The 20S proteasome activator PA28γ controls the compaction of chromatin.

PA28γ (also known as PSME3), a nuclear activator of the 20S proteasome, is involved in the degradation of several proteins regulating cell growth and proliferation and in the dynamics of various nuclear bodies, but its precise cellular functions remain unclear. Here, using a quantitative FLIM-FRET based microscopy assay monitoring close proximity between nucleosomes in living human cells, we show that PA28γ controls chromatin compaction. We find that its depletion induces a decompaction of pericentromeric heterochromatin, which is similar to what is observed upon the knockdown of HP1ß (also known as CBX1), a key factor of the heterochromatin structure. We show that PA28γ is present at HP1ß-containing repetitive DNA sequences abundant in heterochromatin and, importantly, that HP1ß on its own is unable to drive chromatin compaction without the presence of PA28γ. At the molecular level, we show that this novel function of PA28γ is independent of its stable interaction with the 20S proteasome, and most likely depends on its ability to maintain appropriate levels of H3K9me3 and H4K20me3, histone modifications that are involved in heterochromatin formation. Overall, our results implicate PA28γ as a key factor involved in the regulation of the higher order structure of chromatin.

Quantitative proteomic and phosphoproteomic comparison of human colon cancer DLD-1 cells differing in ploidy and chromosome stability.

Although aneuploidy is poorly tolerated during embryogenesis, aneuploidy and whole chromosomal instability (CIN) are common hallmarks of cancer, raising the question of how cancer cells can thrive in spite of chromosome aberrations. Here we present a comprehensive and quantitative proteomics analysis of isogenic DLD-1 colorectal adenocarcinoma cells lines, aimed at identifying cellular responses to changes in ploidy and/or CIN. Specifically, we compared diploid (2N) and tetraploid (4N) cells with posttetraploid aneuploid (PTA) clones and engineered trisomic clones. Our study provides a comparative data set on the proteomes and phosphoproteomes of the above cell lines, comprising several thousand proteins and phosphopeptides. In comparison to the parental 2N line, we observed changes in proteins associated with stress responses and with interferon signaling. Although we did not detect a conspicuous protein signature associated with CIN, we observed many changes in phosphopeptides that relate to fundamental cellular processes, including mitotic progression and spindle function. Most importantly, we found that most changes detectable in PTA cells were already present in the 4N progenitor line. This suggests that activation of mitotic pathways through hyper-phosphorylation likely constitutes an important response to chromosomal burden. In line with this conclusion, cells with extensive chromosome gains showed differential sensitivity toward a number of inhibitors targeting cell cycle kinases, suggesting that the efficacy of anti-mitotic drugs may depend on the karyotype of cancer cells.

Cellular Prion Protein PrPC and Ecto-5'-Nucleotidase Are Markers of the Cellular Stress Response to Aneuploidy.

Aneuploidy is a hallmark of most human tumors, but the molecular physiology of aneuploid cells is not well characterized. In this study, we screened cell surface biomarkers of approximately 300 proteins by multiparameter flow cytometry using multiple aneuploid model systems such as cell lines, patient samples, and mouse models. Several new biomarkers were identified with altered expression in aneuploid cells, including overexpression of the cellular prion protein CD230/PrPC and the immunosuppressive cell surface enzyme ecto-5'-nucleotidase CD73. Functional analyses associated these alterations with increased cellular stress. An increased number of CD73+ cells was observed in confluent cultures in aneuploid cells relative to their diploid counterparts. An elevated expression in CD230/PrPC was observed in serum-deprived cells in association with increased generation of reactive oxygen species. Overall, our work identified biomarkers of aneuploid karyotypes, which suggest insights into the underlying molecular physiology of aneuploid cells. Cancer Res; 77(11); 2914-26. ©2017 AACR.

Evaluation and Improvement of Quantification Accuracy in Isobaric Mass Tag-Based Protein Quantification Experiments.

The multiplexing capabilities of isobaric mass tag-based protein quantification, such as Tandem Mass Tags or Isobaric Tag for Relative and Absolute Quantitation have dramatically increased the scope of mass spectrometry-based proteomics studies. Not only does the technology allow for the simultaneous quantification of multiple samples in a single MS injection, but its seamless compatibility with extensive sample prefractionation methods allows for comprehensive studies of complex proteomes. However, reporter ion-based quantification has often been criticized for limited quantification accuracy due to interference from coeluting peptides and peptide fragments. In this study, we investigate the extent of this problem and propose an effective and easy-to-implement remedy that relies on spiking a 6-protein calibration mixture to the samples. We evaluated our ratio adjustment approach using two large scale TMT 10-plex data sets derived from a human cancer and noncancer cell line as well as E. coli cells grown at two different conditions. Furthermore, we analyzed a complex 2-proteome artificial sample mixture and investigated the precision of TMT and precursor ion intensity-based label free quantification. Studying the protein set identified by both methods, we found that differentially abundant proteins were assigned dramatically higher statistical significance when quantified using TMT. Data are available via ProteomeXchange with identifier PXD003346.

