Differential Structural Features of Two Mutant ADAR1p150 Zα Domains Associated with Aicardi-Goutières Syndrome.

The Zα domain of ADARp150 is critical for proper Z-RNA substrate binding and is a key factor in the type-I interferon response pathway. Two point-mutations in this domain (N173S and P193A), which cause neurodegenerative disorders, are linked to decreased A-to-I editing in disease models. To understand this phenomenon at the molecular level, we biophysically and structurally characterized these two mutated domains, revealing that they bind Z-RNA with a decreased affinity. Less efficient binding to Z-RNA can be explained by structural changes in beta-wing, part of the Z-RNA-protein interface, and alteration of conformational dynamics of the proteins.

cGAS phase separation inhibits TREX1-mediated DNA degradation and enhances cytosolic DNA sensing.

Cyclic GMP-AMP synthase (cGAS) recognition of cytosolic DNA is critical for the immune response to cancer and pathogen infection. Here, we discover that cGAS-DNA phase separation is required to resist negative regulation and allow efficient sensing of immunostimulatory DNA. We map the molecular determinants of cGAS condensate formation and demonstrate that phase separation functions to limit activity of the cytosolic exonuclease TREX1. Mechanistically, phase separation forms a selective environment that suppresses TREX1 catalytic function and restricts DNA degradation to an outer shell at the droplet periphery. We identify a TREX1 mutation associated with the severe autoimmune disease Aicardi-Goutières syndrome that increases penetration of TREX1 into the repressive droplet interior and specifically impairs degradation of phase-separated DNA. Our results define a critical function of cGAS-DNA phase separation and reveal a molecular mechanism that balances cytosolic DNA degradation and innate immune activation.

A phenolic small molecule inhibitor of RNase L prevents cell death from ADAR1 deficiency.

The oligoadenylate synthetase (OAS)-RNase L system is an IFN-inducible antiviral pathway activated by viral infection. Viral double-stranded (ds) RNA activates OAS isoforms that synthesize the second messenger 2-5A, which binds and activates the pseudokinase-endoribonuclease RNase L. In cells, OAS activation is tamped down by ADAR1, an adenosine deaminase that destabilizes dsRNA. Mutation of ADAR1 is one cause of Aicardi-Goutières syndrome (AGS), an interferonopathy in children. ADAR1 deficiency in human cells can lead to RNase L activation and subsequent cell death. To evaluate RNase L as a possible therapeutic target for AGS, we sought to identify small-molecule inhibitors of RNase L. A 500-compound library of protein kinase inhibitors was screened for modulators of RNase L activity in vitro. We identified ellagic acid (EA) as a hit with 10-fold higher selectivity against RNase L compared with its nearest paralog, IRE1. SAR analysis identified valoneic acid dilactone (VAL) as a superior inhibitor of RNase L, with 100-fold selectivity over IRE1. Mechanism-of-action analysis indicated that EA and VAL do not bind to the pseudokinase domain of RNase L despite acting as ATP competitive inhibitors of the protein kinase CK2. VAL is nontoxic and functional in cells, although with a 1,000-fold decrease in potency, as measured by RNA cleavage activity in response to treatment with dsRNA activator or by rescue of cell lethality resulting from self dsRNA induced by ADAR1 deficiency. These studies lay the foundation for understanding novel modes of regulating RNase L function using small-molecule inhibitors and avenues of therapeutic potential.

Absence of RNase H2 triggers generation of immunogenic micronuclei removed by autophagy.

