Regulation by S-nitrosylation of protein post-translational modification

Protein post-translational modification by S-nitrosylation conveys a ubiquitous influence of nitric oxide on signal transduction in eukaryotic cells. The wide functional purview of S-nitrosylation reflects in part the regulation by S-nitrosylation of the principal protein post-translational modifications that play a role in cell signaling, including phosphorylation, acetylation, ubiquitylation and related modifications, palmitoylation, and alternative Cys-based redox modifications. In this minireview, we discuss the mechanisms through which S-nitrosylation exerts its broad pleiotropic influence on protein post-translational modification.     

Non-oxidative modification of lens crystallins by kynurenine: a novel post-translational protein modification with possible relevance to ageing and cataract

In humans, the crystallin proteins of the ocular lens become yellow-coloured and fluorescent with ageing. With the development of senile nuclear cataract, the crystallins become brown and additional fluorophores are formed. The mechanism underlying crystallin colouration is not known but may involve interaction with kynurenine-derived UV filter compounds. We have recently identified a sulphur-linked glutathionyl-3-hydroxykynurenine glucoside adduct in the lens and speculated that kynurenine may also form adducts with GSH and possibly with nucleophilic amino acids of the crystallins (e.g. Cys). Here we show that kynurenine modifies calf lens crystallins non-oxidatively to yield coloured (365 nm absorbing), fluorescent (Ex 380 nm/Em 450-490 nm) protein adducts. Carboxymethylation and succinylation of crystallins inhibited kynurenine-mediated modification by approx. 90%, suggesting that Cys, Lys and possibly His residues may be involved. This was confirmed by showing that kynurenine formed adducts with GSH as well as with poly-His and poly-Lys. NMR studies revealed that the novel poly-Lys-kynurenine covalent linkage was via the epsilon-amino group of the Lys side chain and the betaC of the kynurenine side chain. Analysis of tryptic peptides of kynurenine-modified crystallins revealed that all of the coloured peptides contained either His, Cys or an internal Lys residue. We propose a novel mechanism of kynurenine-mediated crystallin modification which does not require UV light or oxidative conditions as catalysts. Rather, we suggest that the side chain of kynurenine-derived lens UV filters becomes deaminated to yield an alpha,beta-unsaturated carbonyl which is highly susceptible to attack by nucleophilic amino acid residues of the crystallins. The inability of the lens fibre cells to metabolise their constituent proteins results in the accumulation of coloured/fluorescent crystallins with age.     

Heteronuclear NMR studies of the specificity of the post-translational modification of biotinyl domains by biotinyl protein ligase

The lipoyl domains of 2-oxo acid dehydrogenase multienzyme complexes and the biotinyl domains of biotin-dependent enzymes have homologous structures, but the target lysine residue in each domain is correctly selected for posttranslational modification by lipoyl protein ligase and biotinyl protein ligase, respectively. We have applied two-dimensional heteronuclear NMR spectroscopy to investigate the interaction between the apo form of the biotinyl domain of the biotin carboxyl carrier protein of acetyl-CoA carboxylase and the biotinyl protein ligase (BPL) from Escherichia coli. Heteronuclear multiple quantum coherence NMR spectra of the 15N-labelled biotinyl domain were recorded in the presence and absence of the ligase and backbone amide 1H and 15N chemical shifts were evaluated. Small, but significant, changes in chemical shift were found in two regions, including the tight beta-turn that houses the lysine residue targetted for biotinylation, and the beta-strand 2 and the loop that precedes it in the domain. When compared with the three-dimensional structure, sequence alignments of other biotinyl and lipoyl domains, and mutagenesis data, these results give a clear indication of how the biotinyl domain is both recognised by BPL and distinguished from the structurally related lipoyl domain to ensure correct posttranslational modification.     

