Structural and functional modeling of human lysozyme reveals a unique nonapeptide, HL9, with anti-HIV activity

We previously reported that lysozyme accounts for anti-HIV activity associated with the beta-core fraction of human chorionic gonadotropin [Lee-Huang, S., Huang, P. L., Sun, Y., Kung, H. F., Blithe, D. L. & Chen, H. C. (1999) Proc Natl Acad Sci U S A 96, 2678-81]. To define the structural and sequence requirements for anti-HIV activity, we carried out peptide fragmentation and activity mapping of human lysozyme. We identified two peptides that consist of 18 and 9 amino acids of human lysozyme (HL18 and HL9), corresponding to residues 98-115 and 107-115. HL18 and HL9 are potent inhibitors of HIV-1 infection and replication with EC(50)s of 50 to 55 nM, comparable to intact lysozyme. Scrambling the sequence or substitution of key arginine or tryptophan residues results in loss of antiviral activity. HL9, with the sequence RAWVAWRNR, is the smallest peptide we identified with full anti-HIV activity. It forms a pocket with its basic residues on the surface of the molecule. HL9 exists as an alpha-helix in native human lysozyme, in a region of the protein distinct from the muramidase catalytic site. Monte Carlo peptide folding energy minimizing simulation modeling and CD studies indicate that helical propensity does not correlate with antiviral activity. HL9 blocks HIV-1 viral entrance and replication, and modulates gene expression of HIV-infected cells, affecting pathways involved in survival, stress, TGFbeta, p53, NFkappaB, protein kinase C and hedgehog signaling.     

Biological activity of synthetic peptides analogous to heat-stable enterotoxin produced by Yersinia enterocolitica

Abstract 3 peptides were synthesized chemically by following the primary structure of heat-stable enterotoxin (ST) produced by Yersinia enterocolitica . A peptide 1–30, having the whole sequence of 30 amino-acid residues, showed a ST activity similar to that of analogue peptide 15–30 composed of the C-terminal 16 amino acid residues. The c-GMP levels of L cells increased through an interaction with peptide 1–30 but not with peptide 15–30, while membranes isolated from broken L cells responded to both. Peptide 1–11, composed of the N-terminal 11 amino-acid residues, showed no biological activity.     

Cyclic and linear peptides derived from alpha-amylase inhibitory protein tendamistat

Tendamistat is a strong inhibitory protein of porcine pancreatic alpha-amylase (PPA) with a K(i) value of 0.2 nM. To develop potent alpha-amylase inhibitors, we synthesized six odd-length cyclic peptides (5-15 residues) and four even-length cyclic peptides (10 and 12 residues) having the inhibitory sequence of tendamistat. Their PPA inhibitory activities were evaluated, and, among them, the 11-residue cyclic peptide Ten(15-23) (K(i) = 0.27 microM) exhibited the strongest inhibitory activity (K(i) = 0.27-1.41 microM). To examine the effect of cyclic structure on PPA inhibition, ten linear peptides corresponding to the cyclic peptides were also synthesized, and their PPA inhibitory activities were evaluated (K(i) = 0.28-1.00 microM). Interestingly, the 11-residue linear peptide Ten(15-23) exhibited almost the same inhibitory activity (K(i) = 0.28 microM) as that of cyclic Ten(15-23). The results of a circular dichroism study indicated that stabilization of the beta-hairpin structure occurred only for cyclic Ten(15-23). Also, the results of proteolytic digestion experiments of the cyclic and linear Ten(15-23) peptides by trypsin and chymotrypsin suggested no differences in protease resistance between the cyclic and linear structures. Therefore, we demonstrated that both cyclic and linear peptides containing the inhibitory sequence of tendamistat exhibit potent PPA inhibitory activity.     

