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.

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 Role of the unique, non-essential phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt) in Corynebacterium glutamicum

Microbiology ◽  
2020 ◽  
Vol 166 (8) ◽  
pp. 759-776 ◽  
Author(s):  
Nathalie Dautin ◽  
Manuela Argentini ◽  
Niloofar Mohiman ◽  
Cécile Labarre ◽  
David Cornu ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Signal Peptide ◽  
Type Species ◽  
Signal Peptidase ◽  
Covalent Attachment ◽  
Post Translational Modification ◽  
Content Type ◽  
Peptide Cleavage ◽  
Link Type ◽  
Membrane Anchoring

Bacterial lipoproteins are secreted proteins that are post-translationally lipidated. Following synthesis, preprolipoproteins are transported through the cytoplasmic membrane via the Sec or Tat translocon. As they exit the transport machinery, they are recognized by a phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt), which converts them to prolipoproteins by adding a diacylglyceryl group to the sulfhydryl side chain of the invariant Cys+1 residue. Lipoprotein signal peptidase (LspA or signal peptidase II) subsequently cleaves the signal peptide, liberating the α-amino group of Cys+1, which can eventually be further modified. Here, we identified the lgt and lspA genes from Corynebacterium glutamicum and found that they are unique but not essential. We found that Lgt is necessary for the acylation and membrane anchoring of two model lipoproteins expressed in this species: MusE, a C. glutamicum maltose-binding lipoprotein, and LppX, a Mycobacterium tuberculosis lipoprotein. However, Lgt is not required for these proteins’ signal peptide cleavage, or for LppX glycosylation. Taken together, these data show that in C. glutamicum the association of some lipoproteins with membranes through the covalent attachment of a lipid moiety is not essential for further post-translational modification.

Efficient approaches for characterizing ubiquitinated proteins

2008 ◽  
Vol 36 (5) ◽  
pp. 823-827 ◽  
Author(s):  
Roland Hjerpe ◽  
Manuel S. Rodríguez
Keyword(s):  
Affinity Purification ◽  
Large Family ◽  
Cysteine Proteases ◽  
Biochemical Properties ◽  
Covalent Attachment ◽  
Deubiquitinating Enzymes ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Ubiquitinated Proteins ◽  
Ubiquitin Binding Domain

Post-translational modification of proteins offers a rapid route to change the activity of crucial factors within the cell. One of the more drastic post-translational modifications, in terms of effect on biochemical properties, is the covalent attachment of the small protein ubiquitin, to a target factor. The labile nature of some post-translational modifications puts obstacles in the path of attempting to detect modified species of most proteins. Indeed, ubiquitination can be rapidly reversed by the action of a large family of DUBs (deubiquitinating enzymes), most of which are cysteine proteases. This, taken together with the rapid proteasomal degradation of some species of ubiquitinated proteins, results in difficulties in detecting modified targets. In this review, practical approaches developed for the detection, purification and characterization of ubiquitinated proteins are reviewed. After a brief appraisal of the use of histidine-tagged ubiquitin, focus is placed on development of UBD (ubiquitin-binding domain)–ubiquitin affinity purification.

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 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.

scholarly journals System-wide identification and prioritization of enzyme substrates by thermal analysis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Amir Ata Saei ◽  
Christian M. Beusch ◽  
Pierre Sabatier ◽  
Juan Astorga Wells ◽  
Hassan Gharibi ◽  
...  
Keyword(s):  
Thermal Analysis ◽  
Thioredoxin Reductase ◽  
Structural Level ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Enzyme Substrate ◽  
Thioredoxin Reductase 1 ◽  
Enzyme Substrates ◽  
Substrate Proteins

AbstractDespite the immense importance of enzyme–substrate reactions, there is a lack of general and unbiased tools for identifying and prioritizing substrate proteins that are modified by the enzyme on the structural level. Here we describe a high-throughput unbiased proteomics method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that the enzymatic post-translational modification of substrate proteins is likely to change their thermal stability. In our proof-of-concept studies, SIESTA successfully identifies several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, protein kinase B (AKT1) and poly-(ADP-ribose) polymerase-10 systems. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, opening opportunities to investigate the effect of post-translational modifications on signal transduction and facilitate drug discovery.

scholarly journals Role of Host-Mediated Post-Translational Modifications (PTMs) in RNA Virus Pathogenesis

2020 ◽  
Vol 22 (1) ◽  
pp. 323
Author(s):  
Ramesh Kumar ◽  
Divya Mehta ◽  
Nimisha Mishra ◽  
Debasis Nayak ◽  
Sujatha Sunil
Keyword(s):  
Rna Virus ◽  
Viral Proteins ◽  
Post Translational Modification ◽  
Host Proteins ◽  
Viral Protein Synthesis ◽  
Post Translational Modifications ◽  
Small Proteins ◽  
Cellular Machinery ◽  
Chemical Groups

