Direct inactivation of viruses by MCP-1 and MCP-2, natural peptide antibiotics from rabbit leukocytes.

Six homologous peptides were purified to homogeneity from rabbit granulocytes or alveolar macrophages and tested for their ability to inactivate herpes simplex virus type 1 (HSV-1). Two of the peptides, MCP-1 and MCP-2, showed considerable in vitro neutralizing activity, whereas four structurally homologous peptides (NP-3a, NP-3b, NP-4, and NP-5) were relatively ineffective. Inactivation of HSV-1 by MCP-1 or MCP-2 depended on peptide concentration and on the time, temperature, and pH of the incubation. HSV-2, vesicular stomatitis virus, and influenza virus A/WSN were also susceptible to direct neutralization by MCP-1 or MCP-2, whereas cytomegalovirus, echovirus type 11, and reovirus type 3 were not. We speculate that MCP-1 and MCP-2, peptides that are abundant in rabbit granulocytes and lung macrophages, may contribute to antiviral defenses by mediating the direct inactivation of HSV-1 and selected other viruses.     

A cardiac troponin T epitope conserved across phyla.

Troponin T is a thin filament protein that is important in regulating striated muscle contraction. We have raised a monoclonal antibody against rabbit cardiac troponin T, monoclonal (mAb) 13-11, that recognizes its epitope in cardiac troponin T isoforms from fish, bird, and mammal but not from frog. The number of these isoforms expressed in cardiac muscle varies among species and during development. Cardiac troponin T isoforms were not found in adult skeletal muscle, while they were expressed transiently in immature skeletal muscle. We have mapped the epitope recognized by mAb 13-11 using rabbit cardiac troponin T isoforms. Analysis of stepwise cyanogen bromide digestion, which allowed association of the epitope to regions spanning methionine residues, coupled with immunoactivity of synthetic peptides, corresponding to sequences containing methionine residues, indicated that mAb 13-11 recognized its epitope in a 17-residue sequence containing the methionine at position 68, SKPKPRPFMPNLVPPKI. Comparison of skeletal and cardiac troponin T sequences suggested that the epitope was contained within the sequence FMPNLVPPKI. Synthetic peptides PFMPNLVPPKI and FMPNLVPPKI were recognized by mAb 13-11 on slot-blots. Enzyme-linked immunosorbent assay demonstrated mAb 13-11 recognized, in order of descending affinity, the 17-, 11-, and 10-residue sequence. Preabsorption of mAb 13-11 with each of these sequences blocked the recognition of the 17-residue peptide by mAb 13-11. The domain, PFMPNLVPPKI is encoded by the 5' region of the cardiac gene exon 10 and is present in hearts across a broad range of phyla. These findings suggest that this cardiac troponin T-specific sequence confers onto myofilaments structural and functional properties unique to the heart.     

Antibacterial and haemolytic peptides containing D-alloisoleucine from the skin of Bombina variegata.

A family of bombinin-related peptides is present in the skin of Bombina variegata. These peptides contain 27 residues with Gly as N-terminus and display antimicrobial activity. From sequence analysis of the cDNAs encoding for the corresponding peptide precursors, the presence of a novel 20-residue peptide with Ile as N-terminus was predicted. We have now purified a family of hydrophobic peptides named H1-H5, whose sequences correspond to the predicted peptide with some variability in positions 1, 2 and 8. In particular, H3-H5 contain a D-alloisoleucine residue in the second position. All these peptides display antibacterial and haemolytic activity.     

Structure-activity relationships of antimicrobial peptides from the skin of Rana esculenta inhabiting in Korea

The anuran (frogs and toads) skin is a rich source of antimicrobial peptides that can be developed therapeutically. We searched the skin secretions of Korean Rana esculenta for antimicrobial peptides, and isolated two cationic peptides with antimicrobial activity and little hemolytic activity: a 46-residue peptide of the esculentin-1 family and a 24-residue peptide of the brevinin-1 family. Their sequences showed some differences from the esculentins-1 and brevinins-1 of European Rana esculenta, indicating that sequence diversification of anuran skin antimicrobial peptides can arise from differences in habitat as well as from species differences. The 46-residue peptide named esculentin-1c had broad antimicrobial activity, while the 24-residue peptide named brevinin-1Ed exhibited limited activity. The solution structure of brevinin-1Ed was in good agreement with that of other brevinin-1-like peptides, with an amphipathic alpha-helix spanning residues 3-20, stabilized in membrane-mimetic environments. The weak bioactivity of brevinin-1Ed was attributable to the unusual presence of an anionic amino acid in the middle of the helical hydrophilic face. This report contributes to world-wide investigations of the structure-activity relationships and evolutional diversification of anuran-skin antimicrobial peptides.     