Deletion of two C-terminal Gln residues of 12-26-residue fragment of melittin improves its antimicrobial activity

In our previous paper it was shown that the two C-terminal Gln residues of a C-terminal 15-residue fragment, Mel(12-26) (GLPALISWIKRKRQQ-NH2), of melittin and a series of individual substituted analogues might not involved in the interaction with bacterial membranes. In this paper, peptides with one and two Gln residues deletion, respectively, Mel(12-25) and Mel(12-24), were synthesized and characterized. Both of the deletion peptides showed higher antimicrobial activities than the parent peptide, Mel(12-26). If both of the Gln residues of Mel(12-26) were respectively replaced by a hydrophilic amino acid Gly, the antimicrobial activity increased slightly. If the Gln residue of Mel(12-25) was replaced by a hydrophobic amino acid Leu, the antimicrobial activity changed little, although the substituted peptide possessed much higher hydrophobicity and higher alpha-helical conformation percentage in 1,1,1,3,3,3-hexafluoro-2-propanol/water determined by circular dichroism spectroscopy (CD) than the parent peptide. These results indicated that the two C-terminal residues might be indeed not involved in the binding to bacterial membranes. The antimicrobial activity increasing with the residue deletion may be caused by the decrease of the translational and rotational entropic cost of the binding of the peptides to bacterial membranes because of the lower molecular weights of the deletion peptides.     

Substitution of Ser61----Gly61 in human apolipoprotein C-II does not alter its activation of lipoprotein lipase

Lipoprotein lipase (LpL) activity is enhanced by apolipoprotein C-II (apoC-II), a 79 amino acid residue peptide. The minimal apoC-II sequence required for activation of LpL resides between residues 56-79. To determine the possible role of an acyl-apoC-II intermediate involving Ser61 in enzyme catalysis, a synthetic peptide of apoC-II containing residues 56-79 was synthesized and compared to the corresponding peptide with serine at position 61 being substituted with glycine. With two different LpL assay systems, both peptides enhanced enzyme activity. Since glycine does not contain a hydroxyl group, these results rule out the possibility that an acyl-apoC-II intermediate with Ser61 is required for enzyme activation.     

Design,synthesis and activity evaluation of novel peptide fusion inhibitors targeting HIV-1 gp41

Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes about 2 million people to death every year. Fusion inhibitors targeted the envelope protein (gp41) represent a novel and alternative approach for anti-AIDS therapy, which terminates the HIV-1 life cycle at an early stage. Using CP621-652 as a template, a series of peptides were designed, synthesized and evaluated in vitro assays. An interesting phenomenon was found that the substitution of hydrophobic residues at solvent accessible sites could increase the anti-HIV activity when the C-terminal sequence was extended with an enough numbers of amino acids. After the active peptides was synthesized and evaluated, peptide 8 showed the best anti-HIV-1 IIIB whole cell activity (MAGI IC₅₀ = 53.02 nM). Further study indicated that peptide 8 bound with the gp41 NHR helix, and then blocked the conformation of 6-helix, thus inhibited virus–cell membrane fusion. The results would be helpful for the design of peptide fusion inhibitors against HIV-1 infection.     

