Reprogramming G Protein-Coupled Receptor Structure, Function And Signaling By Computational Design

G protein-coupled receptors (GPCRs) are 7-transmembrane alpha-helical integral membrane proteins on which cells heavily rely to receive information regarding their external environment. These receptors are able to transfer information to intracellular downstream effectors upon stimulation from their cognate ligands, which can be considerably diverse. They can include light, small molecules, peptides, protons; Due to its evolutionary success, over millions of years of evolution, this protein family has expanded to include over 800 members. Despite the large size of the family and the diverse inputs they accept, the structure and the mechanism for relaying signals to their downstream partners, G proteins and B-arrestins, remains largely the same. As these receptors are involved in a great deal of biological processes such as sight (rhodopsin), cognition (dopamine, serotonin, opioid receptors), immunity (chemokine receptors), heart function (adrenergic, angiotensin receptors), etc.; a great deal of effort has gone into understanding their structure and function.

Our efforts have gone into understanding their signaling properties and rationally modifying them. Most proteins are fairly flexible entities and their structures tend to oscillate and move around, sampling a number of conformations. Nature has taken advantage of these natural motions to regulate protein function from distant binding sites, in a phenomenon called allostery. GPCRs are unique in the sense that their primary function relies on allostery. By tweaking the allosteric signaling of GPCRs, we hope to have a better understand allostery, use the principles we learn to create designer GPCR biosensors with the desired sensitivity, and to eventually create de novo allosteric proteins.

In this work, I present our progress in this effort, which has been significant. We have created a highly accurate in silico method to allosterically alter relative stabilities of the active and inactive states of the GPCR dopamine D2 receptor, but also to independently control its allosteric signaling strength. With our methodology we have been able to create receptors which have high basal activities by selectively stabilizing the active state of the receptor. This has already facilitated the structure determination of the active, G protein-bound dopamine D2 receptor; a first for this GPCR. Additionally, we have created a number of dopamine D2 variants with roughly 100 times higher sensitivity to dopamine than the WT receptor. These efforts continue to characterize the functional effects of these allosteric mutations.

Furthermore, we are also working on a collaboration to rewire the signaling pathway of the adenosine A2A receptor. Adenosine is a common immunosuppresant in solid tumor micro-environments (TMEs). By rewiring the downstream signaling of the A2A receptor, we hope to reverse the response of T-cells in these TMEs to activate and attack the solid tumor.

Quantitative single cell dynamics of signaling and transcriptional response in mammalian cells

Onur Tidin

Cells live in ever-changing environments, thereby facing a variety of dynamic environmental signals. Environmental stimuli elicit intracellular responses through signaling pathways, which converge on transcriptional activation or repression of target genes. Despite intensive research, dissecting the complex interactions between pathway components that modulate mRNA and protein production still remains a difficult task. To this end, single-cell approaches provide unique insights into intracellular processes and cell responses to environmental stimuli, otherwise inaccessible with traditional bulk studies. Single-cell measurements have revealed that isogenic cells sharing the same environment display substantial heterogeneity in both transcript and protein level. As the initial step in gene expression, transcription is an important source of such variability in eukaryotic cells due to low number of molecules involved, transcription factor dynamics and the discrete nature of biochemical reactions. Notably, production of mRNA during transcription occurs in short periods of activity, termed as transcriptional bursts, followed by longer periods of inactivity. Despite widespread observations of transcriptional dynamics in mammals, upstream molecular mechanisms shaping the eukaryotic transcription remained elusive. In this study, we quantitatively linked upstream factors and transcriptional kinetics by developing an experimental system to temporally monitor, simultaneously, inputs (transcription factors) and outputs (target gene expression) in mammalian cells, in response to stimulus. We established two Tet-On inducible stable cell lines each expressing a fusion protein of a luminescence (Nanoluciferase) reporter to either SMAD4 or SMAD2, the two main transcrip- tion factors downstream of the TGF-Î² pathway. These cell lines that also contain a short-lived firefly luciferase reporter for the expression of the target endogenous connective tissue growth factor (ctgf ) gene allowed us to quantitatively link nuclear accumulation of SMADs upon TGF-Î² stimulation to the target gene expression in real-time single cell measurements. Time- lapse luminescence microscopy and image analysis with the custom developed CAST (Cell Automated Segmentation and Tracking platform) platform provided quantitative single-cell data to link the upstream transcription factor profile to its target gene activity. The data revealed weak single-cell correlations between translocation level and the target gene expression response which suggests a mechanism in which the translocation of SMADs initiates the transcriptional response but affects the response amplitude minimally. However, SMAD4 expression levels influenced the target gene response dynamics such that cells with high SMAD4 abundance favored to respond in a more sustained and even oscillatory manner. This suggests a mechanism consisting of a dynamic interplay between the transcriptional activators and feedback mechanisms in TGF-Î² signaling. We explored further the effect of different factors such as ligand concentration, ligand type on both signaling and target gene response. Taken together, this study proposes an experimental and quantitative framework that dissect mechanisms underlying transcriptional response to stimulus.

EPFL2018

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.