Iron Toxicity in the Retina Requires Alu RNA and the NLRP3 Inflammasome.

Excess iron induces tissue damage and is implicated in age-related macular degeneration (AMD). Iron toxicity is widely attributed to hydroxyl radical formation through Fenton's reaction. We report that excess iron, but not other Fenton catalytic metals, induces activation of the NLRP3 inflammasome, a pathway also implicated in AMD. Additionally, iron-induced degeneration of the retinal pigmented epithelium (RPE) is suppressed in mice lacking inflammasome components caspase-1/11 or Nlrp3 or by inhibition of caspase-1. Iron overload increases abundance of RNAs transcribed from short interspersed nuclear elements (SINEs): Alu RNAs and the rodent equivalent B1 and B2 RNAs, which are inflammasome agonists. Targeting Alu or B2 RNA prevents iron-induced inflammasome activation and RPE degeneration. Iron-induced SINE RNA accumulation is due to suppression of DICER1 via sequestration of the co-factor poly(C)-binding protein 2 (PCBP2). These findings reveal an unexpected mechanism of iron toxicity, with implications for AMD and neurodegenerative diseases associated with excess iron.

Calpain 5 is highly expressed in the central nervous system (CNS), carries dual nuclear localization signals, and is associated with nuclear promyelocytic leukemia protein bodies.

Calpain 5 (CAPN5) is a non-classical member of the calpain family. It lacks the EF hand motif characteristic of classical calpains but retains catalytic and Ca(2+) binding domains, and it contains a unique C-terminal domain. TRA-3, an ortholog of CAPN5, has been shown to be involved in necrotic cell death in Caenorhabditis elegans. CAPN5 is expressed throughout the CNS, but its expression relative to other calpains and subcellular distribution has not been investigated previously. Based on relative mRNA levels, Capn5 is the second most highly expressed calpain in the rat CNS, with Capn2 mRNA being the most abundant. Unlike classical calpains, CAPN5 is a non-cytosolic protein localized to the nucleus and extra-nuclear locations. CAPN5 possesses two nuclear localization signals (NLS): an N-terminal monopartite NLS and a unique bipartite NLS closer to the C terminus. The C-terminal NLS contains a SUMO-interacting motif that contributes to nuclear localization, and mutation or deletion of both NLS renders CAPN5 exclusively cytosolic. Dual NLS motifs are common among transcription factors. Interestingly, CAPN5 is found in punctate domains associated with promyelocytic leukemia (PML) protein within the nucleus. PML nuclear bodies are implicated in transcriptional regulation, cell differentiation, cellular response to stress, viral defense, apoptosis, and cell senescence as well as protein sequestration, modification, and degradation. The roles of nuclear CAPN5 remain to be determined.

Synthesis and functional analysis of novel bivalent estrogens.

The steroid hormone estrogen plays a critical role in female development and homeostasis. Estrogen mediates its effects through binding and activation of specific estrogen receptors alpha (ERalpha) and beta (ERbeta), members of the steroid/nuclear receptor family of ligand-induced transcription factors. Due to their intimate roles in genomic and nongenomic signaling pathways, these hormones and their receptors have been also implicated in the pathologies of a variety of cancers and metabolic disorders, and have been the target of large therapeutic development efforts. The binding of estrogen to its respective receptors initiates a cascade of events that include receptor dimerization, nuclear localization, DNA binding and recruitment of co-regulatory protein complexes. In this manuscript, we investigate the potential for manipulating steroid receptor gene expression activity through the development of bivalent steroid hormones that are predicted to facilitate hormone receptor dimerization events. Data are presented for the development and testing of novel estrogen dimers, linked through their C-17 moiety, that can activate estrogen receptor alpha (ERalpha)-mediated transcription events with efficacy and potency equal to or greater than that of ERalpha's cognate ligand, 17beta-estradiol. These bivalent estrogen structures open the door to the development of a variety of steroid therapeutics that could dramatically impact future drug development in this area.

Tuberin nuclear localization can be regulated by phosphorylation of its carboxyl terminus.

Tuberin, the tuberous sclerosis 2 (TSC2) gene product, has been identified as a tumor suppressor protein genetically implicated in the pathology of tuberous sclerosis and the female-specific lung disease lymphangioleiomyomatosis. Tuberin and its predominant cytoplasmic binding partner hamartin have been shown to complex with a variety of intracellular signaling regulators and affect the processes of protein translation, cellular proliferation, cellular migration, and cellular transcription. In previous studies, we have presented evidence for tuberin binding to the calcium-dependent intracellular signaling protein calmodulin (CaM), overlap of tuberin CaM binding domain with a binding domain for estrogen receptor alpha, and the phosphorylation-associated nuclear localization of tuberin. In the study presented here, we expand our findings on the mechanism of tuberin nuclear localization to show that the CaM-estrogen receptor-alpha binding domain of tuberin can also serve as a tuberin nuclear localization sequence. Furthermore, we identify an Akt/p90 ribosomal S6 kinase-1 phosphorylation site within the carboxyl terminus of tuberin that can regulate tuberin nuclear localization and significantly affect the ability of tuberin to modulate estrogen genomic signaling events. These findings suggest a link between tuberin nuclear localization and a variety of intracellular signaling events that have direct implications with respect to the role of tuberin in the pathology of tuberous sclerosis and lymphangioleiomyomatosis.

Photo-activated affinity-site cross-linking of antibodies using tryptophan containing peptides.

