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Collagen is the most abundant protein in humans. It has a characteristic triple-helix structure and is heavily posttranslationally modified. The complex biosynthesis of collagen involves processing by many enzymes and chaperones in the rough endoplasmic reticulum. Lysyl hydroxylase 1 (LH1) is required to hydroxylate lysine for cross-linking and carbohydrate attachment within collagen triple helical sequences. Additionally, a recent study of prolyl 3-hydroxylase 3 (P3H3) demonstrated that this enzyme may be critical for LH1 activity; however, the details surrounding its involvement remain unclear. If P3H3 is an LH1 chaperone that is critical for LH1 activity, P3H3 and LH1 null mice should display a similar deficiency in lysyl hydroxylation. To test this hypothesis, we compared the amount and location of hydroxylysine in the triple helical domains of type V and I collagen from P3H3 null, LH1 null, and wild-type mice. The amount of hydroxylysine in type V collagen was reduced in P3H3 null mice, but surprisingly type V collagen from LH1 null mice contained as much hydroxylysine as type V collagen from wild-type mice. In type I collagen, our results indicate that LH1 plays a global enzymatic role in lysyl hydroxylation. P3H3 is also involved in lysyl hydroxylation, particularly at cross-link formation sites, but is not required for all lysyl hydroxylation sites. In summary, our study suggests that LH1 and P3H3 likely have two distinct mechanisms to recognize different collagen types and to distinguish cross-link formation sites from other sites in type I collagen.

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 α-(P4H-α(I)) and β-(P4H-β) 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-α(I) or P4H-β by short hairpin RNA and P4H-α(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. [ABSTRACT FROM AUTHOR]

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the hydroxylation of -X-Pro-Gly- sequences and plays a central role in the synthesis of all collagens. The [α(I)]2β2 type I enzyme is effectively inhibited by poly(L-proline), whereas the [α(II)]2β2 type II enzyme is not. We report here that the poly(L-proline) and (Pro-Pro-Gly)10 peptide substrate-binding domain of prolyl 4-hydroxylase is distinct from the catalytic domain and consists of ∼100 amino acids. Peptides of 10–19 kDa beginning around residue 140 in the 517 residue α(I) subunit remained bound to poly(L-proline) agarose after limited proteolysis of the human type I enzyme tetramer. A recombinant polypeptide corresponding to the α(I) subunit residues 138–244 and expressed in Escherichia coli was soluble, became effectively bound to poly(L-proline) agarose and could be eluted with (Pro-Pro-Gly)10. This polypeptide is distinct from the SH3 and WW domains, and from profilin, and thus represents a new type of prolinerich peptide-binding module. Studies with enzyme tetramers containing mutated α subunits demonstrated that the presence of a glutamate and a glutamine in the α(II) subunit in the positions corresponding to Ile182 and Tyr233 in the α(I) subunit explains most of the lack of poly(L-proline) binding of the type II prolyl 4-hydroxylase. [ABSTRACT FROM AUTHOR]

• This work provides molecular level explanation for existence of isoenzymes in collagen prolyl 4-hydroxylation. • X position amino acid in collagen -X-Pro-Gly-triplets is the major determinant for hydroxylation. • Collagen prolyl 4-hydroxylation has sequence specificity. • Deletion of P4ha2 leads to collagen underhydroxylation at specific EPG and DPG sites and leads to reduced collagen melting temperature, reduced collagen fibril diameter and affects basement membranes. • Collagen prolyl 4-hydroxylase mRNA expression is not tightly coordinated with collagen expression. Collagen biosynthesis requires several co- and post-translational modifications of lysine and proline residues to form structurally and functionally competent collagen molecules. Formation of 4-hydroxyproline (4Hyp) in Y-position prolines of the repetitive -X-Y-Gly- sequences provides thermal stability for the triple-helical collagen molecules. 4Hyp formation is catalyzed by a collagen prolyl 4-hydroxylase (C-P4H) family consisting of three isoenzymes. Here we identify specific roles for the two main C-P4H isoenzymes in collagen hydroxylation by a detailed 4Hyp analysis of type I and IV collagens derived from cell and tissue samples. Loss of C-P4H-I results in underhydroxylation of collagen where the affected prolines are not uniformly distributed, but mainly present in sites where the adjacent X-position amino acid has a positively charged or a polar uncharged side chain. In contrast, loss of C-P4H-II results in underhydroxylation of triplets where the X-position is occupied by a negatively charged amino acid glutamate or aspartate. Hydroxylation of these triplets was found to be important as loss of C-P4H-II alone resulted in reduced collagen melting temperature and altered assembly of collagen fibrils and basement membrane. The observed C-P4H isoenzyme differences in substrate specificity were explained by selective binding of the substrate to the active site resulting in distinct differences in Km and Vmax values. Furthermore, our results clearly show that the substrate proline selection is not dependent on the collagen type, but the main determinant is the X-position amino acid of the -X-Pro-Gly- triplet. Although our data clearly shows the necessity of both C-P4H-I and II for normal prolyl 4-hydroxylation and function of collagens, the mRNA expression of the isoenzymes with various procollagens was, surprisingly, not tightly coordinated, suggesting additional levels of control. In conclusion, this study provides a molecular level explanation for the need of multiple C-P4H isoenzymes to generate collagen molecules capable to assemble into intact extracellular matrix structures. [ABSTRACT FROM AUTHOR]

