iTRAQ proteomic identification of pVHL-dependent and -independent targets of Egln1 prolyl hydroxylase knockdown in renal carcinoma cells

Abstract

Summary The large subunit of RNA Polymerase II, Rpb1, undergoes hydroxylation on proline 1465, which in turn triggers Ser5 hydroxylation. While Egln2 prolyl hydroxylase appears to mediate P1465 hydroxylation, Egln1 has an inhibitory activity and its knockdown stimulates constitutive hydroxylation and Ser5 phosphorylation of Rpb1, but only in cells that are VHL(+). In this study we have analyzed protein factors affected by the knockdown of Egln1 in VHL(+) and VHL(−) cells. We found that, in VHL(+) cells, several proteins were inhibited but none were induced by Egln2 knockdown. The function of several of those proteins was related to calcium metabolism and the cytoskeleton. In contrast, in VHL(−) cells Egln1 knockdown caused upregulation of several mitochondrial proteins including subunits of ATP synthase. Several of the proteins repressed in VHL(−) cells by Egln1 knockdown were involved in the function of RNA polymerase II during transcription from chromatin templates. These data suggest that the effects of Egln1 knockdown depend on the status of pVHL and can be correlated with effects on Rpb1.

Identification and validation of TGFBI as a promising prognosis marker of clear cell renal cell carcinoma

“…Moreover, proteomic studies have the advantage to assess the ultimate expression of the malignant phenotype and provide great opportunities to identify new biomarkers. Nowadays, few studies have used the iTRAQ method for RCC biomarker research [7][8][9]. The purpose of this study was to identify prognostic biomarkers in clear cell RCC (ccRCC) by using a proteomic approach and the SSIGN score.…”

Prolyl Hydroxylase Domain (PHD) 2 Affects Cell Migration and F-actin Formation via RhoA/Rho-associated Kinase-dependent Cofilin Phosphorylation

“…For PHD1, PHD2 and PHD3 a regulation of the IB kinase, the large subunit of RNA polymerase II and ATF-4 has been suggested, respectively (18 -20). A recently described iTRAQ proteome approach has additionally led to the suggestion that proteins related to the cytoskeleton are regulated as a function of PHD2 (21).…”

Profiling of Endogenous Peptides by Multidimensional Liquid Chromatography

“…If the flow rate is set to 3 μl/min, and an equal flow of MALDI matrix solution is added post-column (7 mg/ml re-crystallized α-cyanohydroxycinnamic acid, 2 mg/ml ammonium phosphate, 0.1% trifluoroacetic acid, 80% acetonitrile) and the combined eluant is automatically spotted onto a stainless steel MALDI target plate every 6 s (0.6 μl/spot), a total of 370 spots obtained per original SCX fraction (Fort et al, 2009). Haffey demonstrated similar approach and obtained 3828 MALDI-TOF spots from the 12 SCX fractions (Haffey et al, 2009). Such a separation strategy offers enormous discrimination power and imposing peak capacity.…”