Prolyl Hydroxylation Research Articles

First person – Roberta Besio

ABSTRACTFirst Person is a series of interviews with the first authors of a selection of papers published in Disease Models & Mechanisms (DMM), helping early-career researchers promote themselves alongside their papers. Roberta Besio is first author on ‘Cellular stress due to impairment of collagen prolyl hydroxylation complex is rescued by the chaperone 4-phenylbutyrate’, published in DMM. Roberta is a postdoc in the lab of Antonella Forlino at University of Pavia, Italy, investigating collagen and genetic diseases of the connective tissue.

FoxO3 activation in hypoxic tubules prevents chronic kidney disease.

Acute kidney injury (AKI) can lead to chronic kidney disease (CKD) if injury is severe and/or repair is incomplete. However, the pathogenesis of CKD following renal ischemic injury is not fully understood. Capillary rarefaction and tubular hypoxia are common findings during the AKI to CKD transition. We investigated the tubular stress response to hypoxia and demonstrated that a stress responsive transcription factor, FoxO3, was regulated by prolyl hydroxylase. Hypoxia inhibited FoxO3 prolyl hydroxylation and FoxO3 degradation, thus leading to FoxO3 accumulation and activation in tubular cells. Hypoxia-activated Hif-1α contributed to FoxO3 activation and functioned to protect kidneys, as tubular deletion of Hif-1α decreased hypoxia-induced FoxO3 activation, and resulted in more severe tubular injury and interstitial fibrosis following ischemic injury. Strikingly, tubular deletion of FoxO3 during the AKI to CKD transition aggravated renal structural and functional damage leading to a more profound CKD phenotype. We showed that tubular deletion of FoxO3 resulted in decreased autophagic response and increased oxidative injury, which may explain renal protection by FoxO3. Our study indicates that in the hypoxic kidney, stress responsive transcription factors can be activated for adaptions to counteract hypoxic insults, thus attenuating CKD development.

Characterization of PPIB interaction in the P3H1 ternary complex and implications for its pathological mutations.

The P3H1/CRTAP/PPIB complex is essential for prolyl 3-hydroxylation and folding of procollagens in the endoplasmic reticulum (ER). Deficiency in any component of this ternary complex is associated with the misfolding of collagen and the onset of osteogenesis imperfecta. However, little structure information is available about how this ternary complex is assembled and retained in the ER. Here, we assessed the role of the KDEL sequence of P3H1 and probed the spatial interactions of PPIB in the complex. We show that the KDEL sequence is essential for retaining the P3H1 complex in the ER. Its removal resulted in co-secretion of P3H1 and CRTAP out of the cell, which was mediated by the binding of P3H1 N-terminal domain with CRTAP. The secreted P3H1/CRTAP can readily bind PPIB with their C-termini close to PPIB in the ternary complex. Cysteine modification, crosslinking, and mass spectrometry experiments identified PPIB surface residues involved in the complex formation, and showed that the surface of PPIB is extensively covered by the binding of P3H1 and CRTAP. Most importantly, we demonstrated that one disease-associated pathological PPIB mutation on the binding interface did not affect the PPIB prolyl-isomerase activity, but disrupted the formation of P3H1/CRTAP/PPIB ternary complex. This suggests that defects in the integrity of the P3H1 ternary complex are associated with pathological collagen misfolding. Taken together, these results provide novel structural information on how PPIB interacts with other components of the P3H1 complex and indicate that the integrity of P3H1 complex is required for proper collagen formation.

