Yin Hoe Yau

Definition of endotoxin binding sites in horseshoe crab Factor C recombinant sushi proteins and neutralization of endotoxin by sushi peptides

Three truncated fragments, harboring different sushi domains, namely, sushi123, sushil, and sushi3 domains, of Factor C were produced as biologically active secreted recombinant proteins. Sushil and 3 each has a high‐affinity LPS binding site with Kd of 10−9 to 10−10 M. Positive cooperativity in sushi123 resulted in a 1000‐fold increase in Kd2. The core LPS binding region of sushi1 and 3 reside in two 34‐mer peptides, Sl and S3. A rigidly held disulfide‐bonded structure is not essential but is important for LPS binding, as confirmed by a 100‐ to 10000‐fold decrease in affinity. Both S1 and S3 can inhibit LAL reaction and LPS‐induced hTNF‐α secretion with different potency. LAL assay revealed that at least two molecules of S1 bind cooperatively to one LPS molecule, with Hills coefficient of 2.42. The LPS binding by S3 is independent and noncooperative. The modified SΔ1 and SΔ3 peptides exhibited increased LPS neutralization potential although its LPS binding affinities indicated only a 10‐fold improvement. Hence, the structural difference of the four sushi peptides conferred different efficiencies in LPS neutralization without altering their binding affinity for LPS. Circular dichroism spectrometry revealed that the four peptides underwent conformational change in the presence of lipid A, transitioning from a random coil to either an α‐helical or β‐sheet structure. Two factors are critical for the sensitivity of Factor C to LPS: 1) the presence of multiple binding sites for LPS on a single Factor C molecule; and 2) high positive cooperativity in LPS binding. The results showed that in the design of an improved LPS binding and neutralizing peptide, charge balance of the peptide is a critical parameter in addition to its structure.—Tan, N. S., Ng, M. L. P., Yau, Y. H., Chong, P. K. W., Ho, B., Ding, J. L. Definition of endotoxin binding sites in horseshoe crab Factor C recombinant sushi proteins and neutralization of endotoxin by sushi peptides. FASEB J. 14, 1801–1813 (2000)

Angiopoietin-Like 4 Interacts with Integrins β1 and β5 to Modulate Keratinocyte Migration

Adipose tissue secretes adipocytokines for energy homeostasis, but recent evidence indicates that some adipocytokines also have a profound local impact on wound healing. Upon skin injury, keratinocytes use various signaling molecules to promote reepithelialization for efficient wound closure. In this study, we identify a novel function of adipocytokine angiopoietin-like 4 (ANGPTL4) in keratinocytes during wound healing through the control of both integrin-mediated signaling and internalization. Using two different in vivo models based on topical immuno-neutralization of ANGPTL4 as well as ablation of the ANGPTL4 gene, we show that ANGPTL4-deficient mice exhibit delayed wound reepithelialization with impaired keratinocyte migration. Human keratinocytes in which endogenous ANGPTL4 expression was suppressed by either siRNA or a neutralizing antibody show impaired migration associated with diminished integrin-mediated signaling. Importantly, we identify integrins β1 and β5, but not β3, as novel binding partners of ANGPTL4. ANGPTL4-bound integrin β1 activated the FAK-Src-PAK1 signaling pathway, which is important for cell migration. The findings presented herein reveal an unpredicted role of ANGPTL4 during wound healing and demonstrate how ANGPTL4 stimulates intracellular signaling mechanisms to coordinate cellular behavior. Our findings provide insight into a novel cell migration control mechanism and underscore the physiological importance of the modulation of integrin activity in cancer metastasis.

Functional analysis of two cavities in flavivirus NS5 polymerase

Flavivirus NS5 protein encodes methyltransferase and RNA-dependent RNA polymerase (RdRp) activities. Structural analysis of flavivirus RdRp domains uncovered two conserved cavities (A and B). Both cavities are located in the thumb subdomains and represent potential targets for development of allosteric inhibitors. In this study, we used dengue virus as a model to analyze the function of the two RdRp cavities. Amino acids from both cavities were subjected to mutagenesis analysis in the context of genome-length RNA and recombinant NS5 protein; residues critical for viral replication were subjected to revertant analysis. For cavity A, we found that only one (Lys-756) of the seven selected amino acids is critical for viral replication. Alanine substitution of Lys-756 did not affect the RdRp activity, suggesting that this residue functions through a nonenzymatic mechanism. For cavity B, all four selected amino acids (Leu-328, Lys-330, Trp-859, and Ile-863) are critical for viral replication. Biochemical and revertant analyses showed that three of the four mutated residues (Leu-328, Trp-859, and Ile-863) function at the step of initiation of RNA synthesis, whereas the fourth residue (Lys-330) functions by interacting with the viral NS3 helicase domain. Collectively, our results have provided direct evidence for the hypothesis that cavity B, but not cavity A, from dengue virus NS5 polymerase could be a target for rational drug design.

