Fibulin-7 C-terminal fragment and its active synthetic peptide suppress choroidal and retinal neovascularization.

Wet age-related macular degeneration (AMD) and diabetic retinopathy are the leading causes of blindness through increased angiogenesis. Although VEGF-neutralizing proteins provide benefit, inconsistent responses indicate a need for new therapies. We previously identified the Fibulin-7 C-terminal fragment (Fbln7-C) as an angiogenesis inhibitor in vitro. Here we show that Fbln7-C inhibits neovascularization in vivo, in both a model of wet AMD involving choroidal neovascularization (CNV) and diabetic retinopathy involving oxygen-induced ischemic retinopathy. Furthermore, a short peptide sequence from Fbln7-C is responsible for the anti-angiogenic properties of Fbln7-C. Our work suggests Fbln7-C as a therapeutic candidate for wet AMD and ischemic retinopathy.

Development of α-helical calpain probes by mimicking a natural protein-protein interaction.

We have designed a highly specific inhibitor of calpain by mimicking a natural protein-protein interaction between calpain and its endogenous inhibitor calpastatin. To enable this goal we established a new method of stabilizing an α-helix in a small peptide by screening 24 commercially available cross-linkers for successful cysteine alkylation in a model peptide sequence. The effects of cross-linking on the α-helicity of selected peptides were examined by CD and NMR spectroscopy, and revealed structurally rigid cross-linkers to be the best at stabilizing α-helices. We applied this strategy to the design of inhibitors of calpain that are based on calpastatin, an intrinsically unstable polypeptide that becomes structured upon binding to the enzyme. A two-turn α-helix that binds proximal to the active site cleft was stabilized, resulting in a potent and selective inhibitor for calpain. We further expanded the utility of this inhibitor by developing irreversible calpain family activity-based probes (ABPs), which retained the specificity of the stabilized helical inhibitor. We believe the inhibitor and ABPs will be useful for future investigation of calpains, while the cross-linking technique will enable exploration of other protein-protein interactions.

Matrix Gla protein inhibits ectopic calcification by a direct interaction with hydroxyapatite crystals.

Mice lacking the gene encoding matrix gla protein (MGP) exhibit massive mineral deposition in blood vessels and die soon after birth. We hypothesize that MGP prevents arterial calcification by adsorbing to growing hydroxyapatite (HA) crystals. To test this, we have used a combined experimental-computational approach. We synthesized peptides covering the entire sequence of human MGP, which contains three sites of serine phosphorylation and five sites of γ-carboxylation, and studied their effects on HA crystal growth using a constant-composition autotitration assay. In parallel studies, the interactions of these sequences with the {100} and {001} faces of HA were analyzed using atomistic molecular dynamics (MD) simulations. YGlapS (amino acids 1-14 of human MGP) and SK-Gla (MGP43-56) adsorbed rapidly to the {100} and {001} faces and strongly inhibited HA growth (IC(50) = 2.96 µg/mL and 4.96 µg/mL, respectively). QR-Gla (MGP29-42) adsorbed more slowly and was a moderate growth inhibitor, while the remaining three (nonpost-translationally modified) peptides had little or no effect in either analysis. Substitution of gla with glutamic acid reduced the adsorption and inhibition activities of SK-Gla and (to a lesser extent) QR-Gla but not YGlapS; substitution of phosphoserine with serine reduced the inhibitory potency of YGlapS. These studies suggest that MGP prevents arterial calcification by a direct interaction with HA crystals that involves both phosphate groups and gla residues of the protein. The strong correlation between simulated adsorption and measured growth inhibition indicates that MD provides a powerful tool to predict the effects of proteins and peptides on crystal formation.

Ligation events influence ALG-2 dimerization.

