scholarly journals Crystal structures of 2OG oxygenases involved in ribosomal protein hydroxylation

2014 ◽  
Vol 70 (a1) ◽  
pp. C304-C304
Author(s):  
Shoichiro Horita ◽  
John Scotti ◽  
Michael McDonough ◽  
Rok Sekirnik ◽  
Rashed Chowdhury ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Crystal Structures ◽  
Triple Helix ◽  
Hypoxia Inducible Factor ◽  
Post Translational Modification ◽  
Hydrogen Bond Donor ◽  
Post Translational Modifications ◽  
Prolyl Hydroxylases ◽  
Collagen Triple Helix ◽  
Prolyl Hydroxylation

Post-translational modifications play diverse biological functions. Hydroxylation of collagen proteins has long been a recognised post-translational modification in eukaryotes. In the case of collagen, hydroxylation of prolyl residues, by 2-oxoglutarate and iron dependent enzymes (2OG oxygenases), in collagen proteins allows for the stabilisation of the collagen triple helix structure through conformational restraint and through the addition of a hydrogen bond donor. Additionally, hydroxylation of lysine side chains of collagen is required for cross-linking collagen (and possibly other proteins) in the extra-cellular matrix. Post-translational prolyl hydroxylation also plays a pivotal role in transcriptional regulation of the hypoxic response, as catalyzed by the hypoxia inducible factor / HIF prolyl hydroxylases (PHDs or EGLN enzymes). Recently, ribosomal protein hydroxylation catalyzed by 2OG- and Fe(II)-dependent oxygenases has been found to be a highly conserved post-translational modification in eukaryotes and prokaryotes (Ge et al and Loenarz et al). We present several crystal structures of 2OG oxygenases involved in ribosomal protein hydroxylation.

scholarly journals Hydroxylation of Actin Impairs Cell Motility Through Blocking its’ Histidine Methylation

2021 ◽  
Author(s):  
Zhenzhen Zi ◽  
Lin Yuan ◽  
Qing Ding ◽  
Chiho Kim ◽  
Xu-dong Wang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Motility ◽  
Actin Polymerization ◽  
Hypoxia Inducible Factor ◽  
Dependent Manner ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Von Hippel Lindau ◽  
Oxygen Conditions ◽  
Prolyl Hydroxylation

AbstractProtein hydroxylation is a post translational modification happens on various amino acids, which is catalyzed by the oxoglutarate and oxygen dependent dioxygenases. The best characterized hydroxylated protein is the hypoxia inducible factor (HIF), which is degraded by VHL/elongin C/elongin B/cullin 2/RBX1 (VCB/CR) E3 complex under normal oxygen conditions. Hypoxia or inhibitors (including FG4592 and MK8617) of PHDs stabilize HIF1a and regulate its downstream targets. Prolyl hydroxylase, including PHD2 and PHD3 has been reported in regulating actin polymerization and cell motility. Here, we found MK8617 regulated cell motility in Von Hippel Lindau (VHL) dependent manner. Through the protein hydroxylation proteome experiment upon MK8617 treatment, we identified Pro70 in actin could be hydroxylated and near to His73, which has been reported be methylated and stabilize actin polymerization. Using biochemical assay, we found that binding of VHL with hydroxylated actin (Pro70) decrease the His73 methylation by blocking the interaction of actin with SETD3, the His73 methyltransferase, and further regulated actin polymerization and cell motility. In summary, our study revealed that hypoxia and deficiencies in the VHL, in a HIF independent and prolyl hydroxylation dependent manner, regulate actin polymerization and cell motility through the PTM (Post Translational Modifications) crosstalk.

scholarly journals Crystal structures of adenylylated and unadenylylated PII protein GlnK from Corynebacterium glutamicum

2021 ◽  
Vol 77 (3) ◽  
pp. 325-335
Author(s):  
Florian C. Grau ◽  
Andreas Burkovski ◽  
Yves A. Muller
Keyword(s):  
Corynebacterium Glutamicum ◽  
Crystal Structures ◽  
Bound State ◽  
Covalent Attachment ◽  
Signaling Proteins ◽  
Post Translational Modification ◽  
Conformational Switching ◽  
Post Translational Modifications ◽  
Loop Conformations ◽  
Partner Proteins