Affinity-based conjugation methods for antibodies can produce defined and reproducible conjugates. This requires that the target antibody has an affinity site for the ligand and that the ligand has a reactive site. These requirements are critical for the conjugation of antibodies designed for diagnostic and therapeutic application. Our laboratory has discovered a novel affinity of antibodies for the amino acid tryptophan using an azido derivative of tryptophan. Here we show that tryptophan without the azido group can be photo-cross-linked to antibodies. Biotinylated tryptophan peptides are photolysed into monoclonal and polyclonal antibodies and such biotinylated antibodies are used in avidin-based ELISA. With the simple and gentle tryptophan-affinity photo-conjugation of peptides, antibodies can be conjugated with peptides to enhance their potency and expand their targeting range.

Cross-talk between tuberin, calmodulin, and estrogen signaling pathways.

Lymphangioleiomyomatosis (LAM) is a rare disease that occurs primarily in women and has been linked to both estrogen-mediated signaling events and mutations associated with the tuberous sclerosis complex 2 gene product tuberin. These two observations fostered the hypothesis that tuberin's impact on estrogen-mediated signaling might be through a direct interaction with the intracellular receptor for estrogen, estrogen receptor alpha (ERalpha). In the study presented here, tuberin was shown to co-immunoprecipitate and directly bind ERalpha through a domain localized within the carboxyl 73 amino acids of tuberin. This domain had previously been shown to serve as a binding domain for the intracellular calcium signaling molecule calmodulin (CaM). Competition binding studies identified a potential competitive relationship for binding of tuberin by ERalpha and CaM. Additionally, tuberin-ERalpha interactions were found to be modulated by the presence of tuberin's predominant intracellular binding partner hamartin, suggesting that tuberin-hamartin interactions negatively impact the ability of tuberin to modulate ERalpha-mediated gene transcription events. Cumulatively, data presented here support the hypothesis that interactions between tuberin, ERalpha, and CaM may play a critical role in the pathology of LAM disease.

A neuronal-specific differentiation protein that directly modulates retinoid receptor transcriptional activation.

BACKGROUND: The specificity of a nuclear receptor's ability to modulate gene expression resides in its ability to bind a specific lipophilic ligand, associate with specific dimerization partners and bind specific DNA sequences in the promoter regions of genes. This sequence of events appears to be the basis for targeting an additional regulatory complex composed of a variety of protein and RNA components that deliver signals for facilitation or inhibition of the RNA polymerase complex. Characterization of the tissue and cell-specific components of these coregulatory complexes appear to be integral to our understanding of nuclear receptor regulation of transcription. RESULTS: A novel yeast screen sensitive to retinoid-X receptor (RXR) transcriptional activation resulted in the isolation of the rat homologue of the mouse NPDC-1 gene. NPDC-1 has been shown to be involved in the control of neural cell proliferation and differentiation, possibly through interactions with the cell cycle promoting transcription factor E2F-1. Although the amino acid sequence of NPDC-1 is highly conserved between mouse, rat and human homologues, their tissue specific expression was seen to vary. A potential for direct protein:protein interaction between NPDC-1, RXR and retinoic acid receptor beta (RARbeta) was observed in vitro and NPDC-1 facilitated RXR homodimer and RAR-RXR heterodimer DNA binding in vitro. Expression of NPDC-1 was also observed to repress transcription mediated by retinoid receptors as well as by several other nuclear receptor family members, although not in a universal manner. CONCLUSIONS: These results suggest that NPDC-1, through direct interaction with retinoid receptors, functions to enhance the transcription complex formation and DNA binding function of retinoid receptors, but ultimately repress retinoid receptor-mediated gene expression. As with NPDC-1, retinoids and their receptors have been implicated in brain development and these data provide a point of convergence for NPDC-1 and retinoid mediation of neuronal differentiation.

Enhanced anti-B-cell tumor effects with anti-CD20 superantibody.

The data presented here describe a novel approach to enhance the use of antibodies in diagnostic and therapeutic applications. Using a peptide copied from a rare self-binding (autophilic) antibody structure, the authors were able to convert by chemical cross-linking an anti-CD20 antibody to a self-binding (autophilic) structure. The autophilic antibody exhibited better binding to target tumor cells than the naked antibody. By the mechanism of hyper-cross-linking a B-cell receptor (CD20) on tumor cells, the rate of apoptosis is significantly increased, leading to strong inhibition of tumor growth in culture. The demonstration of enhanced binding and apoptosis targeting the CD20 B-cell marker serves as an example for developing second-generation therapeutic antibodies against non-Hodgkin lymphoma.

A calmodulin binding site in the tuberous sclerosis 2 gene product is essential for regulation of transcription events and is altered by mutations linked to tuberous sclerosis and lymphangioleiomyomatosis.

Mutations in the tuberous sclerosis 2 (TSC2) gene product have been genetically linked to the pathology of both tuberous sclerosis (TSC) and the gender-specific lung disease, lymphangioleiomyomatosis (LAM). Both diseases are classified as disorders of cellular migration, proliferation, and differentiation. Earlier studies from our laboratory (1) linked TSC2 with steroid/nuclear receptor signaling. Studies presented here provide evidence for calmodulin (CaM) signaling in the propagation of this TSC2 activity. Far Western screening of a lambda phage human brain cDNA library to identify interacting proteins for the TSC2 gene product (tuberin) yielded multiple clones encoding human CaM. Direct binding with 32P-labeled tuberin demonstrated Ca2+-dependent binding to CaM-Sepharose which was lost upon deletion of the C-terminal 72 residues. The sequence (1740)WIARLRHIKRLRQRIC(1755) was identified as one capable of forming a basic amphipathic helix indicative of CaM binding domains in known calmodulin binding proteins. Studies with a synthetic peptide of this sequence demonstrated very tight Ca2+-dependent binding to CaM as judged by tryptophan fluorescence perturbation studies and phosphodiesterase activation by CaM. Deletion mutagenesis studies further suggested that this CaM binding domain is required for tuberin modulation of steroid receptor function and that mutations in this region may be involved in the pathology of TSC and LAM.