Negative effects of desiccation on the protein sorting and post-translational modification

Bryophytes as the first land plants are believed to have colonized the land from a fresh water origin, requiring adaptive mechanisms that survival of dehydration. Physcomitrella patens is such a non-vascular bryophyte and shows rare desiccation tolerance in its vegetative tissues. Previous studies showed that during the course of dehydration, several related processes are set in motion: plasmolysis, chloroplast remodeling and microtubule depolymerization. And proteomic alteration supported the cellular structural changes in respond to desiccation stress.¹ In this addendum, we report that Golgi bodies are absent and adaptor protein complex AP-1 large subunit is downregulated during the course of dehydration. Those phenomena may be adverse in protein posttranslational modification, protein sorting and cell walls synthesis under the desiccation condition.Key words: AP-1 protein, cell ultrastructure, desiccation, golgi bodies, physcomitrella, proteomeThe plant Golgi apparatus is composed of many small stacks of cisternae, sometimes known as dictyosomes. The Golgi is a complex polarized organelle consisting of both a cis and trans side, containing compartments with functionally different capacities for directing cellular components. The plant Golgi apparatus synthesis a wide range of cell wall polysaccharides and proteoglycans, and also carries out O-linked glycosylation and N-linked glycan processing.^2–5 Moreover, the Golgi is involved in returning escaped proteins back to the endoplasmic reticulum, sorting of proteins and polysaccharides to the cell wall or vacuoles, and in organizing the compartmentation of its own enzymes by retention or retrieval mechanisms.⁶ In conclusion, The Golgi apparatus is central to the growth and division of the plant cell through its roles in protein glycosylation, protein sorting and cell wall synthesis.The transit of proteins and lipids from the trans-Golgi network (TGN) and the plasma membrane to endosomes within eucaryotic cells occurs via the budding and fusion of clathrin-coated vesicles (CCVs).^7,8 At the TGN, this process is mediated by the heterotetrameric AP-1 adaptor complex, which consists of two large subunits, β and γ1; a medium subunit, µ1; and a small σ1 subunit. Recruitment of AP-1 to the TGN membrane is regulated by a small GTPase, ADP-ribosylation factor 1 (ARF1), which cycles between an inactive GDP-bound form in cytosol and an active GTP-bound form that associates with the membrane like other small GTPase.⁹ There is also evidence that phosphorylation/dephosphorylation events are involved in the regulation of the function of AP-1. Ghosh and Kornfeld demostrated that AP-1 recruitment onto the membrane is associated with protein phosphatase 2A (PP2A)-mediated dephosphorylation of its β1 subunit, which enables clathrin assembly. This Golgi-associated isoform of PP2A exhibits specificity for phosphorylated β1 compared with phosphorylated µ1. Once on the membrane, the µ1 subunit undergoes phosphorylation, which results in a conformation change. This conformational change is associated with increased binding to sorting signals on the cytoplasmic tails of cargo molecules. Dephosphorylation of µ1 (and µ2) by another PP2A-like phosphatase reversed the effect and resulted in adaptor release from CCVs. Cyclical phosphorylation/dephosphorylation of the subunits of AP-1 regulate its function from membrane recruitment until its release into cytosol.¹⁰Plants experience desiccation stress either as part of a developmental programme, such as during seed maturation, or because of reductions in air humid and water availability in the soil. Underlying the ability of bryophytes to withstand periods of desiccation are morphological and biochemical adaptations. Plants respond to stress as individual cells and synergistically as a whole organism. Scanning electron microscopy observation showed that the P. patens gametophore cells were shrunk upon the treatment of desiccation, and the shrinking started from the edge of the leaves (Fig. 1). We could clearly observe some dark granula in the untreated cells, but these granula disappeared post-desiccation treatment (Fig. 1). Transmission electron microscopy also revealed that the large stacks of Golgi bodies and numerous coated vesicles are typically visible in the hydrated cells (Fig. 2), but these are absent in the desiccative cells (data not shown). The plant Golgi apparatus plays an important role in protein glycosylation and sorting. Therefore, this event means that the protein sorting and the cargo transporting are disrupted by desiccation stress. During desiccation, the absentness of Golgi bodies reduce the leaf activities of cell, and this is expected to similar to plant dormancy which is a phenomenon in resurrection plants and some drought-tolerant plants. In addition, through two-dimensional gel electrophoresis (2-DE) and LC-MS/MS analysis, AP-1 large subunit was identified as downregulated protein during the course of dehydration (Fig. 3). AP-1 is ubiquitously expressed and participates in the budding of clathrin-coated vesicles from the trans-Golgi network (TGN) and endosomes. AP-1 also recognizes sorting motifs in cargo molecules. Our results suggested that desiccation led to a marked disrupt in protein posttranslational modification, protein sorting and cell walls synthesis.Open in a separate windowFigure 1Scanning Electron microscopy images of normal and dehydrated P. patens gametophores. (A) the fresh leaf; (B) enlargement of the rectangle area of (A); (C) dehydrated gametophores of P. patens. Bar = 5 µm.Open in a separate windowFigure 2Transmission electron microscopy images of cell in fresh game-tophores. The arrows indicate Golgi body, Bar = 2 µm.Open in a separate windowFigure 3Part protein profile of the control and desiccation plants. The arrows indicate the AP-1 large subunit.     

