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Polysorbate is widely used to maintain stability of biotherapeutic proteins in formulation development. Degradation of polysorbate can lead to particle formation in drug products, which is a major quality concern and potential patient risk factor. Enzymatic activity from residual enzymes such as lipases and esterases can cause polysorbate degradation. Their high activity, even at low concentration, constitutes a major analytical challenge. In this study, we evaluated and optimized the activity-based protein profiling (ABPP) approach to identify active enzymes responsible for polysorbate degradation. Using chemical probes to enrich active serine hydrolases, more than 80 proteins were identified in harvested cell culture fluid (HCCF) from monoclonal antibodies (mAb) production. A total of 8 known lipases were identified by ABPP, while only 5 lipases were identified by a traditional abundance-based proteomics (TABP) approach. Interestingly, phospholipase B-like 2 (PLBL2), a well-known problematic HCP was not found to be active in process-intermediates from two different mAbs. In a proof-of-concept study, phospholipase A2 group VII (PLA2G7) and sialic acid acetylesterase (SIAE) were identified by ABPP as possible root causes of polysorbate-80 degradation. The established ABBP approach can fill the gap between lipase abundance and activity, which enables more meaningful polysorbate degradation investigations for biotherapeutic development.

The MYC oncogene is upregulated in human cancers by translocation, amplification, and mutation of cellular pathways that regulate Myc. Myc/Max heterodimers bind to E box sequences in the promoter regions of genes and activate transcription., The MYC oncogene is upregulated in human cancers by translocation, amplification, and mutation of cellular pathways that regulate Myc. Myc/Max heterodimers bind to E box sequences in the promoter regions of genes and activate transcription. The MYC inhibitor Omomyc can reduce the ability of MYC to bind specific box sequences in promoters of MYC target genes by binding directly to E box sequences as demonstrated by chromatin immunoprecipitation (CHIP). Here, we demonstrate by both a proximity ligation assay (PLA) and double chromatin immunoprecipitation (ReCHIP) that Omomyc preferentially binds to Max, not Myc, to mediate inhibition of MYC-mediated transcription by replacing MYC/MAX heterodimers with Omomyc/MAX heterodimers. The formation of Myc/Max and Omomyc/Max heterodimers occurs cotranslationally; Myc, Max, and Omomyc can interact with ribosomes and Max RNA under conditions in which ribosomes are intact. Taken together, our data suggest that the mechanism of action of Omomyc is to bind DNA as either a homodimer or a heterodimer with Max that is formed cotranslationally, revealing a novel mechanism to inhibit the MYC oncogene. We find that in vivo, Omomyc distributes quickly to kidneys and liver and has a short effective half-life in plasma, which could limit its use in vivo.

The development of new antimicrobial drugs is a priority to combat the increasing spread of multidrug-resistant bacteria. This development is especially problematic in gram-negative bacteria due to the outer membrane (OM) permeability barrier and multidrug efflux pumps. Therefore, we screened for compounds that target essential, nonredundant, surface-exposed processes in gram-negative bacteria. We identified a compound, MRL-494, that inhibits assembly of OM proteins (OMPs) by the β-barrel assembly machine (BAM complex). The BAM complex contains one essential surface-exposed protein, BamA. We constructed a bamA mutagenesis library, screened for resistance to MRL-494, and identified the mutation bamA(E470K). BamA(E470K) restores OMP biogenesis in the presence of MRL-494. The mutant protein has both altered conformation and activity, suggesting it could either inhibit MRL-494 binding or allow BamA to function in the presence of MRL-494. By cellular thermal shift assay (CETSA), we determined that MRL-494 stabilizes BamA and BamA(E470K) from thermally induced aggregation, indicating direct or proximal binding to both BamA and BamA(E470K). Thus, it is the altered activity of BamA(E470K) responsible for resistance to MRL-494. Strikingly, MRL-494 possesses a second mechanism of action that kills gram-positive organisms. In microbes lacking an OM, MRL-494 lethally disrupts the cytoplasmic membrane. We suggest that the compound cannot disrupt the cytoplasmic membrane of gram-negative bacteria because it cannot penetrate the OM. Instead, MRL-494 inhibits OMP biogenesis from outside the OM by targeting BamA. The identification of a small molecule that inhibits OMP biogenesis at the cell surface represents a distinct class of antibacterial agents.

