{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T08:34:17Z","timestamp":1786696457317,"version":"build-2736575974"},"reference-count":44,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T00:00:00Z","timestamp":1786665600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>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-1\u0394LIR) 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.<\/jats:p>","DOI":"10.3390\/cells15161459","type":"journal-article","created":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T08:08:29Z","timestamp":1786694909000},"page":"1459","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["HAX-1-Mediated Autophagy Modulation Involves N-Terminal LC3-Interacting Motifs"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9931-7099","authenticated-orcid":false,"given":"Elizabeth","family":"Vafiadaki","sequence":"first","affiliation":[{"name":"Center of Basic Research, Biomedical Research Foundation, Academy of Athens, 11527 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Panagiotis","family":"Papadopoulos","sequence":"additional","affiliation":[{"name":"Center of Basic Research, Biomedical Research Foundation, Academy of Athens, 11527 Athens, Greece"},{"name":"Clinical Genomics and Pharmacogenomics Unit, and Genetic and Pharmacogenetic Counseling Clinic, 4th Department of Internal Medicine, \u201cAttikon\u201d Hospital, Medical School, National and Kapodistrian University of Athens, 11527 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6403-6761","authenticated-orcid":false,"given":"Aristides G.","family":"Eliopoulos","sequence":"additional","affiliation":[{"name":"Department of Biology, Medical School, National and Kapodistrian University of Athens, 11527 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3704-1941","authenticated-orcid":false,"given":"Despina","family":"Sanoudou","sequence":"additional","affiliation":[{"name":"Center of Basic Research, Biomedical Research Foundation, Academy of Athens, 11527 Athens, Greece"},{"name":"Clinical Genomics and Pharmacogenomics Unit, and Genetic and Pharmacogenetic Counseling Clinic, 4th Department of Internal Medicine, \u201cAttikon\u201d Hospital, Medical School, National and Kapodistrian University of Athens, 11527 Athens, Greece"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2736","DOI":"10.4049\/jimmunol.158.6.2736","article-title":"HAX-1, a Novel Intracellular Protein, Localized on Mitochondria, Directly Associates with HS1, a Substrate of Src Family of Tyrosine Kinases","volume":"158","author":"Suzuki","year":"1997","journal-title":"J. Immunol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1139","DOI":"10.1016\/j.bbagen.2009.06.004","article-title":"HAX-1: A multifunctional protein with emerging roles in human disease","volume":"1790","author":"Fadeel","year":"2009","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"119538","DOI":"10.1016\/j.bbamcr.2023.119538","article-title":"HAX1: A versatile, intrinsically disordered regulatory protein","volume":"1870","author":"Wakula","year":"2023","journal-title":"Biochim. Biophys. Acta Mol. Cell Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1016\/j.jmb.2006.10.057","article-title":"Phospholamban interacts with HAX-1, a mitochondrial protein with anti-apoptotic function","volume":"367","author":"Vafiadaki","year":"2007","journal-title":"J. Mol. Biol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1266","DOI":"10.1016\/j.yjmcc.2009.10.028","article-title":"HAX-1: A multifaceted antiapoptotic protein localizing in the mitochondria and the sarcoplasmic reticulum of striated muscle cells","volume":"48","author":"Yap","year":"2010","journal-title":"J. Mol. Cell. Cardiol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"20776","DOI":"10.1073\/pnas.0906998106","article-title":"The anti-apoptotic protein HAX-1 is a regulator of cardiac function","volume":"106","author":"Zhao","year":"2009","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"566","DOI":"10.1038\/nrm1151","article-title":"Phospholamban: A crucial regulator of cardiac contractility","volume":"4","author":"MacLennan","year":"2003","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"306","DOI":"10.1091\/mbc.e08-06-0587","article-title":"The anti-apoptotic protein HAX-1 interacts with SERCA2 and regulates its protein levels to promote cell survival","volume":"20","author":"Vafiadaki","year":"2009","journal-title":"Mol. Biol. Cell"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"359","DOI":"10.1074\/jbc.RA117.000128","article-title":"The antiapoptotic protein HAX-1 mediates half of phospholamban\u2019s inhibitory activity on calcium cycling and contractility in the heart","volume":"293","author":"Bidwell","year":"2018","journal-title":"J. Biol. Chem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"415","DOI":"10.1161\/01.RES.0000237387.05259.a5","article-title":"Overexpression of HAX-1 protects cardiac myocytes from apoptosis through caspase-9 inhibition","volume":"99","author":"Han","year":"2006","journal-title":"Circ. Res."