Serum Lipoproteins Regulate Hypoxia‐Inducible Factors Under Normoxia

Hypoxia‐inducible factor (HIF) transcription factors are central regulators of oxygen homeostasis, controlling energy utilization, erythrogenesis, angiogenesis and cancer metabolism. Oxygen negatively regulates HIF activity by promoting HIFα prolyl hydroxylation and proteasome‐dependent degradation. Using human pancreatic adenocarcinoma cells, we demonstrate that serum lipoproteins are an independent regulator of HIF under normoxia. Decreasing extracellular lipid supply inhibited HIF prolyl hydroxylation, leading to accumulation of HIFα comparable to hypoxia with activation of downstream target genes. Addition of unsaturated fatty acids suppressed this signal, which required an intact mitochondrial respiratory chain. A survey of different cell lines showed that this signaling pathway functions in a wide range of tissues. These studies show that lipoprotein and oxygen supply are distinct signals integrated to control HIF activity and identify serum lipids as a potential modulator of HIF pathway activity.Support or Funding InformationNational Institutes of Health, HL077588 and GM126088This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

4-O-Methylascochlorin inhibits the prolyl hydroxylation of hypoxia-inducible factor-1α, which is attenuated by ascorbate.

4-O-Methylascochlorin (MAC), a methylated derivative of ascochlorin, was previously shown to promote the accumulation of hypoxia-inducible factor (HIF)-1α in human breast adenocarcinoma MCF-7 cells. In the present study, we further investigated the effects of MAC on the expression and function of HIF-1α in human fibrosarcoma HT-1080 cells. MAC promoted the accumulation of the HIF-1α protein without affecting its constitutive mRNA expression and augmented the transcriptional activation of HIF target genes. Ascorbate, but not N-acetylcysteine, attenuated MAC-mediated HIF-1α accumulation. MAC-induced increases in HIF-1α transcriptional activity were also attenuated by ascorbate. MAC inhibited the hydroxylation of HIF-1α at the proline 564 residue, while it was reversed by ascorbate. MAC slightly decreased the intracellular concentration of ascorbate. The present results demonstrated that MAC promoted the accumulation of HIF-1α by preventing prolyl hydroxylation, and ascorbate attenuated the MAC-mediated inhibition of HIF-1α prolyl hydroxylation.

HIF prolyl hydroxylase inhibitor FG-4497 enhances mouse hematopoietic stem cell mobilization via VEGFR2/KDR

AbstractIn normoxia, hypoxia-inducible transcription factors (HIFs) are rapidly degraded within the cytoplasm as a consequence of their prolyl hydroxylation by oxygen-dependent prolyl hydroxylase domain (PHD) enzymes. We have previously shown that hematopoietic stem and progenitor cells (HSPCs) require HIF-1 for effective mobilization in response to granulocyte colony-stimulating factor (G-CSF) and CXCR4 antagonist AMD3100/plerixafor. Conversely, HIF PHD inhibitors that stabilize HIF-1 protein in vivo enhance HSPC mobilization in response to G-CSF or AMD3100 in a cell-intrinsic manner. We now show that extrinsic mechanisms involving vascular endothelial growth factor receptor-2 (VEGFR2), via bone marrow (BM) endothelial cells, are also at play. PTK787/vatalanib, a tyrosine kinase inhibitor selective for VEGFR1 and VEGFR2, and neutralizing anti-VEGFR2 monoclonal antibody DC101 blocked enhancement of HSPC mobilization by FG-4497. VEGFR2 was absent on mesenchymal and hematopoietic cells and was detected only in Sca1+ endothelial cells in the BM. We propose that HIF PHD inhibitor FG-4497 enhances HSPC mobilization by stabilizing HIF-1α in HSPCs as previously demonstrated, as well as by activating VEGFR2 signaling in BM endothelial cells, which facilitates HSPC egress from the BM into the circulation.

Influenza A virus (H1N1) triggers a hypoxic response by stabilizing hypoxia-inducible factor-1α via inhibition of proteasome

Virus reprogramming of host cellular function is a critical strategy for viral survival and replication. A better understanding of virus-host interaction may provide new potential avenues for the treatment of viral diseases. It has been reported that hypoxia-inducible factor-1 (HIF-1) pathway is activated by a range of pathogens via different mechanisms, but the impact of Influenza A virus on HIF-1 signaling is still unclear. In this study, we observed H1N1 infection stabilized HIF-1α under normoxic conditions. In detail, H1N1 did not increase HIF-1α mRNA transcription, nor impaired posttranslational prolyl hydroxylation or ubiquitination of HIF-1α, but inhibited the function of proteasome, resulting in HIF-1α accumulation. Furthermore, a decreased expression of factor inhibiting HIF-1 (FIH-1), which hydroxylates asparagine 803 within HIF-1α to repress HIF-1α activity, was seen after H1N1 infection. Taken together, these findings reveal a previously unrecognized mechanism of viral activation of the HIF-1 pathway, resembling a hypoxic response in normoxia.