Deamination of choline catalyzed by the glycyl radical enzyme choline trimethylamine-lyase (CutC) has emerged as an important route for the production of trimethylamine, a microbial metabolite associated with both human disease and biological methane production. Here, we have determined five high-resolution X-ray structures of wild-type CutC and mechanistically informative mutants in the presence of choline. Within an unexpectedly polar active site, CutC orients choline through hydrogen bonding with a putative general base, and through close interactions between phenolic and carboxylate oxygen atoms of the protein scaffold and the polarized methyl groups of the trimethylammonium moiety. These structural data, along with biochemical analysis of active site mutants, support a mechanism that involves direct elimination of trimethylamine. This work broadens our understanding of radical-based enzyme catalysis and will aid in the rational design of inhibitors of bacterial trimethylamine production., National Science Foundation (U.S.) (Grant 0645960)

Anaerobic choline deamination catalyzed by the glycyl radical enzyme choline trimethylamine-lyase (CutC) has emerged as a major route for trimethylamine (TMA) production within anaerobic environments, including the human gut. The association of this microbial metabolite and its downstream products with diseases such as atherosclerosis and chronic kidney disease has driven the need for a better molecular understanding of TMA-generating enzymes. Our previous work has shown that generating the critical, glycine-centered radical species on CutC requires posttranslational modification by an S-adenosyl-l-methionine (SAM)-dependent radical-activating protein (CutD) harboring an oxygen-sensitive [4Fe-4S] cofactor. In this chapter, we describe our strategy to heterologously express and purify Desulfovibrio alaskensis G20 CutD in Escherichia coli and reconstitute its iron-sulfur center under anaerobic conditions. In addition, we present the methods we have developed to characterize the activity of CutD and utilize this enzyme in conjunction with purified CutC to gain an unprecedented insight into the anaerobic C-N cleavage of choline.

Anaerobic choline deamination catalyzed by the glycyl radical enzyme choline trimethylamine-lyase (CutC) has emerged as a major route for trimethylamine (TMA) production within anaerobic environments, including the human gut. The association of this microbial metabolite and its downstream products with diseases such as atherosclerosis and chronic kidney disease has driven the need for a better molecular understanding of TMA-generating enzymes. Our previous work has shown that generating the critical, glycine-centered radical species on CutC requires posttranslational modification by an S-adenosyl- l -methionine (SAM)-dependent radical-activating protein (CutD) harboring an oxygen-sensitive [4Fe–4S] cofactor. In this chapter, we describe our strategy to heterologously express and purify Desulfovibrio alaskensis G20 CutD in Escherichia coli and reconstitute its iron–sulfur center under anaerobic conditions. In addition, we present the methods we have developed to characterize the activity of CutD and utilize this enzyme in conjunction with purified CutC to gain an unprecedented insight into the anaerobic C–N cleavage of choline.

Elucidation of the molecular mechanisms underlying the human gut microbiota’s effects on health and disease has been complicated by difficulties in linking metabolic functions associated with the gut community as a whole to individual microorganisms and activities. Anaerobic microbial choline metabolism, a disease-associated metabolic pathway, exemplifies this challenge, as the specific human gut microorganisms responsible for this transformation have not yet been clearly identified. In this study, we established the link between a bacterial gene cluster, the choline utilization (cut) cluster, and anaerobic choline metabolism in human gut isolates by combining transcriptional, biochemical, bioinformatic, and cultivation-based approaches. Quantitative reverse transcription-PCR analysis and in vitro biochemical characterization of two cut gene products linked the entire cluster to growth on choline and supported a model for this pathway. Analyses of sequenced bacterial genomes revealed that the cut cluster is present in many human gut bacteria, is predictive of choline utilization in sequenced isolates, and is widely but discontinuously distributed across multiple bacterial phyla. Given that bacterial phylogeny is a poor marker for choline utilization, we were prompted to develop a degenerate PCR-based method for detecting the key functional gene choline TMA-lyase (cutC) in genomic and metagenomic DNA. Using this tool, we found that new choline-metabolizing gut isolates universally possessed cutC. We also demonstrated that this gene is widespread in stool metagenomic data sets. Overall, this work represents a crucial step toward understanding anaerobic choline metabolism in the human gut microbiota and underscores the importance of examining this microbial community from a function-oriented perspective., IMPORTANCE Anaerobic choline utilization is a bacterial metabolic activity that occurs in the human gut and is linked to multiple diseases. While bacterial genes responsible for choline fermentation (the cut gene cluster) have been recently identified, there has been no characterization of these genes in human gut isolates and microbial communities. In this work, we use multiple approaches to demonstrate that the pathway encoded by the cut genes is present and functional in a diverse range of human gut bacteria and is also widespread in stool metagenomes. We also developed a PCR-based strategy to detect a key functional gene (cutC) involved in this pathway and applied it to characterize newly isolated choline-utilizing strains. Both our analyses of the cut gene cluster and this molecular tool will aid efforts to further understand the role of choline metabolism in the human gut microbiota and its link to disease.

The collection of microorganisms that inhabit the human gastrointestinal tract, our gut microbiota, has an extensive set of metabolic capabilities that directly impact human health. Our research se...