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.

Pellino1 specifically binds to phospho-Thr18 of p53 and is recruited to sites of DNA damage.

Pellino1 is an E3 ubiquitin ligase that plays a key role in positive regulation of innate immunity signaling, specifically required for the production of interferon when induced by viral double-stranded RNA. We report the identification of the tumor suppressor protein, p53, as a binding partner of Pellino1. Their interaction has a Kd of 42 ±â€¯2 µM and requires phosphorylation of Thr18 within p53 and association with the forkhead-associated (FHA) domain of Pellino1. We employed laser micro-irradiation and live cell microscopy to show that Pellino1 is recruited to newly occurring DNA damage sites, via its FHA domain. Mutation of a hitherto unidentified nuclear localization signal within the N-terminus of Pellino1 led to its exclusion from the nucleus. This study provides evidence that Pellino1 translocates to damaged DNA in the nucleus and has a functional role in p53 signaling and the DNA damage response.

ANGPTL4 T266M variant is associated with reduced cancer invasiveness.

Angiopoietin-like 4 (ANGPTL4) is a secretory protein that can be cleaved to form an N-terminal and a C-terminal protein. Studies performed thus far have linked ANGPTL4 to several cancer-related and metabolic processes. Notably, several point mutations in the C-terminal ANGPTL4 (cANGPTL4) have been reported, although no studies have been performed that ascribed these mutations to cancer-related and metabolic processes. In this study, we compared the characteristics of tumors with and without wild-type (wt) cANGPTL4 and tumors with cANGPTL4 bearing the T266M mutation (T266M cANGPTL4). We found that T266M cANGPTL4 bound to integrin α5ß1 with a reduced affinity compared to wt, leading to weaker activation of downstream signaling molecules. The mutant tumors exhibited impaired proliferation, anoikis resistance, and migratory capability and had reduced adenylate energy charge. Further investigations also revealed that cANGPTL4 regulated the expression of Glut2. These findings may explain the differences in the tumor characteristics and energy metabolism observed with the cANGPTL4 T266M mutation compared to tumors without the mutation.

Detection of Pathogenic Biofilms with Bacterial Amyloid Targeting Fluorescent Probe, CDy11.

Bacterial biofilms are responsible for a wide range of persistent infections. In the clinic, diagnosis of biofilm-associated infections relies heavily on culturing methods, which fail to detect nonculturable bacteria. Identification of novel fluorescent probes for biofilm imaging will greatly facilitate diagnosis of pathogenic bacterial infection. Herein, we report a novel fluorescent probe, CDy11 (compound of designation yellow 11), which targets amyloid in the Pseudomonas aeruginosa biofilm matrix through a diversity oriented fluorescent library approach (DOFLA). CDy11 was further demonstrated for in vivo imaging of P. aeruginosa in implant and corneal infection mice models.

Small molecule targeting malaria merozoite surface protein-1 (MSP-1) prevents host invasion of divergent plasmodial species.

Malaria causes nearly 1 million deaths annually. Recent emergence of multidrug resistance highlights the need to develop novel therapeutic interventions against human malaria. Given the involvement of sugar binding plasmodial proteins in host invasion, we set out to identify such proteins as targets of small glycans. Combining multidisciplinary approaches, we report the discovery of a small molecule inhibitor, NIC, capable of inhibiting host invasion through interacting with a major invasion-related protein, merozoite surface protein-1 (MSP-1). This interaction was validated through computational, biochemical, and biophysical tools. Importantly, treatment with NIC prevented host invasion by Plasmodium falciparum and Plasmodium vivax--major causative organisms of human malaria. MSP-1, an indispensable antigen critical for invasion and suitably localized in abundance on the merozoite surface represents an ideal target for antimalarial development. The ability to target merozoite invasion proteins with specific small inhibitors opens up a new avenue to target this important pathogen.

The stem region of premembrane protein plays an important role in the virus surface protein rearrangement during dengue maturation.

