Sequential primed kinases create a damage-responsive phosphodegron on Eco1.

Sister-chromatid cohesion is established during S phase when Eco1 acetylates cohesin. In budding yeast, Eco1 activity falls after S phase due to Cdk1-dependent phosphorylation, which triggers ubiquitination by SCF(Cdc4). We show here that Eco1 degradation requires the sequential actions of Cdk1 and two additional kinases, Cdc7-Dbf4 and the GSK-3 homolog Mck1. These kinases recognize motifs primed by previous phosphorylation, resulting in an ordered sequence of three phosphorylation events on Eco1. Only the latter two phosphorylation sites are spaced correctly to bind Cdc4, resulting in strict discrimination between phosphates added by Cdk1 and by Cdc7. Inhibition of Cdc7 by the DNA damage response prevents Eco1 destruction, allowing establishment of cohesion after S phase. This elaborate regulatory system, involving three independent kinases and stringent substrate selection by a ubiquitin ligase, enables robust control of cohesion establishment during normal growth and after stress

protein complexes

Mass spectrometry-based shotgun proteomic analysis of C. elegan

The Deubiquitylase MATH-33 Controls DAF-16 Stability and Function in Metabolism and Longevity

SummaryFOXO family transcription factors are downstream effectors of Insulin/IGF-1 signaling (IIS) and major determinants of aging in organisms ranging from worms to man. The molecular mechanisms that actively promote DAF16/FOXO stability and function are unknown. Here we identify the deubiquitylating enzyme MATH-33 as an essential DAF-16 regulator in IIS, which stabilizes active DAF-16 protein levels and, as a consequence, influences DAF-16 functions, such as metabolism, stress response, and longevity in C. elegans. MATH-33 associates with DAF-16 in cellulo and in vitro. MATH-33 functions as a deubiquitylase by actively removing ubiquitin moieties from DAF-16, thus counteracting the action of the RLE-1 E3-ubiquitin ligase. Our findings support a model in which MATH-33 promotes DAF-16 stability in response to decreased IIS by directly modulating its ubiquitylation state, suggesting that regulated oscillations in the stability of DAF-16 protein play an integral role in controlling processes such as metabolism and longevity

Solubilities of substituted phenols in supercritical carbon dioxide

The binary (solute + CO 2) equilibrium solubilities of six substituted phenols (2,5-dimethyl phenol, 2,3-dimethyl phenol, 2,4,6-trimethyl phenol, 2,3,5-trimethyl phenol, 4-phenyl phenol, and 4-tert-butyl phenol) were determined at a temperature of 308 K in the pressure range of 10.1 to 28.0 MPa. Solubility data were obtained using a dynamic approach with a simple and reliable apparatus. The mole fraction solubilities of substituted phenols ranged from 5.14 × 10 -5 to 2.56 × 10 -2. Solubilities of three ternary (two solutes + CO 2) systems were investigated at a temperature of 308 K in the pressure range of 10.1 to 28.0 MPa. In the 4-phenyl phenol + 2,3,5-trimethyl phenol and 4-phenyl phenol + 2,4,6-trimethyl phenol systems, the solubility of 4-phenyl phenol was enhanced relative to its binary solubility by 22.9 % and 217 %, respectively. The 2,3,5-trimethyl phenol and 2,4,6-trimethyl phenol did not exhibit any solubility enhancements in the two ternary systems. Accurate solubilities could not be measured for the 2,5-dimethyl phenol + 4-tert-butyl phenol ternary system due to the existence of a liquid phase under the conditions studied. © 2006 American Chemical Society

Characterization of a Porous Nano-electrospray Capillary Emitter at Ultra-low Flow Rates

Biopharmaceuticals, especially therapeutic monoclonal antibodies, have emerged as a very promising new generation of protein-based drugs. However, their comprehensive analysis continues to pose new challenges for the bioanalytical field. Hyphenation of capillary electrophoresis with electrospray ionization (CE-MS) is a promising technique to address these challenges. One of the main advantages of CE-MS is the ability to produce stable electrospray at ultra-low flow rates (5–20 nl/min range). In this short communication we report on the characterization of a porous nanoelectrospray capillary emitter focusing on the effects of ultra-low flow rate on ionization efficiency, ion suppression and detection sensitivity. Ion suppression effect of a poorly-ionizable sugar in the presence of an easily-ionized peptide was reduced by almost 2-fold. Intact therapeutic antibody infusion analysis demonstrated that MS detection sensitivity increased by an order of magnitude with the decrease of flow rate from 250 nL/min to 20 nL/min using the nano-electrospray capillary emitter

Sequential primed kinases create a damage-responsive phosphodegron on Eco1.

Sister-chromatid cohesion is established during S phase when Eco1 acetylates cohesin. In budding yeast, Eco1 activity falls after S phase due to Cdk1-dependent phosphorylation, which triggers ubiquitination by SCF(Cdc4). We show here that Eco1 degradation requires the sequential actions of Cdk1 and two additional kinases, Cdc7-Dbf4 and the GSK-3 homolog Mck1. These kinases recognize motifs primed by previous phosphorylation, resulting in an ordered sequence of three phosphorylation events on Eco1. Only the latter two phosphorylation sites are spaced correctly to bind Cdc4, resulting in strict discrimination between phosphates added by Cdk1 and by Cdc7. Inhibition of Cdc7 by the DNA damage response prevents Eco1 destruction, allowing establishment of cohesion after S phase. This elaborate regulatory system, involving three independent kinases and stringent substrate selection by a ubiquitin ligase, enables robust control of cohesion establishment during normal growth and after stress