A method for the isolation of blocked N-terminal peptides.
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Grifola↗
Biomedical subjects
David H Hawke
Publications and source records attributed to David H Hawke.
Tyrosine phosphorylation controls PCNA function through protein stability.
The proliferating cell nuclear antigen (PCNA) is an essential protein for DNA replication and damage repair. How its function is controlled remains an important question. Here, we show that the chromatin-bound PCNA protein is phosphorylated on Tyr 211, which is required for maintaining its function on chromatin and is dependent on the tyrosine kinase activity of EGF receptor (EGFR) in the nucleus. Phosphorylation on Tyr 211 by EGFR stabilizes chromatin-bound PCNA protein and associated functions. Consistently, increased PCNA Tyr 211 phosphorylation coincides with pronounced cell proliferation, and is better correlated with poor survival of breast cancer patients, as well as nuclear EGFR in tumours, than is the total PCNA level. These results identify a novel nuclear mechanism linking tyrosine kinase receptor function with the regulation of the PCNA sliding clamp.
Cell Nucleus↗
Proteomic analysis of nipple aspirate fluid from women with early-stage breast cancer using isotope-coded affinity tags and tandem mass spectrometry reveals differential expression of vitamin D binding protein.
BACKGROUND: Isotope-coded affinity tag (ICAT) tandem mass spectrometry (MS) allows for qualitative and quantitative analysis of paired protein samples. We sought to determine whether ICAT technology could quantify and identify differential expression of tumor-specific proteins in nipple aspirate fluid (NAF) from the tumor-bearing and contralateral disease-free breasts of patients with unilateral early-stage breast cancer. METHODS: Paired NAF samples from 18 women with stage I or II unilateral invasive breast carcinoma and 4 healthy volunteers were analyzed using ICAT labeling, sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), liquid chromatography, and MS. Proteins were identified by sequence database analysis. Western blot analysis of NAF from an independent sample set from 12 women (8 with early-stage breast cancer and 4 healthy volunteers) was also performed. RESULTS: 353 peptides were identified from tandem mass spectra and matched to peptide sequences in the National Center for Biotechnology Information database. Equal numbers of peptides were up- versus down-regulated. Alpha2HS-glycoprotein [Heavy:Light (H:L) ratio 0.63] was underexpressed in NAF from tumor-bearing breasts, while lipophilin B (H:L ratio 1.42), beta-globin (H:L ratio 1.98), hemopexin (H:L ratio 1.73), and vitamin D-binding protein precursor (H:L ratio 1.82) were overexpressed. Western blot analysis of pooled samples of NAF from healthy volunteers versus NAF from women with breast cancer confirmed the overexpression of vitamin D-binding protein in tumor-bearing breasts. CONCLUSION: ICAT tandem MS was able to identify and quantify differences in specific protein expression between NAF samples from tumor-bearing and disease-free breasts. Proteomic screening techniques using ICAT and NAF may be used to find markers for diagnosis of breast cancer.
Biomarkers, Tumor↗
The Set1 methyltransferase opposes Ipl1 aurora kinase functions in chromosome segregation.
A balance in the activities of the Ipl Aurora kinase and the Glc7 phosphatase is essential for normal chromosome segregation in yeast. We report here that this balance is modulated by the Set1 methyltransferase. Deletion of SET1 suppresses chromosome loss in ipl1-2 cells. Conversely, combination of SET1 and GLC7 mutations is lethal. Strikingly, these effects are independent of previously defined functions for Set1 in transcription initiation and histone H3 methylation. We find that Set1 is required for methylation of conserved lysines in a kinetochore protein, Dam1. Biochemical and genetic experiments indicate that Dam1 methylation inhibits Ipl1-mediated phosphorylation of flanking serines. Our studies demonstrate that Set1 has important, unexpected functions in mitosis. Moreover, our findings suggest that antagonism between lysine methylation and serine phosphorylation is a fundamental mechanism for controlling protein function.
Amino Acid Sequence↗
Diagnostic protein discovery using liquid chromatography/mass spectrometry for proteolytic peptide targeting.
A peptide targeting method has been developed for diagnostic protein discovery, which combines proteolytic digestion of fractionated plasma proteins and liquid chromatography coupled to electrospray time-of-flight mass spectrometry (LC/ESI-TOFMS) profiling. Proteolysis prior to profiling overcomes molecular weight limitations and compensates for the poor sensitivity of matrix-assisted laser desorption/ionization (MALDI) protein profiling. LC/MS increases the peak capacity compared to crude fractionation techniques or single sample MALDI analysis. Differentially expressed peptides are targeted in the mass chromatograms using bioinformatic techniques and subsequently sequenced with MALDI tandem MS. In a model study comparing pancreatic cancer patients to controls, 74% of the peptide targets were successfully sequenced. This profiling method was superior to previous experiments using single sample MALDI analysis for protein profiling or proteolytic peptide profiling, because more potential protein markers were identified.
Adult↗