Project description:A number of six proteins were selected during immunoscreening and further analyses. The proteins were divided in silico into overlapping 15-mer oligopeptides with an overlap of 11 residues. The microarrays were incubated with different antibodies to K. pneumoniae, C. jejuni and S. aureus.
Project description:A number of seven proteins were selected during immunoscreening and further analyses. The proteins were in silico divided into overlapping 15-mer oligopeptides with an overlap of 11 residues. The microarrays were incubated with different antibodies to C. jejuni, Escherichia coli and Salmonella enterica.
2013-04-06 | GSE45542 | GEO
Project description:Endogenous RNA bind-n-seq (endo-bind-n-seq): determining RNA interaction motifs of RNA binding proteins isolated from their native environments
Project description:Miz1 is a zinc finger protein that regulates expression of cell cycle inhibitors as part of a complex with Myc. Cell cycle-independent functions of Miz1 are poorly understood. Here, we use a Nestin-Cre transgene to delete an essential domain of Miz1 in the central nervous system (Miz1M-NM-^TPOZNes). Miz1M-NM-^TPOZNes mice display cerebellar neurodegeneration characterized by the progressive loss of Purkinje cells. Chromatin immunoprecipitation sequencing and biochemical analyses show that Miz1 activates transcription upon binding to a non-palindromic sequence present in core promoters. Target genes of Miz1 encode regulators of autophagy and proteins involved in vesicular transport that are required for autophagy. Miz1M-NM-^TPOZ neuronal progenitors and fibroblasts show reduced autophagic flux. Consistently, polyubiquitinated proteins and p62/Sqtm1 accumulate in the cerebella of Miz1M-NM-^TPOZNes mice, characteristic features of defective autophagy. Our data suggest that Miz1 may link cell growth and ribosome biogenesis to the transcriptional regulation of vesicular transport and autophagy. ChIP-Seq with H190 and G18 on an Illumina Genome Analyzer IIx.
Project description:Macroautophagy (hereafter autophagy) is the major pathway by which macromolecules and organelles are degraded. Autophagy is regulated by the mTOR signaling pathway, which is the focal point for integration of metabolic information, with mTORC1 playing a central role in balancing biosynthesis and catabolism. Of the various inputs to mTORC1, the amino acid sensing pathway is among the most potent. Based upon transcriptome analysis of neurons subjected to nutrient deprivation, we identified let-7 as a microRNA capable of promoting neuronal autophagy. We found that let-7 activates autophagy by coordinately down-regulating the amino acid sensing pathway to prevent mTORC1 activation. Let-7 induced autophagy in the brain and greatly reduced protein aggregates in a lentivirus model of polyglutamine disease, establishing the physiological relevance of let-7 for in vivo autophagy modulation. Moreover, peripheral delivery of let-7 anti-miR repressed autophagy in muscle and white fat, suggesting that let-7 autophagy regulation extends beyond the CNS. Hence, let-7 plays a central role in nutrient homeostasis and proteostasis regulation in higher organisms. Using sets of wild-type C57BL/6J mice, we established primary cortical neuron cultures from P0 littermates, and cultured these neurons (n = 3 / set) in CM or NLM for 4 hrs.
Project description:Mutations in the gene encoding Parkin are a major cause of recessive Parkinson's disease. Recent work has shown that Parkin translocates from the cytosol to depolarized mitochondria and induces their autophagic removal (mitophagy). However, the molecular mechanisms underlying Parkin-mediated mitophagy are poorly understood. Here, we investigated whether Parkin interacts with autophagy-regulating proteins. We purified Parkin and associated proteins from HEK293 cells using tandem affinity purification and identified the Parkin interactors using mass spectrometry. We identified the autophagy-promoting protein Ambra1 (activating molecule in Beclin1-regulated autophagy) as a Parkin interactor. Ambra1 activates autophagy in the CNS by stimulating the activity of the class III phosphatidylinositol 3-kinase (PI3K) complex that is essential for the formation of new phagophores. We found Ambra1, like Parkin, to be widely expressed in adult mouse brain, including midbrain dopaminergic neurons. Endogenous Parkin and Ambra1 coimmunoprecipitated from HEK293 cells, SH-SY5Y cells, and adult mouse brain. We found no evidence for ubiquitination of Ambra1 by Parkin. The interaction of endogenous Parkin and Ambra1 strongly increased during prolonged mitochondrial depolarization. Ambra1 was not required for Parkin translocation to depolarized mitochondria but was critically important for subsequent mitochondrial clearance. In particular, Ambra1 was recruited to perinuclear clusters of depolarized mitochondria and activated class III PI3K in their immediate vicinity. These data identify interaction of Parkin with Ambra1 as a key mechanism for induction of the final clearance step of Parkin-mediated mitophagy.
