Kinase Substrate Sensor (KISS), a Mammalian In Situ Protein Interaction Sensor

Probably every cellular process is governed by protein-protein interaction (PPIs), which are often highly dynamic in nature being modulated by in- or external stimuli. Here we present KISS, for KInase Substrate Sensor, a mammalian two-hybrid approach designed to map intracellular PPIs and some of the dynamic features they exhibit. Benchmarking experiments indicate that in terms of sensitivity and specificity KISS is on par with other binary protein interaction technologies while being complementary with regard to the subset of PPIs it is able to detect. We used KISS to evaluate interactions between different types of proteins, including transmembrane proteins, expressed at their native subcellular location. In situ analysis of endoplasmic reticulum stress-induced clustering of the endoplasmic reticulum stress sensor ERN1 and ligand-dependent β-arrestin recruitment to GPCRs illustrated the method''s potential to study functional PPI modulation in complex cellular processes. Exploring its use as a tool for in cell evaluation of pharmacological interference with PPIs, we showed that reported effects of known GPCR antagonists and PPI inhibitors are properly recapitulated. In a three-hybrid setup, KISS was able to map interactions between small molecules and proteins. Taken together, we established KISS as a sensitive approach for in situ analysis of protein interactions and their modulation in a changing cellular context or in response to pharmacological challenges.A protein''s function is largely mediated through its interactions with other proteins, hence the critical importance of protein-protein interaction (PPI)¹ maps for understanding cellular mechanisms of action in health and disease. Whereas many proteins are organized in stable multi-protein complexes, the majority of cellular processes are governed by transient protein encounters, the dynamics of which are directed by a diversity of both intra- and extracellular signals. Our view of protein networks is still, however, mainly a static one (1). Current interactomes consist mainly of data generated by yeast 2-hybrid (Y2H) (2) and (tandem) affinity purification combined with mass spectrometry (3) and should be interpreted as scaffolds of potential PPIs that might occur at a certain time and place in the cell or as snapshots of PPIs taking place under a specific cellular condition. Although very robust and highly efficient, these approaches do not allow studying PPI modulation because they do not offer the proper context for mammalian PPI analysis, e.g. they operate in yeast cells (Y2H) or make use of cell lysates (affinity purification-based methods). Moreover, because these interactome mapping tools are biased against interactions that involve transmembrane proteins, the latter are underrepresented in current interactome network versions (4). Yet, membrane-associated proteins constitute around one third of the entire proteome and their significance is underscored by the fact that over half of currently marketed drugs target membrane proteins (5). These observations support the need for approaches that allow PPIs, including those involving transmembrane proteins, to be assayed in their native cellular environment.Apart from the high-throughput methods mentioned above, a diverse arsenal of other PPI technologies has been developed, a number of which actually operate in mammalian cells. FRET and BRET, which rely on fluorescence or bioluminescence energy transfer between interacting fusion proteins, make assays with high spatiotemporal resolution (6, 7). A variety of PCAs have been reported, including split fluorescent protein or reporter enzyme technologies, that are able to capture aspects of PPI dynamics in a mammalian background (8, 9). A recent addition is an infrared fluorescent PCA that, unlike previous fluorescent PCAs, exhibits reversible complementation, thus enabling spatiotemporal analysis of dynamic PPIs (10). Another binary interaction assay, luminescence-based mammalian interactome mapping (LUMIER), has been applied to map TGFβ induced modulation of PPIs with components of the TGFβ signaling pathway (11). MaMTH, a mammalian version of the split ubiquitin approach, was designed particularly for the analysis of PPIs involving integral membrane proteins, also allowing the detection of functional PPI modulation (12). Efforts to apply purification-based methods for detecting context-dependent PPI modulation recently resulted in the development of AP-SRM (13) and AP-SWATH (14).Our group previously conceived mammalian protein-protein interaction trap (MAPPIT) (supplemental Fig. S1A) (15, 16), a mammalian two-hybrid approach based on complementation of a cytokine receptor that was developed into a broad platform for PPI analysis (17, 18), screening for small molecule PPI disruptors (19, 20) and drug target profiling (21, 22). Although MAPPIT operates in intact human cells, thus providing the natural environment for human protein analysis, the interaction sensor is anchored to the plasma membrane, precluding the analysis of PPIs at their native subcellular localization. In addition, MAPPIT is incompatible with full size transmembrane proteins. Here we describe KInase Substrate Sensor (KISS), a novel binary PPI mapping approach that enables in situ analysis in living mammalian cells of protein interactions and their responses to physiological or pharmacological challenges.     

Effects of Insulin Detemir and NPH Insulin on Body Weight and Appetite-Regulating Brain Regions in Human Type 1 Diabetes: A Randomized Controlled Trial

Studies in rodents have demonstrated that insulin in the central nervous system induces satiety. In humans, these effects are less well established. Insulin detemir is a basal insulin analog that causes less weight gain than other basal insulin formulations, including the current standard intermediate-long acting Neutral Protamine Hagedorn (NPH) insulin. Due to its structural modifications, which render the molecule more lipophilic, it was proposed that insulin detemir enters the brain more readily than other insulins. The aim of this study was to investigate whether insulin detemir treatment differentially modifies brain activation in response to food stimuli as compared to NPH insulin. In addition, cerebral spinal fluid (CSF) insulin levels were measured after both treatments. Brain responses to viewing food and non-food pictures were measured using functional Magnetic Resonance Imaging in 32 type 1 diabetic patients, after each of two 12-week treatment periods with insulin detemir and NPH insulin, respectively, both combined with prandial insulin aspart. CSF insulin levels were determined in a subgroup. Insulin detemir decreased body weight by 0.8 kg and NPH insulin increased weight by 0.5 kg (p = 0.02 for difference), while both treatments resulted in similar glycemic control. After treatment with insulin detemir, as compared to NPH insulin, brain activation was significantly lower in bilateral insula in response to visual food stimuli, compared to NPH (p = 0.02 for right and p = 0.05 for left insula). Also, CSF insulin levels were higher compared to those with NPH insulin treatment (p = 0.003). Our findings support the hypothesis that in type 1 diabetic patients, the weight sparing effect of insulin detemir may be mediated by its enhanced action on the central nervous system, resulting in blunted activation in bilateral insula, an appetite-regulating brain region, in response to food stimuli.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00626080","term_id":"NCT00626080"}}NCT00626080.     

