A simple and traceless solid phase method simplifies the assembly of large peptides and the access to challenging proteins.

Chemical protein synthesis gives access to well-defined native or modified proteins that are useful for studying protein structure and function. The majority of proteins synthesized up to now have been produced using native chemical ligation (NCL) in solution. Although there are significant advantages to assembling large peptides or proteins by solid phase ligation, reports of such approaches are rare. We report a novel solid phase method for protein synthesis which relies on the chemistry of the acetoacetyl group and ketoxime ligation for the attachment of the peptide to the solid support, and on a tandem transoximation/rearrangement process for the detachment of the target protein. Importantly, we show that the combination of solid phase and solution ligation techniques facilitates the production of a challenging and biologically active protein made of 180 amino acids. We show also that the solid phase method enables the purification of complex peptide segments through a chemoselective solid phase capture/release approach.

Carbon nanowalls: a new versatile graphene based interface for the laser desorption/ionization-mass spectrometry detection of small compounds in real samples.

Carbon nanowalls, vertically aligned graphene nanosheets, attract attention owing to their tunable band gap, high conductivity, high mechanical robustness, high optical absorbance and other remarkable properties. In this paper, we report for the first time the use of hydrophobic boron-doped carbon nanowalls (CNWs) for laser desorption/ionization of small compounds and their subsequent detection by mass spectrometry (LDI-MS). The proposed method offers sensitive detection of various small molecules in the absence of an organic matrix. The CNWs were grown by microwave plasma enhanced chemical vapor deposition (MW-PECVD), using a boron-carbon gas flow ratio of 1200 in H2/CH4 plasma, on silicon <100> wafer. The hydrophobicity of the surface offers a straightforward MS sample deposition, consisting of drop casting solutions of analytes and drying in air. Limits of detection in the picomolar and femtomolar ranges (25 fmol µL-1 for neurotensin) were achieved for different types of compounds (fatty acids, lipids, metabolites, saccharides and peptides) having clinical or food industry applications. This rapid and sensitive procedure can also be used for quantitative measurements without internal standards with RSDs <19%, as in the case of glucose in aqueous solutions (LOD = 0.32 ± 0.02 pmol), blood serum or soft drinks. Moreover, melamine (63 ± 8.19 ng µL-1), a toxic compound, together with creatinine and paracetamol, was detected in urine samples, while lecithin was detected in food supplements.

Correction: A simple and traceless solid phase method simplifies the assembly of large peptides and the access to challenging proteins.

[This corrects the article DOI: 10.1039/C7SC01912B.].

Insight into the SEA amide thioester equilibrium. Application to the synthesis of thioesters at neutral pH.

The bis(2-sulfanylethyl)amide (SEA) N,S-acyl shift thioester surrogate has found a variety of useful applications in the field of protein total synthesis. Here we present novel insights into the SEA amide/thioester equilibrium in water which is an essential step in any reaction involving the thioester surrogate properties of the SEA group. We also show that the SEA amide thioester equilibrium can be efficiently displaced at neutral pH for accessing peptide alkylthioesters, i.e. the key components of the native chemical ligation (NCL) reaction.

Necrosis- and apoptosis-related Met cleavages have divergent functional consequences.

Upon activation by its ligand hepatocyte growth factor/scatter factor, the receptor tyrosine kinase Met promotes survival, proliferation, and migration of epithelial cells during embryogenesis. Deregulated Met signaling can also promote cancer progression and metastasis. Met belongs to the functional family of dependence receptors whose activity switches from pro-survival to pro-apoptotic during apoptosis upon caspase cleavage. Although apoptosis resistance is a hallmark of cancer cells, some remain sensitive to other cell death processes, including necrosis induced by calcium stress. The role and fate of Met during necrotic cell death are unknown. Following treatment with calcium ionophores, cell lines and primary cells undergo necrosis, and the full-length Met receptor is efficiently degraded. This degradation is achieved by double cleavage of Met in its extracellular domain by a metalloprotease of the A disintegrin and metalloproteinase (ADAM) family and in its intracellular domain by calpains (calcium-dependent proteases). These cleavages separate the Met extracellular region from its kinase domain, thus preventing Met activity and its potential pro-survival activity. Although the intracellular fragment is very similar to the fragment generated by caspases, it displays no pro-apoptotic property, likely because of the presence of the last few amino acids of Met, known to inhibit this pro-apoptotic function. The fragments identified here are observed in lung tumors overexpressing the Met receptor, along with fragments previously identified, suggesting that proteolytic cleavages of Met are involved in its degradation in tumor tissues. Thus, Met is a modulator of necrosis, able to protect cells when activated by its ligand but efficiently degraded by proteolysis when this process is engaged.