BACKGROUND: Dengue virus surface proteins, envelope (E) and pre-membrane (prM), undergo rearrangement during the maturation process at acidic condition. RESULTS: prM-stem region binds tighter to both E protein and lipid membrane when environment becomes acidic. CONCLUSION: At acidic condition, E proteins are attracted to the membrane-associated prM-stem. SIGNIFICANCE: prM-stem region induces virus structural changes during maturation. Newly assembled dengue viruses (DENV) undergo maturation to become infectious particles. The maturation process involves major rearrangement of virus surface premembrane (prM) and envelope (E) proteins. The prM-E complexes on immature viruses are first assembled as trimeric spikes in the neutral pH environment of the endoplasmic reticulum. When the virus is transported to the low pH environment of the exosomes, these spikes rearrange into dimeric structures, which lie parallel to the virus lipid envelope. The proteins involved in driving this process are unknown. Previous cryoelectron microscopy studies of the mature DENV showed that the prM-stem region (residues 111-131) is membrane-associated and may interact with the E proteins. Here we investigated the prM-stem region in modulating the virus maturation process. The binding of the prM-stem region to the E protein was shown to increase significantly at low pH compared with neutral pH in ELISAs and surface plasmon resonance studies. In addition, the affinity of the prM-stem region for the liposome, as measured by fluorescence correlation spectroscopy, was also increased when pH is lowered. These results suggest that the prM-stem region forms a tight association with the virus membrane and attracts the associated E protein in the low pH environment of exosomes. This will lead to the surface protein rearrangement observed during maturation.

Functional divergence of FimX in PilZ binding and type IV pilus regulation.

Type IV pili (T4P) are polar surface structures that play important roles in bacterial motility, biofilm formation, and pathogenicity. The protein FimX and its orthologs are known to mediate T4P formation in the human pathogen Pseudomonas aeruginosa and some other bacterial species. It was reported recently that FimX(XAC2398) from Xanthomonas axonopodis pv. citri interacts with PilZ(XAC1133) directly through the nonenzymatic EAL domain of FimX(XAC2398). Here we present experimental data to reveal that the strong interaction between FimX(XAC2398) and PilZ(XAC1133) is not conserved in P. aeruginosa and likely other Pseudomonas species. In vitro and in vivo binding experiments showed that the interaction between FimX and PilZ in P. aeruginosa is below the measurable limit. Surface plasmon resonance assays further confirmed that the interaction between the P. aeruginosa proteins is at least more than 3 orders of magnitude weaker than that between the X. axonopodis pv. citri pair. The N-terminal lobe region of FimX(XAC2398) was identified as the binding surface for PilZ(XAC1133) by amide hydrogen-deuterium exchange and site-directed mutagenesis studies. Lack of several key residues in the N-terminal lobe region of the EAL domain of FimX is likely to account for the greatly reduced binding affinity between FimX and PilZ in P. aeruginosa. All together, the results suggest that the interaction between PilZ and FimX in Xanthomonas species is not conserved in P. aeruginosa due to the evolutionary divergence among the FimX orthologs. The precise roles of FimX and PilZ in bacterial motility and T4P biogenesis are likely to vary among bacterial species.

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.

Real-time determination of the activity of ATPase by use of a water-soluble polythiophene.

This contribution introduces a fluorescence assay for real-time determination of the activity of p97/VCP, a 540-kDa homo-hexameric enzyme, belonging to the AAA-ATPase family. A fluorescent reporter "poly 1-(3-((4-methylthiophen-3-yl)oxy)propyl)quinuclidin-1-ium" (poly PTQ) is used to monitor the hydrolysis of ATP to ADP by p97/VCP. The proposed assay relies on the different strength of coordination of ATP and ADP to the polymer backbone. We used recovery of fluorescence intensity on addition of p97/VCP to a poly PTQ/ATP solution to determine the enzymatic activity. The kinetic data K (m) and V (max) were 0.30 mmol L(-1) ATP and 0.134 nmol ATP min(-1) µg(-1) enzyme, respectively. The specificity of the assay was investigated by using an unhydrolyzable ATP analogue and sensitivity against p97 mutagenesis was further examined by detection of the activity of wild type and truncated p97/VCP. Our study demonstrates that determination of the real-time activity of p97/VCP is possible, because of the superior sensitivity and very fast optical response of poly PTQ.

Assembly of subunit d (Vma6p) and G (Vma10p) and the NMR solution structure of subunit G (G(1-59)) of the Saccharomyces cerevisiae V(1)V(O) ATPase.

