Patricia Cortes

Andrographolide interferes with binding of nuclear factor-κB to DNA in HL-60-derived neutrophilic cells

1 Andrographolide, the major active component from Andrographis paniculata, has shown to possess anti‐inflammatory activity. Andrographolide inhibits the expression of several proinflammatory proteins that exhibit a nuclear factor kappa B (NF‐κB) binding site in their gene. 2 In the present study, we analyzed the effect of andrographolide on the activation of NF‐κB induced by platelet‐activating factor (PAF) and N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP) in HL‐60 cells differentiated to neutrophils. 3 PAF (100 nM) and fMLP (100 nM) induced activation of NF‐κB as determined by degradation of inhibitory factor B α (IκBα) using Western blotting in cytosolic extracts and by binding to DNA using electrophoretic mobility shift assay (EMSA) in nuclear extracts. 4 Andrographolide (5 and 50 μM) inhibited the NF‐κB‐luciferase activity induced by PAF. However, andrographolide did not reduce phosphorylation of p38 MAPK or ERK1/2 and did not change IκBα degradation induced by PAF and fMLP. 5 Andrographolide reduced the DNA binding of NF‐κB in whole cells and in nuclear extracts induced by PAF and fMLP. 6 Andrographolide reduced cyclooxygenase‐2 (COX‐2) expression induced by PAF and fMLP in HL‐60/neutrophils. 7 It is concluded that andrographolide exerts its anti‐inflammatory effects by inhibiting NF‐κB binding to DNA, and thus reducing the expression of proinflammatory proteins, such as COX‐2.

The genetic and biochemical basis of Omenn syndrome

Omenn syndrome (OS) is a peculiar, autosomal recessive severe combined immunodeficiency (SCID) associated with early-onset, generalized, exudative erythrodermia; lymphoadenopathy; hepato- and splenomegaly; hypereosinophilia; elevated serum IgE; and normal to high activated, yet non-functional, oligoclonal T cells. Recent investigations have shown that the primum movens of all these puzzling features lies in a defect of the lymphoid-specific V(D)J recombination process. Abnormalities in both alleles of either Rag-1 or -2 genes are found in all OS patients. At variance with T B- SCID, whose Rag mutations represent null alleles, OS mutations maintain a residual recombination activity, allowing limited T-cell receptor gene rearrangements to occur in the thymus. The gene rearrangements are subsequently expanded in the periphery after environmental antigen exposure. Missense mutations detected in OS have been examined in a number of biochemical assays and have contributed to dissect the various functional domains of both Rag-1 and Rag-2 proteins. The examination of a set of mutations occurring in the Rag-1 N-terminal portion has demonstrated that this region plays a fundamental role in vivo. The elucidation of the molecular basis of OS has allowed us to perform early prenatal diagnosis and could be the basis for trials of in utero bone marrow transplantation or gene therapy approaches.

Role of non-homologous end joining in V(D)J recombination.

The pathway of V(D)J recombination was discovered almost three decades ago. Yet it continues to baffle scientists because of its inherent complexity and the multiple layers of regulation that are required to efficiently generate a diverse repertoire of T and B cells. The non-homologous end-joining (NHEJ) DNA repair pathway is an integral part of the V(D)J reaction, and its numerous players perform critical functions in generating this vast diversity, while ensuring genomic stability. In this review, we summarize the efforts of a number of laboratories including ours in providing the mechanisms of V(D)J regulation with a focus on the NHEJ pathway. This involves discovering new players, unraveling unknown roles for known components, and understanding how deregulation of these pathways contributes to generation of primary immunodeficiencies. A long-standing interest of our laboratory has been to elucidate various mechanisms that control RAG activity. Our recent work has focused on understanding the multiple protein–protein interactions and protein–DNA interactions during V(D)J recombination, which allow efficient and regulated generation of the antigen receptors. Exploring how deregulation of this process contributes to immunodeficiencies also continues to be an important area of research for our group.

Artemis C-terminal region facilitates V(D)J recombination through its interactions with DNA Ligase IV and DNA-PKcs

Interactions of Artemis with DNA Ligase IV and DNA-PKcs are required for efficient coding joint formation.

