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↗

PubMed2006

Robustness and modular design of the Drosophila segment polarity network.

Biomolecular networks have to perform their functions robustly. A robust function may have preferences in the topological structures of the underlying network. We carried out an exhaustive computational analysis on network topologies in relation to a patterning function in Drosophila embryogenesis. We found that whereas the vast majority of topologies can either not perform the required function or only do so very fragilely, a small fraction of topologies emerges as particularly robust for the function. The topology adopted by Drosophila, that of the segment polarity network, is a top ranking one among all topologies with no direct autoregulation. Furthermore, we found that all robust topologies are modular-each being a combination of three kinds of modules. These modules can be traced back to three subfunctions of the patterning function, and their combinations provide a combinatorial variability for the robust topologies. Our results suggest that the requirement of functional robustness drastically reduces the choices of viable topology to a limited set of modular combinations among which nature optimizes its choice under evolutionary and other biological constraints.

Animals↗

PubMed2006

A combinatorial score to distinguish biological and nonbiological protein-protein interfaces.

With the large amount of protein-protein complex structural data available, to understand the key features governing the specificity of protein-protein recognition and to define a suitable scoring function for protein-protein interaction predictions, we have analyzed the protein interfaces from geometric and energetic points of view. Atom-based potential of mean force (PMFScore), packing density, contact size, and geometric complementarity are calculated for crystal contacts in 74 homodimers and 91 monomers, which include real biological interactions in dimers and nonbiological contacts in monomers and dimers. Simple cutoffs were developed for single and combinatorial parameters to distinguish biological and nonbiological contacts. The results show that PMFScore is a better discriminator between biological and nonbiological interfaces comparable in size. The combination of PMFScore and contact size is the most powerful pairwise discriminator. A combinatorial score (CFPScore) based on the four parameters was developed, which gives the success rate of the homodimer discrimination of 96.6% and error rate of the monomer discrimination of 6.0% and 19.8% according to Valdar's and our definition, respectively. Compared with other statistical learning models, the cutoffs for the four parameters and their combinations are directly based on physical models, simple, and can be easily applied to protein-protein interface analysis and docking studies.

Binding Sites↗

PubMed2006

Isatin compounds as noncovalent SARS coronavirus 3C-like protease inhibitors.

A series of isatin derivatives were synthesized and tested against SARS CoV 3C-like protease. Substitutions at the N-1 and C-5 positions were examined to elucidate the differences in substrate binding sites of the rhinovirus 3C protease and SARS CoV 3C-like protease. Compound 5f shows significant inhibition with an IC(50) of 0.37 microM. Further study showed that, unlike the irreversible covalent binding of isatin derivatives to human rhinovirus 3C protease, the compounds tested in this study are all noncovalent reversible inhibitors.

Coronavirus 3C Proteases↗

PubMed2006

Subunit exchange of MjHsp16.5 studied by single-molecule imaging and fluorescence resonance energy transfer.

MjHsp16.5 was separately labeled by fluorescent dye Cy3 and Cy5.5. The dissociation event of a single 24-mer MjHsp16.5 molecule was captured by single-molecule imaging (SMI). Temperature-regulated subunit exchange was revealed by the real-time fluorescence resonance energy transfer (FRET). The combination of single-molecular statistics and kinetic parameters from FRET experiments leads to the conclusion that below 75 degrees C the rate-determining step of the subunit exchange was the dissociation of the dye-labeled 24-mer in which the dimer was intact, whereas above 75 degrees C, smaller units emerged in the exchange and the rate-determining step had the character of a bimolecular reaction.

Archaeal Proteins↗

PubMed2006

Structural, biochemical, and dynamic characterizations of the hRPB8 subunit of human RNA polymerases.

