Eleri Hughes

Inhibition of Toxicity in the β-Amyloid Peptide Fragment β-(25–35) Using N-Methylated Derivatives A GENERAL STRATEGY TO PREVENT AMYLOID FORMATION

β-(25–35) is a synthetic derivative of β-amyloid, the peptide that is believed to cause Alzheimers disease. As it is highly toxic and forms fibrillar aggregates typical of β-amyloid, it is suitable as a model for testing inhibitors of aggregation and toxicity. We demonstrate thatN-methylated derivatives of β-(25–35), which in isolation are soluble and non-toxic, can prevent the aggregation and inhibit the resulting toxicity of the wild type peptide.N-Methylation can block hydrogen bonding on the outer edge of the assembling amyloid. The peptides are assayed by Congo red and thioflavin T binding, electron microscopy, and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) toxicity assay on PC12 cells. One peptide (Gly25 N-methylated) has properties similar to the wild type, whereas five have varying effects on prefolded fibrils and fibril assembly. In particular, β-(25–35) with Gly33 N-methylated is able to completely prevent fibril assembly and to reduce the toxicity of prefolded amyloid. With Leu34 N-methylated, the fibril morphology is altered and the toxicity reduced. We suggest that the use of N-methylated derivatives of amyloidogenic peptides and proteins could provide a general solution to the problem of amyloid deposition and toxicity.

The effects of alpha-synuclein on phospholipid vesicle integrity: a study using 31P NMR and electron microscopy

Associations between the 140 amino acid protein α-synuclein (asyn) and presynaptic vesicles may play a role in maintaining synaptic plasticity and neurotransmitter release. These physiological processes may involve disruption and fusion of vesicles, arising from interactions between specific regions of asyn, including the highly basic N-terminal domain, and the surface of vesicles. This work investigates whether asyn affects the integrity of model unilamellar vesicles of varying size and phospholipid composition, by monitoring paramagnetic Mn2+-induced broadening of peaks in the 31P nuclear magnetic resonance spectrum of the lipid head groups. It is shown that asyn increases the permeability to Mn2+ of both large (200nm diameter) and small (50nm diameter) vesicles composed of zwitterionic phosphatidylcholine and anionic phosphatidylglycerol at protein/lipid molar ratios as low as 1:2000. Further experiments on peptides corresponding to sequences in the N-terminal (10–48), C-terminal (120–140) and central hydrophobic (71–82) regions of asyn suggest that single regions of the protein are capable of permeabilizing the vesicles to varying extents. Electron micrographs of the vesicles after addition of asyn indicate that the enhanced permeability is coupled to large-scale disruption or fusion of the vesicles. These results indicate that asyn is able to permeabilize phospholipid vesicles at low relative concentrations, dependent upon the properties of the vesicles. This could have implications for asyn playing a role in vesicle synthesis, maintenance and fusion within synapses.

Solid-state NMR and functional measurements indicate that the conserved tyrosine residues of sarcolipin are involved directly in the inhibition of SERCA1.

The transmembrane protein sarcolipin regulates calcium storage in the sarcoplasmic reticulum of skeletal and cardiac muscle cells by modulating the activity of sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs). The highly conserved C-terminal region (27RSYQY-COOH) of sarcolipin helps to target the protein to the sarcoplasmic reticulum membrane and may also participate in the regulatory interaction between sarcolipin and SERCA. Here we used solid-state NMR measurements of local protein dynamics to illuminate the direct interaction between the Tyr29 and Tyr31 side groups of sarcolipin and skeletal muscle Ca2+-ATPase (SERCA1a) embedded in dioleoylphosphatidylcholine bilayers. Further solid-state NMR experiments together with functional measurements on SERCA1a in the presence of NAc-RSYQY, a peptide representing the conserved region of sarcolipin, suggest that the peptide binds to the same site as the parent protein at the luminal face of SERCA1a, where it reduces Vmax for calcium transport and inhibits ATP hydrolysis with an IC50 of ∼200 μm. The inhibitory effect of NAc-RSYQY is remarkably sequence-specific, with the native aromatic residues being essential for optimal inhibitory activity. This combination of physical and functional measurements highlights the importance of aromatic and polar residues in the C-terminal region of sarcolipin for regulating calcium cycling and muscle contractility.

