Yin-Ming Kuo

Lysine acetyltransferases (KATs) are exquisitely fine-tuned to target specific lysine residues on many proteins, including histones, with aberrant acetylation at distinct lysines implicated in different pathologies. However, researchers face a lack of molecular tools to probe the importance of site-specific acetylation events in vivo. Because of this, there can be a disconnect between the predicted in silico or in vitro effects of a drug and the actual observable in vivo response. We have previously reported on how an in vitro biochemical analysis of the site-specific effects of the compound C646 in combination with the KAT p300 can accurately predict changes in histone acetylation induced by the same compound in cells. Here, we build on this effort by further analyzing a number of reported p300 modulators, while also extending the analysis to correlate the effects of these drugs to developmental and phenotypical changes, utilizing cellular and zebrafish model systems. While this study demonstrates the utility of biochemical models as a starting point for predicting in vivo activity of multi-site targeting KATs, it also highlights the need for the development of new enzyme inhibitors that are more specific to the regulation of KAT activity in vivo.

T cell-mediated adaptive immunity requires naïve, unstimulated T cells to transition from a quiescent metabolic state into a highly proliferative state upon T cell receptor engagement. This complex process depends on transcriptional changes mediated by Ca²⁺-dependent NFAT signaling, mTOR-mediated signaling and increased activity of the guanine nucleotide biosynthetic inosine-5′-monophosphate (IMP) dehydrogenase 1 and 2 enzymes (IMPDH1 and IMPDH2, hereafter IMPDH). Inhibitors of these pathways serve as potent immunosuppressants. Unexpectedly, we discovered that all three pathways converge to promote the assembly of IMPDH protein into micron-scale macromolecular filamentous structures in response to T cell activation. Assembly is post-transcriptionally controlled by mTOR and the Ca²⁺ influx regulator STIM1. Furthermore, IMPDH assembly and catalytic activity were negatively regulated by guanine nucleotide levels, suggesting a negative feedback loop that limits biosynthesis of guanine nucleotides. Filamentous IMPDH may be more resistant to this inhibition, facilitating accumulation of the higher GTP levels required for T cell proliferation.

Acetylation of histones plays a critical role in maintaining the epigenetic state of the eukaryotic cell. One such acetylation site critical for DNA damage repair is H3K56ac. In Saccharomyces cerevisiae , H3K56ac is thought to be driven mainly by Rtt109, a lysine acetyltransferase (KAT) that associates with the histone chaperones Vps75 and Asf1. Both of these chaperones can increase the specificity of histone acetylation by Rtt109, but neither alter the selectivity. It has been shown that histones extracted from cells ( Drosophila ), presumably containing pre-acetylated histones, can incorporate higher amounts of H3K56ac relative to recombinant non-acetylated histones. We hypothesized that histone pre-acetylation and histone chaperones could function together to drive acetylation of H3K56. In the present study, we test this hypothesis using a series of singly acetylated histones to determine the impact of crosstalk on enzyme selectivity. Our data suggest that crosstalk between acetylation sites plays a major role in altering the selectivity of Rtt109-Vps75 and that the histone chaperone Asf1 mediates this crosstalk. Specifically, we show that H3K14ac/H4 functions with Asf1 to drive H3K56ac by Rtt109-Vps75. We identified an acidic patch in Asf1 that mediates this cross-talk and show that mutations to this region can alter the Asf1 mediated crosstalk that changes Rtt109-Vps75 selectivity. These data explain the genetic link between Gcn5, which acetylates H3K14 and Rtt109. More broadly these data demonstrate that acetylation sites can dictate site selectivity even in the absence of a bromodomain and helps to explain the limited complexity that has been observed of the histone post-translational modifications patterns by global proteomic studies.

T cell-mediated adaptive immunity requires naïve, unstimulated T cells to transition from a quiescent metabolic state into a highly proliferative state upon T cell receptor engagement. This complex process depends on transcriptional changes mediated by Ca2+-dependent NFAT signaling, mTOR-mediated signaling and increased activity of the guanine nucleotide biosynthetic enzyme inosine-5'-monophosphate (IMP) dehydrogenase (IMPDH). Inhibitors of these pathways serve as potent immunosuppressants. Unexpectedly, we discovered that all three pathways converge to promote the assembly of IMPDH protein into micron-scale macromolecular filamentous structures in response to T cell activation. Assembly is post-transcriptionally controlled by mTOR and the Ca2+ influx regulator STIM1. Furthermore, IMPDH assembly and catalytic activity were negatively regulated by guanine nucleotide levels, suggesting a negative feedback loop that limits biosynthesis of guanine nucleotides. Filamentous IMPDH may be more resistant to this inhibition, facilitating accumulation of the higher GTP levels required for T cell proliferation.