Structural and biological studies on synthetic peptide analogues of a low-affinity calcium-binding site of skeletal troponin C

We have synthesized four oligopeptides that are structural analogues of a low-affinity Ca2+-specific binding site (site II) of rabbit skeletal troponin C. One analogue (peptide 3) was a dodecapeptide with a sequence corresponding to the 12-residue Ca2+-binding loop (residues 63-74 in troponin C), two (peptides 4 and 5) were 23-residue in length, corresponding to residues 52-74 of the protein, and the fourth (peptide 6) was a 25-residue peptide corresponding to residues 50-74. All four peptides had one amino acid substitution within the 12-residue binding loop in which phenylalanine at position 10 was replaced by tyrosine to provide a marker for spectroscopic studies. In addition, peptides 3 and 4 each had a second substitution within the binding loop where glycine at position 6 was replaced by alanine. The second substitution was motivated by the conservation of glycine at the position in the Ca2+-binding loops of all four Ca2+-binding sites in troponin C. The peptides were characterized by their intrinsic fluorescence, ability to enhance the emission of bound Tb3+, affinity for Ca2+ and Tb3+, and circular dichroism. The affinity for Ca2+ was in the range 10-10(2) M-1, and the affinity for Tb3+ was in the range 10(4)-10(5) M-1. The binding constants of the longer peptides were several-fold larger than that of the dodecapeptide. With peptides 4 and 5, substitution of glycine by alanine at position 6 within the 12-residue loop decreased the affinity for Ca2+ by a factor of four, but had little effect on the affinity for Tb3+. However, the mean residue ellipticity of peptide 4 was substantially higher than that of peptide 5. Since peptide 4 differs from peptide 5 only in the substitution of glycine at position 6 in the loop segment, the conservation of glycine at that position may serve a role in providing a suitable secondary structure of the binding sites for interaction with troponin I. Peptides 4 and 6, when present in a large excess, mimic troponin C in regulating fully reconstituted actomyosin ATPase by showing partial calcium sensitivity and activation of the ATPase. Since these peptides are the smallest peptides containing the Ca2+-binding loop of site II, their biological activity suggests that a Ca2+-dependent binding site of troponin C for troponin I could be as short as the segment comprising residues 52-62.     

NMR studies of defensin antimicrobial peptides. 1. Resonance assignment and secondary structure determination of rabbit NP-2 and human HNP-1.

Two-dimensional nuclear magnetic resonance spectroscopy has been used to make resonance assignments of the proton spectra of two defensin antimicrobial peptides, human neutrophil peptide HNP-1 and rabbit neutrophil peptide NP-2. The secondary structures of these peptides were determined from analysis of the proton-proton NOEs and from the positions of slowly exchanging amide protons. Both peptides contain a long stretch of a double-stranded antiparallel beta-sheet in a hairpin conformation that contains a beta-bulge, a short region of triple-stranded beta-sheet, and several tight turns. The NMR results clearly show that HNP-1 forms a dimer or higher order aggregate in solution and that Pro8 exists as a cis peptide bond. The NMR data on these peptides are compared with NMR data for a homologous peptide NP-5 [Bach, A. C., Selsted, M. E., & Pardi, A. (1987) Biochemistry 26, 4389-4397]. Analysis of the conformation-dependent proton chemical shifts shows that it is not possible to confidently judge the structural similarity of the three defensins from chemical shift data alone. However, comparison of the 3JHN alpha coupling constants in NP-2 and NP-5 indicates that the backbone conformations for these peptides are very similar. A more detailed comparison of the solution conformations of the defensins peptides is made in the following paper in this issue where the NMR data are used as input for distance geometry and molecular dynamics calculations to determine the three-dimensional structures of HNP-1 and NP-2.     