Quantitative proteomic profiling of extracellular matrix and site-specific collagen post-translational modifications in an in vitro model of lung fibrosis

Lung fibrosis is characterized by excessive deposition of extracellular matrix (ECM), in particular collagens, by fibroblasts in the interstitium. Transforming growth factor-β1 (TGF-β1) alters the expression of many extracellular matrix (ECM) components produced by fibroblasts, but such changes in ECM composition as well as modulation of collagen post-translational modification (PTM) levels have not been comprehensively investigated. Here, we performed mass spectrometry (MS)-based proteomics analyses to assess changes in the ECM deposited by cultured lung fibroblasts from idiopathic pulmonary fibrosis (IPF) patients upon stimulation with transforming growth factor β1 (TGF-β1). In addition to the ECM changes commonly associated with lung fibrosis, MS-based label-free quantification revealed profound effects on enzymes involved in ECM crosslinking and turnover as well as multiple positive and negative feedback mechanisms of TGF-β1 signaling. Notably, the ECM changes observed in this in vitro model correlated significantly with ECM changes observed in patient samples. Because collagens are subject to multiple PTMs with major implications in disease, we implemented a new bioinformatic platform to analyze MS data that allows for the comprehensive mapping and site-specific quantitation of collagen PTMs in crude ECM preparations. These analyses yielded a comprehensive map of prolyl and lysyl hydroxylations as well as lysyl glycosylations for 15 collagen chains. In addition, site-specific PTM analysis revealed novel sites of prolyl-3-hydroxylation and lysyl glycosylation in type I collagen. Interestingly, the results show, for the first time, that TGF-β1 can modulate prolyl-3-hydroxylation and glycosylation in a site-specific manner. Taken together, this proof of concept study not only reveals unanticipated TGF-β1 mediated regulation of collagen PTMs and other ECM components but also lays the foundation for dissecting their key roles in health and disease.The proteomic data has been deposited to the ProteomeXchange Consortium via the MassIVE partner repository with the data set identifier MSV000082958.

Pharmacologic Targeting of Hypoxia-Inducible Factors.

Hypoxia-inducible factors (HIFs) control transcriptional responses to reduced O2 availability. HIFs are heterodimeric proteins composed of an O2-regulated HIF-α subunit and a constitutively expressed HIF-1β subunit. HIF-α subunits are subject to prolyl hydroxylation, which targets the proteins for degradation under normoxic conditions. Small molecule prolyl hydroxylase inhibitors, which stabilize the HIF-α subunits and increase HIF-dependent expression of erythropoietin, are in phase III clinical trials for the treatment of anemia in patients with chronic kidney disease. HIFs contribute to the pathogenesis of many cancers, particularly the clear cell type of renal cell carcinoma in which loss of function of the von Hippel-Lindau tumor suppressor blocks HIF-2α degradation. A small molecule inhibitor that binds to HIF-2α and blocks dimerization with HIF-1β is in clinical trials for the treatment of renal cell carcinoma. Targeting HIFs for stabilization or inhibition may improve outcomes in diseases that are common causes of mortality in the US population.

A Fluorescent Benzo[g]isoquinoline-Based HIF Prolyl Hydroxylase Inhibitor for Cellular Imaging.

Prolyl hydroxylation domain (PHD) enzymes catalyze the hydroxylation of the transcription factor hypoxia-inducible factor (HIF) and serve as cellular oxygen sensors. HIF and the PHD enzymes regulate numerous potentially tissue-protective target genes which can adapt cells to metabolic and ischemic stress. We describe a fluorescent PHD inhibitor (1-chloro-4-hydroxybenzo[g]isoquinoline-3-carbonyl)glycine which is suited to fluorescence-based detection assays and for monitoring PHD inhibitors in biological systems. In cell-based assays, application of the fluorescent PHD inhibitor allowed co-localization with a cellular PHD enzyme and led to live cell imaging of processes involved in cellular oxygen sensing.

