The Nucleus-Localized Epidermal Growth Factor Receptor Is SUMOylated

The epidermal growth factor receptor (EGFR) plays important roles in normal and cancer cell growth. The EGFR has principally two different signaling pathways: the canonical kinase route induced at the plasma membrane resulting in an intracellular phosphorylation cascade via MAPKs and PI3K and the more recently discovered pathway by which the receptor functions as a transcriptional co-activator inside the cell nucleus. Full length EGFR translocates to the inner nuclear membrane, via the endoplasmic reticulum, through association with the sec61β translocon. The c-myc (<i>MYC</i>) and cyclin D1 (<i>CNND1</i>) genes represent two target genes for nuclear EGFR (nEGFR). Here we show that EGFR is SUMOylated and that the SUMO-1-modified receptors are almost unexceptionally nuclear. Co-immunoprecipitation experiments suggest that EGFR is multi-SUMOylated. Using two mass spectrometry-based strategies (matrix-assisted laser desorption ionization time of flight and electrospray ionization liquid chromatography with tandem mass spectrometry), lysine 37 was identified as a SUMO-1-modified residue by both methods. A lysine 37 site mutant (K37R) was transfected into EGFR deficient cells. Total SUMOylation of EGFR was not altered in the K37R-transfected cells, confirming the presence of other SUMOylation sites. To gain preliminary insight into the possible functional role of EGFR SUMOylation, we compared the effect of expression of the wild-type EGFR with the K37R mutant on promoter activity and expression of <i>CMYC</i> and <i>CNND1.</i> Our results indicate that SUMO-1 modification may affect the transcriptional activity of EGFR, which might have additional impact on, e.g., cancer progression

Additional file 1: Table S1. of IgG Fc galactosylation predicts response to methotrexate in early rheumatoid arthritis

Demographic characteristics of the nationwide EIRA cohort. Table S2. Characterized complement pathway proteins and IgG-isotype proteins. Table S3. Individual IgG-Fc glycan distribution values in IgG1 and in IgG2 in control subjects, as well as in patients with RA prior to and following MTX treatment. Table S4. Significant differences for the 19 characterized glycan species when comparing healthy versus all, good, moderate, and nonresponding patients prior to and following MTX treatment. Table S5. IgG1 and IgG2 Fc glycan distributions grouped according to structural features, comparing healthy control subjects and patients with RA prior to and following MTX treatment. Table S6. Intra- and interindividual differences in the patients with RA, comparing individual glycan species prior to and following MTX treatment. Table S7. Complete list, ranking, and correlation (with response versus no response to MTX) of the features used in the OPLS-DA model shown in Fig. 3b. Figure S1. Extracted ion chromatograms of IgG1 and IgG2 Fc glycans quantified in a control subject and a patient with RA. Figure S2. Intraindividual changes in galactosylation status on IgG1 and on IgG2 for good and moderate responders and for nonresponders. Figure S3. Intraindividual correlation between the aGal/Gal status of glycans with different types of structural features and of IgG1 and IgG2 substituted glycans. Figure S4. Significant differences between control subjects and patients with early RA at baseline in the classical pathway initiating complements C1 and C9. Figure S5. Intraindividual correlation between the classical and lectin pathway inhibitor C4bBPα versus FA2/(FA2G1 + FA2G2). (DOCX 1180 kb

Proteomics-based predictive model using PLS analysis based on loading plot.

<p>The variables on the right are under-represented proteins and the variables on the left are over-represented proteins in advanced stage of IgA nephropathy (A). Score plot of Predictive model (B). </p

Urinary Prognostic Biomarkers and Classification of IgA Nephropathy by High Resolution Mass Spectrometry Coupled with Liquid Chromatography

<div><p>IgA nephropathy is the most common cause of primary glomerulonephritis. There are different pathologic biopsy-based scoring systems in use, but there is no consensus among nephrologists yet regarding the best classification method. Our aim was to test urine proteomics as a non-invasive method for classification of IgA nephropathy. This aim was pursued by discovering novel prognostic protein biomarkers in urine, and linking them to pathogenesis of the disease through known signaling and metabolic pathways. 13 urine samples of the patients with biopsy-proven IgA nephropathy were analyzed via two proteomics approaches: nanoflow LC-MS/MS and GeLC-MS/MS. The results of label-free quantification were subjected to multivariate statistical analysis, which could classify patients into two groups, broadly corresponding to the primary and advance stages. The proteome classification correlated well with biopsy-based scoring systems, especially endocapillary hypercellularity score of the Oxford’s classification. Differentially excreted candidate proteins were found as potential prognostic biomarkers: afamin, leucine-rich alpha-2-glycoprotein, ceruloplasmin, alpha-1-microgolbulin, hemopexin, apolipoprotein A-I, complement C3, vitamin D-binding protein, beta-2-microglobulin, and retinol-binding protein 4. Pathway analysis suggested impairment of Extra Cellular Matrix (ECM)-Receptor Interaction pathways as well as activation of complement and coagulation pathway in progression of IgA nephropathy.</p> </div

Gene Set Enrichment Analysis of biological Process.

