Heart of glass anchors Rasip1 at endothelial cell-cell junctions to support vascular integrity.

Heart of Glass (HEG1), a transmembrane receptor, and Rasip1, an endothelial-specific Rap1-binding protein, are both essential for cardiovascular development. Here we performed a proteomic screen for novel HEG1 interactors and report that HEG1 binds directly to Rasip1. Rasip1 localizes to forming endothelial cell (EC) cell-cell junctions and silencing HEG1 prevents this localization. Conversely, mitochondria-targeted HEG1 relocalizes Rasip1 to mitochondria in cells. The Rasip1-binding site in HEG1 contains a 9 residue sequence, deletion of which abrogates HEG1's ability to recruit Rasip1. HEG1 binds to a central region of Rasip1 and deletion of this domain eliminates Rasip1's ability to bind HEG1, to translocate to EC junctions, to inhibit ROCK activity, and to maintain EC junctional integrity. These studies establish that the binding of HEG1 to Rasip1 mediates Rap1-dependent recruitment of Rasip1 to and stabilization of EC cell-cell junctions

A rapamycin-sensitive signaling pathway contributes to long-term synaptic plasticity in the hippocampus

Many forms of long-lasting behavioral and synaptic plasticity require the synthesis of new proteins. For example, long-term potentiation (LTIP) that endures for more than an hour requires both transcription and translation. The signal-transduction mechanisms that couple synaptic events to protein translational machinery during long-lasting synaptic plasticity, however, are not well understood. One signaling pathway that is stimulated by growth factors and results in the translation of specific mRNAs includes the rapamycin-sensitive kinase mammalian target of rapamycin (mTOR, also known as FRAP and RAFT-1). Several components of this translational signaling pathway, including mTOR, eukaryotic initiation factor-4E-binding proteins 1 and 2, and eukaryotic initiation factor-4E, are present in the rat hippocampus as shown by Western blot analysis, and these proteins are detected in the cell bodies and dendrites in the hippocampal slices by immunostaining studies. In cultured hippocampal neurons, these proteins are present in dendrites and are often found near the presynaptic protein, synapsin I. At synaptic sites, their distribution completely overlaps with a postsynaptic protein, PSD-95. These observations suggest the postsynaptic localization of these proteins. Disruption of mTOR signaling by rapamycin results in a reduction of late-phase LTP expression induced by high-frequency stimulation; the early phase of LTIP is unaffected. Rapamycin also blocks the synaptic potentiation induced by brain-derived neurotrophic factor in hippocampal slices. These results demonstrate an essential role for rapamycin-sensitive signaling in the expression of two forms of synaptic plasticity that require new protein synthesis. The localization of this translational signaling pathway at postsynaptic sites may provide a mechanism that controls local protein synthesis at potentiated synapses

Analysis of protein complexes through model‐based biclustering of label‐free quantitative AP‐MS data

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102630/1/msb201041.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102630/2/msb201041-sup-0001.pd

PP4 is a γH2AX phosphatase required for recovery from the DNA damage checkpoint

Phosphorylation of histone H2AX on Ser 139 (γH2AX) is one of the earliest events in the response to DNA double-strand breaks; however, the subsequent removal of γH2AX from chromatin is less understood, despite being a process tightly coordinated with DNA repair. Previous studies in yeast have identified the Pph3 phosphatase (the PP4C orthologue) as important for the dephosphorylation of γH2AX. By contrast, work in human cells attributed this activity to PP2A. Here, we report that PP4 contributes to the dephosphorylation of γH2AX, both at the sites of DNA damage and in undamaged chromatin in human cells, independently of a role in DNA repair. Furthermore, depletion of PP4C results in a prolonged checkpoint arrest, most likely owing to the persistence of mediator of DNA damage checkpoint 1 (MDC1) at the sites of DNA lesions. Taken together, these results indicate that PP4 is an evolutionarily conserved γH2AX phosphatase

Rag GTPases and phosphatidylinositol 3-phosphate mediate recruitment of the AP-5/SPG11/SPG15 complex.

Adaptor protein complex 5 (AP-5) and its partners, SPG11 and SPG15, are recruited onto late endosomes and lysosomes. Here we show that recruitment of AP-5/SPG11/SPG15 is enhanced in starved cells and occurs by coincidence detection, requiring both phosphatidylinositol 3-phosphate (PI3P) and Rag GTPases. PI3P binding is via the SPG15 FYVE domain, which, on its own, localizes to early endosomes. GDP-locked RagC promotes recruitment of AP-5/SPG11/SPG15, while GTP-locked RagA prevents its recruitment. Our results uncover an interplay between AP-5/SPG11/SPG15 and the mTORC1 pathway and help to explain the phenotype of AP-5/SPG11/SPG15 deficiency in patients, including the defect in autophagic lysosome reformation

Ydj1 governs fungal morphogenesis and stress response, and facilitates mitochondrial protein import via Mas1 and Mas2