Being opportunistic intracellular pathogens, viruses are dependent on the host for their replication. They hijack host cellular machinery for their replication and survival by targeting crucial cellular physiological pathways, including transcription, translation, immune pathways, and apoptosis. Immediately after translation, the host and viral proteins undergo a process called post-translational modification (PTM). PTMs of proteins involves the attachment of small proteins, carbohydrates/lipids, or chemical groups to the proteins and are crucial for the proteins’ functioning. During viral infection, host proteins utilize PTMs to control the virus replication, using strategies like activating immune response pathways, inhibiting viral protein synthesis, and ultimately eliminating the virus from the host. PTM of viral proteins increases solubility, enhances antigenicity and virulence properties. However, RNA viruses are devoid of enzymes capable of introducing PTMs to their proteins. Hence, they utilize the host PTM machinery to promote their survival. Proteins from viruses belonging to the family: Togaviridae, Flaviviridae, Retroviridae, and Coronaviridae such as chikungunya, dengue, zika, HIV, and coronavirus are a few that are well-known to be modified. This review discusses various host and virus-mediated PTMs that play a role in the outcome during the infection.

scholarly journals Aptamers selected against the unglycosylated EGFRvIII ectodomain and delivered intracellularly reduce membrane-bound EGFRvIII and induce apoptosis

2009 ◽  
Vol 390 (2) ◽  
pp. 137-144 ◽  
Author(s):  
Yingmiao Liu ◽  
Chien-Tsun Kuan ◽  
Jing Mi ◽  
Xiuwu Zhang ◽  
Bryan M. Clary ◽  
...  
Keyword(s):  
Growth Factor Receptor ◽  
Rna Aptamers ◽  
Normal Brain ◽  
Post Translational Modification ◽  
Expression And Purification ◽  
Post Translational Modifications ◽  
Protein Expression And Purification ◽  
Epidermal Growth ◽  
Membrane Bound

Abstract Epidermal growth factor receptor variant III (EGFRvIII) is a glycoprotein uniquely expressed in glioblastoma, but not in normal brain tissues. To develop targeted therapies for brain tumors, we selected RNA aptamers against the histidine-tagged EGFRvIII ectodomain, using an Escherichia coli system for protein expression and purification. Representative aptamer E21 has a dissociation constant (Kd) of 33×10-9 m, and exhibits high affinity and specificity for EGFRvIII in ELISA and surface plasmon resonance assays. However, selected aptamers cannot bind the same protein expressed from eukaryotic cells because glycosylation, a post-translational modification present only in eukaryotic systems, significantly alters the structure of the target protein. By transfecting EGFRvIII aptamers into cells, we find that membrane-bound, glycosylated EGFRvIII is reduced and the percentage of cells undergoing apoptosis is increased. We postulate that transfected aptamers can interact with newly synthesized EGFRvIII, disrupt proper glycosylation, and reduce the amount of mature EGFRvIII reaching the cell surface. Our work establishes the feasibility of disrupting protein post-translational modifications in situ with aptamers. This finding is useful for elucidating the function of proteins of interest with various modifications, as well as dissecting signal transduction pathways.

RegulatING chromatin regulators: post-translational modification of the ING family of epigenetic regulators

2013 ◽  
Vol 450 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Shankha Satpathy ◽  
Arash Nabbi ◽  
Karl Riabowol
Keyword(s):  
Biological Significance ◽  
Type Ii ◽  
Post Translational Modification ◽  
Post Translational Modifications ◽  
Chromatin Regulators ◽  
Cancer Types ◽  
Splicing Isoforms ◽  
Enrichment Techniques ◽  
Affect Cell Growth

The five human ING genes encode at least 15 splicing isoforms, most of which affect cell growth, differentiation and apoptosis through their ability to alter gene expression by epigenetic mechanisms. Since their discovery in 1996, ING proteins have been classified as type II tumour suppressors on the basis of reports describing their down-regulation and mislocalization in a variety of cancer types. In addition to their regulation by transcriptional mechanisms, understanding the range of PTMs (post-translational modifications) of INGs is important in understanding how ING functions are fine-tuned in the physiological setting and how they add to the repertoire of activities affected by the INGs. In the present paper we review the different PTMs that have been reported to occur on INGs. We discuss the PTMs that modulate ING function under normal conditions and in response to a variety of stresses. We also describe the ING PTMs that have been identified by several unbiased MS-based PTM enrichment techniques and subsequent proteomic analysis. Among the ING PTMs identified to date, a subset has been characterized for their biological significance and have been shown to affect processes including subcellular localization, interaction with enzymatic complexes and ING protein half-life. The present review aims to highlight the emerging role of PTMs in regulating ING function and to suggest additional pathways and functions where PTMs may effect ING function.

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