Role of DegP for two-partner secretion in Bordetella.

Sorting of proteins destined to the surface or the extracellular milieu is mediated by specific machineries, which guide the protein substrates towards the proper route of secretion and determine the compartment in which folding occurs. In gram-negative bacteria, the two-partner secretion (TPS) pathway is dedicated to the secretion of large proteins rich in beta-helical structure. The secretion of the filamentous haemagglutinin (FHA), a 230 kDa adhesin of Bordetella pertussis, represents a model TPS system. FHA is exported by the Sec machinery and transits through the periplasm in an extended conformation. From there it is translocated across the outer membrane by its dedicated transporter FhaC to finally fold into a long beta-helix at the cell surface in a progressive manner. In this work, we show that B. pertussis lacking the periplasmic chaperone/protease DegP has a strong growth defect at 37 degrees C, and the integrity of its outer membrane is compromised. While both phenotypes are significantly aggravated by the presence of FHA, the chaperone activity of DegP markedly alleviates the periplasmic stress. In vitro, DegP binds to non-native FHA with high affinity. We propose that DegP chaperones the extended FHA polypeptide in the periplasm and is thus involved in the TPS pathway.

Autocrine induction of invasion and metastasis by tumor-associated trypsin inhibitor in human colon cancer cells.

From the conditioned medium of the human colon carcinoma cells, HT-29 5M21 (CM-5M21), expressing a spontaneous invasive phenotype, tumor-associated trypsin inhibitor (TATI) was identified and characterized by proteomics, cDNA microarray approaches and functional analyses. Both CM-5M21 and recombinant TATI, but not the K18Y-TATI mutant at the protease inhibitor site, trigger collagen type I invasion by several human adenoma and carcinoma cells of the colon and breast, through phosphoinositide-3-kinase, protein kinase C and Rho-GTPases/Rho kinase-dependent pathways. Conversely, the proinvasive action of TATI in parental HT29 cells was alleviated by the TATI antibody PSKAN2 and the K18Y-TATI mutant. Stable expression of K18Y-TATI in HT-29 5M21 cells downregulated tumor growth, angiogenesis and the expression of several metastasis-related genes, including CSPG4 (13.8-fold), BMP-7 (9.7-fold), the BMP antagonist CHORDIN (5.2-fold), IGFBP-2 and IGF2 (9.6- and 4.6-fold). Accordingly, ectopic expression of KY-TATI inhibited the development of lung metastases from HT-29 5M21 tumor xenografts in immunodeficient mice. These findings identify TATI as an autocrine transforming factor potentially involved in early and late events of colon cancer progression, including local invasion of the primary tumor and its metastatic spread. Targeting TATI, its molecular partners and effectors may bring novel therapeutic applications for high-grade human solid tumors in the digestive and urogenital systems.

Biochemical staging of synucleinopathy and amyloid deposition in dementia with Lewy bodies.

The primary feature of dementia with Lewy bodies (DLB) is the aggregation of alpha-synuclein into characteristic lesions: Lewy bodies (LBs) and Lewy neurites. However, in most of DLB cases, LBs are associated with neurofibrillary tangles and amyloid plaques (both Alzheimer disease [AD]-related lesions). We wanted to determine if this overlap of lesions is statistical, as a result of the late onset of both diseases, or results from a specific physiopathological synergy between synucleinopathy and either tauopathy or amyloid pathology. All patients with DLB from our prospective and multidisciplinary study were analyzed. These cases were compared with cases with pure AD and patients with Parkinson disease and controls. All cases were analyzed thoroughly at the neuropathologic and biochemical levels with a biochemical staging of aggregated alpha-synuclein, tau, and Abeta species. All sporadic cases of DLB were associated with abundant deposits of Abeta x-42 that were similar in quality and quantity to those of AD. Amyloid precursor protein (APP) dysfunction is a risk factor for AD as demonstrated by pathogenic mutations and Abeta accumulation. The constant and abundant Abeta x-42 deposition in sporadic DLB suggests that synucleinopathy is also promoted by APP dysfunction. Therefore, we conclude that APP is a therapeutic target for both AD and DLB.

