The acylated Repeats in ToXins (RTX) leukotoxins, the adenylate cyclase toxin (CyaA) or α-hemolysin (HlyA), bind β2 integrins of leukocytes but also penetrate cells lacking these receptors. We show that the indoles of conserved tryptophans in the acylated segments, W876 of CyaA and W579 of HlyA, are crucial for β2 integrin-independent membrane penetration. Substitutions of W876 by aliphatic or aromatic residues did not affect acylation, folding, or the activities of CyaA W876L/F/Y variants on cells expressing high amounts of the β2 integrin CR3. However, toxin activity of CyaA W876L/F/Y on cells lacking CR3 was strongly impaired. Similarly, a W579L substitution selectively reduced HlyA W579L cytotoxicity towards cells lacking β2 integrins. Intriguingly, the W876L/F/Y substitutions increased the thermal stability (Tm) of CyaA by 4 to 8 °C but locally enhanced the accessibility to deuteration of the hydrophobic segment and of the interface of the two acylated loops. W876Q substitution (showing no increase in Tm), or combination of W876F with a cavity-filling V822M substitution (this combination decreasing the Tm closer to that of CyaA), yielded a milder defect of toxin activity on erythrocytes lacking CR3. Furthermore, the activity of CyaA on erythrocytes was also selectively impaired when the interaction of the pyrrolidine of P848 with the indole of W876 was ablated. Hence, the bulky indoles of residues W876 of CyaA, or W579 of HlyA, rule the local positioning of the acylated loops and enable a membrane-penetrating conformation in the absence of RTX toxin docking onto the cell membrane by β2 integrins.

• Klíčová slova
• RTX toxin, acylated segment, adenylate cyclase toxin, cytotoxicity, hydrogen/deuterium exchange, thermal stability, tryptophan residue, α-hemolysin, β(2) integrins,
• MeSH
• adenylátcyklasový toxin * chemie   genetika   metabolismus   MeSH
• antigeny CD18 * genetika   metabolismus   MeSH
• Bordetella pertussis MeSH
• buněčná membrána metabolismus   MeSH
• erytrocyty metabolismus   MeSH
• konzervovaná sekvence MeSH
• tryptofan * chemie   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasový toxin * MeSH
• antigeny CD18 * MeSH
• tryptofan * MeSH

Acylated domains (ADs), like that of the Bordetella pertussis adenylate cyclase toxin (CyaA), are structures found in all pore-forming toxins from the family of Repeat-in-ToXin (RTX) proteins. These AD segments are fatty-acylated on ε-amino groups of conserved lysine residues, such as the K860 and K983 residues of CyaA. The ε-amide-linked acyl chains are essential for toxin activity and promote irreversible membrane insertion of the CyaA molecule, thus enabling the toxin to translocate its N-terminal adenyl cyclase enzyme domain into the host cell cytoplasm. In parallel, the membrane-inserted CyaA molecules can oligomerize into cation-selective pores in the plasma membrane. Here, we show that the attached acyl chains are not only crucial for membrane insertion of the toxin but also play an important role in CyaA folding. We demonstrate that assembly of the noncanonical β-roll structure in the C-terminal segment of the AD of CyaA is cooperatively directed by the Ca2+-driven folding of the adjacent RTX domain. In contrast, the N-terminal AD segment consists of an α-helical structure that folds independently of Ca2+ ion binding and may form one or two acyl binding site(s) accommodating the acyl chains protruding from the C-terminal AD segment. This acyl-mediated interaction between the N- and C-terminal segments promotes local structural rearrangements within the AD that significantly enhances the stability of the toxin molecule. These findings highlight the critical role of the acyl modification in membrane interaction capacity and structural stability of the CyaA toxin.