Phosphate homeostasis in Bartter syndrome: a case-control study.

BACKGROUND: Bartter patients may be hypercalciuric. Additional abnormalities in the metabolism of calcium, phosphate, and calciotropic hormones have occasionally been reported. METHODS: The metabolism of calcium, phosphate, and calciotropic hormones was investigated in 15 patients with Bartter syndrome and 15 healthy subjects. RESULTS: Compared to the controls, Bartter patients had significantly reduced plasma phosphate {mean [interquartile range]:1.29 [1.16-1.46] vs. 1.61 [1.54-1.67] mmol/L} and maximal tubular phosphate reabsorption (1.16 [1.00-1.35] vs. 1.41 [1.37-1.47] mmol/L) and significantly increased parathyroid hormone (PTH) level (6.1 [4.5-7.7] vs. 2.8 [2.2-4.4] pmol/L). However, patients and controls did not differ in blood calcium, 25-hydroxyvitamin D, alkaline phosphatase, and osteocalcin levels. In patients, an inverse correlation (P < 0.05) was noted between total plasma calcium or glomerular filtration rate and PTH concentration. A positive correlation was also noted between PTH and osteocalcin concentrations (P < 0.005), as well as between chloriduria or natriuria and phosphaturia (P < 0.001). No correlation was noted between calciuria and PTH concentration or between urinary or circulating phosphate and PTH. CONCLUSIONS: The results of this study demonstrate a tendency towards renal phosphate wasting and elevated circulating PTH levels in Bartter patients.

FilmArray® respiratory panel performance in respiratory samples from neonatal care units.

FilmArray Respiratory Panel (RP) (Idaho Technology, Inc., Salt Lake City, UT, USA) performance was retrospectively evaluated in respiratory samples collected from neonates in 2 reference neonatology units. Using the FilmArray RP assay, 121/152 (79.6%) samples were positive for at least 1 respiratory virus, while 31/152 (20.4%) were negative. FilmArray RP results were concordant in 68/72 (94.4%) respiratory samples tested with laboratory-developed real-time PCR assays, while in 4/72 (5.6%) samples, the FilmArray RP assay detected an additional virus (2 human rhinovirus/enterovirus and 2 bocavirus). In addition, FilmArray RP results for 70 of 80 (87.5%) respiratory samples tested were concordant with the Seegene Seeplex RV15® detection assay (Seegene, Inc., Seoul, South Korea), while 10/80 (12.5%) were discordant. The advantages of the FilmArray RP are the rapid detection of respiratory viruses (1 hour), the wide number of pathogens detectable in a single assay, and the reduced hands-on time.

Renal phosphate handling in Gitelman syndrome--the results of a case-control study.

BACKGROUND: Patients with Gitelman syndrome, a hereditary salt-wasting tubulopathy, have loss-of-function mutations in the SLC12A3 gene coding for the thiazide-sensitive sodium chloride co-transporter in the distal convoluted tubule. Since the bulk of filtered phosphate is reabsorbed in the proximal tubule, renal phosphate wasting is considered exceptional in Gitelman syndrome. METHODS: We investigated the renal handling of inorganic phosphate in 12 unselected Italian patients affected with Gitelman syndrome (5 females and 7 males, aged 6.0-18 years, median age 12 years) and in 12 healthy subjects matched for gender and age (controls). The diagnosis of Gitelman syndrome among the patients had been made clinically and confirmed by molecular biology studies. RESULTS: The biochemical hallmarks of Gitelman syndrome, namely hypochloremia, hypokalemia, hypomagnesemia, increased urinary excretion of sodium, chloride, potassium and magnesium and reduced urinary excretion of calcium, were present in the 12 patients. In addition, both the plasma inorganic phosphate concentration (median and interquartile range: 1.28 [1.12-1.36] vs. 1.61 [1.51-1.66)] mmol/L) and the maximal tubular reabsorption of inorganic phosphate (1.08 [0.99-1.22] vs. 1.41 [1.38-1.47] mmol/L) were significantly lower (P < 0.001) in Gitelman patients than in control subjects. Circulating levels of 25-hydroxyvitamin D, intact parathyroid hormone and osteocalcin were similar in patients and controls. CONCLUSIONS: The results of our case-control study disclose a hitherto unrecognized tendency towards renal phosphate wasting with mild to moderate hypophosphatemia in Gitelman syndrome.