Hydrolysis of alpha(s, 1)-Casein B by Streptococcus lactis Membrane Proteinase

The membrane-associated proteinase of Streptococcus lactis strain 3 hydrolyzed α_{s, 1}-casein B into 11 peptide fragments. Eight of the 11 peptides were purified and partially characterized. Each peptide contained several, but not all six, essential amino acids required for growth. The culture was able to utilize one peptide as the sole source for the essential amino acid leucine. Leucine, serine, valine, and glycine were found to be NH₂-terminal residues. Two of the peptides were phosphopeptides. The data support the functional role of the membrane-associated proteinase as being involved in the initial breakdown of proteins to peptides.     

Investigating the tolerance of coiled-coil peptides to nonheptad sequence inserts

Coiled-coil motifs foster a wide variety of protein-protein interactions. Canonical coiled coils are based on 7-residue repeats, which guide the folding and assembly of amphipathic alpha-helices. In many cases such repeats remain unbroken for tens to hundreds of residues. However, the sequences of an increasing number of putative and characterised coiled coils digress from this pattern. We probed the consequences of nonheptad inserts using a designed leucine-zipper system. The parent peptide, SKIP0, which had four contiguous heptads, was confirmed as a parallel homodimer by circular dichroism spectroscopy and analytical ultracentrifugation. Seven daughter peptides were constructed in which 1 to 7 alanine residues were inserted between the central heptads of SKIP0. Like SKIP0, SKIP7 formed a stable helical dimer, but the other peptides were highly destabilised, with the order of dimer stability SKIP4 > SKIP5 > SKIP6 > SKIP3 > SKIP2 > SKIP1. These results are consistent with an extended theory of coiled-coil assembly in which coiled-coil-compatible motifs are based on 3- and 4-residue spacings and most notably heptad (7-residue) and hendecad (11-residue) repeats. Thus, they help explain why in natural sequences, inserts after canonical heptad repeats most commonly comprise 4 residues. Possible biological roles for nonheptad inserts are discussed.     

Structure of dog and rabbit precursors of atrial natriuretic polypeptides deduced from nucleotide sequence of cloned cDNA

The structure of precursors of dog and rabbit atrial natriuretic polypeptides was determined by nucleotide sequence analysis of cloned cDNA of mRNA encoding the peptides. The dog and rabbit precursors are 149 and 153 residues long having 23- and 25-residue putative signal peptides at their N-termini respectively. The 28-residue peptide with identical sequence to that of human, which has potent natriuretic activity, is located at the C-terminus of the dog precursor. The 28-residue peptide of identical sequence to that of mouse/rat is located at C-terminus of rabbit precursor followed by additional Arg-Arg sequence which is also found in rat/mouse precursors and is apparently removed during processing.     

大熊猫乳酸脱氢酶同工酶M_4胰酶水解后的HPLC肽谱分析

 比较了大熊猫与猪LDH-M_4用胰酶水解后的HPLC肽谱;对分离出的各个肽段测定了氨基酸组成与N-末端。经分析,在两者各有的35个肽段中,22个肽段有相同的氨基酸组成与N-末端且在HPLC图谱上有相同的保留时间。另外有13个肽段在氨基酸组成与保留时间上存在差异。对大熊猫LDH-M中部分肽段测定了氨基酸残基序列。结果表明,与结合NAD~+有关的12肽的序列与一级结构已知的猪LDH-M含有Cys165的相应肽段完全一样;在与底物结合部位含有His191的35肽中,两者只有一个氨基酸残基的差异。在N-端的21肽中,有3个残基出现差异;而在C-端的14肽中,仅出现一个残基的差异。     

