title: ANKRD1 specifically binds CASQ2 in heart extracts and both proteins are co-enriched in piglet cardiac Purkinje cells.
abstract: It has been suggested that the cardiac ankyrin repeat domain 1 protein (ANKRD1), also known as CARP, can play a pathophysiological role in the contractile responsiveness of myocardium. Here, we study the potential functional roles of ANKRD1 by searching for endogenous cardiac proteins that interact preferentially with ANKRD1 in the heart-tissue extract from neonatal piglets, using non-biased pull-down approaches. These approaches identified, for the first time, a selective interaction between ANKRD1 and endogenous cardiac calsequestrin-2 (CASQ2) that is important for Ca2+ release and excitation-contraction coupling. Blot-overlay and co-immunoprecipitation assays provided further confirmation of the direct and specific interaction between the two proteins. Mapping of the peptides involved in the interaction revealed five non-overlapping binding sequences for CASQ2 on ANKRD1, as well as, three binding peptides for ANKRD1 in CASQ2. For the first time, we show by immunohistochemistry that endogenous ANKRD1 and CASQ2 are co-enriched in piglet cardiac Purkinje cells. Collectively, the results provide the first sing of a possible functional interaction between ANKRD1 and CASQ2 and suggest a potentially novel role for both proteins in cardiac Purkinje fibers.
authors.affiliation: Developmental Biology Unit, Institute of Health Sciences, University of La Coruña, Campus de Oza, Building El Fortín, As Xubias s/n, 15006 La Coruña, Spain.
authors.forename: Mario
authors.initials: M
authors.lastname: Torrado
authors.forename: Beatriz
authors.initials: B
authors.lastname: Nespereira
authors.forename: Eduardo
authors.initials: E
authors.lastname: López
authors.forename: Alberto
authors.initials: A
authors.lastname: Centeno
authors.forename: Alfonso
authors.initials: A
authors.lastname: Castro-Beiras
authors.forename: Alexander T
authors.initials: AT
authors.lastname: Mikhailov
mesh_terms: D000375:Aging / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002155:Calsequestrin / Q000737:chemistry / Q000378:metabolism*; D002457:Cell Extracts; D004285:Dogs; D006321:Heart / Q000187:drug effects; D006801:Humans; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011689:Purkinje Cells / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine; D014020:Tissue Extracts
chemical_list: D002155:Calsequestrin; D002457:Cell Extracts; D009687:Nuclear Proteins; D012097:Repressor Proteins; D014020:Tissue Extracts; D002118:Calcium