The RPB8 subunit is present in all three types of eukaryotic RNA polymerases and is highly conserved during evolution. It is an essential subunit required for the transcription of nuclear genes, but the detailed mechanism including its interactions with different subunits and oligonucleotides remains largely unclear. Herein, we report the three-dimensional structure of human RPB8 (hRPB8) at high resolution determined by NMR spectroscopy. The protein fold comprises an eight-stranded beta-barrel, six short helices, and a large unstructured Omega-loop. The overall structure of hRPB8 is similar to that of yRPB8 from Saccharomyces cerevisiae and belongs to the oligonucleotide/oligosaccharide-binding fold. However, several features of the tertiary structures are notably different between the two proteins. In particular, hRPB8 has a more clustered positively charged binding interface with the largest subunit RPB1 of the RNA polymerases. We employed biochemical methods to detect its interactions with different single-stranded DNA sequences. In addition, single-stranded DNA titration experiments were performed to identify the residues involved in nonspecific binding with different DNA sequences. Furthermore, we characterized the millisecond time scale conformational flexibility of hRPB8 upon its binding to single-stranded DNA. The current results demonstrate that hRPB8 interacts with single-stranded DNA nonspecifically and adopts significant conformational changes, and the hRPB8/single-stranded DNA complex is a fast exchanging system. The solution structure in conjunction with the biochemical and dynamic studies reveal new aspects of this subunit in the molecular assembly and the biological function of the human nuclear RNA polymerases.

Amino Acid Sequence↗

PubMed2006

Only one protomer is active in the dimer of SARS 3C-like proteinase.

The severe acute respiratory syndrome coronavirus 3C-like protease has been proposed to be a key target for structurally based drug design against SARS. The enzyme exists as a mixture of dimer and monomer, and only the dimer was considered to be active. In this report, we have investigated, using molecular dynamics simulation and mutational studies, the problems as to why only the dimer is active and whether both of the two protomers in the dimer are active. The molecular dynamics simulations show that the monomers are always inactive, that the two protomers in the dimer are asymmetric, and that only one protomer is active at a time. The enzyme activity of the hybrid severe acute respiratory syndrome coronavirus 3C-like protease of the wild-type protein and the inactive mutant proves that the dimerization is important for enzyme activity and only one active protomer in the dimer is enough for the catalysis. Our simulations also show that the right conformation for catalysis in one protomer can be induced upon dimer formation. These results suggest that the enzyme may follow the association, activation, catalysis, and dissociation mechanism for activity control.

Binding Sites↗

PubMed2006

PSI-DOCK: towards highly efficient and accurate flexible ligand docking.

We have developed a new docking method, Pose-Sensitive Inclined (PSI)-DOCK, for flexible ligand docking. An improved SCORE function has been developed and used in PSI-DOCK for binding free energy evaluation. The improved SCORE function was able to reproduce the absolute binding free energies of a training set of 200 protein-ligand complexes with a correlation coefficient of 0.788 and a standard error of 8.13 kJ/mol. For ligand binding pose exploration, a unique searching strategy was designed in PSI-DOCK. In the first step, a tabu-enhanced genetic algorithm with a rapid shape-complementary scoring function is used to roughly explore and store potential binding poses of the ligand. Then, these predicted binding poses are optimized and compete against each other by using a genetic algorithm with the accurate SCORE function to determine the binding pose with the lowest docking energy. The PSI-DOCK 1.0 program is highly efficient in identifying the experimental binding pose. For a test dataset of 194 complexes, PSI-DOCK 1.0 achieved a 67% success rate (RMSD < 2.0 A) for only one run and a 74% success rate for 10 runs. PSI-DOCK can also predict the docking binding free energy with high accuracy. For a test set of 64 complexes, the correlation between the experimentally observed binding free energies and the docking binding free energies for 64 complexes is r = 0.777 with a standard deviation of 7.96 kJ/mol. Moreover, compared with other docking methods, PSI-DOCK 1.0 is extremely easy to use and requires minimum docking preparations. There is no requirement for the users to add hydrogen atoms to proteins because all protein hydrogen atoms and the flexibility of the terminal protein atoms are intrinsically taken into account in PSI-DOCK. There is also no requirement for the users to calculate partial atomic charges because PSI-DOCK does not calculate an electrostatic energy term. These features are not only convenient for the users but also help to avoid the influence of different preparation methods.

