The mechanism of vault opening from the high resolution structure of the N-terminal repeats of MVP

Vaults are ubiquitous ribonucleoprotein complexes involved in a diversity of cellular processes, including multidrug resistance, transport mechanisms and signal transmission. The vault particle shows a barrel-shaped structure organized in two identical moieties, each consisting of 39 copies of the major vault protein MVP. Earlier data indicated that vault halves can dissociate at acidic pH. The crystal structure of the vault particle solved at 8 Å resolution, together with the 2.1-Å structure of the seven N-terminal domains (R1–R7) of MVP, reveal the interactions governing vault association and provide an explanation for a reversible dissociation induced by low pH. The structural comparison with the recently published 3.5 Å model shows major discrepancies, both in the main chain tracing and in the side chain assignment of the two terminal domains R1 and R2

Novel CCL21-Vault Nanocapsule Intratumoral Delivery Inhibits Lung Cancer Growth

Based on our preclinical findings, we are assessing the efficacy of intratumoral injection of dendritic cells (DC) transduced with an adenoviral vector expressing the secondary lymphoid chemokine (CCL21) gene (Ad-CCL21-DC) in a phase I trial in advanced non-small cell lung cancer (NSCLC). While this approach shows immune enhancement, the preparation of autologous DC for CCL21 genetic modification is cumbersome, expensive and time consuming. We are evaluating a non-DC based approach which utilizes vault nanoparticles for intratumoral CCL21 delivery to mediate antitumor activity in lung cancer.Here we describe that vault nanocapsule platform for CCL21 delivery elicits antitumor activity with inhibition of lung cancer growth. Vault nanocapsule packaged CCL21 (CCL21-vaults) demonstrated functional activity in chemotactic and antigen presenting activity assays. Recombinant vaults impacted chemotactic migration of T cells and this effect was predominantly CCL21 dependent as CCL21 neutralization abrogated the CCL21 mediated enhancement in chemotaxis. Intratumoral administration of CCL21-vaults in mice bearing lung cancer enhanced leukocytic infiltrates (CXCR3(+)T, CCR7(+)T, IFNγ(+)T lymphocytes, DEC205(+) DC), inhibited lung cancer tumor growth and reduced the frequencies of immune suppressive cells [myeloid derived suppressor cells (MDSC), T regulatory cells (Treg), IL-10 T cells]. CCL21-vaults induced systemic antitumor responses by augmenting splenic T cell lytic activity against parental tumor cells.This study demonstrates that the vault nanocapsule can efficiently deliver CCL21 to sustain antitumor activity and inhibit lung cancer growth. The vault nanocapsule can serve as an "off the shelf" approach to deliver antitumor cytokines to treat a broad range of malignancies

Selection and characterisation of a phage-displayed human antibody (Fab) reactive to the lung resistance-related major vault protein

The major vault protein is the main component on multimeric vault particles, that are likely to play an essential role in normal cell physiology and to be associated with multidrug resistance of tumour cells. In order to unravel the function of vaults and their putative contribution to multidrug resistance, specific antibodies are invaluable tools. Until now, only conventional major vault protein-reactive murine monoclonal antibodies have been generated, that are most suitable for immunohistochemical analyses. The phage display method allows for selection of human antibody fragments with potential use in clinical applications. Furthermore, cDNA sequences encoding selected antibody fragments are readily identified, facilitating various molecular targeting approaches. In order to obtain such human Fab fragments recognising major vault protein we used a large non-immunized human Fab fragment phage library. Phages displaying major vault protein-reactive Fabs were obtained through several rounds of selection on major vault protein-coated immunotubes and subsequent amplification in TG1 E coli bacteria. Eventually, one major vault protein-reactive clone was selected and further examined. The anti-major vault protein Fab was found suitable for immunohistochemical and Western blot analysis of tumour cell lines and human tissues. BIAcore analysis showed that the binding affinity of the major vault protein-reactive clone almost equalled that of the murine anti-major vault protein Mabs. The cDNA sequence of this human Fab may be exploited to generate an intrabody for major vault protein-knock out studies. Thus, this human Fab fragment should provide a valuable tool in elucidating the contribution(s) of major vault protein/vaults to normal physiology and cellular drug resistance mechanisms

Recruitment of the Major Vault Protein by InlK: A Listeria monocytogenes Strategy to Avoid Autophagy

L. monocytogenes is a facultative intracellular bacterium responsible for listeriosis. It is able to invade, survive and replicate in phagocytic and non-phagocytic cells. The infectious process at the cellular level has been extensively studied and many virulence factors have been identified. Yet, the role of InlK, a member of the internalin family specific to L. monocytogenes, remains unknown. Here, we first show using deletion analysis and in vivo infection, that InlK is a bona fide virulence factor, poorly expressed in vitro and well expressed in vivo, and that it is anchored to the bacterial surface by sortase A. We then demonstrate by a yeast two hybrid screen using InlK as a bait, validated by pulldown experiments and immunofluorescence analysis that intracytosolic bacteria via an interaction with the protein InlK interact with the Major Vault Protein (MVP), the main component of cytoplasmic ribonucleoproteic particules named vaults. Although vaults have been implicated in several cellular processes, their role has remained elusive. Our analysis demonstrates that MVP recruitment disguises intracytosolic bacteria from autophagic recognition, leading to an increased survival rate of InlK over-expressing bacteria compared to InlK− bacteria. Together these results reveal that MVP is hijacked by L. monocytogenes in order to counteract the autophagy process, a finding that could have major implications in deciphering the cellular role of vault particles

Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets

<p>Abstract</p> <p>Background</p> <p>MeCP2, methyl-CpG-binding protein 2, binds to methylated cytosines at CpG dinucleotides, as well as to unmethylated DNA, and affects chromatin condensation. <it>MECP2 </it>mutations in females lead to Rett syndrome, a neurological disorder characterized by developmental stagnation and regression, loss of purposeful hand movements and speech, stereotypic hand movements, deceleration of brain growth, autonomic dysfunction and seizures. Most mutations occur <it>de novo </it>during spermatogenesis. Located at Xq28, <it>MECP2 </it>is subject to X inactivation, and affected females are mosaic. Rare hemizygous males suffer from a severe congenital encephalopathy.</p> <p>Methods</p> <p>To identify the pathways mis-regulated by MeCP2 deficiency, microarray-based global gene expression studies were carried out in cerebellum of <it>Mecp2 </it>mutant mice. We compared transcript levels in mutant/wildtype male sibs of two different MeCP2-deficient mouse models at 2, 4 and 8 weeks of age. Increased transcript levels were evaluated by real-time quantitative RT-PCR. Chromatin immunoprecipitation assays were used to document <it>in vivo </it>MeCP2 binding to promoter regions of candidate target genes.</p> <p>Results</p> <p>Of several hundred genes with altered expression levels in the mutants, twice as many were increased than decreased, and only 27 were differentially expressed at more than one time point. The number of misregulated genes was 30% lower in mice with the exon 3 deletion (<it>Mecp2</it>^tm1.1Jae) than in mice with the larger deletion (<it>Mecp2</it>^tm1.1Bird). Between the mutants, few genes overlapped at each time point. Real-time quantitative RT-PCR assays validated increased transcript levels for four genes: <it>Irak1</it>, interleukin-1 receptor-associated kinase 1; <it>Fxyd1</it>, phospholemman, associated with Na, K-ATPase;<it>Reln</it>, encoding an extracellular signaling molecule essential for neuronal lamination and synaptic plasticity; and <it>Gtl2/Meg3</it>, an imprinted maternally expressed non-translated RNA that serves as a host gene for C/D box snoRNAs and microRNAs. Chromatin immunoprecipitation assays documented <it>in vivo </it>MeCP2 binding to promoter regions of <it>Fxyd1, Reln</it>, and <it>Gtl2</it>.</p> <p>Conclusion</p> <p>Transcriptional profiling of cerebellum failed to detect significant global changes in <it>Mecp2</it>-mutant mice. Increased transcript levels of <it>Irak1, Fxyd1, Reln</it>, and <it>Gtl2 </it>may contribute to the neuronal dysfunction in MeCP2-deficient mice and individuals with Rett syndrome. Our data provide testable hypotheses for future studies of the regulatory or signaling pathways that these genes act on.</p

Wdr74 Is Required for Blastocyst Formation in the Mouse

Preimplantation is a dynamic developmental period during which a combination of maternal and zygotic factors program the early embryo resulting in lineage specification and implantation. A reverse genetic RNAi screen in mouse embryos identified the WD Repeat Domain 74 gene (Wdr74) as being required for these critical first steps of mammalian development. Knockdown of Wdr74 results in embryos that develop normally until the morula stage but fail to form blastocysts or properly specify the inner cell mass and trophectoderm. In Wdr74-deficient embryos, we find activated Trp53-dependent apoptosis as well as a global reduction of RNA polymerase I, II and III transcripts. In Wdr74-deficient embryos blocking Trp53 function rescues blastocyst formation and lineage differentiation. These results indicate that Wdr74 is required for RNA transcription, processing and/or stability during preimplantation development and is an essential gene in the mouse

Lung resistance-related protein as a predictor of clinical outcome in advanced testicular germ-cell tumours

This study was undertaken to investigate the expression and predictive value for outcome of multidrug resistance-associated (MDR) proteins P-glycoprotein (Pgp), MRP1, BCRP, and LRP, in advanced testicular germ-cell tumours (TGCT). Paraffin-embedded sections from 56 previously untreated patients with metastatic TGCT were immunostained for Pgp, MRP1, BCRP, and LRP. All patients received platinum-based chemotherapy after orchidectomy. Immunostaining was related to clinicopathological parameters, response to chemotherapy, and outcome. Strong and intermediate expressions of the different MDR-related proteins were: 27 and 41% (Pgp), 54 and 37% (MRP1), 86 and 7% (BCRP), and 14 and 29% (LRP). P-glycoprotein and MRP1 associated, respectively, to low AFP (P=0.026) and high LDH levels (P=0.014), whereas LRP expression associated with high beta-hCG levels (P=0.003) and stage IV tumours (P=0.029). No correlation was found between Pgp, MRP1, and BCRP expression and response to chemotherapy and survival. In contrast, patients with LRP-positive tumours (strong or intermediate expression) had shorter progression-free (P=0.0006) and overall survival (P=0.0116) than LRP-negative patients, even after individual log-rank adjustments by statistically associated variables. Our data suggest that a positive LRP immunostaining at the time of diagnosis in metastatic TGCT is associated with an adverse clinical outcome