The mechanism of vault opening from the high resolution structure of the N-terminal repeats of MVP.
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ABSTRACT:
SUBMITTER: Querol-Audi J
PROVIDER: S-EPMC2752026 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cellular vaults are ubiquitous 13 mega Da multi-subunit ribonuceloprotein particles that may have a role in nucleocytoplasmic transport. Seventy percent of the vault's mass consists of a ?100 kDa protein, the major vault protein (MVP). In humans, a drug resistance-associated protein, originally identified as lung resistance protein in metastatic lung cancer, was ultimately shown to be the previously described MVP. In this study, a partial MVP sequence was cloned from channel catfish. Recombinant MVP (rMVP) was used to generate a monoclonal antibody that recognizes full length protein in distantly related fish species, as well as mice. MVP is expressed in fish spleen, liver, anterior kidney, renal kidney, and gills, with a consistent expression in epithelial cells, macrophages, or endothelium at the interface of the tissue and environment or vasculature. We show that vaults are distributed throughout cells of fish lymphoid cells, with nuclear and plasma membrane aggregations in some cells. Protein expression studies were extended to liver neoplastic lesions in Atlantic killifish collected in situ at the Atlantic Wood USA-EPA superfund site on the southern branch of the Elizabeth River, VA. MVP is highly expressed in these lesions, with intense staining at the nuclear membrane, similar to what is known about MVP expression in human liver neoplasia. Additionally, MVP mRNA expression was quantified in channel catfish ovarian cell line following treatment with different classes of pharmacological agents. Notably, mRNA expression is induced by ethidium bromide, which damages DNA. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:1981-1992, 2017. © 2017 Wiley Periodicals, Inc.
| S-EPMC5742863 | biostudies-literature
Project description:Rationale: Bone homeostasis is maintained by a balanced interplay of osteoblasts and osteoclasts. Osteoclasts are derived from monocyte/macrophage lineage. Major vault protein (MVP) is known to promote apoptosis and prevent metabolic diseases in macrophage. However, whether MVP is involved in osteoclastogenesis is unknown. Here, we identified an important function of MVP as a negative regulator of osteoclastogenesis and its therapeutic potential in preventing bone loss. Methods: Expression of MVP in osteoclasts was investigated in human tumor tissues with immunohistochemical staining. Next, we generated total body (Mvp-/- ) and monocyte-specific (Mvpf/fLyz2-Cre) MVP gene knockout mice to observe bone phenotype and osteoclastogenesis using micro-CT and bone histomorphometry. Moreover, we examined the effects of MVP on osteoclast differentiation, bone resorption, NFATc1 activation and calcium oscillations in vitro. Finally, we explored the clinical potential of targeting MVP in two osteoporosis mouse models and used an adeno-associated virus (AAV) gene to overexpress MVP locally in mice. Results: We found that Mvp-/- and Mvpf/fLyz2-Cre mice both exhibited osteoporosis-like phenotypes. MVP-deficiency also enhanced calcineurin-NFATc1 signaling and promoted NFATc1 activity, which led to enhanced osteoclastogenesis and bone resorption. Calcineurin inhibition using the small molecule inhibitor FK506 corrected the enhanced osteoclastogenesis in Mvpf/fLyz2-Cre group. Additionally, MVP reexpression in Mvpf/fLyz2-Cre group rescued calcineurin expression. MVP overexpression in wild-type mice prevented pathologic bone loss in mouse models of ovariectomized (OVX) and calvaria-adjacent lipopolysaccharide (LPS)-injected. Conclusions: Our data suggested that MVP negatively regulates osteoclast differentiation and bone resorption via inhibition of calcineurin-NFATc1 signaling. In osteoclast-related bone diseases such as osteoporosis, manipulation of MVP activity may be an attractive therapeutic target.
| S-EPMC8210610 | biostudies-literature
Project description:Vaults are the largest known cytoplasmic ribonucleoprotein structures and may function in innate immunity. The vault shell self-assembles from 96 copies of major vault protein and encapsulates two other proteins and a small RNA. We crystallized rat liver vaults and several recombinant vaults, all among the largest non-icosahedral particles to have been crystallized. The best crystals thus far were formed from empty vaults built from a cysteine-tag construct of major vault protein (termed cpMVP vaults), diffracting to about 9-A resolution. The asymmetric unit contains a half vault of molecular mass 4.65 MDa. X-ray phasing was initiated by molecular replacement, using density from cryo-electron microscopy (cryo-EM). Phases were improved by density modification, including concentric 24- and 48-fold rotational symmetry averaging. From this, the continuous cryo-EM electron density separated into domain-like blocks. A draft atomic model of cpMVP was fit to this improved density from 15 domain models. Three domains were adapted from a nuclear magnetic resonance substructure. Nine domain models originated in ab initio tertiary structure prediction. Three C-terminal domains were built by fitting poly-alanine to the electron density. Locations of loops in this model provide sites to test vault functions and to exploit vaults as nanocapsules.