Hypomorphic mutations in the DNA repair enzyme RNase H2 cause the neuroinflammatory autoimmune disorder Aicardi-Goutières syndrome (AGS). Endogenous nucleic acids are believed to accumulate in patient cells and instigate pathogenic type I interferon expression. However, the underlying nucleic acid species amassing in the absence of RNase H2 has not been established yet. Here, we report that murine RNase H2 knockout cells accumulated cytosolic DNA aggregates virtually indistinguishable from micronuclei. RNase H2-dependent micronuclei were surrounded by nuclear lamina and most of them contained damaged DNA. Importantly, they induced expression of interferon-stimulated genes (ISGs) and co-localized with the nucleic acid sensor cGAS. Moreover, micronuclei associated with RNase H2 deficiency were cleared by autophagy. Consequently, induction of autophagy by pharmacological mTOR inhibition resulted in a significant reduction of cytosolic DNA and the accompanied interferon signature. Autophagy induction might therefore represent a viable therapeutic option for RNase H2-dependent disease. Endogenous retroelements have previously been proposed as a source of self-nucleic acids triggering inappropriate activation of the immune system in AGS. We used human RNase H2-knockout cells generated by CRISPR/Cas9 to investigate the impact of RNase H2 on retroelement propagation. Surprisingly, replication of LINE-1 and Alu elements was blunted in cells lacking RNase H2, establishing RNase H2 as essential host factor for the mobilisation of endogenous retrotransposons.

Rho-associated protein kinase 2 (ROCK2): a new target of autoimmunity in paraneoplastic encephalitis.

Onconeural antibodies are associated with cancer and paraneoplastic encephalitis. While their pathogenic role is still largely unknown, their high diagnostic value is undisputed. In this study we describe the discovery of a novel target of autoimmunity in an index case of paraneoplastic encephalitis associated with urogenital cancer.A 75-year-old man with a history of invasive bladder carcinoma 6 years ago with multiple recurrences and a newly discovered renal cell carcinoma presented with seizures and progressive cognitive decline followed by super-refractory status epilepticus. Clinical and ancillary findings including brain biopsy suggested paraneoplastic encephalitis. Immunohistochemistry of the brain biopsy was used to characterize the inflammatory response. Indirect immunofluorescence assay (IFA) was used for autoantibody screening. The autoantigen was identified by histo-immunoprecipitation and mass spectrometry and was validated by expressing the recombinant antigen in HEK293 cells and neutralization tests. Sera from 125 control patients were screened using IFA to test for the novel autoantibodies.IFA analysis of serum revealed a novel autoantibody against brain tissue. An intracellular enzyme, Rho-associated protein kinase 2 (ROCK2), was identified as target-antigen. ROCK2 was expressed in affected brain tissue and archival bladder tumor samples of this patient. Brain histopathology revealed appositions of cytotoxic CD8+ T cells on ROCK2-positive neurons. ROCK2 antibodies were not found in the sera of 20 patients with bladder cancer and 17 with renal cancer, both without neurological symptoms, 49 healthy controls, and 39 patients with other antineuronal autoantibodies. In conclusion, novel onconeural antibodies targeting ROCK2 are associated with paraneoplastic encephalitis and should be screened for when paraneoplastic neurological syndromes, especially in patients with urogenital cancers, occur.

Low-Titre GAD Antibody-Associated Late-Onset Cerebellar Ataxia with a Significant Clinical Response to Intravenous Immunoglobulin Treatment.

Antiglutamic acid decarboxylase antibody-associated cerebellar ataxia (GAD-Abs CA) is a rare, but increasingly detected, autoimmune neurological disorder characterized by the clinical presence of a cerebellar syndrome concomitant with positive GAD-Abs levels in serum and cerebrospinal fluid (CSF). It represents 3% of all immune-mediated sporadic CAs. Low-titre GAD-Abs CA is an even rarer subtype of GAD-Abs CA. We report on a 68-year-old woman with a 3-year history of progressive gait ataxia. In addition to the modified Rankin Scale (mRS), we used two other objective scales to evaluate CA severity, i.e. the International Cooperative Ataxia Rating Scale (ICARS) and the Scale for Assessment and Rating of Ataxia (SARA). Series of CT and MRI showed atrophy of the cerebellum. Except for the glycated haemoglobin (HbA1c) levels, all other routine laboratory examinations were within normal limits. Autoimmune laboratory examinations showed positive (25.8 U/mL) serum GAD-Abs levels. The GAD antibody index was <1.0. The CSF analysis showed no oligoclonal immunoglobulin bands. Intravenous immunoglobulin (IVIg) therapy was started and significant improvement was observed. The diagnosis of low-titre GAD-Abs CA was established.

SAMHD1, the Aicardi-Goutières syndrome gene and retroviral restriction factor, is a phosphorolytic ribonuclease rather than a hydrolytic ribonuclease.

