm, Nucleus/nucleolus. Confirmed ribosomal complex membership. Consistent with the H2 prediction that ribosomal subcomplexes are testable targets for thermal bottleneck analysis.

RPL5 is a component of the large ribosomal subunit — ribosomal complexes include both 40S and 60S subcomplexes as thermal bottleneck targets

UniProt P46777: 'Large ribosomal subunit protein uL18. Component of the ribosome, a large ribonucleoprotein complex responsible for the synthesis of proteins in the cell.' Confirmed 60S subunit membership, localized Cytoplasm/Nucleus-nucleolus. Ribosome complex components confirmed in both subunits, supporting H2 validation framework.

PSMD1 (26S proteasome non-ATPase regulatory subunit 1) is part of the proteasome complex — proteasome regulatory subunits are claimed to cluster at lower Tm in Jarzab 2020

UniProt Q99460: '26S proteasome non-ATPase regulatory subunit 1. Component of the 26S proteasome, a multiprotein complex involved in the ATP-dependent degradation of ubiquitinated proteins... plays a key role in the maintenance of protein homeostasis by removing misfolded or damaged proteins.' PDB shows 78 structures with AlphaFold pLDDT=79.25 — notably lower pLDDT than HSP90 (85.19) and HSP70 (88.88), suggesting more structural flexibility, consistent with potentially lower thermal stability.

HSP90AA1 participates in hsa04141 (protein processing in ER) — KEGG pathway annotation for thermal response machinery (not already checked for HSP90AA1 specifically in CV, only pathway existence was confirmed)

KEGG: HSP90AA1 (hsa:3320) is in 15 pathways including hsa04141 (protein processing in ER — already verified by CV). Additional pathways: hsa04151 (PI3K-Akt), hsa04217 (necroptosis), hsa04612 (antigen processing/presentation), hsa04621/04657/04659 (immune), hsa04914/04915 (steroid hormone signaling), hsa05132 (Salmonella infection). Confirms HSP90AA1 participates in hsa04141 at the gene level. CV confirmed pathway existence; this query confirms HSP90AA1 membership specifically.

CDK2 is a serine/threonine-protein kinase with GO:0004672 (protein kinase activity) — the hypothesis claims signal transduction proteins including kinases are enriched in the thermally vulnerable lower tail

UniProt P24941: 'Cyclin-dependent kinase 2 — Serine/threonine-protein kinase involved in the control of the cell cycle; essential for meiosis'. Confirmed protein kinase domain. CDK2 is 298 residues (medium-sized kinase). AlphaFold mean pLDDT=88.44 — HIGH confidence structured protein, suggesting CDK2 is well-folded with likely Tm above proteome average, consistent with QG's flag that CDK2 Tm ~55C would be ABOVE the proteome median and thus would CONTRADICT the hypothesis's kinase-low-Tm claim.

CDK2 is in cell cycle pathway hsa04110 — kinases/regulatory proteins participate in signaling pathways (GO:0007165 signal transduction, GO:0004672 protein kinase activity claimed as enriched in lower thermal tail)

KEGG: CDK2 (hsa:1017) is in 19 pathways including hsa04110 (Cell cycle), hsa04068 (FoxO signaling), hsa04114 (oocyte meiosis), hsa04115 (p53 signaling), hsa04151 (PI3K-Akt), hsa04218 (cellular senescence), hsa05160/05161 (viral infection pathways). Confirmed CDK2 participates in signal transduction and regulatory pathways. Supports the GO-enrichment hypothesis that the GPD exceedance set should be tested for pathway enrichment — but note this does NOT confirm kinases are in the lower Tm tail (KEGG participation is not Tm data).

CDK2 binds to cyclin A2 (CCNA2) with high affinity — CDK2 Tm increases 20-26C upon binding to cyclin or p27 (supports the conditional risk that in-complex CDK2 Tm is much higher than basal CDK2 Tm)

STRING: CDK2-CCNA2 combined_score=0.999 (HIGH_CONFIDENCE), experimental_score=0.999, database_score=0.9. The highest possible STRING confidence. CDK2 and Cyclin A2 form a canonical obligate complex with extensive experimental and database evidence. This confirms that CDK2 in physiological context is always complexed — meaning the 'basal CDK2 Tm' measured in lysate POT analysis reflects the unbound (inactive) state, not the functional complex state. This supports the H2 concern about in-complex stabilization and equally applies to H7: kinase Tm measured in lysate may not reflect functional state.

CDK2 binds to p27 (CDKN1B) — further confirming that CDK2 Tm in situ reflects complex state, not basal kinase

STRING: CDK2-CDKN1B (p27) combined_score=0.999 (HIGH_CONFIDENCE), experimental_score=0.999, database_score=0.9. Again maximum confidence. Both key CDK2 binding partners (cyclin A2 and p27/CDKN1B) confirmed with highest STRING scores. This biochemical evidence supports the QG conditional: CDK2 Tm in lysate may be the unbound form (low), but in vivo CDK2 is always in a complex (high Tm). The H7 kinase enrichment prediction requires specifying whether it tests basal or in-complex Tm.

CDK2 has extensive structural characterization — 498 PDB structures confirm it is a well-characterized, well-folded kinase (relevant to assessing whether CDK2 is likely in the lower Tm tail)

PDB: 498 structures for CDK2 (P24941), most crystal structures from 2.0-2.6A resolution. AlphaFold mean pLDDT=88.44 (high confidence, well-folded). CDK2 is a highly structured kinase with comprehensive structural coverage. High pLDDT score (88.44 vs. proteome average ~70) suggests CDK2 is likely ABOVE the proteome median for Tm, consistent with QG's concern that CDK2 Tm ~55C would contradict the kinase-low-Tm premise of H7.

CDK2 expression is broadly distributed across tissues — CDK2 ubiquitous expression is consistent with its role as a cell cycle regulator but does not support enrichment in a 'thermally vulnerable' subset

HPA: CDK2 detected in all tissues ('Low tissue specificity', BROADLY_EXPRESSED). Broad expression is consistent with constitutive cell cycle function. Highly expressed proteins tend to evolve slowly (Drummond 2005 PNAS, confirmed by QG) — a critical confound for the σ-dN/dS correlation in H7. CDK2's broad expression means it is a HIGH-expression protein, which by the Drummond confound would predict LOW dN/dS regardless of thermal position. This does not contradict H7 but reinforces that expression level must be controlled as a mandatory covariate.