Analysis of post-translational modification of mycobacterial proteins using a cassette expression system

A recombinant expression system was developed to analyse sequence determinants involved in O-glycosylation of proteins in mycobacteria. By expressing peptide sequences corresponding to known glycosylation sites within a chimeric lipoprotein construct, amino acids flanking modified threonine residues were found to have an important influence on glycosylation. The expression system was used to screen mycobacterial sequences selected using a neural network (NetOglyc) trained on eukaryotic O-glycoproteins. Evidence of glycosylation was obtained for eight of 11 proteins tested. The results suggest that sites involved in O-glycosylation of mycobacterial and eukaryotic proteins share similar structural features.     

Effects of a post-translational modification mutation on different developmentally regulated glycosidases in Dictyostelium discoideum.

We have studied the effect of a post-translational modification mutation upon four developmentally regulated glycosidases of Dictyostelium discoideum. The presence of the modA mutation affects the intracellular level of these multimeric enzymes differently. The level of alpha-glucosidase is unaffected in the modA mutant. The mutant cell contains only a very small fraction of the wild type beta-glucosidase-1 activity. The alteration in modification renders beta-glucosidase-1 holoenzyme thermolabile and susceptible to degradation in vivo. alpha-Mannosidase-1 and N-acetylglucosaminidase are found at approximately 1/3 of the wild type level in the modA mutant. Degradation of holoenzyme does not appear to be responsible for the low level of these activities. We propose that alpha-mannosidase-1 and N-acetylglucosaminidase subunits are being degraded prior to subunit assembly. We conclude the modification bestows different properties upon the various glycosidases.     

Polyglutamylation is a post-translational modification with a broad range of substrates

Polyglutamylation is a post-translational modification that generates lateral acidic side chains on proteins by sequential addition of glutamate amino acids. This modification was first discovered on tubulins, and it is important for several microtubule functions. Besides tubulins, only the nucleosome assembly proteins NAP1 and NAP2 have been shown to be polyglutamylated. Here, using a proteomic approach, we identify a large number of putative substrates for polyglutamylation in HeLa cells. By analyzing a selection of these putative substrates, we show that several of them can serve as in vitro substrates for two of the recently discovered polyglutamylases, TTLL4 and TTLL5. We further show that TTLL4 is the main polyglutamylase enzyme present in HeLa cells and that new substrates of polyglutamylation are indeed modified by TTLL4 in a cellular context. No clear consensus polyglutamylation site could be defined from the primary sequence of the here-identified new substrates of polyglutamylation. However, we demonstrate that glutamate-rich stretches are important for a protein to become polyglutamylated. Most of the newly identified substrates of polyglutamylation are nucleocytoplasmic shuttling proteins, including many chromatin-binding proteins. Our work reveals that polyglutamylation is a much more widespread post-translational modification than initially thought and thus that it might be a regulator of many cellular processes.     

Absolute quantification of protein and post-translational modification abundance with stable isotope-labeled synthetic peptides

In the analysis of biological systems, it is of interest to identify the components of the system and to monitor their changes in abundance under different conditions. The AQUA (for 'absolute quantification') method allows sensitive and specific targeted quantification of protein and post-translational modifications in complex protein mixtures using stable isotope-labeled peptides as internal standards. Each AQUA experiment is composed of two stages: method development and application to a biological scenario. In the method development stage, peptides from the protein of interest are chosen and then synthesized with stable isotopes such as (13)C, (2)H or (15)N. The abundance of these internal standards and their endogenous counterparts can be measured by mass spectrometry with selected reaction monitoring or selected ion monitoring methods. Once an AQUA method is established, it can be rapidly applied to a wide range of biological samples, from tissue culture cells to human plasma and tissue. After AQUA peptide synthesis, the development, optimization and application of AQUA analyses to a specific biological problem can be achieved in ~1 week. Here we demonstrate the usefulness of this method by monitoring both Polo-like kinase 1 (Plk1) protein abundance in multiple lung cancer cell lines and the extent of Plk1 activation loop phosphorylation (pThr-210) during release from S phase.     