Collagen Prolyl Hydroxylases Are Bifunctional Growth Regulators in Melanoma

Appropriate post-translational processing of collagen requires prolyl hydroxylation, catalyzed by collagen prolyl 3-hydroxylase and collagen prolyl 4-hydroxylase, and is essential for normal cell function. Here wehave investigated the expression, transcriptional regulation, and function of the collagen prolyl 3-hydroxylase and collagen prolyl 4-hydroxylase families in melanoma. We show that the collagen prolyl3-hydroxylase family exemplified by Leprel1 and Leprel2 is subject to methylation-dependent transcriptional silencing in primary and metastatic melanoma consistent with a tumor suppressor function. In contrast, although there is transcriptional silencing of P4HA3 in a subset of melanomas, the collagen prolyl 4-hydroxylase familymembers P4HA1, P4HA2, and P4HA3 are often overexpressed in melanoma, expression being prognostic of worse clinical outcomes. Consistent with tumor suppressor function, ectopic expression of Leprel1 and Leprel2 inhibits melanoma proliferation, whereas P4HA2 and P4HA3 increase proliferation, and particularly invasiveness, of melanoma cells. Pharmacological inhibition with multiple selective collagen prolyl 4-hydroxylase inhibitors reduces proliferation and inhibits invasiveness of melanoma cells. Together,our data identify the collagen prolyl 3-hydroxylase and collagen prolyl 4-hydroxylase families as potentially important regulators of melanoma growth and invasiveness and suggest that selective inhibitionof collagen prolyl 4-hydroxylase is an attractive strategy to reduce the invasive properties of melanoma cells.

Loss of the Prolyl Hydroxylase OGFOD1 In Vivo Leads to Metabolic Alterations

Prolyl hydroxylation is a post-translational modification that modulates protein stability, turnover, and activity, as well as cell survival. There is a large family of proteins that catalyze proline hydroxylation, including the hypoxia-inducible factor alpha (HIFα) prolyl hydroxylase domain enzymes (HIF-PHDs). Although the HIF-PHDs have been well studied, very little is known about the endogenous function of the prolyl hydroxylase 2-oxoglutarate- and Fe2+-dependent oxygenase 1 (OGFOD1), which in contrast to HIF-PHDs remains active under hypoxic conditions. We investigated whether OGFOD1 has a role in augmenting or suppressing myocardial injury following I/R using OGFOD1 knockout mice, which are viable and fertile. We subjected perfused hearts from 6 wildtype (WT) and 6 OGFOD1 knockout (KO) to ischemia and reperfusion, and found that OGFOD1 loss was protective against I/R injury, with OGFOD1-KO hearts showing a ~33% (p < 0.01) reduction in infarct size. To better understand the mechanism responsible for the protection in the OGFOD1-KO hearts, we isolated hearts from 5 OGFOD1-WT mice and 5 OGFOD1-KO mice and used Tandem Mass Tag (TMT) Liquid Chromatography and tandem Mass Spectrometry (LC-MS/MS) to identify proteomic changes. We found that OGFOD1 loss leads to up-regulation of enzymes in fatty acid and ketone oxidation. These results were supported by metabolomics data of cardiac tissue, which showed a significant decrease in the ketone body β-hydroxybutyrate in OGFOD1-KO hearts, despite comparable levels of β-hydroxybutyrate in serum from OGFOD1-WT and OGFOD1-KO mice. Altogether, these data support the hypothesis that OGFOD1 ablation alters myocardial metabolism, which may contribute to protection against I/R injury.

