Combined proteomic and biochemical analyses redefine the consensus sequence requirement for epidermal growth factor-like domain hydroxylation.

Epidermal growth factor-like domains (EGFDs) have important functions in cell-cell signaling. Both secreted and cell surface human EGFDs are subject to extensive modifications, including aspartate and asparagine residue C3-hydroxylations catalyzed by the 2-oxoglutarate oxygenase aspartate/asparagine-ß-hydroxylase (AspH). Although genetic studies show AspH is important in human biology, studies on its physiological roles have been limited by incomplete knowledge of its substrates. Here, we redefine the consensus sequence requirements for AspH-catalyzed EGFD hydroxylation based on combined analysis of proteomic mass spectrometric data and mass spectrometry-based assays with isolated AspH and peptide substrates. We provide cellular and biochemical evidence that the preferred site of EGFD hydroxylation is embedded within a disulfide-bridged macrocycle formed of 10 amino acid residues. This definition enabled the identification of previously unassigned hydroxylation sites in three EGFDs of human fibulins as AspH substrates. A non-EGFD containing protein, lymphocyte antigen-6/plasminogen activator urokinase receptor domain containing protein 6B (LYPD6B) was shown to be a substrate for isolated AspH, but we did not observe evidence for LYPD6B hydroxylation in cells. AspH-catalyzed hydroxylation of fibulins is of particular interest given their important roles in extracellular matrix dynamics. In conclusion, these results lead to a revision of the consensus substrate requirements for AspH and expand the range of observed and potential AspH-catalyzed hydroxylation in cells, which will enable future study of the biological roles of AspH.

Fount, fate, features, and function of renal erythropoietin-producing cells.

Renal erythropoietin (Epo)-producing (REP) cells represent a rare and incompletely understood cell type. REP cells are fibroblast-like cells located in close proximity to blood vessels and tubules of the corticomedullary border region. Epo mRNA in REP cells is produced in a pronounced "on-off" mode, showing transient transcriptional bursts upon exposure to hypoxia. In contrast to "ordinary" fibroblasts, REP cells do not proliferate ex vivo, cease to produce Epo, and lose their identity following immortalization and prolonged in vitro culture, consistent with the loss of Epo production following REP cell proliferation during tissue remodelling in chronic kidney disease. Because Epo protein is usually not detectable in kidney tissue, and Epo mRNA is only transiently induced under hypoxic conditions, transgenic mouse models have been developed to permanently label REP cell precursors, active Epo producers, and inactive descendants. Future single-cell analyses of the renal stromal compartment will identify novel characteristic markers of tagged REP cells, which will provide novel insights into the regulation of Epo expression in this unique cell type.

Hydroxyproline in animal metabolism, nutrition, and cell signaling.

trans-4-Hydroxy-L-proline is highly abundant in collagen (accounting for about one-third of body proteins in humans and other animals). This imino acid (loosely called amino acid) and its minor analogue trans-3-hydroxy-L-proline in their ratio of approximately 100:1 are formed from the post-translational hydroxylation of proteins (primarily collagen and, to a much lesser extent, non-collagen proteins). Besides their structural and physiological significance in the connective tissue, both trans-4-hydroxy-L-proline and trans-3-hydroxy-L-proline can scavenge reactive oxygen species and have both structural and physiological significance in animals. The formation of trans-4-hydroxy-L-proline residues in protein kinases B and DYRK1A, eukaryotic elongation factor 2 activity, and hypoxia-inducible transcription factor plays an important role in regulating their phosphorylation and catalytic activation as well as cell signaling in animal cells. These biochemical events contribute to the modulation of cell metabolism, growth, development, responses to nutritional and physiological changes (e.g., dietary protein intake and hypoxia), and survival. Milk, meat, skin hydrolysates, and blood, as well as whole-body collagen degradation provide a large amount of trans-4-hydroxy-L-proline. In animals, most (nearly 90%) of the collagen-derived trans-4-hydroxy-L-proline is catabolized to glycine via the trans-4-hydroxy-L-proline oxidase pathway, and trans-3-hydroxy-L-proline is degraded via the trans-3-hydroxy-L-proline dehydratase pathway to ornithine and glutamate, thereby conserving dietary and endogenously synthesized proline and arginine. Supplementing trans-4-hydroxy-L-proline or its small peptides to plant-based diets can alleviate oxidative stress, while increasing collagen synthesis and accretion in the body. New knowledge of hydroxyproline biochemistry and nutrition aids in improving the growth, health and well-being of humans and other animals.

