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The leukocyte nuclear envelope proteome varies with cell activation and contains novel transmembrane proteins that affect genome architecture.

Korfali N, Wilkie GS, Swanson SK, Srsen V, Batrakou DG, Fairley EA, Malik P, Zuleger N, Goncharevich A, de Las Heras J, Kelly DA, Kerr AR, Florens L, Schirmer EC - Mol. Cell Proteomics (2010)

Bottom Line: Several known proteins identified in both data sets have functions in chromatin organization and gene regulation.To test whether the novel NETs identified might include those that also regulate chromatin, nine were run through two screens for different chromatin effects.The variation in the protein milieu with pharmacological activation of the same cell population and consequences for gene regulation suggest that the nuclear envelope is a complex regulatory system with significant influences on genome organization.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH93JR, United Kingdom.

ABSTRACT
A favored hypothesis to explain the pathology underlying nuclear envelopathies is that mutations in nuclear envelope proteins alter genome/chromatin organization and thus gene expression. To identify nuclear envelope proteins that play roles in genome organization, we analyzed nuclear envelopes from resting and phytohemagglutinin-activated leukocytes because leukocytes have a particularly high density of peripheral chromatin that undergoes significant reorganization upon such activation. Thus, nuclear envelopes were isolated from leukocytes in the two states and analyzed by multidimensional protein identification technology using an approach that used expected contaminating membranes as subtractive fractions. A total of 3351 proteins were identified between both nuclear envelope data sets among which were 87 putative nuclear envelope transmembrane proteins (NETs) that were not identified in a previous proteomics analysis of liver nuclear envelopes. Nuclear envelope localization was confirmed for 11 new NETs using tagged fusion proteins and antibodies on spleen cryosections. 27% of the new proteins identified were unique to one or the other of the two leukocyte states. Differences in expression between activated and resting leukocytes were confirmed for some NETs by RT-PCR, and most of these proteins appear to only be expressed in certain types of blood cells. Several known proteins identified in both data sets have functions in chromatin organization and gene regulation. To test whether the novel NETs identified might include those that also regulate chromatin, nine were run through two screens for different chromatin effects. One screen found two NETs that can recruit a specific gene locus to the nuclear periphery, and the second found a different NET that promotes chromatin condensation. The variation in the protein milieu with pharmacological activation of the same cell population and consequences for gene regulation suggest that the nuclear envelope is a complex regulatory system with significant influences on genome organization.

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Fraction purity. A, Coomassie-stained gel of NE and microsome fractions analyzed. B, Western blot of the above fractions stained with organelle markers. ER markers calreticulin and calnexin were absent from NEs, whereas NE markers lamin B2 and the NET LAP2β were absent from microsomes. Similar amounts of total protein were loaded. MM, microsomal membranes.
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Figure 3: Fraction purity. A, Coomassie-stained gel of NE and microsome fractions analyzed. B, Western blot of the above fractions stained with organelle markers. ER markers calreticulin and calnexin were absent from NEs, whereas NE markers lamin B2 and the NET LAP2β were absent from microsomes. Similar amounts of total protein were loaded. MM, microsomal membranes.

Mentions: To increase lamina solubility, PBMC or liver NEs were incubated on ice in 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 2 mm MgCl2, 0.2% Nonidet P-40 in the presence of protease inhibitor mixture (Roche Applied Science, 11 873 580 001) for 15 min and then sonicated in a 4 °C sonic bath. Microsomes were treated similarly. Protein concentrations of liver NEs and microsomes (see Fig. 5) were determined by Bradford assay before adding sample buffer (100 mm Tris, pH 6.8, 4 m urea, 2% SDS, 50 mm DTT, 15% sucrose), heating at 65 °C, and then sonicating again in a 4 °C sonic bath. PBMC NEs and microsomes were adjusted for equal loading based on Coomassie Blue staining on polyacrylamide gels (see Fig. 3A). Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membrane (LI-COR Biosciences). Membranes were blocked in PBS, 5% milk, 0.2% Tween. Primary antibodies were diluted in this buffer (1:200 for Millipore NET peptide antibodies, 1:500 for calreticulin, 1:200 for calnexin, and 1:2000 for lamin A) and allowed to incubate overnight at 4 °C. Secondary IR800-conjugated goat anti-rabbit antibodies (LI-COR Biosciences) were added at 1:5000 dilution at RT for 2 h. Visualization and quantification were performed using a LI-COR Odyssey system and software (Odyssey 3.0.16) using median background subtraction. Three independent blots were run for each NET and control, and averages from all three are presented in Fig. 5B.


