Biochemical characterization of bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI)

<p>Abstract</p> <p>Background</p> <p>TAFI is a plasma protein assumed to be an important link between coagulation and fibrinolysis. The three-dimensional crystal structures of authentic mature bovine TAFI (TAFIa) in complex with tick carboxypeptidase inhibitor, authentic full lenght bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI), and recombinant human TAFI have recently been solved. In light of these recent advances, we have characterized authentic bovine TAFI biochemically and compared it to human TAFI.</p> <p>Results</p> <p>The four N-linked glycosylation sequons within the activation peptide were all occupied in bovine TAFI, similar to human TAFI, while the sequon located within the enzyme moiety of the bovine protein was non-glycosylated. The enzymatic stability and the kinetic constants of TAFIa differed somewhat between the two proteins, as did the isoelectric point of TAFI, but not TAFIa. Equivalent to human TAFI, bovine TAFI was a substrate for transglutaminases and could be proteolytically cleaved by trypsin or thrombin/solulin complex, although small differences in the fragmentation patterns were observed. Furthermore, bovine TAFI exhibited intrinsic activity and TAFIa attenuated tPA-mediated fibrinolysis similar to the human protein.</p> <p>Conclusion</p> <p>The findings presented here suggest that the properties of these two orthologous proteins are similar and that conclusions reached using the bovine TAFI may be extrapolated to the human protein.</p

SILAC-MS Based Characterization of LPS and Resveratrol Induced Changes in Adipocyte Proteomics:Resveratrol as Ameliorating Factor on LPS Induced Changes

Adipose tissue inflammation is believed to play a pivotal role in the development obesity-related morbidities such as insulin resistance. However, it is not known how this (low-grade) inflammatory state develops. It has been proposed that the leakage of lipopolysaccharides (LPS), originating from the gut microbiota, through the gut epithelium could drive initiation of inflammation. To get a better understanding of which proteins and intracellular pathways are affected by LPS in adipocytes, we performed SILAC proteomic analysis and identified proteins that were altered in expression. Furthermore, we tested the anti-inflammatory compound resveratrol. A total of 927 proteins were quantified by the SILAC method and of these 57- and 64 were significantly up- and downregulated by LPS, respectively. Bioinformatic analysis (GO analysis) revealed that the upregulated proteins were especially involved in the pathways of respiratory electron transport chain and inflammation. The downregulated proteins were especially involved in protein glycosylation. One of the latter proteins, GALNT2, has previously been described to regulate the expression of liver lipases such as ANGPTL3 and apoC-III affecting lipid metabolism. Furthermore, LPS treatment reduced the protein levels of the insulin sensitizing adipokine, adiponectin, and proteins participating in the final steps of triglyceride- and cholesterol synthesis. Generally, resveratrol opposed the effect induced by LPS and, as such, functioning as an ameliorating factor in disease state. Using an unbiased proteomic approach, we present novel insight of how the proteome is altered in adipocytes in response to LPS as seen in obesity. We suggest that LPS partly exerts its detrimental effects by altering glycosylation processes of the cell, which is starting to emerge as important posttranscriptional regulators of protein expression. Furthermore, resveratrol could be a prime candidate in ameliorating dysfunctioning adipose tissue induced by inflammatory stimulation

Genomic and exoproteomic analyses of cold- and alkaline-adapted bacteria reveal an abundance of secreted subtilisin-like proteases

Proteases active at low temperature or high pH are used in many commercial applications, including the detergent, food and feed industries, and bacteria specifically adapted to these conditions are a potential source of novel proteases. Environments combining these two extremes are very rare, but offer the promise of proteases ideally suited to work at both high pH and low temperature. In this report, bacteria from two cold and alkaline environments, the ikaite columns in Greenland and alkaline ponds in the McMurdo Dry Valley region, Antarctica, were screened for extracellular protease activity. Two isolates, Arsukibacterium ikkense from Greenland and a related strain, Arsukibacterium sp. MJ3, from Antarctica, were further characterized with respect to protease production. Genome sequencing identified a range of potential extracellular proteases including a number of putative secreted subtilisins. An extensive liquid chromatography–tandem mass spectrometry analysis of proteins secreted by A. ikkense identified six subtilisin‐like proteases as abundant components of the exoproteome in addition to other peptidases potentially involved in complete degradation of extracellular protein. Screening of Arsukibacterium genome libraries in Escherichia coli identified two orthologous secreted subtilisins active at pH 10 and 20°C, which were also present in the A. ikkense exoproteome. Recombinant production of both proteases confirmed the observed activity

BE Newsletter Issue #12 December 2014

Inside this issue: Editorial – Ronny Billen Playing XBOX@CTF3 – Jose Luis Navarro Quirante & Frank Tecker, BE-OP-PS/CTF3 Independence for Controls – Stefan Lüders, IT-DI-CSO The Smooth Upgrades Working Group – Vito Baggiolini, BE-CO, Leader of the SUWG A Cryogenic Beam Current Monitor for Low-Energy Antiproton Facilities – Miguel Abreu Fernandes, BE-BI-PI Les EIS : Vous connaissez ? – Anne Funken, BE-ASR-SU Safety Column – Safety Unit Life in BE – Juan F. Esteban Müller, BE-RF-BR Responsibility changes & Newsletter contact

Characterization of the gila monster (Heloderma suspectum suspectum) venom proteome

The data presented here is related to the research article entitled “Characterization of the gila monster (Heloderma suspectum suspectum) venom proteome” by Sanggaard et al. in Journal of Proteomics [1]. The gila monster venom was collected, analyzed by 2D-gel electrophoresis and after Coomassie-Brilliant Blue staining the major spots were excised, subjected to in-gel trypsin digestion, and analyzed by LC–MS/MS. Subsequently, the venom proteins were identified based on de novo sequencing and homology searching. The mass spectrometry proteomics data have been deposited to the ProteomeXchange (dataset identifier PXD0001343), and in the present article we present an overview of the identified proteins. Protein identification failed for three of the selected spots, with the method described above. Instead, an iterative process, based on de novo sequencing, was employed

GO analysis of regulated proteins by LPS.

<p>(A) Upregulated proteins by LPS treatment belonged to the GO classes: respiratory electron transport chain and generation of precursor metabolites and energy processes. (B) Downregulated proteins especially belonged to the GO class protein glycosylation and to a smaller degree lipid metabolic processes. (C and D) Schematic overview of the distribution of upregulated (C) and downregulated (D) proteins in different GO classes represented here by gene name. Abbreviations: please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159747#pone.0159747.s001" target="_blank">S1 Table</a>.</p

STAT1 expression measured by qPCR and SILAC-MS.

<p>(A) STAT1 gene expression measured by qPCR in 3T3-L1 cells incubated with LPS and resveratrol. (B) IFIT1 protein expression measured by SILAC-MS in 3T3-L1 cells incubated with LPS and resveratrol.</p

Thirty most downregulated proteins.

<p>Thirty most downregulated proteins.</p

IFIT1 expression in whole adipose tissue measured by qPCR and in 3T3-L1 cells measured by and SILAC-MS.

<p>(A) Mice treated with LPS for 28 days showed increased gene expression of IFIT1, which was ameliorated by resveratrol delivered through the diet. (B) IFIT1 protein expression measured by SILAC-MS in 3T3-L1 cells incubated with LPS and resveratrol.</p