Hydroxylation of the Acetyltransferase NAA10 Trp38 Is Not an Enzyme-Switch in Human Cells.

NAA10 is a major N-terminal acetyltransferase (NAT) that catalyzes the cotranslational N-terminal (Nt-) acetylation of 40% of the human proteome. Several reports of lysine acetyltransferase (KAT) activity by NAA10 exist, but others have not been able to find any NAA10-derived KAT activity, the latter of which is supported by structural studies. The KAT activity of NAA10 towards hypoxia-inducible factor 1α (HIF-1α) was recently found to depend on the hydroxylation at Trp38 of NAA10 by factor inhibiting HIF-1α (FIH). In contrast, we could not detect hydroxylation of Trp38 of NAA10 in several human cell lines and found no evidence that NAA10 interacts with or is regulated by FIH. Our data suggest that NAA10 Trp38 hydroxylation is not a switch in human cells and that it alters its catalytic activity from a NAT to a KAT.

Now a Nobel gas: oxygen.

The recent bestowal of the Nobel Prize 2019 in Physiology or Medicine to Gregg L. Semenza, Sir Peter J. Ratcliffe, and William G. Kaelin Jr. celebrates a series of remarkable discoveries that span from the physiological research question on how oxygen deficiency (hypoxia) induces the red blood cell forming hormone erythropoietin (Epo) to the first clinical application of a novel family of Epo-inducing drugs to treat patients suffering from renal anemia. This review looks back at the most important findings made by the three Nobel laureates, highlights current research trends, and sheds an eye on future perspectives of hypoxia research, including emerging and potential clinical applications.

Adventures in Defining Roles of Oxygenases in the Regulation of Protein Biosynthesis.

The 2-oxoglutarate (2OG) dependent oxygenases were first identified as having roles in the post-translational modification of procollagen in animals. Subsequently in plants and microbes, they were shown to have roles in the biosynthesis of many secondary metabolites, including signalling molecules and the penicillin/cephalosporin antibiotics. Crystallographic studies of microbial 2OG oxygenases and related enzymes, coupled to DNA sequence analyses, led to the prediction that 2OG oxygenases are widely distributed in aerobic biology. This personal account begins with examples of the roles of 2OG oxygenases in antibiotic biosynthesis, and then describes efforts to assign functions to other predicted 2OG oxygenases. In humans, 2OG oxygenases have been found to have roles in small molecule metabolism, as well as in the epigenetic regulation of protein and nucleic acid biosynthesis and function. The roles and functions of human 2OG oxygenases are compared, focussing on discussion of their substrate and product selectivities. The account aims to emphasize how scoping the substrate selectivity of, sometimes promiscuous, enzymes can provide insights into their functions and so enable therapeutic work.

New horizons in hypoxia signaling pathways.

Investigation into the regulation of the erythropoietin gene by oxygen led to the discovery of a process of direct oxygen sensing that transduces many cellular and systemic responses to hypoxia. The oxygen-sensitive signal is generated through the catalytic action of a series of 2-oxoglutarate-dependent oxygenases that regulate the transcription factor hypoxia-inducible factor (HIF) by the post-translational hydroxylation of specific amino acid residues. Here we review the implications of the unforeseen complexity of the HIF transcriptional cascade for the physiology and pathophysiology of hypoxia, and consider the origins of post-translational hydroxylation as a signaling process.

OH-PRED: prediction of protein hydroxylation sites by incorporating adapted normal distribution bi-profile Bayes feature extraction and physicochemical properties of amino acids.

Hydroxylation of proline or lysine residues in proteins is a common post-translational modification event, and such modifications are found in many physiological and pathological processes. Nonetheless, the exact molecular mechanism of hydroxylation remains under investigation. Because experimental identification of hydroxylation is time-consuming and expensive, bioinformatics tools with high accuracy represent desirable alternatives for large-scale rapid identification of protein hydroxylation sites. In view of this, we developed a supporter vector machine-based tool, OH-PRED, for the prediction of protein hydroxylation sites using the adapted normal distribution bi-profile Bayes feature extraction in combination with the physicochemical property indexes of the amino acids. In a jackknife cross validation, OH-PRED yields an accuracy of 91.88% and a Matthew's correlation coefficient (MCC) of 0.838 for the prediction of hydroxyproline sites, and yields an accuracy of 97.42% and a MCC of 0.949 for the prediction of hydroxylysine sites. These results demonstrate that OH-PRED increased significantly the prediction accuracy of hydroxyproline and hydroxylysine sites by 7.37 and 14.09%, respectively, when compared with the latest predictor PredHydroxy. In independent tests, OH-PRED also outperforms previously published methods.

