Reprogramming the Genetic Code
The information for synthesizing the molecules that allow organisms to survive and replicate is encoded in genomic DNA. In the cell, DNA is copied to messenger RNA, and triplet codons (64) in the messenger RNA are decoded - in the process of translation - to synthesize polymers of the natural 20 amino acids. This process (DNA RNA protein) describes the central dogma of molecular biology and is conserved in terrestrial life. We are interested in rewriting the central dogma to create organisms that synthesize proteins containing unnatural amino acids and polymers composed of monomer building blocks beyond the 20 natural amino acids. I will discuss our invention and synthetic evolution of new 'orthogonal' translational components (including ribosomes and aminoacyl-tRNA synthetases) to address the major challenges in re-writing the central dogma of biology. I will discuss the application of the approaches we have developed for incorporating unnatural amino acids into proteins and investigating and synthetically controlling diverse biological processes, with a particular emphasis on understanding the role of post-translational modifications.
Structural mapping and labeling of GPCRs via genetically encoded chemical tools
We apply non-canonical amino acids (ncAAs) to address general questions about functioning of G protein-coupled receptors (GPCRs) directly from the natural environment of the live mammalian cell. On one hand, we use photo-and chemical crosslinking amino acids to define the topology of GPCR interactions both with ligands (especially peptide ligands) and intracellular partners. On the other hand, we have engineered enhanced tRNAs that have enabled efficient incorporation of last generation ncAAs for bioorthogonal chemistry into challenging protein targets. In this way, we could achieve quantitative single-residue labeling of sensitive GPCR regions, such as the loops, with small organic fluorescent probes and put the basis for the development of small-size fluorescent sensors for in-cell studies of GPCR dynamics.
The roles of lipids in cell biology
Although lipids are essential contributors to numerous cellular functions, they are understudied relative to other biological molecules like proteins. While lipids have been connected to many diseases, their therapeutic potential has not yet been realised, in part due to our poor understanding of their metabolism and functions. Lipids are fundamentally small molecules and as such chemical biology approaches are essential to investigate their roles. A primary interest of my laboratory is to understand the cell biology of lipids. We are investigating the roles of lipids in a range of biological processes, including cell division, cell-cell interactions and organelle structure, using lipidomics, imaging, cell biology and chemical biology.
Spying on cells with glowing chemical-genetic hybrids
Cells and organisms are complex machines driven by a set of dynamic biological events tightly orchestrated in space and time. Our understanding of their inner workings is intricately related to our ability to observe how their constituents organize and interact. During this talk, I will present the development of chemical-genetic tools for the observation of biomolecules and dynamic biochemical events in live cells and tissues. These hybrid systems are composed of a protein module and a synthetic small molecule. The advantage of using a protein module is that instructions for its manufacture can be easily and specifically introduced into cells in the form of DNA. In addition, its properties can be adjusted using protein evolution techniques. The interest of using a small synthetic molecule, on the other hand, is to be able to use molecular engineering to refine its properties, and thus benefit from the power of modern chemistry to explore biological processes. I will detail how such hybrid approach allowed us to design innovative fluorescent reporters and biosensors for various applications in fluorescence imaging and cell biology.
Bacterial superglues, and nano-assemblies to tickle the immune syste
A special feature of the bacterium Streptococcus pyogenes enables spontaneous isopeptide bond formation within its surface proteins. We re-engineered this system to generate an irreversible peptide-protein interaction (SpyTag/SpyCatcher). This reaction is rapid, genetically-encodable and specific in diverse biological environments. Latest advances include accelerated reactivity and a toolbox of modules for rapidly controlling protein architectures. Cyclizing enzymes using SpyTag conferred resilience to boiling, for applications in biotransformation and nutrition. SpyTag and its related superglue SnoopTag allows programmable synthesis of multi-functional teams or biomaterials, to modulate precisely cancer cell signaling. Vaccines are one of the most successful medical interventions and an important frontier for chemical biology. Virus-like particles (VLPs) are nano-assemblies with many attractive features for vaccination. However, decorating VLPs with target antigens by genetic fusion or standard chemical coupling is often unsuccessful. We demonstrated 100% reaction to SpyCatcher-VLPs after mixing with SpyTag linked to a range of malaria antigens and cancer targets. Spy-VLPs efficiently induced antibody responses after only a single immunization and without adjuvant. Modular and programmable assembly using SpyTag shows promise in accelerating vaccine development against a range of existing and emerging diseases.
Opto-chemical control of immune cell activation
Cellular signalling networks are an intricate system of biological components and great progress has been made in identifying the constituents of these networks. Nonetheless, for a comprehensive description of any system, there must also be an understanding of how information actually flows through the network but our knowledge of these dynamic interconnections for most signalling networks remains very limited. This is especially true for T cells, an essential white blood cell type of our immune response, which we focus on. To directly interrogate T-cell signalling dynamics, we engineer synthetic receptors that can be opto-chemically modulated, providing unprecedented spatio-temporal control over both the intensity and frequency of T-cell activation. Using this approach, we have investigated the biochemical ‘memory’ of T-cell activation that persists after cessation of signalling and whether the signalling network can encode both the frequency and amplitude of receptor activation. Our eventual goal is to uncover points in the network that could be fine-tuned therapeutically to alleviate immune diseases caused by aberrant cell signalling.
