Przemek A. Gorski

Sarco(endo)plasmic Reticulum Calcium ATPase (SERCA) Inhibition by Sarcolipin Is Encoded in Its Luminal Tail

Background: Sarcolipin is a regulator of SERCA in skeletal and atrial muscle with inhibitory properties thought to be similar to phospholamban. Results: Residues critical for SERCA inhibition reside in the luminal extension of sarcolipin. Conclusion: The luminal extension of sarcolipin is a distinct and transferrable domain that encodes most of its inhibitory properties. Significance: Sarcolipin and phospholamban use different inhibitory mechanisms to regulate SERCA. The sarco(endo)plasmic reticulum calcium ATPase (SERCA) is regulated in a tissue-dependent manner via interaction with the short integral membrane proteins phospholamban (PLN) and sarcolipin (SLN). Although defects in SERCA activity are known to cause heart failure, the regulatory mechanisms imposed by PLN and SLN could have clinical implications for both heart and skeletal muscle diseases. PLN and SLN have significant sequence homology in their transmembrane regions, suggesting a similar mode of binding to SERCA. However, unlike PLN, SLN has a conserved C-terminal luminal tail composed of five amino acids (27RSYQY), which may contribute to a distinct SERCA regulatory mechanism. We have functionally characterized alanine mutants of the C-terminal tail of SLN using co-reconstituted proteoliposomes of SERCA and SLN. We found that Arg27 and Tyr31 are essential for SLN function. We also tested the effect of a truncated variant of SLN (Arg27stop) and extended chimeras of PLN with the five luminal residues of SLN added to its C terminus. The Arg27stop form of SLN resulted in loss of function, whereas the PLN chimeras resulted in superinhibition with characteristics of both PLN and SLN. Based on our results, we propose that the C-terminal tail of SLN is a distinct, essential domain in the regulation of SERCA and that the functional properties of the SLN tail can be transferred to PLN.

Transmembrane Helix 11 Is a Genuine Regulator of the Endoplasmic Reticulum Ca2+ Pump and Acts as a Functional Parallel of β-Subunit on α-Na+,K+-ATPase

Background: The ubiquitous sarco/endoplasmic reticulum Ca2+ ATPase SERCA2b has a C-terminal extension that increases the Ca2+ affinity. It consists of a transmembrane helix (TM11) and a luminal extension. Results: Both parts control Ca2+ affinity independently. Conclusion: TM11 is an independent and highly conserved functional region of SERCA2b. Significance: This study shows that TM11 acts as a genuine regulator of the Ca2+ pump. The housekeeping sarco(endo)plasmic reticulum Ca2+ ATPase SERCA2b transports Ca2+ across the endoplasmic reticulum membrane maintaining a vital Ca2+ gradient. Compared with the muscle-specific isoforms SERCA2a and SERCA1a, SERCA2b houses an 11th transmembrane segment (TM11) and a short luminal extension (LE) at its C terminus (2b-tail). The 2b-tail imposes a 2-fold higher apparent Ca2+ affinity and lower Vmax. Previously, we assumed that LE is the sole functional region of the 2b-tail and that TM11 is a passive element providing an additional membrane passage. However, here we show that peptides corresponding to the TM11 region specifically modulate the activity of the homologous SERCA1a in co-reconstituted proteoliposomes and mimic the 2b-tail effect (i.e. lower Vmax and higher Ca2+ affinity). Using truncated 2b-tail variants we document that TM11 regulates SERCA1a independently from LE, confirming that TM11 is a second, previously unrecognized functional region of the 2b-tail. A phylogenetic analysis further indicates that TM11 is the oldest and most conserved feature of the 2b-tail, found in the SERCA pump of all Bilateria, whereas LE is only present in Nematoda and vertebrates. Considering remarkable similarities with the Na+,K+-ATPase α-β interaction, we now propose a model for interaction of TM11 with TM7 and TM10 in the anchoring subdomain of the Ca2+ pump. This model involves a TM11-induced helix bending of TM7. In conclusion, more than just a passive structural feature, TM11 acts as a genuine regulator of Ca2+ transport through interaction with the pump.

Regulation of the sarcoplasmic reticulum calcium pump by divergent phospholamban isoforms in zebrafish.

Background: Zebrafish possess multiple phospholamban isoforms, one of which has a unique luminal domain. Results: Zebrafish and human PLN have comparable effects on SERCA activity, and both can be reversed by phosphorylation. Conclusion: Despite similar function, the zebrafish sequence variations are context-dependent and not synonymous with human phospholamban. Significance: The different forms of phospholamban in zebrafish may provide a novel SERCA regulatory mechanism. The sarcoplasmic reticulum calcium pump (SERCA) is regulated by the small integral membrane proteins phospholamban (PLN) and sarcolipin (SLN). These regulators have homologous transmembrane regions, yet they differ in their cytoplasmic and luminal domains. Although the sequences of PLN and SLN are practically invariant among mammals, they vary in fish. Zebrafish (zf) appear to harbor multiple PLN isoforms, one of which contains 18 sequence variations and a unique luminal extension. Characterization of this isoform (zfPLN) revealed that SERCA inhibition and reversal by phosphorylation were comparable with human PLN. To understand the sequence variations in zfPLN, chimeras were created by transferring the N terminus, linker, and C terminus of zfPLN onto human PLN. A chimera containing the N-terminal domain resulted in a mild loss of function, whereas a chimera containing the linker domain resulted in a gain of function. This latter effect was due to changes in basic residues in the linker region of PLN. Removing the unique luminal domain of zfPLN (53SFHGM) resulted in loss of function, whereas adding this domain to human PLN had a minimal effect on SERCA inhibition. We conclude that the luminal extension contributes to SERCA inhibition but only in the context of zfPLN. Although this domain is distinct from the SLN luminal tail, zfPLN appears to use a hybrid PLN-SLN inhibitory mechanism. Importantly, the different zebrafish PLN isoforms raise the interesting possibility that sarcoplasmic reticulum calcium handling and cardiac contractility may be regulated by the differential expression of PLN functional variants.