We thank Zhen-Yuan Lin for help in the preparation of the AP-MS samples, and Cathy Collins for technical assistance. MDL is supported by a Sir Henry Wellcome Postdoctoral Fellowship (Wellcome Trust 096072), LEC is supported by a Canada Research Chair in Microbial Genomics and Infectious Disease and by Cana-dian Institutes of Health Research (CIHR) Grants MOP-119520 and MOP-86452. OK is supported by National Insti-tutes of Health grant 5R01GM108975. A-CG is supported by a CIHR Foundation Grant (FDN143301), Genome Cana-da Genomics Innovation Network (GIN) Node and Tech-nical Development Grants, and a Canada Research Chair in Functional Proteomics. J-PL was supported by a TD Bank Health Research Fellowship at the Lunenfeld-Tanenbaum Research Institute and by a Scholarship for the Next Gen-eration of Scientists from the Cancer Research Society. JLX is supported by a CIHR – Frederick Banting and Charles Best Canada Graduate Scholarship. The funding agencies had no role in the study design, data collection and inter-pretation, or the decision to submit the work for publication.Peer reviewedPublisher PD

The bromodomain-containing protein Ibd1 links multiple chromatin related protein complexes to highly expressed genes in Tetrahymena thermophila

Background: The chromatin remodelers of the SWI/SNF family are critical transcriptional regulators. Recognition of lysine acetylation through a bromodomain (BRD) component is key to SWI/SNF function; in most eukaryotes, this function is attributed to SNF2/Brg1. Results: Using affinity purification coupled to mass spectrometry (AP-MS) we identified members of a SWI/SNF complex (SWI/SNFTt) in Tetrahymena thermophila. SWI/SNFTt is composed of 11 proteins, Snf5Tt, Swi1Tt, Swi3Tt, Snf12Tt, Brg1Tt, two proteins with potential chromatin interacting domains and four proteins without orthologs to SWI/SNF proteins in yeast or mammals. SWI/SNFTt subunits localize exclusively to the transcriptionally active macronucleus (MAC) during growth and development, consistent with a role in transcription. While Tetrahymena Brg1 does not contain a BRD, our AP-MS results identified a BRD-containing SWI/SNFTt component, Ibd1 that associates with SWI/SNFTt during growth but not development. AP-MS analysis of epitope-tagged Ibd1 revealed it to be a subunit of several additional protein complexes, including putative SWRTt, and SAGATt complexes as well as a putative H3K4-specific histone methyl transferase complex. Recombinant Ibd1 recognizes acetyl-lysine marks on histones correlated with active transcription. Consistent with our AP-MS and histone array data suggesting a role in regulation of gene expression, ChIP-Seq analysis of Ibd1 indicated that it primarily binds near promoters and within gene bodies of highly expressed genes during growth. Conclusions: Our results suggest that through recognizing specific histones marks, Ibd1 targets active chromatin regions of highly expressed genes in Tetrahymena where it subsequently might coordinate the recruitment of several chromatin remodeling complexes to regulate the transcriptional landscape of vegetatively growing Tetrahymena cells.Comment: Published on BMC Epigenetics & Chromati

Label-free quantitative proteomics and SAINT analysis enable interactome mapping for the human Ser/Thr protein phosphatase 5

Affinity purification coupled to mass spectrometry (AP-MS) represents a powerful and proven approach for the analysis of protein–protein interactions. However, the detection of true interactions for proteins that are commonly considered background contaminants is currently a limitation of AP-MS. Here using spectral counts and the new statistical tool, Significance Analysis of INTeractome (SAINT), true interaction between the serine/threonine protein phosphatase 5 (PP5) and a chaperonin, heat shock protein 90 (Hsp90), is discerned. Furthermore, we report and validate a new interaction between PP5 and an Hsp90 adaptor protein, stress-induced phosphoprotein 1 (STIP1; HOP). Mutation of PP5, replacing key basic amino acids (K97A and R101A) in the tetratricopeptide repeat (TPR) region known to be necessary for the interactions with Hsp90, abolished both the known interaction of PP5 with cell division cycle 37 homolog and the novel interaction of PP5 with stress-induced phosphoprotein 1. Taken together, the results presented demonstrate the usefulness of label-free quantitative proteomics and statistical tools to discriminate between noise and true interactions, even for proteins normally considered as background contaminants.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/83479/1/1508_ftp.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/83479/2/pmic_201000770_sm_SupplInfo.pd

Gold nanoparticles prepared by laser ablation in aqueous biocompatible solutions: assessment of safety and biological identity for nanomedicine applications

International audienceGold nanoparticles prepared by laser ablation in aqueous biocompatible solutions: assessment of safety and biological identity for nanomedicine applications Abstract: Due to excellent biocompatibility, chemical stability, and promising optical properties , gold nanoparticles (Au-NPs) are the focus of research and applications in nanomedicine. Au-NPs prepared by laser ablation in aqueous biocompatible solutions present an essentially novel object that is unique in avoiding any residual toxic contaminant. This paper is conceived as the next step in development of laser-ablated Au-NPs for future in vivo applications. The aim of the study was to assess the safety, uptake, and biological behavior of laser-synthesized Au-NPs prepared in water or polymer solutions in human cell lines. Our results showed that laser ablation allows the obtaining of stable and monodisperse Au-NPs in water, polyethylene glycol, and dextran solutions. The three types of Au-NPs were internalized in human cell lines, as shown by transmission electron microscopy. Biocompatibility and safety of Au-NPs were demonstrated by analyzing cell survival and cell morphology. Furthermore, incubation of the three Au-NPs in serum-containing culture medium modified their physicochemical characteristics , such as the size and the charge. The composition of the protein corona adsorbed on Au-NPs was investigated by mass spectrometry. Regarding composition of complement C3 proteins and apolipoproteins, Au-NPs prepared in dextran solution appeared as a promising drug carrier. Altogether, our results revealed the safety of laser-ablated Au-NPs in human cell lines and support their use for theranostic applications