Cell Wall Trapping of Autocrine Peptides for Human G-Protein-Coupled Receptors on the Yeast Cell Surface

G-protein-coupled receptors (GPCRs) regulate a wide variety of physiological processes and are important pharmaceutical targets for drug discovery. Here, we describe a unique concept based on yeast cell-surface display technology to selectively track eligible peptides with agonistic activity for human GPCRs (Cell Wall Trapping of Autocrine Peptides (CWTrAP) strategy). In our strategy, individual recombinant yeast cells are able to report autocrine-positive activity for human GPCRs by expressing a candidate peptide fused to an anchoring motif. Following expression and activation, yeast cells trap autocrine peptides onto their cell walls. Because captured peptides are incapable of diffusion, they have no impact on surrounding yeast cells that express the target human GPCR and non-signaling peptides. Therefore, individual yeast cells can assemble the autonomous signaling complex and allow single-cell screening of a yeast population. Our strategy may be applied to identify eligible peptides with agonistic activity for target human GPCRs

A theoretical entropy score as a single value to express inhibitor selectivity

<p>Abstract</p> <p>Background</p> <p>Designing maximally selective ligands that act on individual targets is the dominant paradigm in drug discovery. Poor selectivity can underlie toxicity and side effects in the clinic, and for this reason compound selectivity is increasingly monitored from very early on in the drug discovery process. To make sense of large amounts of profiling data, and to determine when a compound is sufficiently selective, there is a need for a proper quantitative measure of selectivity.</p> <p>Results</p> <p>Here we propose a new theoretical entropy score that can be calculated from a set of IC₅₀data. In contrast to previous measures such as the 'selectivity score', Gini score, or partition index, the entropy score is non-arbitary, fully exploits IC₅₀data, and is not dependent on a reference enzyme. In addition, the entropy score gives the most robust values with data from different sources, because it is less sensitive to errors. We apply the new score to kinase and nuclear receptor profiling data, and to high-throughput screening data. In addition, through analyzing profiles of clinical compounds, we show quantitatively that a more selective kinase inhibitor is not necessarily more drug-like.</p> <p>Conclusions</p> <p>For quantifying selectivity from panel profiling, a theoretical entropy score is the best method. It is valuable for studying the molecular mechanisms of selectivity, and to steer compound progression in drug discovery programs.</p

Computational analysis of the evolutionarily conserved Missing In Metastasis/Metastasis Suppressor 1 gene predicts novel interactions, regulatory regions and transcriptional control

Missing in Metastasis (MIM), or Metastasis Suppressor 1 (MTSS1), is a highly conserved protein, which links the plasma membrane to the actin cytoskeleton. MIM has been implicated in various cancers, however, its modes of action remain largely enigmatic. Here, we performed an extensive in silico characterisation of MIM to gain better understanding of its function. We detected previously unappreciated functional motifs including adaptor protein (AP) complex interaction site and a C-helix, pointing to a role in endocytosis and regulation of actin dynamics, respectively. We also identified new functional regions, characterised with phosphorylation sites or distinct hydrophilic properties. Strong negative selection during evolution, yielding high conservation of MIM, has been combined with positive selection at key sites. Interestingly, our analysis of intra-molecular co-evolution revealed potential regulatory hotspots that coincided with reduced potentially\ua0pathogenic polymorphisms. We explored databases for the mutations and expression levels of MIM in cancer. Experimentally, we focused on chronic lymphocytic leukaemia (CLL), where MIM showed high overall expression, however, downregulation on poor prognosis samples. Finally, we propose strong conservation of MTSS1 also on the transcriptional level and predict novel transcriptional regulators. Our data highlight important targets for future studies on the role of MIM in different tissues and cancers

Recognition of sorting signals by clathrin adaptors

Sorting of membrane proteins is generally mediated by cytosolic coats, which create a scaffold to form coated buds and vesicles and to selectively concentrate cargo by interacting with cytosolic signals. The classical paradigm is the interaction between clathrin coats and associated adaptor proteins, which cluster receptors with characteristic tyrosine and dileucine motifs during endocytosis. Clathrin in association with different sets of adaptors is found in addition at the trans-Golgi network and endosomes. Sequences similar to internalization signals also direct lysosomal and basolateral sorting, which implicates related clathrinadaptor coats in the respective sorting pathways. This review concentrates on the recognition of sorting signals by clathrin-associated adaptor proteins, an area of significant recent progress due to new methodological and conceptual approaches

In vitro binding of clathrin adaptors to sorting signals correlates with endocytosis and basolateral sorting.

To analyze the interaction of sorting signals with clathrin-associated adaptor complexes, we developed an in vitro assay based on surface plasmon resonance analysis. This method monitors the binding of purified adaptors to immobilized oligopeptides in real time and determines binding kinetics and affinities. A peptide corresponding to the cytoplasmic domain of wild-type influenza hemagglutinin, an apical membrane protein that is not endocytosed, did not significantly bind adaptor complexes. However, peptide sequences containing a tyrosine residue that has previously been shown to induce endocytosis and basolateral sorting were specifically recognized by adaptor complexes. The in vitro rates of adaptor association with these peptides correlated with the internalization rates of the corresponding hemagglutinin variants in vivo. Binding was observed both for purified AP-2 adaptors of the plasma membrane and for AP-1 adaptors of the Golgi, with similar apparent equilibrium dissociation constants in the range 10(-7)-10(-6) M. Adaptor binding was also demonstrated for a sequence containing a C-terminal di-leucine sequence, the second major motif of endocytosis/basolateral sorting signals. These results confirm the concept that interaction of cytoplasmic signals with plasma membrane adaptors determines the endocytosis rate of membrane proteins, and suggest the model that clathrin-coated vesicles of the trans-Golgi network are involved in basolateral sorting