Algorithms↗

PubMed2006

A continuous fluorescence assay for phospholipase A2 with nontagged lipid.

Human nonpancreatic secreted phospholipase A2 (hnps PLA2) is considered to be an important drug target for antiinflammation therapy. We have established a new fluorescence assay by using 1-anilinonaphthalene-8-sulfonate (ANS) as an interfacial probe for hydrophobic environment detection. The fitted apparent k(cat)/K(m) of hnps PLA2 is 0.0181 +/- 0.0005 RFU/microMs. Tests on known synthesized inhibitor gave IC50 values similar to those from isotope-labeled assay. Because ANS is a commonly used probe for hydrophobic environment detection that needs no modification in the current assay, this strategy may be widely applicable for interfacial catalytic reactions.

Anilino Naphthalenesulfonates↗

PubMed2006

Quaternary structure, substrate selectivity and inhibitor design for SARS 3C-like proteinase.

The SARS coronavirus 3C-like proteinase is recognized as a potential drug design target for the treatment of severe acute respiratory syndrome. In the past few years, much work has been done to understand the catalytic mechanism of this target protein and to design its selective inhibitors. The protein exists as a dimer/monomer mixture in solution and the dimer was confirmed to be the active species for the enzyme reaction. Quantitative dissociation constants have been reported for the dimer by using analytic ultracentrifuge, gel filtration and enzyme assays. Though the enzyme is a cysteine protease with a chymotrypsin fold, SARS 3C-like proteinase follows the general base catalytic mechanism similar to chymotrypsin. As the enzyme can cut eleven different sites on the viral polyprotein, the substrate specificity has been studied by synthesized peptides corresponding or similar to the cleavage sites on the polyprotein. Predictive model was built for substrate structure and activity relationships and can be applied in inhibitor design. Due to the lack of potential drugs for the treatment of SARS, the discovery of inhibitors against SARS 3C-like proteinase, which can potentially be optimized as drugs appears to be highly desirable. Various groups have been working on inhibitor discovery by virtual screening, compound library screening, modification of existing compounds or natural products. High-throughput in vitro assays, auto-cleavage assays and viral replication assays have been developed for inhibition activity tests. Inhibitors with IC50 values as low as 60 nM have been reported.

Animals↗

PubMed2005

The N-terminal octapeptide acts as a dimerization inhibitor of SARS 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. Accurate determination of the dimer dissociation constant and the role of the N-finger (residues 1-7) will provide more insights into the enzyme catalytic mechanism of SARS 3CL proteinase. The dimer dissociation constant of the wild-type protein was determined to be 14.0microM by analytical ultracentrifugation method. The N-finger fragment of the enzyme plays an important role in enzyme dimerization as shown in the crystal structure. Key residues in the N-finger have been studied by site-directed mutagenesis, enzyme assay, and analytical ultracentrifugation. A single mutation of M6A was found to be critical to maintain the dimer structure of the enzyme. The N-terminal octapeptide N8 and its mutants were also synthesized and tested for their potency as dimerization inhibitors. Peptide cleavage assay confirms that peptide N8 is a dimerization inhibitor with a K(i) of 2.20mM. The comparison of the inhibitory activities of N8 and its mutants indicates that the hydrophobic interaction of Met-6 and the electrostatic interaction of Arg-4 contribute most for inhibitor binding. This study describes the first example of inhibitors targeting the dimeric interface of SARS 3CL proteinase, providing a novel strategy for drug design against SARS and other coronaviruses.

Binding Sites↗

PubMed2005

The interaction between severe acute respiratory syndrome coronavirus 3C-like proteinase and a dimeric inhibitor by capillary electrophoresis.

