Molecular Mechanisms of DNA Sequence Selectivity in V(D)J Recombination.

Antigen receptor (AgR) diversity is central to the ability of adaptive immunity in jawed vertebrates to protect against pathogenic agents. The production of highly diverse AgR repertoires is initiated during B and T cell lymphopoiesis by V(D)J recombination, which assembles the receptor genes from component gene segments in a cut-and-paste recombination reaction. Recombination activating proteins, RAG1 and RAG2 (RAG1/2), catalyze V(D)J recombination by cleaving adjacent to recombination signal sequences (RSSs) that flank AgR gene segments.

An updated definition of V(D)J recombination signal sequences revealed by high-throughput recombination assays.

In the adaptive immune system, V(D)J recombination initiates the production of a diverse antigen receptor repertoire in developing B and T cells. Recombination activating proteins, RAG1 and RAG2 (RAG1/2), catalyze V(D)J recombination by cleaving adjacent to recombination signal sequences (RSSs) that flank antigen receptor gene segments. Previous studies defined the consensus RSS as containing conserved heptamer and nonamer sequences separated by a less conserved 12 or 23 base-pair spacer sequence.

Mechanism of Anti-Inflammatory Activity of TLR4-Interacting SPA4 Peptide.

The TLR4-interacting SPA4 peptide suppresses inflammation. We assessed the structural and physicochemical properties and binding of SPA4 peptide to TLR4-MD2. We also studied the changes at the whole transcriptome level, cell morphology, viability, secreted cytokines and chemokines, and cell influx in cell systems and mouse models challenged with LPS and treated with SPA4 peptide.

Full length RAG2 expression enhances the DNA damage response in pre-B cells.

V(D)J recombination by the RAG1 and RAG2 protein complex in developing lymphocytes includes DNA double strand break (DSB) intermediates. RAG2 undergoes export from the nucleus and enrichment at the centrosome minutes following production of DSBs by genotoxic stress, suggesting that RAG2 participates in cellular responses to DSBs such as those generated during V(D)J recombination. To determine the effect of RAG2 expression on cell viability following DSB generation, we measured pre-B cells that expressed either full length (FL) wild-type RAG2, or a T490A mutant of RAG2 that has increased stability and fails to undergo nuclear export following generation of DSBs.

RAG2 localization and dynamics in the pre-B cell nucleus.

RAG2 of the V(D)J recombinase is essential for lymphocyte development. Within the RAG2 noncore region is a plant homeodomain (PHD) that interacts with the modified histone H3K4me3, and this interaction is important for relieving inhibition of the RAG recombinase for V(D)J recombination. However, the effect of the noncore region on RAG2 localization and dynamics in cell nuclei is poorly understood.

Riches in RAGs: Revealing the V(D)J Recombinase through High-Resolution Structures.

Development of the adaptive immune system is dependent on V(D)J recombination, which forms functional antigen receptor genes through rearrangement of component gene segments. The V(D)J recombinase, comprising recombination-activating proteins RAG1 and RAG2, guides the initial DNA cleavage events to the recombination signal sequence (RSS), which flanks each gene segment. Although the enzymatic steps for RAG-mediated endonucleolytic activity were established over two decades ago, only recently have high-resolution structural studies of the catalytically active core regions of the RAG proteins shed light on conformational requirements for the reaction.

The B3 Subunit of the Cone Cyclic Nucleotide-gated Channel Regulates the Light Responses of Cones and Contributes to the Channel Structural Flexibility.

Cone photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in cone phototransduction, which is a process essential for daylight vision, color vision, and visual acuity. Mutations in the cone channel subunits CNGA3 and CNGB3 are associated with human cone diseases, including achromatopsia, cone dystrophies, and early onset macular degeneration. Mutations in CNGB3 alone account for 50% of reported cases of achromatopsia.

Structure of a TLR4-interacting SPA4 peptide.

We have recently identified a Toll-like receptor (TLR4)-interacting SPA4 peptide encoding amino acids: GDFRYSDGTPVNYTNWYRGE, a shorter region of human surfactant protein-A (SP-A). The SPA4 peptide suppressed lipopolysaccharide-induced inflammation (). In this report, we examined the structure of synthetic SPA4 peptide in solution by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy.