Localization of the calmodulin- and the actin-binding sites of caldesmon

Expression of the C-terminal third of chicken gizzard caldesmon in Escherichia coli, using the Nagai vector (Nagai, K., and Th?gersen, H.V. (1987) Methods Enzmol. 153, 461-481), produces a cII-caldesmon fusion protein (27 kDa) with caldesmon sequence beginning at Lys579. Degradation during purification yields five peptides with molecular masses of 24, 22, 19 (two peptides), and 15 kDa. The 24-kDa peptide begins at Phe581; the 22-kDa peptide begins at Leu597, the two 19-kDa peptides begin at Phe581 and Val629, respectively; the 15-kDa peptide also begins at Val629. We estimate that the 15-kDa and one of the 19-kDa peptides end near Leu710. Site-directed mutagenesis was used to produce truncated peptides with known C termini; one peptide (17 kDa) terminates at Asn675. Digestion of the fragments with chymotrypsin generates a second 15-kDa fragment that begins at Ser666 (15K'). All of the peptides, with the exception of 15K', bind Ca(2+)-calmodulin-Sepharose and share a common 37-amino acid peptide between Val629 and Ser666, suggesting this contains the calmodulin binding site. Comparison with published sequences (Takagi, T., Yazawa, M., Ueno, T., Suzuki, S., and Yagi, K. (1989) J. Biochem. (Tokyo) 106, 778-783 and Bartegi, A., Fattoum, A., Derancourt, J., and Kassab, R. (1990) J. Biol. Chem. 265, 15231-15238) for other calmodulin-binding fragments further restricts the binding site to 7 residues, Trp-Glu-Lys-Gly-Asn-Val-Phe, between Trp659 and Ser666. All of the fragments, except the two 15-kDa peptides, co-sediment with F-actin, indicating that there are two segments in the C-terminal third of caldesmon that can interact with F-actin: one between Leu597 and Val629, the other between Arg711 and Pro756. Although separated in the primary sequence, these domains may interact with the calmodulin-binding region in the folded structure.     

The N-terminal nucleophile serine of cephalosporin acylase executes the second autoproteolytic cleavage and acylpeptide hydrolysis

Cephalosporin acylase (CA) precursor is translated as a single polypeptide chain and folds into a self-activating pre-protein. Activation requires two peptide bond cleavages that excise an internal spacer to form the mature αβ heterodimer. Using Q-TOF LC-MS, we located the second cleavage site between Glu(159) and Gly(160), and detected the corresponding 10-aa spacer (160)GDPPDLADQG(169) of CA mutants. The site of the second cleavage depended on Glu(159): moving Glu into the spacer or removing 5-10 residues from the spacer sequence resulted in shorter spacers with the cleavage at the carboxylic side of Glu. The mutant E159D was cleaved more slowly than the wild-type, as were mutants G160A and G160L. This allowed kinetic measurements showing that the second cleavage reaction was a first-order, intra-molecular process. Glutaryl-7-aminocephalosporanic acid is the classic substrate of CA, in which the N-terminal Ser(170) of the β-subunit, is the nucleophile. Glu and Asp resemble glutaryl, suggesting that CA might also remove N-terminal Glu or Asp from peptides. This was indeed the case, suggesting that the N-terminal nucleophile also performed the second proteolytic cleavage. We also found that CA is an acylpeptide hydrolase rather than a previously expected acylamino acid acylase. It only exhibited exopeptidase activity for the hydrolysis of an externally added peptide, supporting the intra-molecular interaction. We propose that the final CA activation is an intra-molecular process performed by an N-terminal nucleophile, during which large conformational changes in the α-subunit C-terminal region are required to bridge the gap between Glu(159) and Ser(170).     

Identification of downstream target genes of latent membrane protein 1 in nasopharyngeal carcinoma cells by suppression subtractive hybridization

Proteolytic digestion of bovine beta-lactoglobulin by trypsin yielded four peptide fragments with bactericidal activity. The peptides were isolated and their sequences were found as follows: VAGTWY (residues 15-20), AASDISLLDAQSAPLR (residues 25-40), IPAVFK (residues 78-83) and VLVLDTDYK (residues 92-100). The four peptides were synthesized and found to exert bactericidal effects against the Gram-positive bacteria only. In order to understand the structural requirements for antibacterial activity, the amino acid sequence of the peptide VLVLDTDYK was modified. The replacement of the Asp (98) residue by Arg and the addition of a Lys residue at the C-terminus yielded the peptide VLVLDTRYKK which enlarged the bactericidal activity spectrum to the Gram-negative bacteria Escherichia coli and Bordetella bronchiseptica and significantly reduced the antibacterial capacity of the peptide toward Bacillus subtilis. By data base searches with the sequence VLVLDTRYKK a high homology was found with the peptide VLVATLRYKK (residues 55-64) of human blue-sensitive opsin, the protein of the blue pigment responsible for color vision. A peptide with this sequence was synthesized and assayed for bactericidal activity. VLVATLRYKK was strongly active against all the bacterial strains tested. Our results suggest a possible antimicrobial function of beta-lactoglobulin after its partial digestion by endopeptidases of the pancreas and show moreover that small targeted modifications in the sequence of beta-lactoglobulin could be useful to increase its antimicrobial function.     