Recombination-activating gene 1 (Rag1)–deficient mice with severe combined immunodeficiency treated with lentiviral gene therapy demonstrate autoimmune Omenn-like syndrome

BACKGROUND Recombination-activating gene 1 (RAG1) deficiency results in severe combined immunodeficiency (SCID) caused by a complete lack of T and B lymphocytes. If untreated, patients succumb to recurrent infections. OBJECTIVES We sought to develop lentiviral gene therapy for RAG1-induced SCID and to test its safety. METHODS Constructs containing the viral spleen-focus-forming virus (SF), ubiquitous promoters, or cell type-restricted promoters driving sequence-optimized RAG1 were compared for efficacy and safety in sublethally preconditioned Rag1(-/-) mice undergoing transplantation with transduced bone marrow progenitors. RESULTS Peripheral blood CD3(+) T-cell reconstitution was achieved with SF, ubiquitous promoters, and cell type-restricted promoters but 3- to 18-fold lower than that seen in wild-type mice, and with a compromised CD4(+)/CD8(+) ratio. Mitogen-mediated T-cell responses and T cell-dependent and T cell-independent B-cell responses were not restored, and T-cell receptor patterns were skewed. Reconstitution of mature peripheral blood B cells was approximately 20-fold less for the SF vector than in wild-type mice and often not detectable with the other promoters, and plasma immunoglobulin levels were abnormal. Two months after transplantation, gene therapy-treated mice had rashes with cellular tissue infiltrates, activated peripheral blood CD44(+)CD69(+) T cells, high plasma IgE levels, antibodies against double-stranded DNA, and increased B cell-activating factor levels. Only rather high SF vector copy numbers could boost T- and B-cell reconstitution, but mRNA expression levels during T- and B-cell progenitor stages consistently remained less than wild-type levels. CONCLUSIONS These results underline that further development is required for improved expression to successfully treat patients with RAG1-induced SCID while maintaining low vector copy numbers and minimizing potential risks, including autoimmune reactions resembling Omenn syndrome.

Analysis of mutations from SCID and Omenn syndrome patients reveals the central role of the Rag2 PHD domain in regulating V(D)J recombination

Rag2 plays an essential role in the generation of antigen receptors. Mutations that impair Rag2 function can lead to severe combined immunodeficiency (SCID), a condition characterized by complete absence of T and B cells, or Omenn syndrome (OS), a form of SCID characterized by the virtual absence of B cells and the presence of oligoclonal autoreactive T cells. Here, we present a comparative study of a panel of mutations that were identified in the noncanonical plant homeodomain (PHD) of Rag2 in patients with SCID or OS. We show that PHD mutant mouse Rag2 proteins that correspond to those found in these patients greatly impaired endogenous recombination of Ig gene segments in a Rag2-deficient pro-B cell line and that this correlated with decreased protein stability, impaired nuclear localization, and/or loss of the interaction between Rag2 and core histones. Our results demonstrate that point mutations in the PHD of Rag2 compromise the functionality of the entire protein, thus explaining why the phenotype of cells expressing PHD point mutants differs from those expressing core Rag2 protein that lacks the entire C-terminal region and is therefore devoid of the regulation imposed by the PHD. Together, our findings reveal the various deleterious effects of PHD Rag2 mutations and demonstrate the crucial role of this domain in regulating antigen receptor gene assembly. We believe these results reveal new mechanisms of immunodeficiency in SCID and OS.

Structural Basis of DNA Ligase IV-Artemis Interaction in Nonhomologous End-Joining

DNA ligase IV (LigIV) and Artemis are central components of the nonhomologous end-joining (NHEJ) machinery that is required for V(D)J recombination and the maintenance of genomic integrity in mammalian cells. We report here crystal structures of the LigIV DNA binding domain (DBD) in both its apo form and in complex with a peptide derived from the Artemis C-terminal region. We show that LigIV interacts with Artemis through an extended hydrophobic surface. In particular, we find that the helix α2 in LigIV-DBD is longer than in other mammalian ligases and presents residues that specifically interact with the Artemis peptide, which adopts a partially helical conformation on binding. Mutations of key residues on the LigIV-DBD hydrophobic surface abolish the interaction. Together, our results provide structural insights into the specificity of the LigIV-Artemis interaction and how the enzymatic activities of the two proteins may be coordinated during NHEJ.