An interdomain boundary in RAG1 facilitates cooperative binding to RAG2 in formation of the V(D)J recombinase complex.

V(D)J recombination assembles functional antigen receptor genes during lymphocyte development. Formation of the recombination complex containing the recombination activating proteins, RAG1 and RAG2, is essential for the site-specific DNA cleavage steps in V(D)J recombination. However, little is known concerning how complex formation leads to a catalytically-active complex.

Spatio-temporal regulation of RAG2 following genotoxic stress.

V(D)J recombination of lymphocyte antigen receptor genes occurs via the formation of DNA double strand breaks (DSBs) through the activity of RAG1 and RAG2. The co-existence of RAG-independent DNA DSBs generated by genotoxic stressors potentially increases the risk of incorrect repair and chromosomal abnormalities. However, it is not known whether cellular responses to DSBs by genotoxic stressors affect the RAG complex.

Glycosylation of Skp1 affects its conformation and promotes binding to a model f-box protein.

In the social amoeba Dictyostelium, Skp1 is hydroxylated on proline 143 and further modified by three cytosolic glycosyltransferases to yield an O-linked pentasaccharide that contributes to O2 regulation of development. Skp1 is an adapter in the Skp1/cullin1/F-box protein family of E3 ubiquitin ligases that targets specific proteins for polyubiquitination and subsequent proteasomal degradation. To investigate the biochemical consequences of glycosylation, untagged full-length Skp1 and several of its posttranslationally modified isoforms were expressed and purified to near homogeneity using recombinant and in vitro strategies.

Structural characterization of the second intra-discal loop of the photoreceptor tetraspanin RDS.

Vertebrate photoreceptors contain a unique tetraspanin protein known as 'retinal degeneration slow' (RDS). Mutations in the RDS gene have been identified in a variety of human retinal degenerative diseases, and more than 70% of these mutations are located in the second intra-discal (D2) loop, highlighting the importance of this region. Here we examined the conformational and thermal stability properties of the D2 loop of RDS, as well as interactions with ROM-1, a non-glycosylated homolog of RDS.

A unique loop structure in oncostatin M determines binding affinity toward oncostatin M receptor and leukemia inhibitory factor receptor.

Oncostatin M (OSM) and leukemia inhibitory factor are pleiotropic cytokines that belong to the interleukin-6 (IL-6) family. These cytokines play a crucial role in diverse biological events like inflammation, neuroprotection, hematopoiesis, metabolism, and development. The family is grouped together based on structural similarities and their ability to activate the transmembrane receptor glycoprotein 130 (gp130).

Mechanism involved in the modulation of photoreceptor-specific cyclic nucleotidegated channel by the tyrosine kinase adapter protein Grb14.

We recently found that growth factor receptor-bound (Grb) protein 14 is a novel physiological modulator of photoreceptor specific cyclic nucleotide-gated channel alpha subunit (CNGA1). Grb14 promotes the CNG channel closure through its Ras-associating (RA) domain. In the current study we show that this RA domain-mediated inhibition of rod CNG channel is electrostatic in nature.

Elucidating the domain architecture and functions of non-core RAG1: the capacity of a non-core zinc-binding domain to function in nuclear import and nucleic acid binding.

Background: The repertoire of the antigen-binding receptors originates from the rearrangement of immunoglobulin and T-cell receptor genetic loci in a process known as V(D)J recombination. The initial site-specific DNA cleavage steps of this process are catalyzed by the lymphoid specific proteins RAG1 and RAG2. The majority of studies on RAG1 and RAG2 have focused on the minimal, core regions required for catalytic activity.

Binding to lipid membrane induces conformational changes in RPE65: implications for its isomerohydrolase activity.

The visual cycle is a multi-step pathway to recycle 11-cis retinal, the chromophore for both rod and cone visual pigments. The isomerohydrolase RPE65, a membrane-associated enzyme, converts atRE (all-trans-retinyl ester) to 11-cis-retinol, a key step in the visual cycle. Previously, it has been shown that membrane association of RPE65 is essential for its catalytic activity.