PII proteins are ubiquitous signaling proteins that are involved in the regulation of the nitrogen/carbon balance in bacteria, archaea, and some plants and algae. Signal transduction via PII proteins is modulated by effector molecules and post-translational modifications in the PII T-loop. Whereas the binding of ADP, ATP and the concomitant binding of ATP and 2-oxoglutarate (2OG) engender two distinct conformations of the T-loop that either favor or disfavor the interaction with partner proteins, the structural consequences of post-translational modifications such as phosphorylation, uridylylation and adenylylation are far less well understood. In the present study, crystal structures of the PII protein GlnK from Corynebacterium glutamicum have been determined, namely of adenylylated GlnK (adGlnK) and unmodified unadenylylated GlnK (unGlnK). AdGlnK has been proposed to act as an inducer of the transcription repressor AmtR, and the adenylylation of Tyr51 in GlnK has been proposed to be a prerequisite for this function. The structures of unGlnK and adGlnK allow the first atomic insights into the structural implications of the covalent attachment of an AMP moiety to the T-loop. The overall GlnK fold remains unaltered upon adenylylation, and T-loop adenylylation does not appear to interfere with the formation of the two major functionally important T-loop conformations, namely the extended T-loop in the canonical ADP-bound state and the compacted T-loop that is adopted upon the simultaneous binding of Mg-ATP and 2OG. Thus, the PII-typical conformational switching mechanism appears to be preserved in GlnK from C. glutamicum, while at the same time the functional repertoire becomes expanded through the accommodation of a peculiar post-translational modification.

Stronger and Longer Synthetic Collagen

2007 ◽  
Vol 1062 ◽  
Author(s):  
Ronald T. Raines
Keyword(s):  
Self Assembly ◽  
Triple Helix ◽  
Conformational Stability ◽  
Human Proteome ◽  
Steric Effects ◽  
Post Translational Modification ◽  
Abundant Protein ◽  
Stereoelectronic Effects ◽  
Collagen Triple Helix ◽  
Gauche Effect

ABSTRACTCollagen is the most abundant protein in the human proteome. The post-translational modification of collagen by the enzyme prolyl 4-hydroxylase increases markedly the conformational stability of the collagen triple helix. We have discovered that a previously unappreciated force—stereoelectronic effects—is responsible for this increased stability. By exploiting these stereoelectronic effects (e.g., the gauche effect and n→π* interaction) and reciprocal steric effects, we have created synthetic collagen of unprecedented stability. We have also used the molecular self-assembly of triple-helical fragments to create synthetic collagen of unprecedented length. These synthetic collagens have numerous applications in biotechnology and biomedicine.

Investigating the dependence of the hypoxia-inducible factor hydroxylases (factor inhibiting HIF and prolyl hydroxylase domain 2) on ascorbate and other reducing agents

2010 ◽  
Vol 427 (1) ◽  
pp. 135-142 ◽  
Author(s):  
Emily Flashman ◽  
Sarah L. Davies ◽  
Kar Kheng Yeoh ◽  
Christopher J. Schofield
Keyword(s):  
Initial Rate ◽  
Hypoxia Inducible Factor ◽  
Reducing Agents ◽  
Side Chain ◽  
Prolyl Hydroxylases ◽  
Repeat Domain ◽  
Ankyrin Repeat Domain ◽  
Prolyl Hydroxylation ◽  
Domain Peptide ◽  
Stimulation Of

The HIF (hypoxia-inducible factor) hydroxylases [PHDs or EGLNs (prolyl hydroxylases), which in humans are PHD isoforms 1–3, and FIH (factor inhibiting HIF)] regulate HIF levels and activity. These enzymes are Fe(II)/2-oxoglutarate-dependent oxygenases, many of which are stimulated by ascorbate. We have investigated the ascorbate dependence of PHD2-catalysed hydroxylation of two prolyl hydroxylation sites in human HIF-1α, and of FIH-catalysed hydroxylation of asparaginyl hydroxylation sites in HIF-1α and in a consensus ankyrin repeat domain peptide. The initial rate and extent of hydroxylation was increased in the presence of ascorbate for each of these reactions. When ascorbate was replaced with structural analogues, the results revealed that the ascorbate side chain was not important in its contribution to HIF hydroxylase catalysis, whereas modifications to the ene-diol portion of the molecule negated the ability to promote hydroxylation. We investigated whether alternative reducing agents (glutathione and dithiothreitol) could be used to promote HIF hydroxylase activity, and found partial stimulation of hydroxylation in an apparently enzyme- and substrate-specific manner. The results raise the possibility of developing reducing agents targeted to specific HIF hydroxylase-catalysed reactions.

scholarly journals Re-evaluation of lysyl hydroxylation in the collagen triple helix: lysyl hydroxylase 1 and prolyl 3-hydroxylase 3 have site-differential and collagen type-dependent roles in lysine hydroxylation

