{"doi":"10.1101/2020.09.18.303644","title":"The transmembrane domain of DWORF activates SERCA directly; P15 and W22 residues are essential","abstract":"Abstract The Ca-ATPase isoform 2a (SERCA2a) re-sequesters cytosolic Ca 2+ into the sarcoplasmic reticulum (SR) of cardiac myocytes, enabling muscle relaxation during diastole. A central factor in heart failure is abnormally high cytosolic [Ca 2+ ], resulting in pathophysiology and decreased cardiac performance. Therefore, augmentation of the SERCA2a Ca 2+ transport activity is a promising therapeutic approach. A novel transmembrane peptide, dwarf open reading frame (DWORF), is proposed to enhance SR Ca 2+ uptake and myocyte contractility by displacing the protein phospholamban (PLB) from its inhibitory site on SERCA2a. In the present study, we have developed several cell-based FRET biosensor systems for time-resolved FRET (TR-FRET) measurements of the protein-protein interactions and structural changes in SERCA2a complexes with PLB and/or DWORF. To test the hypothesis that DWORF competes with PLB to occupy the putative SERCA2a binding site, we transiently transfected DWORF into a stable cell line expressing SERCA2a labeled with green fluorescent protein (GFP, the FRET donor) and PLB labeled with red fluorescent protein (RFP, the FRET acceptor). We observed a significant decrease in FRET efficiency, consistent with a decrease in the fraction of SERCA2a bound to PLB. Functional analysis demonstrates that DWORF activates SERCA in both the presence and absence of PLB. Furthermore, using site-directed mutagenesis, we generated DWORF variants that do not activate SERCA, thus identifying residues that are necessary for functional SERCA2a-DWORF interactions. This work advances our mechanistic understanding of the regulation of SERCA2a by small transmembrane proteins and sets the stage for future therapeutic development in heart failure research.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2020,"id":131008,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9573,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":584958,"name":"Daniel R. Stroik","orcid":null,"position":1,"is_corresponding":false},{"id":459047,"name":"Samantha L. Yuen","orcid":"0000-0002-1871-5544","position":2,"is_corresponding":false},{"id":493917,"name":"Evan Kleinboehl","orcid":"0000-0002-5174-1314","position":3,"is_corresponding":false},{"id":417548,"name":"Rǎzvan L. Cornea","orcid":"0000-0001-5739-0992","position":4,"is_corresponding":false},{"id":417547,"name":"David D. Thomas","orcid":"0000-0002-8822-2040","position":5,"is_corresponding":false},{"id":584306,"name":"Ang Li","orcid":"0009-0008-2964-288X","position":0,"is_corresponding":true}],"reference_count":28,"raw_metadata":null,"created_at":"2026-07-18T23:16:00.235845Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}