Project description:Nix is a membrane-anchored outer mitochondrial protein that induces mitophagy. While Nix has an LC3-interacting (LIR) motif that binds to ATG8 proteins, it also contains a minimal essential region (MER) that induces mitophagy through an unknown mechanism. We used chemically induced dimerization (CID) to probe the mechanism of Nix-mediated mitophagy and found that both the LIR and MER are required for robust mitophagy. We find that the Nix MER interacts with the autophagy effector WIPI2 and recruits WIPI2 to mitochondria. The Nix LIR motif is also required for robust mitophagy and converts a homogeneous WIPI2 distribution on the surface of the mitochondria into puncta, even in the absence of ATG8s. Together, this work reveals unanticipated mechanisms in Nix-induced mitophagy and the elusive role of the MER, while also describing an interesting example of autophagy induction that acts downstream of the canonical initiation complexes.
Project description:Many human monoclonal antibodies that neutralize multiple clades of HIV-1 are polyreactive and bind avidly to mammalian autoantigens. Indeed, the generation of neutralizing antibodies to the 2F5 and 4E10 epitopes of HIV-1 gp41 in man may be proscribed by immune tolerance since mice expressing the VH and VL regions of 2F5 have a block in B-cell development characteristic of central tolerance. This developmental blockade implies the presence of tolerizing autoantigens that are mimicked by the membrane-proximal external region of HIV-1 gp41. Here we identify human kynureninase (KYNU) and splicing factor 3b subunit 3 (SF3B3) as the primary conserved, vertebrate self-antigens recognized by the 2F5 and 4E10 antibodies, respectively. 2F5 binds the H4 domain of KYNU which contains the complete 2F5 linear epitope (ELDKWA). 4E10 recognizes a conformational epitope of SF3B3 that is strongly dependent on hydrophobic interactions. Opossums carry a rare KYNU H4 domain that abolishes 2F5 binding, but retain all SF3B3 4E10 epitopes. Immunization of opossums with HIV-1 gp140 induced extraordinary titers of serum antibody to the 2F5 ELDKWA epitope but little or nothing to the 4E10 determinant. Identification of structural motif shared by vertebrates and HIV-1 provides direct evidence that immunological tolerance can impair humoral responses to HIV-1. The invitrogen protoarray that contains >9,400 recombinant human proteins was used to identify self-ligands that are recognized by broadly neutralizing HIV-1 antibodies 2F5 and 4E10. An isotype-matched human myeloma protein (151K, Southern Biotech) was used as control.
Project description:Nix is a membrane-anchored outer mitochondrial protein that induces mitophagy. While Nix has an LC3-interacting (LIR) motif that binds to ATG8 proteins, it also contains a minimal essential region (MER) that induces mitophagy through an unknown mechanism. We used chemically-induced dimerization (CID) to probe the mechanism of Nix-mediated mitophagy and find that both the LIR and MER are required for robust mitophagy. We find that the Nix MER interacts with the autophagy effector WIPI2 and recruits WIPI2 to mitochondria. The Nix LIR motif is also required for robust mitophagy and converts a homogeneous WIPI2 distribution on the surface of the mitochondria into puncta, even in the absence of ATG8s. Together, this work reveals unanticipated mechanisms in Nix-induced mitophagy and the elusive role of the MER, while also describing an interesting example of autophagy induction that acts downstream of the canonical initiation complexes.
Project description:Although methyl CpG binding domain protein-2 (MeCP2) is commonly understood to function as a silencing factor at methylated DNA sequences, recent studies also show that MeCP2 can bind unmethylated sequences and coordinate gene activation. MeCP2 displays broad binding patterns throughout the genome, with high expression levels similar to histone H1 in neurons. Despite its significant presence in the brain, only subtle gene expression changes occur in the absence of MeCP2. This may reflect a more complex regulatory mechanism of MeCP2 to complement chromatin binding. Using an RNA immunoprecipitation of native chromatin technique, we identify MeCP2 interacting microRNAs in mouse primary cortical neurons. In addition, comparison with mRNA sequencing data from Mecp2-null mice suggests that differentially expressed genes may indeed be targeted by MeCP2-interacting microRNAs. These findings highlight the MeCP2 interaction with microRNAs that may modulate its binding with chromatin and regulate gene expression.