Cytoplasmic residues of phospholamban interact with membrane surfaces in the presence of SERCA: A new role for phospholipids in the regulation of cardiac calcium cycling?

The 52-amino acid transmembrane protein phospholamban (PLB) regulates calcium cycling in cardiac cells by forming a complex with the sarco(endo)plasmic reticulum calcium ATPase (SERCA) and reversibly diminishing the rate of calcium uptake by the sarcoplasmic reticulum. The N-terminal cytoplasmic domain of PLB interacts with the cytoplasmic domain of SERCA, but, in the absence of the enzyme, can also associate with the surface of anionic phospholipid membranes. This work investigates whether the cytoplasmic domain of PLB can also associate with membrane surfaces in the presence of SERCA, and whether such interactions could influence the regulation of the enzyme. It is shown using solid-state NMR and isothermal titration calorimetry (ITC) that an N-terminally acetylated peptide representing the first 23 N-terminal amino acids of PLB (PLB1-23) interacts with membranes composed of zwitterionic phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) lipids in the absence and presence of SERCA. Functional measurements of SERCA in sarcoplasmic reticulum (SR) vesicles, planar SR membranes and reconstituted into PC/PG membranes indicate that PLB1-23 lowers the maximal rate of ATP hydrolysis by acting at the cytoplasmic face of the enzyme. A small, but statistically significant, reduction in the inhibitory effect of the peptide is observed for SERCA reconstituted into PC/PG membranes compared to SERCA in membranes of PC alone. It is suggested that interactions between the cytoplasmic domain of PLB and negatively charged phospholipids might play a role in moderating the regulation of SERCA, with implications for cardiac muscle contractility.

The Conformation of ATP within the Na,K-ATPase Nucleotide Site: A Statistically Constrained Analysis of REDOR Solid-State NMR Data†

P-type ATPases are membrane-embedded ion pumps that are present in all animal cells. ATPases utilize the energy released from adenosine triphosphate (ATP) hydrolysis to drive the vectorial transport of ions. Na,K-ATPase (NKA) contributes to the maintenance of cellular potentials by actively exchanging intracellular Na for K ions; this exchange is facilitated by two enzyme conformations referred to as E1 and E2. [1] When sufficient Na ions are present to induce the E1 conformation, NKA exhibits high affinity towards ATP and adenosine diphosphate (ADP). Some 50 years after the discovery of NKA, two structures of the enzyme in the absence of a nucleotide were recently determined by X-ray crystallography. The conformation of the ATP substrate in the high-affinity site is unknown. Solid-state nuclear magnetic resonance (SSNMR) is a powerful technique for the structural analysis of organic ligands complexed with membrane-embedded receptors. Rotational-echo double resonance (REDOR) is an SSNMR method for measuring heteronuclear (e.g., C–N, C–P) dipolar couplings that encode interatomic distances. REDOR may be exploited to measure structurally diagnostic distances between carbon sites and the three phosphorus sites (Pa, Pb, and Pg) of ATP when complexed with NKA. However, the quality of REDOR data is compromised by the poor sensitivity of NMR spectroscopy and the low abundance of membrane receptors. We have alleviated these difficulties by using statistical information to assist the analysis of REDOR data, and present the first experimentally derived conformation of ATP in the high-affinity nucleotide site of NKA in native membranes. Figure 1a shows a P NMR spectrum of [U-C,N]ATP complexed with approximately 16 nmol NKA and stabilized by freeze-trapping. The equilibrium binding curves are consistent with rapid nucleotide binding to a single site, and dissociation constants are in the range of 0.2–0.5 mm and a capacity of about 2.9 nmolmg . 6] Under these conditions, we estimate that approximately 90% of the nucleotide is bound to NKA. The procedure to form the complex prior to freeze-trapping took less than 10 min, during which less than 10 mmol ATP were hydrolyzed out of the 160 mmol present. This result is confirmed by the P NMR spectrum, which shows signals for the three phosphate groups of ATP in approximately equal intensity. Figure 1b shows a cross-polarization magic-angle spinning (CP-MAS) C NMR spectrum after subtracting the background signals of the lipids and the protein. We elected to measure Pa–C8, Pb–C8, and Pg–C8 dipolar couplings simultaneously in a frequency-selective DANTE-P(C)-REDOR experiment with P observation and dephasing selective for C8. The C8 signal is well resolved and favorable for selective inversion in the REDOR experiment. Couplings between P and C5’ or C2, which are also resolved in the spectrum, could provide additional structural restraints, but were not measured because of the lengthy data collection times involved. REDOR dephasing (SD) was monitored from the Pa, Pb, and Pg signal intensities at three echo times (26 days of data Figure 1. Structure of ATP and SSNMR spectra of [U-C,N]ATP complexed with NKA (at 25 8C). Dotted lines on the structure of ATP denote the three C–P distances measured here. a) P MAS spectrum of bound ATP. Pi denotes inorganic phosphate ions. Peaks are assigned according to Ref. [5]. b) C CP-MAS spectrum of the same sample, obtained after subtraction of the background signals of the lipids and the protein. Peaks are assigned according to Ref. [13].

