Arcle Newly Discovered Micropeptide Regulators of SERCA Form Oligomers but Bind to the Pump as Monomers Deo R. Singh 1,y, Michael P. Dalton 1,y, Ellen E. Cho 1, Marsha P. Pribadi 1, 1 1 2 2 Taylor J. Zak , Jaroslava Seflova , Catherine A. Makarewich , Eric N. Olson and Seth L. Robia 1 1 - Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, IL 60153, USA 2 - Department of Molecular Biology and Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA Correspondence to Seth L. Robia: [email protected]. https://doi.org/10.1016/j.jmb.2019.07.037 Edited by Daniel L. Minor Abstract The recently-discovered single-span transmembrane proteins endoregulin (ELN), dwarf open reading frame (DWORF), myoregulin (MLN), and another-regulin (ALN) are reported to bind to the SERCA calcium pump in a manner similar to that of known regulators of SERCA activity, phospholamban (PLB) and sarcolipin (SLN). To determine how micropeptide assembly into oligomers affects the availability of the micropeptide to bind to SERCA in a regulatory complex, we used co-immunoprecipitation and fluorescence resonance energy transfer (FRET) to quantify micropeptide oligomerization and SERCA-binding. Micropeptides formed avid homo-oligomers with high-order stoichiometry (n > 2 protomers per homo-oligomer), but it was the monomeric form of all micropeptides that interacted with SERCA. In view of these two alternative binding interactions, we evaluated the possibility that oligomerization occurs at the expense of SERCA-binding. However, even the most avidly oligomeric micropeptide species still showed robust FRET with SERCA, and there was a surprising positive correlation between oligomerization affinity and SERCA-binding. This comparison of micropeptide family members suggests that the same structural determinants that support oligomerization are also important for binding to SERCA. Moreover, the unique oligomerization/SERCA-binding profile of DWORF is in harmony with its distinct role as a PLB-competing SERCA activator, in contrast to the inhibitory function of the other SERCA-binding micropeptides. © 2019 Elsevier Ltd. All rights reserved. Introduction regulatory “micropeptides”, which interact with the transmembrane domain of SERCA. These include The sarco(endo)plasmic reticulum calcium the well-known SERCA regulatory partners phos- ATPase is the principal transport mechanism for pholamban (PLB) [1] and sarcolipin (SLN) [2], which sequestration of calcium (Ca) in the endoplasmic are inhibitors of SERCA in cardiac and skeletal reticulum (ER). These intracellular Ca stores are the muscle. New additions to this family include dwarf foundation of signals that coordinate diverse cellular open reading frame (DWORF), myoregulin (MLN), functions. For example, in muscle cells, sarcoplas- endoregulin (ELN), expressed in cardiac muscle, mic reticulum (SR) Ca release and reuptake are the skeletal muscle, and endo/epithelial tissues, respec- signals for contraction and relaxation. Different tively [3e5], and another-regulin (ALN), expressed tissues vary in their Ca uptake requirements, and ubiquitously. With these newly identified micropep- differential expression of distinct SERCA isoforms tides, SERCA regulatory complexity is beginning to provides some tissue-specific specialization of Ca resemble that of a related ion transporter, the Naþ/ handling. In addition, the kinetics and Ca sensitivity K þ -ATPase (NKA), which has many tissue-speci- of transport are modulated by single-span proteins, fic, species-specific regulatory peptides [6,7]. The 0022-2836/© 2019 Elsevier Ltd. All rights reserved. Journal of Molecular Biology (2019) 431, 4429e4443 4430 (a) (b) 100 SERCA 80 alone 60 max DWORF SLN % V MLN 40 ELN ALN 20 PLB 0 10-8 10-7 10-6 [Ca2+] (M) (c) +Myc +Myc-SERCA (d) +Myc +Myc-peptides HA Peptide: - - HA peptide: kDa kDa Myc (SERCA) Myc (peptides) -15 -100 Myc IP Myc IP HA (peptides) -10 HA (peptides) -10 Newly Discovered Micropeptide Regulators of SERCA Form Oligomers Myc (peptides) -15 Myc (SERCA) -100 Input -10 Input HA (peptides) -10 HA (peptides) GAPDH -37 GAPDH -37 Fig. 1. Regulation of SERCA by micropeptides. A) Sequence alignment of micropeptides, with key conserved residues highlighted. B) Compared to control (SERCA alone, empty squares, black dotted line), the apparent Ca affinity of transport activity was decreased when SERCA was co-expressed with PLB (black circles, black solid line), SLN (red), ALN (blue), ELN (purple), and MLN (gray). The Ca sensitivity of SERCA co-expressed with DWORF (green) was not significantly decreased. C) and D) Pull down of Myc-SERCA (C) or Myc-micropeptide (D) resulted in coimmunoprecipitation of HA-tagged micropeptide, suggesting a physical interaction in transfected HEK