Modeling (not so) rare developmental disorders associated with mutations in the protein-tyrosine phosphatase SHP2.

Src homology region 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2) is a highly conserved protein tyrosine phosphatase (PTP), which is encoded by and is indispensable during embryonic development. Mutations in in human patients cause aberrant signaling of SHP2, resulting in multiple rare hereditary diseases, including Noonan Syndrome (NS), Noonan Syndrome with Multiple Lentigines (NSML), Juvenile Myelomonocytic Leukemia (JMML) and Metachondromatosis (MC). Somatic mutations in have been found to cause cancer.

Targeting Oncogenic Src Homology 2 Domain-Containing Phosphatase 2 (SHP2) by Inhibiting Its Protein-Protein Interactions.

We developed a new class of inhibitors of protein-protein interactions of the SHP2 phosphatase, which is pivotal in cell signaling and represents a central target in the therapy of cancer and rare diseases. Currently available SHP2 inhibitors target the catalytic site or an allosteric pocket but lack specificity or are ineffective for disease-associated SHP2 mutants. Considering that pathogenic lesions cause signaling hyperactivation due to increased levels of SHP2 association with cognate proteins, we developed peptide-based molecules with nanomolar affinity for the N-terminal Src homology domain of SHP2, good selectivity, stability to degradation, and an affinity for pathogenic variants of SHP2 that is 2-20 times higher than for the wild-type protein.

The sixth international RASopathies symposium: Precision medicine-From promise to practice.

The RASopathies are a group of genetic disorders that result from germline pathogenic variants affecting RAS-mitogen activated protein kinase (MAPK) pathway genes. RASopathies share RAS/MAPK pathway dysregulation and share phenotypic manifestations affecting numerous organ systems, causing lifelong and at times life-limiting medical complications. RASopathies may benefit from precision medicine approaches.

Targeting prohibitins at the cell surface prevents Th17-mediated autoimmunity.

T helper (Th)17 cells represent a unique subset of CD4 T cells and are vital for clearance of extracellular pathogens including bacteria and fungi. However, Th17 cells are also involved in orchestrating autoimmunity. By employing quantitative surface proteomics, we found that the evolutionarily conserved prohibitins (PHB1/2) are highly expressed on the surface of both murine and human Th17 cells.

Galectin-1 dimers can scaffold Raf-effectors to increase H-ras nanoclustering.

Galectin-1 (Gal-1) dimers crosslink carbohydrates on cell surface receptors. Carbohydrate-derived inhibitors have been developed for cancer treatment. Intracellularly, Gal-1 was suggested to interact with the farnesylated C-terminus of Ras thus specifically stabilizing GTP-H-ras nanoscale signalling hubs in the membrane, termed nanoclusters.

Specific cancer-associated mutations in the switch III region of Ras increase tumorigenicity by nanocluster augmentation.

Hotspot mutations of Ras drive cell transformation and tumorigenesis. Less frequent mutations in Ras are poorly characterized for their oncogenic potential. Yet insight into their mechanism of action may point to novel opportunities to target Ras.

The efficacy of Raf kinase recruitment to the GTPase H-ras depends on H-ras membrane conformer-specific nanoclustering.

Solution structures and biochemical data have provided a wealth of mechanistic insight into Ras GTPases. However, information on how much the membrane organization of these lipid-modified proteins impacts on their signaling is still scarce. Ras proteins are organized into membrane nanoclusters, which are necessary for Ras-MAPK signaling.

Nanoclustering and heterogeneous membrane diffusion of Ras studied by FRAP and RICS analysis.

Fluorescence Recovery After Photobleaching (FRAP) and Raster Image Correlation Spectroscopy (RICS) are two powerful techniques to study the diffusion dynamics of fluorescently labeled proteins. FRAP and RICS can be easily applied on any commercial confocal microscope. In this chapter, we describe the principles of these methods and provide the reader with a detailed guide on how to apply these methods in the study of Ras nanoclustering and diffusion in the plasma membrane of live cells.

Cellular FRET-Biosensors to Detect Membrane Targeting Inhibitors of N-Myristoylated Proteins.

Hundreds of eukaryotic signaling proteins require myristoylation to functionally associate with intracellular membranes. N-myristoyl transferases (NMT) responsible for this modification are established drug targets in cancer and infectious diseases. Here we describe NANOMS (NANOclustering and Myristoylation Sensors), biosensors that exploit the FRET resulting from plasma membrane nanoclustering of myristoylated membrane targeting sequences of Gαi2, Yes- or Src-kinases fused to fluorescent proteins.