Trypanosoma cruzi prolyl oligopeptidase Tc80 is involved in nonphagocytic mammalian cell invasion by trypomastigotes.

Trypanosoma cruzi is an intracellular protozoan parasite able to invade a wide variety of mammalian cells. To have access to the target organs/cells, the parasite must cross the basal laminae and the extracellular matrix (ECM). We previously characterized an 80-kDa proteinase (Tc80) secreted by the infective trypomastigotes that hydrolyzes native collagens and might be involved in infection by degrading ECM components. Here, we present evidence indicating a role for Tc80 in the invasion of nonphagocytic cells. Tc80 was classified as a member of the prolyl oligopeptidase (POP) family of serine proteases and was also found to hydrolyze fibronectin. Selective inhibitors for POP Tc80 were synthesized that blocked parasite entry into cells. Blockage occurred when trypomastigotes were preincubated with irreversible inhibitors but not after host cell preincubation, and the blockage correlated with inhibition of POP Tc80 activity in treated parasites. These data and the enzyme location inside a vesicular compartment close to the flagellar pocket, a specialized domain in endocytosis/exocytosis, strongly suggest a role for POP Tc80 in the maturation of parasite protein(s) and/or, after secretion, in a local action on parasite or host cell/ECM components required for invasion.

Subtilisin-like autotransporter serves as maturation protease in a bacterial secretion pathway.

Proteins of Gram-negative bacteria destined to the extracellular milieu must cross the two cellular membranes and then fold at the appropriate time and place. The synthesis of a precursor may be a strategy to maintain secretion competence while preventing aggregation or premature folding (especially for large proteins). The secretion of 230 kDa filamentous haemagglutinin (FHA) of Bordetella pertussis requires the synthesis and the maturation of a 367 kDa precursor that undergoes the proteolytic removal of its approximately 130 kDa C-terminal intramolecular chaperone domain. We have identified a specific protease, SphB1, responsible for the timely maturation of the precursor FhaB, which allows for extracellular release of FHA. SphB1 is a large exported protein with a subtilisin-like domain and a C-terminal domain typical of bacterial autotransporters. SphB1 is the first described subtilisin-like protein that serves as a specialized maturation protease in a secretion pathway of Gram-negative bacteria. This is reminiscent of pro-protein convertases of eukaryotic cells.

Expression and purification of recombinant mouse Ets-1 transcription factor.

Ets-1 is a transcription factor which belongs to the ETS family. Its mRNA is expressed in the embryo during normal development and also in tumors. In order to sort out functional Ets-1-binding sites among those present in gene promoters, we constructed an expression vector and designed a purification protocol for the production of the 440-amino-acid form of mouse Ets-1, based on heparin-Sepharose affinity and anion-exchange chromatographies. This protocol allows the purification of large amounts of pure recombinant protein as assessed by SDS-PAGE, C18 reverse-phase HPLC, amino-terminal sequencing, and mass spectrometry. The purified protein is recognized by specific anti-Ets-1 antibodies and binds to DNA ETS-binding sites.

1H NMR study on the binding of Pin1 Trp-Trp domain with phosphothreonine peptides.

