John Paul Glaves

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.

Rewiring AMPK and Mitochondrial Retrograde Signaling for Metabolic Control of Aging and Histone Acetylation in Respiratory-Defective Cells

Abnormal respiratory metabolism plays a role in numerous human disorders. We find that regulation of overall histone acetylation is perturbed in respiratory-incompetent (ρ(0)) yeast. Because histone acetylation is highly sensitive to acetyl-coenzyme A (acetyl-CoA) availability, we sought interventions that suppress this ρ(0) phenotype through reprogramming metabolism. Nutritional intervention studies led to the discovery that genetic coactivation of the mitochondrion-to-nucleus retrograde (RTG) response and the AMPK (Snf1) pathway prevents abnormal histone deacetylation in ρ(0) cells. Metabolic profiling of signaling mutants uncovered links between chromatin-dependent phenotypes of ρ(0) cells and metabolism of ATP, acetyl-CoA, glutathione, branched-chain amino acids, and the storage carbohydrate trehalose. Importantly, RTG/AMPK activation reprograms energy metabolism to increase the supply of acetyl-CoA to lysine acetyltransferases and extend the chronological lifespan of ρ(0) cells. Our results strengthen the framework for rational design of nutrient supplementation schemes and drug-discovery initiatives aimed at mimicking the therapeutic benefits of dietary interventions.

Wild-type Shadoo proteins convert to amyloid-like forms under native conditions

J. Neurochem. (2010) 10.1111/j.1471‐4159.2010.06575.x

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.

Chapter Seven - Helical Crystallization of Two Example Membrane Proteins: MsbA and the Ca2+-ATPase

Helical crystallization is a powerful tool for the moderate resolution structure determination of integral membrane proteins, where the insight gained often includes domain architecture and the disposition of α-helical segments. A necessary first step toward helical crystallization involves membrane protein reconstitution, which itself is a powerful technique for structure-function studies of integral membrane proteins. The correct insertion of a detergent-solubilized, purified membrane protein into lipid vesicles (proteoliposomes) can facilitate the functional characterization of the protein in a well-defined, chemically pure environment without interference from other membrane-associated components. In addition, the lipid-to-protein ratio can be controlled during reconstitution to generate a high concentration of a particular membrane protein in the proteoliposomes, which are then suitable for both functional assays and crystallization trials. Traditional approaches to two-dimensional crystallization for electron microscopy rely on dialysis methods for the simultaneous reconstitution and crystallization of a membrane protein [Kühlbrandt, W. (1992). Two-dimensional crystallization of membrane proteins. Q. Rev. Biophys.25, 1-49.], yet some systems allow these two steps to be experimentally separated and independently considered. Some examples of integral membrane proteins that have been reconstituted and crystallized in a helical lattice include cytochrome bc1 complex from bovine heart [Akiba, T., et al. (1996). Three-dimensional structure of bovine cytochrome bc(1) complex by electron cryomicroscopy and helical image reconstruction. Nat. Struct. Biol.3, 553-561.], Escherichia coli melibiose permease [Rigaud, J. L., et al. (1997). Bio-beads: An efficient strategy for two-dimensional crystallization of membrane proteins. J. Struct. Biol.118, 226-235.], a bacterial ATP-binding cassette transporter MsbA [Ward, A., et al. (2009). Nucleotide dependent packing differences in helical crystals of the ABC transporter MsbA. J. Struct. Biol.165, 169-175.], and the sarcoplasmic reticulum Ca(2+)-ATPase [Young, H. S., et al. (1997). How to make tubular crystals by reconstitution of detergent-solubilized Ca(2+)-ATPase. Biophys. J.72, 2545-2558.]. The reconstitution and helical crystallization of MsbA and Ca(2+)-ATPase will be the focus of this chapter.

The phospholamban pentamer interacts with the sarcoplasmic reticulum calcium pump SERCA.

