Isolation and determination of PI(4,5)P2 proximal proteins
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Epithelial Cell
SUBMITTER: 
Yoshikazu Nakamura
LAB HEAD: Yoshikazu nakamura
PROVIDER: PXD031266 | Pride | 2022-03-26
REPOSITORIES: Pride
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| MSB38429APEX2Band_01.raw | Raw | |||
| MSB38429APEX2Band_02.raw | Raw | |||
| MSB38429APEX2Band_03.raw | Raw | |||
| MSB38429APEX2Band_04.raw | Raw | |||
| MSB38429APEX2Band_05.raw | Raw |
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Project description:While thousands of previously unannotated small and alternative open reading frames (alt-ORFs) have recently been revealed in the human genome, the functions of only a handful are currently known, leaving open the question of their biological significance as a class. Using a proteomic strategy for discovery of unannotated short open reading frames in human cells, we report the detection of alt-RPL36, a 148-amino acid protein co-encoded with and overlapping human RPL36. Alt-RPL36 interacts with TMEM24, which transports the phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] precursor phosphatidylinositol from the endoplasmic reticulum to the plasma membrane. Knock-out of alt-RPL36 in HEK 293T cells increased PI(4,5)P2 levels in the plasma membrane and upregulated the PI3K-AKT-mTOR signaling pathway. Remarkably, we find that four serine residues of alt-RPL36 are phosphorylated, and mutation of these four serines to alanine abolished the interaction with TMEM24 and regulation of PI3K signaling. These results implicate alt-RPL36 as a novel regulator of PI(4,5)P2 synthesis upstream of the PI3K-AKT-mTOR signaling pathway, and the first example of a phosphorylated alt-ORF product. More broadly, both alt-RPL36 and RPL36 regulate protein synthesis and cell growth via different molecular mechanisms – AKT signaling and ribosome composition, respectively. One human transcript can therefore express two sequence-independent polypeptides from overlapping ORFs that regulate the same processes via distinct mechanisms.
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Project description:Aging is characterized by the loss of homeostasis and the general decline of physiological functions, accompanied by various degenerative diseases and higher rates of mortality. Anti-aging targeted small molecule screens have been carried outperformed many times, however, few have focused on endogenous metabolic intermediates—metabolites. Here, using C. elegans lifespan assays, we conducted a worm metabolite screen and identified an conserved metabolite through eEukaryotes-conserved metabolite, myo-inositol (MI), to extend lifespan, increase mobility and reduce fat content. Genetic analysis of enzymes in MI metabolic pathway suggestsed that MI alleviates aging through its derivative PI(4,5)P2. MI and PI(4,5)P2 are precursors of PI(3,4,5)P3, which is negatively related to longevity. However, the mechanism study shows itslongevity effects could be fully blocked by a null-allele mutation of the tumor suppressor gene, daf-18 (homologous to mouse Pten), areand was independent of its classical pathway downstream elements, akt or daf-16., but Instead, we found MI effects on aging were dependent on its downstream mitophagy regulator pink-1. MI’s anti-aging effect is was also conserved in mouse model, implicatingindicating a conserved mechanism in mammals that may extend to human.
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Project description:Phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) is a lipid second messenger produced by class I phosphoinositide 3-kinases (PI 3-kinases) phosphorylation of phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2). Although typically associated with the plasma membrane, it is now appreciated that PI(3,4,5)P3 generation also occurs in the nucleus to regulate a variety of biological functions. Using a PI(3,4,5)P3-specific GST-GRP1PH domain reporter and immunofluorescence microscopy, we have found that DLD1 and SW480 colorectal cancer (CRC) cells exhibit pronounced nuclear staining of PI(3,4,5)P3. To characterise the nuclear PI(3,4,5)P3 interactome in DLD1 and SW480 cells, we performed affinity pull-down experiments on nuclear extracts using a ω-amino analogue of phosphatidylinositol 3,4,5 -triphosphate (PI(3,4,5)P3 immobilised onto Affi-gel10 beads. Gel fractionation and LC/MS-MS analysis was performed to reduce sample complexity and increase resolution, with samples run on a LTQ Orbitrap Elite mass spectrometer. DLD1 and SW480 cells were both analysed in four independent biological replicates. A protein was considered a member of the nuclear PI(3,4,5)P3 interactome if it was identified in at least two experiments (with at least two peptides, FDR of 1%) in both cell lines, yielding a total of 1237 unique proteins (representing 1103 genes for DLD1 and 1001 genes for SW480 cells).
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Project description:Phosphatase activities on PI(3,4,5)P3 and
PI(3,4)P2
This model describes the action of various phosphatases on
PI(3,4,5)P3 and PI(3,4)P2, in response to a stimulation by EGF. It
contains boolean switches to simulate knock-down and knock-out of
phosphatases as well as inhibition of PI3 kinase.
This model is described in the article:
PTEN Regulates PI(3,4)P2
Signaling Downstream of Class I PI3K
Mouhannad Malek, Anna Kielkowska,
Tamara Chessa, Karen E. Anderson, David Barneda, P?nar Pir,
Hiroki Nakanishi, Satoshi Eguchi, Atsushi Koizumi, Junko Sasaki,
Véronique Juvin, Vladimir Y. Kiselev, Izabella Niewczas,
Alexander Gray, Alexandre Valayer, Dominik Spensberger, Marine
Imbert, Sergio Felisbino, Tomonori Habuchi, Soren Beinke, Sabina
Cosulich, Nicolas Le Novère, Takehiko Sasaki, Jonathan
Clark, Phillip T. Hawkins and Len R. Stephens
Molecular Cell
Abstract:
The PI3K signaling pathway regulates cell growth and
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PI(3,4)P2 3-phosphatase, both in vitro and in vivo. PTEN is a
major PI(3,4)P2 phosphatase in Mcf10a cytosol, and loss of PTEN
and INPP4B, a known PI(3,4)P2 4-phosphatase, leads to
synergistic accumulation of PI(3,4)P2, which correlated with
increased invadopodia in epidermal growth factor
(EGF)-stimulated cells. PTEN deletion increased PI(3,4)P2
levels in a mouse model of prostate cancer, and it inversely
correlated with PI(3,4)P2 levels across several EGF-stimulated
prostate and breast cancer lines. These results point to a role
for PI(3,4)P2 in the phenotype caused by loss-of-function
mutations or deletions in PTEN.
This model is hosted on
BioModels Database
and identified by:
MODEL1704190000.
To cite BioModels Database, please use:
Chelliah V et al. BioModels: ten-year
anniversary. Nucl. Acids Res. 2015, 43(Database
issue):D542-8.
To the extent possible under law, all copyright and related or
neighbouring rights to this encoded model have been dedicated to
the public domain worldwide. Please refer to
CC0
Public Domain Dedication for more information.
| MODEL1704190000 | BioModels
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