The molecular characterization, expression pattern and alternative initiation of Megalobrama amblycephala Hif prolyl hydroxylase Phd1

HIF prolyl hydroxylase 1 (PHD1) functions in prolyl hydroxylation on mammal hypoxia-inducible factors (HIF), important transcription factors involved in hypoxia, however the roles of Phd1 in fish remain unclear. In this study, the full-length cDNA and promoter sequences of blunt snout bream (Megalobrama amblycephala) phd1 gene were isolated by a modified RACE strategy. The phd1 cDNA was 2672 bp for encoding 481 amino acid residues. In silico assays indicated that phd1 had 5 exons, and a 348 bp CpG island in the exon1, and several transcription factor binding sites (CAAT box, HRE, ARNT, FOX, etc) were also found on the promoter. The quantitative real-time PCR results suggested that phd1 mRNA was constitutively expressed in all detected tissues, and higher in the blood, brain and heart in normoxia, but significantly decreased after hypoxia in all detected tissues except for gill. Western blot assays indicated that two Phd1 isoforms were generated by alternative translation initiation. Moreover, these two isoforms were both localized in the nucleus, therein only the senior isoform promoted cell proliferation. Taken together, the present study firstly describes the functions of M. amblycephala two Phd1 isoforms in hypoxia and cell proliferation.

Characterization of Angiotensin-Converting Enzyme Inhibitory Activity of X-Hyp-Gly-Type Tripeptides: Importance of Collagen-Specific Prolyl Hydroxylation.

Hydroxyproline (Hyp) is a collagen-specific amino acid formed by post-translational hydroxylation of Pro residues. Various Hyp-containing oligopeptides are transported into the blood at high concentrations after oral ingestion of collagen hydrolysate. Here we investigated the angiotensin-converting enzyme (ACE) inhibitory activity of X-Hyp-Gly-type tripeptides. In an in vitro assay, ginger-degraded collagen hydrolysate enriched with X-Hyp-Gly-type tripeptides dose-dependently inhibited ACE and various synthetic X-Hyp-Gly-type tripeptides showed ACE-inhibitory activity. In particular, strong inhibition was observed for Leu-Hyp-Gly, Ile-Hyp-Gly, and Val-Hyp-Gly with IC50 values of 5.5, 9.4, and 12.8 μM, respectively. Surprisingly, substitution of Hyp with Pro dramatically decreased inhibitory activity of X-Hyp-Gly, indicating that Hyp is important for ACE inhibition. This finding was supported by molecular docking experiments using Leu-Hyp-Gly/Leu-Pro-Gly. We further demonstrated that prolyl hydroxylation significantly enhanced resistance to enzymatic degradation by incubation with mouse plasma. The strong ACE-inhibitory activity and high stability of X-Hyp-Gly-type tripeptides highlight their potential for hypertension control.

Expression characterization and functional implication of the collagen-modifying Leprecan proteins in mouse gonadal tissue and mature sperm.

The Leprecan protein family which includes the prolyl 3-hydroxylase enzymes (P3H1, P3H2, and P3H3), the closely related cartilage-associated protein (CRTAP), and SC65 (Synaptonemal complex 65, aka P3H4, LEPREL4), is involved in the post-translational modification of fibrillar collagens. Mutations in CRTAP, P3H1 and P3H2 cause human genetic diseases. We recently showed that SC65 forms a stable complex in the endoplasmic reticulum with P3H3 and lysyl hydroxylase 1 and that loss of this complex leads to defective collagen lysyl hydroxylation and causes low bone mass and skin fragility. Interestingly, SC65 was initially described as a synaptonemal complex-associated protein, suggesting a potential additional role in germline cells. In the present study, we describe the expression of SC65, CRTAP and other Leprecan proteins in postnatal mouse reproductive organs. We detect SC65 expression in peritubular cells of testis up to 4 weeks of age but not in cells within seminiferous tubules, while its expression is maintained in ovarian follicles until adulthood. Similar to bone and skin, SC65 and P3H3 are also tightly co-expressed in testis and ovary. Moreover, we show that CRTAP, a protein normally involved in collagen prolyl 3-hydroxylation, is highly expressed in follicles and stroma of the ovary and in testes interstitial cells at 4 weeks of age, germline cells and mature sperm. Importantly, CrtapKO mice have a mild but significant increase in morphologically abnormal mature sperm (17% increase compared to WT). These data suggest a role for the Leprecans in the post-translational modification of collagens expressed in the stroma of the reproductive organs. While we could not confirm that SC65 is part of the synaptonemal complex, the expression of CRTAP in the seminiferous tubules and in mature sperm suggest a role in the testis germ cell lineage and sperm morphogenesis.