• Klíčová slova
• Bordetella pertussis, RTX toxin, acylation, adenylate cyclase toxin, protein folding,
• MeSH
• acylace MeSH
• adenylátcyklasový toxin * metabolismus   chemie   genetika   MeSH
• Bordetella pertussis * metabolismus   enzymologie   genetika   MeSH
• buněčná membrána * metabolismus   MeSH
• lidé MeSH
• proteinové domény MeSH
• sbalování proteinů MeSH
• vápník metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenylátcyklasový toxin * MeSH
• vápník MeSH

Pore-forming repeats in toxins (RTX) are key virulence factors of many Gram-negative pathogens. We have recently shown that the aromatic side chain of the conserved tyrosine residue 940 within the acylated segment of the RTX adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) plays a key role in target cell membrane interaction of the toxin. Therefore, we used a truncated CyaA-derived RTX719 construct to analyze the impact of Y940 substitutions on functional folding of the acylated segment of CyaA. Size exclusion chromatography combined with CD spectroscopy revealed that replacement of the aromatic side chain of Y940 by the side chains of alanine or proline residues disrupted the calcium-dependent folding of RTX719 and led to self-aggregation of the otherwise soluble and monomeric protein. Intriguingly, corresponding alanine substitutions of the conserved Y642, Y643 and Y639 residues in the homologous RtxA, HlyA and ApxIA hemolysins from Kingella kingae, Escherichia coli and Actinobacillus pleuropneumoniae, affected the membrane insertion, pore-forming (hemolytic) and cytotoxic capacities of these toxins only marginally. Activities of these toxins were impaired only upon replacement of the conserved tyrosines by proline residues. It appears, hence, that the critical role of the aromatic side chain of the Y940 residue is highly specific for the functional folding of the acylated domain of CyaA and determines its capacity to penetrate target cell membrane.

• MeSH
• adenylátcyklasový toxin genetika   MeSH
• Bordetella bronchiseptica * genetika   metabolismus   MeSH
• Bordetella pertussis * genetika   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• hemolýza MeSH
• infekce bakteriemi rodu Bordetella mikrobiologie   MeSH
• lidé MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• THP-1 buňky MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasový toxin MeSH

BACKGROUND: High-density lipoprotein plays a key role in reverse cholesterol transport. In addition, high-density lipoprotein particles may be cardioprotective and reduce infarct size in the setting of myocardial injury. Lecithin-cholesterol acyltransferase is a rate-limiting enzyme in reverse cholesterol transport. MEDI6012 is a recombinant human lecithin-cholesterol acyltransferase that increases high-density lipoprotein cholesterol. Administration of lecithin-cholesterol acyltransferase has the potential to reduce infarct size and regress coronary plaque in acute ST-segment-elevation myocardial infarction. METHODS: REAL-TIMI 63B (A Randomized, Placebo‑controlled Phase 2b Study to Evaluate the Safety and Efficacy of MEDI6012 in Acute ST Elevation Myocardial Infarction) was a phase 2B multinational, placebo-controlled, randomized trial. Patients with ST-segment-elevation myocardial infarction within 6 hours of symptom onset and planned for percutaneous intervention were randomly assigned 2:1 to MEDI6012 (2- or 6-dose regimen) or placebo and followed for 12 weeks. The primary outcome was infarct size as a percentage of left ventricular mass by cardiac MRI at 10 to 12 weeks, with the primary analysis in patients with TIMI Flow Grade 0 to 1 before percutaneous intervention who received at least 2 doses of MEDI6012. The secondary outcome was change in noncalcified plaque volume on coronary computed tomographic angiography from baseline to 10 to 12 weeks with the primary analysis in patients who received all 6 doses of MEDI6012. RESULTS: A total of 593 patients were randomly assigned. Patients were a median of 62 years old, 77.9% male, and 95.8% statin naive. Median time from symptom onset to randomization was 146 (interquartile range [IQR], 103-221) minutes and from hospitalization to randomization was 12.7 (IQR, 6.6-24.0) minutes, and the first dose of drug was administered a median of 8 (IQR, 3-13) minutes before percutaneous intervention. The index myocardial infarction was anterior in 69.6% and TIMI Flow Grade 0 to 1 in 65.1% of patients. At 12 weeks, infarct size did not differ between treatment groups (MEDI6012: 9.71%, IQR 4.79-16.38; placebo: 10.48%, [IQR, 4.92-16.61], 1-sided P=0.79. There was also no difference in noncalcified plaque volume (geometric mean ratio, 0.96 [95% CI, NA-1.10], 1-sided P=0.30). There was no significant difference in treatment emergent serious adverse events. CONCLUSIONS: Administration of MEDI6012 in patients with acute ST-segment-elevation myocardial infarction did not result in a significant reduction in infarct size or noncalcified plaque volume at 12 weeks. MEDI6012 was well tolerated with no excess in overall serious adverse events. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT03578809.

