Biomedical subjects

Wuhong Pei

Publications and source records attributed to Wuhong Pei.

6 recordsLinked to original sources

PubMed2005

Identification of the putative staphylococcal AgrB catalytic residues involving the proteolytic cleavage of AgrD to generate autoinducing peptide.

The P2 operon of the staphylococcal accessory gene regulator (agr) encodes four genes (agrA, -B, -C, and -D) whose products compose a quorum sensing system: AgrA and AgrC resemble a two-component signal transduction system of which AgrC is a sensor kinase and AgrA is a response regulator; AgrD, a polypeptide that is integrated into the cytoplasmic membrane via an amphipathic alpha-helical motif in its N-terminal region, is the propeptide for an autoinducing peptide that is the ligand for AgrC; and AgrB is a novel membrane protein that involves in the processing of AgrD propeptide and possibly the secretion of the mature autoinducing peptide. In this study, we demonstrated that AgrB had endopeptidase activity, and identified 2 amino acid residues in AgrB (cysteine 84 and histidine 77) that might form a putative cysteine endopeptidase catalytic center in the proteolytic cleavage of AgrD at its C-terminal processing site. Computer analysis revealed that the cysteine and histidine residues were conserved among the potential AgrB homologous proteins, suggesting that the Agr quorum sensing system homologues might also exist in other Gram-positive bacteria.

Amino Acid Sequence↗

PubMed2005

Staphylococcus intermedius produces a functional agr autoinducing peptide containing a cyclic lactone.

The agr system is a global regulator of accessory functions in staphylococci, including genes encoding exoproteins involved in virulence. The agr locus contains a two-component signal transduction module that is activated by an autoinducing peptide (AIP) encoded within the agr locus and is conserved throughout the genus. The AIP has an unusual partially cyclic structure that is essential for function and that, in all but one case, involves an internal thiolactone bond between a conserved cysteine and the C-terminal carboxyl group. The exceptional case is a strain of Staphylococcus intermedius that has a serine in place of the conserved cysteine. We demonstrate here that the S. intermedius AIP is processed by the S. intermedius AgrB protein to generate a cyclic lactone, that it is an autoinducer as well as a cross-inhibitor, and that all of five other S. intermedius strains examined also produce serine-containing AIPs.

Amino Acid Sequence↗

PubMed2002

Redesign of a four-helix bundle protein by phage display coupled with proteolysis and structural characterization by NMR and X-ray crystallography.

To test whether it is practical to use phage display coupled with proteolysis for protein design, we used this approach to convert a partially unfolded four-helix bundle protein, apocytochrome b(562), to a stably folded four-helix bundle protein. Four residues expected to form a hydrophobic core were mutated. One residue was changed to Trp to provide a fluorescence probe for studying the protein's physical properties and to partially fill the void left by the heme. The other three positions were randomly mutated. In addition, another residue in the region to be redesigned was substituted with Arg to provide a specific cutting site for protease Arg-c. This library of mutants was displayed on the surface of phage and challenged with protease Arg-c to select stably folded proteins. The consensus sequence that emerged from the selection included hydrophobic residues at only one of the three positions and non-hydrophobic residues at the other two. Nevertheless, the selected proteins were thermodynamically very stable. The structure of a selected protein was characterized using multi-dimensional NMR. All four helices were formed in the structure. Further, site-directed mutagenesis was used to change one of the two non-hydrophobic residues to a hydrophobic residue, which increased the stability of the protein, indicating that the selection result was not based solely on the protein's global stability and that local structural characteristics may also govern the selection. This conclusion is supported by the crystal structure of another mutant that has two hydrophobic residues substituted for the two non-hydrophobic residues. These results suggest that the hydrophobic interactions in the core are not sufficient to dictate the selection and that the location of the cutting site of the protease also influences the selection of structures.

Amino Acid Sequence↗

PubMed2002

Populating partially unfolded forms by hydrogen exchange-directed protein engineering.

The native-state hydrogen exchange of a redesigned apocytochrome b(562) suggests that at least two partially unfolded forms (PUFs) exist for this four-helix bundle protein under native conditions. The more stable PUF has the N-terminal helix unfolded. To verify the conclusion further and obtain more detailed structural information about this PUF, five hydrophobic core residues in the N-terminal helix were mutated to Gly and Asp to destabilize the native state selectively and populate the PUF for structural studies. The secondary structure and the backbone dynamics of this mutant were characterized using multidimensional NMR. Consistent with the prediction, the N-terminal region of the mutant was found to be unfolded while other parts of the proteins remained folded. These results suggest that native-state hydrogen exchange-directed protein engineering can be a useful approach to populating partially unfolded forms for detailed structural studies.

Amyloid↗

PubMed2002

Relationship between the native-state hydrogen exchange and folding pathways of a four-helix bundle protein.

The hydrogen exchange behavior of a four-helix bundle protein in low concentrations of denaturant reveals some partially unfolded forms that are significantly more stable than the fully unfolded state. Kinetic folding of the protein, however, is apparently two-state in the absence of the accumulation of early folding intermediates. The partially unfolded forms are either as folded as or more folded than the rate-limiting transition state and appear to represent the major intermediates in a folding and unfolding reaction. These results are consistent with the suggestion that partially unfolded intermediates may form after the rate-limiting step for small proteins with apparent two-state folding kinetics.

Amides↗

PubMed2000

[The Comparison between the Killing Effects of Two Anti-T Immunotoxins on Target Cells]

The key to killing target cells by immunotoxin depends on the specific recognition of antibody to target cell and the cytotoxic effect of toxin. The comparative study of the killing effects of two anti-T immunotoxins, CD5:Ricin and CD5:rRA, on target cells was performed. The elimination rate of immunotoxins was analysed by flow cytometry and MLR. The effect of immunotoxins on the proliferation of hematopoiesis was evaluted by CFU-GM. The results showed that (1) CD5(+) T cells were eliminated and CD25(+) CD3(+) activated T cells were concentration-dependently inhibited by the two immunotoxins in the range of 10(-9) - 10(-11) mol/L; (2) both immunotoxins significantly inhibited the mixed lymphocyte reaction, and the inhibiting effect of CD5:rRA to T cell proliferation was markedly lower than that of CD5:Ricin in the range of 10(-10) - 10(-11) mol/L; (3) the combination of CD5:rRA with 10 mmol/L NH(4)Cl increased the T cell elimination rate; and (4) the two immunotoxins and the combination of NH(4)Cl and CD5:rRA did not suppressed proliferation of granulocyte-macrophage progenitors in the range of concentrations with killing effect. It was concluded that T cell and activated T cell could be eliminated effectively by immunotoxins, the proliferation of granulocyte-macrophage progenitor was not inhibited significantly.

Journal Article↗