On a mouse monoclonal antibody that neutralizes all four dengue virus serotypes.

The flavivirus envelope glycoprotein (E) is responsible for viral attachment and entry by membrane fusion. Its ectodomain is the primary target of the humoral immune response. In particular, the C-terminal Ig-like domain III of E, which is exposed at the surface of the viral particle, forms an attractive antigen for raising protective monoclonal antibodies (mAb). 9F12, a mouse mAb raised against a dengue virus (DENV) serotype 2 recombinant domain III, cross-reacts with corresponding domains from the other three DENV serotypes and also with West Nile virus. mAb 9F12 binds with nanomolar affinity to a conserved epitope that maps to the viral surface comprising residues 305, 307, 310 and 330 of the E protein. mAb 9F12 neutralizes all four DENV serotypes in plaque reduction assays. We expressed a single-chain Fv from 9F12 that retains the binding activity of the parent mAb. Adsorption and fusion inhibition assays indicate that mAb 9F12 prevents early steps of viral entry. Its virus inhibition activity and broad cross-reactivity makes mAb 9F12 a suitable candidate for optimization and humanization into a therapeutic antibody to treat severe infections by dengue.

A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease

In drug discovery, the occurrence of false positives is a major hurdle in the search for lead compounds that can be developed into drugs. A small-molecular-weight compound that inhibits dengue virus protease at low micromolar levels was identified in a screening campaign. Binding to the enzyme was confirmed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). However, a structure-activity relationship study that ensued did not yield more potent leads. To further characterize the parental compound and its analogues, we developed a high-speed, low-cost, quantitative fluorescence quenching assay. We observed that specific analogues quenched dengue protease fluorescence and showed variation in IC(50) values. In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC(50) values with steep dose-response curves. We validated the assay using single Trp-to-Ala protease mutants and the competitive protease inhibitor aprotinin. Specific compounds detected in the binding assay were further analyzed by competitive ITC, NMR, and surface plasmon resonance, and the assays utility in comparison with these biophysical methods is discussed. The sensitivity of this assay makes it highly useful for hit finding and validation in drug discovery. Furthermore, the technique can be readily adapted for studying other protein-ligand interactions.

Competing targets of microRNA-608 affect anxiety and hypertension

MicroRNAs (miRNAs) can repress multiple targets, but how a single de-balanced interaction affects others remained unclear. We found that changing a single miRNA–target interaction can simultaneously affect multiple other miRNA–target interactions and modify physiological phenotype. We show that miR-608 targets acetylcholinesterase (AChE) and demonstrate weakened miR-608 interaction with the rs17228616 AChE allele having a single-nucleotide polymorphism (SNP) in the 3′-untranslated region (3′UTR). In cultured cells, this weakened interaction potentiated miR-608-mediated suppression of other targets, including CDC42 and interleukin-6 (IL6). Postmortem human cortices homozygote for the minor rs17228616 allele showed AChE elevation and CDC42/IL6 decreases compared with major allele homozygotes. Additionally, minor allele heterozygote and homozygote subjects showed reduced cortisol and elevated blood pressure, predicting risk of anxiety and hypertension. Parallel suppression of the conserved brain CDC42 activity by intracerebroventricular ML141 injection caused acute anxiety in mice. We demonstrate that SNPs in miRNA-binding regions could cause expanded downstream effects changing important biological pathways.