• Klíčová slova
• HDL, ST elevation myocardial infarction, acyltransferases, cholesterol ester transfer proteins, lipoproteins,
• MeSH
• cholesterol MeSH
• cholesterolacyltransferasa terapeutické užití   MeSH
• infarkt myokardu přední stěny * MeSH
• infarkt myokardu s elevacemi ST úseků * diagnostické zobrazování   farmakoterapie   MeSH
• lecitincholesterolacyltransferasa * terapeutické užití   MeSH
• lecitiny terapeutické užití   MeSH
• lidé středního věku MeSH
• lidé MeSH
• lipoproteiny HDL terapeutické užití   MeSH
• statiny * terapeutické užití   MeSH
• výsledek terapie MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• klinické zkoušky, fáze II MeSH
• práce podpořená grantem MeSH
• randomizované kontrolované studie MeSH
• Názvy látek
• cholesterol MeSH
• cholesterolacyltransferasa MeSH
• lecitincholesterolacyltransferasa * MeSH
• lecitiny MeSH
• lipoproteiny HDL MeSH
• statiny * MeSH

The Bordetella adenylate cyclase toxin-hemolysin (CyaA) and the α-hemolysin (HlyA) of Escherichia coli belong to the family of cytolytic pore-forming Repeats in ToXin (RTX) cytotoxins. HlyA preferentially binds the αLβ2 integrin LFA-1 (CD11a/CD18) of leukocytes and can promiscuously bind and also permeabilize many other cells. CyaA bears an N-terminal adenylyl cyclase (AC) domain linked to a pore-forming RTX cytolysin (Hly) moiety, binds the complement receptor 3 (CR3, αMβ2, CD11b/CD18, or Mac-1) of myeloid phagocytes, penetrates their plasma membrane, and delivers the AC enzyme into the cytosol. We constructed a set of CyaA/HlyA chimeras and show that the CyaC-acylated segment and the CR3-binding RTX domain of CyaA can be functionally replaced by the HlyC-acylated segment and the much shorter RTX domain of HlyA. Instead of binding CR3, a CyaA1-710/HlyA411-1024 chimera bound the LFA-1 receptor and effectively delivered AC into Jurkat T cells. At high chimera concentrations (25 nm), the interaction with LFA-1 was not required for CyaA1-710/HlyA411-1024 binding to CHO cells. However, interaction with the LFA-1 receptor strongly enhanced the specific capacity of the bound CyaA1-710/HlyA411-1024 chimera to penetrate cells and deliver the AC enzyme into their cytosol. Hence, interaction of the acylated segment and/or the RTX domain of HlyA with LFA-1 promoted a productive membrane interaction of the chimera. These results help delimit residues 400-710 of CyaA as an "AC translocon" sufficient for translocation of the AC polypeptide across the plasma membrane of target cells.

• Klíčová slova
• AC domain translocation, AC translocon, Bordetella pertussis, CyaA, Escherichia coli (E. coli), HlyA, RTX toxin, acylation, acyltransferase, bacterial toxin, complement receptor 3 (CR3,), fatty acid, fatty acyl, integrin, protein acylation, protein translocation,
• MeSH
• adenylátcyklasový toxin metabolismus   MeSH
• antigen-1 spojený s lymfocytární funkcí metabolismus   MeSH
• Bordetella * MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• cytosol metabolismus   MeSH
• Jurkat buňky MeSH
• lidé MeSH
• makrofágový antigen 1 metabolismus   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• THP-1 buňky MeSH
• transport proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasový toxin MeSH
• antigen-1 spojený s lymfocytární funkcí MeSH
• makrofágový antigen 1 MeSH