ALG-2 dimerization was studied using Förster resonance-energy-transfer. D162C variants of ALG-2des23 were covalently modified with Alexa Fluor 488 and Alexa Fluor 647. When samples of the two labeled protein-preparations are combined, the sensitized emission from AF647 serves as a sensitive probe of dimer formation. At 25 °C, in the absence of divalent ions, the wild-type homodimer, ΔGF122 homodimer, and heterodimer display dissociation constants of 7.1, 26, and 4.5 nM, respectively. At 35 °C, subunit interaction is weaker, indicating that dimer formation is exothermic. Binding of Mg2+ in the C-terminal EF-hand (EF5) dramatically enhances ALG-2 dimer stability. Although occupation of EF5 by Ca2+ likewise has a stabilizing effect, its direct influence on dimer stability would be negligible at cytosolic Ca2+ levels. However, dimer stability is substantially increased by the Ca2+-dependent binding of ALG-2 target-peptides, suggesting that the occupation-status of the target-protein binding site is communicated to the dimer interface. Tween 20 is commonly used to improve ALG-2 solubility, the micelles ostensibly acting as target-protein surrogates. Paradoxically, however, the detergent markedly destabilizes ALG-2 dimers, particularly in the presence of Ca2+.

Helical structure of phospholamban in membrane bilayers.

The regulation of calcium levels across the membrane of the sarcoplasmic reticulum involves the complex interplay of several membrane proteins. Phospholamban is a 52 residue integral membrane protein that is involved in reversibly inhibiting the Ca(2+) pump and regulating the flow of Ca ions across the sarcoplasmic reticulum membrane during muscle contraction and relaxation. The structure of phospholamban is central to its regulatory role. Using homonuclear rotational resonance NMR methods, we show that the internuclear distances between [1-(13)C]Leu7 and [3-(13)C]Ala11 in the cytoplasmic region, between [1-(13)C]Pro21 and [3-(13)C]Ala24 in the juxtamembrane region and between [1-(13)C]Leu42 and [3-(13)C]Cys46 in the transmembrane domain of phospholamban are consistent with alpha-helical secondary structure. Additional heteronuclear rotational-echo double-resonance NMR measurements confirm that the secondary structure is helical in the region of Pro21 and that there are no large conformational changes upon phosphorylation. These results support the model of the phospholamban pentamer as a bundle of five long alpha-helices. The long extended helices provide a mechanism by which the cytoplasmic region of phospholamban interacts with residues in the cytoplasmic domain of the Ca(2+) pump.

Total chemical synthesis of human matrix Gla protein.

Human matrix Gla protein (MGP) is a vitamin K-dependent extracellular matrix protein that binds Ca2+ ions and that is involved in the prevention of vascular calcification. MGP is a 10.6-kD protein (84 amino acids) containing five gamma-carboxyglutamic acid (Gla) residues and one disulfide bond. Studies of the mechanism by which MGP prevents calcification of the arterial media are hampered by the low solubility of the protein (<10 microg/mL). Because of solubility problems, processing of a recombinantly expressed MGP-fusion protein chimera to obtain MGP was unsuccessful. Here we describe the total chemical synthesis of MGP by tBoc solid-phase peptide synthesis (SPPS) and native chemical ligation. Peptide Tyr1-Ala53 was synthesized on a derivatized resin yielding a C-terminal thioester group. Peptide Cys54-Lys84 was synthesized on Lys-PAM resin yielding a C-terminal carboxylic acid. Subsequent native chemical ligation of the two peptides resulted in the formation of a native peptide bond between Ala53 and Cys54. Folding of the 1-84-polypeptide chain in 3 M guanidine (pH 8) resulted in a decrease of molecular mass from 10,605 to 10,603 (ESI-MS), representing the loss of two protons because of the formation of the Cys54-Cys60 internal disulfide bond. Like native MGP, synthetic MGP had the same low solubility when brought into aqueous buffer solutions with physiological salt concentrations, confirming its native like structure. However, the solubility of MGP markedly increased in borate buffer at pH 7.4 in the absence of sodium chloride. Ca2+-binding to MGP was confirmed by analytical HPLC, on which the retention time of MGP was reduced in the presence of CaCl2. Circular dichroism studies revealed a sharp increase in alpha-helicity at 0.2 mM CaCl2 that may explain the Ca2+-dependent shift in high-pressure liquid chromatography (HPLC)-retention time of MGP. In conclusion, facile and efficient chemical synthesis in combination with native chemical ligation yielded MGP preparations that can aid in unraveling the mechanism by which MGP prevents vascular calcification.

