- Chemical Biology and Medicinal Chemistry
- Discovering Enzyme Substrates and Functions
- Discovering Protein Ligands to Probe and Alter Function
- Discovering Enzyme Activators
- Analyzing Mechanisms of Drug Resistance via Chemical Biology
- Analyzing Enzyme Conformational Dynamics, Substrate Binding, and Catalysis
- Effective Drug Targeting of Pathogens via Medicinal Chemistry
- Computational Chemistry and Biology
- Physical Biology
- Protein and Cellular Engineering
- Monitoring enzyme activity and disease biomarkers
- Generating human proteome antibodies via phage display and directed evolution
- Globally analyzing and dissecting apoptosis
- Proximity tagging of protein-protein interactions
- Investigating cellular interactions in tissues
- Creating fluorescent probes targeting the genome and key bio-pathways
- De novo design of catalytic and membrane proteins
- Probing and modulating membrane proteins
- Chemical Biology and Medicinal Chemistry
Creating fluorescent probes targeting the genome and key bio-pathways
Example of our methods and research include
New probes for fluorescence microscopy of epignetics and intracellular signalling
Department scientists are improving upon fluorescent reporting of cellular processes, using protein, nucleic acid, and cellular engineering approaches to develop new probes. Specifically, they are adapting new techniques to probe specific DNA sequences for the visualization of their dynamics and are synthesizing smaller, more precise immuno-labeling for tracking key intracellular signal transmissions—in both cases, without disturbing the targeted processes. Thus they are converting key biological questions into imaging processes.
“Imagenomics”: genome dynamics via CRISPR/dCas9 visualization
While the genetic sequences in our cells’ nuclei are fixed, gene expression (the encoding of proteins that carry out the activities of life) is not. The genome’s functional output is regulated by its dynamic interactions with proteins and RNAs, including enzymes’ chemical modifications to histones—spool-like proteins that DNA is wound around.
Such interactions can alter the spatial arrangement and packaging of the chromatin (the complex of DNA and proteins), which, in turn, can regulate which genes are accessible for activation. Such conformational change by the genome, altering gene activity, underlies both the cell’s healthy ability to respond to endogenous signals and environmental stimuli and, when it goes awry, the inappropriate genetic expression underlying many diseases.
While some epigenetic modifications may be detected, identified, and located via methods such as mass spectrometry, scientists have not been able to directly and visually monitor these dynamic processes of chromatin remodeling in living cells and map them to overall DNA sequences and dynamics in real time. This would fundamentally advance our understanding of genetic regulation toward potential therapeutic interventions.
In collaboration with UCSF colleagues, department scientists are developing methods to view, monitor, and map genomic modifications and dynamics (epigenomics) in living cells and even living animals via fluorescence microscopy. This involves adapting a recently developed genome engineering technique, based on the CRISPR system, for visualization.
CRISPR stands for Clustered Regularly Interspaced Palindromic Repeats. It is a mechanism used by bacteria to immunize themselves against viruses (bacteriophages) by taking part of an invader’s DNA sequence and inserting it into its own for future recognition and resistance. When the guide RNA transcribed from these repeats combines with the Cas9 nuclease enzyme, the CRISPR system can cut DNA sequences complimentary to the guide RNA.
While this method has been used to edit and regulate specific genes, department research is applying it to visualization of specific genomic loci by deactivating the DNA-cutting (endonuclease) aspect of the Cas9, making dCas9, and fusing it with fluorescent proteins.
Department scientists and their collaborators have demonstrated broad applications for an imaging-optimized CRISPR system as a platform for viewing specific genomic elements and their dynamics in living mammalian cells. Their work and findings include:
- Establishing comparable target sequence flexibility and detection accuracy/efficiency to the fluorescence situ hybridization (FISH) technique, but with the advantage of being able to label features in living cells.
- Generation of cloned human cell lines expressing fused proteins combining nuclease-deactivated Cas9 (dCas9) and enhanced green fluorescent protein (EGFP).
- Visualization of human telomeres in different cell types: These stabilizing caps at the ends of eukaryotic chromosomes are composed to 5,000 to 15,000 base pairs with a repeated nucleotide sequence of TTAGGG (human), thus allowing the recruitment of numerous dCas9-EGFP proteins with a single sgRNA sequence.