Specific modulators of protein phosphatase-1 and their application in heart failure
Protein serine/threonine-specific phosphatases have in the past been considered to be housekeeping enzymes, undruggable, and challenging to study due to their multiple roles and the conservation of the catalytic subunits. However, this view is currently changing. Of these, protein phosphatase-1 (PP1) is an important ubiquitous phosphatase that is estimated to remove phosphate groups from about a third of all phosphorylated serines and threonines in eukaryotic cells, counteracting more than a hundred kinases. PP1 catalytic subunit (PP1c) has broad substrate specificity but is restrained in vivo by numerous PP1c-interacting proteins that impart high substrate specificity to it and function for example as activity-modulating or localization-determining factors. These so-called PP1-holoenzymes play roles in many different diseases such as cancer, diabetes, and cardiovascular diseases. The lack of selective modulators of PP1 has in the past been a limiting factor in its research. We have addressed this challenge by designing peptides that target PP1c and disrupts its protein–protein interactions with regulatory proteins, leading to the release of free, active PP1c inside cells. I will report on the probe development and describe their application to study PP1 activity in the pathomechanism of heart failure.
Quantitative chemical imaging in live cells
The chemical milieu within an organelle has been evolutionarily optimized to enable the biochemistry that occurs within. My lab studies how organelle function impacts cell function by mapping chemicals within the organelle lumens using a new, quantitative chemical imaging technology based on DNA. DNA self-assembles into molecularly precise, synthetic assemblies, commonly referred to as DNA nanodevices. My lab creates DNA nanodevices that are chemically responsive, fluorescent probes that can be targeted to specific organelles. I will discuss how we get these probes to interface with cells in programmable and targeted ways to localize in specific organelles. I will show how we use these reporters to quantitatively image chemical messengers in organelles of cells in culture, in live multicellular organisms as well as in cells obtained from blood draws or skin biopsies from human patients. I will focus on two recent findings. One, where we solved a thirty-year problem in molecular sensing by mapping lumenal calcium in acidic organelles and in doing so, identified the first example of a lysosomal Ca2+ importer in the animal kingdom. In the second, I will describe a DNA-based voltmeter using which, we have measured the membrane potentials of several organelles in situ in live cells, many of which were unknown till now.
Tackling Sweet Questions with Small Probes
Glycans constitute the outermost layer of every living cell. The “glyco-code” that fine-tunes molecular interactions is manufactured by >250 glycosyltransferases (GTs), and is thus not a direct gene product. Alterations of the glyco-code are strongly associated with human disease, particularly tumorigenesis. As GTs functionally interact with each other, traditional methods of molecular and cell biology are unlikely to reveal the full picture of cellular glycosylation. Synthetic tools are uniquely suited to complement these approaches. Chemically modified monosaccharides can serve as GT substrates to be incorporated into glycoproteins and characterised by virtue of bioorthogonal chemistry. However, these substrates are often used by multiple GTs or interconverted and thus non-specific by default. We are combining synthetic chemistry with protein engineering, structural biology and modern methods of cell biology, proteomics and genome engineering to generate “precision tools” that allow us to probe the glyco-code in unprecedented detail.
Chemical probing of natural product assemblies: insights and opportunities
Natural products constitute an abundant and highly diverse source of compounds of biomedical interest, which comprise antibiotic, anticancer, antiviral and anti-inflammatory agents amongst others. A detailed chemical elucidation of biosynthetic pathways leading to natural products is of the utmost importance as it paves the way towards novel bioactive product generation and diversification via organic synthesis as well as synthetic biology. In our lab we have been developing a range of chemical probes for the ‘capture’ and the elucidation of biosynthetic intermediates leading to natural product assembly in vitro and in live microorganisms. The probes mimic the building blocks utilised by natural product enzymatic assembly lines (e.g. polyketide synthases and nonribosomal peptide synthetases) and intercept biosynthetic intermediates throughout whole natural product formation, thereby unveiling unprecedented mechanistic details and novel opportunities for chemoenzymatic product diversification. I will present and discuss our latest developments in the design and use of chemical probes for biosynthetic investigations and their implications for novel compound development.
Illuminating the Biochemical Activity Architecture of the Cell
The complexity and specificity of many forms of signal transduction are widely suspected to require spatial microcompartmentation and dynamic modulation of the activities of signaling molecules, such as protein kinases, phosphatases and second messengers. We have developed a series of fluorescent biosensors to probe the compartmentalized signaling activities in living cells. In this talk, I will present several new fluorescent biosensors that we recently developed; I will then focus on cAMP/PKA and PI3K/Akt/mTORC1 signaling pathways and present studies where we combined genetically encoded fluorescent biosensors, superresolution imaging, targeted biochemical perturbations and mathematical modeling to probe the biochemical activity architecture of the cell.