The role of tryptophan in the antibacterial activity of a 15-residue bovine lactoferricin peptide.

Bovine lactoferricin is a 25-residue antibacterial peptide isolated after gastric cleavage of the iron transporting protein lactoferrin. A 15-residue fragment, FKCRRWQWRMKKLGA of this peptide sustains most of the antibacterial activity. In this truncated sequence, the two Trp residues are found to be essential for antibacterial activity. The anchoring properties of Trp, as have been observed in membrane proteins, are believed to be important for the interaction of Trp containing antibacterial peptides with bacterial cell membranes. We have investigated the molecular properties which make Trp important for the antibacterial activity of the 15-residue peptide by replacing Trp with natural and unnatural aromatic amino acids. This series of peptides was tested for antibacterial activity against Echerichia coli and Staphylococcus aureus. We found that neither the hydrogen bonding ability nor the amphipathicity of the indole system are essential properties for the effect of Trp on the antibacterial activity of the peptides. Replacement of Trp with residues containing aromatic hydrocarbon side chains gave the most active peptides. We propose that aromatic hydrocarbon residues are able to position themselves deeper into the bacterial cell membrane, making the peptide more efficient in disrupting the bacterial cell membrane. From our results the size, shape and aromatic character of Trp seem to be the most important features for the activity of this class of Trp containing antibacterial peptides.     

Isolation and characterization of four bactericidal domains in the bovine β-lactoglobulin

Proteolytic digestion of bovine β-lactoglobulin by trypsin yielded four peptide fragments with bactericidal activity. The peptides were isolated and their sequences were found as follows: VAGTWY (residues 15–20), AASDISLLDAQSAPLR (residues 25–40), IPAVFK (residues 78–83) and VLVLDTDYK (residues 92–100). The four peptides were synthesized and found to exert bactericidal effects against the Gram-positive bacteria only. In order to understand the structural requirements for antibacterial activity, the amino acid sequence of the peptide VLVLDTDYK was modified. The replacement of the Asp (98) residue by Arg and the addition of a Lys residue at the C-terminus yielded the peptide VLVLDTRYKK which enlarged the bactericidal activity spectrum to the Gram-negative bacteria Escherichia coli and Bordetella bronchiseptica and significantly reduced the antibacterial capacity of the peptide toward Bacillus subtilis. By data base searches with the sequence VLVLDTRYKK a high homology was found with the peptide VLVATLRYKK (residues 55–64) of human blue-sensitive opsin, the protein of the blue pigment responsible for color vision. A peptide with this sequence was synthesized and assayed for bactericidal activity. VLVATLRYKK was strongly active against all the bacterial strains tested. Our results suggest a possible antimicrobial function of β-lactoglobulin after its partial digestion by endopeptidases of the pancreas and show moreover that small targeted modifications in the sequence of β-lactoglobulin could be useful to increase its antimicrobial function.     