1,25-Dihydroxyvitamin D3 and Its Analog TX527 Promote a Stable Regulatory T Cell Phenotype in T Cells from Type 1 Diabetes Patients

The emergence of regulatory T cells (Tregs) as central mediators of peripheral tolerance in the immune system has led to an important area of clinical investigation to target these cells for the treatment of autoimmune diseases such as type 1 diabetes. We have demonstrated earlier that in vitro treatment of T cells from healthy individuals with TX527, a low-calcemic analog of bioactive vitamin D, can promote a CD4⁺CD25^highCD127^low regulatory profile and imprint a migratory signature specific for homing to sites of inflammation. Towards clinical application of vitamin D-induced Tregs in autologous adoptive immunotherapy for type 1 diabetes, we show here that 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] and TX527 similarly imprint T cells from type 1 diabetes patients with a CD4⁺CD25^highCD127^low regulatory profile, modulate surface expression of skin- and inflammation-homing receptors, and increase expression of CTLA-4 and OX-40. Also, 1,25(OH)₂D₃ and TX527 treatment inhibit the production of effector cytokines IFN-γ, IL-9, and IL-17. Importantly, 1,25(OH)₂D₃ and TX527 promote the induction of IL-10-producing CD4⁺CD25^highCD127^low T cells with a stable phenotype and the functional capacity to suppress proliferation of autologous responder T cells in vitro. These findings warrant additional validation of vitamin D-induced Tregs in view of future autologous adoptive immunotherapy in type 1 diabetes.     

Fine mapping and imprinting analysis for fatness trait QTLs in pigs

Quantitative trait loci (QTL) for fatness traits were reported recently in an experimental Meishan × Large White and Landrace F₂ cross. To further investigate the regions on pig Chr 2 (SSC2), SSC4, and SSC7, 25 additional markers from these regions were typed on 800 animals (619 F₂ animals, their F₁ parents, and F₀ grandfathers). Compared with the published maps, a modified order of markers was observed for SSC4 and SSC7. QTL analyses were performed both within the half-sib families as well as across families (line cross). Furthermore, a QTL model accounting for imprinting effects was tested. Information content could be increased considerably on all three chromosomes. Evidence for the backfat thickness QTL on SSC7 was increased, and the location could be reduced to a 33-cM confidence interval. The QTL for intramuscular fat on SSC4 could not be detected in this half-sib analysis, whereas under the line cross model a suggestive QTL on a different position on SSC4 was detected. For SSC2, in the half-sib analysis, a suggestive QTL for backfat thickness was detected with the best position at 26 cM. Imprinting analysis, however, revealed a genome-wise, significant, paternally expressed QTL on SSC2 with the best position at 63 cM. Our results suggest that this QTL is different from the previously reported paternally expressed QTL for muscle mass and fat deposition on the distal tip of SSC2p. Received: 15 October 1999 / Accepted: 21 February 2000     

Short-Term Summer Inundation as a Measure to Counteract Acidification in Rich Fens

In regions with intensive agriculture, water level fluctuation in wetlands has generally become constricted within narrow limits. Water authorities are, however, considering the re-establishment of fluctuating water levels as a management tool in biodiverse, base-rich fens (‘rich fens’). This includes temporary inundation with surface water from ditches, which may play an important role in counteracting acidification in order to conserve and restore biodiversity. Inundation may result in an increased acid neutralizing capacity (ANC) for two reasons: infiltration of base-rich inundation water into peat soils, and microbial alkalinity generation under anaerobic conditions. The main objectives of this study were to test whether short-term (2 weeks) summer inundation is more effective than short-term winter inundation to restore the ANC in the upper 10 cm of non-floating peat soils, and to explain potential differences. Large-scale field experiments were conducted for five years in base-rich fens and Sphagnum-dominated poor fens. Winter inundation did not result in increased porewater ANC, because infiltration was inhibited in the waterlogged peat and evapotranspiration rates were relatively low. Also, low temperatures limit microbial alkalinity generation. In summer, however, when temperature and evapotranspiration rates are higher, inundation resulted in increased porewater Ca and HCO₃ ^- concentrations, but only in areas with characteristic rich fen bryophytes. This increase was not only due to stronger infiltration into the soil, but also to higher microbial alkalinity generation under anaerobic conditions. In contrast, porewater ANC did not increase in Sphagnum-plots as a result of the ability of Sphagnum spp. to acidify their environment. In both rich and poor fens, flooding-induced P-mobilization remained sufficiently low to safeguard P-limited vegetation. NO₃ ^- and NH₄ ⁺ dynamics showed no considerable changes either. In conclusion, short-term summer inundation with base-rich and nutrient-poor surface water is considered beneficial in the management of non-floating rich fens, and much more effective than winter inundation.