Chemoproteomics Yields a Selective Molecular Host for Acetyl-CoA.

Chemoproteomic profiling is a powerful approach to define the selectivity of small molecules and endogenous metabolites with the human proteome. In addition to mechanistic studies, proteome specificity profiling also has the potential to identify new scaffolds for biomolecular sensing. Here, we report a chemoproteomics-inspired strategy for selective sensing of acetyl-CoA. First, we use chemoproteomic capture experiments to validate the N-terminal acetyltransferase NAA50 as a protein capable of differentiating acetyl-CoA and CoA. A Nanoluc-NAA50 fusion protein retains this specificity and can be used to generate a bioluminescence resonance energy transfer (BRET) signal in the presence of a CoA-linked fluorophore. This enables the development of a ligand displacement assay in which CoA metabolites are detected via their ability to bind the Nanoluc-NAA50 protein "host" and compete binding of the CoA-linked fluorophore "guest". We demonstrate that the specificity of ligand displacement reflects the molecular recognition of the NAA50 host, while the window of dynamic sensing can be controlled by tuning the binding affinity of the CoA-linked fluorophore guest. Finally, we show that the method's specificity for acetyl-CoA can be harnessed for gain-of-signal optical detection of enzyme activity and quantification of acetyl-CoA from cellular samples. Overall, our studies demonstrate the potential of harnessing insights from chemoproteomics for molecular sensing and provide a foundation for future applications in target engagement and selective metabolite detection.

Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions.

Prenylated proteins play key roles in several human diseases including cancer, atherosclerosis and Alzheimer's disease. KRAS4b, which is frequently mutated in pancreatic, colon and lung cancers, is processed by farnesylation, proteolytic cleavage and carboxymethylation at the C-terminus. Plasma membrane localization of KRAS4b requires this processing as does KRAS4b-dependent RAF kinase activation. Previous attempts to produce modified KRAS have relied on protein engineering approaches or in vitro farnesylation of bacterially expressed KRAS protein. The proteins produced by these methods do not accurately replicate the mature KRAS protein found in mammalian cells and the protein yield is typically low. We describe a protocol that yields 5-10 mg/L highly purified, farnesylated, and methylated KRAS4b from insect cells. Farnesylated and methylated KRAS4b is fully active in hydrolyzing GTP, binds RAF-RBD on lipid Nanodiscs and interacts with the known farnesyl-binding protein PDEδ.

Insulin resistance and postprandial triglyceride levels in primary renal disease.

BACKGROUND: Renal failure is associated with a range of metabolic abnormalities including insulin resistance and dyslipidemia. We examined the role of creatinine clearance (CrCl) and body composition in the development of insulin resistance in patients with primary renal disease and a variable degree of renal failure. We also determined the effect of a high-fat meal on postprandial triglyceride levels in a subgroup of these patients. METHODS: Forty-four patients with primary renal disease (men, 25; women, 19; age, 21-75 years) were compared to 44 controls matched for age, sex, and body composition. Renal biochemistry, plasma glucose, insulin, lipids, and nonesterified fatty acids were measured in the fasting state. Insulin sensitivity was calculated using the Homeostasis Model Assessment for Insulin Resistance (HOMA-R), and pancreatic beta-cell secretory capacity by HOMA- beta . Fourteen normotriglyceridemic subjects from each group consumed an 80-g fat meal to examine their postprandial metabolic response. RESULTS: Although there was no significant difference between HOMA-R for the controls and the entire patient group ( P = .06), HOMA-R was significantly higher in patients with CrCl less than 60 mL/min than those with CrCl greater than 60 mL/min or control subjects ( P < .01 for each pair). Exponential analysis of the relationship between CrCl and HOMA-R and - beta showed a line of best fit that was superior to that obtained by linear regression analysis ( P < .01 and P < .005, respectively). HOMA-R in renal patients was correlated with several parameters of body composition, including central fat (kilogram) ( P < .005). There was no difference in body fat parameters or HOMA-R for the patient and control subgroups undergoing a fat meal challenge. However, the patient subgroup showed a greater postprandial incremental rise in plasma triglycerides compared to controls ( P < .02). CONCLUSION: Patients with renal disease exhibit metabolic features typically associated with the metabolic syndrome. Insulin resistance increased with decline in renal function and was significantly higher in patients with CrCl less than 60 mL/min compared to subjects with CrCl greater than 60 mL/min or carefully matched controls. Renal patients also showed significant postprandial hypertriglyceridemia.

Novel applications of recombinant factor VIIa for the management of pediatric coagulopathic diseases.

Recombinant factor VIIa (rFVIIa) was envisioned for the treatment of bleeding in hemophilia patients with inhibitors. In response to tissue factor expressed upon vessel wall injury, rFVIIa amplifies the thrombin burst primarily on membrane surfaces, including activated platelets. Because it is functional at a key point in the clotting cascade, rFVIIa shows promise as a therapeutic option for various bleeding situations. The prothrombin time (PT) is often used as a surrogate to monitor rFVIIa therapy. Using laboratory and clinical measures of outcome, the use of rVIIa as a therapeutic option in treating several different pediatric coagulopathic conditions is described.