The recent crystal structure of Pin1 protein bound to a doubly phosphorylated peptide from the C-terminal domain of RNA polymerase II revealed that binding interactions between Pin1 and its substrate take place through its Trp-Trp (WW) domain at the level of the loop Ser(11)-Arg(12) and the aromatic pair Tyr(18)-Trp(29), and showed a trans conformation for both pSer-Pro peptide bonds. However, the orientation of the ligand in the aromatic recognition groove still could be sequence-specific, as previously observed in SH3 domains complexed by peptide ligands or for different class of WW domains (Zarrinpar, A., and Lim, W. A. (2000) Nat. Struct. Biol. 7, 611-613). Because the bound peptide conformation could also differ as observed for peptide ligands bound to the 14-3-3 domain, ligand orientation and conformation for two other biologically relevant monophosphate substrates, one derived from the Cdc25 phosphatase of Xenopus laevis (EQPLpTPVTDL) and another from the human tau protein (KVSVVRpTPPKSPS) in complex with the WW domain are here studied by solution NMR methods. First, the proton resonance perturbations on the WW domain upon complexation with both peptide ligands were determined to be essentially located in the positively charged beta-hairpin Ser(11)-Gly(15) and around the aromatic Trp(29). Dissociation equilibrium constants of 117 and 230 microm for Cdc25 and tau peptides, respectively, were found. Several intermolecular nuclear Overhauser effects between WW domain and substrates were obtained from a ligand-saturated solution and were used to determine the structures of the complexes in solution. We found a similar N to C orientation as the one observed in the crystal complex structure of Pin1 and a trans conformation for the pThr-Pro peptidic bond in both peptide ligands, thereby indicating a unique binding scheme for the Pin1 WW domain to its multiple substrates.

Mycobacterium smegmatis laminin-binding glycoprotein shares epitopes with Mycobacterium tuberculosis heparin-binding haemagglutinin.

Mycobacterium tuberculosis, the causative agent of tuberculosis, produces a heparin-binding haemagglutinin adhesin (HBHA), which is involved in its epithelial adherence. To ascertain whether HBHA is also present in fast-growing mycobacteria, Mycobacterium smegmatis was studied using anti-HBHA monoclonal antibodies (mAbs). A cross-reactive protein was detected by immunoblotting of M. smegmatis whole-cell lysates. However, the M. tuberculosis HBHA-encoding gene failed to hybridize with M. smegmatis chromosomal DNA in Southern blot analyses. The M. smegmatis protein recognized by the anti-HBHA mAbs was purified by heparin-Sepharose chromatography, and its amino-terminal sequence was found to be identical to that of the previously described histone-like protein, indicating that M. smegmatis does not produce HBHA. Biochemical analysis of the M. smegmatis histone-like protein shows that it is glycosylated like HBHA. Immunoelectron microscopy demonstrated that the M. smegmatis protein is present on the mycobacterial surface, a cellular localization inconsistent with a histone-like function, but compatible with an adhesin activity. In vitro protein interaction assays showed that this glycoprotein binds to laminin, a major component of basement membranes. Therefore, the protein was called M. smegmatis laminin-binding protein (MS-LBP). MS-LBP does not appear to be involved in adherence in the absence of laminin but is responsible for the laminin-mediated mycobacterial adherence to human pneumocytes and macrophages. Homologous laminin-binding adhesins are also produced by virulent mycobacteria such as M. tuberculosis and Mycobacterium leprae, suggesting that this adherence mechanism may contribute to the pathogenesis of mycobacterial diseases.

p13(SUC1) and the WW domain of PIN1 bind to the same phosphothreonine-proline epitope.

The WW domain of the human PIN1 and p13(SUC1), a subunit of the cyclin-dependent kinase complex, were previously shown to be involved in the regulation of the cyclin-dependent kinase complex activity at the entry into mitosis, by an unresolved molecular mechanism. We report here experimental evidence for the direct interaction of p13(SUC1) with a model CDC25 peptide, dependent on the phosphorylation state of its threonine. Chemical shift perturbation of backbone (1)H(N), (15)N, and (13)Calpha resonances during NMR titration experiments allows accurate identification of the binding site, primarily localized around the anion-binding site, occupied in the crystal structure of the homologous p9(CKSHs2) by a sulfate molecule. The epitope recognized by p13(SUC1) includes the proline at position +1 of the phosphothreonine, as was shown by the decrease in affinity for a mutated CDC25 phosphopeptide, containing an alanine/proline substitution. No direct interaction between the PIN1 WW domain or its catalytic proline cis/trans-isomerase domain and p13(SUC1) was detected, but our study showed that in vitro the WW domain of the human PIN1 antagonizes the binding of the p13(SUC1) to the CDC25 phosphopeptide, by binding to the same phosphoepitope. We thus propose that the full cyclin-dependent kinase complex stimulates the phosphorylation of CDC25 through binding of its p13(SUC1) module to the phosphoepitope of the substrate and that the reported WW antagonism of p13(SUC1)-stimulated CDC25 phosphorylation is caused by competitive binding of both protein modules to the same phosphoepitope.

