ABSTRACT: Posttranslational modifications can cause profound changes in protein function. Typically, these modifications are reversible, and thus provide a biochemical on-off switch. In contrast, proline residues are the substrates for an irreversible reaction that is the most common posttranslational modification in humans. This reaction, which is catalyzed by prolyl 4-hydroxylase (P4H), yields (2S,4R)-4-hydroxyproline (Hyp). The protein substrates for P4Hs are diverse. Likewise, the biological consequences of prolyl hydroxylation vary widely, and include altering protein conformation and protein-protein interactions, and enabling further modification. The best known role for Hyp is in stabilizing the collagen triple helix. Hyp is also found in proteins with collagen-like domains, as well as elastin, conotoxins, and argonaute 2. A prolyl hydroxylase domain protein acts on the hypoxia inducible factor alpha, which plays a key role in sensing molecular oxygen, and could act on inhibitory kappaB kinase and RNA polymerase II. P4Hs are not unique to animals, being found in plants and microbes as well. Here, we review the enzymic catalysts of prolyl hydroxylation, along with the chemical and biochemical consequences of this subtle but abundant posttranslational modification.
Project description:Proline hydroxylation is the most prevalent post-translational modification in collagen. The resulting product trans-4-hydroxyproline (Hyp) is of critical importance for the stability and thus function of collagen, with defects leading to several diseases. Prolyl 4-hydroxylases (P4Hs) are mononuclear non-heme iron ?-ketoglutarate (?KG)-dependent dioxygenases that catalyze Hyp formation. Although animal and plant P4Hs target peptidyl proline, prokaryotes have been known to use free l-proline as a precursor to form Hyp. The P4H from Bacillus anthracis (BaP4H) has been postulated to act on peptidyl proline in collagen peptides, making it unusual within the bacterial clade, but its true physiological substrate remains enigmatic. Here we use mass spectrometry, fluorescence binding, x-ray crystallography, and docking experiments to confirm that BaP4H recognizes and acts on peptidyl substrates but not free l-proline, using elements characteristic of an Fe(II)/?KG-dependent dioxygenases. We further show that BaP4H can hydroxylate unique peptidyl proline sites in collagen-derived peptides with asymmetric hydroxylation patterns. The cofactor-bound crystal structures of BaP4H reveal active site conformational changes that define open and closed forms and mimic "ready" and "product-released" states of the enzyme in the catalytic cycle. These results help to clarify the role of BaP4H as well as provide broader insights into human collagen P4H and proteins with poly-l-proline type II helices.
Project description:Collagen prolyl-4-hydroxylase (C-P4H) catalyzes the hydroxylation of specific proline residues in procollagen, which is an essential step in collagen biosynthesis. A new form of P4H from Bacillus anthracis (anthrax-P4H) that shares many characteristics with the type I C-P4H from human has recently been characterized. The structure of anthrax-P4H could provide important insight into the chemistry of C-P4Hs and into the function of this unique homodimeric P4H. X-ray diffraction data of selenomethionine-labeled anthrax-P4H recombinantly expressed in Escherichia coli have been collected to 1.4 A resolution.
Project description: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.
Project description:BACKGROUND: Collagens require the hydroxylation of proline (Pro) residues in their triple-helical domain repeating sequence Xaa-Pro-Gly to function properly as a main structural component of the extracellular matrix in animals at physiologically relevant conditions. The regioselective proline hydroxylation is catalyzed by a specific prolyl 4-hydroxylase (P4H) as a posttranslational processing step. RESULTS: A recombinant human collagen type I ?-1 (rCI?1) with high percentage of hydroxylated prolines (Hyp) was produced in transgenic maize seeds when co-expressed with both the ?- and ?- subunits of a recombinant human P4H (rP4H). Germ-specific expression of rCI?1 using maize globulin-1 gene promoter resulted in an average yield of 12 mg/kg seed for the full-length rCI?1 in seeds without co-expression of rP4H and 4 mg/kg seed for the rCI?1 (rCI?1-OH) in seeds with co-expression of rP4H. High-resolution mass spectrometry (HRMS) analysis revealed that nearly half of the collagenous repeating triplets in rCI?1 isolated from rP4H co-expressing maize line had the Pro residues changed to Hyp residues. The HRMS analysis determined the Hyp content of maize-derived rCI?1-OH as 18.11%, which is comparable to the Hyp level of yeast-derived rCI?1-OH (17.47%) and the native human CIa1 (14.59%), respectively. The increased Hyp percentage was correlated with a markedly enhanced thermal stability of maize-derived rCI?1-OH when compared to the non-hydroxylated rCI?1. CONCLUSIONS: This work shows that maize has potential to produce adequately modified exogenous proteins with mammalian-like post-translational modifications that may be require for their use as pharmaceutical and industrial products.