Translocating proline-rich peptides from the antimicrobial peptide bactenecin 7

The intracellular delivery of most peptides, proteins, and nucleotides to the cytoplasm and nucleus is impeded by the cell membrane. To allow simplified, noninvasive delivery of attached cargo, cell-permeant peptides that are either highly cationic or hydrophobic have been utilized. Because cell-permeable peptides share half of the structural features of antimicrobial peptides containing clusters of charge and hydrophobic residues, we have explored antimicrobial peptides as templates for designing cell-permeant peptides. We prepared synthetic fragments of Bac 7, an antimicrobial peptide with four 14-residue repeats from the bactenecin family. The dual functions of cell permeability and antimicrobial activity of Bac 7 were colocalized at the N-terminal 24 residues of Bac 7. In general, long fragments of Bac(1-24) containing both regions were bactericidal and cell-permeable, whereas short fragments with only a cationic or hydrophobic region were cell-permeant without the attendant microbicidal activity when measured in a fluorescence quantitation assay and by confocal microscopy. In addition, the highly cationic fragments were capable of traversing the cell membrane and residing within the nucleus. A common characteristic shared by the cell-permeant Bac(1-24) fragments, irrespective of their number of charged cationic amino acids, is their high proline content. A 10-residue proline-rich peptide with two arginine residues was capable of delivering a noncovalently linked protein into cells. Thus, the proline-rich peptides represent a potentially new class of cell-permeant peptides for intracellular delivery of protein cargo. Furthermore, our results suggest that antimicrobial peptides may represent a rich source of templates for designing cell-permeant peptides.     

Sequence motifs important for peptide binding to the human MHC class I molecule, HLA-A2.

Previous studies have indicated that most HLA-A2-binding peptides are 9 amino acid (aa) residues long, with a Leu at position 2 (P2), and a Val or Leu at P9. We compared the binding properties of different peptides by measuring the rate of dissociation of beta 2-microglobulin from peptide-specific HLA-A2 complexes. The simplest peptide that we identified that could form HLA-A2 complexes had the sequence (in single letter aa code) GLFGGGGGV, indicating that three nonglycine aa are sufficient for binding to HLA-A2. To determine whether most nonapeptides that contained Leu at P2 and Val or Leu at P9 could bind to HLA-A2, we tested the binding of nonapeptides selected from published HIV and melanoma protein sequences, and found that six of seven tested formed stable HLA-A2 complexes. We identified an optimal antigenic undecapeptide from the cytomegalovirus gB protein that could form stable HLA-A2 complexes that contained apparent anchor residues at P2 and P11 (sequence FIAGN-SAYEYV), indicating that the spacing between anchor residues can be somewhat variable. Finally, we tested the importance of every aa in the influenza A matrix peptide 58-66 (sequence GILGFVFTL) for binding to HLA-A2, by using Ala-substituted and Lys-substituted peptides. We found that multiple positions were important for stable binding, including P2, P3, P5-P7, and P9. We conclude that the P2 and P9 anchor residues are of prime importance for peptide binding to HLA-A2. However, other peptide side chains (especially at P3) contribute to the stability of the interaction. In certain cases, the optimal length for peptide binding can be longer than 9 residues.     

Purification of cationic cystine-rich peptides from rat bone marrow. Primary structures and biological activity of the rat corticostatin family of peptides.

Seven cationic, cystine-rich peptides of 29 to 32 amino acid residues have been purified from extracts of rat bone marrow (R-1, R-1a, R-1b, R-2, R-3, R-4 and R-5). Structural analysis clearly indicated that all seven peptides belong to the corticostatin/defensin family of leukocyte-derived peptides known to participate in oxygen-independent killing of phagocytosed bacteria. For R-1 to R-5, six cysteine residues were found at characteristic and highly conserved positions. R-1a and R-1b were partially characterized and appear to be structural variants of R-1. Aside from the conserved cysteines, there is a remarkable degree of structural diversity evident within the sequences of those members of the corticostatin/defensin family characterized so far. The structures of the peptides that we have purified can be compared directly with the sequences obtained for rat defensins isolated from extracts of peritoneal neutrophils (Lehrer, Ganz and Selsted, Cell, 64 (1991) 229-230). Some discrepancies are apparent which can be explained in terms of proteolytic cleavage of several of these peptides at both amino- and carboxyl-termini. The corticostatins owe their bioactivity to their ability to compete with corticotropin for occupancy of the corticotropin receptor (Zhu, Hu, Mulay, Esch, Shimasaki and Solomon, Proc. Natl. Acad. Sci. USA, 85 (1988) 592-596). The potency of these peptides can be expressed in terms of their capacity to inhibit the steroidogenic response of isolated rat adrenocrotical cells half-maximally stimulated by corticotropin (i.e., at the ED50 concentration for corticotropin in this assay, namely 33 pM). In this assay, the rat peptides R-1, R-2 and R-3 were shown to be inactive. In contrast, the more cationic peptides R-4 and R-5 were found to inhibit steroidogenesis. R-4 was somewhat less active than rabbit corticostatin (IC50 25 nM) showing an IC50 value of 50 nM. R-5 appeared to be significantly less potent than R-4. The lower yield of R-5 precluded an accurate estimate of the corticostatic potency of this peptide. R-4 differs in structure from R-5 in having an arginine to serine substitution at position 7. It can be concluded that an arginine at this position accounts, at least in part, for the corticostatic activity of R-4.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of staphylococcal delta-toxin and synthetic analogues with erythrocytes and phospholipid vesicles. Biological and physical properties of the amphipathic peptides

