A functional proteomics approach for the detection of nuclear proteins based on derepressed importin alpha

Blazek, Erik; Meisterernst, Michael

doi:10.1002/pmic.200500449

Cited by 9 publications

(6 citation statements)

References 27 publications

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“…In the context of other studies and T cell proteomics data sets, large scale measurements from the nucleus of human T cells have mostly used the Jurkat cell lines (34, 35, 59). Han and co-workers (34) have studied nuclear fractionation methods and reported 1174 proteins associated with the nuclear fraction from Jurkat cells during apoptosis.…”

Section: Methodsmentioning

confidence: 99%

Quantitative Proteomics Analysis of the Nuclear Fraction of Human CD4+ Cells in the Early Phases of IL-4-induced Th2 Differentiation

Moulder

Lönnberg

Elo

et al. 2010

Molecular & Cellular Proteomics

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We used stable isotope labeling with 4-plex iTRAQ (isobaric tags for relative and absolute quantification) reagents and LC-MS/MS to investigate proteomic changes in the nucleus of activated human CD4+ cells during the early stages of Th2 cell differentiation. The effects of IL-4 stimulation upon activated naïve CD4+ cells were measured in the nuclear fractions from 6 and 24 h in three biological replicates, each using pooled cord blood samples derived from seven or more individuals. In these analyses, in the order of 800 proteins were detected with two or more peptides and quantified in three biological replicates. In addition to consistent differences observed with the nuclear localization/expression of established human Th2 and Th1 markers, there were changes that suggested the involvement of several proteins either only recently reported or otherwise not known in this context. These included SATB1 and among the novel changes detected and validated an IL-4-induced increase in the level of YB1. This unique data set from human cord blood CD4+ T cells details an extensive list of protein determinations that compares with and complements previous data determined from the Jurkat cell nucleus.

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Section: Methodsmentioning

confidence: 99%

Quantitative Proteomics Analysis of the Nuclear Fraction of Human CD4+ Cells in the Early Phases of IL-4-induced Th2 Differentiation

Moulder

Lönnberg

Elo

et al. 2010

Molecular & Cellular Proteomics

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“…Systematic searches for cargo proteins have been performed, especially for those dependent on Imp-␣ to associate with Imp-␤, using Far Western-based cellular proteomics (7) and the screening of in vitro virus libraries (8) and oriented peptide libraries (9). The experiments using synthetic libraries helped to refine the classical nuclear localization signal (cNLS) sequences to which Imp-␣ binds, but they did not detect natural cargo proteins directly.…”

mentioning

confidence: 99%

Identification of Cargo Proteins Specific for the Nucleocytoplasmic Transport Carrier Transportin by Combination of an in Vitro Transport System and Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Quantitative Proteomics

Kimura

Kose

Okumura

et al. 2013

Molecular & Cellular Proteomics

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The human importin-␤ family consists of 21 nucleocytoplasmic transport carrier proteins that carry proteins and RNAs across the nuclear envelope through nuclear pores in specific directions. These transport carriers are responsible for the nucleocytoplasmic transport of thousands of proteins, but the cargo allocation of each carrier, which is necessary information if one wishes to understand the physiological context of transport, is poorly characterized. To address this issue, we developed a high-throughput method to identify the cargoes of transport carriers by applying stable isotope labeling by amino acids in cell culture to construct an in vitro transport system. Our method can be outlined in three steps. Most genetic processes, including chromosome replication and transcription, occur in the nucleus, and thus the selection of proteins that enter the nucleus and act there is crucial for cellular processes (1). During interphase, all nuclear proteins cross the nuclear envelope via nuclear pores, channels that constitute a selective permeability barrier for macromolecules. Only a fraction of proteins traverse these channels by means of free diffusion; most nuclear proteins are imported into or exported out of the nucleus with the assistance of importin-␤ (Imp-␤) 1 family proteins (Imp-␤s) (2, 3). Imp-␤s interact with the nuclear pore complex in a way that allows them to travel in and out of the nucleus. To drive cargo From the ‡Cellular Dynamics Laboratory, Advanced Science Institute, RIKEN, 2-1 Hirosawa,

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“…Six KPNA paralogs exist in mouse with which the corresponding human homologs share 80–90% amino acid identity. Each KPNA paralog likely transports a large number of cargo proteins . Furthermore, each KPNA paralog has differential affinity for a subset of cargo proteins with the binding affinity and concentration of the specific KPNA being determinants of the rate of protein transport .…”

Section: Introductionmentioning

confidence: 99%

Karyopherin Alpha 1 Regulates Satellite Cell Proliferation and Survival by Modulating Nuclear Import

Choo¹,

Cutler

Rother

et al. 2016

Stem Cells

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Satellite cells are stem cells with an essential role in skeletal muscle repair. Precise regulation of gene expression is critical for proper satellite cell quiescence, proliferation, differentiation and self -renewal. Nuclear proteins required for gene expression are dependent on the nucleocytoplasmic transport machinery to access to nucleus, however little is known about regulation of nuclear transport in satellite cells. The best characterized nuclear import pathway is classical nuclear import which depends on a classical nuclear localization signal (cNLS) in a cargo protein and the heterodimeric import receptors, karyopherin alpha (KPNA) and beta (KPNB). Multiple KPNA1 paralogs exist and can differ in importing specific cNLS proteins required for cell differentiation and function. We show that transcripts for six Kpna paralogs underwent distinct changes in mouse satellite cells during muscle regeneration accompanied by changes in cNLS proteins in nuclei. Depletion of KPNA1, the most dramatically altered KPNA, caused satellite cells in uninjured muscle to prematurely activate, proliferate and undergo apoptosis leading to satellite cell exhaustion with age. Increased proliferation of satellite cells led to enhanced muscle regeneration at early stages of regeneration. In addition, we observed impaired nuclear localization of two key KPNA1 cargo proteins: p27, a cyclin-dependent kinase inhibitor associated with cell cycle control and lymphoid enhancer factor 1, a critical co-transcription factor for β-catenin. These results indicate that regulated nuclear import of proteins by KPNA1 is critical for satellite cell proliferation and survival and establish classical nuclear import as a novel regulatory mechanism for controlling satellite cell fate.

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A functional proteomics approach for the detection of nuclear proteins based on derepressed importin alpha

Cited by 9 publications

References 27 publications

Quantitative Proteomics Analysis of the Nuclear Fraction of Human CD4+ Cells in the Early Phases of IL-4-induced Th2 Differentiation

Quantitative Proteomics Analysis of the Nuclear Fraction of Human CD4+ Cells in the Early Phases of IL-4-induced Th2 Differentiation

Identification of Cargo Proteins Specific for the Nucleocytoplasmic Transport Carrier Transportin by Combination of an in Vitro Transport System and Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Quantitative Proteomics

Karyopherin Alpha 1 Regulates Satellite Cell Proliferation and Survival by Modulating Nuclear Import

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