A Novel Immuno-Competitive Capture Mass Spectrometry Strategy for Protein–Protein Interaction Profiling Reveals That LATS Kinases Regulate HCV Replication Through NS5A Phosphorylation*

Mapping protein–protein interactions is essential to fully characterize the biological function of a protein and improve our understanding of diseases. Affinity purification coupled to mass spectrometry (AP-MS) using selective antibodies against a target protein has been commonly applied to study protein complexes. However, one major limitation is a lack of specificity as a substantial part of the proposed binders is due to nonspecific interactions. Here, we describe an innovative immuno-competitive capture mass spectrometry (ICC-MS) method to allow systematic investigation of protein–protein interactions. ICC-MS markedly increases the specificity of classical immunoprecipitation (IP) by introducing a competition step between free and capturing antibody prior to IP. Instead of comparing only one experimental sample with a control, the methodology generates a 12-concentration antibody competition profile. Label-free quantitation followed by a robust statistical analysis of the data is then used to extract the cellular interactome of a protein of interest and to filter out background proteins. We applied this new approach to specifically map the interactome of hepatitis C virus (HCV) nonstructural protein 5A (NS5A) in a cellular HCV replication system and uncovered eight new NS5A-interacting protein candidates along with two previously validated binding partners. Follow-up biological validation experiments revealed that large tumor suppressor homolog 1 and 2 (LATS1 and LATS2, respectively), two closely related human protein kinases, are novel host kinases responsible for NS5A phosphorylation at a highly conserved position required for optimal HCV genome replication. These results are the first illustration of the value of ICC-MS for the analysis of endogenous protein complexes to identify biologically relevant protein–protein interactions with high specificity.The exploration of a protein''s interactome in a given biological system is often critical to understand its function. Since the introduction of yeast two-hybrid experiments, alternative methods to explore protein–protein interactions have emerged (1–3). In particular, the combination of affinity-purification with mass spectrometry (AP-MS)¹ (4) has shown great promise for the identification of protein complexes directly in mammalian cell lines (5). This approach typically involves capturing the protein of interest either through an epitope tag or using a selective antibody. The main challenge with AP-MS is to discern bona fide interactors from highly abundant cellular proteins e.g. cytoskeletal or ribosomal proteins that bind nonspecifically to the affinity matrix (6). This can be partially addressed by including a negative control, such as IP with an antibody of the same isotype against an irrelevant protein or using samples where the target protein is absent (4). More recently, the introduction of quantitative MS (7–9), involving either isotope labeling or label-free strategies (for a review see (9, 10)), have led to a better distinction between true and false-positive interactions. While most of the recent efforts to reduce false positive rates have concentrated on refining data analysis (11), very few attempts have been made to improve the selectivity at the IP step (12). Consequently, classical quantitative side-by-side comparison of a sample with its control (wild type versus knockout cell lysates or capturing antibody versus control isotype) still suffers from the fact that the control sample is not identical to the probed one and both samples can lead to the association of different nonspecific binders.In this study, we present an innovative approach, termed immuno-competitive capture MS (ICC-MS), which involves a competition step between free and bound antibody in the same cellular extract and quantitation using label-free MS. Instead of comparing only one IP with a control, the methodology generates a 12-concentration antibody competition profile. Combined with a robust statistical analysis of the quantified MS signals, the cellular endogenous interactome of a protein of interest can be extracted out of the background of hundreds of proteins. We used this new approach to specifically map the interactome of the HCV NS5A protein, an essential viral regulatory protein for both genome replication and modulation of the host environment (13). Proteins interacting with NS5A have been previously identified using yeast two-hybrid (14) or classical co-expression and co-immunoprecipitation methods (15). In this study, we use a human hepatocyte-derived cellular model of HCV genome replication and uncover eight new NS5A-interacting protein candidates in addition to other well-known partners. In particular, we highlight LATS1 and LATS2, two closely related human serine/threonine protein kinases, and demonstrate that they are new host kinases responsible for NS5A phosphorylation and optimal HCV replication.     

Changes of Body Mass Index in Relation to Mortality: Results of a Cohort of 42,099 Adults

Background

High Body-Mass-Index (BMI) is associated with increased all-cause mortality, but little is known about the effect of short- and long-term BMI change on mortality. The aim of the study was to determine how long-term weight change affects mortality.