SAMHD1 plays diverse roles in innate immunity, autoimmune diseases and HIV restriction, but the mechanisms involved are still unclear. SAMHD1 has been reported to have both dNTPase and RNase activities. However, whether SAMHD1 possesses RNase activity remains highly controversial. Here, we found that, unlike conventional hydrolytic exoribonucleases, SAMHD1 requires inorganic phosphate to degrade RNA substrates and produces nucleotide diphosphates rather than nucleoside monophosphates, which indicated that SAMHD1 is a phosphorolytic but not hydrolytic 3'-5' exoribonuclease. Furthermore, SAMHD1 preferentially cleaved single-stranded RNAs comprising A20 or U20, whereas neither C20 nor G20 was susceptible to SAMHD1-mediated degradation. Our findings will facilitate more advanced studies into the role of the SAMHD1 RNase function in the cellular pathogenesis implicated in nucleic acid-triggered inflammatory responses and the anti-retroviral function of SAMHD1.

[Phenotypic variations in Aicardi-Goutieres syndrome caused by RNASEH2B gene mutations: report of two new cases]. / Variaciones fenotípicas en el síndrome de Aicardi-Goutières causado por mutaciones en el gen RNASEH2B: presentación de dos nuevos casos.

INTRODUCTION: Aicardi-Goutieres syndrome is a rare immune disorder due to mutations in seven different genes that encode proteins called TREX1, ribonuclease H2 complex, SAMHD1, ADAR and IDIH1 (MDA5), which are involved in acid nucleic metabolism. Two cases are described in detail below caused by RNASEH2B gene mutation, one of which displays a mutation no described to date. CASE REPORTS: Case 1: male consulting because from 5-month-old shows loss of maturity items acquired until then, coming with several fever episodes. Case 2: a 4-month-old boy showing since 2-month-old great irritability and oral-feeding trouble with severe psychomotor impairment. In both cases it was found an increase of pterines in the cerebrospinal fluid, mainly neopterine, with calcifications in the basal ganglia. The diagnosis was proved by sequencing RNASEH2B gene, founding in case 2 a new mutation not described previously. CONCLUSIONS: The reported cases belong to the description already done by Aicardi-Goutieres, it should be noticed this syndrome in a patient with a subacute encephalopathy of debut in the first year of life, dystonia/spasticity in variable degree and important affectation/regression of psychomotor development, particularly in those with increase of pterines (neopterine) in the cerebrospinal fluid and calcifications in the basal ganglia.

TITLE: Variaciones fenotipicas en el sindrome de Aicardi-Goutieres causado por mutaciones en el gen RNASEH2B: presentacion de dos nuevos casos.Introduccion. El sindrome de Aicardi-Goutieres es un trastorno inmunitario raro debido a mutaciones en siete genes que codifican proteinas llamadas TREX1, el complejo ribonucleasa H2, SAMHD1, ADAR e IFIH1 (MAD5), las cuales estan implicadas en el metabolismo de los acidos nucleicos. A continuacion se presentan dos nuevos casos por mutacion en el gen RNASEH2B, uno de los cuales presenta una mutacion no descrita hasta la fecha. Casos clinicos. Caso 1: varon que consulto porque desde los 5 meses, coincidiendo con cuadros febriles de repeticion, presentaba perdida de los items madurativos adquiridos hasta la fecha. Caso 2: niño de 4 meses que desde los 2 meses mostraba gran irritabilidad con dificultades en la alimentacion, asociado a un grave retraso psicomotor. En ambos casos se constato un aumento de las pterinas en el liquido cefalorraquideo, principalmente de la neopterina, con calcificaciones en los ganglios basales. El diagnostico se confirmo mediante secuenciacion del gen RNASEH2B; el caso 2 presentaba una mutacion no descrita en la literatura medica. Conclusiones. Los casos corresponden a la descripcion clasica realizada por Aicardi-Goutieres. Debe tenerse en cuenta este sindrome ante un paciente con un cuadro de encefalopatia subaguda de comienzo en el primer año de vida, distonia/espasticidad en grado variable e importante afectacion/regresion del desarrollo psicomotor, especialmente si asocia aumento de las pterinas (neopterina) en el liquido cefalorraquideo y calcificaciones en los ganglios basales.

