Selective Targeting of SH2 Domain–Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies

The binding of Src-homology 2 (SH2) domains to phosphotyrosine (pY) sites is critical for the autoinhibition and substrate recognition of the eight Src family kinases (SFKs). The high sequence conservation of the 120 human SH2 domains poses a significant challenge to selectively perturb the interactions of even the SFK SH2 family against the rest of the SH2 domains. We have developed synthetic binding proteins, termed monobodies, for six of the SFK SH2 domains with nanomolar affinity. Most of these monobodies competed with pY ligand binding and showed strong selectivity for either the SrcA (Yes, Src, Fyn, Fgr) or SrcB subgroup (Lck, Lyn, Blk, Hck). Interactome analysis of intracellularly expressed monobodies revealed that they bind SFKs but no other SH2-containing proteins. Three crystal structures of monobody-SH2 complexes unveiled different and only partly overlapping binding modes, which rationalized the observed selectivity and enabled structure-based mutagenesis to modulate inhibition mode and selectivity. In line with the critical roles of SFK SH2 domains in kinase autoinhibition and T-cell receptor signaling, monobodies binding the Src and Hck SH2 domains selectively activated respective recombinant kinases, whereas an Lck SH2-binding monobody inhibited proximal signaling events downstream of the T-cell receptor complex. Our results show that SFK SH2 domains can be targeted with unprecedented potency and selectivity using monobodies. They are excellent tools for dissecting SFK functions in normal development and signaling and to interfere with aberrant SFK signaling networks in cancer cells. (C) 2017 The Authors. Published by Elsevier Ltd.

Photopatterning of Synthetic Hydrogels

Katarzyna Mosiewicz

In recent years sophisticated biomaterials have made a significant impact on our understanding of cellular behavior, particularly in the fields of stem cell research and tissue engineering. Nevertheless, the promising potential of stem cells for regenerative medicine has remained largely unmet. One reason for such limitations could be that there is still incomplete understanding of stem cell behavior due to a lack of advanced in vitro tools suitable to achieve adequate control over cellular processes. By mimicking the dynamic and complex biophysical and biochemical interactions between cells and their natural extracellular matrixes (ECMs), it is possible that we may bridge this gap between current limited in vitro models and complex in vivo organisms. In this thesis, an innovative class of biomaterials is presented that enables the flexible in vitro recapitulation of the dynamic biochemical and biophysical signaling involved in controlling cell fate. Such systems of increasing fidelity to real physiological processes could bring new insights into fundamental cell biology as well as bring us to closer towards cell-based therapeutics applications. In order to achieve a dynamic artificial ECM (aECM), we designed chemical schemes which would allow us to control the biochemical and biophysical properties of the hydrogel in space, time and intensity. The chosen concept is built on a synthetic hydrogel providing static physicochemical support, while spatiotemporal control over a single or multiple signals is achieved by spatiotemporal photoactivation reactions within this hydrogel. Indeed, by modulating the duration, location and intensity of illumination, desired heterogeneities in biochemical and biophysical signal can be introduced in the otherwise homogeneous and static hydrogel network. To implement the designed hydrogel photo-patterning tool, new chemical components were synthesized. Synthetic hydrogels based on poly (ethylene glycol) (PEG) were utilized due to their inert properties, cell biocompatibility and lack of unspecific protein binding. Biochemical signal tethering was rendered spatiotemporally controllable by adding photosensitivity to the existing enzyme-based crosslinking scheme. This was achieved by functionalizing one of enzymatic substrate with a photo-labile “caging” moiety, thereby preventing crosslinking to occur until light illumination. This caged substrate was incorporated into the hydrogel network and served as a light-controllable and specific ligand-binding site. Importantly, it was possible to photo-pattern proteins, which are much complex than peptide-based biomolecules, and their bioactivity was fully preserved with this site-specific immobilization scheme. It was also possible to pattern biophysical cues by making the Michael-type (MT) addition reaction for crosslinking PEG-based hydrogel photosensitive. In this case, the thiol moieties of one of the reactive PEG macromers undergoing crosslinking, were equipped with a caging group which prevented its susceptibility to the counter-reactive functionality, the vinyl sulfone group. Thus, the crosslinking density of the hydrogel backbone could be controlled by light, which was directly translated into differential patterns of hydrogel stiffness. Using this approach, user-defined biochemical and biophysical patterns and even gradients could be obtained in the spatiotemporal manner within the same hydrogel. The crosslinking reaction is the critical parameter in the success of hydrogel photo-patterning, and must rely on a well-controlled and highly selective chemical scheme. Therefore, we also developed a novel enzyme-based cross-linking scheme for synthetic PEG hydrogels. The reaction originates from the naturally occurring post-translational modification of proteins by phosphopantetheine (Ppant) group performed by a family of enzymes known as Ppant transferases (PPTases). By synthesizing biochemical components of this reaction, we implemented a PPTase-catalyzed reaction to form and bio-functionalize PEG hydrogel. The utility of the developed hydrogel platforms was determined in the context of 2D and 3D cell culture. Photo-directed patterns of a cell-adhesive fibronectin fragment triggered adhesion and spreading of muscle progenitor cells, a phenomenon which occurred only within the areas of patterned signaling protein. A photo-patterned elasticity gradient was used to direct mesenchymal stem cell spreading and migration. Finally, the PPTase-based hydrogel was proven to be suitable for 3D culture, as shown by encapsulation of primary human fibroblasts. In this thesis, we demonstrated that a combination of chemical schemes could be assembled to achieve a functional materials toolbox which would begin to reconstruct the complexity of the dynamic in vivo ECM. Such a tool is expected to contribute to the field of cell biology by allowing us to study questions which previously could not be addressed in vitro, and could ultimately contribute to cell-based regenerative medicine.

