Leptin: linking adipocyte metabolism with cardiovascular and autoimmune diseases

Leptin was originally discovered as an adipocyte-derived hormone involved in the central control of body weight and energy homeostasis. It is now clear that leptin is a pleiotropic cytokine, with activities on many peripheral cell types. These findings may help explain the surprising role of leptin in pathophysiological processes. Recent evidence suggests that leptin contributes to atherosclerosis and to the increased risk of cardiovascular disease in obese people. Leptin also appears to be involved in T-cell-dependent immunity and possibly in the development and maintenance of certain autoimmune diseases. Here, we review the role of leptin in cardiovascular and autoimmune diseases, and also briefly address the potential therapeutic use of leptin antagonists.     

Habitat and laying date of Great and Blue Tit Parus major and P. caeruleus

Using 19 year's data from nine study areas at Ghent we investigated the effect of habitat on laying date, and found it to be quite different for Great and Blue Tits. In Great Tits we found a clear gradient in laying dates from urban over suburban to rural habitats. Blue Tits laid earliest in a suburban habitat and latest in a rural habitat. All other areas, including urban, suburban and rural ones, formed one intermediate group. The overall average laying date for the Great Tit, 18.9 April, was slightly earlier than that of the Blue Tit, 19.4 April. In urban areas Great Tits laid significantly earlier than Blue Tits, in optimal oak habitat and in one suburban area Blue Tits laid earlier, whereas in the five remaining areas no significant differences between the two species were found.
We argue that the differences in laying dates, although correlated to temperature, are probably caused by differences in the timing of food availability. We suggest that differences in laying dates of Great and Blue Tits are caused by a different response to environmental variations through differences in feeding ecology.     

Reverse MAPPIT detects disruptors of protein-protein interactions in human cells

Reverse mammalian protein-protein interaction trap (MAPPIT) is a mammalian reverse two-hybrid technology. The method is adapted from the forward MAPPIT technique, a two-hybrid complementation system in which the interaction between a bait-fusion protein and a prey-fusion protein restores ligand-dependent cytokine receptor signaling. In the reverse mode described in detail here, a positive readout is generated on disruption of the designated protein-protein interactions. Reverse MAPPIT functions in intact human cells, facilitating simultaneous analysis of disruption, toxicity and permeability of the tested compounds, making it particularly suitable for screening for molecules that target therapeutically interesting protein-protein interactions or for mapping the interaction interface between proteins. The total handling time of a typical reverse MAPPIT experiment is approximately 9 h and is spread over 4-5 d.     

The C-terminus of CIS defines its interaction pattern

Proteins of the SOCS (suppressors of cytokine signalling) family are characterized by a conserved modular structure with pre-SH2 (Src homology 2), SH2 and SOCS-box domains. Several members, including CIS (cytokine-inducible SH2 protein), SOCS1 and SOCS3, are induced rapidly upon cytokine receptor activation and function in a negative-feedback loop, attenuating signalling at the receptor level. We used a recently developed mammalian two-hybrid system [MAPPIT (mammalian protein-protein interaction trap)] to analyse SOCS protein-interaction patterns in intact cells, allowing direct comparison with biological function. We find that, besides the SH2 domain, the C-terminal part of the CIS SOCS-box is required for functional interaction with the cytokine receptor motifs examined, but not with the N-terminal death domain of the TLR (Toll-like receptor) adaptor MyD88. Mutagenesis revealed that one single tyrosine residue at position 253 is a critical binding determinant. In contrast, substrate binding by the highly related SOCS2 protein, and also by SOCS1 and SOCS3, does not require their SOCS-box.     

Heteromeric MAPPIT: a novel strategy to study modification-dependent protein–protein interactions in mammalian cells

We recently reported a two-hybrid trap for detecting protein–protein interactions in intact mammalian cells (MAPPIT). The bait protein was fused to a STAT recruitment-deficient, homodimeric cytokine receptor and the prey protein to functional STAT recruitment sites. In such a configuration, STAT-dependent responses can be used to monitor a given bait–prey interaction. Using this system, we were able to demonstrate both modification-independent and tyrosine phosphorylation- dependent interactions. Protein modification in this approach is, however, strictly dependent on the receptor-associated JAK tyrosine kinases. We have now extended this concept by using extracellular domains of the heteromeric granulocyte/macrophage colony-stimulating factor receptor (GM-CSFR). Herein, the bait was fused to the βc chain and its modifying enzyme to the GM-CSFRα chain (or vice versa). We demonstrate several serine phosphorylation-dependent interactions in the TGFβ/Smad pathway using the catalytic domains of the ALK4 or ALK6 serine/threonine kinase receptors. In all cases tested, STAT-dependent signaling was completely abolished when mutant baits were used wherein critical serine residues were replaced by alanines. This approach operates both in transient and stable expression systems and may not be limited to serine phosphorylation but has the potential for studying various different types of protein modification-dependent interactions in intact cells.     

Methods to map protein interactions in mammalian cells: different tools to address different questions

In the post-genome era, functional annotation of the predicted gene-sets will be one of the most important upcoming challenges. So-called interactome analysis positions a protein in its subcellular environment by mapping its interaction partners. Such interaction maps are essential for an accurate insight into protein function since many cellular processes are organised to operate in protein complexes. These assemblies have dynamic structures and can interact with each other, two properties which are often controlled by regulated protein expression and modification. Various methods exist to unravel protein interaction circuitries, which can be roughly divided into biochemical and genetic strategies. In this review we focus on the different strategies to study protein-protein interactions in living mammalian cells. Recently developed analytical and screening methods are also addressed.     

Large-scale protein interactome mapping: strategies and opportunities

Interactions between proteins are central to any cellular process, and mapping these into a protein network is informative both for the function of individual proteins and the functional organization of the cell as a whole. Many strategies have been developed that are up to this task, and the last 10 years have seen the high-throughput application of a number of those in large-scale, sometimes proteome-wide, interactome mapping efforts. Although initially the quality of the data produced in these screening campaigns has been questioned, quality standards and empirical validation schemes are now in place to ensure high-quality data generation. Through their integration with other 'omics' data, interactomics datasets have proven highly valuable towards applications in different areas of clinical importance.     

Analysis of Tyr to Phe and fa/fa leptin receptor mutations in the PC12 cell line

Weight regulation through body-fat content and energy homeostasis, is regulated mainly through the actions of leptin. Herein, we analyse the effect of mutations in the mouse leptin receptor using the PC12 pheochromocytoma cell line as a model system. Both the induction of pancreatitis associated protein 1 and metallothionein-II, two leptin regulated genes in PC12, was evaluated. Tyr to Phe mutations in the cytoplasmic tail of the mouse leptin receptor confirmed the critical role of Tyr1138 (a YxxQ motif) and STAT-3 activation for induction of leptin-induced genes in PC12. In addition, the Tyr985Phe mutation showed enhanced responsiveness to leptin, which was even more pronounced in combination with Tyr1077Phe. The short isoform of the leptin receptor showed complete loss of stimulation of both genes. In contrast, a leptin receptor devoid of all Tyr residues in its cytoplasmic tail was still capable of a limited induction of the PAP 1 gene. A mutant mouse leptin receptor containing the fa/fa mutation showed constitutive signalling and impaired responsiveness to leptin. Treatment with the adenylate cyclase activator forskolin alone, in the absence of leptin was sufficient to obtain full induction of both genes.