Is the role of human RNase H2 restricted to its enzyme activity?

In human cells, ribonuclease (RNase) H2 complex is the predominant source of RNase H activities with possible roles in nucleic acid metabolism to preserve genome stability and to prevent immune activation. Dysfunction mutations in any of the three subunits of human RNase H2 complex can result in embryonic/perinatal lethality or cause Aicardi-Goutières syndrome (AGS). Most recently, increasing findings have shown that human RNase H2 proteins play roles beyond the RNase H2 enzymatic activities in health and disease. Firstly, the biochemical and structural properties of human RNase H2 proteins allow their interactions with various partner proteins that may support functions other than RNase H2 enzymatic activities. Secondly, the disparities of clinical presentations of AGS with different AGS-mutations and the biochemical and structural analysis of AGS-mutations, especially the results from both AGS-knockin and RNase H2-null mouse models, suggest that human RNase H2 complex has certain cellular functions beyond the RNase H2 enzymatic activities to prevent the innate-immune-mediated inflammation. Thirdly, the subunit proteins RNASEH2A and RNASEH2B respectively, not related to the RNase H2 enzymatic activities, have been shown to play a certain role in the pathophysiological processes of different cancer types. In this minireview, we aims to provide a brief overview of the most recent investigations into the biological functions of human RNase H2 proteins and the underlying mechanisms of their actions, emphasizing on the new insights into the roles of human RNase H2 proteins playing beyond the RNase H2 enzymatic activities in health and disease.

Ribonuclease H2 in health and disease.

Innate immune sensing of nucleic acids provides resistance against viral infection and is important in the aetiology of autoimmune diseases. AGS (Aicardi-Goutières syndrome) is a monogenic autoinflammatory disorder mimicking in utero viral infection of the brain. Phenotypically and immunologically, it also exhibits similarities to SLE (systemic lupus erythaematosus). Three of the six genes identified to date encode components of the ribonuclease H2 complex. As all six encode enzymes involved in nucleic acid metabolism, it is thought that pathogenesis involves the accumulation of nucleic acids to stimulate an inappropriate innate immune response. Given that AGS is a monogenic disorder with a defined molecular basis, we use it as a model for common autoimmune disease to investigate cellular processes and molecular pathways responsible for nucleic-acid-mediated autoimmunity. These investigations have also provided fundamental insights into the biological roles of the RNase H2 endonuclease enzyme. In the present article, we describe how human RNase H2 and its role in AGS were first identified, and give an overview of subsequent structural, biochemical, cellular and developmental studies of this enzyme. These investigations have culminated in establishing this enzyme as a key genome-surveillance enzyme required for mammalian genome stability.

Altered spatio-temporal dynamics of RNase H2 complex assembly at replication and repair sites in Aicardi-Goutières syndrome.

Ribonuclease H2 plays an essential role for genome stability as it removes ribonucleotides misincorporated into genomic DNA by replicative polymerases and resolves RNA/DNA hybrids. Biallelic mutations in the genes encoding the three RNase H2 subunits cause Aicardi-Goutières syndrome (AGS), an early-onset inflammatory encephalopathy that phenotypically overlaps with the autoimmune disorder systemic lupus erythematosus. Here we studied the intracellular dynamics of RNase H2 in living cells during DNA replication and in response to DNA damage using confocal time-lapse imaging and fluorescence cross-correlation spectroscopy. We demonstrate that the RNase H2 complex is assembled in the cytosol and imported into the nucleus in an RNase H2B-dependent manner. RNase H2 is not only recruited to DNA replication foci, but also to sites of PCNA-dependent DNA repair. By fluorescence recovery after photobleaching, we demonstrate a high mobility and fast exchange of RNase H2 at sites of DNA repair and replication. We provide evidence that recruitment of RNase H2 is not only PCNA-dependent, mediated by an interaction of the B subunit with PCNA, but also PCNA-independent mediated via the catalytic domain of the A subunit. We found that AGS-associated mutations alter complex formation, recruitment efficiency and exchange kinetics at sites of DNA replication and repair suggesting that impaired ribonucleotide removal contributes to AGS pathogenesis.

