An extra dimension in protein tagging by quantifying universal proteotypic peptides using targeted proteomics

The use of protein tagging to facilitate detailed characterization of target proteins has not only revolutionized cell biology, but also enabled biochemical analysis through efficient recovery of the protein complexes wherein the tagged proteins reside. The endogenous use of these tags for detailed protein characterization is widespread in lower organisms that allow for efficient homologous recombination. With the recent advances in genome engineering, tagging of endogenous proteins is now within reach for most experimental systems, including mammalian cell lines cultures. In this work, we describe the selection of peptides with ideal mass spectrometry characteristics for use in quantification of tagged proteins using targeted proteomics. We mined the proteome of the hyperthermophile Pyrococcus furiosus to obtain two peptides that are unique in the proteomes of all known model organisms (proteotypic) and allow sensitive quantification of target proteins in a complex background. By combining these 'Proteotypic peptides for Quantification by SRM' (PQS peptides) with epitope tags, we demonstrate their use in co-immunoprecipitation experiments upon transfection of protein pairs, or after introduction of these tags in the endogenous proteins through genome engineering. Endogenous protein tagging for absolute quantification provides a powerful extra dimension to protein analysis, allowing the detailed characterization of endogenous proteins

Trapping mammalian protein complexes in viral particles

Cell lysis is an inevitable step in classical mass spectrometry-based strategies to analyse protein complexes. Complementary lysis conditions, in situ cross-linking strategies and proximal labelling techniques are currently used to reduce lysis effects on the protein complex. We have developed Virotrap, a viral particle sorting approach that obviates the need for cell homogenization and preserves the protein complexes during purification. By fusing a bait protein to the HIV-1 GAG protein, we show that interaction partners become trapped within virus-like particles (VLPs) that bud from mammalian cells. Using an efficient VLP enrichment protocol, Virotrap allows the detection of known binary interactions and MS-based identification of novel protein partners as well. In addition, we show the identification of stimulus-dependent interactions and demonstrate trapping of protein partners for small molecules. Virotrap constitutes an elegant complementary approach to the arsenal of methods to study protein complexes

Lifestyle behaviour and risk factor control in coronary patients : Belgian results from the cross-sectional EUROASPIRE surveys

Objective: The aim of this study was to assess lifestyle behaviour as well as risk factor management across Belgian coronary patients who participated in the cross-sectional European Action on Secondary Prevention through Intervention to Reduce Events (EUROASPIRE) surveys. Methods: Analyses are based on a series of coronary patients by combining data from the Belgian participants in the EUROASPIRE III (328 patients; in 2006-2007) and EUROASPIRE IV (343 patients; in 2012-2013) surveys. Four hospitals located in the Ghent area participated in the surveys. Patients included in the analyses were >= 18 years old and had been hospitalised for a coronary event. Information on cardiovascular risk factors, lifestyle behaviour and medical treatment were obtained. Results: Overall, the proportion of smokers was 11% with 40% persistent smokers. Adequate physical activity levels were reported by 17%, 28% of patients were obese, 47% was central obese and known diabetes was prevalent in 21% of patients. Hypertension was observed in 46% of patients and 20% had a total cholesterol >= 5 mmol/L. About 80% had participated in a cardiac rehabilitation programme and the majority of patients were treated with blood pressure (92%) or lipid-lowering drugs (92%). Anxiety and depressive symptoms were reported by 30% and 24%, respectively. Differences between EUROASPIRE III and IV were limited. Conclusions: Compared to the overall EUROASPIRE results in Europe, Belgian CHD patients seem to do slightly better. However, tackling obesity, physical inactivity, hypertension and psychosocial distress remains an important challenge in the management of coronary patients

The impact of drop-out in cardiac rehabilitation on outcome among coronary artery disease patients

Background: The effect of adherence to cardiac rehabilitation (CR) on outcome is not clear. Therefore, we aimed to assess the impact of drop-out for non-medical reasons of CR on event-free survival in coronary artery disease (CAD). Methods: A total of 876 patients who attended CR after acute coronary syndrome (ACS), percutaneous coronary intervention (PCI) or coronary artery bypass graft (CABG) were included. Drop-out was defined as attending 50% of the training sessions. A combined endpoint of all-cause mortality and rehospitalization for a cardiovascular event was used to specify event-free survival. Differences in clinical characteristics were assessed and parameters with p<0.10 were entered in a multiple Cox regression analysis. Results: A total of 15% died or had a cardiovascular event during a median follow-up period of 33 months (interquartile range 24, 51). Overall, 17% dropped out before finishing half of the program. Patients who withdrew prematurely had a risk twice as high for a cardiovascular event or death (hazard ratio 1.92, 95% confidence interval 1.28-2.90) than those who attended more than half of the sessions. Both ACS (2.36, 1.47-3.58) and PCI (2.20, 1.22-3.96), as primary indicators for CR, were associated with an adverse outcome and also a prior history of chronic heart failure (CHF) remained negatively associated with event-free survival (3.67, 1.24-10.91).Finally, the presence of hyperlipidemia was independently related to a worse outcome (1.48, 1.02-2.16). Conclusions: Drop-out for non-medical reasons was independently associated with a negative outcome in CAD. Therefore, underlying factors for drop-out should gain more attention in future research and should be taken into account when organizing CR

FO‐SPR biosensor calibrated with recombinant extracellular vesicles enables specific and sensitive detection directly in complex matrices