The leukocyte nuclear envelope proteome varies with cell activation and contains novel transmembrane proteins that affect genome architecture.

Korfali N, Wilkie GS, Swanson SK, Srsen V, Batrakou DG, Fairley EA, Malik P, Zuleger N, Goncharevich A, de Las Heras J, Kelly DA, Kerr AR, Florens L, Schirmer EC - Mol. Cell Proteomics (2010)

Fraction purity. A, Coomassie-stained gel of NE and microsome fractions analyzed. B, Western blot of the above fractions stained with organelle markers. ER markers calreticulin and calnexin were absent from NEs, whereas NE markers lamin B2 and the NET LAP2β were absent from microsomes. Similar amounts of total protein were loaded. MM, microsomal membranes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3101955&req=5

Figure 3: Fraction purity. A, Coomassie-stained gel of NE and microsome fractions analyzed. B, Western blot of the above fractions stained with organelle markers. ER markers calreticulin and calnexin were absent from NEs, whereas NE markers lamin B2 and the NET LAP2β were absent from microsomes. Similar amounts of total protein were loaded. MM, microsomal membranes.
Mentions: To increase lamina solubility, PBMC or liver NEs were incubated on ice in 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 2 mm MgCl2, 0.2% Nonidet P-40 in the presence of protease inhibitor mixture (Roche Applied Science, 11 873 580 001) for 15 min and then sonicated in a 4 °C sonic bath. Microsomes were treated similarly. Protein concentrations of liver NEs and microsomes (see Fig. 5) were determined by Bradford assay before adding sample buffer (100 mm Tris, pH 6.8, 4 m urea, 2% SDS, 50 mm DTT, 15% sucrose), heating at 65 °C, and then sonicating again in a 4 °C sonic bath. PBMC NEs and microsomes were adjusted for equal loading based on Coomassie Blue staining on polyacrylamide gels (see Fig. 3A). Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membrane (LI-COR Biosciences). Membranes were blocked in PBS, 5% milk, 0.2% Tween. Primary antibodies were diluted in this buffer (1:200 for Millipore NET peptide antibodies, 1:500 for calreticulin, 1:200 for calnexin, and 1:2000 for lamin A) and allowed to incubate overnight at 4 °C. Secondary IR800-conjugated goat anti-rabbit antibodies (LI-COR Biosciences) were added at 1:5000 dilution at RT for 2 h. Visualization and quantification were performed using a LI-COR Odyssey system and software (Odyssey 3.0.16) using median background subtraction. Three independent blots were run for each NET and control, and averages from all three are presented in Fig. 5B.

Bottom Line: Several known proteins identified in both data sets have functions in chromatin organization and gene regulation.To test whether the novel NETs identified might include those that also regulate chromatin, nine were run through two screens for different chromatin effects.The variation in the protein milieu with pharmacological activation of the same cell population and consequences for gene regulation suggest that the nuclear envelope is a complex regulatory system with significant influences on genome organization.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH93JR, United Kingdom.

ABSTRACT
A favored hypothesis to explain the pathology underlying nuclear envelopathies is that mutations in nuclear envelope proteins alter genome/chromatin organization and thus gene expression. To identify nuclear envelope proteins that play roles in genome organization, we analyzed nuclear envelopes from resting and phytohemagglutinin-activated leukocytes because leukocytes have a particularly high density of peripheral chromatin that undergoes significant reorganization upon such activation. Thus, nuclear envelopes were isolated from leukocytes in the two states and analyzed by multidimensional protein identification technology using an approach that used expected contaminating membranes as subtractive fractions. A total of 3351 proteins were identified between both nuclear envelope data sets among which were 87 putative nuclear envelope transmembrane proteins (NETs) that were not identified in a previous proteomics analysis of liver nuclear envelopes. Nuclear envelope localization was confirmed for 11 new NETs using tagged fusion proteins and antibodies on spleen cryosections. 27% of the new proteins identified were unique to one or the other of the two leukocyte states. Differences in expression between activated and resting leukocytes were confirmed for some NETs by RT-PCR, and most of these proteins appear to only be expressed in certain types of blood cells. Several known proteins identified in both data sets have functions in chromatin organization and gene regulation. To test whether the novel NETs identified might include those that also regulate chromatin, nine were run through two screens for different chromatin effects. One screen found two NETs that can recruit a specific gene locus to the nuclear periphery, and the second found a different NET that promotes chromatin condensation. The variation in the protein milieu with pharmacological activation of the same cell population and consequences for gene regulation suggest that the nuclear envelope is a complex regulatory system with significant influences on genome organization.

Show MeSH
Related in: MedlinePlus