Serum proteomic analysis in esophagectomy patients with postoperative delirium: A case-control study.

BACKGROUND: Postoperative delirium occurs in up to 80% of patients undergoing esophagectomy. We performed an exploratory proteomic analysis to identify protein pathways that may be associated with delirium post-esophagectomy. OBJECTIVES: Identify proteins associated with delirium and delirium severity in a younger and higher-risk surgical population. METHODS: We performed a case-control study using blood samples collected from patients enrolled in a negative, randomized, double-blind clinical trial. English speaking adults aged 18 years or older, undergoing esophagectomy, who had blood samples obtained were included. Cases were defined by a positive delirium screen after surgery while controls were patients with negative delirium assessments. Delirium was assessed using Richmond Agitation Sedation Scale and Confusion Assessment Method for the Intensive Care Unit, and delirium severity was assessed by Delirium Rating Scale-Revised-98. Blood samples were collected pre-operatively and on post-operative day 1, and discovery proteomic analysis was performed. Between-group differences in median abundance ratios were reported using Wilcoxon-Mann-Whitney Odds (WMWodds1) test. RESULTS: 52 (26 cases, 26 controls) patients were included in the study with a mean age of 64 (SD 9.6) years, 1.9% were females and 25% were African American. The median duration of delirium was 1 day (IQR: 1-2), and the median delirium/coma duration was 2.5 days (IQR: 2-4). Two proteins with greater relative abundance ratio in patients with delirium were: Coagulation factor IX (WMWodds: 1.89 95%CI: 1.0-4.2) and mannosyl-oligosaccharide 1,2-alpha-mannosidase (WMWodds: 2.4 95%CI: 1.03-9.9). Protein abundance ratios associated with mean delirium severity at postoperative day 1 were Complement C2 (Spearman rs = -0.31, 95%CI [-0.55, -0.02]) and Mannosyl-oligosaccharide 1,2-alpha-mannosidase (rs = 0.61, 95%CI = [0.29, 0.81]). CONCLUSIONS: We identified changes in proteins associated with coagulation, inflammation, and protein handling; larger, follow-up studies are needed to confirm our hypothesis-generating findings.

A novel GCN5b lysine acetyltransferase complex associates with distinct transcription factors in the protozoan parasite Toxoplasma gondii.

Toxoplasma gondii is a protozoan parasite that has a tremendous impact on human health and livestock. High seroprevalence among humans and other animals is facilitated by the conversion of rapidly proliferating tachyzoites into latent bradyzoites that are housed in tissue cysts, which allow transmission through predation. Epigenetic mechanisms contribute to the regulation of gene expression events that are crucial in both tachyzoites as well as their development into bradyzoites. Acetylation of histones is one of the critical histone modifications that is linked to active gene transcription. Unlike most early-branching eukaryotes, Toxoplasma possesses two GCN5 homologues, one of which, GCN5b, is essential for parasite viability. Surprisingly, GCN5b does not associate with most of the well-conserved proteins found in the GCN5 complexes of other eukaryotes. Of particular note is that GCN5b interacts with multiple putative transcription factors that have plant-like DNA-binding domains denoted as AP2. To understand the function of GCN5b and its role(s) in epigenetic gene regulation of stage switching, we performed co-immunoprecipitation of GCN5b under normal and bradyzoite induction conditions. We report the greatest resolution of the GCN5b complex to date under these various culture conditions. Moreover, reciprocal co-IPs were performed with distinct GCN5b-interacting AP2 factors (AP2IX-7 and AP2XII-4) to delineate the interactomes of each putative transcription factor. Our findings suggest that GCN5b is associated with at least two distinct complexes that are characterized by two different pairs of AP2 factors, and implicate up to four AP2 proteins to be involved with GCN5b-mediated gene regulation.

Quantitative phosphoproteomic analysis identifies novel functional pathways of tumor suppressor DLC1 in estrogen receptor positive breast cancer.