The Arg-62 residues of the TREX1 exonuclease act across the dimer interface contributing to catalysis in the opposing protomers.

TREX1 is a 3'-deoxyribonuclease that degrades single- and double-stranded DNA (ssDNA and dsDNA) to prevent inappropriate nucleic acid-mediated immune activation. More than 40 different disease-causing TREX1 mutations have been identified exhibiting dominant and recessive genetic phenotypes in a spectrum of autoimmune disorders. Mutations in TREX1 at positions Asp-18 and Asp-200 to His and Asn exhibit dominant autoimmune phenotypes associated with the clinical disorders familial chilblain lupus and Aicardi-Goutières syndrome. Our previous biochemical studies showed that the TREX1 dominant autoimmune disease phenotype depends upon an intact DNA-binding process coupled with dysfunctional active site chemistry. Studies here show that the TREX1 Arg-62 residues extend across the dimer interface into the active site of the opposing protomer to coordinate substrate DNA and to affect catalysis in the opposing protomer. The TREX1(R62A/R62A) homodimer exhibits ∼50-fold reduced ssDNA and dsDNA degradation activities relative to TREX1(WT). The TREX1 D18H, D18N, D200H, and D200N dominant mutant enzymes were prepared as compound heterodimers with the TREX1 R62A substitution in the opposing protomer. The TREX1(D18H/R62A), TREX1(D18N/R62A), TREX1(D200H/R62A), and TREX1(D200N/R62A) compound heterodimers exhibit higher levels of ss- and dsDNA degradation activities than the homodimers demonstrating the requirement for TREX1 Arg-62 residues to provide necessary structural elements for full catalytic activity in the opposing TREX1 protomer. This concept is further supported by the loss of dominant negative effects in the TREX1 D18H, D18N, D200H, and D200N compound heterodimers. These data provide compelling evidence for the required TREX1 dimeric structure for full catalytic function.

Aicardi-Goutières syndrome: clues from the RNase H2 knock-out mouse.

Ribonuclease H2 (RNase H2) belongs to the family of RNase H enzymes, which process RNA/DNA hybrids. Apart from cleaving the RNA moiety of a plain RNA/DNA hybrid, RNase H2 participates in the removal of single ribonucleotides embedded in a DNA duplex. Mutations in RNase H2 lead to the chronic inflammatory disorder Aicardi-Goutières syndrome (AGS), which has significant phenotypic overlaps with the autoimmune disease systemic lupus erythematosus. RNase H2 knock-out mice are embryonic lethal. Mouse embryos lacking RNase H2 accumulate DNA damage and exhibit a p53-mediated growth arrest commencing at gastrulation. On a molecular level, the knock-out mice reveal that RNase H2 represents an essential DNA repair enzyme, whose main cellular function is the removal of accidentally misincorporated ribonucleotides from genomic DNA. Ribonucleotides strongly accumulate within the genomic DNA of RNase H2-deficient cells, in turn resulting in a massive build-up of DNA damage in these cells. The DNA lesions that arise from misincorporated ribonucleotides constitute the by far most frequent type of naturally occurring DNA damage. AGS-causing mutations have also been found in the genes of the 3'-exonuclease TREX1, the dNTP triphosphatase SAMHD1, as well as the RNA-editing enzyme ADAR1, defining defects in nucleic acid metabolism pathways as a common hallmark of AGS pathology. However, recent evidence gathered from RNase H2 knock-out mice might provide additional insight into the molecular mechanisms underlying AGS development and a potential role of DNA damage as a trigger of autoimmunity is discussed.

Synonymous mutations in RNASEH2A create cryptic splice sites impairing RNase H2 enzyme function in Aicardi-Goutières syndrome.