- Imaging of protein-coding genes in different cell types: Researchers targeted the MUC4 and MUC1 genes, which encode mucin glycoproteins that combine with others to form protective mucus gels and have been associated with cancers and other diseases. They demonstrated they could detect gene copy numbers in living cells by targeting a tandem repetitive region as well as a specific five kilo-base non-repetitive region of MUC4.
- Using CRISPR imaging to monitor telomere length and dynamics in living cells. This work included detection of experimentally induced elongation. (Telomere shortening has been associated with cellular aging and with cancer-related genomic instability.) In addition, time-lapse microscopy tracked the movement of telomeres in living cells and confirmed that the labeling did not disrupt such dynamics.
- Using time-lapse CRISPR imaging of the labels on specific genomic loci to track chromosome dynamics and reorganization during cell division.
Discovering new locations and timing for signalling by G-protein-coupled receptors
G-protein-coupled receptors (GPCRs) are biologically and pharmacologically vital subjects of study. This family of transmembrane proteins bind with extracellular signaling molecules such as hormones or neurotransmitters, then transmit information into cells (signal transduction), via changes in their shape (conformation) that release the G-protein from a complex attached to a part of the GPCR inside the cell. The G-protein (specifically its Gs alpha subunit) then activates specific enzymes that, in turn, trigger biochemical sequences (pathways) that can greatly alter cell activities such as metabolism, division, or gene expression.
Such prime biological roles make GPCRs relevant to the treatment of many disorders, including cancers, diabetes, heart disease, asthma, and allergies. Indeed, one published analysis notes that 30 percent of all marketed drugs act on them; another that they are targeted by 40 percent of all prescription drugs.
GPCRs have traditionally been seen as generating their G-protein-coupled signaling strictly from their locations in cells’ outermost walls (plasma membranes) —with the signaling thought to end after intracellular proteins called arrestins bind to the GPCRs, inhibit them, and target them for internalization. In the latter process, known as endocytosis, the receptors are drawn into pockets formed in the cell membrane (clathrin-coated pits) that are pinched off into containers (vesicles) called endosomes for transport, breakdown, and/or recycling to the cell membrane.
However, in recent years a number of studies proposed that the GPCRs for certain ligands (e.g., thyroid-stimulating hormone, dopamine) continue their signaling activity from within endosomes. This could suggest additional or different effects and locations at which to target GPCRs with drugs.
That hypothesis had not been directly tested until department scientists in collaboration with UCSF, national, and international colleagues applied recently developed fluorescence microscopy probes to obtain photographic and video evidence that GPCRs are, indeed, signaling from endosomes after their internalization.
Specifically, the researchers examined beta-adrenergic receptors (β2-AR)—prototypical GPCRs that bind extracellular ligands such as adrenaline (epinephrine). The β2-AR-associated G-proteins released from complex inside cells activate specific enzymes to stimulate the cAMP-dependent pathway, which ultimately dilates blood vessels and respiratory bronchi, increases heart rate, increases the breakdown of energy stored as fat or glycogen—which can all be part of the body’s stress response.
Scientists here used nanobodies—the binding domains of antibodies from camels (which are single rather than double chain, thus especially small and flexible), genetically expressed by bacteria. The nanobodies were engineered to selectively bind to either the active conformations of the beta-adrenergic receptors or to an intermediate of the activated β2-AR-associated G-proteins. The nanobodies were, in turn, fused to enhanced green fluorescent protein.
Researchers used the adrenergic receptor activator (agonist) isoprenaline in combination with the fluorescent activity reporters to probe and track the activation of the β2 adrenergic receptors and their associated G-proteins in living mammalian cells.
The scientists found the receptors were, indeed, not only activated in the cells’ plasma membranes after the initial application of the agonist but also, in a second phase several minutes later—after arrestin inhibition and internalization. The GPCR is activated again in vesicles known as early endosomes, as detected via β2-AR-associated G-proteins active on those endosomes’ surfaces. Thus, those internalized receptors’ activity contribute to a temporally distinct and significant further activation of the cells’ cAMP pathway.
The findings not only provided direct support for GPCR signaling from endosomes, thereby revising a long-held tenet of molecular pharmacology, but also demonstrated a strategy for probing the location and timing of dynamic protein conformational changes in living cells.