<p>This analysis was done by "DAVID" using under-represented proteins obtained from both methods.</p

Quality factor Q against number of proteins N included in the model.

<p>The proteins were sorted according to the number of detected peptides per protein, and N top proteins were retained for the model. The highest Q corresponds to a model N=38 proteins, which was accepted as the best model (A). Unsupervised PCA scores plot based on 38 quantified proteins across all 13 samples. All the replicates were close to each other and there was no statistical outlier (B).</p

Quality factor plot for 1D GeLC-MS/MS.

<p>The best model corresponded to 230 proteins (A). Score plot of PCA using 1D GeLC-MS/MS (B).</p

Complement-coagulation pathway from KEGG database [57, 58].

<p>The proteins over-represented in our data set are marked by an arrow.</p

DataSheet_3_Protein domain-dependent vesiculation of Lipoprotein A, a protein that is important in cell wall synthesis and fitness of the human respiratory pathogen Haemophilus influenzae.docx

The human pathogen Haemophilus influenzae causes respiratory tract infections and is commonly associated with prolonged carriage in patients with chronic obstructive pulmonary disease. Production of outer membrane vesicles (OMVs) is a ubiquitous phenomenon observed in Gram-negative bacteria including H. influenzae. OMVs play an important role in various interactions with the human host; from neutralization of antibodies and complement activation to spread of antimicrobial resistance. Upon vesiculation certain proteins are found in OMVs and some proteins are retained at the cell membrane. The mechanism for this phenomenon is not fully elucidated. We employed mass spectrometry to study vesiculation and the fate of proteins in the outer membrane. Functional groups of proteins were differentially distributed on the cell surface and in OMVs. Despite its supposedly periplasmic and outer membrane location, we found that the peptidoglycan synthase-activator Lipoprotein A (LpoA) was accumulated in OMVs relative to membrane fractions. A mutant devoid of LpoA lost its fitness as revealed by growth and electron microscopy. Furthermore, high-pressure liquid chromatography disclosed a lower concentration (55%) of peptidoglycan in the LpoA-deficient H. influenzae compared to the parent wild type bacterium. Using an LpoA-mNeonGreen fusion protein and fluorescence microscopy, we observed that LpoA was enriched in “foci” in the cell envelope, and further located in the septum during cell division. To define the fate of LpoA, C-terminally truncated LpoA-variants were constructed, and we found that the LpoA C-terminal domain promoted optimal transportation to the OMVs as revealed by flow cytometry. Taken together, our study highlights the importance of LpoA for H. influenzae peptidoglycan biogenesis and provides novel insights into cell wall integrity and OMV production.</p

DataSheet_6_Protein domain-dependent vesiculation of Lipoprotein A, a protein that is important in cell wall synthesis and fitness of the human respiratory pathogen Haemophilus influenzae.docx

The human pathogen Haemophilus influenzae causes respiratory tract infections and is commonly associated with prolonged carriage in patients with chronic obstructive pulmonary disease. Production of outer membrane vesicles (OMVs) is a ubiquitous phenomenon observed in Gram-negative bacteria including H. influenzae. OMVs play an important role in various interactions with the human host; from neutralization of antibodies and complement activation to spread of antimicrobial resistance. Upon vesiculation certain proteins are found in OMVs and some proteins are retained at the cell membrane. The mechanism for this phenomenon is not fully elucidated. We employed mass spectrometry to study vesiculation and the fate of proteins in the outer membrane. Functional groups of proteins were differentially distributed on the cell surface and in OMVs. Despite its supposedly periplasmic and outer membrane location, we found that the peptidoglycan synthase-activator Lipoprotein A (LpoA) was accumulated in OMVs relative to membrane fractions. A mutant devoid of LpoA lost its fitness as revealed by growth and electron microscopy. Furthermore, high-pressure liquid chromatography disclosed a lower concentration (55%) of peptidoglycan in the LpoA-deficient H. influenzae compared to the parent wild type bacterium. Using an LpoA-mNeonGreen fusion protein and fluorescence microscopy, we observed that LpoA was enriched in “foci” in the cell envelope, and further located in the septum during cell division. To define the fate of LpoA, C-terminally truncated LpoA-variants were constructed, and we found that the LpoA C-terminal domain promoted optimal transportation to the OMVs as revealed by flow cytometry. Taken together, our study highlights the importance of LpoA for H. influenzae peptidoglycan biogenesis and provides novel insights into cell wall integrity and OMV production.</p