Total synthesis and functional properties of the membrane-intrinsic protein phospholamban.

The membrane-intrinsic protein phospholamban (PLN), the regulatory protein of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, was chemically synthesized. The synthesis was accomplished by double couplings and efficient capping procedures, thus eliminating hydrophobic failure sequences. The crude peptide was purified by high-performance liquid chromatographic ion exchange and gel permeation chromatography in chloroform-methanol mixtures. Ion spray mass spectroscopy showed that the product had the correct molecular mass. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis runs produced the typical monomer-pentamer structural pattern. A predominantly helical CD spectrum was obtained in 0.075% C12E8 (67.9% helix, 1.8% beta, 12.2% turn, 18.1% random coil). Synthetic PLN was phosphorylated in detergent solutions by protein kinase A with a stoichiometry close to 1:1 (Pi to PLN monomer). Reconstitution of the isolated skeletal muscle SR Ca2+ ATPase in phosphatidylcholine membranes in the presence of PLN using the freezing and thawing technique yielded a preparation with lower Ca(2+)-dependent ATPase activity. The inhibition was mainly due to a decrease in the affinity (Km(Ca)) of the ATPase for Ca2+ and was partially reversed by PLN phosphorylation with protein kinase A. By contrast, addition of PLN to diluted intact SR vesicles uncoupled the Ca(2+)-transport reaction, suggesting an ionophoric effect of PLN. Because this effect was observed at very high PLN-to-SR vesicle ratios and was not influenced by PLN phosphorylation, its biological function is doubtful.

A novel peptide designed for sensitization of terbium (III) luminescence.

Several synthetic peptides, modelled from a Ca(2+)-binding loop of the EF-hand family of proteins, were prepared containing cysteine residues. The peptide, GDKNADGFICFEEL, was labelled covalently at the cysteine residue (loop position 9) with iodoacetamidosalicylic acid. This novel conjugate is a metal-binding loop containing a salicylic acid side chain that could not only chelate Tb3+ in conjunction with the other chelating groups in the sequence, but could also sensitize Tb3+ luminescence. The loop had a high Tb3+ affinity, with stoichiometric binding observed under experimental conditions. The luminescence from the Tb(3+)-peptide complex was more than 10-fold greater than the luminescence reported from a related peptide which contained Trp as the Tb3+ donor at loop position 7. This peptide has significant potential for use in lanthanide-based time-resolved luminescence immunoassays.

Total chemical synthesis and chemotactic activity of human S100A12 (EN-RAGE).

Human S100A12 (extracellular newly identified RAGE (receptor for advanced glycosylation end products)-binding protein), a new member of the S100 family of EF-hand calcium-binding proteins, was chemically synthesised using highly optimised 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/tert-butoxycarbonyl in situ neutralisation solid-phase chemistry. Circular dichroism studies indicated that CaCl(2) decreased the helical content by 27% whereas helicity was marginally increased by ZnCl(2). The propensity of S100A12 to dimerise was examined by electrospray ionisation time-of-flight mass spectrometry which clearly demonstrated the prevalence of the non-covalent homodimer (20890 Da). Importantly, synthetic human S100A12 in the nanomolar range was chemotactic for neutrophils and macrophages in vitro.

Poly-arginine-fused calpastatin peptide, a living cell membrane-permeable and specific inhibitor for calpain.

Calpain, a Ca2+-dependent neutral protease, is highly related to the pathogenesis of a variety of disorders and its inhibitors offer potential for therapeutic intervention. General calpain inhibitors, however, have the disadvantage of a lack of specificity or poor cellular permeability or oxidization under physiological conditions. Here, we developed a membrane-permeable specific calpain inhibitor by fusing calpastatin peptide (CS) and 11 poly-arginine peptides (11R). The 11R-fused CS (11R-CS) effectively penetrated across the plasma membrane of living neurons and significantly inhibited calpain activity in the cells.

Gelsolin immunoreactivity in corneal amyloid, wound healing, and macular and granular dystrophies.