VDJ Recombination: Artemis and Its In Vivo Role in Hairpin Opening

Artemis is the newest player in VDJ recombination and double strand break repair. First identified in radiation-sensitive and immune-deficient patients, it was recently shown to interact with DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and have nuclease activity, becoming the most popular candidate for the opening of hairpin coding ends. Reports presented in this issue (1) and in the December issue of Molecular Cell (2) address the role of Artemis in vivo by studying the effect of its deletion on the generation of intermediates and products of VDJ recombination in mouse ES cells, MEFs, and thymocytes. In all three systems analyzed, a defect in coding joint formation was observed, which was highly dependent on the cellular background and included inefficient and partially defective opening of hairpin coding ends. In addition, Artemis-deficient ES cells and Artemis-deficient MEFs show spontaneous chromosomal abnormalities, including telomere fusions, indicating that Artemis is also required for maintenance of genomic stability. This commentary will focus on the results regarding the role of Artemis in VDJ recombination. VDJ recombination, also known as antigen receptor gene rearrangement, is the process that assembles the variable domain of immunoglobulin and TCR genes. The hallmark of this reaction is the production of a large repertoire of antigen receptors with different specificities, a characteristic that is essential to the normal functioning of the immune system. VDJ recombination is directed to the immunoglobulin and TCR loci by highly conserved recombination signal sequences (RSSs) comprised of a heptamer and a nonamer motif with an intervening 12- or 23-bp spacer. Efficient recombination in vivo occurs almost exclusively between RSSs with different spacers, which is referred to as the 12/23 rule (3). Several proteins that mediate VDJ recombination in vivo have been identified (4). The lymphoid-specific components of the recombination machinery are RAG-1, RAG-2, and terminal deoxynucleotidyl transferase (TdT). RAG-1 and RAG-2 together constitute the recombinase. TdT, although not essential for catalysis, plays an important role increasing diversity by mediating the incorporation of nontemplate-dependent nucleotides. The nonlymphoid-restricted components identified so far are DNA-PKcs, Ku70, Ku80, XRCC4, ligase 4, and the most recently identified Artemis. All these proteins are involved in DNA double strand break repair as well as VDJ recombination. These nonlymphoid-specific components are likely to participate in the processing and joining steps of VDJ recombination, but their specific architectural or catalytic roles in the reaction remain largely unclear. Two other nonlymphoid-specific components, HMG1 and HMG2, have been implicated in VDJ recombination. In vitro experiments showed that these proteins increase the efficiency of cleavage by RAGs (5, 6). The role of HMG1 and HMG2 in VDJ recombination in vivo remains to be investigated. During the initial stages of the reaction, RAG-1/RAG-2 form a complex with the RSS, which is in part stabilized by the interactions between the nonamer binding domain of RAG-1 and the nonamer motif. Bridging of 12 and 23 RSSs in a synaptic complex is critical for DNA cleavage and it seems to be facilitated by the DNA bending proteins HMG1 and HMG2 as well as other yet unidentified cellular factors (Fig. 1) . Within the synaptic complex, RAG-1/RAG-2 efficiently introduce a nick at each RSS via a hydrolysis reaction at the heptamer/coding flank border, generating a 3′ hydroxyl end. Subsequently, a transesterification reaction that resembles the mechanism of transpositional recombination creates a double strand break as the free 3′ hydroxyl of the nicked strand is used in a nucleophilic attack on the opposing strand generating a covalently sealed hairpin intermediate, known as the hairpin coding end (5, 6). Opening and processing of this hairpin intermediate is a critical step in the generation of junctional diversity and has been the topic of intensive analysis in the last few years. Figure 1. Schematic representation of protein–DNA complexes in VDJ recombination. Blue and yellow rectangles represent the coding segments to be recombined (exemplified as Vλ and Jλ). The 12-RSS and 23-RSS are represented as white and black ... In vitro experiments demonstrated that after RSS cleavage, RAG-1/RAG-2 and HMG1 proteins remain bound in a complex with the resulting 5′ phosphorylated blunt signal end and the hairpin coding end in what is known as the postcleavage complex (7). At this stage, the generation of junctional diversity is initiated by opening of hairpin coding ends leading to the formation of open double strand ends, which may contain single stranded overhangs. Also at this stage, the open coding ends are potential substrates for nontemplate nucleotide additions by TdT, polymerase extension leading to palindromic nucleotides, nuclease attack generating small coding element deletions, and microhomology-directed nonhomologous end joining (NHEJ) via annealing of short complementary stretches of the overhangs. These coding end intermediates, arising at this late stage of VDJ recombination, have common branched structures characterized by duplex/single strand junctions lacking consensus binding motifs (8). Many of these recombination intermediates could be processed by the nuclease activity of Artemis, RAG-1/RAG-2, or the Mre11/Rad50/NBS1 (MRN) complex. Evidence supporting the participation of these protein complexes in hairpin coding end opening and processing will be further discussed. Ultimately, the VDJ recombination reaction is concluded with the joining of the processed coding ends and the unprocessed signal ends by the ubiquitously expressed double strand break repair proteins. Although it is not clearly known when the double strand break repair factors are incorporated in the recombination complex, it is plausible that they coexist with RAG-1/RAG-2 during the opening and processing of the coding ends as suggested in the scheme presented in Fig. 1. Furthermore, evidence accumulated so far suggests that coding and signal joints are formed independently (4), which argues for the existence of different protein–DNA complexes during the later stages of VDJ recombination.