| S-EPMC2229873 | biostudies-literature
Project description:Ribosomal RNA (rRNA) carries extensive 2'-O-methyl marks at functionally important sites. This simple chemical modification is thought to confer stability, promote RNA folding, and contribute to generate a heterogenous ribosome population with a yet-uncharacterized function. 2'-O-methylation occurs both in archaea and eukaryotes and is accomplished by the Box C/D RNP enzyme in an RNA-guided manner. Extensive and partially conflicting structural information exists for the archaeal enzyme, while no structural data is available for the eukaryotic enzyme. The yeast Box C/D RNP consists of a guide RNA, the RNA-primary binding protein Snu13, the two scaffold proteins Nop56 and Nop58, and the enzymatic module Nop1. Here we present the high-resolution structure of the eukaryotic Box C/D methyltransferase Nop1 from Saccharomyces cerevisiae bound to the amino-terminal domain of Nop56. We discuss similarities and differences between the interaction modes of the two proteins in archaea and eukaryotes and demonstrate that eukaryotic Nop56 recruits the methyltransferase to the Box C/D RNP through a protein-protein interface that differs substantially from the archaeal orthologs. This study represents a first achievement in understanding the evolution of the structure and function of these proteins from archaea to eukaryotes.
| S-EPMC7962484 | biostudies-literature
Project description:Here we attempt to describe the structure of Hs21 at high resolution using proximity ligation assays in combination with target enrichment in several conditions (yet to be determined). We will also generate additional epigenetic information that will help with the interpretation of the data.This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
2015-04-08 | E-ERAD-349 | biostudies-arrayexpress
Project description:Osteoclasts (OCs), derived from monocyte/macrophage lineage, are key orchestrators in bone remodeling. Targeting osteoclast apoptosis is a promising approach to cut down excessive osteoclast numbers, and thus slow down the rate of bone mass loss that inevitably occurs during aging. However, the therapeutic target of apoptosis in osteoclasts has not been fully studied. Our previous work generated Mvpf/fLyz2-Cre mice, conditionally depleting major vault protein (MVP) in monocyte lineage, and identified MVP as a bone protector for its negative role in osteoclastogenesis in vivo and in vitro. Here, we observed a notable decline of MVP in osteoclasts with aging in mice, encouraging us to further investigate the regulatory role of osteoclast MVP. Then, Mvpf/fLyz2-Cre mice were exploited in two osteoporosis contexts, aging and abrupt loss of estrogen, and we revealed that conditional knockout of MVP inhibited osteoclast apoptosis in vivo and in vitro. Moreover, we reported the interaction between MVP and death receptor Fas, and MVP-Fas signaling cascade was identified to positively regulate the apoptosis of osteoclasts, thus preventing osteoporosis. Collectively, our comprehensive discovery of MVP's regulatory role in osteoclasts provides new insight into osteoclast biology and therapeutic targets for osteoporosis.
| S-EPMC10500014 | biostudies-literature
Project description:IntroductionClinical studies suggest that obesity, in addition to promoting breast cancer aggressiveness, is associated with a decrease in chemotherapy efficacy, although the mechanisms involved remain elusive. As chemotherapy is one of the main treatments for aggressive or metastatic breast cancer, we investigated whether adipocytes can mediate resistance to doxorubicin (DOX), one of the main drugs used to treat breast cancer, and the mechanisms associated.MethodsWe used a coculture system to grow breast cancer cells with in vitro differentiated adipocytes as well as primary mammary adipocytes isolated from lean and obese patients. Drug cellular accumulation, distribution, and efflux were studied by immunofluorescence, flow cytometry, and analysis of extracellular vesicles. Results were validated by immunohistochemistry in a series of lean and obese patients with cancer.ResultsAdipocytes differentiated in vitro promote DOX resistance (with cross-resistance to paclitaxel and 5-fluorouracil) in a large panel of human and murine breast cancer cell lines independently of their subtype. Subcellular distribution of DOX was altered in cocultivated cells with decreased nuclear accumulation of the drug associated with a localized accumulation in cytoplasmic vesicles, which then are expelled into the extracellular medium. The transport-associated major vault protein (MVP), whose expression was upregulated by adipocytes, mediated both processes. Coculture with human mammary adipocytes also induced chemoresistance in breast cancer cells (as well as the related MVP-induced DOX efflux) and their effect was amplified by obesity. Finally, in a series of human breast tumors, we observed a gradient of MVP expression, which was higher at the invasive front, where tumor cells are at close proximity to adipocytes, than in the tumor center, highlighting the clinical relevance of our results. High expression of MVP in these tumor cells is of particular interest since they are more likely to disseminate to give rise to chemoresistant metastases.ConclusionsCollectively, our study shows that adipocytes induce an MVP-related multidrug-resistant phenotype in breast cancer cells, which could contribute to obesity-related chemoresistance.
