Prolyl-4-hydroxylase Α subunit 2 (P4HA2) expression is a predictor of poor outcome in breast ductal carcinoma in situ (DCIS)

© 2018, Cancer Research UK. Background: Extracellular matrix (ECM) plays a crucial role in tumour behaviour. Prolyl-4-hydroxlase-A2 (P4HA2) is a key enzyme in ECM remodelling. This study aims to evaluate the prognostic significance of P4HA2 in breast ductal carcinoma in situ (DCIS). Methods: P4HA2 expression was assessed immunohistochemically in malignant cells and surrounding stroma of a large DCIS cohort comprising 481 pure DCIS and 196 mixed DCIS and invasive carcinomas. Outcome analysis was evaluated using local recurrence free interval (LRFI). Results: High P4HA2 expression was detected in malignant cells of half of pure DCIS whereas its expression in stroma was seen in 25% of cases. Higher P4HA2 expression was observed in mixed DCIS cases compared to pure DCIS both in tumour cells and in stroma. High P4HA2 was associated with features of high risk DCIS including younger age, higher grade, comedo necrosis, triple negative and HER2-positive phenotypes. Interaction between P4HA2 and radiotherapy was also observed regarding the outcome. High P4HA2 expression was an independent prognostic factor in predicting shorter LRFI. Conclusion: P4HA2 plays a role in DCIS progression and can potentially be used to predict DCIS outcome. Incorporation of P4HA2 with other clinicopathological parameters could refine DCIS risk stratification that can potentially guide management decisions

Prolyl and lysyl hydroxylases in collagen synthesis

Abstract Collagens are the most abundant proteins in the extracellular matrix. They provide a framework to build organs and tissues and give structural support to make them resistant to mechanical load and forces. Several intra‐ and extracellular modifications are needed to make functional collagen molecules, intracellular post‐translational modifications of proline and lysine residues having key roles in this. In this article, we provide a review on the enzymes responsible for the proline and lysine modifications, that is collagen prolyl 4‐hydroxylases, 3‐hydroxylases and lysyl hydroxylases, and discuss their biological functions and involvement in diseases

Role of prolyl hydroxylation in the molecular interactions of collagens

Abstract Co- and post-translational hydroxylation of proline residues is critical for the stability of the triple helical collagen structure. In this review, we summarise the biology of collagen prolyl 4-hydroxylases and collagen prolyl 3-hydroxylases, the enzymes responsible for proline hydroxylation. Furthermore, we describe the potential roles of hydroxyproline residues in the complex interplay between collagens and other proteins, especially integrin and discoidin domain receptor type cell adhesion receptors. Qualitative and quantitative regulation of collagen hydroxylation may have remarkable effects on the properties of the extracellular matrix and consequently on the cell behaviour

Proline hydroxylation in collagen supports integrin binding by two distinct mechanisms

Abstract Collagens are the most abundant extracellular matrix proteins in vertebrates and have a characteristic triple-helix structure. Hydroxylation of proline residues is critical for helix stability, and diminished prolyl hydroxylase activity causes wide-spread defects in connective tissues. Still, the role of proline hydroxylation in the binding of collagen receptors such as integrins is unclear. Here, we isolated skin collagen from genetically modified mice having reduced prolyl 4-hydroxylase activity. At room temperature, the reduced proline hydroxylation did not affect interactions with the recombinant integrin α2I domain, but at 37 °C, collagen hydroxylation correlated with the avidity of α2I domain binding. Of note, LC–MS/MS analysis of isolated skin collagens revealed no major changes in the hydroxyproline content of the main integrin-binding sites. Thus, the disrupted α2I domain binding at physiological temperatures was most likely due to structural destabilization of the collagenous helix. Integrin α2I binding to the triple-helical GFPGER motif was slightly weaker than to GFOGER (O = hydroxyproline). This phenomenon was more prominent when α1 integrin was tested. Integrin α1β1 expressed on CHO cells and recombinant α1I domain showed remarkably slower binding velocity and weaker avidity to GFPGER when compared with GFOGER. Structural modeling revealed the critical interaction between Arg-218 in α1I and the hydroxyproline residue in the integrin-binding motif. The role of Arg-218 was further validated by testing a variant R218D α1I domain in solid-phase binding assays. Thus, our results show that the lack of proline hydroxylation in collagen can affect integrin binding by a direct mechanism and via structural destabilization of the triple helix