Evidence for phosphorylation of serine 753 in CFTR using a novel metal-ion affinity resin and matrix-assisted laser desorption mass spectrometry.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes an apical membrane Cl- channel regulated by protein phosphorylation. To identify cAMP-dependent protein kinase (PKA)-phosphorylated residues in full-length CFTR, immobilized metal-ion affinity chromatography (IMAC) was used to selectively purify phosphopeptides. The greater specificity of iron-loaded (Fe3+) nitrilotriacetic (NTA). Sepharose compared to iminodiacetic acid (IDA) metal-chelating matrices was demonstrated using a PKA-phosphorylated recombinant NBD1-R protein from CFTR. Fe(3+)-loaded NTA Sepharose preferentially bound phosphopeptides, whereas acidic and poly-His-containing peptides were co-purified using the conventional IDA matrices. IMAC using NTA Sepharose enabled the selective recovery of phosphopeptides and identification of phosphorylated residues from a complex proteolytic digest. Phosphopeptides from PKA-phosphorylated full-length CFTR, generated in Hi5 insect cells using a baculovirus expression system, were purified using NTA Sepharose. Phosphopeptides were identified using matrix-assisted laser desorption mass spectrometry (MALDI/MS) with post-source decay (PSD) analysis and collision-induced dissociation (CID) experiments. Phosphorylated peptides were identified by mass and by the metastable loss of HPO3 and H3PO4 from the parent ions. Peptide sequence and phosphorylation at CFTR residues 660Ser, 737Ser, and 795Ser were confirmed using MALDI/PSD analysis. Peptide sequences and phosphorylation at CFTR residues 700Ser, 712Ser, 768Ser, and 813Ser were deduced from peptide mass, metastable fragment ion formation, and PKA consensus sequences. Peptide sequence and phosphorylation at residue 753Ser was confirmed using MALDI/CID analysis. This is the first report of phosphorylation of 753Ser in full-length CFTR.     

JmjC-domain-containing histone demethylases of the JMJD1B type as putative precursors of endogenous DSIP

Delta sleep inducing peptide (WAGGDASGE, DSIP) is a well known multifunctional regulatory peptide. Numerous studies have confirmed its stress-protective and adaptive activity which is independent of the origin or nature of the stress or other harmful factors. However, the biosynthetic origin of DSIP remains obscure, since nothing is known of its protein precursor(s) and their encoding gene(s). We have performed a comprehensive analysis of available gene and protein databases for homologous peptide sites within mammalian resources including man. A family of Jumonji C (JmjC)-domain-containing histone demethylases was shown to contain a sequence fragment closely homologous to DSIP. One type of these ubiquitous and phylogenetically ancient proteins encoded by JMJD1B gene includes the WKGGNASGE sequence that differs from DSIP by only 2 amino acid residues in positions 2 and 5. The respective peptide was synthesized and its biological effects were evaluated in a preliminary way in the forced swimming and antitoxic tests. We suggest that the histone demethylases of the JmjC-group containing DSIP-related region can be considered as possible protein precursors of endogenous peptides with DSIP-like activity.     

MALDI-TOF mass spectrometry analysis of substrate specificity of lebetase, a direct-acting fibrinolytic metalloproteinase from Vipera lebetina snake venom

Lebetase is a direct-acting fibrinolytic zinc metalloendopeptidase related in amino acid sequence to reprolysins which include both hemorrhagic and non-hemorrhagic proteinases. Despite apparent structural similarities, fibrinolytic and hemorrhagic proteinases differ significantly in substrate specificity. In this study, we have examined the activity of lebetase I against biologically active peptides (bradykinin, kallidin, substance P) and 6-10 amino acid residues containing peptides synthesized according to cleavage regions of alpha(2)-macroglobulin, pregnancy zone protein (PZP) and fibrinogen. Lebetase was found to have no activity against studied hexapeptides. Surprisingly, the best substrates for lebetase were substance P, and peptide fragment of PZP, both were cleaved at position Pro-Gln. Identification of the hydrolysis products of 15 peptides by MALDI-TOF mass spectrometry analysis indicates that lebetase possesses broad substrate specificity. The MALDI-TOF MS technique was proven to be highly efficient for the recovery and identification of the peptides released by lebetase hydrolysis.     

Determinants on simian virus 40 large T antigen are important for recognition and phosphorylation by casein kinase I.

