A gene coding for a thermostable esterase was isolated by functional screening of Escherichia coli cells that had been transformed with fosmid environmental DNA libraries constructed with metagenomes from thermal environmental samples. The gene conferring esterase activity on E. coli grown on tributyrin agar was composed of 936 bp, corresponding to 311 amino acid residues with a molecular mass of 34 kDa. The enzyme showed significant amino acid similarity (64%) to the enzyme from a hyperthermophilic archaeon, Pyrobaculum calidifontis. An amino acid sequence comparison with other esterases and lipases revealed that the enzyme should be classified as a new member of the hormone-sensitive lipase family. The recombinant esterase that was overexpressed and purified from E. coli was active above 30°C up to 95°C and had a high thermal stability. It displayed a high degree of activity in a pH range of 5.5 to 7.5, with an optimal pH of approximately 6.0. The best substrate for the enzyme among the p-nitrophenyl esters (C 4 to C 16 ) examined was p-nitrophenyl caproate (C 6 ), and no lipolytic activity was observed with esters containing an acyl chain length of longer than 10 carbon atoms, indicating that the enzyme is an esterase and not a lipase.

In a fluorescence polarization screen for the MYC-MAX interaction, we have identified a novel small-molecule inhibitor of MYC, KJ-Pyr-9, from a Kröhnke pyridine library. The K d of KJ-Pyr-9 for MYC in vitro is 6.5 ± 1.0 nM, as determined by backscattering interferometry; KJ-Pyr-9 also interferes with MYC-MAX complex formation in the cell, as shown in a protein fragment complementation assay. KJ-Pyr-9 specifically inhibits MYC-induced oncogenic transformation in cell culture; it has no or only weak effects on the oncogenic activity of several unrelated oncoproteins. KJ-Pyr-9 preferentially interferes with the proliferation of MYC-overexpressing human and avian cells and specifically reduces the MYC-driven transcriptional signature. In vivo, KJ-Pyr-9 effectively blocks the growth of a xenotransplant of MYC-amplified human cancer cells.M YC is a transcriptional regulator that occupies an apex position in the organizational hierarchy of the cell (1-3). It belongs to a family of basic helix-loop-helix leucine zipper (bHLH-LZ) proteins that dimerize with the small bHLH-LZ protein MAX to become functional (4). The MYC-MAX heterodimer preferentially binds to the palindromic DNA sequence CACGTG, referred to as the E-box motif. As a transcription factor, MYC can bind to the promoters of target genes to stimulate or repress transcriptional activity (5-7). The human genome contains three MYC genes, c-MYC, N-MYC, and L-MYC. Throughout this paper, we will use "MYC" to indicate the protein product of the c-MYC gene.MYC is involved in almost all cancers (8, 9). It is rarely mutated, but achieves gain of function through overexpression or amplification. Because of this broad pathogenic significance, MYC is an important cancer target. However, both conceptual and practical difficulties have stood in the way of identifying potent and effective small-molecule inhibitors of MYC. The conceptual obstacles reflect concern about inhibiting a gene that controls essential cellular activities. Because MYC plays an important role in cell proliferation (10, 11), it is often argued that inhibition of this function would lead to broad and unacceptable side effects in vivo. However, studies with the dominant-negative MYC construct Omomyc have shown that inhibiting MYC has only mild and rapidly reversible effects on normal, fast-proliferating tissues (8,12,13). The main practical difficulty in targeting MYC is the absence of pockets or grooves that could serve as binding sites for small molecules (14).The preferred strategy for the identification of potential MYC inhibitors has been interference with MYC-MAX dimerization (15-18). The formation of the MYC-MAX heterodimer involves the bHLH-LZ domains of the two partner molecules with a protein-protein interaction (PPI) surface of ∼3,200 Å 2 . This surface lacks well-defined binding sites for small molecules and therefore is widely considered as "undruggable." However, despite the large interaction surface, a single-amino acid substitution can completely disrupt the dimerization of MYC with MAX (14...

Qβ virus-like particles encapsulating multiple copies of fluorescent proteins were generated in high yields using a modular system enhanced by specific engineered RNA-protein interactions. The resulting particles were structurally indistinguishable from recombinant Qβ alone. The encapsidated proteins were nearly identical in photochemical properties to monomeric analogues, were more stable toward thermal degradation, and were protected from proteolytic cleavage. Residues on the outer capsid surface were chemically derivatized by acylation and azide-alkyne cycloaddition without affecting the fluorescence properties of the packaged proteins. A high affinity carbohydrate-based ligand of the CD22 receptor was thereby attached, and specific cell labeling by the particles was successfully detected by flow cytometry and confocal laser microscopy.

Background: EstE1 is a hyperthermophilic esterase belonging to the hormone-sensitive lipase family and was originally isolated by functional screening of a metagenomic library constructed from a thermal environmental sample. Dimers and oligomers may have been evolutionally selected in thermophiles because intersubunit interactions can confer thermostability on the proteins. The molecular mechanisms of thermostabilization of this extremely thermostable esterase are not well understood due to the lack of structural information.

Virus-like particles (VLPs) have proven to be versatile platforms for chemical and functionalization for a variety of purposes in biomedicine, catalysis, and materials science. We here the simultaneous modification of the bacteriophage Qβ VLP with a metalloporphyrin derivative photodynamic therapy and a glycan ligand for specific targeting of cells bearing the CD-22 receptor. This application benefits from the presence of the targeting function and the delivery of a high local concentration of singlet oxygen-generating payload.

The fragment indole-6-carboxylic acid (1F1), previously identified as a flap site binder in a fragment-based screen against HIV protease (PR), has been co-crystallized with pepstatin-inhibited PR and with apo-PR. Another fragment, 3-indolepropionic acid (1F1-N), predicted by AutoDock calculations and confirmed in a novel ‘inhibition of nucleation’ crystallization assay, exploits the same interactions in the flap site in two crystal structures. Both 1F1 and 1F1-N bind to the closed form of apo-PR and to pepstatin:PR. In solution, 1F1 and 1F1-N raise the Tm of apo-PR by 3.5–5 °C as assayed by differential scanning fluorimetry (DSF), and show equivalent low-micromolar binding constants to both apo-PR and pepstatin:PR, assayed by backscattering interferometry (BSI). The observed signal intensities in BSI are greater for each fragment upon binding to apo-PR than to pepstatin-bound PR, consistent with greater conformational change in the former binding event. Together, these data indicate that fragment binding in the flap site favors a closed conformation of HIV PR.

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