Solid-State NMR Studies of Adenosine 5′-Triphosphate Freeze-Trapped in the Nucleotide Site of Na,K-ATPase

The Na,K-ATPase (NKA) maintains the cellular potentials of all animal cells by driving the vectorial transport of Na and K across the plasma membrane through two different enzyme conformations (E1 and E2). [1] NKA exhibits high affinity towards the ATP substrate when sufficient Na is present to induce the E1 conformation. [2, 3] More than fifty years after the discovery of NKA, the details of how ATP interacts with the enzyme in the E1 conformation remain unknown, and the only X-ray crystal structure of NKA is for the E2 conformation in the absence of nucleotide. Solid-state NMR spectroscopy (SSNMR) is a valuable alternative to X-ray diffraction for providing atomistic details of ligands bound to receptors within their native membranes. SSNMR studies of bound nucleotide in membranous preparations of NKA from pig kidney and shark salt glands are hampered by a contaminating enzyme activity that rapidly hydrolyses ATP, ADP and AMP in the absence and presence of Mg + . 10] Hydrolysis is not inhibited by ouabain, vanadate or adenylate kinase inhibitor. This problem is overcome here by including a Mg chelator and rapidly freeze-trapping uniformly C/N-labelled ATP ([U-C,N]ATP) complexed with NKA in membrane preparations. SSNMR spectroscopy experiments at 25 8C detect C signals from bound ATP and provide preliminary insights into the nucleotide contacts with the high-affinity site. A critical requirement for the SSNMR experiments is to saturate the high-affinity nucleotide site with ATP whilst avoiding excess free or unspecifically bound nucleotide, because SSNMR spectroscopy cannot readily distinguish these from specifically bound nucleotide. Biochemical measurements of radiolabelled nucleotide binding were therefore carried out to establish such a regime. Equilibrium binding curves show nucleotide binding to a single site with dissociation constants in the range of 0.2–0.5 mm, and a capacity of about 2.9 nmol mg 1 protein for both shark and pig NKA in the buffer used for SSNMR experiments (Figure S1 in the Supporting Information). Samples for SSNMR analysis contain 130 mm NKA, so we can safely assume that the nucleotide site is saturated by adding 160 mm [U-C,N]ATP to the membranes; this gives a free nucleotide concentration of about 30 mm. The binding assay also demonstrates the absence of additional unspecific binding to NKA. We thus confirm that at these concentrations more than 80 % of the C NMR signal for the nucleotide will correspond to ATP bound specifically to shark or kidney enzyme. Figure 1 A shows C cross-polarisation magic-angle spinning (CP-MAS) SSNMR spectra at 25 8C for a control shark NKA