Deleted in Liver Cancer-1 (DLC1), a member of the RhoGAP family of proteins, functions as a tumor suppressor in several cancers including breast cancer. However, its clinical relevance is unclear in breast cancer. In this study, expression of DLC1 was correlated with prognosis using publicly available breast cancer gene expression datasets and quantitative Reverse Transcription PCR in cohorts of Estrogen Receptor-positive (ER+) breast cancer. Low expression of DLC1 correlates with poor prognosis in patients with ER+ breast cancer with further decrease in metastatic lesions. The Cancer Genome Atlas (TCGA) data showed that down regulation of DLC1 is not due to methylation or mutations. To seek further insights in understanding the role of DLC1 in ER+ breast cancer, we stably overexpressed DLC1-full-length (DLC1-FL) in T-47D breast cancer cells; this inhibited cell colony formation significantly in vitro compared to its control counterpart. Label-free global proteomic and TiO2 phosphopeptide enrichment assays (ProteomeXchange identifier PXD008220) showed that 205 and 122 phosphopeptides were unique to DLC1-FL cells and T-47D-control cells, respectively, whereas 6,726 were quantified by phosphoproteomics analysis in both conditions. The top three significant clusters of differentially phosphopeptides identified by DAVID pathway analysis represent cell-cell adhesion, mRNA processing and splicing, and transcription regulation. Phosphoproteomics analysis documented an inverse relation between DLC1 expression and several phosphopeptides including epithelial cell transforming sequence 2 (ECT2). Decreased phosphorylation of ECT2 at the residue T359, critical for its active conformational change, was validated by western blot. In addition, the ECT2 T359-containing phosphopeptide was detected in both basal and luminal patient-derived breast cancers breast cancer phosphoproteomics data on the Clinical Proteomic Tumor Analysis Consortium (CPTAC) Assay portal. Together, for the first time, this implicates ECT2 phosphorylation in breast cancer, which has been proposed as a therapeutic target in lung cancer. In conclusion, this data suggests that low expression of DLC1 is associated with poor prognosis. Targeting ECT2 phosphopeptides could provide a promising mechanism for controlling poor prognosis seen in DLC1low ER+ breast cancer.

Molecular Mechanism of Mot1, a TATA-binding Protein (TBP)-DNA Dissociating Enzyme.

The essential Saccharomyces cerevisiae ATPase Mot1 globally regulates transcription by impacting the genomic distribution and activity of the TATA-binding protein (TBP). In vitro, Mot1 forms a ternary complex with TBP and DNA and can use ATP hydrolysis to dissociate the TBP-DNA complex. Prior work suggested an interaction between the ATPase domain and a functionally important segment of DNA flanking the TATA sequence. However, how ATP hydrolysis facilitates removal of TBP from DNA is not well understood, and several models have been proposed. To gain insight into the Mot1 mechanism, we dissected the role of the flanking DNA segment by biochemical analysis of complexes formed using DNAs with short single-stranded gaps. In parallel, we used a DNA tethered cleavage approach to map regions of Mot1 in proximity to the DNA under different conditions. Our results define non-equivalent roles for bases within a broad segment of flanking DNA required for Mot1 action. Moreover, we present biochemical evidence for two distinct conformations of the Mot1 ATPase, the detection of which can be modulated by ATP analogs as well as DNA sequence flanking the TATA sequence. We also show using purified complexes that Mot1 dissociation of a stable, high affinity TBP-DNA interaction is surprisingly inefficient, suggesting how other transcription factors that bind to TBP may compete with Mot1. Taken together, these results suggest that TBP-DNA affinity as well as other aspects of promoter sequence influence Mot1 function in vivo.

The Modifier of Transcription 1 (Mot1) ATPase and Spt16 Histone Chaperone Co-regulate Transcription through Preinitiation Complex Assembly and Nucleosome Organization.

Modifier of transcription 1 (Mot1) is a conserved and essential Swi2/Snf2 ATPase that can remove TATA-binding protein (TBP) from DNA using ATP hydrolysis and in so doing exerts global effects on transcription. Spt16 is also essential and functions globally in transcriptional regulation as a component of the facilitates chromatin transcription (FACT) histone chaperone complex. Here we demonstrate that Mot1 and Spt16 regulate a largely overlapping set of genes in Saccharomyces cerevisiae. As expected, Mot1 was found to control TBP levels at co-regulated promoters. In contrast, Spt16 did not affect TBP recruitment. On a global scale, Spt16 was required for Mot1 promoter localization, and Mot1 also affected Spt16 localization to genes. Interestingly, we found that Mot1 has an unanticipated role in establishing or maintaining the occupancy and positioning of nucleosomes at the 5' ends of genes. Spt16 has a broad role in regulating chromatin organization in gene bodies, including those nucleosomes affected by Mot1. These results suggest that the large scale overlap in Mot1 and Spt16 function arises from a combination of both their unique and shared functions in transcription complex assembly and chromatin structure regulation.