Bruck syndrome is a rare autosomal recessive connective tissue disorder characterized by fragile bones, joint contractures, scoliosis, and osteoporosis. The telopeptides of bone collagen I are underhydroxylated in these patients, leading to abnormal collagen cross-linking. Three point mutations in lysyl hydroxylase (LH) 2, the enzyme responsible for the hydroxylation of collagen telopeptides, have been identified in Bruck syndrome. As none of them affects the residues known to be critical for LH activity, we studied their consequences at the molecular level by analyzing the folding and catalytic properties of the corresponding mutant recombinant polypeptides. Folding and oligomerization of the R594H and G597V mutants were abnormal, and their activity was reduced by >95% relative to the wild type. The T604I mutation did not affect the folding properties, although the mutant retained only ∼8% activity under standard assay conditions. As the reduced activity was caused by a 10-fold increase in the Km for 2-oxoglutarate, the mutation interferes with binding of this cosubstrate. In the presence of a saturating 2-oxoglutarate concentration, the activity of the T6041 mutant was ∼30% of that of the wild type. However, the T6041 mutant did not generate detectable amounts of hydroxylysine in the N-terminal telopeptide of a recombinant procollagen I chain when coexpressed in insect cells. The low activity of the mutant LH2 polypeptides is in accordance with the markedly reduced extent of collagen telopeptide hydroxylation in Bruck syndrome, with consequent changes in the cross-linking of collagen fibrils and severe abnormalities in the skeletal structures. [ABSTRACT FROM AUTHOR]

Cellular energy demands are met by uptake and metabolism of nutrients like glucose. The principal transcriptional regulator for adapting glycolytic flux and downstream pathways like de novo lipogenesis to glucose availability in many cell types is carbohydrate response element-binding protein (ChREBP). ChREBP is activated by glucose metabolites and post-translational modifications, inducing nuclear accumulation and regulation of target genes. Here we report that ChREBP is modified by proline hydroxylation at several residues. Proline hydroxylation targets both ectopically expressed ChREBP in cells and endogenous ChREBP in mouse liver. Functionally, we found that specific hydroxylated prolines were dispensable for protein stability but required for the adequate activation of ChREBP upon exposure to high glucose. Accordingly, ChREBP target gene expression was rescued by re-expressing WT but not ChREBP that lacks hydroxylated prolines in ChREBP-deleted hepatocytes. Thus, proline hydroxylation of ChREBP is a novel posttranslational modification that may allow for therapeutic interference in metabolic diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Prolyl 4-hydroxylases (P4Hs) are 2-oxoglutarate dioxygenases that catalyze the hydroxylation of peptidyl prolines. They play an important role in collagen synthesis, oxygen homeostasis, and plant cell wall formation. We describe four structures of a P4H from the green alga Chlamydomonas reinhardtii, two of the apoenzyme at 1.93 and 2.90 Å resolution, one complexed with the competitive inhibitor Zn2+, and one with Zn2+ and pyridine 2,4-dicarboxylate (which is an analogue of 2-oxoglutarate) at 1.85 Å resolution. The structures reveal the double-stranded ß-helix core fold (jellyroll motif), typical for 2-oxoglutarate dioxygenases. The catalytic site is at the center of an extended shallow groove lined by two flexible loops. Mutagenesis studies together with the crystal-lographic data indicate that this groove participates in the binding of the proline-rich peptide-substrates. It is discussed that the algal P4H and the catalytic domain of collagen P4Hs have notable structural similarities, suggesting that these enzymes form a separate structural subgroup of P4Hs different from the hypoxia-inducible factor P4Hs. Key structural differences between these two subgroups are described. These studies provide first insight into the structure-function relationships of the collagen P4Hs, which unlike the hypoxia-inducible factor P4Hs use proline-rich peptides as their substrates. [ABSTRACT FROM AUTHOR]

