Biomedical subjects

Luhua Lai

Publications and source records attributed to Luhua Lai.

At least 19 recordsLinked to original sources

PubMed2023

Photoaffinity labeling coupled with proteomics identify PDI-ADAM17 module is targeted by (-)-vinigrol to induce TNFR1 shedding and ameliorate rheumatoid arthritis in mice.

Various biological agents have been developed to target tumor necrosis factor alpha (TNF-α) and its receptor TNFR1 for the rheumatoid arthritis (RA) treatment, whereas small molecules modulating such cytokine receptors are rarely reported in comparison to the biologicals. Here, by revealing the mechanism of action of vinigrol, a diterpenoid natural product, we show that inhibition of the protein disulfide isomerase (PDI, PDIA1) by small molecules activates A disintegrin and metalloprotease 17 (ADAM17) and then leads to the TNFR1 shedding on mouse and human cell membranes. This small-molecule-induced receptor shedding not only effectively blocks the inflammatory response caused by TNF-α in cells, but also reduces the arthritic score and joint damage in the collagen-induced arthritis mouse model. Our study indicates that targeting the PDI-ADAM17 signaling module to regulate the shedding of cytokine receptors by the chemical approach constitutes a promising strategy for alleviating RA.

Mice↗

PubMed2004

Estimating protein-ligand binding free energy: atomic solvation parameters for partition coefficient and solvation free energy calculation.

Solvation energy calculation is one of the main difficulties for the estimation of protein-ligand binding free energy and the correct scoring in docking studies. We have developed a new solvation energy estimation method for protein-ligand binding based on atomic solvation parameter (ASP), which has been shown to improve the power of protein-ligand binding free energy predictions. The ASP set, designed to handle both proteins and organic compounds and derived from experimental n-octanol/water partition coefficient (log P) data, contains 100 atom types (united model that treats hydrogen atoms implicitly) or 119 atom types (all-atom model that treats hydrogen atoms explicitly). By using this unified ASP set, an algorithm was developed for solvation energy calculation and was further integrated into a score function for predicting protein-ligand binding affinity. The score function reproduced the absolute binding free energies of a test set of 50 protein-ligand complexes with a standard error of 8.31 kJ/mol. As a byproduct, a conformation-dependent log P calculation algorithm named ASPLOGP was also implemented. The predictive results of ASPLOGP for a test set of 138 compounds were r = 0.968, s = 0.344 for the all-atom model and r = 0.962, s = 0.367 for the united model, which were better than previous conformation-dependent approaches and comparable to fragmental and atom-based methods. ASPLOGP also gave good predictive results for small peptides. The score function based on the ASP model can be applied widely in protein-ligand interaction studies and structure-based drug design.

Databases, Protein↗

PubMed2004

Suppression of SARS-CoV entry by peptides corresponding to heptad regions on spike glycoprotein.

Heptad repeat regions (HR1 and HR2) are highly conserved sequences located in the glycoproteins of enveloped viruses. They form a six-helix bundle structure and are important in the process of virus fusion. Peptides derived from the HR regions of some viruses have been shown to inhibit the entry of these viruses. SARS-CoV was also predicted to have HR1 and HR2 regions in the S2 protein. Based on this prediction, we designed 25 peptides and screened them using a HIV-luc/SARS pseudotyped virus assay. Two peptides, HR1-1 and HR2-18, were identified as potential inhibitors, with EC(50) values of 0.14 and 1.19microM, respectively. The inhibitory effects of these peptides were validated by the wild-type SARS-CoV assay. HR1-1 and HR2-18 can serve as functional probes for dissecting the fusion mechanism of SARS-CoV and also provide the potential of further identifying potent inhibitors for SARS-CoV entry.

Amino Acid Sequence↗

PubMed2004

3C-like proteinase from SARS coronavirus catalyzes substrate hydrolysis by a general base mechanism.