DNA Ligase IV regulates XRCC4 nuclear localization

DNA Ligase IV, along with its interacting partner XRCC4, are essential for repairing DNA double strand breaks by non-homologous end joining (NHEJ). Together, they complete the final ligation step resolving the DNA break. Ligase IV is regulated by XRCC4 and XLF. However, the mechanism(s) by which Ligase IV control the NHEJ reaction and other NHEJ factor(s) remains poorly characterized. Here, we show that a C-terminal region of Ligase IV (aa 620-800), which encompasses a NLS, the BRCT I, and the XRCC4 interacting region (XIR), is essential for nuclear localization of its co-factor XRCC4. In Ligase IV deficient cells, XRCC4 showed deregulated localization remaining in the cytosol even after induction of DNA double strand breaks. DNA Ligase IV was also required for efficient localization of XLF into the nucleus. Additionally, human fibroblasts that harbor hypomorphic mutations within the Ligase IV gene displayed decreased levels of XRCC4 protein, implicating that DNA Ligase IV is also regulating XRCC4 stability. Our results provide evidence for a role of DNA Ligase IV in controlling the cellular localization and protein levels of XRCC4.

Structure and dynamics of an intrinsically disordered protein region that partially folds upon binding by chemical-exchange NMR

Many intrinsically disordered proteins (IDPs) and protein regions (IDRs) engage in transient, yet specific, interactions with a variety of protein partners. Often, if not always, interactions with a protein partner lead to partial folding of the IDR. Characterizing the conformational space of such complexes is challenging: in solution-state NMR, signals of the IDR in the interacting region become broad, weak, and often invisible, while X-ray crystallography only provides information on fully ordered regions. There is thus a need for a simple method to characterize both fully and partially ordered regions in the bound state of IDPs. Here, we introduce an approach based on monitoring chemical exchange by NMR to investigate the state of an IDR that folds upon binding through the observation of the free state of the protein. Structural constraints for the bound state are obtained from chemical shifts, and site-specific dynamics of the bound state are characterized by relaxation rates. The conformation of the interacting part of the IDR was determined and subsequently docked onto the structure of the folded partner. We apply the method to investigate the interaction between the disordered C-terminal region of Artemis and the DNA binding domain of Ligase IV. We show that we can accurately reproduce the structure of the core of the complex determined by X-ray crystallography and identify a broader interface. The method is widely applicable to the biophysical investigation of complexes of disordered proteins and folded proteins.