RESUMEN
As the genetic landscape of cardiomyopathies continues to expand, the identification of missense variants in disease-associated genes frequently leads to a classification of variant of uncertain significance (VUS). For the proper reclassification of such variants, functional characterization is an important contributor to the proper assessment of pathogenic potential. Several missense variants in the calcium transport regulatory protein phospholamban have been associated with dilated cardiomyopathy. However, >40 missense variants in this transmembrane peptide are currently known and most remain classified as VUS with little clinical information. Similarly, missense variants in cardiac myosin binding protein have been associated with hypertrophic cardiomyopathy. However, hundreds of variants are known and many have low penetrance and are often found in control populations. Herein, we focused on novel missense variants in phospholamban, an Ala15-Thr variant found in a 4-year-old female and a Pro21-Thr variant found in a 60-year-old female, both with a family history and clinical diagnosis of dilated cardiomyopathy. The patients also harbored a Val896-Met variant in cardiac myosin binding protein. The phospholamban variants caused defects in the function, phosphorylation, and dephosphorylation of this calcium transport regulatory peptide, and we classified these variants as potentially pathogenic. The variant in cardiac myosin binding protein alters the structure of the protein. While this variant has been classified as benign, it has the potential to be a low-risk susceptibility variant because of the structural change in cardiac myosin binding protein. Our studies provide new biochemical evidence for missense variants previously classified as benign or VUS.
Asunto(s)
Proteínas de Unión al Calcio , Cardiomiopatía Dilatada , Preescolar , Femenino , Humanos , Persona de Mediana Edad , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Cardiomiopatía Dilatada/diagnóstico , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/metabolismo , Péptidos/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismoRESUMEN
The calcium pump (a.k.a. Ca2+-ATPase or SERCA) is a membrane transport protein ubiquitously found in the endoplasmic reticulum (ER) of all eukaryotic cells. As a calcium transporter, SERCA maintains the low cytosolic calcium level that enables a vast array of signaling pathways and physiological processes (e.g. synaptic transmission, muscle contraction, fertilization). In muscle cells, SERCA promotes relaxation by pumping calcium ions from the cytosol into the lumen of the sarcoplasmic reticulum (SR), the main storage compartment for intracellular calcium. X-ray crystallographic studies have provided an extensive understanding of the intermediate states that SERCA populates as it progresses through the calcium transport cycle. Historically, SERCA is also known to be regulated by small transmembrane peptides, phospholamban (PLN) and sarcolipin (SLN). PLN is expressed in cardiac muscle, whereas SLN predominates in skeletal and atrial muscle. These two regulatory subunits play critical roles in cardiac contractility. While our understanding of these regulatory mechanisms are still developing, SERCA and PLN are one of the best understood examples of peptide-transporter regulatory interactions. Nonetheless, SERCA appeared to have only two regulatory subunits, while the related sodium pump (a.k.a. Na+, K+-ATPase) has at least nine small transmembrane peptides that provide tissue specific regulation. The last few years have seen a renaissance in our understanding of SERCA regulatory subunits. First, structures of the SERCA-SLN and SERCA-PLN complexes revealed molecular details of their interactions. Second, an array of micropeptides concealed within long non-coding RNAs have been identified as new SERCA regulators. This chapter will describe our current understanding of SERCA structure, function, and regulation.
Asunto(s)
Señalización del Calcio/fisiología , Calcio , Retículo Endoplásmico , Células Musculares/enzimología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Animales , Calcio/química , Calcio/metabolismo , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Cristalografía por Rayos X , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/genética , Humanos , Proteínas Musculares/química , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteolípidos/química , Proteolípidos/genética , Proteolípidos/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/química , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
The sarcoplasmic reticulum Ca2+-ATPase SERCA promotes muscle relaxation by pumping calcium ions from the cytoplasm into the sarcoplasmic reticulum. SERCA activity is regulated by a variety of small transmembrane peptides, most notably by phospholamban in cardiac muscle and sarcolipin in skeletal muscle. However, how phospholamban and sarcolipin regulate SERCA is not fully understood. In the present study, we evaluated the effects of phospholamban and sarcolipin on calcium translocation and ATP hydrolysis by SERCA under conditions that mimic environments in sarcoplasmic reticulum membranes. For pre-steady-state current measurements, proteoliposomes containing SERCA and phospholamban or sarcolipin were adsorbed to a solid-supported membrane and activated by substrate concentration jumps. We observed that phospholamban altered ATP-dependent calcium translocation by SERCA within the first transport cycle, whereas sarcolipin did not. Using pre-steady-state charge (calcium) translocation and steady-state ATPase activity under substrate conditions (various calcium and/or ATP concentrations) promoting particular conformational states of SERCA, we found that the effect of phospholamban on SERCA depends on substrate preincubation conditions. Our results also indicated that phospholamban can establish an inhibitory interaction with multiple SERCA conformational states with distinct effects on SERCA's kinetic properties. Moreover, we noted multiple modes of interaction between SERCA and phospholamban and observed that once a particular mode of association is engaged it persists throughout the SERCA transport cycle and multiple turnover events. These observations are consistent with conformational memory in the interaction between SERCA and phospholamban, thus providing insights into the physiological role of phospholamban and its regulatory effect on SERCA transport activity.