NDUFAF5 Hydroxylates NDUFS7 at an Early Stage in the Assembly of Human Complex I.

Complex I (NADH ubiquinone oxidoreductase) in mammalian mitochondria is an L-shaped assembly of 45 proteins. One arm lies in the inner membrane, and the other extends about 100 Å into the matrix of the organelle. The extrinsic arm contains binding sites for NADH, the primary electron acceptor FMN, and seven iron-sulfur clusters that form a pathway for electrons linking FMN to the terminal electron acceptor, ubiquinone, which is bound in a tunnel in the region of the junction between the arms. The membrane arm contains four antiporter-like domains, energetically coupled to the quinone site and involved in pumping protons from the matrix into the intermembrane space contributing to the proton motive force. Seven of the subunits, forming the core of the membrane arm, are translated from mitochondrial genes, and the remaining subunits, the products of nuclear genes, are imported from the cytosol. Their assembly is coordinated by at least thirteen extrinsic assembly factor proteins that are not part of the fully assembled complex. They assist in insertion of co-factors and in building up the complex from smaller sub-assemblies. One such factor, NDUFAF5, belongs to the family of seven-ß-strand S-adenosylmethionine-dependent methyltransferases. However, similar to another family member, RdmB, it catalyzes the introduction of a hydroxyl group, in the case of NDUFAF5, into Arg-73 in the NDUFS7 subunit of human complex I. This modification occurs early in the pathway of assembly of complex I, before the formation of the juncture between peripheral and membrane arms.

Hydroxylated histidine of human ribosomal protein uL2 is involved in maintaining the local structure of 28S rRNA in the ribosomal peptidyl transferase center.

Protein uL2 is essential for the catalytic activity of the ribosome and has a conserved shape in ribosomes from all domains of life. However, the sequence of its unstructured C-terminal loop apex that contacts the conserved 23S/28S rRNA helix (H) 93 near the ribosomal peptidyl transferase center differs in bacteria, archaea and eukaryotes. Eukaryote-specific residue His216 located in this loop in mammalian uL2 is hydroxylated in ribosomes. We used a set of chemical probes to explore the structure of an RNA that mimicked a segment of 28S rRNA domain V containing part of the uL2 binding site including H93, complexed with either natural (hydroxylated) or recombinant (unmodified) human uL2. It was found that both protein forms engage H93 during binding, but only natural uL2 (uL2n) protects it from hydroxyl radicals. The association of uL2n with RNA leads to changes in its structure at U4532 adjacent to the universally conserved U4531 (U2585, Escherichia coli numbering) involved in peptidyl transferase center formation, and at the universally conserved C4447 (2501) located in the ribosome near A4397 (2451) and C3909 (2063) belonging to the peptidyl transferase center. As a result, both nucleotides become strongly exposed to hydroxyl radicals. Our data argue that the hydroxyl group at His216 in the C-terminal loop apex of mammalian uL2 contributes to stabilization of a protein conformation that is favorable for binding to H93 of 28S rRNA and that this binding induces structural rearrangement in the regions close to the peptidyl transferase center in the mature ribosome.

Post-translational modifications near the quinone binding site of mammalian complex I.

Complex I (NADH:ubiquinone oxidoreductase) in mammalian mitochondria is an L-shaped assembly of 44 protein subunits with one arm buried in the inner membrane of the mitochondrion and the orthogonal arm protruding about 100 Å into the matrix. The protruding arm contains the binding sites for NADH, the primary acceptor of electrons flavin mononucleotide (FMN), and a chain of seven iron-sulfur clusters that carries the electrons one at a time from FMN to a coenzyme Q molecule bound in the vicinity of the junction between the two arms. In the structure of the closely related bacterial enzyme from Thermus thermophilus, the quinone is thought to bind in a tunnel that spans the interface between the two arms, with the quinone head group close to the terminal iron-sulfur cluster, N2. The tail of the bound quinone is thought to extend from the tunnel into the lipid bilayer. In the mammalian enzyme, it is likely that this tunnel involves three of the subunits of the complex, ND1, PSST, and the 49-kDa subunit. An arginine residue in the 49-kDa subunit is symmetrically dimethylated on the ω-N(G) and ω-N(G') nitrogen atoms of the guanidino group and is likely to be close to cluster N2 and to influence its properties. Another arginine residue in the PSST subunit is hydroxylated and probably lies near to the quinone. Both modifications are conserved in mammalian enzymes, and the former is additionally conserved in Pichia pastoris and Paracoccus denitrificans, suggesting that they are functionally significant.