Project description:<h4>Background</h4>Prolyl hydroxylation is a post-translational modification that affects the structure, stability and function of proteins including collagen by catalysing hydroxylation of proline to hydroxyproline through action of collagen prolyl hydroxylases3 (C-P3H) and 4 (C-P4H). Three C-P3Hs (nomenclature was amended according to approval by the HGNC symbols and names at http://www.genenames.org/ and Entrez database at http://www.ncbi.nlm.nih.gov/gene) leucineproline-enriched proteoglycan (leprecan) 1 (Lepre1), leprecan-like 1 (Leprel1), leprecan-like 2 (Leprel2) and two paralogs Cartilage-Related Protein (CRTAP) and leprecan-like 4 (Leprel4) are found in humans. The C-P4Hs are tetrameric proteins comprising a variable ? subunit, encoded by the P4HA1, P4HA2 and P4HA3 genes and a constant ? subunit encoded by P4HB.<h4>Methods</h4>We used RT-PCR, qPCR, pyrosequencing, methylation-specific PCR, western blotting and immunohistochemistry to investigate expression and regulation of the C-P3H and C-P4H genes in B lymphomas and normal bone marrow.<h4>Results</h4>C-P3H and C-P4H are downregulated in lymphoma. Down-regulation is associated with methylation in the CpG islands and is detected in almost all common types of B-cell lymphoma, but the CpG islands are unmethylated or methylated at lower levels in DNA isolated from normal bone marrow and lymphoblastoid cell lines. Methylation of multiple C-P3H and C-P4H genes is present in some lymphomas, particularly Burkitt's lymphoma.<h4>Conclusions</h4>Methylation of C-P3H and C-P4H is common in B lymphomas and may have utility in differentiating disease subtypes.
Project description:Recombinant production of pharmaceutical proteins is crucial, not only for personalized medicine. While most biopharmaceuticals are currently produced in mammalian cell culture, plant-made pharmaceuticals gain momentum. Post-translational modifications in plants are similar to those in humans, however, existing differences may affect quality, safety and efficacy of the products. A frequent modification in higher eukaryotes is prolyl-4-hydroxylase (P4H)-catalysed prolyl-hydroxylation. P4H sequence recognition sites on target proteins differ between humans and plants leading to non-human posttranslational modifications of recombinant human proteins produced in plants. The resulting hydroxyprolines display the anchor for plant-specific O-glycosylation, which bears immunogenic potential for patients. Here we describe the identification of a plant gene responsible for non-human prolyl-hydroxylation of human erythropoietin (hEPO) recombinantly produced in plant (moss) bioreactors. Targeted ablation of this gene abolished undesired prolyl-hydroxylation of hEPO and thus paves the way for plant-made pharmaceuticals humanized via glyco-engineering in moss bioreactors.
Project description:Plant and algal prolyl 4-hydroxylases (P4Hs) are key enzymes in the synthesis of cell wall components. These monomeric enzymes belong to the 2-oxoglutarate dependent superfamily of enzymes characterized by a conserved jelly-roll framework. This algal P4H has high sequence similarity to the catalytic domain of the vertebrate, tetrameric collagen P4Hs, whereas there are distinct sequence differences with the oxygen-sensing hypoxia-inducible factor P4H subfamily of enzymes. We present here a 1.98-A crystal structure of the algal Chlamydomonas reinhardtii P4H-1 complexed with Zn(2+) and a proline-rich (Ser-Pro)(5) substrate. This ternary complex captures the competent mode of binding of the peptide substrate, being bound in a left-handed (poly)l-proline type II conformation in a tunnel shaped by two loops. These two loops are mostly disordered in the absence of the substrate. The importance of these loops for the function is confirmed by extensive mutagenesis, followed up by enzyme kinetic characterizations. These loops cover the central Ser-Pro-Ser tripeptide of the substrate such that the hydroxylation occurs in a highly buried space. This novel mode of binding does not depend on stacking interactions of the proline side chains with aromatic residues. Major conformational changes of the two peptide binding loops are predicted to be a key feature of the catalytic cycle. These conformational changes are probably triggered by the conformational switch of Tyr(140), as induced by the hydroxylation of the proline residue. The importance of these findings for understanding the specific binding and hydroxylation of (X-Pro-Gly)(n) sequences by collagen P4Hs is also discussed.