Aicardi-Goutières syndrome is an inflammatory disorder resulting from mutations in TREX1, RNASEH2A/2B/2C, SAMHD1, or ADAR1. Here, we provide molecular, biochemical, and cellular evidence for the pathogenicity of two synonymous variants in RNASEH2A. Firstly, the c.69G>A (p.Val23Val) mutation causes the formation of a splice donor site within exon 1, resulting in an out of frame deletion at the end of exon 1, leading to reduced RNase H2 protein levels. The second mutation, c.75C>T (p.Arg25Arg), also introduces a splice donor site within exon 1, and the internal deletion of 18 amino acids. The truncated protein still forms a heterotrimeric RNase H2 complex, but lacks catalytic activity. However, as a likely result of leaky splicing, a small amount of full-length active protein is apparently produced in an individual homozygous for this mutation. Recognition of the disease causing status of these variants allows for diagnostic testing in relevant families.

Nuclease activity of the human SAMHD1 protein implicated in the Aicardi-Goutieres syndrome and HIV-1 restriction.

The human HD domain protein SAMHD1 is implicated in the Aicardi-Goutières autoimmune syndrome and in the restriction of HIV-1 replication in myeloid cells. Recently, this protein has been shown to possess dNTP triphosphatase activity, which is proposed to inhibit HIV-1 replication and the autoimmune response by hydrolyzing cellular dNTPs. Here, we show that the purified full-length human SAMHD1 protein also possesses metal-dependent 3'→5' exonuclease activity against single-stranded DNAs and RNAs in vitro. In double-stranded substrates, this protein preferentially cleaved 3'-overhangs and RNA in blunt-ended DNA/RNA duplexes. Full-length SAMHD1 also exhibited strong DNA and RNA binding to substrates with complex secondary structures. Both nuclease and dNTP triphosphatase activities of SAMHD1 are associated with its HD domain, but the SAM domain is required for maximal activity and nucleic acid binding. The nuclease activity of SAMHD1 could represent an additional mechanism contributing to HIV-1 restriction and suppression of the autoimmune response through direct cleavage of viral and endogenous nucleic acids. In addition, we demonstrated the presence of dGTP triphosphohydrolase and nuclease activities in several microbial HD domain proteins, suggesting that these proteins might contribute to antiviral defense in prokaryotes.

Glutamic acid decarboxylase autoantibody syndrome presenting as schizophrenia.

INTRODUCTION: Glutamic acid decarboxylase (GAD) is the rate-limiting enzyme converting glutamate into γ-aminobutyric acid. Impaired GAD function can alter motor, cognitive, and behavioral function. Anti-GAD antibodies (GADAbs) can cause several neurological disorders. However, the association between anti-GADAbs and pure psychosis, without seizures or focal neurological deficits, is not well defined. CASE REPORT: A 19-year-old woman with recent-onset psychotic disorder was diagnosed with schizophrenia. Brain magnetic resonance imaging and cerebrospinal fluid analysis were normal. Serum anti-GADAb titers were elevated. Brain biopsy showed subcortical gliosis and microglia-macrophage infiltration. The clinical syndrome improved with immune therapy. CONCLUSIONS: Severe psychosis and mild cognitive decline without other neurological features, meeting the Diagnostic and Statistical Manual of Mental Disorders, 4th edition, text revision diagnostic criteria for schizophrenia, can result from brain inflammation associated with elevated serum anti-GADAbs.

The TREX1 exonuclease R114H mutation in Aicardi-Goutières syndrome and lupus reveals dimeric structure requirements for DNA degradation activity.

