Large scale structural rearrangement of a serine hydrolase from Francisella tularensis facilitates catalysis.

Tularemia is a deadly, febrile disease caused by infection by the gram-negative bacterium, Francisella tularensis. Members of the ubiquitous serine hydrolase protein family are among current targets to treat diverse bacterial infections. Herein we present a structural and functional study of a novel bacterial carboxylesterase (FTT258) from F. tularensis, a homologue of human acyl protein thioesterase (hAPT1). The structure of FTT258 has been determined in multiple forms, and unexpectedly large conformational changes of a peripheral flexible loop occur in the presence of a mechanistic cyclobutanone ligand. The concomitant changes in this hydrophobic loop and the newly exposed hydrophobic substrate binding pocket suggest that the observed structural changes are essential to the biological function and catalytic activity of FTT258. Using diverse substrate libraries, site-directed mutagenesis, and liposome binding assays, we determined the importance of these structural changes to the catalytic activity and membrane binding activity of FTT258. Residues within the newly exposed hydrophobic binding pocket and within the peripheral flexible loop proved essential to the hydrolytic activity of FTT258, indicating that structural rearrangement is required for catalytic activity. Both FTT258 and hAPT1 also showed significant association with liposomes designed to mimic bacterial or human membranes, respectively, even though similar structural rearrangements for hAPT1 have not been reported. The necessity for acyl protein thioesterases to have maximal catalytic activity near the membrane surface suggests that these conformational changes in the protein may dually regulate catalytic activity and membrane association in bacterial and human homologues.

The structural basis for the narrow substrate specificity of an acetyl esterase from Thermotoga maritima.

Acetyl esterases from carbohydrate esterase family 7 exhibit unusual substrate specificity. These proteins catalyze the cleavage of disparate acetate esters with high efficiency, but are unreactive to larger acyl groups. The structural basis for this distinct selectivity profile is unknown. Here, we investigate a thermostable acetyl esterase (TM0077) from Thermotoga maritima using evolutionary relationships, structural information, fluorescent kinetic measurements, and site directed mutagenesis. We measured the kinetic and structural determinants for this specificity using a diverse series of small molecule enzyme substrates, including novel fluorogenic esters. These experiments identified two hydrophobic plasticity residues (Pro228, and Ile276) surrounding the nucleophilic serine that impart this specificity of TM0077 for small, straight-chain esters. Substitution of these residues with alanine imparts broader specificity to TM0077 for the hydrolysis of longer and bulkier esters. Our results suggest the specificity of acetyl esterases have been finely tuned by evolution to catalyze the removal of acetate groups from diverse substrates, but can be modified by focused amino acid substitutions to yield enzymes capable of cleaving larger ester functionalities.

Selective, bead-based global peptide capture using a bifunctional cross-linker.

Peptides are important species for a variety of biological functions. Detection and analysis of these molecules can be complicated by the presence of background matrix or contaminants. Therefore, a selective method to capture peptides could provide researchers with an option to isolate these remarkable species. Our goal was to perform a set of experiments that would validate the concept of a novel, selective peptide capture, whereby peptides are isolated on functionalized magnetic beads through the use of the heterobifunctional cross-linker, Sulfo-LC-SPDP. Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to monitor mass changes associated with the cross-linking reaction. MALDI-TOF MS was then used to monitor conjugation between the cross-linked peptides and sulfhydryl magnetic beads by analyzing supernatant solutions for the presence or absence of cross-linked peptide. Through these experiments, we have proof of concept data confirming that peptides can be isolated on sulfhydryl magnetic beads by using Sulfo-LC-SPDP. This method is a suitable selective global peptide isolation strategy to separate the molecules from contaminating species or sample matrix. This novel method has a variety of potential applications and detection methods.

Comparative LC-MS/MS analysis of optimal cutting temperature (OCT) compound removal for the study of mammalian proteomes.

Primary tissue samples are valuable resources for investigators interested in understanding disease. In order to maximize the information content that can be gained from these precious samples, proper storage, handling, and preparation are essential. Some tissue preservation techniques utilize the cryopreservation medium, optimal cutting temperature (OCT) compound. While this medium provides benefits for traditional molecular studies, certain components can interfere with mass spectrometric analyses. Mass spectrometry based proteomics is a growing field with many applications for disease research. Our goal is to determine a reliable method for separating the proteins from the contaminating species in OCT embedded samples, thus making these samples compatible with mass spectrometric analyses. The novel applications of ether-methanol precipitation, filter-aided sample preparation (FASP), and SDS-PAGE provide researchers with protocols for removing OCT contaminating species from valuable samples. The results presented in this study show that all three methods reproducibly remove OCT; however, precipitation and FASP outperform SDS-PAGE by common proteomic metrics.

Induced Chromosomal Aneuploidy Results in Global and Consistent Deregulation of the Transcriptome of Cancer Cells.

Chromosomal aneuploidy is a defining feature of epithelial cancers. The pattern of aneuploidies is cancer-type specific. For instance, the gain of chromosome 13 occurs almost exclusively in colorectal cancer. We used microcell-mediated chromosome transfer to generate gains of chromosome 13 in the diploid human colorectal cancer cell line DLD-1. Extra copies of chromosome 13 resulted in a significant and reproducible up-regulation of transcript levels of genes on chromosome 13 (P = .0004, FDR = 0.01) and a genome-wide transcriptional deregulation in all 8 independent clones generated. Genes contained in two clusters were particularly affected: the first cluster on cytoband 13q13 contained 7 highly up-regulated genes (NBEA, MAB21L1, DCLK1, SOHLH2, CCDC169, SPG20 and CCNA1, P = .0003) in all clones. A second cluster was located on 13q32.1 and contained five upregulated genes (ABCC4, CLDN10, DZIP1, DNAJC3 and UGGT2, P = .003). One gene, RASL11A, localized on chromosome band 13q12.2, escaped the copy number-induced overexpression and was reproducibly and significantly down-regulated on the mRNA and protein level (P = .0001, FDR = 0.002). RASL11A expression levels were also lower in primary colorectal tumors as compared to matched normal mucosa (P = .0001, FDR = 0.0001. Overexpression of RASL11A increases cell proliferation and anchorage independent growth while decreasing cell migration in +13 clones. In summary, we observed a strict correlation of genomic copy number and resident gene expression levels, and aneuploidy dependent consistent genome-wide transcriptional deregulation.

Comparison of bottom-up proteomic approaches for LC-MS analysis of complex proteomes.

Discovery-based proteomic studies aim to answer important biological questions by identifying as many proteins as possible. In order to accomplish this lofty goal, an effort must be placed on determining an optimal workflow that maximizes protein identifications. In this study, we compare protein extraction, digestion and fractionation methods for bottom-up proteomics using a human colon cancer cell line as our model system. Four different buffers for protein extraction, two digestion approaches, as well as three sample fractionation methods were evaluated in order to determine an accessible workflow that gives maximal protein identifications. Samples comparing these workflows were analyzed via UPLC paired with tandem MS on a Q-Exactive mass spectrometer. Our goal is to determine an optimal workflow to enable users to maximize protein identifications. Our results show that an increased number of confident protein identifications are attained with a filter-aided digestion approach as compared to an in-solution digestion. Overall SDS-PAGE fractionation leads to higher numbers of identifications than SCX SpinTip and reverse phased cartridge platforms. The novel aspect of this work is the comparison of two readily available, offline platforms for fractionation in reference to a traditional technique, SDS-PAGE.