Characterization of the post-translational modification of recombinant human BMP-15 mature protein

Bone morphogenetic protein-15 (BMP-15) is an oocyte-secreted factor critical for the regulation of ovarian physiology. When recombinant human BMP-15 (rhBMP-15) produced in human embryonic kidney 293 cells was subjected to SDS-PAGE analysis, two mature protein forms corresponding to 16 kDa (P16) and 17 kDa (P17) were observed. Despite the physiological relevance and critical function of BMP-15 in female reproduction, little is known about the structure of rhBMP-15. Here, we have analyzed the structure of the rhBMP-15 mature proteins (P16 and P17) using state-of-the-art proteomics technology. Our findings are as follows: (1) the N-terminal amino acid of P16 and P17 is pyroglutamic acid; (2) the Ser residue at the sixth position of P16 is phosphorylated; (3) P17 is O-glycosylated at Thr10; and (4) the C-terminal amino acid of P16 and P17 is truncated. These findings are the first knowledge of the structure of rhBMP-15 mature protein toward understanding the molecular basis of BMP-15 function and could provide an important contribution to the rapidly progressing research area involving oocyte-specific growth factors in modulation of female fertility.     

MAPRes: an efficient method to analyze protein sequence around post-translational modification sites

Functional switches are often regulated by dynamic protein modifications. Assessing protein functions, in vivo, and their functional switches remains still a great challenge in this age of development. An alternative methodology based on in silico procedures may facilitate assessing the multifunctionality of proteins and, in addition, allow predicting functions of those proteins that exhibit their functionality through transitory modifications. Extensive research is ongoing to predict the sequence of protein modification sites and analyze their dynamic nature. This study reports the analysis performed on phosphorylation, Phospho.ELM (version 3.0) and glycosylation, OGlycBase (version 6.0) data for mining association patterns utilizing a newly developed algorithm, MAPRes. This method, MAPRes (Mining Association Patterns among preferred amino acid residues in the vicinity of amino acids targeted for post-translational modifications), is based on mining association among significantly preferred amino acids of neighboring sequence environment and modification sites themselves. Association patterns arrived at by association pattern/rule mining were in significant conformity with the results of different approaches. However, attempts to analyze substrate sequence environment of phosphorylation sites catalyzed for Tyr kinases and the sequence data for O-GlcNAc modification were not successful, due to the limited data available. Using the MAPRes algorithm for developing an association among PTM site with its vicinal amino acids is a valid method with many potential uses: this is indeed the first method ever to apply the association pattern mining technique to protein post-translational modification data.     

Conversion of a transmembrane to a water-soluble protein complex by a single point mutation

Proteins exist in one of two generally incompatible states: either membrane associated or soluble. Pore-forming proteins are exceptional because they are synthesized as a water-soluble molecule but end up being located in the membrane -- that is, they are nonconstitutive membrane proteins. Here we report the pronounced effect of the single point mutation Y221G of the pore-forming toxin aerolysin. This mutation blocks the hemolytic activity of the toxin but does not affect its initial structure, its ability to bind to cell-surface receptors or its capacity to form heptamers, which constitute the channel-forming unit. The overall structure of the Y221G protein as analyzed by cryo-negative staining EM and three-dimensional reconstruction is remarkably similar to that of the wild type heptamer. The mutant protein forms a mushroom-shaped complex whose stem domain is thought to be within the membrane in the wild type toxin. In contrast to the wild type heptamer, which is a hydrophobic complex, the Y221G heptamer is fully hydrophilic. This point mutation has, therefore, converted a normally membrane-embedded toxin into a soluble complex.     

Factors influencing expression and post-translational modification of recombinant protein C.

Factors affecting the production of recombinant human protein C were investigated. When recombinant cells producing human protein C were cultured with microcarrier in two different scales, we found that (1) as cells grew to confluence, specific productivity of the protein was decreased and that (2) the efficiency of gamma-carboxylation of the generated protein C was lower in a larger culture than in a smaller culture. Higher cell density was shown to influence the specific productivity unfavorably. On the other hand, the amount of oxygen supply as demonstrated by oxygen consumption rate in the Opticell culture system correlated well with the efficiency of gamma-carboxylation, suggesting that oxygen metabolism is somehow implicated in the post-translational modification of recombinant protein C.     