Three hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs) regulate the HIFs by hydroxylating prolines at two separate sites in the oxygen-dependent degradation domain (ODDD) of their a subunits. We compared in vitro hydroxylation by purified recombinant human HIF-P4Hs of 19 -20- and 35-residue peptides corresponding to the two sites in HIF-αs and purified recombinant HIF-1α and HIF-2α ODDDs of 248 and 215 residues. The increase in the length of peptides representing the C-terminal site from 19 to 20 to 35 residues reduced the Km values to 90-800 nM, i.e. to 0.7-11% of those for the shorter peptides, whereas those representing the N-terminal site were 10-470 µM, i.e. 10-135%. The Km values of HIF-P4H-1 for the recombinant HIF-α ODDDs were 10-20 nM, whereas those of HIF-P4H-2 and -3 were 60-140 nM, identical values being found for the wild-type HIF-1α ODDD and its N site mutant. The Km, values for the C site mutant were about 5-10 times higher but only 0.2-3% of those for the 35-residue N site peptides, and this marked difference suggested that the HIF- P4Hs may become bound first to the C-terminal site of an ODDD and that this binding may enhance subsequent binding to the N-terminal site. The Km values of HIF-P4H-2 for oxygen determined with the HIF-1α ODDD and both its mutants as substrates were all about 100 µM, being 40% of those reported for the three HIF-P4Hs with a 19-residue peptide. Even this value is high compared with tissue O2 levels, indicating that HIF-P4Hs are effective oxygen sensors. [ABSTRACT FROM AUTHOR]

Collagen prolyl 4-hydroxylases catalyze the formation of 4-hydroxyproline in -X-Pro-Gly- sequences and have an essential role in collagen synthesis. The vertebrate enzymes are α2β2 tetramers in which the catalytic α-sub-units contain separate peptide-substrate-binding and catalytic domains. We report on the crystal structure of the peptide-substrate-binding domain of the human type I enzyme refined at 2.3 Å resolution. It was found to belong to a family of tetratricopeptide repeat domains that are involved in many protein-protein interactions and consist of five α-helices forming two tetratricopeptide repeat motifs plus the solvating helix. A prominent feature of its concave surface is a deep groove lined by tyrosines, a putative binding site for proline-rich peptides. Solvent-exposed side chains of three of the tyrosines have a repeat distance similar to that of a polyL-proline type II helix. The aromatic surface ends at one of the tyrosines, where the groove curves almost 90° away from the linear arrangement of the three tyrosine side chains, possibly inducing a bent conformation in the bound peptide. This finding is consistent with previous suggestions by others that a minimal structural requirement for proline 4-hydroxylation may be a sequence in the poly-L-proline type II conformation followed by a β-turn in the Pro-Gly segment. Site-directed mutagenesis indicated that none of the tyrosines was critical for tetramer assembly, whereas most of them were critical for the binding of a peptide substrate and inhibitor both to the domain and the α2β2 enzyme tetramer. [ABSTRACT FROM AUTHOR]

