PIP protein binding in CD4+ T cells
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Velos
ORGANISM(S): Mus Musculus (mouse)
TISSUE(S): T Cell
SUBMITTER: 
Richard Cattley
LAB HEAD: William Hawse
PROVIDER: PXD046124 | Pride | 2024-12-16
REPOSITORIES: Pride
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Project description:To directly observe the conformational changes in Akt1 3P elicited by PIP 3 binding, we performed HDX-MS analysis of Akt1 3P in the presence of plasma membrane-mimicking liposomes (20% cholesterol, 30% phosphatidylcholine, 15% phosphatidylserine, 35% phosphatidylethanolamine) that contained either 0% or 5% PIP 3 . The sequence coverage of Akt1 was excellent, with 160 peptides spanning ~97% of all exchangeable amides (Supplementary Table 2). With 5% PIP 3 liposomes there was extensive protection of the PH domain, similar to what had been previously observed for non-phosphorylated Akt1. In addition to protection of the PH domain elicited by PIP 3 binding, Akt1 3P exhibited significant deprotection of the C-lobe of the kinase domain, including the activation loop, that corresponds to the interface between the PH and kinase domains observed in our structure of autoinhibited Akt1. We also carriedout HDX-MS experiments in the presence and absence of ATPS, for which we observed no significant differences. The likely binding pocket for the hydrophobic motif is the so-called PDK1- interacting fragment (PIF) pocket in Akt that binds the phosphorylated hydrophobic motif in the active conformation (21, 22) . Previous hydrogen- deuterium exchange mass spectrometry (HDX-MS) analysis of Akt1 DrLink indicated small, but significant exposure of the PIF pocket upon PI(3,4,5)P 3 binding (26) (Figure 5D, deuterium incorporation plots reproduced with permission). We confirmed this observation by comparing the deuterium incorporation rates for Akt1 DrLink and Akt1 ΔC in solution. Sequence coverage of the truncated Akt1 ΔC comprised 85 peptides spanning ~94% of all exchangeable amides (Supplementary Table 2). Two peptides in Akt1 ΔC , corresponding to β3-αB in the N-lobe and the catalytic loop in the C-lobe of the kinase domain exhibited a modest, but significant, 6% increase in deuterium incorporation (Figure 5E). These changes indicate exposure of the PIF pocket and consequent local disordering of the N-lobe in the absence of the hydrophobic motif. These observationsis areis consistent with the lack of electron density observed for theC helix and activation loop and overall higher temperature factors for the N- lobe of the kinase domain (Supplementary Figure S5CD). In order to test whether the unphosphorylated hydrophobic motif indeed binds to the PIF pocket in the inactive conformation, we measured the binding affinity of a tail peptide 19 containing the missing C-terminal tail residues in Akt1 ΔC (residues 457-480 of human Akt1) by fluorescence anisotropy. The binding constant was estimated to be approximately 0.5 mM from three independent titrations (Figure 5F), although it was not possible to reach saturation due to limiting Akt1 ΔC concentration. Whilst this is a relatively weak interaction, it is sufficient in the context of an intramolecular interaction to sequester the hydrophobic motif more than 99% of the time at equilibrium due to the almost infinite local concentration.
2021-09-01 | PXD022061 | Pride
Project description:We used PIP-seq with a CROP-seq library to evaluate the feability of PIP-seq for large pooled CRISPR screens
2023-01-12 | GSE202915 | GEO
Project description:The study of the compond PIP-RBPJ-1‘ function on neural stem cell. Synthetic DNA-binding inhibitors capable of gaining precise control over neurogenesis factors could obviate the current clinical barriers associated with small molecule use in regenerative medicine. Here, we show the design and bio-efficacy of a synthetic ligand PIP-RBPJ-1 to cause promoter-specific suppression of neurogenesis-associated HES1, and its downstream genes. Furthermore, PIP-RBPJ-1 alone could alter the neural system-associated notch signaling factors and remarkably induce neurogenesis with an efficiency that is comparable to a conventional approach. Here is one day treatment of the PIP-RBPJ-1 on neural stem cells.
2018-07-19 | GSE109916 | GEO
Project description:We have previously shown that the resistance increase observed after biological systemic acquired resistance (SAR) induction in plants can be mimicked by exogenous plant treatment with (pipecolic acid) Pip (Návarová et al., 2012; Bernsdorff et al., 2016). In order to investigate whether elevations of Pip are sufficient for a SAR-like transcriptional reprogramming in leaf tissue, we supplied individual Arabidopsis Col-0 and fmo1 mutant plants with doses of 10 µmol Pip and determined the transcriptional response in leaves one day later on the whole genome level by RNA-sequencing analyses in relation to untreated plants. Arabidopsis thaliana plants were grown individually in pots containing a mixture of soil, vermiculite and sand (8:1:1) in a controlled cultivation chamber with a 10-h day (9 AM to 7 PM; photon flux density 100 mol m-2 s-1) / 14-h night cycle and a relative humidity of 70 %. Day and night temperatures were set to 21°C and 18°C, respectively. Experiments were performed with 5- to 6-week-old, naive plants exhibiting a uniform appearance.Treatments with Pip were essentially performed as detailed in Návarová et al. (2012).10 ml of a 1 mM aqueous solution of Pip (equates to 10 µmol) were pipetted onto the soil of individually cultivated plants. 10 ml of water were applied in the control treatments. Three biologically independent, replicate experiments were performed. In each experiment, at least 4 full-grown leaves from 6 different plants were pooled 24 hours after the treatment with Pip or water for one biological replicate. In this way, 3 biologically independent, replicate samples per treatment and plant genotype were obtained.