Structure of a putative lipoate protein ligase from Thermoplasma acidophilum and the mechanism of target selection for post-translational modification

Lipoyl-lysine swinging arms are crucial to the reactions catalysed by the 2-oxo acid dehydrogenase multienzyme complexes. A gene encoding a putative lipoate protein ligase (LplA) of Thermoplasma acidophilum was cloned and expressed in Escherichia coli. The recombinant protein, a monomer of molecular mass 29 kDa, was catalytically inactive. Crystal structures in the absence and presence of bound lipoic acid were solved at 2.1 A resolution. The protein was found to fall into the alpha/beta class and to be structurally homologous to the catalytic domains of class II aminoacyl-tRNA synthases and biotin protein ligase, BirA. Lipoic acid in LplA was bound in the same position as biotin in BirA. The structure of the T.acidophilum LplA and limited proteolysis of E.coli LplA together highlighted some key features of the post-translational modification. A loop comprising residues 71-79 in the T.acidophilum ligase is proposed as interacting with the dithiolane ring of lipoic acid and discriminating against the entry of biotin. A second loop comprising residues 179-193 was disordered in the T.acidophilum structure; tryptic cleavage of the corresponding loop in the E.coli LplA under non-denaturing conditions rendered the enzyme catalytically inactive, emphasizing its importance. The putative LplA of T.acidophilum lacks a C-terminal domain found in its counterparts in E.coli (Gram-negative) or Streptococcus pneumoniae (Gram-positive). A gene encoding a protein that appears to have structural homology to the additional domain in the E.coli and S.pneumoniae enzymes was detected alongside the structural gene encoding the putative LplA in the T.acidophilum genome. It is likely that this protein is required to confer activity on the LplA as currently purified, one protein perhaps catalysing the formation of the obligatory lipoyl-AMP intermediate, and the other transferring the lipoyl group from it to the specific lysine residue in the target protein.     

Capillary affinity electrophoresis for the screening of post-translational modification of proteins with carbohydrates

Glycosylation is one of the most important post-translational events for proteins, affecting their functions in health and disease, and plays significant roles in various information traffics for intracellular and intercellular biological events (Hancock, W. S. J. Proteome Res. 2002, 1, 297). We have attempted to obtain the information on the numbers and amounts of carbohydrate chains. Interaction between carbohydrate chains and proteins that recognize them is a target to understand the biological roles of glycosylation. To date, there have been a few strategies for simultaneous analysis of the interactions between complex mixtures of carbohydrates and proteins. Here, we report an approach to categorize carbohydrate chains using a few glycoprotein samples as models for the studies on the analysis of post-translational modification of proteins with carbohydrates. A combination of some specific lectins was used as carbohydrate-binding proteins. The method is based on high-resolution separation of fluorescent-labeled carbohydrates by capillary electrophoresis with laser-induced fluorescent detection in the presence of carbohydrate-binding proteins at different concentrations. The present technique affords (1) simultaneous determination of carbohydrate chains, (2) binding specificity of the constituent carbohydrate chains to specific proteins, and (3) kinetic data such as the association constant of each carbohydrate. We found that the lectins employed in the present study could discriminate subtle difference in linkages and resolved the carbohydrate mixtures. The results will be useful, for example, to understand the biological events expressed with carbohydrate changes on the cell surface.     

Differential post-translational modification of human type I keratins synthesized in a rabbit reticulocyte cell-free system

The translation products synthesized in a rabbit reticulocyte lysate system, from total cellular mRNA from the human epithelial cell-line ME-180, have been examined. Keratin proteins are prominent among these translation products, and they precisely coelectrophorese in sodium dodecyl sulfate-polyacrylamide gels with keratins purified from the cells. Type-I, acidic, keratins which are acetylated in vivo, are also acetylated by the reticulocyte lysate. Examination by two-dimensional electrophoresis, of two acidic keratins known to be phosphorylated in vivo reveals that only one of these proteins is phosphorylated in the lysate system. Phosphorylation of this protein occurs after release of the completed polypeptide chain from the ribosome. The protein phosphorylated by the lysate is known to be the only ME-180 phosphokeratin modulated by cyclic AMP, reflecting in vitro the differential modification of ME-180 keratins in vivo.