Lipids form a significant part of animal organs and they are responsible for important biological functions, such as semi-permeability and fluidity of membranes, signaling activity, anti-inflammatory processes, etc. We have performed a comprehensive nontargeted lipidomic characterization of porcine brain, heart, kidney, liver, lung, spinal cord, spleen, and stomach using hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization mass spectrometry (ESI/MS) to describe the representation of individual lipid classes in these organs. Detailed information on identified lipid species inside classes are obtained based on relative abundances of deprotonated molecules [M-H](-) in the negative-ion ESI mass spectra, which provides important knowledge on phosphatidylethanolamines and their different forms of fatty acyl linkage (ethers and plasmalogens), phosphatidylinositols, and hexosylceramides containing nonhydroxy- and hydroxy-fatty acyls. The detailed analysis of identified lipid classes using reversed-phase liquid chromatography in the second dimension was performed for porcine brain to determine more than 160 individual lipid species containing attached fatty acyls of different acyl chain length, double-bond number, and positions on the glycerol skeleton. The fatty acid composition of porcine organs is determined by gas chromatography with flame ionization detection after the transesterification with sodium methoxide.

• MeSH
• chromatografie kapalinová metody   MeSH
• chromatografie plynová metody   MeSH
• fosfatidylethanolaminy analýza   MeSH
• fosfolipidy analýza   MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací metody   MeSH
• hydrofobní a hydrofilní interakce MeSH
• játra chemie   MeSH
• ledviny chemie   MeSH
• lipidy analýza   chemie   MeSH
• mastné kyseliny analýza   MeSH
• mícha chemie   MeSH
• mozek - chemie MeSH
• myokard chemie   MeSH
• plasmalogeny analýza   MeSH
• plíce chemie   MeSH
• prasata MeSH
• slezina chemie   MeSH
• žaludek chemie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfatidylethanolaminy MeSH
• fosfolipidy MeSH
• lipidy MeSH
• mastné kyseliny MeSH
• plasmalogeny MeSH

Acyl derivatives of hyaluronan (acyl-HA) are promising materials for biomedical applications. Depending on the acyl length and the degree of substitution, these derivatives range from self-assembling water-soluble polymers to materials insoluble in aqueous environments. The behaviour of acyl-HA was studied in solution, but little attention was paid to the solid state, despite its importance for applications such as medical device fabrication. We thus used X-ray scattering and electron microscopy to explore the solid-state nano-structure of acyl-HA. The set of samples included various substituents, substitution degrees and molecular weights. The obtained data showed that all studied acyl-HA materials contained structures with dimensions on the order of nanometres that were not present in unmodified HA. The size of the nanostructures increased with the acyl length, while the degree of substitution and molecular weight had negligible effects. We suggest that the observed nanostructure corresponds to a distribution of hydrophobic domains in a hydrophilic matrix of unmodified HA segments.

• Klíčová slova
• Electron microscopy, Hyaluronan, Hydrophobization, Nanostructure, Small-angle X-ray scattering, Solid-state,
• Publikační typ
• časopisecké články MeSH

BACKGROUND: The human mitochondrial alpha-ketoglutarate dehydrogenase complex (hKGDHc) converts KG to succinyl-CoA and NADH. Malfunction of and reactive oxygen species generation by the hKGDHc as well as its E1-E2 subcomplex are implicated in neurodegenerative disorders, ischemia-reperfusion injury, E3-deficiency and cancers. METHODS: We performed cryo-EM, cross-linking mass spectrometry (CL-MS) and molecular modeling analyses to determine the structure of the E2 component of the hKGDHc (hE2k); hE2k transfers a succinyl group to CoA and forms the structural core of hKGDHc. We also assessed the overall structure of the hKGDHc by negative-stain EM and modeling. RESULTS: We report the 2.9 Å resolution cryo-EM structure of the hE2k component. The cryo-EM map comprises density for hE2k residues 151-386 - the entire (inner) core catalytic domain plus a few additional residues -, while residues 1-150 are not observed due to the inherent flexibility of the N-terminal region. The structure of the latter segment was also determined by CL-MS and homology modeling. Negative-stain EM on in vitro assembled hKGDHc and previous data were used to build a putative overall structural model of the hKGDHc. CONCLUSIONS: The E2 core of the hKGDHc is composed of 24 hE2k chains organized in octahedral (8 × 3 type) assembly. Each lipoyl domain is oriented towards the core domain of an adjacent chain in the hE2k homotrimer. hE1k and hE3 are most likely tethered at the edges and faces, respectively, of the cubic hE2k assembly. GENERAL SIGNIFICANCE: The revealed structural information will support the future pharmacologically targeting of the hKGDHc.