Mutations in the TREX1 gene cause Aicardi-Goutières syndrome (AGS) and are linked to the autoimmune disease systemic lupus erythematosus. The TREX1 protein is a dimeric 3' DNA exonuclease that degrades DNA to prevent inappropriate immune activation. One of the most common TREX1 mutations, R114H, causes AGS as a homozygous and compound heterozygous mutation and is found as a heterozygous mutation in systemic lupus erythematosus. The TREX1 proteins containing R114H and the insertion mutations aspartate at position 201 (D201ins) and alanine at position 124 (A124ins), found in compound heterozygous AGS with R114H, were prepared and the DNA degradation activities were tested. The homodimer TREX1(R114H/R114H) exhibits a 23-fold reduced single-stranded DNA (ssDNA) exonuclease activity relative to TREX1(WT). The TREX1(D201ins/D201ins) and TREX1(A124ins/A124ins) exhibit more than 10,000-fold reduced ssDNA degradation activities. However, the TREX1(R114H/D201ins) and TREX1(R114H/A124ins) compound heterodimers exhibit activities 10-fold greater than the TREX1(R114H/R114H) homodimer during ssDNA and double-stranded DNA (dsDNA) degradation. These higher levels of activities measured in the TREX1(R114H/D201ins) and TREX1(R114H/A124ins) compound heterodimers are attributed to Arg-114 residues of TREX1(D201ins) and TREX1(A124ins) positioned at the dimer interface contributing to the active sites of the opposing TREX1(R114H) protomer. This interpretation is further supported by exonuclease activities measured for TREX1 enzymes containing R114A and R114K mutations. These biochemical data provide direct evidence for TREX1 residues in one protomer contributing to DNA degradation catalyzed in the opposing protomer and help to explain the dimeric TREX1 structure required for full catalytic competency.

Dominant mutation of the TREX1 exonuclease gene in lupus and Aicardi-Goutieres syndrome.

TREX1 is a potent 3' → 5' exonuclease that degrades single- and double-stranded DNA (ssDNA and dsDNA). TREX1 mutations at amino acid positions Asp-18 and Asp-200 in familial chilblain lupus and Aicardi-Goutières syndrome elicit dominant immune dysfunction phenotypes. Failure to appropriately disassemble genomic DNA during normal cell death processes could lead to persistent DNA signals that trigger the innate immune response and autoimmunity. We tested this concept using dsDNA plasmid and chromatin and show that the TREX1 exonuclease locates 3' termini generated by endonucleases and degrades the nicked DNA polynucleotide. A competition assay was designed using TREX1 dominant mutants and variants to demonstrate that an intact DNA binding process, coupled with dysfunctional chemistry in the active sites, explains the dominant phenotypes in TREX1 D18N, D200N, and D200H alleles. The TREX1 residues Arg-174 and Lys-175 positioned adjacent to the active sites act with the Arg-128 residues positioned in the catalytic cores to facilitate melting of dsDNA and generate ssDNA for entry into the active sites. Metal-dependent ssDNA binding in the active sites of the catalytically inactive dominant TREX1 mutants contributes to DNA retention and precludes access to DNA 3' termini by active TREX1 enzyme. Thus, the dominant disease genetics exhibited by the TREX1 D18N, D200N, and D200H alleles parallel precisely the biochemical properties of these TREX1 dimers during dsDNA degradation of plasmid and chromatin DNA in vitro. These results support the concept that failure to degrade genomic dsDNA is a principal pathway of immune activation in TREX1-mediated autoimmune disease.

Functional consequences of the RNase H2A subunit mutations that cause Aicardi-Goutieres syndrome.

Mutations in the three genes encoding the heterotrimeric RNase H2 complex cause Aicardi-Goutières Syndrome (AGS). Our mouse RNase H2 structure revealed that the catalytic RNase H2A subunit interfaces mostly with the RNase H2C subunit that is intricately interwoven with the RNase H2B subunit. We mapped the positions of AGS-causing RNase H2A mutations using the mouse RNase H2 structure and proposed that these mutations cause varied effects on catalytic potential. To determine the functional consequences of these mutations, heterotrimeric human RNase H2 complexes containing the RNase H2A subunit mutations were prepared, and catalytic efficiencies and nucleic acid binding properties were compared with the wild-type (WT) complex. These analyses reveal a dramatic range of effects with mutations at conserved positions G37S, R186W, and R235Q, reducing enzymatic activities and substrate binding affinities by as much as a 1000-fold, whereas mutations at non-conserved positions R108W, N212I, F230L, T240M, and R291H reduced activities and binding modestly or not at all. All mutants purify as three-subunit complexes, further supporting the required heterotrimeric structure in eukaryotic RNase H2. These kinetic properties reveal varied functional consequences of AGS-causing mutations in the catalytic RNase H2A subunit and reflect the complex mechanisms of nuclease dysfunction that include catalytic deficiencies and altered protein-nucleic acid interactions relevant in AGS.