Cloning of Plasmodium falciparum protein disulfide isomerase homologue by affinity purification using the antiplasmodial inhibitor 1,4-bis[3-[N-(cyclohexyl methyl)amino]propyl]piperazine..

A series of 10 1,4-bis(3-aminopropyl)piperazine compounds was found to display antiplasmodial activity with 50% growth inhibition between 30 and 250 nM, on three Plasmodium falciparum strains differently sensitive to chloroquine. By affinity chromatography using one of these compounds, a 52-kDa protein was isolated from P. falciparum, microsequenced and cloned. It corresponded to a single copy gene encoding a 453 amino acid protein displaying the typical features of protein disulfide isomerases, a thiol metabolizing enzyme belonging to the thiol: disulfide oxidoreductase superfamily, which was not previously described in malarial species.

Novel topological features of FhaC, the outer membrane transporter involved in the secretion of the Bordetella pertussis filamentous hemagglutinin.

Many pathogenic Gram-negative bacteria secrete virulence factors across the cell envelope into the extracellular milieu. The secretion of filamentous hemagglutinin (FHA) by Bordetella pertussis depends on the pore-forming outer membrane protein FhaC, which belongs to a growing family of protein transporters. Protein alignment and secondary structure predictions indicated that FhaC is likely to be a beta-barrel protein with an odd number of transmembrane beta-strands connected by large surface loops and short periplasmic turns. The membrane topology of FhaC was investigated by random insertion of the c-Myc epitope and the tobacco etch virus protease-specific cleavage sequence. FhaC was fairly permissive to short linker insertions. Furthermore, FhaC appeared to undergo conformational changes upon FHA secretion. Surface detection of the inserted sequences indicated that several predicted loops in the C-terminal moiety as well as the N terminus of the protein are exposed. However, a large surface-predicted region in the N-terminal moiety of FhaC was inaccessible from the surface. In addition, the activity and the stability of the protein were affected by insertions in that region, indicating that it may have important structural and/or functional roles. The surface exposure of the N terminus and the presence of an odd number of beta-strands are novel features for beta-barrel outer membrane proteins.

Inflammation-induced systemic proteolysis of inter-alpha-inhibitor in plasma from patients with sepsis.

Inter-alpha-inhibitor (IalphaI) is a human plasma serine proteinase inhibitor. It contains one light peptide chain called bikunin that exerts antiproteinase activity and other antiinflammatory functions. Bikunin is covalently linked to two heavy chains that, after tissular diffusion, stabilize the extracellular matrix. Owing to its negative acute-phase reactant character and its susceptibility to proteolysis, IalphaI has been implicated in the pathophysiology of sepsis. Moreover, IalphaI has been shown to exert a protective effect on a pig model of endotoxic shock. Twenty patients admitted to the intensive care unit (ICU) for a septic syndrome were included in the present study. IalphaI and antithrombin III (ATIII) levels were measured on admission. Sequential measurements of IalphaI could be done in 4 patients. We demonstrate that IalphaI levels are significantly decreased in plasma samples collected on admission from patients with sepsis (59 +/- 32 mg/L vs 241 +/- 70 mg/L; P < .0001). This decrease was greater in severe sepsis and septic shock than in sepsis. Death was not predictable from initiol IalphaI levels. In 2 patients with a favorable course, IalphaI values regularly increased during the ICU stay. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot analysis and microsequencing, we characterized IalphaI-related components in plasma from several patients; they obviously arise from IalphaI through proteolytic cleavage. Thus, systemic proteolysis and decreased biosynthesis both contribute to the fall in the plasma level of IalphaI. Because IalphaI is very sensitive to proteolysis by polymorphonuclear granulocytes (PMNs) that are stimulated during sepsis, we suggest that IalphaI plasma level would be a useful marker for neutrophil proteinase activity. ATIII, as well as IalphaI, is considered a negative acute phase protein. Because in vitro ATIII is less susceptible than IalphaI to proteolysis by PMNs and because their relative levels weakly correlated, we suggest that an unspecific systemic proteolysis is not significantly involved in the ATIII deficiency occurring in sepsis.

