HSPA4 (Apg-2) is a cytosolic member of the Hsp110 branch of the Hsp70 superfamily. Hsp110 proteins have an Hsp70-like N-terminal nucleotide-binding domain, a beta-sandwich substrate-binding domain with an acidic insertion, and an extended alpha-helical C-terminal region. Rather than acting mainly as ATP-driven folding chaperones in their own right, they act as nucleotide-exchange factors for cytosolic Hsp70s such as Hsc70 (HSPA8), releasing ADP so that a new Hsp70 cycle can start. Through this activity HSPA4 lets Hsc70 and J-domain proteins such as DNAJB1 disaggregate and refold aggregated proteins. The exchange activity depends on concentration, and the acidic subdomain and the C-terminal extension tune how tightly HSPA4 binds Hsc70. The protein described here, A0A9L0S5Z5, is the product of an alternative horse transcript (Ensembl ENSECAT00000106098, 800 aa) of the HSPA4 gene. It skips one 120-nt coding exon and so lacks a 40-residue segment of the conserved C-terminal helical region, corresponding to human residues 679-718. The full-length 840-aa product of the same gene is F6YZJ4, from the Ensembl canonical transcript.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005524 ATP binding | IEA GO_REF:0000002 | ACCEPT | Summary: ATP binding by the Hsp70-like nucleotide-binding domain, which this horse model retains in full. In Hsp110 proteins, ATP binding is what allows the protein to work as a nucleotide-exchange factor. Reason: A0A9L0S5Z5 belongs to PANTHER PTHR45639:SF6 (HSPA4 orthologs), together with human P34932. Its N-terminal nucleotide-binding domain matches the human sequence in full; the only segment missing is in the C-terminal helical region (RESULTS-rereview.md). In the Hsp110 family, exchange activity needs a working ATP-binding domain (Sse1p G223D, Dragovic et al. 2006). ATP-bound Hsp110 is the form that engages Hsp70 (Cabrera et al. 2019). ATP binding is therefore part of the core exchange mechanism, not a side activity. The InterPro2GO mapping is consistent with this. Supporting Evidence: PMID:16688212 Surprisingly, the release rate of MABA-ADP was reduced more than 95% relative to wild-type (wt) Sse1p in the presence of Sse1p-G223D, suggesting that the nucleotide exchange activity of Sse1p requires a functional ATP binding domain. PMID:30521813 Our data support a mechanism in which Apg2 arrests the chaperone cycle through an interaction with Hsc70(ATP) that might lead to premature ATP dissociation before hydrolysis. file:HORSE/HSPA4/HSPA4-bioinformatics/RESULTS-rereview.md | A0A9L0S5Z5 | 800 | 800 | 0.97 | [[679, 718]] | |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | MODIFY | Summary: The location is correct but too general. Apg2/HSPA4 is one of the cytosolic Hsp110 proteins, and the more precise term is cytosol (GO:0005829). Human HSPA4 carries cytosol by IDA and by IBA. Reason: Cytoplasm is true but broad. Apg2 is described as one of the three cytosolic Hsp110 chaperones in humans. It works with the cytosolic Hsc70/HSPA8 machinery, and PAINT node PTN000452854, above the HSPA4 subfamily, carries cytosol. The horse protein is in the same PANTHER subfamily (SF6) and has no targeting signal (no signal peptide or transmembrane segment is annotated), so cytosol is the supported specific term. The missing C-terminal exon segment does not add or remove a targeting signal. Proposed replacements: cytosol Supporting Evidence: PMID:36167183 Apg2, one of the three cytosolic Hsp110 chaperones in humans, supports reactivation of unordered and ordered protein aggregates by Hsc70 (HspA8). PMID:16688212 Various cytosolic Hsp110 isoforms have been described including Hsp110 (also known as Hsp105), Apg-1, and Apg-2 in mammalian cytosol |
| GO:0016887 ATP hydrolysis activity | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: This is a family-wide InterPro2GO transfer of the catalytic ATPase of conventional Hsp70s. In Hsp110 proteins, including Apg2, the biologically relevant activity is nucleotide exchange on Hsc70, not ATP turnover by the protein itself. Reason: IPR013126 covers the whole Hsp70 superfamily, so this mapping assigns the canonical Hsp70 ATPase to every member. In PAINT, ATP hydrolysis activity (GO:0016887) is placed at node PTN000452648. Its seeds are conventional Hsp70s (DnaK, HSPA1A/B, HSPA8, HSPA5, Ssa/Ssb/Kar2, mortalin), and no Hsp110 protein is among them. Human HSPA4 in SF6 gets no IBA for this term, only the same IEA mapping. The PAINT HSPA4 IBA set (exchange factor activity, cytosol, nucleus, protein folding) leaves ATP hydrolysis out, and we treat that omission as a deliberate decision. Experimentally, the Hsp110 contribution to disaggregation depends on nucleotide exchange and not on ATPase activity. The cited Apg2 work measures how Apg2 changes the ATPase cycle of Hsc70; it does not show that Apg2 hydrolyses ATP itself. The weak intrinsic ATPase of Hsp110s has not been shown for HSPA4, so the term is not certainly false, but it misstates the protein's function. Supporting Evidence: PMID:22990239 This Hsp110 effect relies on nucleotide exchange, not on ATPase activity, implying ATP-driven chaperoning is not required. PMID:30521813 Apg2 shows a biphasic behavior: at low concentration, it stimulates the ATPase cycle of Hsc70 |
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Download this section (compressed HTML)Q: Is the 800-aa horse transcript ENSECAT00000106098, which skips the exon encoding human HSPA4 residues 679-718, supported by full-length transcript evidence? Does the encoded protein keep Hsc70 nucleotide-exchange activity and the ability to support disaggregation?
Experiment: Express full-length horse HSPA4 (F6YZJ4) and the exon-skipped form (A0A9L0S5Z5). Compare their stimulation of Hsc70 ADP release, Hsc70 ATPase turnover, and Hsc70/DNAJB1-dependent refolding of aggregated luciferase, across a range of concentrations.
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