Staphylococcal delta-toxin, a 26-residue amphiphilic peptide is lytic for cells and phospholipid vesicles and is assumed to insert as an amphipathic helix and oligomerize in membranes. For the first time, the relationship between these properties and toxin structure is investigated by means of eight synthetic peptides, one identical in sequence to the natural toxin, five 26-residue analogues and two shorter peptides corresponding to residues 1-11 and 11-26. These peptides were designed by the Edmundson wheel axial projection in order to maintain: (a) the hydrophilic/hydrophobic balance while rationalizing the sequence, (b) the alpha-helical configuration and (c) the common epitopic structure. The fluorescence of the single Trp residue was used to monitor the behaviour of the natural toxin and analogues. All 26-residue analogues were hemolytically active although to a lesser extent than natural toxin. The peptide of residues 11-26 bound lipids weakly and was hemolytic at high concentration. The peptide of residues 1-11 did not bind lipids and was hemolytically inactive. All peptides except the latter cross-reacted in immunoprecipitation tests with the natural toxin. The study of a 26-residue analogue by circular dichroism revealed an alpha-helical configuration in both the free and lipid-bound state. Changes in the fluorescence of the peptides in the presence of lipid micelles and bilayers varied according to the position of the reporter group. When bound to lipids, Trp5, Trp16 and the Fmoc-1 positions of the analogues became buried while Trp15 of the natural toxin and its synthetic replicate remained more exposed. All changes are rationalized by the proposal of an amphipathic helix whose hydrophobic face is embedded within the apolar core of bilayers while the hydrophilic and charged face remains more exposed to solvent.     

The complete amino acid sequence of rabbit muscle phosphoglucomutase

The complete amino acid sequence of rabbit muscle phosphoglucomutase has been determined by isolating the 11 peptide fragments produced by the cyanogen bromide cleavage reaction and subjecting these to automated sequencing procedures. Products produced by treatment of some of these fragments with hydroxylamine, iodosobenzoic acid, mild acid, cyanogen bromide in formic and heptafluorobutyric acids, Staphylococcus aureus V8 protease, and trypsin (with or without blocking at lysine residues) were used to complete the sequence for each of the cyanogen bromide fragments. The cyanogen bromide fragments were ordered by isolating the four tryptic peptides produced by a limited tryptic digest of the native enzyme in the presence of its substrates and its bivalent metal ion activator, Mg2+, degrading these by means of trypsin, after blocking digestion at lysine residues, and isolating and identifying all fragments thus produced that contained 10 or more residues. The 561-residue sequence thus obtained is one of the longest that has been determined by chemical means. There is excellent agreement between this sequence and published compositions after appropriate normalization. The absorbance of the enzyme is about 7.0 at 278 nm for a 1% solution; this value is 9% lower than that previously used.     