• Klíčová slova
• 2-Oxoglutarate dehydrogenase complex, Cross-linking mass spectrometry, Cryo-electron microscopy, Dihydrolipoamide succinyltransferase, α-Ketoglutarate dehydrogenase complex,
• MeSH
• acylkoenzym A metabolismus   MeSH
• acyltransferasy chemie   metabolismus   MeSH
• elektronová kryomikroskopie metody   MeSH
• hmotnostní spektrometrie metody   MeSH
• ketoglutarátdehydrogenasový komplex chemie   metabolismus   MeSH
• konformace proteinů MeSH
• kyseliny ketoglutarové metabolismus   MeSH
• lidé MeSH
• molekulární modely MeSH
• NAD metabolismus   MeSH
• reagencia zkříženě vázaná chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• acylkoenzym A MeSH
• acyltransferasy MeSH
• dihydrolipoamide succinyltransferase MeSH Prohlížeč
• ketoglutarátdehydrogenasový komplex MeSH
• kyseliny ketoglutarové MeSH
• NAD MeSH
• reagencia zkříženě vázaná MeSH
• succinyl-coenzyme A MeSH Prohlížeč

• MeSH
• arginin chemie   fyziologie   MeSH
• buněčná membrána enzymologie   MeSH
• histidin chemie   fyziologie   MeSH
• hydrolýza MeSH
• mutageneze cílená MeSH
• protonové ATPasy chemie   genetika   metabolismus   MeSH
• protonové pumpy metabolismus   MeSH
• Saccharomyces cerevisiae enzymologie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• arginin MeSH
• histidin MeSH
• protonové ATPasy MeSH
• protonové pumpy MeSH

The synthesis of peptide bonds catalysed by subtilisin Carlsberg was studied in different hydrophilic organic solvents with variable H2O concentration. Z-Val-Trp-OMe and Z-Ala-Phe-OMe were used as acyl donors, and a series of amino acid derivatives, di- and tripeptides of the general structure Xaa-Gly, Gly-Xaa, Gly-Gly-Xaa (Xaa represents all natural L-amino acids except cysteine) and other peptides were used as nucleophiles. A comparative study of the enzymatic synthesis in aqueous DMF (50%, v/v) and acetonitrile containing 10% (v/v) of H2O demonstrated that the yields of peptide products were higher in most cases when acetonitrile with low H2O concentration was used. The acylation of weak nucleophiles was improved in organic solvents with very low H2O concentration (2%). The reactions in anhydrous Bu(t)-OH proceeded with substantially lower velocity. Generally, the restricted nucleophile specificity of the enzyme for glycine and hydrophilic amino acid residues in P1' position, as well as numerous side reactions, limit the utilization of subtilisin in peptide synthesis, especially in the case of the segment condensations. Contrary to the published data, we have proved that proline derivatives were not acylated in any media with the help of subtilisin Carlsberg. Effective ester hydrolysis of a protected nonapeptide corresponding to the N-terminal sequence of dicarba-eel-calcitonin catalysed by subtilisin was achieved.

• MeSH
• acetonitrily chemie   MeSH
• biosyntéza peptidů * MeSH
• biotechnologie metody   MeSH
• chemické modely MeSH
• hydrolýza MeSH
• peptidy chemická syntéza   MeSH
• subtilisiny chemie   MeSH
• voda chemie   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetonitrile MeSH Prohlížeč
• acetonitrily MeSH
• peptidy MeSH
• subtilisiny MeSH
• voda MeSH