HSP70CA2 is associated with cytoskeletal, membrane repair, and chaperone responses during acute heat shock in Urechis unicinctus.

Heat shock proteins (HSPs) are essential for cellular homeostasis and thermal adaptation, yet their interaction networks and regulatory mechanisms under high temperature stress remain largely unexplored in aquatic organisms. Here, the expression profiles of HSPs in Urechis unicinctus under high temperature stress were systematically characterized. Among 85 identified HSPs, a member of the HSP70 family, HSP70CA2, was selected for further study due to its sustained and significant differential expression. Sequence analysis revealed that HSP70CA2 has an open reading frame (ORF) of 1917 bp, encoding a 638 amino acid protein. Western blotting confirmed a rapid and significant increase in HSP70CA2 protein levels in both body wall and midgut tissues during early high temperature stress, indicating its active involvement in the initial stress response. Crucially, five novel interacting proteins of HSP70CA2 were screened and identified as cytoskeletal (CIFP), membrane repair (ANXA7), ER-associated (CRT-like), and chaperone (HSP90A1, HSPA8) factors by pull-down assays coupled with LC-MS. These interactions were validated by point to point yeast two-hybrid assays. Our findings suggest that HSP70CA2 acts as an early chaperone related responder associated with cytoskeletal maintenance, membrane repair, and protein folding processes during the acute heat shock response in U. unicinctus. This study provides new evidence for the involvement of a single HSP70 member in multiple cellular protective processes in an aquatic invertebrate, offering fundamental insights into thermal stress adaptation and a potential target for thermotolerance breeding.