2018-03-23 | E-MTAB-6243 | biostudies-arrayexpress
Project description:Background The PIP (prolactin-inducible protein) gene has been shown to be expressed in breast cancers, with contradictory results concerning its implication. As both the physiological role and the molecular pathways in which PIP is involved are poorly understood, we conducted a combined gene expression profiling and network analysis studies on selected breast cancer cell lines presenting distinct PIP expression levels and hormonal receptor status, to explore the functional and regulatory network of PIP co-modulated genes. Results Microarray analysis allowed identification of genes co-modulated with PIP independently of modulations resulting from hormonal treatment or cell line heterogeneity. Relevant clusters of genes that can discriminate between [PIP+] and [PIP-] cells were identified. Functional and regulatory network analyses based on knowledge database revealed a master network of PIP co-modulated genes, including many interconnecting oncogenes and tumor suppressor genes, half of which were detected as differentially expressed through high-precision measurements. The networks identified appear associated with an inhibition of proliferation coupled with an increase of apoptosis and an enhancement of cell adhesion in breast cancer cell lines. Finally, the STAT5 motif was identified in promoters of an important part of genes belonging to the PIP networks. Conclusion Our global exploratory approach was found to be an effective strategy to identify the biological pathways modulated along with the PIP expression, thus supporting good prognostic value of disease-free survival time in breast cancer based on previous reports focusing on PIP’s favorable signature. Moreover, our data allowed us to provide the first insight in its regulatory subnetwork in which STAT5 appears as a potential key regulator.
2009-03-06 | GSE11627 | GEO
Project description:Proteins were pulled down using CD91 to determine what proteins may be interacting
2024-12-19 | PXD051580 | Pride
Project description:In the HPLC high-resolution ESI-MS profiles of both multiple sclerosis subjects and healthy controls saliva samples three proteins, with exp. monoisotopic ion [M+H]+ at 13493.9 ± 0.2 m/z, 13696.9 ± 0.2 m/z (+203 Da, with respect to 13493.9), and 13843.1 ± 0.2 m/z (+147 Da, with respect to 13696.9) eluting between 37.8-38.2 minutes, were detected. The mass difference of 203 Da suggested that the protein with exp. monoisotopic [M+H]+ at 13696.9 ± 0.2 m/z could correspond to the N-acetylhexosamine (theor. monoisotopic ion at 203.1 m/z) derivative of the 13493.9 ± 0.2 m/z protein. On the other hand, the mass difference of 146 Da between the proteins with exp. monoisotopic [M+H]+ at 13843.1 ± 0.2 m/z and 13696.9 ± 0.2 m/z was in agreement with an additional deoxyhesose moiety (theor. monoisotopic ion at 146.1 m/z). Manual inspection of the high-resolution MS/MS spectra of the [M+10H]+10 ions at 1351.00, 1371.32 and 1386.11 m/z allowed to establish that the three proteins were different proteoforms of PIP with the N-terminal glutamine residue converted to pyro-glutamic acid and with two disulfide bonds, but not to assign the glycosylation site. The presence in the MS/MS spectra of low-molecular weight saccharide oxoniun ions at 204.087 and 138.106 m/z confirmed the presence of an N-acetylhexosamine moiety in the protein with [M+H]+ at 13696.9 ± 0.2 m/z.
2018-08-10 | PXD009440 | Pride
Project description:Plasma membrane intrinsic proteins (PIPs) are one subfamily of aquaporins that mediate the transmembrane transport of water. To reveal their function in poplar, we generated transgenic poplar plants in which the translation of PIP genes was downregulated by RNA interference investigated these plants with a comprehensive leaf plasma membrane proteome and physiome analysis. First, inhibition of PIP synthesis strongly altered the leaf plasma membrane protein composition. Strikingly, several signaling components and transporters involved in the regulation of stomatal movement were differentially regulated in transgenic poplars. Furthermore, hormonal crosstalk related to abscisic acid, auxin and brassinosteroids was altered, in addition to cell wall biosynthesis/cutinization, the organization of cellular structures and membrane trafficking. A physiological analysis confirmed the proteomic results. The leaves had wider opened stomata and higher net CO2 assimilation and transpiration rates as well as greater mesophyll conductance for CO2 (gm) and leaf hydraulic conductance (Kleaf). Based on these results, we conclude that PIP proteins not only play essential roles in whole leaf water and CO2 flux but have important roles in the regulation of stomatal movement.
2015-08-10 | PXD002544 | Pride
Project description:PIP variable region deep sequencing from Enterococcal wastewater isolates
| PRJEB13161 | ENA