Neotuberostemonine inhibits the differentiation of lung fibroblasts into myofibroblasts in mice by regulating HIF-1α signaling.

Pulmonary fibrosis may be partially the result of deregulated tissue repair in response to chronic hypoxia. In this study we explored the effects of hypoxia on lung fibroblasts and the effects of neotuberostemonine (NTS), a natural alkaloid isolated from Stemona tuberosa, on activation of fibroblasts in vitro and in vivo. PLFs (primary mouse lung fibroblasts) were activated and differentiated after exposure to 1% O2 or treatment with CoCl2 (100 μmol/L), evidenced by markedly increased protein or mRNA expression of HIF-1α, TGF-β, FGF2, α-SMA and Col-1α/3α, which was blocked after silencing HIF-1α, suggesting that the activation of fibroblasts was HIF-1α-dependent. NTS (0.1-10 μmol/L) dose-dependently suppressed hypoxia-induced activation and differentiation of PLFs, whereas the inhibitory effect of NTS was abolished by co-treatment with MG132, a proteasome inhibitor. Since prolyl hydroxylation is a critical step in initiation of HIF-1α degradation, we further showed that NTS treatment reversed hypoxia- or CoCl2-induced reduction in expression of prolyl hydroxylated-HIF-1α. With hypoxyprobe immunofiuorescence staining, we showed that NTS treatment directly reversed the lower oxygen tension in hypoxia-exposed PLFs. In a mouse model of lung fibrosis, oral administration of NTS (30 mg·kg-1·d-1, for 1 or 2 weeks) effectively attenuated bleomycin-induced pulmonary fibrosis by inhibiting the levels of HIF-1α and its downstream profibrotic factors (TGF-β, FGF2 and α-SMA). Taken together, these results demonstrate that NTS inhibits the protein expression of HIF-1α and its downstream factors TGF-β, FGF2 and α-SMA both in hypoxia-exposed fibroblasts and in lung tissues of BLM-treated mice. NTS with anti-HIF-1α activity may be a promising pharmacological agent for the treatment of pulmonary fibrosis.

Unveiled cold sensitivity of TRPA1 by the prolyl hydroxylation inhibition-induced sensitization to ROS in oxaliplatin-induced acute peripheral neuropathy

Unveiled cold sensitivity of TRPA1 by the prolyl hydroxylation inhibition-induced sensitization to ROS in oxaliplatin-induced acute peripheral neuropathy

Inositol Polyphosphate Multikinase Inhibits Angiogenesis via Inositol Pentakisphosphate-Induced HIF-1α Degradation.

Inositol polyphosphate multikinase (IPMK) and its major product inositol pentakisphosphate (IP5) regulate a variety of cellular functions, but their role in vascular biology remains unexplored. We have investigated the role of IPMK in regulating angiogenesis. Deletion of IPMK in fibroblasts induces angiogenesis in both in vitro and in vivo models. IPMK deletion elicits a substantial increase of VEGF (vascular endothelial growth factor), which mediates the regulation of angiogenesis by IPMK. The regulation of VEGF by IPMK requires its catalytic activity. IPMK is predominantly nuclear and regulates gene transcription. However, IPMK does not apparently serve as a transcription factor for VEGF. HIF (hypoxia-inducible factor)-1α is a major determinant of angiogenesis and induces VEGF transcription. IPMK deletion elicits a major enrichment of HIF-1α protein and thus VEGF. HIF-1α is constitutively ubiquitinated by pVHL (von Hippel-Lindau protein) followed by proteasomal degradation under normal conditions. However, HIF-1α is not recognized and ubiquitinated by pVHL in IPMK KO (knockout) cells. IP5 reinstates the interaction of HIF-1α and pVHL. HIF-1α prolyl hydroxylation, which is prerequisite for pVHL recognition, is interrupted in IPMK-deleted cells. IP5 promotes HIF-1α prolyl hydroxylation and thus pVHL-dependent degradation of HIF-1α. Deletion of IPMK in mouse brain increases HIF-1α/VEGF levels and vascularization. The increased VEGF in IPMK KO disrupts blood-brain barrier and enhances brain blood vessel permeability. IPMK, via its product IP5, negatively regulates angiogenesis by inhibiting VEGF expression. IP5 acts by enhancing HIF-1α hydroxylation and thus pVHL-dependent degradation of HIF-1α.