Immunohistologic studies of tissue sections obtained from patients with type 1 or type 2 lattice corneal dystrophy, polymorphic amyloid degeneration, or gelatinous amyloid degeneration were performed by using a monoclonal antibody raised to a chymotryptic fragment inclusive of the carboxy-terminal half of plasma gelsolin, and also with a series of polyclonal antibodies specific for synthetic peptides corresponding to immunogenic epitopes of gelsolin. These epitopes are parts of sequences at the amino- and carboxy-terminal ends of gelsolin, as well as adjacent to and inclusive of the codon 187 mutant 7-11 kD fragment that has been shown to be the subunit protein of amyloid fibrils occurring systemically in patients affected by Finnish type familial amyloidosis. These antibodies were also tested on tissue sections obtained from patients with granular and macular corneal dystrophy, corneal wounds, and normal control corneas. Specificity of staining was established by absorption with gelsolin purified from plasma, or the appropriate synthetic peptide. Gelsolin immunoreactivity was detected in the conjunctival and skin amyloid in familial amyloidosis by using familial amyloid (Finnish type) antibody. In other types of corneal amyloid, including lattice dystrophy type 1, immunoreactivity with gelsolin and synthetic peptides was observed adjacent to the deposits, but rarely within them. In macular dystrophy, variable staining of the deposits could result from the association of subunit proteins with glycosaminoglycans.

[Synthesis of tetracontapeptide--a hypothetical evolutionary precursor of calcium-binding proteins. I. Synthesis of (1-9), (10-14), (15-20), (21-26), (29-33), (34-40) fragments]. / Sintez tetrakontapeptida--gipoteticheskogo évoliutsionnogo predshestvennika kal'tsiisviazyvaiushchikh belkov. I. Sintez fragmentov (1-9), (10-14), (15-20), (21-26), (29-33), (34-40).

Fragments (1-9), (10-14), (15-20), (21-26), (29-33) and (34-40) of a tetracontapeptide hypothetical ancestor of calcium-binding proteins were synthesised with the use of pentafluorophenyl esters. Formation of a succinimide derivative was detected during synthesis of fragment (15-20) containing Asp(OBzl)-Gly sequence. To avoid this side process, tert-butylprotecting group was used instead of benzyl group. alpha-Carboxyls of C-terminal amino acids were protected by phenacyl group.

[Synthesis of tetracontapeptide--a hypothetical evolutionary precursor of calcium-binding proteins. II. Condensation of fragments]. / Sintez tetrakontapeptida--gipoteticheskogo évoliutsionnogo predshestvennika kal'tsiisviazyvaiushchikh belkov. II. Kondensatsiia fragmentov.

A tetracontapeptide was obtained by condensation of synthetic fragments (see the preceding paper) with the use of pentafluorophenyl esters as well as with carbodiimide and hydroxybenzotriazole. Racemization during fragment condensation was 1-2 per cent. Deblocking of the protected tetracontapeptide was carried out by treatment with trifluoroacetic acid and hydrogen fluoride with thioanisole. The obtained peptide was purified by gel-chromatography and HPLC.

Discovery and structural characterization of a phospholamban-binding cyclic peptide and design of novel inhibitors of phospholamban.

The interplay between cardiac sarcoplasmic Ca(2+)ATPase and phospholamban is a key regulating factor of contraction and relaxation in the cardiac muscle. In heart failure, aberrations in the inhibition of sarcoplasmic Ca(2+)ATPase by phospholamban are associated with anomalies in cardiac functions. In experimental heart failure models, modulation of the interaction between these two proteins has been shown to be a potential therapeutic approach. The aim of our research was to find molecules able to interfere with the inhibitory activity of phospholamban on sarcoplasmic Ca(2+)ATPase. For this purpose, a portion of phospholamban was synthesized and used as target for a phage-display peptide library screening. The cyclic peptide C-Y-W-E-L-E-W-L-P-C-A was found to bind to phospholamban (1-36) with high specificity. Its functional activity was tested in Ca(2+)uptake assays utilizing preparations from cardiac sarcoplasmic reticulum. By synthesizing and testing a series of alanine point-mutated cyclic peptides, we identified which amino acid was important for the inhibition of the phospholamban function. The structures of active and inactive alanine-mutated cyclic peptides, and of phospholamban (1-36), were determined by NMR. This structure-activity analysis allowed building a model of phospholamban -cyclic peptide complex. Thereafter, a simple pharmacophore was defined and used for the design of small molecules. Finally, examples of such molecules were synthesized and characterized as phospholamban inhibitors.