Distinct morphological and electrophysiological properties of an elk prion peptide

A key event in prion diseases is the conversion of the prion protein (PrP) from its native α-helical conformation to a misfolded, β-sheet rich conformation. Thus, preventing or reversing PrP misfolding could provide a means to disrupt prion disease progression and transmission. However, determining the structure of misfolded PrP has been notoriously difficult due to its inherent heterogeneity and aggregation behavior. For these reasons, simplified peptide fragments have been used as models that recapitulate characteristics of full-length PrP, such as amyloid-like aggregation and fibril formation, and in vitro toxicity. We provide a biochemical and structural comparison of PrP(127-147) peptides from elk, bovine and hamster using electrophysiology, electron microscopy and fluorescence. Our results demonstrate that the PrP(127-147) peptides adopt distinct populations of fibril structures. In addition, the elk PrP(127-147) peptide is unique in its ability to enhance Thioflavin T fluorescence and its ability to modulate neuronal ion channel conductances.

Structure-Function Relationship of the SERCA Pump and Its Regulation by Phospholamban and Sarcolipin

Calcium is a universal second messenger involved in diverse cellular processes, including excitation-contraction coupling in muscle. The contraction and relaxation of cardiac muscle cells are regulated by the cyclic movement of calcium primarily between the extracellular space, the cytoplasm and the sarcoplasmic reticulum (SR). The rapid removal of calcium from the cytosol is primarily facilitated by the sarco(endo)plasmic reticulum calcium ATPase (SERCA) which pumps calcium back into the SR lumen and thereby controls the amount of calcium in the SR. The most studied member of the P-type ATPase family, SERCA has multiple tissue- and cell-specific isoforms and is primarily regulated by two peptides in muscle, phospholamban and sarcolipin. The multifaceted regulation of SERCA via these peptides is exemplified in the biological fine-tuning of their independent oligomerization and regulation. In this chapter, we overview the structure-function relationship of SERCA and its peptide modulators, detailing the regulation of the complexes and summarizing their physiological and disease relevance.

Two dimensional crystallization of calcium transport regulatory complexes

Phospholamban and sarcolipin physically interact with the sarcoplasmic reticulum calcium pump (SERCA) and differentially regulate contractility in smooth, skeletal, and cardiac muscle. While mutagenesis and cross-linking studies have provided insight into the mechanism of interaction, we lack a molecular understanding of these regulatory complexes. We have compared two crystal forms of SERCA in the absence and presence of phospholamban by electron cryo-microscopy. Our previous studies with phospholamban utilized both small helical crystals [1] and large two-dimensional crystals [2], where the fundamental units of both crystal forms were found to be anti-parallel dimer ribbons of SERCA molecules. The SERCA dimer ribbons have been known for decades as a rigid assembly of calcium-free SERCA molecules induced by the addition of decavanadate. While the lattice formed by the SERCA dimer ribbons is different in the helical (p2) and two-dimensional crystals (p22121), we now show that a phospholamban oligomer interacts with SERCA in a similar manner in both crystal types. With this information, we next undertook a structural investigation of SERCA and sarcolipin in the large two-dimensional crystals. Both wild-type and a gain-of-function mutant (Asn4-to-Ala) mutant of sarcolipin were utilized. Projection maps were determined for SERCA in the presence of sarcolipin to a resolution of 8.5 Å and were most consistent with a pentameric state for sarcolipin. While both phospholamban and sarcolipin interacted with transmembrane segment M3 of SERCA, the interaction of the sarcolipin pentamer was mediated by an additional density consistent with a sarcolipin monomer.

Species-Dependent Differences in Fibril Morphology and the Effects on Basal Forebrain Neurons of a Prion Protein Fragment (Residues 127-147)

Prion protein mis-folding is central to the transmission and pathobiology of transmissible spongiform encephalopathies. Unfortunately, the heterogeneous and aggregation-prone nature of the disease-associated mis-folded prion protein has complicated its study. Fragments of the prion protein, such as residues 106-126 and 127-147, exhibit amyloid-like fibril formation and neurotoxicity in cell culture. These prion protein fragments serve as simplified models for the study of the mis-folded prion protein and its role in disease. We have characterized a fibril-forming peptide (residues 127-147) from elk, hamster and bovine prion proteins. At neutral pH, the elk and hamster prion peptides form distinct fibril morphologies. The hamster fibrils are ∼20 nm in width, Thioflavin T-negative, and generally lack a repetitive helical structure. The elk fibrils are thinner (∼15 nm in width), Thioflavin T-positive, and have a repetitive helical structure. Notably, the elk fibrils appear more homogeneous in their morphology and helical periodicity, whereas the hamster fibrils appear more heterogeneous and non-periodic. As observed for the elk peptide, the bovine fibrils appear homogeneous in their morphology and periodicity. Three-dimensional reconstructions from negative-stain transmission electron microscopy images of representative elk, hamster, and bovine fibrils highlight their distinct morphologies. Interestingly, the elk prion peptide, but not the hamster peptide, significantly decreased whole cell currents in rat basal forebrain neurons, similar to previous studies of a human prion peptide (residues 106-126; Alier et al., Journal of Neuroscience Research, 2010).