Human pre-alpha-inhibitor is a positive acute-phase protein that is more susceptible than inter-alpha-inhibitor to proteolysis by stimulated neutrophils.

BACKGROUND: Pre-alpha-inhibitor (PalphaI) is a human plasma serine-proteinase inhibitor that is structurally related to inter-alpha-inhibitor (IalphaI). It is composed of a heavy chain named H3 covalently linked to bikunin by means of a glycosaminoglycan chain. We developed an ELISA procedure making it possible to measure PalphaI for the first time and we investigated its levels in sera from patients with inflammatory diseases. MATERIALS AND METHODS: We generated rabbit anti-H3 immunoglobulins, which were used on solid phase and biotinylated antibikunin immunoglobulins to detect trapped PalphaI. RESULTS: We demonstrate that PalphaI is more susceptible than IalphaI to in vitro proteolysis by stimulated neutrophils. However, the degradation products thus released as well as the other members of the IalphaI family present in serum do not affect the ELISA test. In a panel of control sera we observed PalphaI concentrations of 25.6 +/- 7.8 mg L-1 (mean +/- SD; n = 30). These values increased to 64.2 +/- 16.06 mg L-1 (mean +/- SD; n = 15) in patients with inflammatory diseases, concording with the positive acute-phase protein nature of PalphaI. However, for all these patients, the serum concentrations of PalphaI and C-reactive protein poorly correlated (r = 0.476; P = 0.076). Indeed, four patients had a relatively weaker increase in their PalphaI level than that of C-reactive protein. More often than not their plasma elastase content was then elevated. CONCLUSION: During inflammatory diseases plasma PalphaI levels may be dependent on increased synthesis in combination with enhanced catabolism, perhaps implicating neutrophil or other proteinases.

Synthesis of polyamine derivatives for the preparation of affinity chromatography columns for the search of new Trypanosoma cruzi targets.

The most potent trypanocidal compound of a series of symmetrically substituted 1,4-bis(3-aminopropylpiperazines) which displayed an IC50 value of 5 microM on Trypanosoma cruzi trypomastigotes, was inactive on trypanothione reductase. Two derivatives 6 and 12 of this compound, one symmetrical and one dissymmetrical, were synthesized via a reductive amination reaction, to prepare affinity chromatography columns, which allowed us to isolate three parasitic proteins. Among these, the major ligand 6- and 12-binding protein having an apparent molecular weight of 52 kDa has been identified as the thiol-disulfide oxido-reductase Tc52, previously characterized in Trypanosoma cruzi.

Divalent cations differentially regulate integrin alphaIIb cytoplasmic tail binding to beta3 and to calcium- and integrin-binding protein.

We have used recombinant or synthetic alphaIIb and beta3 integrin cytoplasmic peptides to study their in vitro complexation and ligand binding capacity by surface plasmon resonance. alpha.beta heterodimerization occurred in a 1:1 stoichiometry with a weak KD in the micromolar range. Divalent cations were not required for this association but stabilized the alpha.beta complex by decreasing the dissociation rate. alpha.beta complexation was impaired by the R995A substitution or the KVGFFKR deletion in alphaIIb but not by the beta3 S752P mutation. Recombinant calcium- and integrin-binding protein (CIB), an alphaIIb-specific ligand, bound to the alphaIIb cytoplasmic peptide in a Ca2+- or Mn2+-independent, one-to-one reaction with a KD value of 12 microM. In contrast, in vitro liquid phase binding of CIB to intact alphaIIbbeta3 occurred preferentially with Mn2+-activated alphaIIbbeta3 conformers, as demonstrated by enhanced coimmunoprecipitation of CIB with PAC-1-captured Mn2+-activated alphaIIbbeta3, suggesting that Mn2+ activation of intact alphaIIbbeta3 induces the exposure of a CIB-binding site, spontaneously exposed by the free alphaIIb peptide. Since CIB did not stimulate PAC-1 binding to inactive alphaIIbbeta3 nor prevented activated alphaIIbbeta3 occupancy by PAC-1, we conclude that CIB does not regulate alphaIIbbeta3 inside-out signaling, but rather is involved in an alphaIIbbeta3 post-receptor occupancy event.