The activity of hypoxia-inducible transcription factor HIF, an αβ heterodimer that has an essential role in adaptation to low oxygen availability, is regulated by two oxygen-dependent hydroxylation events. Hydroxylation of specific proline residues by HIF prolyl 4-hydroxylases targets the HIF-α subunit for proteasomal destruction, whereas hydroxylation of an asparagine in the C-terminal transactivation domain prevents its interaction with the transcriptional coactivator p300. The HIF asparaginyl hydroxylase is identical to a previously known factor inhibiting HIF (FIH). We report here that recombinant FIH has unique catalytic and inhibitory properties when compared with those of the HIF prolyl 4-hydroxylases. FIH was found to require particularly long peptide substrates so that omission of only a few residues from the N or C terminus of a 35-residue HIF-1α sequence markedly reduced its substrate activity. Hydroxylation of two HIF-2α peptides was far less efficient than that of the corresponding HIF-1α peptides. The Km of FIH for O2 was about 40% of its atmospheric concentration, being about one-third of those of the HIF prolyl 4-hydroxylases but 2.5 times that of the type I collagen prolyl 4-hydroxylase. Several 2-oxoglutarate analogs were found to inhibit FIH but with distinctly different potencies from the HIF prolyl 4-hydroxylases. For exampie, the two most potent HIF prolyl 4-hydroxylase inhibitors among the compounds studied were the least effective ones for FIH. It should therefore be possible to develop specific small molecule inhibitors for the two enzyme classes involved in the hypoxia response. [ABSTRACT FROM AUTHOR]

The collagen prolyl 4-hydroxylases (C-P4Hs) catalyze the formation of 4-hydroxyproline by the hydroxylation of proline residues in -Xaa-Pro-Gly-sequences. The vertebrate enzymes are α[sub 2]β[sub 2] tetramers in which protein-disulfide isomerase serves as the β subunit. Two isoforms of the catalytic α subunit have been identified and shown to form [α(I)][sub 2]]β[sub 2] and [α(II)][sub 2]β[sub 2] tetramers, the type I and type II C-P4Hs, respectively. The peptide-substrate-binding domain of type I C-P4H has been shown to be located between residues 138 and 244 in the 517-residue α(I) subunit and to be distinct from the catalytic domain that is located in the C-terminal region. We report here that a recombinant human C-P4H α(I) polypeptide Phe[sup 144]-Ser[sup 244] forms a folded domain consisting of five α helices and one short β strand. This structure is quite different from those of other proline-rich peptide-binding modules, which consist mainly of β strands. Binding of the peptide (Pro-Pro-Gly)[sub 2] to this domain caused major chemical shifts in many backbone amide resonances, the residues showing the largest shifts being mainly hydrophobic, including three tyrosines. The K[sub d] values determined by surface plasmon resonance and isothermal titration calorimetry for the binding of several synthetic peptides to the α(I) and the corresponding α(II) domain were very similar to the K[sub m] and K[sub i] values for these peptides as substrates and inhibitors of the type I and type II C-P4H tetramers. The K[sub d] values of the α(I) and α(II) domains for (Gly-Pro-4Hyp)[sub 5] were much higher than those for (Pro-Pro-Gly)[sub 5], indicating a marked decrease in the affinity of hydroxylated peptides for the domain. Many characteristic features of the binding of peptides to the type I and type II C-P4H tetramers can thus be explained by the properties of binding to this domain rather than the catalytic domain. [ABSTRACT FROM AUTHOR]

The hypoxia-inducible factors (HIFs) play a central role in oxygen homeostasis. Hydroxylation of one or two critical prolines by specific hydroxylases (P4Hs) targets their HIF-α subunits for proteasomal degradation. By studying the three human HIF-P4Hs, we found that the longest and shortest isoenzymes have major transcripts encoding inactive polypeptides, which suggest novel regulation by alternative splicing. Recombinant HIFP4Hs expressed in insect cells required peptides of more than 8 residues, distinct differences being found between isoenzymes. All the HIF-P4Hs hydroxylated peptides corresponding to Pro[sup 564] in HIF-1α, whereas a Pro[sup 402] peptide had 20-50-fold K[sub m] values for two isoenzymes but was not hydroxylated by the shortest isoenzyme at all; this difference was not explained by the two prolines being in a -Pro[sup 402]-Ala- and -Pro[sup 564]-Tyr-sequence. All the HIF-P4Hs-hydroxylated peptides corresponding to two of three potential sites in HIF-2α and one in HIF-3α. The K[sub m] values for O[sub 2] were slightly above its atmospheric concentration, indicating that the HIFP4Hs are effective oxygen sensors. Small molecule inhibitors of collagen P4Hs also inhibited the HIF-P4Hs, but with distinctly different K[sub i] values, indicating that it should be possible to develop specific inhibitors for each class of P4Hs and possibly even for the individual HIF-P4Hs. [ABSTRACT FROM AUTHOR]