Extracellular vesicles (EVs) have drawn huge attention for diagnosing myriad of diseases, including cancer. However, the EV detection and analyses procedures often lack much desired sample standardization. To address this, we used well-characterized recombinant EVs (rEVs) for the first time as a biological reference material in developing a fiber optic surface plasmon resonance (FO-SPR) bioassay. In this context, EV binding on the FO-SPR probes was achieved only with EV-specific antibodies (e.g. anti-CD9 and anti-CD63) but not with non-specific anti-IgG. To increase detection sensitivity, we tested six different combinations of EV-specific antibodies in a sandwich bioassay. Calibration curves were generated with two most effective combinations (anti-CD9/(B)anti-CD81 and anti-CD63/(B)anti-CD9), resulting in 10(3) and 10(4) times higher sensitivity than the EV concentration in human blood plasma from healthy or cancer patients, respectively. Additionally, by using anti-CD63/(B)anti-CD9, we detected rEVs spiked in cell culture medium and HEK293 endogenous EVs in the same matrix without any prior EV purification or enrichment. Lastly, we selectively captured breast cancer cell EVs spiked in blood plasma using anti-EpCA M antibody on the FO-SPR surface. The obtained results combined with FO-SPR real-time monitoring, fast response time and ease of operation, demonstrate its outstanding potential for EV quantification and analysis

IRE1β negatively regulates IRE1α signaling in response to endoplasmic reticulum stress

IRE1β is an ER stress sensor uniquely expressed in epithelial cells lining mucosal surfaces. Here, we show that intestinal epithelial cells expressing IRE1β have an attenuated unfolded protein response to ER stress. When modeled in HEK293 cells and with purified protein, IRE1β diminishes expression and inhibits signaling by the closely related stress sensor IRE1α. IRE1β can assemble with and inhibit IRE1α to suppress stress-induced XBP1 splicing, a key mediator of the unfolded protein response. In comparison to IRE1α, IRE1β has relatively weak XBP1 splicing activity, largely explained by a nonconserved amino acid in the kinase domain active site that impairs its phosphorylation and restricts oligomerization. This enables IRE1β to act as a dominant-negative suppressor of IRE1α and affect how barrier epithelial cells manage the response to stress at the host–environment interface

Proof-of-concept trial results of the HeartMan mobile personal health system for self-management in congestive heart failure

This study tested the effectiveness of HeartMan—a mobile personal health system offering decisional support for management of congestive heart failure (CHF)—on health-related quality of life (HRQoL), self-management, exercise capacity, illness perception, mental and sexual health. A randomized controlled proof-of-concept trial (1:2 ratio of control:intervention) was set up with ambulatory CHF patients in stable condition in Belgium and Italy. Data were collected by means of a 6-min walking test and a number of standardized questionnaire instruments. A total of 56 (34 intervention and 22 control group) participants completed the study (77% male; mean age 63 years, sd 10.5). All depression and anxiety dimensions decreased in the intervention group (p &lt; 0.001), while the need for sexual counselling decreased in the control group (p &lt; 0.05). Although the group differences were not significant, self-care increased (p &lt; 0.05), and sexual problems decreased (p &lt; 0.05) in the intervention group only. No significant intervention effects were observed for HRQoL, self-care confidence, illness perception and exercise capacity. Overall, results of this proof-of-concept trial suggest that the HeartMan personal health system significantly improved mental and sexual health and self-care behaviour in CHF patients. These observations were in contrast to the lack of intervention effects on HRQoL, illness perception and exercise capacity

Robust generation of knock-in cell lines using CRISPR-Cas9 and rAAV-assisted repair template delivery

The programmable Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease 9 (Cas9) technology revolutionized genome editing by providing an efficient way to cut the genome at a desired location (Ledford, 2015). In mammalian cells, DNA lesions trigger the error-prone non-homologous end joining (NHEJ) DNA repair mechanism. However, in presence of a DNA repair template, Homology-Directed Repair (HDR) can occur leading to precise repair of the lesion site. This last process can be exploited to enable precise knock-in changes by introducing the desired genomic alteration on the repair template. In this protocol, we describe the delivery of long repair templates (>200 nucleotides) using recombinant Adeno Associated Virus (rAAV) for CRISPR-Cas9-based knock-in of a C-terminal tag sequence in a human cell line

High-confidence interactome for RNF41 built on multiple orthogonal assays

Ring finger protein 41 (RNF41) is an E3 ubiquitin ligase involved in the ubiquitination and degradation of many proteins including ErbB3 receptors, BIRC6, and parkin. Next to this, RNF41 regulates the intracellular trafficking of certain JAK2-associated cytokine receptors by ubiquitinating and suppressing USP8, which, in turn, destabilizes the ESCRT-0 complex. To further elucidate the function of RNF41 we used different orthogonal approaches to reveal the RNF41 protein complex: affinity purification mass spectrometry, BioID, and Virotrap. We combined these results with known data sets for RNF41 obtained with microarray MAPPIT and Y2H screens. This way, we establish a comprehensive high-resolution interactome network comprising 175 candidate protein partners. To remove potential methodological artifacts from this network, we distilled the data into a high-confidence interactome map by retaining a total of 19 protein hits identified in two or more of the orthogonal methods. AP2S1, a novel RNF41 interaction partner, was selected from this high-confidence interactome for further functional validation. We reveal a role for AP2S1 in leptin and LIF receptor signaling and show that RNF41 stabilizes and relocates AP2S1