Heparin-derived oligosaccharides interact with the phospholamban cytoplasmic domain and stimulate SERCA function

The association between the cardiac transmembrane proteins phospholamban and sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) regulates the active transport of Ca(2+) into the sarcoplasmic reticulum (SR) lumen and controls the contraction and relaxation of the heart. Heart failure (HF) and cardiac hypertrophy have been linked to defects in Ca(2+) uptake by the cardiac SR and stimulation of calcium transport by modulation of the PLB-SERCA interaction is a potential therapy. This work is part of an effort to identify compounds that destabilise the PLB-SERCA interaction in well-defined membrane environments. It is shown that heparin-derived oligosaccharides (HDOs) interact with the cytoplasmic domain of PLB and consequently stimulate SERCA activity. These results indicate that the cytoplasmic domain of PLB is functionally important and could be a valid target for compounds with drug-like properties.

A study of the membrane association and regulatory effect of the phospholemman cytoplasmic domain

Phospholemman (PLM) is a single-span transmembrane protein belonging to the FXYD family of proteins. PLM (or FXYD1) regulates the Na,K-ATPase (NKA) ion pump by altering its affinity for K(+) and Na(+) and by reducing its hydrolytic activity. Structural studies of PLM in anionic detergent micelles have suggested that the cytoplasmic domain, which alone can regulate NKA, forms a partial helix which is stabilized by interactions with the charged membrane surface. This work examines the membrane affinity and regulatory function of a 35-amino acid peptide (PLM(38-72)) representing the PLM cytoplasmic domain. Isothermal titration calorimetry and solid-state NMR measurements confirm that PLM(38-72) associates strongly with highly anionic phospholipid membranes, but the association is weakened substantially when the negative surface charge is reduced to a more physiologically relevant environment. Membrane interactions are also weakened when the peptide is phosphorylated at S68, one of the substrate sites for protein kinases. PLM(38-72) also lowers the maximal velocity of ATP hydrolysis (V(max)) by NKA, and phosphorylation of the peptide at S68 gives rise to a partial recovery of V(max). These results suggest that the PLM cytoplasmic domain populates NKA-associated and membrane-associated states in dynamic equilibrium and that phosphorylation may alter the position of the equilibrium. Interestingly, peptides representing the cytoplasmic domains of two other FXYD proteins, Mat-8 (FXYD3) and CHIF (FXYD4), have little or no interaction with highly anionic phospholipid membranes and have no effect on NKA function. This suggests that the functional and physical properties of PLM are not conserved across the entire FXYD family.

Solid-state NMR measurements of the kinetics of the interaction between phospholamban and Ca2 + -ATPase in lipid bilayers

Phospholamban (PLB) is a small transmembrane protein that regulates calcium transport across the sarcoplasmic reticulum (SR) of cardiac cells via a reversible inhibitory interaction with Ca2 + -ATPase. In this work solid-state NMR methods have been used to investigate the dynamics of the inhibitory association between PLB and Ca2 + -ATPase. Skeletal muscle Ca2 + -ATPase was incorporated into phosphatidylcholine membranes together with a ten-fold excess of a null-cysteine mutant of PLB labelled with 13C at Leu-44 in the transmembrane domain ([α-13C-L44]AAA-PLB). In these membranes the PLB variant was found to partially inhibit Ca2 + -ATPase by reducing the affinity of the enzyme for calcium. Cross-polarization magic angle spinning (CP-MAS) 13C NMR spectra of the membranes exhibited a signature peak from [α-13C-L44]AAA-PLB at 56 ppm. Changes in the intensity of the peak were observed at different temperatures, which was diagnostic of direct interaction between [α-13C-L44]AAA-PLB and Ca2 + -ATPase. Measurements of dipolar couplings between the 13C label and neighbouring protons were analysed to show that the mean residency time for the association of AAA-PLB with Ca2 + -ATPase was on the order of 2.5 ms at temperatures between 0°C and 30°C. This new NMR approach will be useful for examining how the association of the two proteins is affected by physiological stimuli such as kinases and the elevation of calcium concentration.