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1161\/CIRCRESAHA.112.279935","article-title":"Novel role of HAX-1 in ischemic injury protection involvement of heat shock protein 90","volume":"112","author":"Lam","year":"2013","journal-title":"Circ. Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"432","DOI":"10.1089\/dna.2017.3873","article-title":"Identification of the Functional Autophagy-Regulatory Domain in HCLS1-Associated Protein X-1 That Resists Against Oxidative Stress","volume":"37","author":"Li","year":"2018","journal-title":"DNA Cell Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"594","DOI":"10.1038\/s41419-026-08805-y","article-title":"Granulocyte differentiation arrest in HAX1-deficient cells, demonstrated in a new in vitro model of a certain phenotypic aspects of Kostmann disease, is caused by ineffective lipid droplet autophagy and fatty acids uptake","volume":"17","author":"Wakula","year":"2026","journal-title":"Cell Death Dis."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1007\/s00018-025-05604-0","article-title":"IGF2BP1-HAX-1 positive feedback loop-mediated HAX-1 overexpression blocks autophagic flux and promotes chemoresistance in nasopharyngeal carcinoma","volume":"82","author":"Zhang","year":"2025","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1080\/15548627.2020.1797280","article-title":"Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1","volume":"17","author":"Klionsky","year":"2021","journal-title":"Autophagy"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1812","DOI":"10.1161\/CIRCRESAHA.117.311082","article-title":"Autophagy and Mitophagy in Cardiovascular Disease","volume":"120","author":"Kroemer","year":"2017","journal-title":"Circ. Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"934","DOI":"10.1093\/cvr\/cvab158","article-title":"The role of autophagy in cardiovascular pathology","volume":"118","author":"Gatica","year":"2022","journal-title":"Cardiovasc. Res."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2299","DOI":"10.1093\/cvr\/cvaf203","article-title":"Recent progress regarding the role of autophagy in cardiac disease","volume":"121","author":"Maejima","year":"2025","journal-title":"Cardiovasc. Res."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Santovito, D., Steffens, S., Barachini, S., and Madonna, R. (2023). Autophagy, innate immunity, and cardiac disease. Front. Cell Dev. Biol., 11.","DOI":"10.3389\/fcell.2023.1149409"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"118432","DOI":"10.1016\/j.bbamcr.2019.01.013","article-title":"Autophagy in cardiomyopathies","volume":"1867","author":"Zech","year":"2020","journal-title":"Biochim. Biophys. Acta Mol. Cell Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"e55192","DOI":"10.15252\/embr.202255192","article-title":"ER-phagy: Selective autophagy of the endoplasmic reticulum","volume":"23","author":"Mochida","year":"2022","journal-title":"EMBO Rep."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"42","DOI":"10.1007\/s00395-023-01009-x","article-title":"Mitophagy for cardioprotection","volume":"118","author":"Titus","year":"2023","journal-title":"Basic Res. Cardiol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"450","DOI":"10.1007\/s00018-024-05471-1","article-title":"The phospholamban R14del generates pathogenic aggregates by impairing autophagosome-lysosome fusion","volume":"81","author":"Vafiadaki","year":"2024","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Vafiadaki, E., Haghighi, K., Arvanitis, D.A., Kranias, E.G., and Sanoudou, D. (2022). Aberrant PLN-R14del Protein Interactions Intensify SERCA2a Inhibition, Driving Impaired Ca2+ Handling and Arrhythmogenesis. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23136947"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"671","DOI":"10.1038\/nmeth.2089","article-title":"NIH Image to ImageJ: 25 years of image analysis","volume":"9","author":"Schneider","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Yoshii, S.R., and Mizushima, N. (2017). Monitoring and Measuring Autophagy. Int. J. Mol. Sci., 18.","DOI":"10.3390\/ijms18091865"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1007\/978-1-59745-157-4_4","article-title":"LC3 and Autophagy","volume":"445","author":"Tanida","year":"2008","journal-title":"Methods Mol. Biol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1435","DOI":"10.1080\/15548627.2018.1474314","article-title":"Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion","volume":"14","author":"Mauthe","year":"2018","journal-title":"Autophagy"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"BSR20203094","DOI":"10.1042\/BSR20203094","article-title":"The interactome of multifunctional HAX1 protein suggests its role in the regulation of energy metabolism, de-aggregation, cytoskeleton organization and RNA-processing","volume":"40","author":"Wakula","year":"2020","journal-title":"Biosci. Rep."