Casein kinase I has been shown to phosphorylate Ser123 and possibly Thr124, in simian virus 40 (SV40) large T antigen; the same sites are also modified in cultured cells incubated with 32Pi [Friedrich A. Gr?sser, Karl H. Scheidtmann, Polygena T. Tuazon, Jolinda A. Traugh & Gernot Walter (1988) Virology 165, 13-22]. The peptide, A-D-S-Q-H-S-T-P-P, which corresponds to the amino acid sequence 118-125 of SV40 large T antigen, was synthesized together with peptides containing changes in specific amino acid residues on either side of Ser123. These peptides were used as model substrates to determine the amino acids in the SV40 large T antigen important for recognition by casein kinase I. The native peptide identified above, with aspartate at the -4 position, was a poor substrate for casein kinase I in vitro. Peptides with acidic residues added at the -2 and -3 positions, preceding Ser123, were phosphorylated by casein kinase I with apparent Km values around 2 mM and Vmax values up to 500 pmol.min-1.ml-1. When acidic residues were added at both sides of the phosphorylatable serine, the peptide had a first-order rate constant over 20-fold higher than peptides with acidic amino acid residues at the N-terminus only; the apparent Km value was 0.65 mM with a Vmax of 2900 pmol.min-1.ml-1. The effects of modifying Ser120 to phosphoserine were examined by addition of a recognition sequence for the cAMP-dependent protein kinase prior to Ser120. Prior phosphorylation of the peptide at Ser120 lowered the apparent Km to 0.061 mM and increased the Vmax to 360 pmol.min-1.ml-1, a 50-fold decrease in Km for casein kinase I and a 6-fold increase in Vmax as compared to the non-phosphorylated peptide. This indicates that Ser120, which has been shown to be phosphorylated in vivo, provides an appropriate recognition determinant for casein kinase I.     

长叶车前花叶病毒上海分离株(HRVsh)外壳蛋白免疫多肽的研究

长叶车前花叶病毒上海分离株(HRVsh)的外壳蛋白中含有4个甲硫氨酸残基,本文采用溴化氰裂解,并结合葡聚糖凝胶G-100柱层析、高压纸电泳及纸层析等方法,分离纯化了5个多肽片段,经~(125)I标记抗体对免疫多肽的鉴定,表明其中二段多肽与~(125)-IgG的结合能力接近完整病毒的水平,说明这二段多肽具有HRVsh的抗原专一性,决定HRVsh抗原性的抗原决定簇主要分布于这二个肽段中。 外壳蛋白的胰蛋白酶酶解肽谱及多肽氨基酸序列分析的结果,表明HRVsh和HRV标准株系间在氨基酸序列上有很大相似性,这就决定了两者密切的血清学亲缘关系。     

Design and conformation of non-Aib synthetic peptides enjoying alamethicin-like ionophore activity

Analogues of alamethicin, a 20-mer amphipathic helical peptide with ionophore activity, in the sequence of which all Aib residues were substituted by Ala (A1) or Leu (L1), were synthesized by the solid phase method, purified by high performance liquid chromatography and characterized by fast atomic bombardment mass spectrometry. Infrared and CD studies showed that A1 easily underwent a transconformation to beta-structure whereas L1 displayed a predominant alpha-helical character, thus being a potential ionophore model. Its voltage-dependent multistate activity in model membranes showed that Aib is not a requisite residue to observe an alamethicin-like behavior. However, as the lifetime of the single channels was much shorter than for alamethicin, the peptide chain was lengthened by a Leu (LL1) or a Ser (SL1) residue. The last peptide gave an increased channel lifetime, but the design of other non-Aib peptides, taking into account the hydroxyl C-terminus and side-chain interactions between helices in a barrel-stave bundle, is desirable to approach more closely the alamethicin activity.     

Enhanced antitumour activity of 15-residue bovine lactoferricin derivatives containing bulky aromatic amino acids and lipophilic N-terminal modifications.