Methods and findings

Within a population-based prospective cohort of 42,099 Austrian men and women (mean age 43 years) with at least three BMI measurements we investigated the relationship of BMI at baseline and two subsequent BMI change intervals of five years each with all-cause mortality using Cox proportional Hazard models. During median follow-up of 12 years 4,119 deaths were identified. The lowest mortalities were found in persons with normal weight or overweight at baseline and stable BMI over 10 years. Weight gain (≥0.10 kg/m²/year) during the first five years was associated with increased mortality in overweight and obese people. For weight gain during both time intervals mortality risk remained significantly increased only in overweight (Hazard Ratio (HR): 1.39 (95% confidence interval: 1.01; 1.92)) and obese women (1.85 (95% confidence interval: 1.18; 2.89)). Weight loss (< −0.10 kg/m²/year) increased all-cause mortality in men and women consistently. BMI change over time assessed using accepted World Health Organisation BMI categories showed no increased mortality risk for people who remained in the normal or overweight category for all three measurements. In contrast, HRs for stable obese men and women were 1.57 (95% CI: 1.31; 1.87) and 1.46 (95% CI: 1.25; 1.71) respectively.

Conclusion

Our findings highlight the importance of weight stability and obesity avoidance in prevention strategy.     

Referate

Ohne Zusammenfassung     

Die Wirkung der α-Bestrahlung auf Protoplasma und Chloroplasten

Ohne Zusammenfassung     

Wirkung der UV-Strahlen auf die Plasmapermeabilität

Ohne Zusammenfassung     

Fermentation of glycerol to 1,3-propanediol by Klebsiella and Citrobacter strains

Summary Glycerol-fermenting anaerobes were enriched with glycerol at low and high concentrations in order to obtain strains that produce 1,3-propanediol. Six isolates were selected for more detailed characterization; four of them were identified as Citrobacter freundii, one as Klebsiella oxytoca and one as K. pneumoniae. The Citrobacter strains formed 1.3-propanediol and acetate and almost no by-products, while the Klebsiella strains produced varying amounts of ethanol in addition and accordingly less 1,3-propanediol. Enterobacterial strains of the genera Enterobacter, Klebsiella, and Citrobacter from culture collections showed similar product patterns except for one group which formed limited amounts of ethanol, but no propanediol. Seven strains were grown in pH-controlled batch cultures to determine the parameters necessary to evaluate their capacity for 1,3-propanediol production. K. pneumoniae DSM 2026 exhibited the highest final concentration (61 g/l) and the best productivity (1.7 g/l h) whereas C. freundii Zu and K2 achieved only 35 g/l and 1.4 g/l h, respectively. The Citrobacter strains on the other hand gave somewhat better yields which were very close to the theoretical optimum of 65 mol %. Offprint requests to: H. Biebl     

Phosphorylation and dephosphorylation of distinct isoforms of the heavy neurofilament protein NF-H

Summary 1. Previous immunohistochemical studies led to the suggestion that distinctly phosphorylated neurofilament isoforms exist in different types of neurons. We have recently examined this hypothesis by direct biochemical experiments, which revealed that the heavy neurofilament protein NF-H of bovine ventral root cholinergic neurons is more acidic and markedly more phosphorylated than that of bovine dorsal root neurons.2. In the present study we employed this system to study the degree to which distinctly phosphorylated NF-H isoforms differ in the extents to which they can be phosphorylated and dephosphorylatedin vitro. This was performed utilizing alkaline phosphatase and protein kinase PK40^ERK, which is specific to serines of Lys-Ser-Pro (KSP) repeats. The results obtained reveal that:3. The more extensively phosphorylated ventral root NF-H is dephosphorylated more rapidly than dorsal root NF-H.4. Ventral root NF-H and dorsal root NF-H in their native form are both poor substrates of PK40^ERK.5. Following dephosphorylation, ventral root and dorsal root NF-H are phosphorylated extensively and differentially by this kinase. Under these conditions, PK40^ERK catalyzes the incorporation of, respectively, 4.2±1.3 and 2.8±0.6 mol of phosphate per molecule of ventral root NF-H and dorsal root NF-H. The ratio of phosphates incorporated into ventral root NF-H to those incorporated into dorsal root NF-H is 1.46±0.17.6. These findings support the hypothesis that different classes of neurons contain distinctly phosphorylated neurofilaments and show that ventral root and dorsal root neurons are a useful model system for studying the distinct characteristics of neurofilament phosphorylation in different types of neurons.