ABSTRACT
An organic solvent-stable alkaline hydrolase (PA27) from Pseudomonas aeruginosa MH38 was expressed, characterized, and immobilized for biotechnological applications. Recombinant PA27 was expressed in Escherichia coli as a 27 kDa soluble protein and was purified by standard procedures. PA27 was found to be stable at pH 8-11 and below 50 °C. It maintained more than 80% of its activity under alkaline conditions (pH 8.0-11.0). Furthermore, PA27 exhibited remarkable stability in benzene and n-hexane at concentrations of 30% and 50%. Based on these properties, immobilization of PA27 for biotechnological applications was explored. Scanning electron microscopy revealed a very smooth spherical structure with numerous large pores. Interestingly, immobilized PA27 displayed improved thermal/chemical stabilities and high reusability. Specifically, immobilized PA27 has improved thermal stability, maintaining over 90% of initial activity after 1 h of incubation at 80 °C, whereas free PA27 had only 35% residual activity. Furthermore, immobilized PA27 showed higher residual activity than the free enzyme biocatalysts against detergents, urea, and phenol. Immobilized PA27 could be recycled 20 times with retention of ~60% of its initial activity. Furthermore, macroscopic hydrogel formation of PA27 was also investigated. These characteristics make PA27 a great candidate for an industrial biocatalyst with potential applications.
Subject(s)
Enzymes, Immobilized/chemistry , Hydrolases/biosynthesis , Pseudomonas aeruginosa/enzymology , Cloning, Molecular , Enzyme Stability , Enzymes, Immobilized/biosynthesis , Escherichia coli , Gene Expression Regulation, Enzymologic , Hydrogen-Ion Concentration , Hydrolases/chemistry , Hydrolases/genetics , Organic Chemicals/chemistry , Pseudomonas aeruginosa/chemistry , Solvents/chemistry , Substrate Specificity , TemperatureABSTRACT
While intercellular adhesion molecule-1 (ICAM-1) is a transmembrane protein, two types of extracellular ICAM-1 have been detected in cell culture supernatants as well as in the serum: a soluble form of ICAM-1 (sICAM-1) and a membranous form of ICAM-1 (mICAM-1) associated with exosomes. Previous observations have demonstrated that sICAM-1 cannot exert potent immune modulatory activity due to its low affinity for leukocyte function-associated antigen-1 (LFA-1) or membrane attack complex-1. In this report, we initially observed that human cancer cells shed mICAM-1(+)-exosomes but were devoid of vascular cell adhesion molecule-1 and E-selectin. We demonstrate that mICAM-1 on exosomes retained its topology similar to that of cell surface ICAM-1, and could bind to leukocytes. In addition, we show that exosomal mICAM-1 exhibits potent anti-leukocyte adhesion activity to tumor necrosis factor-alpha-activated endothelial cells compared to that of sICAM-1. Taken together with previous findings, our results indicate that mICAM-1 on exosomes exhibits potent immune modulatory activity.
Subject(s)
Endothelial Cells/immunology , Exosomes/immunology , Inflammation/immunology , Intercellular Adhesion Molecule-1/metabolism , Leukocytes/immunology , Cell Adhesion , Cell Line , E-Selectin/genetics , E-Selectin/metabolism , Endothelial Cells/metabolism , Exosomes/metabolism , Humans , Intercellular Adhesion Molecule-1/genetics , Leukocytes/metabolism , Vascular Cell Adhesion Molecule-1/genetics , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
The Ig superfamily (IgSF) intercellular adhesion molecule-1 (ICAM-1) equilibrates between monomeric and dimeric forms on the cell surface, and dimerization enhances cell adhesion. A crystal structure of ICAM-1 IgSF domains (D) 3-5 revealed a unique dimerization interface in which D4s of two protomers fuse through edge beta-strands to form a single super beta-sandwich domain. Here, we describe a crystal structure at 2.7-A resolution of monomeric ICAM-1 D3-D5, stabilized by the monomer-specific Fab CA7. CA7 binds to D5 in a region that is buried in the dimeric interface and is distal from the dimerization site in D4. In monomeric ICAM-1 D3-D5, a 16-residue loop in D4 that is disordered in the dimeric structure could clearly be traced as a BC loop, a short C strand, and a CE meander with a cis-Pro followed by a solvent-exposed, flexible four-residue region. Deletions of 6 or 10 residues showed that the C-strand is essential for monomer stability, whereas a distinct six-residue deletion showed little contribution of the CE meander. Mutation of two inward-pointing Leu residues in edge beta-strand E to Lys increased monomer stability, confirming the hypothesis that inward-pointing charged side chains on edge beta-strands are an important design feature to prevent beta-supersheet formation. Overall, the studies reveal that monomer-dimer transition is associated with a surprisingly large, physiologically relevant, IgSF domain rearrangement.
Subject(s)
Cell Membrane/metabolism , Immunoglobulin Subunits/chemistry , Intercellular Adhesion Molecule-1/chemistry , Amino Acid Sequence , Animals , CHO Cells , Cricetinae , Cricetulus , Crystallography, X-Ray , Immunoglobulin Fab Fragments/chemistry , Molecular Conformation , Molecular Sequence Data , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino AcidABSTRACT
We have analyzed a series of peptides derived from the C-terminus of alpha-synuclein for chaperone-like activity. Specifically, a cyclic peptide generated by introducing a disulfide bond was observed to increase chaperone-like activity. This is the first example of a disulfide-crosslinked peptide that exhibits activity against protein aggregation and activity loss.
Subject(s)
Molecular Chaperones/pharmacology , Peptides, Cyclic/pharmacology , alpha-Synuclein/chemistry , Alcohol Dehydrogenase/metabolism , Amino Acid Sequence , Molecular Sequence Data , Monophenol Monooxygenase , Peptide Fragments/pharmacology , Protein Denaturation/drug effects , Thiosulfate Sulfurtransferase/pharmacologyABSTRACT
Protein phosphatase inhibitor-1 (PPI-1) is a major inhibitor of protein phosphatase 1 (PP1), which regulates signal transduction in many eukaryotic cellular processes. Biophysical studies have shown that PPI-1 has a large Stokes radius and is heat stable, suggesting that it lacks extensive secondary structures. The unfolded structure of PPI-1 may enable it to interact with many proteins or ligands during stress conditions. Here we show that PPI-1 can act as a protective molecule, inhibiting protein aggregation and guarding E. coli cells against various stresses. Therefore, PPI-1 seems to have a physiological function as a protective molecule as well as regulator of protein serine/threonine phosphatases.
Subject(s)
Escherichia coli/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Amino Acid Sequence , Dopamine and cAMP-Regulated Phosphoprotein 32/chemistry , Dopamine and cAMP-Regulated Phosphoprotein 32/genetics , Dopamine and cAMP-Regulated Phosphoprotein 32/metabolism , Escherichia coli Proteins/metabolism , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Molecular Sequence Data , Oxidative Stress , Protein Conformation , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Signal TransductionABSTRACT
The aggregation and fibrillization of alpha-synuclein, a major component of Lewy bodies, is a key event in Parkinson's disease. Although the mechanisms of fibrils formation are largely investigated, physiological function of alpha-synuclein is not yet clearly elucidated. Here, we showed that C-terminal region of alpha-synuclein is similar to alpha-crystalline domain of small heat shock proteins. In our experiments, alpha-synuclein, like small heat shock proteins, protected cellular proteins from denaturation, and confer Escherichia coli cellular tolerances against thermal- and oxidative-stresses.
