Site-specific identification and quantitation of endogenous SUMO modifications under native conditions.

Small ubiquitin-like modifier (SUMO) modification regulates numerous cellular processes. Unlike ubiquitin, detection of endogenous SUMOylated proteins is limited by the lack of naturally occurring protease sites in the C-terminal tail of SUMO proteins. Proteome-wide detection of SUMOylation sites on target proteins typically requires ectopic expression of mutant SUMOs with introduced tryptic sites. Here, we report a method for proteome-wide, site-level detection of endogenous SUMOylation that uses α-lytic protease, WaLP. WaLP digestion of SUMOylated proteins generates peptides containing SUMO-remnant diglycyl-lysine (KGG) at the site of SUMO modification. Using previously developed immuno-affinity isolation of KGG-containing peptides followed by mass spectrometry, we identified 1209 unique endogenous SUMO modification sites. We also demonstrate the impact of proteasome inhibition on ubiquitin and SUMO-modified proteomes using parallel quantitation of ubiquitylated and SUMOylated peptides. This methodological advancement enables determination of endogenous SUMOylated proteins under completely native conditions

Diffusion-weighted imaging of the breast-a consensus and mission statement from the EUSOBI International Breast Diffusion-Weighted Imaging working group.

Funder: Radboud University Medical CenterThe European Society of Breast Radiology (EUSOBI) established an International Breast DWI working group. The working group consists of clinical breast MRI experts, MRI physicists, and representatives from large vendors of MRI equipment, invited based upon proven expertise in breast MRI and/or in particular breast DWI, representing 25 sites from 16 countries. The aims of the working group are (a) to promote the use of breast DWI into clinical practice by issuing consensus statements and initiate collaborative research where appropriate; (b) to define necessary standards and provide practical guidance for clinical application of breast DWI; (c) to develop a standardized and translatable multisite multivendor quality assurance protocol, especially for multisite research studies; (d) to find consensus on optimal methods for image processing/analysis, visualization, and interpretation; and (e) to work collaboratively with system vendors to improve breast DWI sequences. First consensus recommendations, presented in this paper, include acquisition parameters for standard breast DWI sequences including specifications of b values, fat saturation, spatial resolution, and repetition and echo times. To describe lesions in an objective way, levels of diffusion restriction/hindrance in the breast have been defined based on the published literature on breast DWI. The use of a small ROI placed on the darkest part of the lesion on the ADC map, avoiding necrotic, noisy or non-enhancing lesion voxels is currently recommended. The working group emphasizes the need for standardization and quality assurance before ADC thresholds are applied. The working group encourages further research in advanced diffusion techniques and tailored DWI strategies for specific indications. Key Points • The working group considers breast DWI an essential part of a multiparametric breast MRI protocol and encourages its use. • Basic requirements for routine clinical application of breast DWI are provided, including recommendations on b values, fat saturation, spatial resolution, and other sequence parameters. • Diffusion levels in breast lesions are defined based on meta-analysis data and methods to obtain a reliable ADC value are detailed

Role of angiotensin II AT1 receptor activation in cardiovascular diseases

Numerous clinical studies and experimental investigations using cell culture and animal models suggest that angiotensin II (AngII) via AT1 receptor activation might induce cardiovascular hypertrophy, fibrosis and atherosclerosis resulting in vascular events such as myocardial infarction, heart failure or stroke and in end-organ damages. However, a question still remains: which part of these damages is due to a direct effect of AngII on its target tissues and which is due to AngII-induced hypertension? In an attempt to answer this question, a new model of transgenic mice, expressing a constitutively activated AT1A receptor instead of the wild type receptor has been obtained by homologous recombination. These mice present with a moderate increase of blood pressure (20mm Hg), hypertrophy of the small kidney arteries but not cardiac hypertrophy. The major phenotypic trait of these mice is the early and progressive development of a cardiovascular fibrosis. In light of these results and those from the literature, there is more and more evidence that in human hypertension, activation of the renin angiotensin system plays a minor role in the development of cardiovascular hypertrophy, but clearly participates to the development of cardiovascular fibrosis

Regulation of Angiotensinogen Gene

has introduced new directions for the study of the angiotensinogen molecule. The cloning and se-quencing of the human and rat cDNAs demonstrate the similarity of angiotensinogen to various serine protease inhibitors produced by the liver and was the beginning of studies looking for new physio-logical roles of angiotensinogen, in addition to the substrate for renin. The determination of the nucleotide sequence of these cDNAs also allowed the identification of angiotensinogen mRNA in many tissues in addition to the liver that is the major site of synthesis. This multilocalization of angiotensinogen is one of the arguments for the presence and the function of local renin-angioten-sin systems. Finally, the hepatic biosynthesis of angiotensinogen is regulated by many different hormonal factors including glucocorticoid, estrogen, thyroid hormone, insulin, and angiotensin II. The cloning of the angiotensinogen gene offers the opportunity to study this regulation at the tran-scriptional level. We present in this paper a review of the literature concerning the new aspects of angiotensinogen using molecular biological tools and its regulation together with the characteriza-tion of the human angiotensinogen gene. Am

Expression and characterization of recombinant human angiotensinogen in a heterologous eukaryotic cell line

Transfection of Chinese hamster ovary cells with an expression plasmid containing a full length human angiotensinogen cDNA has provided cell lines that secrete recombinant angiotensinogen in large quantities. This angiotensinogen is immunologically identical to plasma angiotensinogen and can be cleaved by human kidney renin (EC 3.4.23.15.). The peptide liberated by renin cleavage is immunologically identical to standard angiotensin I and shows a retention time on isocratic reversed-phase high-pressure liquid chromatography identical to that of standard angiotensin I. The heterogeneity of recombinant angiotensinogen on sodium dodecyl sulfate-polyacrylamide gel electrophoresis differs from that of plasma angiotensinogen. Treatment with endoglycosidases demonstrated that this difference is restricted to that of N-glycans and that N-glycans correspond to the quasi-totality of the carbohydrate content of both recombinant and plasma angiotensinogens. The development of a system capable of expressing human angiotensinogen cDNA in mammalian cells and the ability to obtain the corresponding angiotensinogen in large quantities will allow new studies on structure-function relationships of this protein

Internalization of the rat AT1a and AT1b receptors: pharmacological and functional requirements

AbstractThe capacity of the angiotensin II (AngII) agonist [Sar1]AngII, the antagonist [Sar1-I1e8]AngII and the non-peptidic antagonist DuP753 to undergo receptor internalization were studied in Chinese hamster ovary cells expressing rat AngII type 1a or 1b receptors (AT1a or AT1b) or a mutant of AT1a (Asn74) unable to couple G-protein. In this expression system, the ligand-induced internalization of rat AT1a and AT1b are similar. Moreover, peptidic ligands, either the agonist or antagonist, induce a significant internalization of AT1 receptors, but the non-peptidic antagonist DuP753 is far less potent. Finally, the normal internalization of the mutant Asn74 demonstrates that receptor activation and G-protein coupling are not required for AT1ainternalization