Lysine acetyltransferases (KATs) play a unique role in regulating gene transcription as well as maintaining the epigenetic state of the cell. KATs such as Gcn5 and p300/CBP can modify multiple residues on a single histone; however, order and specificity of acetylation can be altered by factors such as histone chaperones, subunit proteins or external stimulus. While the importance of acetylation is well documented, it has been difficult to quantitatively measure the specificity and selectivity of acetylation at different residues within a histone. In this paper, we demonstrate a label-free quantitative high throughput mass spectrometry-based assay capable of quantitatively monitoring all known acetylation sites of H3 simultaneously. Using this assay, we are able to analyze the steady-state enzyme kinetics of Gcn5, an evolutionarily conserved KAT. In doing so, we measured Gcn5-mediated acetylation at six residues (K14>K9 ≈ K23> K18> K27 ≈ K36) and the catalytic efficiency (k_cat/K_m) for K9, K14, K18, and K23 as well as the nonenzymatic acetylation rate. We observed selectivity differences of up to −4 kcal/mol between K14 and K18, the highest and lowest measurable k_cat/K_m. These data provide a first look at quantitating the specificity and selectivity of multiple lysines on a single substrate (H3) by Gcn5.

We propose a novel application of secondary electrospray ionization-mass spectrometry (SESI-MS) as a real-time clinical diagnostic tool for bacterial infection. It is known that volatile organic compounds (VOCs), produced in different combinations and quantities by bacteria as metabolites, generate characteristic odors for certain bacteria. These VOCs comprise a specific metabolic profile that can be used for species or serovar identification, but rapid and sensitive analytical methods are required for broad utility. In this study, the VOC profiles of five bacterial groups from four genera, Pseudomonas aeruginosa , Staphylococcus aureus , Escherichia coli , Salmonella enterica serovar Typhimurium, and Salmonella enterica serovar Pullorum, were characterized by SESI-MS. Thirteen compounds were identified from these bacterial cultures, and the combination of these VOCs creates a unique pattern for each genus. In addition, principal component analysis (PCA) was applied for the purpose of species or serovar discrimination. The first three principal components exhibit a clear separation between the metabolic volatile profiles of these five bacterial groups that is independent of the growth medium. As a first step toward addressing the complexity of clinical application, in vitro tests for mixed cultures were conducted. The results show that individual species or serovars in a mixed culture are identifiable among a biological VOC background, and the ratios of the detected volatiles reflect the proportion of each bacterium in the mixture. Our data confirm the utility of SESI-MS in real-time identification of bacterial species or serovars in vitro , which, in the future, may play a promising clinical role in diagnosing infections.

Decabromodiphenyl ether (BDE 209) is the main congener in the commonly used commercial flame retardant mixture, “deca-BDE”. There is evidence showing that fish can debrominate BDE 209 into potentially more toxic congeners. The objective of this study was to evaluate BDE 209 uptake and its potential effects on juvenile lake whitefish (Coregonus clupeaformis). Lake whitefish were fed BDE 209 at four nominal concentrations (control, 0.1, 1, and 2 μg/g-diet) for 30 days. Livers and carcasses were analyzed for 11 polybrominated diphenyl ether (PBDE) congeners (BDE 47, 99, 100, 153, 154, 196, 197, 206, 207, 208, and 209) and daily otolith increment width was measured as an estimate of growth before and after exposure. Four congeners (BDE 206, 207, 208, and 209) were detected in livers and carcasses. Hepatic BDE 209 concentrations in the 1 and 2 μg/g treatments were significantly higher than in the control group (1.25 and 5.80 nmol/g-lipid compared to 0.183 nmol/g-lipid). The concentration of BDE 209 detected in the tissues of the control group resulted from BDE 209 in the base diets. Concentrations of all congeners from the 1 and 2 μg/g groups were higher in livers than carcasses, indicating the liver was the primary organ of BDE 209 accumulation. Compared to the fraction in diets, the molar fraction of BDE 209 was lower in livers and carcasses, whereas the fractions of BDE 206, 207, and 208 were higher. These different distributions of PBDE congeners resulted from differential adsorption and metabolism. One congener, BDE 206, could be a major metabolite from BDE 209 debromination. Otolith increment widths were narrower in fish from the highest diet concentration administered, suggesting BDE 209 may have affected growth rates. In conclusion, this in vivo study with lake whitefish showed that BDE 209 was debrominated into lower PBDE congeners and that exposure to 2 μg/g may have affected fish growth.