3C-like proteinase of severe acute respiratory syndrome (SARS) coronavirus has been demonstrated to be a key target for drug design against SARS. The interaction between SARS coronavirus 3C-like (3CL) proteinase and an octapeptide interface inhibitor was studied by affinity capillary electrophoresis (ACE). The binding constants were estimated by the change of migration time of the analytes in the buffer solution containing different concentrations of SARS 3CL proteinase. The results showed that SARS 3CL proteinase was able to complex with the octapeptide competitively, with binding constants of 2.44 x 10(4) M(-1) at 20 degrees C and 2.11 x 10(4)M(-1) at 37 degrees C. In addition, the thermodynamic parameters deduced reveal that hydrophobic interaction might play major roles, along with electrostatic force, in the binding process. The ACE method used here could be developed to be an effective and simple way of applying large-scale drug screening and evaluation.

3C Viral Proteases↗

PubMed2005

Specificity of trypsin and chymotrypsin: loop-motion-controlled dynamic correlation as a determinant.

Trypsin and chymotrypsin are both serine proteases with high sequence and structural similarities, but with different substrate specificity. Previous experiments have demonstrated the critical role of the two loops outside the binding pocket in controlling the specificity of the two enzymes. To understand the mechanism of such a control of specificity by distant loops, we have used the Gaussian network model to study the dynamic properties of trypsin and chymotrypsin and the roles played by the two loops. A clustering method was introduced to analyze the correlated motions of residues. We have found that trypsin and chymotrypsin have distinct dynamic signatures in the two loop regions, which are in turn highly correlated with motions of certain residues in the binding pockets. Interestingly, replacing the two loops of trypsin with those of chymotrypsin changes the motion style of trypsin to chymotrypsin-like, whereas the same experimental replacement was shown necessary to make trypsin have chymotrypsin's enzyme specificity and activity. These results suggest that the cooperative motions of the two loops and the substrate-binding sites contribute to the activity and substrate specificity of trypsin and chymotrypsin.

Amino Acid Sequence↗

PubMed2005

The substrate specificity of SARS coronavirus 3C-like proteinase.

The 3C-like proteinase of severe acute respiratory syndrome coronavirus (SARS) has been proposed to be a key target for structural based drug design against SARS. We have designed and synthesized 34 peptide substrates and determined their hydrolysis activities. The conserved core sequence of the native cleavage site is optimized for high hydrolysis activity. Residues at position P4, P3, and P3' are critical for substrate recognition and binding, and increment of beta-sheet conformation tendency is also helpful. A comparative molecular field analysis (CoMFA) model was constructed. Based on the mutation data and CoMFA model, a multiply mutated octapeptide S24 was designed for higher activity. The experimentally determined hydrolysis activity of S24 is the highest in all designed substrates and is close to that predicted by CoMFA. These results offer helpful information for the research on the mechanism of substrate recognition of coronavirus 3C-like proteinase.

Amino Acid Sequence↗

PubMed2004

Expression, purification, crystallization and preliminary crystallographic study of a potential metal-dependent hydrolase with cyclase activity from Thermoanaerobacter tengcongensis.

The putative metal-dependent hydrolase gene TTE1006 from Thermoanaerobacter tengcongensis strain MB4T (T = type strain; Genbank accession No. AE008691) was heterologously expressed in Escherichia coli. The 205-amino-acid gene product was purified and crystallized. The crystal used for data collection belongs to space group P2(1), with unit-cell parameters a = 85.2, b = 62.1, c = 172.4 A, beta = 104.2 degrees. Using a synchrotron-radiation source, the resolution limit of the data reached 1.87 A. Eight molecules were estimated to be present in the asymmetric unit, with a solvent content of 48%. Structure determination is ongoing using the multiple-wavelength anomalous diffraction (MAD) method and also the molecular-replacement (MR) method.

Amino Acid Sequence↗

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↗