The interaction of phospholamban with the sarcoplasmic reticulum calcium pump (SERCA) is a major regulatory axis in cardiac muscle contractility. The prevailing model involves reversible inhibition of SERCA by monomeric phospholamban and storage of phospholamban as an inactive pentamer. However, this paradigm has been challenged by studies demonstrating that phospholamban remains associated with SERCA and that the phospholamban pentamer is required for cardiac contractility. We have previously used two-dimensional crystallization and electron microscopy to study the interaction between SERCA and phospholamban. To further understand this interaction, we compared small helical crystals and large two-dimensional crystals of SERCA in the absence and presence of phospholamban. In both crystal forms, SERCA molecules are organized into identical anti-parallel dimer ribbons. The dimer ribbons pack together with distinct crystal contacts in the helical versus large two-dimensional crystals, which allow phospholamban differential access to potential sites of interaction with SERCA. Nonetheless, we show that a phospholamban oligomer interacts with SERCA in a similar manner in both crystal forms. In the two-dimensional crystals, a phospholamban pentamer interacts with transmembrane segments M3 of SERCA and participates in a crystal contact that bridges neighboring SERCA dimer ribbons. In the helical crystals, an oligomeric form of phospholamban also interacts with M3 of SERCA, though the phospholamban oligomer straddles a SERCA-SERCA crystal contact. We conclude that the pentameric form of phospholamban interacts with SERCA, and that it plays distinct structural and functional roles in SERCA regulation.

Residue-specific mobility changes in soluble oligomers of the prion protein define regions involved in aggregation

Prion (PrP) diseases are neurodegenerative diseases characterized by the formation of β-sheet rich, insoluble and protease resistant protein deposits (called PrPSc) that occur throughout the brain. Formation of synthetic or in vitro PrPSc can occur through on-pathway toxic oligomers. Similarly, toxic and infectious oligomers identified in cell and animal models of prion disease indicate that soluble oligomers are likely intermediates in the formation of insoluble PrPSc. Despite the critical role of prion oligomers in disease progression, little is known about their structure. In order, to obtain structural insight into prion oligomers, we generated oligomers by shaking-induced conversion of recombinant, monomeric prion protein PrPc (spanning residues 90-231). We then obtained two-dimensional solution NMR spectra of the PrPc monomer, a 40% converted oligomer, and a 94% converted oligomer. Heteronuclear single-quantum correlation (1H-15N) studies revealed that, in comparison to monomeric PrPc, the oligomer has intense amide peak signals in the N-terminal (residues 90-114) and C-terminal regions (residues 226-231). Furthermore, a core region with decreased mobility is revealed from residues ~127 to 225. Within this core oligomer region with decreased mobility, there is a pocket of increased amide peak signal corresponding to the middle of α-helix 2 and the loop between α-helices 2 and 3 in the PrPc monomer structure. Using high-resolution solution-state NMR, this work reveals detailed and divergent residue-specific changes in soluble oligomeric models of PrP.

A Common Phenotype Polymorphism in Mammalian Brains Defined by Concomitant Production of Prolactin and Growth Hormone

Pituitary Prolactin (PRL) and Growth Hormone (GH) are separately controlled and sub-serve different purposes. Surprisingly, we demonstrate that extra-pituitary expression in the adult mammalian central nervous system (CNS) is coordinated at mRNA and protein levels. However this was not a uniform effect within populations, such that wide inter-individual variation was superimposed on coordinate PRL/GH expression. Up to 44% of individuals in healthy cohorts of mice and rats showed protein levels above the norm and coordinated expression of PRL and GH transcripts above baseline occurred in the amygdala, frontal lobe and hippocampus of 10% of human subjects. High levels of PRL and GH present in post mortem tissue were often presaged by altered responses in fear conditioning and stress induced hyperthermia behavioral tests. Our data define a common phenotype polymorphism in healthy mammalian brains, and, given the pleiotropic effects known for circulating PRL and GH, further consequences of coordinated CNS over-expression may await discovery.

Two-Dimensional Crystallization of the Ca 2+ -ATPase for Electron Crystallography

Electron crystallography of two-dimensional crystalline arrays is a powerful alternative for the structure determination of membrane proteins. The advantages offered by this technique include a native membrane environment and the ability to closely correlate function and dynamics with crystalline preparations and structural data. Herein, we provide a detailed protocol for the reconstitution and two-dimensional crystallization of the sarcoplasmic reticulum calcium pump (also known as Ca(2+)-ATPase or SERCA) and its regulatory subunits phospholamban and sarcolipin.

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.