cells. Newly Discovered Micropeptide Regulators of SERCA Form Oligomers 4431 differential expression of diverse micropeptides [4] family. To define the relative affinities of the may precisely tune the function of ion transporter micropeptide regulatory complexes, we used FRET isoforms to the needs of muscle and non-muscle to measure the dissociation constant of the micro- cells. Thus, peptide-transporter regulatory interac- peptide homo-oligomers (KD1) and the micropeptide- tions are relevant to a variety of disciplines including SERCA complexes (KD2). We focused on the cardiology, neuroscience, and endocrinology. cardiac isoform, SERCA2a, as this transporter is of There is a great deal of excitement about these particular interest as a possible point of intervention new modes of SERCA regulation as they may against heart disease. Besides its central role in represent new opportunities for therapeutic interven- normal cardiac physiology, the SERCA pump has tion, but at this early stage the structure/function been implicated in the impaired calcium handling mechanisms of micropeptides are still poorly under- that underlies many types of heart failure [29,30]. stood. One key functional determinant must be the While the etiology of heart failure is complex, stability of the regulatory complex of the micropep- mutations in calcium-handling proteins cause cardi- tide with the target transporter. Different micropep- omyopathy [31e34], and restoring calcium home- tides co-expressed in the same tissue may compete ostasis has improved function and survival in animal for SERCA [5,8], with a net effect depending on the models of heart failure [35,36]. This has focused relative expression and binding affinity of each considerable attention on SERCA as a therapeutic species. Despite decades of study of the prototypical target [30,35e37]. Though SERCA gene delivery micropeptide, PLB, it has been difficult to determine has yielded only modest gains in SERCA expression precisely the extent to which SERCA is regulated by in patients [38], efforts to deliver exogenous SERCA this interaction in vivo. As reviewed by Ceholski et al. to the failing heart are continuing. In addition, there is [9], there are a wide range of estimates of interest in parallel approaches based on improving PLB:SERCA ratios reported in the literature, from the function of the endogenous SERCA remaining in 1:5to4:1[10e14]. We think that the larger the diseased myocardium. For this, we need a better PLB:SERCA ratios are more likely to be correct, understanding of fundamental functional but even a molar excess of PLB does not necessarily determinants. reflect stoichiometric regulation of SERCA. Over- expression of PLB in a transgenic mouse model resulted in increased SERCA functional regulation, Results suggesting some SERCA was not regulated at native expression levels of PLB [15]. Nevertheless, this apparent substoichiometric regulation is still Micropeptide-SERCA Regulatory Interactions physiologically significant, and relief of inhibition results in robust enhancement of calcium handling to A comparison of the sequences of micropeptides support increased cardiac function during exercise. is shown in Fig. 1A, with key conserved residues One reason for substoichiometric regulation of highlighted. To assess SERCA regulation by micro- SERCA in spite of molar excess of PLB is that the peptides, we performed oxalate-supported 45Ca- micropeptide forms pentamers that do not regulate uptake measurements and determined all micropep- the transporter. More generally, oligomerization of tides decreased the apparent affinity of SERCA for micropeptides may indirectly modulate transporter Ca, with the exception of DWORF (Fig. 1B). DWORF function by altering micropeptide availability, thereby yielded a Ca-dependence of transport that was not tuning overall functional potency. This concept of distinguishable from control (SERCA alone). Values linked equilibria of SERCA-binding and oligomeriza- of KCa for Ca uptake are provided in Table 1. tion was developed from PLB mutagenesis experi- Previous studies have suggested a direct physical ments in which destabilization of the PLB pentamer interaction between SERCA and micropeptides increased SERCA binding and inhibition [16e19].In [3,4,8,21,39]. Here we observed comparable effi- a reciprocal experiment, increasing the proportion of ciencies of coimmunoprecipitation of HA-tagged SERCA resulted in decreased PLB oligomerization micropeptides PLB, SLN, DWORF, ALN, ELN, and [20], suggesting that SERCA sequestered mono- MLN with Myc-SERCA (Fig. 1C). The data are meric PLB. Also, phosphorylation [21e23] or oxida- consistent with the model in which DWORF binds tive crosslinking [24,25] of