Inhibition of human mu-calpain by conformationally constrained calpastatin peptides.

The 27-mer peptide CP1B-[1-27] derived from exon 1B of calpastatin stands out among the known inhibitors for mu- and m-calpain due to its high potency and selectivity. By systematical truncation, a 20-mer peptide, CP1B-[4-23], was identified as the core sequence required to maintain the affinity/selectivity profile of CP1B-[1-27]. Starting with this peptide, the turn-like region Glu(10)(i)-Leu(11)(i+1)-Gly(12)(i+2)-Lys(13)(i+3) was investigated. Sequence alignment of subdomains 1B, 2B, 3B and 4B from different mammalians revealed that the amino acid residues in position i+1 and i+2 are almost invariably flanked by oppositely charged residues, pointing towards a turn-like conformation stabilized by salt bridge/H-bond interaction. Accordingly, using different combinations of acidic and basic residues in position i and i+3, a series of conformationally constrained variants of CP1B-[4-23] were synthesized by macrolactamization utilizing the side chain functionalities of these residues. With the combination of Glu(i)/Dab(i+3), the maximum of conformational rigidity without substantial loss in affinity/selectivity was reached. These results clearly demonstrate that the linear peptide chain corresponding to subdomain 1B reverses its direction in the region Glu(10)-Lys(13) upon binding to mu-calpain, and thereby adopts a loop-like rather than a tight turn conformation at this site.

Backbone dynamics determined by electron paramagnetic resonance to optimize solid-phase peptide synthesis of TOAC-labeled phospholamban.

Electron paramagnetic resonance (EPR) was used to optimize the solid-phase peptide synthesis of a membrane-bound peptide labeled with TOAC (2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxylic acid). The incorporation of this paramagnetic amino acid results in a nitroxide spin label coupled rigidly to the alpha-carbon, providing direct detection of peptide backbone dynamics by EPR. We applied this approach to phospholamban, which regulates cardiac calcium transport. The synthesis of this amphipathic 52-amino-acid membrane peptide including TOAC is a challenge, especially in the addition of TOAC and the next several amino acids. Therefore, EPR of synthetic intermediates, reconstituted into lipid bilayers, was used to ensure complete coupling and 9-fluorenylmethoxycarbonyl (Fmoc) deprotection. The attachment of Fmoc-TOAC-OH leads to strong immobilization of the spin label, whereas Fmoc deprotection dramatically mobilizes it, producing an EPR spectral peak that is completely resolved from that observed before deprotection. Similarly, coupling of the next amino acid (Ser) restores the spin label to strong immobilization, giving a peak that is completely resolved from that of the preceding step. For several subsequent steps, the effect of coupling and deprotection is similar but less dramatic. Thus, the sensitivity and resolution of EPR provides a quantitative monitor of completion at each of these critical steps in peptide synthesis. Mass spectrometry, circular dichroism, and Edman degradation were used in concert with EPR to verify the chemistry and characterize the secondary structure. In conclusion, the application of conventional analytical methods in combination with EPR offers an improved approach to optimize the accurate synthesis of TOAC spin-labeled membrane peptides.

Rational design of peptide inhibitors of the sarcoplasmic reticulum calcium pump.