LC-MS peptide hydroxylation assay data files: PHD3 31-70

LC-MS raw data files acquired on an ACQUITY Xevo G2-S QToF mass spectrometer (Waters) which correspond to peptide hydroxylation assays presented in Figure 1 (and supplement). See "Peptide Raw file index" pdf for index of MS runs (referenced to enzyme and substrate)

Activation of the hypoxia response protects mice from amyloid-β accumulation

Abstract Alzheimer’s disease (AD) is the most common cause of dementia with limited treatment options affecting millions of people and the prevalence increasing with the aging population. The current knowledge on the role of the hypoxia/hypoxia-inducible factor (HIF) in the AD pathology is restricted and controversial. We hypothesized based on benefits of the genetic long-term inactivation of HIF prolyl 4-hydroxylase-2 (HIF-P4H-2) on metabolism, vasculature and inflammatory response that prolonged moderate activation of the hypoxia response could hinder AD pathology. We used an aging model to study potential spontaneous accumulation of amyloid-β (Aβ) in HIF-P4H-2-deficient mice and a transgenic APP/PSEN1 mouse model subjected to prolonged sustained environmental hypoxia (15% O2 for 6 weeks) at two different time points of the disease; at age of 4 and 10 months. In both settings, activation of the hypoxia response reduced brain protein aggregate levels and this associated with higher vascularity. In the senescent HIF-P4H-2-deficient mice metabolic reprogramming also contributed to less protein aggregates while in APP/PSEN1 mice lesser Aβ associated additionally with hypoxia-mediated favorable responses to neuroinflammation and amyloid precursor protein processing. In conclusion, continuous, non-full-scale activation of the HIF pathway appears to mediate protection against neurodegeneration via several mechanisms and should be studied as a treatment option for AD

Notch downregulation and extramedullary erythrocytosis in hypoxia-inducible factor prolyl 4-hydroxylase 2-deficient mice

Abstract Erythrocytosis is driven mainly by erythropoietin, which is regulated by hypoxia-inducible factor (HIF). Mutations in HIF prolyl 4-hydroxylase 2 (HIF-P4H-2) (PHD2/EGLN1), the major downregulator of HIFα subunits, are found in familiar erythrocytosis, and large-spectrum conditional inactivation of HIF-P4H-2 in mice leads to severe erythrocytosis. Although bone marrow is the primary site for erythropoiesis, spleen remains capable of extramedullary erythropoiesis. We studied HIF-P4H-2-deficient (Hif-p4h-2gt/gt) mice, which show slightly induced erythropoiesis upon aging despite nonincreased erythropoietin levels, and identified spleen as the site of extramedullary erythropoiesis. Splenic hematopoietic stem cells (HSCs) of these mice exhibited increased erythroid burst-forming unit (BFU-E) growth, and the mice were protected against anemia. HIF-1α and HIF-2α were stabilized in the spleens, while the Notch ligand genes Jag1, Jag2, and Dll1 and target Hes1 became downregulated upon aging HIF-2α dependently. Inhibition of Notch signaling in wild-type spleen HSCs phenocopied the increased BFU-E growth. HIFα stabilization can thus mediate non-erythropoietin-driven splenic erythropoiesis via altered Notch signaling

LC-MS peptide hydroxylation assay data files: PHD2 31-70

LC-MS raw data files acquired on an ACQUITY Xevo G2-S QToF mass spectrometer (Waters) which correspond to peptide hydroxylation assays presented in Figure 1 (and supplement). See "Peptide Raw file index" pdf for index of MS runs (referenced to enzyme and substrate)

LC-MS peptide hydroxylation assay data files PHD1 31-70

LC-MS raw data files acquired on an ACQUITY Xevo G2-S QToF mass spectrometer (Waters) which correspond to peptide hydroxylation assays presented in Figure 1 (and supplement). See "Peptide Raw file index" pdf for index of MS runs (referenced to enzyme and substrate)