Evidence for the expression of three genes encoding homologous atrial gland peptides that cause egg laying in Aplysia

Three peptide complexes which can induce egg laying in Aplysia were isolated from the atrial gland of the marine mollusc Aplysia californica and chemically characterized. Amino acid sequence analyses established the covalent structures, including disulfide assignments, of all three dimeric complexes. Each complex consisted of an identical 18-residue peptide (A-AP) which was disulfide-bonded to a 36-residue peptide that was homologous to bag cell egg-laying hormone (ELH). The primary structure of A-AP was determined to be: NH2-Asp-Ser-Asp-Val-Ser-Leu-Phe-Asn-Gly-Asp-Leu-Leu-Pro-Asn-Gly-Arg-Cys- Ser-COOH. The primary structure of one of the three ELH-related peptides (A-ELH) was determined to be NH2-Ile-Ser-Ile-Asn-Gln-Asp-Leu-Lys-Ala-Ile-Thr-Asp-Met-Leu-Leu-Thr-Glu- Gln-Ile-Gln-Ala-Arg-Arg-Arg-Cys-Leu-Asp-Ala-Leu-Arg-Gln-Arg-Leu-Leu-Asp- -Leu-COOH. The two other ELH-related peptides, [Ala27]A-ELH and [Gln23, Ala27]A-ELH, differed from A-ELH at 1 and 2 residues, respectively. Both [Ala27] A-ELH and [Gln23, Ala27]A-ELH were novel peptide sequences representing products of as yet uncharacterized genes within the ELH family. These structural studies provide the first direct chemical evidence that an 18-residue peptide (A-AP) derived from a polypeptide precursor encoded by the A gene, as predicted from nucleotide sequence analysis, occurs in the atrial gland; the Cys17 residue of A-AP is disulfide-bonded to Cys25 of A-ELH; and A-AP also occurs disulfide-bonded to two additional, previously undescribed ELH-related peptides, [Ala27]A-ELH and [Gln23, Ala27]A-ELH.     

Phosphorylation of rabbit skeletal muscle glycogen synthase by casein kinase 1: Evidence of phosphorylation sites specific for casein kinase 1

Casein kinase 1 phosphorylated rabbit skeletal muscle glycogen synthase at both seryl and threonyl residues. With glycogen synthase phosphorylated up to 7.5 mol phosphate/mol subunit, about 26% of the phosphate was present in the N-terminal cyanogen bromide fragment (CB1) and 74% in the C-terminal fragment (CB2). Both fragments contained phosphothreonine (11 to 14%) in addition to phosphoserine. When ³²P-labeled glycogen synthase was totally digested with trypsin and chromatographed on reversephase high-performance liquid chromatography, seven phosphopeptides were observed. Peptide I eluted in the vicinity of the peptide containing site 1a, peptide II coincided with sites 4 + 5, peptides III and IV eluted in the region corresponding to sites 3a + 3b + 3c, peptide V appeared slightly after the peptide containing site 1b and peptide VII behaved as the peptide containing site 2, whereas peptide VI did not coincide with any of the known phosphopeptides. Limited trypsinization prior to analysis by HPLC led to the disappearance of peaks V and VI without altering peaks I to IV and VII. Only peaks I and VII remained when limited chymotrypsinization was performed prior to HPLC analysis. Chromatography on HPLC of the fragments derived from complete trypsinization of CB2 showed the presence of peaks II to VI. Phosphoamino acid analysis of the different peptides demonstrated the presence of quantitative amounts of phosphothreonine in peptides V, VI, and VII. These results indicate that multiple phosphorylation sites for casein kinase 1 must exist in both the N-terminal and C-terminal regions of glycogen synthase, some of which would only be labeled by casein kinase 1.     

Influence of glycine residues on peptide conformation in membrane environments.

