Lingyin Li, Doctoral Dissertation Advisor (AC)
LRRC8A:C/E Heteromeric Channels Are Ubiquitous Transporters of cGAMP.
Extracellular 2'3'-cyclic-GMP-AMP (cGAMP) is an immunotransmitter exported by diseased cells and imported into host cells to activate the innate immune STING pathway. We previously identified SLC19A1 as a cGAMP importer, but its use across human cell lines is limited. Here, we identify LRRC8A heteromeric channels, better known as volume-regulated anion channels (VRAC), as widely expressed cGAMP transporters. LRRC8A forms complexes with LRRC8C and/or LRRC8E, depending on their expression levels, to transport cGAMP and other 2'3'-cyclic dinucleotides. In contrast, LRRC8D inhibits cGAMP transport. We demonstrate that cGAMP is effluxed or influxed via LRRC8 channels, as dictated by the cGAMP electrochemical gradient. Activation of LRRC8A channels, which can occur under diverse stresses, strongly potentiates cGAMP transport. We identify activator sphingosine 1-phosphate and inhibitor DCPIB as chemical tools to manipulate channel-mediated cGAMP transport. Finally, LRRC8A channels are key cGAMP transporters in resting primary human vasculature cells and universal human cGAMP transporters when activated.
View details for DOI 10.1016/j.molcel.2020.10.021
View details for PubMedID 33171122
Structure-Aided Development of Small-Molecule Inhibitors of ENPP1, the Extracellular Phosphodiesterase of the Immunotransmitter cGAMP.
Cell chemical biology
Cancer cells initiate an innate immune response by synthesizing and exporting the small-molecule immunotransmitter cGAMP, which activates the anti-cancer Stimulator of Interferon Genes (STING) pathway in the host. An extracellular enzyme, ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1), hydrolyzes cGAMP and negatively regulates this anti-cancer immune response. Small-molecule ENPP1 inhibitors are much needed as tools to study the basic biology of extracellular cGAMP and as investigational cancer immunotherapy drugs. Here, we surveyed structure-activity relationships around a series of cell-impermeable and thus extracellular-targeting phosphonate inhibitors of ENPP1. In addition, we solved the crystal structure of an exemplary phosphonate inhibitor to elucidate the interactions that drive potency. This study yielded several best-in-class inhibitors with Ki< 2nM and excellent physicochemical and pharmacokinetic properties. Finally, we demonstrate that an ENPP1 inhibitor delays tumor growth in a breast cancer mouse model. Together, we have developed ENPP1 inhibitors that are excellent tool compounds and potential therapeutics.
View details for DOI 10.1016/j.chembiol.2020.07.007
View details for PubMedID 32726585
Investigation of Natural and Synthetic Aggregation Inhibitors using Microfluidic Applications
CELL PRESS. 2020: 337A
View details for Web of Science ID 000513023202181
Development of cGAMP-Luc, a sensitive and precise coupled enzyme assay to measure cGAMP in complex biological samples.
The Journal of biological chemistry
2'5'/3'5' cyclic GMP-AMP (cGAMP) is a second messenger produced in response to cytosolic dsDNA that activates the stimulator of interferon genes (STING) pathway. We recently discovered that cGAMP is exported by cancer cells and that this extracellular signal is an immunotransmitter key to tumor detection and elimination by the innate immune system. The enhancement of extracellular cGAMP levels therefore holds great promise for managing cancer. However, there is still much more to understand about the basic biology of cGAMP before its full therapeutic potential can be realized. To answer these questions, we must be able to detect and quantitate cGAMP with an assay that is high throughput, sensitive, and precise. Existing assays fall short of these needs. Here, we describe the development of cGAMP-Luc, a coupled enzyme assay that relies on the degradation of cGAMP to AMP by ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) and an optimized assay for the detection of AMP by luciferase. We also developed STING-CAP, a STING-mediated method to concentrate and purify cGAMP from any type of biological sample. We conclude that cGAMP-Luc is an economical high throughput assay that matches the accuracy of and surpasses the detection limit of mass spectrometry, the current gold standard of cGAMP quantitation. We propose that cGAMP-Luc is a powerful tool that may enable discoveries that advance insights into extracellular cGAMP levels in healthy and diseased tissues, such as cancer.