| S-EPMC6337862 | biostudies-literature
Project description:BackgroundLong terminal repeats (LTRs, consisting of U3-R-U5 portions) are important elements of retroviruses and related retrotransposons. They are difficult to analyse due to their variability.The aim was to obtain a more comprehensive view of structure, diversity and phylogeny of LTRs than hitherto possible.ResultsHidden Markov models (HMM) were created for 11 clades of LTRs belonging to Retroviridae (class III retroviruses), animal Metaviridae (Gypsy/Ty3) elements and plant Pseudoviridae (Copia/Ty1) elements, complementing our work with Orthoretrovirus HMMs. The great variation in LTR length of plant Metaviridae and the few divergent animal Pseudoviridae prevented building HMMs from both of these groups.Animal Metaviridae LTRs had the same conserved motifs as retroviral LTRs, confirming that the two groups are closely related. The conserved motifs were the short inverted repeats (SIRs), integrase recognition signals (5´TGTTRNR…YNYAACA 3´); the polyadenylation signal or AATAAA motif; a GT-rich stretch downstream of the polyadenylation signal; and a less conserved AT-rich stretch corresponding to the core promoter element, the TATA box. Plant Pseudoviridae LTRs differed slightly in having a conserved TATA-box, TATATA, but no conserved polyadenylation signal, plus a much shorter R region.The sensitivity of the HMMs for detection in genomic sequences was around 50% for most models, at a relatively high specificity, suitable for genome screening.The HMMs yielded consensus sequences, which were aligned by creating an HMM model (a 'Superviterbi' alignment). This yielded a phylogenetic tree that was compared with a Pol-based tree. Both LTR and Pol trees supported monophyly of retroviruses. In both, Pseudoviridae was ancestral to all other LTR retrotransposons. However, the LTR trees showed the chromovirus portion of Metaviridae clustering together with Pseudoviridae, dividing Metaviridae into two portions with distinct phylogeny.ConclusionThe HMMs clearly demonstrated a unitary conserved structure of LTRs, supporting that they arose once during evolution. We attempted to follow the evolution of LTRs by tracing their functional foundations, that is, acquisition of RNAse H, a combined promoter/ polyadenylation site, integrase, hairpin priming and the primer binding site (PBS). Available information did not support a simple evolutionary chain of events.
| S-EPMC3601003 | biostudies-literature
Project description:WRKY proteins, defined by the conserved WRKYGQK sequence, are comprised of a large superfamily of transcription factors identified specifically from the plant kingdom. This superfamily plays important roles in plant disease resistance, abiotic stress, senescence as well as in some developmental processes. In this study, the Arabidopsis WRKY1 was shown to be involved in the salicylic acid signaling pathway and partially dependent on NPR1; a C-terminal domain of WRKY1, AtWRKY1-C, was constructed for structural studies. Previous investigations showed that DNA binding of the WRKY proteins was localized at the WRKY domains and these domains may define novel zinc-binding motifs. The crystal structure of the AtWRKY1-C determined at 1.6 A resolution has revealed that this domain is composed of a globular structure with five beta strands, forming an antiparallel beta-sheet. A novel zinc-binding site is situated at one end of the beta-sheet, between strands beta4 and beta5. Based on this high-resolution crystal structure and site-directed mutagenesis, we have defined and confirmed that the DNA-binding residues of AtWRKY1-C are located at beta2 and beta3 strands. These results provided us with structural information to understand the mechanism of transcriptional control and signal transduction events of the WRKY proteins.
| S-EPMC1851648 | biostudies-literature
Project description:Clostridium difficile is a major nosocomial pathogen that produces two large protein toxins [toxin A (TcdA) and toxin B (TcdB)] capable of disrupting intestinal epithelial cells. Both belong to the family of large clostridial cytotoxins, which are characterized by the presence of a repetitive C-terminal repetitive domain (CRD). In TcdA, the CRD is composed of 39 repeats that are responsible for binding to cell surface carbohydrates. To understand the molecular structural basis of cell binding by the toxins from C. difficile, we have determined a 1.85-A resolution crystal structure of a 127-aa fragment from the C terminus of the toxin A CRD. This structure reveals a beta-solenoid fold containing five repeats, with each repeat consisting of a beta-hairpin followed by a loop of 7-10 residues in short repeats (SRs) or 18 residues in long repeats (LRs). Adjacent pairs of beta-hairpins are related to each other by either 90 degree or 120 degree screw-axis rotational relationships, depending on the nature of the amino acids at key positions in adjacent beta-hairpins. Models of the complete CRDs of toxins A and B suggest that each CRD contains straight stretches of beta-solenoid composed of three to five SRs that are punctuated by kinks introduced by the presence of a single LR. These structural features provide a framework for understanding how large clostridial cytotoxins bind to cell surfaces and suggest approaches for developing novel treatments for C. difficile-associated diseases by blocking the binding of toxins to cell surfaces.
| S-EPMC1317924 | biostudies-literature