SARS 3C-like proteinase has been proposed to be a key enzyme for drug design against SARS. Lack of a suitable assay has been a major hindrance for enzyme kinetic studies and a large-scale inhibitor screen for SARS 3CL proteinase. Since SARS 3CL proteinase belongs to the cysteine protease family (family C3 in clan CB) with a chymotrypsin fold, it is important to understand the catalytic mechanism of SARS 3CL proteinase to determine whether the proteolysis proceeds through a general base catalysis mechanism like chymotrypsin or an ion pair mechanism like papain. We have established a continuous colorimetric assay for SARS 3CL proteinase and applied it to study the enzyme catalytic mechanism. The proposed catalytic residues His41 and Cys145 were confirmed to be critical for catalysis by mutating to Ala, while the Cys145 to Ser mutation resulted in an active enzyme with a 40-fold lower activity. From the pH dependency of catalytic activity, the pK(a)'s for His41 and Cys145 in the wild-type enzyme were estimated to be 6.38 and 8.34, while the pK(a)'s for His41 and Ser145 in the C145S mutant were estimated to be 6.15 and 9.09, respectively. The C145S mutant has a normal isotope effect in D(2)O for general base catalysis, that is, reacts slower in D(2)O, while the wild-type enzyme shows an inverse isotope effect which may come from the lower activation enthalpy. The pK(a) values measured for the active site residues and the activity of the C145S mutant are consistent with a general base catalysis mechanism and cannot be explained by a thiolate-imidazolium ion pair model.

Alanine↗

PubMed2004

Formation of amyloid fibrils from fully reduced hen egg white lysozyme.

The fully reduced hen egg white lysozyme (HEWL), which is a good model of random coil structure, has been converted to highly organized amyloid fibrils at low pH by adding ethanol. In the presence of 90% (v/v) ethanol, the fully reduced HEWL adopts beta-sheet secondary structure at pH 4.5 and 5.0, and an alpha-to-beta transition is observed at pH 4.0. A red shift of the Congo red absorption spectrum caused by the precipitation of the fully reduced HEWL in the presence of 90% (v/v) ethanol is typical of the presence of amyloid aggregation. EM reveals unbranched fibrils with a diameter of 2-5 nm and as long as 1-2 microm. The pH dependence of the initial structure of the fully reduced HEWL in the presence of 90% (v/v) ethanol suggests that Asp and His residues may play an important role.

Amyloid↗

PubMed2004

Chlorobenzylidine-calf thymus DNA interaction II: circular dichroism and nuclear magnetic resonance studies.

The binding of chlorobenzylidine to calf thymus DNA has been studied in detail by means of circular dichroism (CD), nuclear magnetic resonance (1H NMR), viscosimetry and denaturation temperature (Tm). Chlorobenzylidine is found to intercalate between base pairs of DNA as evidence by: (1) induced circular dichroism; (2) broadened 1H NMR signals; (3) enhanced viscosity; and (4) increased denaturation temperature of the DNA helix. In addition, Scatchard plot from CD titration data gives a binding constant of 2.6 x 10(4) M(-1) and a binding site size of 4 base pairs at 25 degrees C. Matrix rank analysis shows that there are three main components contributing to the observed CD spectra. The nuclear magnetic resonance experiment shows that the planar aromatic ring of tetrahydroberberine group in chlorobenzylidine intercalates between the base pairs of DNA when chlorobenzylidine is bound to DNA.

Animals↗

PubMed2003

Structure-based method for analyzing protein-protein interfaces.

Hydrogen bond, hydrophobic and vdW interactions are the three major non-covalent interactions at protein-protein interfaces. We have developed a method that uses only these properties to describe interactions between proteins, which can qualitatively estimate the individual contribution of each interfacial residue to the binding and gives the results in a graphic display way. This method has been applied to analyze alanine mutation data at protein-protein interfaces. A dataset containing 13 protein-protein complexes with 250 alanine mutations of interfacial residues has been tested. For the 75 hot-spot residues (deltadelta G > or = 1.5 kcal mol(-1)), 66 can be predicted correctly with a success rate of 88%. In order to test the tolerance of this method to conformational changes upon binding, we utilize a set of 26 complexes with one or both of their components available in the unbound form. The difference of key residues exported by the program is 11% between the results using complexed proteins and those from unbound ones. As this method gives the characteristics of the binding partner for a particular protein, in-depth studies on protein-protein recognition can be carried out. Furthermore, this method can be used to compare the difference between protein-protein interactions and look for correlated mutation.

Alanine↗

PubMed2003

Biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3C-like proteinase.