In a structure-antibacterial activity relationship study of a peptide fragment of bovine lactoferricin consisting of FKCRRWQWRMKKLGA (LFB 17-31), it was revealed that the two Trp residues were important for antibacterial activity. It has further been demonstrated that the size, shape and the aromatic character of the side chains were even more important than the Trp itself. In this study the antitumour effect of a series of LFB 17-31 derivatives are reported, in which the two Trp residues in position 6 and 8 were replaced with the larger non-coded aromatic amino acids Tbt, Tpc, Bip and Dip. The counterproductive Cys in position 3 was also substituted with these larger aromatic residues. In addition, the effect of introducing lipophilic groups of different size and shape in the N-terminal of the LFB 17-31 sequence was addressed. The resulting peptide derivatives were tested for activity against three human tumour cell lines and against normal human umbilical vein endothelial cells and fibroblasts. High antitumour activity by several of the peptides demonstrated that Trp successfully could be substituted by the bulky aromatic residues, and peptides containing the large and rigid Tbt residue in position 6 and/or 8 in LFB 17-31 were the most active candidates. The antitumour effect was even more increased by the Tbt-modified peptides when the three counterproductive amino acids Cys3, Gln7 and Gly14 were replaced by Ala. Enhanced antitumour activity was also obtained by modifying the N-terminal of LFB 17-31 with either long-chained fatty acids or bulky moieties. Thus, our results revealed that the size and shape of the lipophilic groups and their position in the peptide sequence were important for antitumour activity.     

Conjugates of catecholamines. VII. Synthesis and beta-adrenergic activity of peptide catecholamine conjugates

A series of novel catecholamine derivatives has been prepared in which one of the N-methyl substituents of isoproterenol has been extended by a spacer consisting of a chain of four methylenes which terminates with an amide linkage to a peptide, the point of attachment being via the aromatic amino group of p-aminophenylalanine. In one of the derivatives, two catecholamines are attached to the same peptide in this manner. The peptides, which range in size from three to eight amino acid residues and contain phenylalanine, glycine, and L-alpha-amino-delta-hydroxyvaleric acid, were synthesized via stepwise and fragment condensation techniques. The beta-adrenergic agonist activities of the derivatives were evaluated in vitro by measuring the intracellular accumulation of cyclic AMP in S49 mouse lymphoma cells.     

A preferred AMPK phosphorylation site adjacent to the inhibitory loop of cardiac and skeletal troponin I

5'-AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is activated when cellular AMP to ATP ratios rise, potentially serving as a key regulator of cellular energetics. Among the known targets of AMPK are catabolic and anabolic enzymes, but little is known about the ability of this kinase to phosphorylate myofilament proteins and thereby regulating the contractile apparatus of striated muscles. Here, we demonstrate that troponin I isoforms of cardiac (cTnI) and fast skeletal (fsTnI) muscles are readily phosphorylated by AMPK. For cTnI, two highly conserved serine residues were identified as AMPK sites using a combination of high-resolution top-down electron capture dissociation mass spectrometry, (32) P-incorporation, synthetic peptides, phospho-specific antibodies, and site-directed mutagenesis. These AMPK sites in cTnI were Ser149 adjacent to the inhibitory loop and Ser22 in the cardiac-specific N-terminal extension, at the level of cTnI peptides, the intact cTnI subunit, whole cardiac troponin complexes and skinned cardiomyocytes. Phosphorylation time-course experiments revealed that Ser149 was the preferred site, because it was phosphorylated 12-16-fold faster than Ser22 in cTnI. Ser117 in fsTnI, analogous to Ser149 in cTnI, was phosphorylated with similar kinetics as cTnI Ser149. Hence, the master energy-sensing protein AMPK emerges as a possibly important regulator of cardiac and skeletal contractility via phosphorylation of a preferred site adjacent to the inhibitory loop of the thin filament protein TnI.