2019 ◽  
Author(s):  
Yoshihiro Ishikawa ◽  
Yuki Taga ◽  
Keith Zientek ◽  
Nobuyo Mizuno ◽  
Antti M. Salo ◽  
...  
Keyword(s):  
Triple Helix ◽  
Type I Collagen ◽  
Type I ◽  
Post Translational Modifications ◽  
Type V Collagen ◽  
Collagen Triple Helix ◽  
Type V ◽  
Lysyl Hydroxylase 1 ◽  
Lysine Hydroxylation ◽  
Molecular Ensemble

AbstractCollagen is the most abundant protein in humans and is heavily post-translationally modified. Its biosynthesis is very complex and requires three different types of hydroxylation (two for proline and one for lysine) that are generated in the rough endoplasmic reticulum (rER). These processes involve many enzymes and chaperones which were collectively termed the molecular ensemble for collagen biosynthesis. However, the function of some of the proteins in this molecular ensemble is controversial. While prolyl 3-hydroxylase 1 and 2 (P3H1, P3H2) are bona fide collagen prolyl 3-hydroxylases, the function of prolyl 3-hydroxylase 3 (P3H3) is less clear. A recent study of P3H3 null mice demonstrated that this enzyme had no activity as prolyl 3-hydroxylase but may instead act as a chaperone for lysyl hydroxylase 1 (LH1). LH1 is required to generate hydroxylysine for crosslinking within collagen triple helical sequences. If P3H3 is a LH1 chaperone that is critical for LH1 activity, P3H3 and LH1 null mice should have similar deficiency in lysyl hydroxylation. To test this hypothesis, we compared lysyl hydroxylation in type I and V collagen from P3H3 and LH1 null mice. Our results indicate LH1 plays a global role for lysyl hydroxylation in triple helical domain of type I collagen while P3H3 is indeed involved in lysyl hydroxylation particularly at crosslink formation sites but is not required for all lysyl hydroxylation sites in type I collagen triple helix. Furthermore, although type V collagen from LH1 null mice surprisingly contained as much hydroxylysine as type V collagen from wild type, the amount of hydroxylysine in type V collagen was clearly suppressed in P3H3 null mice. In summary, our study suggests that P3H3 and LH1 likely have two distinct mechanisms to distinguish crosslink formation sites from other sites in type I collagen and to recognize different collagen types in the rER.Author summaryCollagen is one of the most heavily post-translationally modified proteins in the human body and its post-translational modifications provide biological functions to collagen molecules. In collagen post-translational modifications, crosslink formation on a collagen triple helix adds important biomechanical properties to the collagen fibrils and is mediated by hydroxylation of very specific lysine residues. LH1 and P3H3 show the similar role in lysine hydroxylation for specific residues at crosslink formation sites of type I collagen. Conversely, they have very distinct rules in lysine hydroxylation at other residues in type I collagen triple helix. Furthermore, they demonstrate preferential recognition and modification of different collagen types. Our findings provide a better understanding of the individual functions of LH1 and P3H3 in the rER and also offer new directions for the mechanism of lysyl hydroxylation followed by crosslink formation in different tissues and collagens.

Dicarbonyl derived post-translational modifications: chemistry bridging biology and aging-related disease

2020 ◽  
Vol 64 (1) ◽  
pp. 97-110
Author(s):  
Christian Sibbersen ◽  
Mogens Johannsen
Keyword(s):  
Mass Spectrometry ◽  
Future Research ◽  
Amino Acid Residues ◽  
Post Translational Modification ◽  
Dicarbonyl Compounds ◽  
Protein Targets ◽  
Post Translational Modifications ◽  
Reactive Protein ◽  
Glycation End Products ◽  
Direct Mass Spectrometry

Abstract In living systems, nucleophilic amino acid residues are prone to non-enzymatic post-translational modification by electrophiles. α-Dicarbonyl compounds are a special type of electrophiles that can react irreversibly with lysine, arginine, and cysteine residues via complex mechanisms to form post-translational modifications known as advanced glycation end-products (AGEs). Glyoxal, methylglyoxal, and 3-deoxyglucosone are the major endogenous dicarbonyls, with methylglyoxal being the most well-studied. There are several routes that lead to the formation of dicarbonyl compounds, most originating from glucose and glucose metabolism, such as the non-enzymatic decomposition of glycolytic intermediates and fructosyl amines. Although dicarbonyls are removed continuously mainly via the glyoxalase system, several conditions lead to an increase in dicarbonyl concentration and thereby AGE formation. AGEs have been implicated in diabetes and aging-related diseases, and for this reason the elucidation of their structure as well as protein targets is of great interest. Though the dicarbonyls and reactive protein side chains are of relatively simple nature, the structures of the adducts as well as their mechanism of formation are not that trivial. Furthermore, detection of sites of modification can be demanding and current best practices rely on either direct mass spectrometry or various methods of enrichment based on antibodies or click chemistry followed by mass spectrometry. Future research into the structure of these adducts and protein targets of dicarbonyl compounds may improve the understanding of how the mechanisms of diabetes and aging-related physiological damage occur.

