HAX-1-Mediated Autophagy Modulation Involves N-Terminal LC3-Interacting Motifs

HS-1-associated protein X-1 (HAX-1) is a ubiquitously expressed, multifunctional protein that regulates Ca2+ homeostasis and cell survival in cardiac muscle. In addition to its well-established anti-apoptotic function, HAX-1 has recently been implicated in autophagy regulation. In the present study, we explored the molecular mechanisms underlying HAX-1-mediated autophagy modulation in cellular models, including cardiac-derived H9c2 myotubes. HAX-1 overexpression enhanced autophagic activity, as evidenced by decreased sequestosome-1 (p62), increased microtubule-associated protein light chain 3-II (LC3-II), enhanced LC3 puncta formation, and elevated autophagic flux. Conversely, HAX-1 knockdown attenuated autophagic activity. Mechanistically, co-immunoprecipitation assays showed that HAX-1 associates with both p62 and LC3. Bioinformatic analysis of the HAX-1 protein sequence identified two conserved LC3-interacting region (LIR) motifs within its N-terminal domain. Deletion of this LIR-containing region (HAX-1ΔLIR) reduced LC3 association, decreased autophagic activity, and impaired autophagy-dependent clearance of HAX-1 itself following autophagy induction, suggesting the importance of these motifs. At a cardiac-relevant level, HAX-1 promoted a chloroquine-sensitive reduction in protein levels of its known binding partner, phospholamban (PLN), a key regulator of sarcoplasmic reticulum (SR) Ca2+ cycling. These findings indicate a previously unrecognized LC3/LIR-dependent mechanism underlying HAX-1-mediated autophagy modulation and suggest a potential role for HAX-1 in SR protein proteostasis.