The 3C-like proteinase of severe acute respiratory syndrome (SARS) coronavirus has been proposed to be a key target for structural-based drug design against SARS. In order to understand the active form and the substrate specificity of the enzyme, we have cloned, expressed, and purified SARS 3C-like proteinase. Analytic gel filtration shows a mixture of monomer and dimer at a protein concentration of 4 mg/ml and mostly monomer at 0.2 mg/ml, which correspond to the concentration used in the enzyme assays. The linear decrease of the enzymatic-specific activity with the decrease of enzyme concentration revealed that only the dimeric form is active and the dimeric interface could be targeted for structural-based drug design against SARS 3C-like proteinase. By using a high pressure liquid chromatography assay, SARS 3C-like proteinase was shown to cut the 11 peptides covering all of the 11 cleavage sites on the viral polyprotein with different efficiency. The two peptides corresponding to the two self-cleavage sites are the two with highest cleavage efficiency, whereas peptides with non-canonical residues at P2 or P1' positions react slower. The P2 position of the substrates seems to favor large hydrophobic residues. Secondary structure studies for the peptide substrates revealed that substrates with more beta-sheetlike structure tend to react fast. This study provides a basic understanding of the enzyme catalysis and a full substrate specificity spectrum for SARS 3C-like proteinase, which are helpful for structural-based inhibitor design against SARS and other coronavirus.

Catalysis↗

PubMed2003

On the mechanism of chaperone activity of the small heat-shock protein of Methanococcus jannaschii.

The small heat-shock protein (sHSP) from Methanococcus jannaschii (Mj HSP16.5) forms a homomeric complex of 24 subunits and has an overall structure of a multiwindowed hollow sphere with an external diameter of approximately 120 A and an internal diameter of approximately 65 A with six square "windows" of approximately 17 A across and eight triangular windows of approximately 30 A across. This sHSP has been known to protect other proteins from thermal denaturation. Using purified single-chain monellin as a substrate and a series of methods such as protease digestion, antibody binding, and electron microscopy, we show that the substrates bind to Mj HSP16.5 at a high temperature (80 degrees C) on the outside surface of the sphere and are prevented from forming insoluble substrate aggregates in vitro. Circular dichroism studies suggest that a very small, if any, conformational change occurs in sHSP even at 80 degrees C, but substantial conformational changes of the substrate are required for complex formation at 80 degrees C. Furthermore, deletion mutation studies of Mj HSP16.5 suggest that the N-terminal region of the protein has no structural role but may play an important kinetic role in the assembly of the sphere by "preassembly condensation" of multiple monomers before final assembly of the sphere.

Archaeal Proteins↗

PubMed2003

[Modeling of the SARS coronavirus main proteinase and conformational flexibility of the active site].

SARS coronavirus 3CL proteinase is the key enzyme for virus replication which may serve as the target for drug discovery against SARS. A 3D structure model has been built for SARS coronavirus 3CL proteinase by comparative protein modeling. A homodimer model of the proteinase was also built. Analysis of the dimeric interface suggests the 3CL proteinase may have dimer form in solution. The conformational flexibility of the active site has been simulated by molecular dynamics combined with multi-canonical sampling. The active site loops have two typical conformations which may be related to the conformational movement associated with the enzymatic reaction.

Amino Acid Sequence↗

PubMed2003

Direct observations of the cleavage reaction of an L-DPPC monolayer catalyzed by phospholipase A2 and inhibited by an indole inhibitor at the air/water interface.

The enzymatic hydrolysis of an L-dipalmitoylphosphatidylcholine (L-DPPC) monolayer at the air/water interface, catalyzed by phospholipase A(2) (PLA(2)), serves as a model for biospecific interfacial reactions. The cleavage of L-DPPC was investigated by Brewster angle microscopy. Different types of domain defects were observed to form in the coexisting liquid expanded and liquid condensed phases during the hydrolysis reaction. The adsorption of the enzyme was quantitatively recorded as the increase of the surface pressure over a fixed molecular area with time. In the case of L-DPPC, the surface pressure first increases and then starts to decrease, which indicates that a soluble product (lysolipid) is produced during the catalytic cleavage reaction. The increase and decrease of the surface pressure, which corresponds to the change of shape and number density of domains, indicated the occurrence of the following processes: adsorption of PLA(2), cleavage reaction, and rearrangement of substrate and product molecules at the interface. Addition of a PLA(2) inhibitor to the lipid monolayer leads to a fast surface pressure increase after enzyme injection. The surface pressure reaches a maximum value and then does not change for a long time. During this period, no change in the domain shape and number density was observed, which indicates that the enzyme is inhibited for a certain period of time. The experimental results provide the possibility of a direct way to prove inhibitor activity.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2003

Metal ion binding and enzymatic mechanism of Methanococcus jannaschii RNase HII.