scholarly journals Immunological discrimination of β-tubulin isoforms in developing mouse brain. Post-translational modification of non-class-III β-tubulins

1992 ◽  
Vol 288 (3) ◽  
pp. 919-924 ◽  
Author(s):  
I Linhartová ◽  
P Dráber ◽  
E Dráberová ◽  
V Viklický
Keyword(s):  
Mouse Brain ◽  
Specific Class ◽  
Class Iii ◽  
Post Translational Modification ◽  
Beta Tubulin ◽  
Developmentally Regulated ◽  
Post Translational Modifications ◽  
Tubulin Isotypes ◽  
Tubulin Isoforms ◽  
Developing Mouse Brain

Individual beta-tubulin isoforms in developing mouse brain were characterized using immunoblotting, after preceding high-resolution isoelectric focusing, with monoclonal antibodies against different structural regions of beta-tubulin. Some of the antibodies reacted with a limited number of tubulin isoforms in all stages of brain development and in HeLa cells. The epitope for the TU-14 antibody was located in the isotype-defining domain and was present on the beta-tubulin isotypes of classes I, II and IV, but absent on the neuron-specific class-III isotype. The data suggest that non-class-III beta-tubulins in mouse brain are substrates for developmentally regulated post-translational modifications and that beta-tubulins of non-neuronal cells are also post-translationally modified.

scholarly journals Roles of HIF and 2-Oxoglutarate-Dependent Dioxygenases in Controlling Gene Expression in Hypoxia

Cancers ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 350
Author(s):  
Julianty Frost ◽  
Mark Frost ◽  
Michael Batie ◽  
Hao Jiang ◽  
Sonia Rocha
Keyword(s):  
Gene Expression ◽  
Hypoxia Inducible Factor ◽  
Transcriptional Regulator ◽  
Hydroxylase Activity ◽  
Control Of Gene Expression ◽  
Prolyl Hydroxylases ◽  
Chromatin Organisation ◽  
Sense And Respond ◽  
Almost All ◽  
And Function

Hypoxia—reduction in oxygen availability—plays key roles in both physiological and pathological processes. Given the importance of oxygen for cell and organism viability, mechanisms to sense and respond to hypoxia are in place. A variety of enzymes utilise molecular oxygen, but of particular importance to oxygen sensing are the 2-oxoglutarate (2-OG) dependent dioxygenases (2-OGDs). Of these, Prolyl-hydroxylases have long been recognised to control the levels and function of Hypoxia Inducible Factor (HIF), a master transcriptional regulator in hypoxia, via their hydroxylase activity. However, recent studies are revealing that dioxygenases are involved in almost all aspects of gene regulation, including chromatin organisation, transcription and translation. We highlight the relevance of HIF and 2-OGDs in the control of gene expression in response to hypoxia and their relevance to human biology and health.

scholarly journals Post translational modifications of milk proteins in geographically diverse goat breeds

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
P. K. Rout ◽  
M. Verma
Keyword(s):  
Protein Function ◽  
Whey Proteins ◽  
Milk Proteins ◽  
Goat Milk ◽  
Peptide Sequence ◽  
Cow Milk ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Functional Roles ◽  
Dpp Iv

AbstractGoat milk is a source of nutrition in difficult areas and has lesser allerginicity than cow milk. It is leading in the area for nutraceutical formulation and drug development using goat mammary gland as a bioreactor. Post translational modifications of a protein regulate protein function, biological activity, stabilization and interactions. The protein variants of goat milk from 10 breeds were studied for the post translational modifications by combining highly sensitive 2DE and Q-Exactive LC-MS/MS. Here we observed high levels of post translational modifications in 201 peptides of 120 goat milk proteins. The phosphosites observed for CSN2, CSN1S1, CSN1S2, CSN3 were 11P, 13P, 17P and 6P, respectively in 105 casein phosphopeptides. Whey proteins BLG and LALBA showed 19 and 4 phosphosites respectively. Post translational modification was observed in 45 low abundant non-casein milk proteins mainly associated with signal transduction, immune system, developmental biology and metabolism pathways. Pasp is reported for the first time in 47 sites. The rare conserved peptide sequence of (SSSEE) was observed in αS1 and αS2 casein. The functional roles of identified phosphopeptides included anti-microbial, DPP-IV inhibitory, anti-inflammatory and ACE inhibitory. This is first report from tropics, investigating post translational modifications in casein and non-casein goat milk proteins and studies their interactions.

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