Understanding the structural traits of subunit G is essential, as it is needed for V(1)V(O) assembly and function. Here solution NMR of the recombinant N- (G(1-59)) and C-terminal segment (G(61-114)) of subunit G, has been performed in the absence and presence of subunit d of the yeast V-ATPase. The data show that G does bind to subunit d via its N-terminal part, G(1-59) only. The residues of G(1-59) involved in d binding are Gly7 to Lys34. The structure of G(1-59) has been solved, revealing an alpha-helix between residues 10 and 56, whereby the first nine- and the last three residues of G(1-59) are flexible. The surface charge distribution of G(1-59) reveals an amphiphilic character at the N-terminus due to positive and negative charge distribution at one side and a hydrophobic surface on the opposite side of the structure. The C-terminus exhibits a strip of negative residues. The data imply that G(1-59)-d assembly is accomplished by hydrophobic interactions and salt-bridges of the polar residues. Based on the recently determined NMR structure of segment E(18-38) of subunit E of yeast V-ATPase and the presently solved structure of G(1-59), both proteins have been docked and binding epitopes have been analyzed.

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.

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 assay's 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.

Analysis of affinity of dengue virus protein interaction using Biacore.

Surface plasmon resonance (SPR) biosensors have become the mainstream method for biomolecular interaction analysis. It offers many advantages over conventional methods by its label-free, real-time monitoring, low sample consumption, high throughput, and remarkable sensitivity. We have examined dengue virus protein interactions in the context of antibody affinity measurement, protein-protein interaction, and in the screening of small molecule inhibitors as well as the characterization of the interactions between the small molecule binders and the relevant dengue protein. Here we describe the basic methods involved in performing SPR assays as well as in data processing and evaluation using some examples of dengue proteins.

Comprehensive analysis and identification of the human STIM1 domains for structural and functional studies.

STIM1 is a Ca(2+) sensor within the ER membrane known to activate the plasma membrane store-operated Ca(2+) channel upon depletion of its target ion in the ER lumen. This activation is a crucial step to initiate the Ca(2+) signaling cascades within various cell types. Human STIM1 is a 77.4 kDa protein consisting of various domains that are involved in Ca(2+) sensing, oligomerization, and channel activation and deactivation. In this study, we identify the domains and boundaries in which functional and stable recombinant human STIM1 can be produced in large quantities. To achieve this goal, we cloned nearly 200 constructs that vary in their initial and terminal residues, length and presence of the transmembrane domain, and we conducted expression and purification analyses using these constructs. The results revealed that nearly half of the constructs could be expressed and purified with high quality, out of which 25% contained the integral membrane domain. Further analyses using surface plasmon resonance, nuclear magnetic resonance and a thermostability assay verified the functionality and integrity of these constructs. Thus, we have been able to identify the most stable and well-behaved domains of the hSTIM1 protein, which can be used for future in vitro biochemical and biophysical studies.

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.

Association of the eukaryotic V1VO ATPase subunits a with d and d with A.

Owing to the complex nature of V(1)V(O) ATPases, identification of neighboring subunits is essential for mechanistic understanding of this enzyme. Here, we describe the links between the V(1) headpiece and the V(O)-domain of the yeast V(1)V(O) ATPase via subunit A and d as well as the V(O) subunits a and d using surface plasmon resonance and fluorescence correlation spectroscopy. Binding constants of about 60 and 200 nM have been determined for the a-d and d-A assembly, respectively. The data are discussed in light of subunit a and d forming a peripheral stalk, connecting the catalytic A(3)B(3) hexamer with V(O).

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.

Crystallographic structure of the tetratricopeptide repeat domain of Plasmodium falciparum FKBP35 and its molecular interaction with Hsp90 C-terminal pentapeptide.

Plasmodium falciparum FK506-binding protein 35 (PfFKBP35) that binds to FK506 contains a conserved tetratricopeptide repeat (TPR) domain. Several known TPR domains such as Hop, PPP5, CHIP, and FKBP52 are structurally conserved and are able to interact with molecular chaperones such as Hsp70/Hsp90. Here, we present the crystal structure of PfFKBP35-TPR and demonstrate its interaction with Hsp90 C-terminal pentapeptide (MEEVD) by surface plasmon resonance and nuclear magnetic resonance spectroscopy-based binding studies. Our sequence and structural analyses reveal that PfFKBP35 is similar to Hop and PPP5 in possessing all the conserved residues which are important for carboxylate clamping with Hsp90. Mutational studies were carried out on positively charged clamp residues that are crucial for binding to carboxylate groups of aspartate, showing that all the mutated residues are important for Hsp90 binding. Molecular docking and electrostatic calculations demonstrated that the MEEVD peptide of Hsp90 can form aspartate clamp unlike FKBP52. Our results provide insightful information and structural basis about the molecular interaction between PfFKBP35-TPR and Hsp90.