The methyltransferase Ezh2 controls cell adhesion and migration through direct methylation of the extranuclear regulatory protein talin

A cytosolic role for the histone methyltransferase Ezh2 in regulating lymphocyte activation has been suggested, but the molecular mechanisms underpinning this extranuclear function have remained unclear. Here we found that Ezh2 regulated the integrin signaling and adhesion dynamics of neutrophils and dendritic cells (DCs). Ezh2 deficiency impaired the integrin-dependent transendothelial migration of innate leukocytes and restricted disease progression in an animal model of multiple sclerosis. Direct methylation of talin, a key regulatory molecule in cell migration, by Ezh2 disrupted the binding of talin to F-actin and thereby promoted the turnover of adhesion structures. This regulatory effect was abolished by targeted disruption of the interactions of Ezh2 with the cytoskeletal-reorganization effector Vav1. Our studies reveal an unforeseen extranuclear function for Ezh2 in regulating adhesion dynamics, with implications for leukocyte migration, immune responses and potentially pathogenic processes.

Kindlin-3 Mediates Integrin αLβ2 Outside-in Signaling, and It Interacts with Scaffold Protein Receptor for Activated-C Kinase 1 (RACK1)

Background: Kindlin-3 is a cytoplasmic protein that binds and modulates the ligand binding property of integrin αLβ2. Results: Kindlin-3 induces integrin αLβ2 clustering, and it interacts with the scaffold protein RACK1. Conclusion: Kindlin-3 is involved in integrin αLβ2 outside-in signaling. Significance: This study presents important findings in understanding the role of kindlin-3 in integrin signaling. Integrins are heterodimeric type I membrane cell adhesion molecules that are involved in many biological processes. Integrins are bidirectional signal transducers because their cytoplasmic tails are docking sites for cytoskeletal and signaling molecules. Kindlins are cytoplasmic molecules that mediate inside-out signaling and activation of the integrins. The three kindlin paralogs in humans are kindlin-1, -2, and -3. Each of these contains a 4.1-ezrin-radixin-moesin (FERM) domain and a pleckstrin homology domain. Kindlin-3 is expressed in platelets, hematopoietic cells, and endothelial cells. Here we show that kindlin-3 is involved in integrin αLβ2 outside-in signaling. It also promotes micro-clustering of integrin αLβ2. We provide evidence that kindlin-3 interacts with the receptor for activated-C kinase 1 (RACK1), a scaffold protein that folds into a seven-blade propeller. This interaction involves the pleckstrin homology domain of kindlin-3 and blades 5–7 of RACK1. Using the SKW3 human T lymphoma cells, we show that integrin αLβ2 engagement by its ligand ICAM-1 promotes the association of kindlin-3 with RACK1. We also show that kindlin-3 co-localizes with RACK1 in polarized SKW3 cells and human T lymphoblasts. Our findings suggest that kindlin-3 plays an important role in integrin αLβ2 outside-in signaling.

Solid-phase synthesis of BODIPY dyes and development of an immunoglobulin fluorescent sensor

The diversification of the BODIPY scaffold has been hindered by its controversial adaptability to solid-phase chemistry. Herein we report the first solid-phase synthesis of a BODIPY library in high purities. We screened the library against a set of proteins, identified an immunoglobulin fluorescent sensor (Ig Orange) and confirmed its binding by SPR experiments.

Residual Structure in Islet Amyloid Polypeptide Mediates Its Interactions with Soluble Insulin

Islet amyloid polypeptide (IAPP), a 37-amino acid polypeptide hormone of the calcitonin family, is colocalized and cosecreted with insulin in secretory granules of pancreatic islet beta cells. IAPP can assemble into toxic oligomers and amyloid fibrils, a hallmark of type 2 diabetes. Its interactions with insulin in the secretory granules might influence the formation of cytotoxic oligomers and amyloid fibrils. Presented NMR analysis shows that IAPP, free in solution and in complex with insulin, retains elements of residual secondary structure. NMR chemical shifts and (15)N relaxation data as well as 49 ns replica exchange molecular dynamic simulations indicate that the transiently populated helical structure in residues 11-18 is essential for interactions with insulin. These interactions are mediated by salt bridges between positively charged residues Arg11 or Arg18 of rat IAPP and Glu13 of insulin B chain as well as by hydrophobic interactions flanking the salt bridges. The insulin binding region is composed of the same amino acids in amyloidogenic human IAPP and soluble rat IAPP (with the sole exception of His/Arg-18), implying the same binding mode for both hormones. This His/Arg-18 mutation results in reduced affinity binding of human IAPP to insulin in comparison to rat IAPP as it is detected by surface plasmon resonance biosensor analysis. Implications of the described interactions between soluble forms of IAPP and insulin in preventing oligomerization of human IAPP are discussed.