Measuring TREX1 and TREX2 exonuclease activities

Abstract

Three-prime Repair Exonuclease (TREX1) degrades ssDNA and dsDNA. TREX1 localizes to the perinuclear space in cells and degrades cytosolic DNA to prevent aberrant nucleic acid sensing and immune activation in humans and mice. Mutations in the TREX1 gene cause a spectrum of human autoimmune diseases including Aicardi-Goutières syndrome, familial chilblain lupus, retinal vasculopathy with cerebral leukodystrophy, and are associated with systemic lupus erythematosus. More than 60 disease-causing TREX1 variants have been identified including dominant and recessive, missense, and frameshift mutations that map to the catalytic core region and to the Cterminal cell localization region. The TREX1-disease causing mutations affect exonuclease activity at varied levels. In this chapter, we describe methods to purify variant recombinant TREX1 enzymes and measure the exonuclease activity using ssDNA and dsDNA substrates. The relationships between TREX1 activities, types of TREX1 mutations, and TREX1-associated autoimmune diseases are considered.

TREX1 as a Novel Immunotherapeutic Target

“…Detailed protocols for hT1, mT1, and hT2 enzyme purification have been published ( 101 ) and summarized here. For homodimers, pLM303x constructs encoding the recombinant TREX enzyme fused to an N-terminal maltose-binding protein (MBP) are transformed into Rosetta II cells, and the MBP-linker-TREX fusion protein is overexpressed.…”

“…Our detailed protocol for this assay is published ( 101 ). Reaction mixture was prepared containing variable concentrations of a dsDNA substrate, 5 mM MgCl 2 , 2 mM DTT, 20 mM Tris base (pH 7.5).…”

“…A high-throughput screening (HTS) strategy is critical to identifying candidate small molecule TREX1 inhibitors. We have reported methodology to successfully purify large quantities of recombinant TREX1 enzyme ( 101 ) to facilitate a scalable biochemical assay. Since the desired therapeutic effect from TREX1 inhibition is cGAS stimulation ( 26 , 27 , 97 , 102 ) and because cGAS has specificity for dsDNA ( 103 ), TREX1 exonuclease activity on dsDNA is the appropriate biochemical metric for an inhibitor’s potential.…”

“…Since the desired therapeutic effect from TREX1 inhibition is cGAS stimulation ( 26 , 27 , 97 , 102 ) and because cGAS has specificity for dsDNA ( 103 ), TREX1 exonuclease activity on dsDNA is the appropriate biochemical metric for an inhibitor’s potential. We have described a fluorescence-based exonuclease assay to measure TREX1’s degradation of dsDNA ( 101 ) that is scalable to a 384-well microplate HTS assay. Importantly, this assay utilizes substrate concentrations at or below the TREX1 dsDNA K m of ~15 nM 2 , allowing the assay to readily detect small molecules with a broad range of inhibition kinetics ( 104 ).…”

“…In this capacity, TREX1 is well suited for rational drug design. We have published a detailed protocol for generating large quantities of high purity TREX1 enzyme ( 101 ) and multiple structures of the mTREX1 enzyme solved by x-ray diffraction ( 6 , 11 , 16 , 92 ), including apoenzyme and co-crystallizations with TREX1 substrates and product. These structures demonstrate the capacity for mTREX1 to be co-crystallized with a variety of molecules and can also be used in computational approaches to model the binding mechanisms of candidate inhibitors ( 111 , 112 ).…”