RNase H degrades the RNA moiety in DNA:RNA hybrid in a divalent metal ion dependent manner. It is essential to understand the role of metal ion in enzymatic mechanism. One of the key points in this study is how many metal ions are involved in the enzyme catalysis. Accordingly, either one-metal binding mechanism or two-metal binding mechanism is proposed. We have studied the thermodynamic properties of four metal ions (Mg(2+), Mn(2+), Ca(2+), and Ba(2+)) binding to Methanococcus jannaschii RNase HII using isothermal titration calorimetry. All of the four metal ions were found to bind Mj RNase HII with 1:1 stoichiometry in the absence of substrate. Together with enzymatic activity assay data, we propose that only one metal ion binding to the enzyme in catalytic process. We also studied the pH dependence of metal binding and enzyme activity and found that at pH 6.5, Mg(2+) did not bind to the enzyme without the substrate but still activated the enzyme to about 2% of its maximum activity (in 10 mM Mn(2+) at pH 8). This implies that the substrate may also be incorporated in metal ion binding and help to position the metal ion. To find which acidic residues correspond to metal ion binding, we also studied the binding thermodynamics and enzymatic activity assay of four mutants: D7N, E8Q, D112N, and D149N in the presence of Mn(2+). The thermodynamic parameters are least affected for the D149N mutant, which has a very low enzymatic activity. This indicates that Asp149 is essential for the enzymatic activity. On the basis of all these observations, we suggest a metal binding model in which D7, E8, and D112 bind the metal ion and D149 activates a water molecule to attack the P-O bond in the RNA chain of the substrate.

Archaeal Proteins↗

PubMed2003

Beyond the rotamer library: genetic algorithm combined with the disturbing mutation process for upbuilding protein side-chains.

The disturbing genetic algorithm, incorporating the disturbing mutation process into the genetic algorithm flow, has been developed to extend the searching space of side-chain conformations and to improve the quality of the rotamer library. Moreover, the growing generation amount idea, simulating the real situation of the natural evolution, is introduced to improve the searching speed. In the calculations using the pseudo energy scoring function of the root mean squared deviation, the disturbing genetic algorithm method has been shown to be highly efficient. With the real energy function based on AMBER force field, the program has been applied to rebuilding side-chain conformations of 25 high-quality crystallographic structures of single-protein and protein-protein complexes. The averaged root mean standard deviation of atom coordinates in side-chains and veracities of the torsion angles of chi(1) and chi(1) + chi(2) are 1.165 A, 88.2 and 72.9% for the buried residues, respectively, and 1.493 A, 79.2 and 64.7% for all residues, showing that the method has equal precision to the program SCWRL, whereas it performs better in the prediction of buried residues and protein-protein interfaces. This method has been successfully used in redesigning the interface of the Basnase-Barstar complex, indicating that it will have extensive application in protein design, protein sequence and structure relationship studies, and research on protein-protein interaction.

Algorithms↗

PubMed2003

Thermodynamic characterization of the folding coupled DNA binding by the monomeric transcription activator GCN4 peptide.

Dimerization is a widely believed critical requirement for the yeast transcriptional activator GCN4 specifically recognizing its DNA target sites. Nonetheless, the binding of the monomeric GCN4 to DNA target sites AP-1 and ATF/CREB was recently detected by kinetic studies. Here, for the first time, we present a detailed description of the thermodynamics of a monomeric peptide GCN4-br, the basic region (226-252) of GCN4, binding to AP-1, and ATF/CREB. GCN4 specifically binds to AP-1 and ATF/CREB in the monomeric form as shown by our circular dichroism thermal unfolding measurements. Isothermal titration calorimetry experiments indicate that the binding process of GCN4-br with DNA is enthalpically driven, accompanied by an unfavorable entropy change. The temperature dependence of DeltaH(0) reveals negative changes in heat capacity DeltaC(p): DeltaC(p) = -0.92 kJ. mol(-1) K(-1) and DeltaC(p) = -0.95 kJ. mol(-1) K(-1) for GCN4-br binding to AP-1 and ATF/CREB, respectively, which is a striking manifestation of GCN4-br specifically recognizing DNA target sites. These thermodynamic characteristics may give new insight into the mechanism by which GCN4 protein binds to DNA target sites for its transcriptional regulation.