The sequence of phospholamban (PLB) is practically invariant among mammalian species. The hydrophobic transmembrane domain has 10 leucine and 8 isoleucine residues. Two roles have been proposed for the leucines; one subset stabilizes PLB oligomers, while a second subset physically interacts with SERCA. On the basis of the sequence of the PLB transmembrane domain, we chemically synthesized a series of peptides and tested their ability to regulate SERCA in reconstituted membranes. In all, eight peptides were studied: a peptide corresponding to the null-cysteine transmembrane domain of PLB (TM-Ala-PLB), two polyleucine peptides (Leu18 and Leu24), polyalanine peptides containing 4, 7, and 12 leucine residues (Leu4, Leu7, and Leu12, respectively), and a polyalanine peptide containing the 9 leucine residues present in the transmembrane domain of PLB with and without the essential Asn34 residue (Asn1Leu9 and Leu9, respectively). With the exception of Leu18, co-reconstitution of the peptides revealed effects on the apparent calcium affinity of SERCA. The TM-Ala-PLB peptide possessed approximately 70% of the inhibitory function of wild-type PLB. The remaining peptides exhibited significant inhibitory activity decreasing in the following order: Leu12, Leu9, Leu24, Leu7, and Leu4. Replacing Asn34 of PLB in the Leu9 peptide resulted in superinhibition of SERCA. On the basis of these observations, we conclude that a partial requirement for SERCA inhibition is met by a simple hydrophobic surface on a transmembrane alpha-helix. In addition, the superinhibition observed for the Asn34-containing peptide suggests that the model peptides mimic the inhibitory properties of PLB. A model is presented in which surface complementarity around key amino acid positions is enhanced in the interaction with SERCA.

Total chemical synthesis of human psoriasin by native chemical ligation.

Human psoriasin (S100A7), a member of the S100 family of calcium-binding proteins, is richly expressed in keratinocytes of patients suffering from psoriasis. To date, the exact physiological function of psoriasin abundant in many human cell types remains unclear. A recent report by Schröder and colleagues suggests that psoriasin, purified from human stratum corneum extracts, selectively kills Escherichia coli by sequestering Zn(2+) ions essential for bacterial growth, indicative of an important role in innate immune defense against microbial infection. We chemically synthesized the N-terminally acetylated psoriasin of 100 amino acid residues using solid phase peptide synthesis in combination with native chemical ligation. More than 140 mg of highly pure and correctly folded synthetic psoriasin was obtained from a single synthesis on a 0.25 mmol scale. Analysis of synthetic psoriasin by size exclusion chromatography showed that the protein forms a homodimer in solution. Circular dichroism analysis indicated that the alpha-helicity of psoriasin increases by more than 20% in the presence of CaCl(2) or ZnCl(2), suggesting a metal ion binding induced conformational change. Circular dichroism based titration further established that the synthetic protein binds two Ca(2+) and two Zn(2+) ions per dimer, in agreement with the published structural findings. Importantly, the ability of the synthetic protein to kill E. coli and the inhibition of the killing by ZnCl(2) is comparable to that of psoriasin isolated from its natural source. The robust synthetic access to large quantities of human psoriasin should facilitate studies of its biological functions as well as its mode of action.

The effects of Ca2+ binding on the dynamic properties of a designed Ca2+-binding protein.

The effects of Ca(2+) binding on the dynamic properties of Ca(2+)-binding proteins are important in Ca(2+) signaling. To understand the role of Ca(2+) binding, we have successfully designed a Ca(2+)-binding site in the domain 1 of rat CD2 (denoted as Ca.CD2) with the desired structure and retained function. In this study, the backbone dynamic properties of Ca.CD2 have been investigated using (15)N spin relaxation NMR spectroscopy to reveal the effect of Ca(2+) binding on the global and local dynamic properties without the complications of multiple interactive Ca(2+) binding and global conformational change. Like rat CD2 (rCD2) and human CD2 (hCD2), residues involved in the recognition of the target molecule CD48 exhibit high flexibility. Mutations N15D and N17D that introduce the Ca(2+) ligands increase the flexibility of the neighboring residues. Ca(2+)-induced local dynamic changes occur mainly at the residues proximate to the Ca(2+)-binding pocket or the residues in loop regions. The beta-strand B of Ca.CD2 that provides two Asp for the Ca(2+) undergoes an S(2) decrease upon the Ca(2+) binding, while the DE-loop that provides one Asn and one Asp undergoes an S(2) increase. Our study suggests that Ca(2+) binding has a differential effect on the rigidity of the residues depending on their flexibility and location within the secondary structure.