Anatomy of a discovery

For hundreds of millions of years, plants and animals have been engaged in constant battle against microbial and viral pathogens. For higher metazoan organisms, the adaptive and innate arms of the immune system represent our dual lines of defense against an extensive and rapidly evolving bastion of enemies. Gerald Edelman and Susumu Tonegawa won Nobel prizes in successive generations decades ago for their discoveries showing the nature of antibodies and the elaborate means by which they are put together to facilitate adaptive immunity. More recently, Bruce Beutler and Jules Hoffmann won the Nobel prize for their groundbreaking work on Toll-like receptors as the operational nuts and bolts of the innate immune system.

Despite being blessed with these gorgeous pinnacles of scientific achievement, plenty of mysteries remain. Over the past several years, Zhijian “James” Chen and his colleagues here at the University of Texas Southwestern Medical Center have cracked open a new set of discoveries further clarifying our understanding of how innate immunity works.

Certain pathogens expose their genetic blueprint, sometimes in the form of duplex DNA, to the cytoplasmic compartment of host cells. Our own DNA belongs within either the nucleus or the mitochondrion – not in the cytoplasm. Indeed, DNA was found as an immune-response stimulant even before it was found to carry the genetic blueprint.

How did Chen and his team track down the mechanism by which cytoplasmic DNA stimulates the innate immune system? They, of course, started by standing on the shoulders of others who already had discovered that cytoplasmic DNA triggers activation of an endoplasmic reticulum protein designated as STING, MITA, MPYS or ERIS. The STING protein, when magically activated by cytoplasmic DNA, recruits the IKK and TBK1 kinase enzymes, which respectively activate the NF-κB and IRF3 transcription factors. Activation of NF-κB and IRF3 already was understood to trigger cells to activate the expression of genes vital for stimulation of the immune response.

To find out what might happen upstream of STING activation, Chen’s team crafted a cell line missing the STING protein. They could then expose the cells to cytoplasmic DNA and prepare extracts in search of molecules responsible for propagating the pro-immune stimulatory signal. Vital to this game plan, Chen’s team used permeabilized macrophage cells blessed with an intact STING pathway. By applying extracts from STING-deficient cells exposed to cytoplasmic DNA to the permeabilized macrophage cells, they could monitor IRF3 activation. This was their assay. No matter how arduous, biochemists always need a reliable assay.

Plenary lecturer
and ASBMB-Merck award winner

Chen

Zhijian "James" Chen, University of Texas Southwestern Medical Center at Dallas 
Annual meeting lecture: "Enemy within: immune and autoimmune responses to the cytosolic DNA and RNA"                        
When: 8 a.m. to 8:45 a.m. Monday, March 30
Where: Ballroom West, Third Level, Boston Convention Center

The substance produced by STING-deficient cells that activated STING in the recipient macrophage cells turned out to be heat-stable and small – a metabolite, not a protein. By use of an arduous combination of purification, analytical chemistry and synthetic chemistry, Chen and his team discovered the activating substance to be cyclic GMP-AMP, abbreviated as cGAMP The precise nature of the phosphodiester linkages subsequently was found to be mixed – one was between the 2'-OH of GMP and the 5'-phosphate of AMP, and the other was between the 3'-OH of AMP and the 5'-phosphate of GMP. That cGAMP is the STING activator has since been nailed unequivocally, including resolution of the X-ray crystal structure of metabolite-bound STING.

Having discovered the cGAMP metabolite as the signaling message, the Chen team turned to its mode of synthesis. They incubated cytoplasmic material from cultured cells with ATP and GTP in the presence of DNA. By applying these reaction products to permeabilized macrophages (after DNAase digestion and heat treatment), Chen’s team could track fractions capable of producing the cGAMP stimulant of STING activity. Again, by use of a sophisticated array of chromatographic steps, the team was able to isolate, purify and identify the cGAMP synthase enzyme (now designated cGAS).

These breakthrough studies revealed the precise identity of the cGAS enzyme and its selective activation by DNA. Subsequent X-ray crystallographic experiments showed mechanistically how DNA binds and activates cGAS and offered a compelling understanding of how the enzyme converts ATP and GTP into its cGAMP product. Those studies positioned the Chen team to generate mice selectively missing a functional gene encoding the cGAS enzyme. These cGAS-deficient mice are considerably more susceptible to lethal infection by herpes simplex virus than wild-type littermates, and studies of immune adjuvant effects of cGAMP have yielded vivid evidence of the importance of the cGAS pathway for proper deployment of the immune system. For-profit biotechnology companies, large and small, now are pursuing cGAS inhibitors as potential treatments for autoimmune diseases and cGAMP-related molecules for immunotherapies and vaccines.

I recount this story as a celebration of our discipline – the discipline of biochemistry. Once Chen’s team threaded the needle in the discovery of cGAMP and the enzyme that makes it, other scientists could contribute rapidly. Crystallization of the cGAS enzyme was accomplished within months of the Chen team’s discoveries, and nearly any molecular biologist could have made knockout mice lacking the enzyme for studies of its role in innate immunity.

What could not have been achieved without hardcore biochemistry were the rate-limiting discoveries of the cGAMP metabolite as a second message produced in response to cytoplasmic DNA as well as the enzyme that synthesizes cGAMP in response to exposure to cytoplasmic DNA. As members of the American Society for Biochemistry and Molecular Biology, we should take pride in the dominance of our scientific discipline: Textbooks are filled with the fruits of our field. Future textbooks covering disciplines ranging from immunology to cell biology to biochemistry undoubtedly will feature the contributions of Chen and his team briefly summarized in this essay. Boy, should we be proud of our field, including its illustrious past, vibrant present and promising future. Biochemistry rules!

Steven McKnight Steven McKnight is president of the American Society for Biochemistry and Molecular Biology and chairman of the biochemistry department at the University of Texas-Southwestern Medical Center at Dallas.