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"80","DOI":"10.1016\/j.jmb.2019.07.016","article-title":"Selective Autophagy: ATG8 Family Proteins, LIR Motifs and Cargo Receptors","volume":"432","author":"Johansen","year":"2020","journal-title":"J. Mol. Biol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"268","DOI":"10.1016\/j.molcel.2019.09.005","article-title":"A Diversity of Selective Autophagy Receptors Determines the Specificity of the Autophagy Pathway","volume":"76","author":"Kirkin","year":"2019","journal-title":"Mol. Cell"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1146\/annurev-cellbio-120219-035530","article-title":"Mechanisms of Selective Autophagy","volume":"37","author":"Lamark","year":"2021","journal-title":"Annu. Rev. Cell Dev. Biol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"7165","DOI":"10.1073\/pnas.1508815112","article-title":"Metformin increases degradation of phospholamban via autophagy in cardiomyocytes","volume":"112","author":"Teng","year":"2015","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Eijgenraam, T.R., Stege, N.M., Oliveira Nunes Teixeira, V., de Brouwer, R., Schouten, E.M., Grote Beverborg, N., Sun, L., Spater, D., Knoll, R., and Hansson, K.M. (2022). Antisense Therapy Attenuates Phospholamban p. (Arg14del) Cardiomyopathy in Mice and Reverses Protein Aggregation. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23052427"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"19094","DOI":"10.1074\/jbc.M111.322933","article-title":"Microtubule-associated protein 1 light chain 3 (LC3) interacts with Bnip3 protein to selectively remove endoplasmic reticulum and mitochondria via autophagy","volume":"287","author":"Hanna","year":"2012","journal-title":"J. Biol. Chem."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"7527","DOI":"10.1038\/ncomms8527","article-title":"Bcl-2-like protein 13 is a mammalian Atg32 homologue that mediates mitophagy and mitochondrial fragmentation","volume":"6","author":"Murakawa","year":"2015","journal-title":"Nat. Commun."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1038\/embor.2009.256","article-title":"Nix is a selective autophagy receptor for mitochondrial clearance","volume":"11","author":"Novak","year":"2010","journal-title":"EMBO Rep."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"600","DOI":"10.4161\/auto.6260","article-title":"Bcl-2 family members: Dual regulators of apoptosis and autophagy","volume":"4","author":"Levine","year":"2008","journal-title":"Autophagy"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"489","DOI":"10.1161\/CIRCRESAHA.116.303791","article-title":"Therapeutic targeting of autophagy: Potential and concerns in treating cardiovascular disease","volume":"116","author":"Orogo","year":"2015","journal-title":"Circ. Res."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"671","DOI":"10.1016\/j.jaccao.2023.08.004","article-title":"ER-Specific Autophagy or ER-Phagy in Cardiac Myocytes Protects the Heart Against Doxorubicin-Induced Cardiotoxicity","volume":"5","author":"Glembotski","year":"2023","journal-title":"JACC CardioOncol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1006","DOI":"10.1161\/CIRCRESAHA.123.323304","article-title":"DWORF Extends Life Span in a PLN-R14del Cardiomyopathy Mouse Model by Reducing Abnormal Sarcoplasmic Reticulum Clusters","volume":"133","author":"Stege","year":"2023","journal-title":"Circ. Res."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"E6466\u201375","DOI":"10.1073\/pnas.1508760112","article-title":"HAX-1 regulates cyclophilin-D levels and mitochondria permeability transition pore in the heart","volume":"112","author":"Lam","year":"2015","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"220","DOI":"10.1016\/j.yjmcc.2017.11.014","article-title":"HAX-1 regulates SERCA2a oxidation and degradation","volume":"114","author":"Bidwell","year":"2018","journal-title":"J. Mol. Cell. Cardiol."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Yokoe, S., and Asahi, M. (2017). Phospholamban Is Downregulated by pVHL-Mediated Degradation through Oxidative Stress in Failing Heart. Int. J. Mol. Sci., 18.","DOI":"10.3390\/ijms18112232"}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/16\/1459\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T08:23:48Z","timestamp":1786695828000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/16\/1459"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,14]]},"references-count":44,"journal-issue":{"issue":"16","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["cells15161459"],"URL":"https:\/\/doi.org\/10.3390\/cells15161459","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,14]]}}}