Secretory (S) IgA antibodies against severe acute respiratory syndrome (SARS)-CoV-2 are induced in saliva and upper respiratory tract (URT) secretions by natural infection and may be critical in determining the outcome of initial infection. Secretory IgA1 (SIgA1) is the predominant isotype of antibodies in these secretions. Neutralization of SARS-CoV-2 is most effectively accomplished by polymeric antibodies such as SIgA. We hypothesize that cleavage of SIgA1 antibodies against SARS-CoV-2 by unique bacterial IgA1 proteases to univalent Fabα antibody fragments with diminished virus neutralizing activity would facilitate the descent of the virus into the lungs to cause serious disease and also enhance its airborne transmission to others. Recent studies of the nasopharyngeal microbiota of patients with SARS-CoV-2 infection have revealed significant increases in the proportions of IgA1 protease-producing bacteria in comparison with healthy subjects. Similar considerations might apply also to other respiratory viral infections including influenza, possibly explaining the original attribution of influenza to Haemophilus influenzae, which produces IgA1 protease.

• MeSH
• Bacteria enzymologie   MeSH
• COVID-19 * přenos   imunologie   virologie   mikrobiologie   MeSH
• Haemophilus influenzae enzymologie   imunologie   MeSH
• imunoglobulin A sekreční * metabolismus   MeSH
• imunoglobulin A imunologie   MeSH
• lidé MeSH
• nazofarynx mikrobiologie   virologie   MeSH
• neutralizující protilátky imunologie   MeSH
• protilátky virové imunologie   MeSH
• SARS-CoV-2 * imunologie   MeSH
• serinové endopeptidasy metabolismus   imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Rhomboid proteases are intramembrane enzymes that hydrolyze peptide bonds of transmembrane proteins in the lipid bilayer. They play a variety of roles in key biological events and are linked to several disease states. Over the last decade a great deal of structural and functional knowledge has been generated on this fascinating class of proteases. Both structural and kinetic analyses require milligram amounts of protein, which may be challenging for membrane proteins such as rhomboids. Here, we present a detailed protocol for optimization of expression and purification of three rhomboid proteases from Escherichia coli (ecGlpG), Haemophilus influenzae (hiGlpG), and Providencia stuartii (AarA). We discuss the optimization of expression conditions, such as concentration of inducing agent, induction time, and temperature, as well as purification protocol with precise details for each step. The provided protocol yields 1-2.5mg of rhomboid enzyme per liter of bacterial culture and can assist in structural and functional studies of intramembrane proteases.

• MeSH
• DNA vazebné proteiny biosyntéza   chemie   genetika   izolace a purifikace   MeSH
• endopeptidasy biosyntéza   chemie   genetika   izolace a purifikace   MeSH
• Escherichia coli enzymologie   MeSH
• Haemophilus influenzae enzymologie   MeSH
• kinetika MeSH
• lipidové dvojvrstvy chemie   MeSH
• membránové proteiny biosyntéza   chemie   genetika   izolace a purifikace   MeSH
• molekulární biologie metody   MeSH
• proteiny z Escherichia coli biosyntéza   chemie   genetika   izolace a purifikace   MeSH
• Providencia enzymologie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH

Thirteen mono-N-acyl derivatives of 2,6-diaminopimelic acid (DAP)-new potential inhibitors of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE; EC 3.5.1.18)-were analyzed and characterized by infrared (IR) and nuclear magnetic resonance (NMR) spectroscopies and two capillary electromigration methods: capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). Structural features of DAP derivatives were characterized by IR and NMR spectroscopies, whereas CZE and MEKC were applied to evaluate their purity and to investigate their electromigration properties. Effective electrophoretic mobilities of these compounds were determined by CZE in acidic and alkaline background electrolytes (BGEs) and by MEKC in acidic and alkaline BGEs containing a pseudostationary phase of anionic detergent sodium dodecyl sulfate (SDS) or cationic detergent cetyltrimethylammonium bromide (CTAB). The best separation of DAP derivatives, including diastereomers of some of them, was achieved by MEKC in an acidic BGE (500 mM acetic acid [pH 2.54] and 60mM SDS). All DAP derivatives were examined for their ability to inhibit catalytic activity of DapE from Haemophilus influenzae (HiDapE) and ArgE from Escherichia coli (EcArgE). None of these DAP derivatives worked as an effective inhibitor of HiDapE, but one derivative-N-fumaryl, Me-ester-DAP-was found to be a moderate inhibitor of EcArgE, thereby providing a promising lead structure for further studies on ArgE inhibitors.

• MeSH
• amidohydrolasy antagonisté a inhibitory   MeSH
• elektroforéza kapilární metody   MeSH
• Escherichia coli enzymologie   MeSH
• Haemophilus influenzae enzymologie   MeSH
• inhibitory enzymů chemie   farmakologie   MeSH
• kyselina diaminopimelová chemie   MeSH
• magnetická rezonanční spektroskopie metody   MeSH
• spektrofotometrie infračervená metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

• MeSH
• alosterická regulace * MeSH
• buněčná membrána enzymologie   MeSH
• Escherichia coli enzymologie   MeSH
• Haemophilus influenzae enzymologie   MeSH
• membránové proteiny metabolismus   MeSH
• Providencia enzymologie   MeSH
• serinové proteasy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• komentáře MeSH
• práce podpořená grantem MeSH

• MeSH
• beta-laktamasy analýza   fyziologie   MeSH
• beta-laktamová rezistence MeSH
• Haemophilus influenzae enzymologie   izolace a purifikace   účinky léků   MeSH
• hemofilové infekce krev   MeSH
• lidé MeSH
• Check Tag
• lidé MeSH
• Geografické názvy
• Česká republika MeSH