O2 sensing–associated glycosylation exposes the F-box–combining site of the Dictyostelium Skp1 subunit in E3 ubiquitin ligases

Skp1 is a conserved protein linking cullin-1 to F-box proteins in SCF (Skp1/Cullin-1/F-box protein) E3 ubiquitin ligases, which modify protein substrates with polyubiquitin chains that typically target them for 26S proteasome-mediated degradation. In Dictyostelium (a social amoeba), Toxoplasma gondii (the agent for human toxoplasmosis), and other protists, Skp1 is regulated by a unique pentasaccharide attached to hydroxylated Pro-143 within its C-terminal F-box-binding domain. Prolyl hydroxylation of Skp1 contributes to O2-dependent Dictyostelium development, but full glycosylation at that position is required for optimal O2 sensing. Previous studies have shown that the glycan promotes organization of the F-box-binding region in Skp1 and aids in Skp1's association with F-box proteins. Here, NMR and MS approaches were used to determine the glycan structure, and then a combination of NMR and molecular dynamics simulations were employed to characterize the impact of the glycan on the conformation and motions of the intrinsically flexible F-box-binding domain of Skp1. Molecular dynamics trajectories of glycosylated Skp1 whose calculated monosaccharide relaxation kinetics and rotational correlation times agreed with the NMR data indicated that the glycan interacts with the loop connecting two α-helices of the F-box-combining site. In these trajectories, the helices separated from one another to create a more accessible and dynamic F-box interface. These results offer an unprecedented view of how a glycan modification influences a disordered region of a full-length protein. The increased sampling of an open Skp1 conformation can explain how glycosylation enhances interactions with F-box proteins in cells.

Bacillus anthracis Prolyl 4-Hydroxylase Interacts with and Modifies Elongation Factor Tu

Prolyl hydroxylation is a very common post-translational modification and plays many roles in eukaryotes such as collagen stabilization, hypoxia sensing, and controlling protein transcription and translation. There is a growing body of evidence that suggests that prokaryotes contain prolyl 4-hydroxylases (P4Hs) homologous to the hypoxia-inducible factor (HIF) prolyl hydroxylase domain (PHD) enzymes that act on elongation factor Tu (EFTu) and are likely involved in the regulation of bacterial translation. Recent biochemical and structural studies with a PHD from Pseudomonas putida (PPHD) determined that it forms a complex with EFTu and hydroxylates a prolyl residue of EFTu. Moreover, while animal, plant, and viral P4Hs act on peptidyl proline, most prokaryotic P4Hs have been known to target free l-proline; the exceptions include PPHD and a P4H from Bacillus anthracis (BaP4H) that modifies collagen-like proline-rich peptides. Here we use biophysical and mass spectrometric methods to demonstrate that BaP4H recognizes full-length BaEFTu and a BaEFTu 9-mer peptide for site-specific proline hydroxylation. Using size-exclusion chromatography coupled small-angle X-ray scattering (SEC-SAXS) and binding studies, we determined that BaP4H forms a 1:1 heterodimeric complex with BaEFTu. The SEC-SAXS studies reveal dissociation of BaP4H dimeric subunits upon interaction with BaEFTu. While BaP4H is unusual within bacteria in that it is structurally and functionally similar to the animal PHDs and collagen P4Hs, respectively, this work provides further evidence of its promiscuous substrate recognition. It is possible that the enzyme might have evolved to hydroxylate a universally conserved protein in prokaryotes, similar to the PHDs, and implies a functional role in B. anthracis.