EPFL2012

The Principles of Ligand Specificity on beta-2-adrenergic receptor

Shuguang Yuan

G protein-coupled receptors are recognized as one of the largest families of membrane proteins. Despite sharing a characteristic seven-transmembrane topology, G protein-coupled receptors regulate a wide range of cellular signaling pathways in response to various physical and chemical stimuli, and prevail as an important target for drug discovery. Notably, the recent progress in crystallographic methods led to a breakthrough in elucidating the structures of membrane proteins. The structures of beta(2)-adrenergic receptor bound with a variety of ligands provide atomic details of the binding modes of agonists, antagonists and inverse agonists. In this study, we selected four representative molecules from each functional class of ligands and investigated their impacts on beta(2)-adrenergic receptor through a total of 12 x 100 ns molecular dynamics simulations. From the obtained trajectories, we generated molecular fingerprints exemplifying propensities of protein-ligand interactions. For each functional class of compounds, we characterized and compared the fluctuation of the protein backbone, the volumes in the intracellular pockets, the water densities in the receptors, the domain interaction networks as well as the movements of transmembrane helices. We discovered that each class of ligands exhibits a distinct mode of interactions with mainly TM5 and TM6, altering the shape and eventually the state of the receptor. Our findings provide insightful prospective into GPCR targeted structure-based drug discoveries.

Nature Publishing Group2016

Analysis of expression of PDCP and MAL13P1.308 of Plasmodium falciparum employing a quantitative proteomics approach based on SILAC

Céline Freymond

Plasmodium falciparum is a deadly parasite that causes malaria in humans. This disease causes the death of one million people every year. To find new means to fight this parasite, it is important to learn more about its biology. As the pathways of protein expression are better understood, it becomes easier to find out how to block these mechanisms. The completion of the genome sequencing has opened new perspective of genome wide analysis. One of these studies was done by LaCount et al. who used the yeast1two1hybrid system to map the complete interaction network between proteins of P. falciparum. ¨ From this network of interaction an interesting protein was studied further by Daubenberger et al. which is PDCP. This protein is closely related to MSP11 in this protein network, a protein involved in erythrocyte invasion. PDCP is a CCCH1type zinc finger protein, a family of proteins that are involved in protein1protein interaction, nucleic acid binding and binding in small ligands. It was shown that its expression was dependant on the density of the parasites. In this study we used the SILAC technology to confirm these previous results. A culture is grown with isotopically heavy isoleucine in the medium as a control sample. After a few cell cycles, almost all the natural isoleucines are replaced with the labeled ones. Three cultures with parasitemia of 2, 5 and 10% are grown and mixed with an equal amount of the control culture. When the proteins are analyzed by mass spectrometry, the peptides that contained isoleucine will be detected as two separate peaks. After normalization of each peptide area with this internal control, the quantity of PDCP can be compared between cultures of different parasitemias. 11 peptides of PDCP have been detected by mass spectrometry, proving its existence for the first time. These peptides were labeled to more than 95%, allowing the comparison of PDCP expression between the cultures. But because of difficulty of reproducibility in in vitro cultures, the regulation of PDCP by the parasitemia has not been observed by SILAC. We wanted to study further the protein interaction network in which PDCP is involved. MAL13P1.308, a protein directly interacting with PDCP and MSP11 in the protein network map, was analyzed by IFA. It was found to be expressed during all stages of the asexual blood stage cycle and it was not imported in the host cell. The next step will be to see if it interacts with PDCP by co1localization studies