The disease-causing mutations in the carboxyl terminus of the cone cyclic nucleotide-gated channel CNGA3 subunit alter the local secondary structure and interfere with the channel active conformational change.

The cone photoreceptor cyclic nucleotide-gated (CNG) channel plays a pivotal role in phototransducton. Mutations in the channel subunits are associated with achromatopsia and progressive cone dystrophy in humans. More than 50 mutations have been identified in the channel CNGA3 subunit, with 50% of them located in the carboxyl (C) terminus.

A zinc site in the C-terminal domain of RAG1 is essential for DNA cleavage activity.

The recombination-activating protein, RAG1, a key component of the V(D)J recombinase, binds multiple Zn(2+) ions in its catalytically required core region. However, the role of zinc in the DNA cleavage activity of RAG1 is not well resolved. To address this issue, we determined the stoichiometry of Zn(2+) ions bound to the catalytically active core region of RAG1 under various conditions.

A non-sequence-specific DNA binding mode of RAG1 is inhibited by RAG2.

RAG1 and RAG2 proteins catalyze site-specific DNA cleavage reactions in V(D)J recombination, a process that assembles antigen receptor genes from component gene segments during lymphocyte development. The first step towards the DNA cleavage reaction is the sequence-specific association of the RAG proteins with the conserved recombination signal sequence (RSS), which flanks each gene segment in the antigen receptor loci. Questions remain as to the contribution of each RAG protein to recognition of the RSS.

Thermal dependency of RAG1 self-association properties.

Background: Functional immunoglobulin and T cell receptor genes are produced in developing lymphocytes by V(D)J recombination. The initial site-specific DNA cleavage steps in this process are catalyzed by the V(D)J recombinase, consisting of RAG1 and RAG2, which is directed to appropriate DNA cleavage sites by recognition of the conserved recombination signal sequence (RSS). RAG1 contains both the active site and the RSS binding domains, although RAG2 is also required for DNA cleavage activity.

DNA cleavage of a cryptic recombination signal sequence by RAG1 and RAG2. Implications for partial V(H) gene replacement.

Antibody and T cell receptor genes are assembled from gene segments by V(D)J recombination to produce an almost infinitely diverse repertoire of antigen specificities. Recombination is initiated by cleavage of conserved recombination signal sequences (RSS) by RAG1 and RAG2 during lymphocyte development. Recent evidence demonstrates that recombination can occur at noncanonical RSS sites within Ig genes or at other loci, outside the context of normal lymphocyte receptor gene rearrangement.

DNA cleavage activity of the V(D)J recombination protein RAG1 is autoregulated.

RAG1 and RAG2 catalyze the first DNA cleavage steps in V(D)J recombination. We demonstrate that the isolated central domain of RAG1 has inherent single-stranded (ss) DNA cleavage activity, which does not require, but is enhanced by, RAG2. The central domain, therefore, contains the active-site residues necessary to perform hydrolysis of the DNA phosphodiester backbone.

Putting the pieces together: identification and characterization of structural domains in the V(D)J recombination protein RAG1.

V(D)J recombination generates functional immunoglobulin and T-cell receptor genes in developing lymphocytes. The recombination-activating gene 1 (RAG1) and RAG2 proteins catalyze site-specific DNA cleavage in this recombination process. Biochemical studies have identified catalytically active regions of each protein, referred to as the core regions.

Biochemical and structural characterization of the interaction of memapsin 2 (beta-secretase) cytosolic domain with the VHS domain of GGA proteins.

Memapsin 2 (beta-secretase) is a membrane-associated aspartic protease that initiates the hydrolysis of beta-amyloid precursor protein (APP) leading to the production of amyloid-beta and the onset of Alzheimer's disease (AD). Both memapsin 2 and APP are transported from the cell surface to endosomes where APP hydrolysis takes place. Thus, the intracellular transport mechanism of memapsin 2 is important for understanding the pathogenesis of AD.