Transmembrane (TM) segments of integral membrane proteins are putatively alpha-helical in conformation, yet their primary sequences are rich in residues known in globular proteins as helix-breakers (Gly) and beta-sheet promoters (Ile, Val, Thr). To examine the specific 2 degrees structure propensities of such residues in membrane environments, we have now designed and synthesized a series of model 20-residue peptides with "guest" hydrophobia segments embedded in "host" N- and C-terminal hydrophilic matrices. Molecular design was based on the prototypical sequence NH2-(Ser-Lys)2-Ala5-Leu6-x7-Ala8-Leu9-y10-Trp 11-Ala12-Leu13-z14-(Lys-Ser)3-OH. The 10-residue hydrophobic mid-segment 5-14 is expected to act as ca. three turns of an alpha-helix. In the present work, we compare the 20-residue peptide having three "helix-forming" Ala residues [x = y = z = Ala (peptide 3A)] to the corresponding peptide 3G (x = y = z = Gly) which contains three "helix-breaking" Gly residues. Trp was inserted to provide a measure of aromatic character typical of TM segments; Ser and Lys enhanced solubility in aqueous media. Circular dichroism studies in water, in a membrane-mimetic [sodium dodecylsulfate (SDS)], medium, and in methanol solutions, demonstrated the exquisite sensitivity of the conformations of these peptides to environment, and proved that despite its backbone flexibility, Gly can be accommodated as readily as Ala into a hydrophobic alpha-helix in a membrane. Nevertheless, the relative stability of Ala- vs. Gly-containing helices emerged in methanol solvent titration and temperature dependence experiments in SDS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vitamin K-dependent carboxylation. A synthetic peptide based upon the gamma-carboxylation recognition site sequence of the prothrombin propeptide is an active substrate for the carboxylase in vitro

The vitamin K-dependent blood-clotting proteins contain a gamma-carboxylation recognition site in the propeptide, between the signal peptide and the mature protein, that directs gamma-carboxylation of specific glutamic acid residues. To develop a better substrate for the in vitro assay of the vitamin K-dependent gamma-carboxylase and to understand the substrate recognition requirements of the carboxylase, we prepared synthetic peptides based upon the structure of human proprothrombin. These peptides were employed as substrates for in vitro carboxylation using a partially purified form of the bovine liver carboxylase. A 28-residue peptide (HVFLAPQQARSLLQRVRRANTFLEEVRK), based on residues -18 to +10 in proprothrombin, includes the complete propeptide and the first 10 residues of acarboxyprothrombin. Carboxylation of this peptide is characterized by a Km of 3.6 microM. In contrast, FLEEL is carboxylated with a Km of about 2200 microM. A 10-residue peptide (ANTFLEEVRK), based on residues +1 to +10 in prothrombin, and a 20-residue peptide (ARSLLQRVRRANTFLEEVRK), based on residues -10 to +10 in proprothrombin, are also poor substrates for the carboxylase. Replacement of phenylalanine with alanine at residue 3 (equivalent to position -16 in proprothrombin) in the 28-residue peptide significantly alters the Km to 200 microM. A synthetic propeptide (HVFLAPQQARSLLQRVRRY), homologous to residues -18 to -1 in proprothrombin, inhibited carboxylation of the 28-residue peptide substrate with a Ki of 3.5 microM, but modestly stimulated the carboxylation of the 5- and 10-residue peptide substrates. These results indicate that an intact carboxylation recognition site is required for efficient in vitro carboxylation and that this site includes critical residues in region -18 to -11 of proprothrombin. The carboxylation recognition site in the propeptide binds directly to the carboxylase or to a closely associated protein.     

Identification of a novel Mcl-1 protein binding motif

Recent characterization of Mcl-1 as the primary anti-apoptotic Bcl-2 family member expressed in solid tumors, coupled with its ability to enable therapeutic resistance, has provided the impetus for further study into how Mcl-1 is involved in apoptosis signaling. Here, we employ Sabutoclax, a potent and effective Mcl-1 antagonist, as a competing agent to screen a randomized 12-residue phage display library for peptides that bind strongly to the Bcl-2 homology 3 (BH3) binding groove of Mcl-1. Although the screen identified a number of α-helical peptides with canonical BH3 domain sequences, it also isolated a pair of unique peptide sequences. These sequences exhibit a reverse organization of conserved hydrophobic and acidic residues when compared with canonical BH3 sequences, and we therefore refer to them as reverse BH3 (rBH3) peptides. Furthermore, studies of the rBH3 peptides using NMR spectroscopy, fluorescence polarization displacement assays, and alanine scanning data all suggest that they bind to the BH3 binding groove of Mcl-1 selectively over Bcl-x(L). A search for proteins containing the rBH3 motif has identified a number of interesting Mcl-1 protein partners, some of which have previously been associated with apoptosis regulation involving Mcl-1. These findings provide insights into the development of more specific Mcl-1 antagonists and open the way to the identification of a previously unknown family of apoptosis-regulating and Mcl-1 interacting proteins.