Project description:Prolyl 4-hydroxylases (P4H) catalyze the post-translational hydroxylation of proline residues and play a role in collagen production, hypoxia response, and cell wall development. P4Hs belong to the group of Fe(II)/alphaKG oxygenases and require Fe(II), alpha-ketoglutarate (alphaKG), and O(2) for activity. We report the 1.40 A structure of a P4H from Bacillus anthracis, the causative agent of anthrax, whose immunodominant exosporium protein BclA contains collagen-like repeat sequences. The structure reveals the double-stranded beta-helix core fold characteristic of Fe(II)/alphaKG oxygenases. This fold positions Fe-binding and alphaKG-binding residues in what is expected to be catalytically competent orientations and is consistent with proline peptide substrate binding at the active site mouth. Comparisons of the anthrax P4H structure with Cr P4H-1 structures reveal similarities in a peptide surface groove. However, sequence and structural comparisons suggest differences in conformation of adjacent loops may change the interaction with peptide substrates. These differences may be the basis of a substantial disparity between the K(M) values for the Cr P4H-1 compared to the anthrax and human P4H enzymes. Additionally, while previous structures of P4H enzymes are monomers, B. anthracis P4H forms an alpha(2) homodimer and suggests residues important for interactions between the alpha(2) subunits of alpha(2)beta(2) human collagen P4H. Thus, the anthrax P4H structure provides insight into the structure and function of the alpha-subunit of human P4H, which may aid in the development of selective inhibitors of the human P4H enzyme involved in fibrotic disease.
Project description:Collagen prolyl 4-hydroxylase (P4H) expression and collagen hydroxylation in cancer cells are necessary for breast cancer progression. Here, we show that P4H alpha 1 subunit (P4HA1) protein expression is induced in triple-negative breast cancer (TNBC) and HER2 positive breast cancer. By modulating alpha ketoglutarate (?-KG) and succinate levels P4HA1 expression reduces proline hydroxylation on hypoxia-inducible factor (HIF) 1?, enhancing its stability in cancer cells. Activation of the P4HA/HIF-1 axis enhances cancer cell stemness, accompanied by decreased oxidative phosphorylation and reactive oxygen species (ROS) levels. Inhibition of P4HA1 sensitizes TNBC to the chemotherapeutic agent docetaxel and doxorubicin in xenografts and patient-derived models. We also show that increased P4HA1 expression correlates with short relapse-free survival in TNBC patients who received chemotherapy. These results suggest that P4HA1 promotes chemoresistance by modulating HIF-1-dependent cancer cell stemness. Targeting collagen P4H is a promising strategy to inhibit tumor progression and sensitize TNBC to chemotherapeutic agents.
Project description:Human Argonaute (Ago) proteins are essential components of the RNA-induced silencing complexes (RISCs). Argonaute 2 (Ago2) has a P-element-induced wimpy testis (PIWI) domain, which folds like RNase H and is responsible for target RNA cleavage in RNA interference. Proteins such as Dicer, TRBP, MOV10, RHA, RCK/p54 and KIAA1093 associate with Ago proteins and participate in small RNA processing, RISC loading and localization of Ago proteins in the cytoplasmic messenger RNA processing bodies. However, mechanisms that regulate RNA interference remain obscure. Here we report physical interactions between Ago2 and the alpha-(P4H-alpha(I)) and beta-(P4H-beta) subunits of the type I collagen prolyl-4-hydroxylase (C-P4H(I)). Mass spectrometric analysis identified hydroxylation of the endogenous Ago2 at proline 700. In vitro, both Ago2 and Ago4 seem to be more efficiently hydroxylated than Ago1 and Ago3 by recombinant human C-P4H(I). Importantly, human cells depleted of P4H-alpha(I) or P4H-beta by short hairpin RNA and P4H-alpha(I) null mouse embryonic fibroblast cells showed reduced stability of Ago2 and impaired short interfering RNA programmed RISC activity. Furthermore, mutation of proline 700 to alanine also resulted in destabilization of Ago2, thus linking Ago2 P700 and hydroxylation at this residue to its stability regulation. These findings identify hydroxylation as a post-translational modification important for Ago2 stability and effective RNA interference.