View details for DOI 10.1074/jbc.RA119.012170
View details for PubMedID 32127400
Extracellular cGAMP is a cancer cell-produced immunotransmitter involved in radiation-induced anti-cancer immunity.
2020; 1 (2): 184–96
2'3'-cyclic GMP-AMP (cGAMP) is an intracellular second messenger that is synthesized in response to cytosolic double-stranded DNA and activates the innate immune STING pathway. Our previous discovery of its extracellular hydrolase ENPP1 hinted at the existence of extracellular cGAMP. Here, we detected that cGAMP is continuously exported but then efficiently cleared by ENPP1, explaining why it has previously escaped detection. By developing potent, specific, and cell impermeable ENPP1 inhibitors, we found that cancer cells continuously export cGAMP in culture at steady state and at higher levels when treated with ionizing radiation (IR). In mouse tumors, depletion of extracellular cGAMP decreased tumor-associated immune cell infiltration and abolished the curative effect of IR. Boosting extracellular cGAMP with ENPP1 inhibitors synergized with IR to delay tumor growth. In conclusion, extracellular cGAMP is an anti-cancer immunotransmitter that could be harnessed to treat cancers with low immunogenicity.
View details for DOI 10.1038/s43018-020-0028-4
View details for PubMedID 33768207
View details for PubMedCentralID PMC7990037
Real-Time Intrinsic Fluorescence Visualization and Sizing of Proteins and Protein Complexes in Microfluidic Devices
2018; 90 (6): 3849–55
Optical detection has become a convenient and scalable approach to read out information from microfluidic systems. For the study of many key biomolecules, however, including peptides and proteins, which have low fluorescence emission efficiencies at visible wavelengths, this approach typically requires labeling of the species of interest with extrinsic fluorophores to enhance the optical signal obtained - a process which can be time-consuming, requires purification steps, and has the propensity to perturb the behavior of the systems under study due to interactions between the labels and the analyte molecules. As such, the exploitation of the intrinsic fluorescence of protein molecules in the UV range of the electromagnetic spectrum is an attractive path to allow the study of unlabeled proteins. However, direct visualization using 280 nm excitation in microfluidic devices has to date commonly required the use of coherent sources with frequency multipliers and devices fabricated out of materials that are incompatible with soft lithography techniques. Here, we have developed a simple, robust, and cost-effective 280 nm LED platform that allows real-time visualization of intrinsic fluorescence from both unlabeled proteins and protein complexes in polydimethylsiloxane microfluidic channels fabricated through soft lithography. Using this platform, we demonstrate intrinsic fluorescence visualization of proteins at nanomolar concentrations on chip and combine visualization with micron-scale diffusional sizing to measure the hydrodynamic radii of individual proteins and protein complexes under their native conditions in solution in a label-free manner.
View details for DOI 10.1021/acs.analchem.7b04523
View details for Web of Science ID 000428219600030
View details for PubMedID 29451779
beta-Synuclein suppresses both the initiation and amplification steps of alpha-synuclein aggregation via competitive binding to surfaces
2016; 6: 36010
α-Synuclein is an intrinsically disordered protein that is associated with the pathogenesis of Parkinson's disease through the processes involved in the formation of amyloid fibrils. α and β-synuclein are homologous proteins found at comparable levels in presynaptic terminals but β-synuclein has a greatly reduced propensity to aggregate and indeed has been found to inhibit α-synuclein aggregation. In this paper, we describe how sequence differences between α- and β-synuclein affect individual microscopic processes in amyloid formation. In particular, we show that β-synuclein strongly suppresses both lipid-induced aggregation and secondary nucleation of α-synuclein by competing for binding sites at the surfaces of lipid vesicles and fibrils, respectively. These results suggest that β-synuclein can act as a natural inhibitor of α-synuclein aggregation by reducing both the initiation of its self-assembly and the proliferation of its aggregates.
View details for DOI 10.1038/srep36010
View details for Web of Science ID 000386845400001
View details for PubMedID 27808107
View details for PubMedCentralID PMC5093550