The TREX1 Double-stranded DNA Degradation Activity Is Defective in Dominant Mutations Associated with Autoimmune Disease

Abstract

Mutations in TREX1 have been linked to a spectrum of human autoimmune diseases including Aicardi-Goutières syndrome (AGS), familial chilblain lupus (FCL), systemic lupus erythematosus, and retinal vasculopathy and cerebral leukodystrophy. A common feature in these conditions is the frequent detection of antibodies to double-stranded DNA (dsDNA). TREX1 participates in a cell death process implicating this major 3 3 5 exonuclease in genomic DNA degradation to minimize potential immune activation by persistent self DNA. The TREX1 D200N and D18N dominant heterozygous mutations were identified in AGS and FCL, respectively. TREX1 enzymes containing the D200N and D18N mutations were compared using nicked dsDNA and single-stranded DNA (ssDNA) degradation assays. The TREX1WT/D200N and TREX1 WT/D18N heterodimers are completely deficient at degrading dsDNA and degrade ssDNA at an expected ϳ2-fold lower rate than TREX1 WT enzyme. Further, the D200N-and D18N-containing TREX1 homo-and heterodimers inhibit the dsDNA degradation activity of TREX1 WT enzyme, providing a likely explanation for the dominant phenotype of these TREX1 mutant alleles in AGS and FCL. By comparison, the TREX1 R114H homozygous mutation causes AGS and is found as a heterozygous mutation in systemic lupus erythematosus. The TREX1 R114H/R114H homodimer has dysfunctional dsDNA and ssDNA degradation activities and does not detectibly inhibit the TREX1 WT enzyme, whereas the TREX1heterodimer has a functional dsDNA degradation activity, supporting the recessive genetics of TREX1 R114H in AGS. The dysfunctional dsDNA degradation activities of these disease-related TREX1 mutants could account for persistent dsDNA from dying cells leading to an aberrant immune response in these clinically related disorders.The TREX1 gene encodes the major 3Ј 3 5Ј exonuclease detected in mammalian cells. TREX1 is a single open reading frame located on chromosome 3p21.31 and encodes a 314-amino acid polypeptide (1-3). TREX1 is closely related to TREX2 (located on chromosome Xq28) except TREX1 contains additional coding sequence for a C-terminal region not found in TREX2 (4). The N-terminal 242 amino acids of TREX1 contain the catalytically robust 3Ј 3 5Ј exonuclease that is active on ssDNA 2 and dsDNA (5) with the highest activity detected using a partial duplex DNA containing unpaired 3Ј nucleotides (1-3). The C-terminal 72 amino acids contain a transmembrane region that localizes TREX1 to the endoplasmic reticulum in the perinuclear space of cells (6). Activation of a cell death pathway and treatment of cells with DNA-damaging agents cause TREX1 to relocate to the nucleus where it acts on DNA 3Ј termini (6 -8).The structure of TREX1 catalytic domain with bound DNA reveals the protein-polynucleotide interactions that explain the preference for multiple unpaired 3Ј termini in TREX1 exonuclease action and highlights the extensive interface contacts in the stable dimeric enzyme (9). A short ssDNA of approximately four nucleotides is accommodated in the TREX1 active site, and the Arg-1...

Dominant Mutations of the TREX1 Exonuclease Gene in Lupus and Aicardi-Goutières Syndrome

“…Heterozygous de novo and inherited mutations in the highly conserved TREX1 Asp-18 and Asp-200 metal-binding residues exhibit dominant FCL and AGS (12,14,19,24). The disease phenotypes in dominant FCL and AGS patients correlate best with the dsDNA degradation activities measured in the TREX1 D18N and D200N enzymes and not with the ssDNA degradation activities (23). We have proposed that the inability to perform chemistry of phosphodiester bond cleavage resulting from the D18N or D200N mutation might trap the TREX1 mutant enzyme onto the dsDNA in a nonproductive enzyme-DNA complex at the site of the nick.…”

“…Additions of up to 10-fold higher concentrations of the TREX1 mutant heterodimers resulted in no detectable dsDNA degradation (Ref. 23 and data not shown). These data indicate that the dominant TREX1 D18N, D200N, and D200H heterodimers exhibit at least a 200-fold decreased level of dsDNA degradation activity relative to TREX1…”

“…Enzyme Preparation-The human recombinant TREX1 enzymes were expressed in bacteria and purified as stable homo-or heterodimers as described (23). Briefly, mutant TREX1-containing plasmids were produced using a PCR sitedirected mutagenesis strategy and confirmed by DNA sequencing.…”

“…The TREX1 AGS-causing mutations locate predominantly to the catalytic core region with a few notable exceptions positioned in the C-terminal hydrophobic region (22). Some TREX1 AGS-causing mutants exhibit dramatically lower levels of catalytic function, whereas others show more modest effects on the ssDNA degradation activities, yet all yield similar human pathologies (21,23). The TREX1 systemic lupus erythematosus-associated mutations are located mostly in the C-terminal region with a few variants positioned in the catalytic core.…”

Early onset of autoimmune disease by the retroviral integrase inhibitor raltegravir

“…The processed ends would identify the DNA as generated by reverse transcriptase and distinguish it from external plasmid DNA, which is not degraded by Trex1 (1). Although it is active on single-stranded and double-stranded DNA, Trex1 is most active on double-stranded DNA with unpaired 3Ј termini (30)(31)(32)(33). Recessed ends, as provided by the integrase processing, may thus be an in vivo target for Trex1.…”