Activating Transcription Factor 2↗

PubMed2002

Monitoring the kinetics and thermodynamics of interfacial enzymatic catalysis by differential scanning calorimetry.

Using phase transition profile as an indicator of thermodynamic property and phase transition heat as the second indicator of the percentage of substrates unhydrolyzed, differential scanning calorimetry has been used to observe in detail the kinetics and thermodynamics of phospholipase A(2)-catalyzed 1,2-dipalmitoyl-sn-glycero-3-phosphocholine large unilamellar vesicle (LUV) hydrolysis. Phase transition profiles show that the original LUV almost completely changes into a novel aggregate at the end of the latency, followed by an abrupt activation of the reaction. The phase transition profiles are asymmetric between the heating and cooling curves, indicating a thermodynamic mesostatic property of the system. The reaction in activated phase follows a single first-order kinetics and all of the substrates in vesicles can be hydrolyzed. All these evidences indicate that the products and substrates can freely exchange between the outer and the inner layers of the vesicles and the membrane of the vesicle in the activated phase is permeable. This permeability favors the exchange of the substrates and products, thus, resulting in the activation of the fast reaction.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2002

CH...O hydrogen bonds at protein-protein interfaces.

For the first time, a statistical potential has been developed to quantitatively describe the CH.O hydrogen bonding interaction at the protein-protein interface. The calculated energies of the CH.O pair interaction show a favorable valley at approximately 3.3 A, exhibiting a feature typical of an H-bond and similar to the ab initio quantum calculation result (Scheiner, S., Kar, T., and Gu, Y. (2001) J. Biol. Chem. 276, 9832-9837). The potentials have been applied to a set of 469 protein-protein complexes to calculate the contribution of different types of interactions to each protein complex: the average energy contribution of a conventional H-bond is approximately 30%; that of a CH.O H-bond is 17%; and that of a hydrophobic interaction is 50%. In some protein-protein complexes, the contribution of the CH.O H-bond can reach as high as approximately 40-50%, indicating the importance of the CH.O H-bond at the protein interface. At the interfaces of these complexes, C(alpha)H.O H-bonds frequently occur between adjacent strands in both parallel and antiparallel orientations, having the obvious structural motif of bifurcated H-bonds. Our study suggests that the weak CH.O H-bond makes an important contribution to the association and stability of protein complexes and needs more attention in protein-protein interaction studies.

Amino Acid Sequence↗

PubMed2002

Linear correlation between thermal stability and folding kinetics of lysozyme.

We have studied the refolding and thermal denaturation of hen egg white lysozyme in a wide range of pH values (from 1.5 to 9.4) using stopped-flow circular dichroism (CD) and differential scanning calorimetry (DSC). A linear correlation was found between the thermal denaturation temperature (T(m)) and the logarithm of the refolding rate of the slow folding phase of hen egg white lysozyme (lnk(2)).

Animals↗

PubMed2002

Potential of mean force for protein-protein interaction studies.

Calculating protein-protein interaction energies is crucial for understanding protein-protein associations. On the basis of the methodology of mean-field potential, we have developed an empirical approach to estimate binding free energy for protein-protein interactions. This knowledge-based approach has been used to derive distance-dependent free energies of protein complexes from a nonredundant training set in the Protein Data Bank (PDB), with a careful treatment of homology. We calculate atom pair potentials for 16 pair interactions, which can reflect the importance of hydrophobic interactions and specific hydrogen-bonding interactions. The derived potentials for hydrogen-bonding interactions show a valley of favorable interactions at a distance of approximately 3 A, corresponding to that of an established hydrogen bond. For the test set of 28 protein complexes, the calculated energies have a correlation coefficient of 0.75 compared with experimental binding free energies. The performance of the method in ranking the binding energies of different protein-protein complexes shows that the energy estimation can be applied to value binding free energies for protein-protein associations.

Databases, Protein↗