Post-translational Modification Of Specific Proteins Research Articles

Acetylation and Phosphorylation Regulate the Role of Pyruvate Kinase as a Glycolytic Enzyme or a Protein Kinase in Lamb.

Protein post-translational modifications (PTMs) play an essential role in meat quality development. However, the effect of specific PTM sites on meat proteins has not been investigated yet. The characteristics of pyruvate kinase M (PKM) were found to exhibit a close correlation with final meat quality, and thus, serine 99 (S99) and lysine 137 (K137) in PKM were mutated to study their effect on PKM function. The structural and functional properties of five lamb PKM variants, including wild-type PKM (wtPKM), PKM_S99D (S99 phosphorylation), PKM_S99A (PKM S99 dephosphorylation), PKM_K137Q (PKM K137 acetylation), and PKM_K137R (PKM K137 deacetylation), were evaluated. The results showed that the secondary structure, tertiary structure, and polymer formation were affected among different PKM variants. In addition, the glycolytic activity of PKM_K137Q was decreased because of its weakened binding with phosphoenolpyruvate. In the PKM_K137R variant, the actin phosphorylation level exhibited a decrease, suggesting a low kinase activity of PKM_K137R. The results of molecular simulation showed a 42% reduction in the interface area between PKM_K137R and actin, in contrast to wtPKM and actin. These findings are significant for revealing the mechanism of how PTMs regulate PKM function and provide a theoretical foundation for the development of precise meat quality preservation technology.

Systematic Characterization of Lysine Post-translational Modification Sites Using MUscADEL.

Among various types of protein post-translational modifications (PTMs), lysine PTMs play an important role in regulating a wide range of functions and biological processes. Due to the generation and accumulation of enormous amount of protein sequence data by ongoing whole-genome sequencing projects, systematic identification of different types of lysine PTM substrates and their specific PTM sites in the entire proteome is increasingly important and has therefore received much attention. Accordingly, a variety of computational methods for lysine PTM identification have been developed based on the combination of various handcrafted sequence features and machine-learning techniques. In this chapter, we first briefly review existing computational methods for lysine PTM identification and then introduce a recently developed deep learning-based method, termed MUscADEL (Multiple Scalable Accurate Deep Learner for lysine PTMs). Specifically, MUscADEL employs bidirectional long short-term memory (BiLSTM) recurrent neural networks and is capable of predicting eight major types of lysine PTMs in both the human and mouse proteomes. The web server of MUscADEL is publicly available at http://muscadel.erc.monash.edu/ for the research community to use.

PTMdyna: exploring the influence of post-translation modifications on protein conformational dynamics.

Protein post-translational modifications (PTM) play vital roles in cellular regulation, modulating functions by driving changes in protein structure and dynamics. Exploring comprehensively the influence of PTM on conformational dynamics can facilitate the understanding of the related biological function and molecular mechanism. Currently, a series of excellent computation tools have been designed to analyze the time-dependent structural properties of proteins. However, the protocol aimed to explore conformational dynamics of post-translational modified protein is still a blank. To fill this gap, we present PTMdyna to visually predict the conformational dynamics differences between unmodified and modified proteins, thus indicating the influence of specific PTM. PTMdyna exhibits an AUC of 0.884 tested on 220 protein-protein complex structures. The case of heterochromatin protein 1α complexed with lysine 9-methylated histone H3, which is critical for genomic stability and cell differentiation, was used to demonstrate its applicability. PTMdyna provides a reliable platform to predict the influence of PTM on protein dynamics, making it easier to interpret PTM functionality at the structure level. The web server is freely available at http://ccbportal.com/PTMdyna.

Methods to Study Posttranslational Modification Patterns in Cytotoxic T-Cells and Cancer.

Protein posttranslational modifications (PTMs) regulate intracellular signaling associated with development and progression of many diseases; thus, they are key to understanding pathological mechanisms and set up more tailored therapies. In addition, many posttranslationally modified proteins are released into biological fluids and can be used as new and more specific biomarkers. Based on this evidence, we analyzed the role of some PTMs in cancer and described the correlation between specific PTMs and T-cells activation/inhibition in cancer microenvironment. In the second part of this chapter, we analyzed the most commonly used approaches for qualitative and quantitative determination of PTMs. The comparison of three distinct but often complementary methodologies such as immunoblotting, mass spectrometry, and ELISA assays has allowed to highlight the pros and cons of each approach with a focus on their current application and their future developments to obtain more confident biomarkers and therapeutic targets useful for diagnosis, prognosis, and monitoring of the response to therapy.

Reticulocyte and red blood cell deformation triggers specific phosphorylation events.

The capacity to undergo substantial deformation is a defining characteristic of the red blood cell (RBC), facilitating transit through the splenic interendothelial slits and microvasculature. Establishment of this remarkable property occurs during a process of reticulocyte maturation that begins with egress through micron-wide pores in the bone marrow and is completed within the circulation. The requirement to undertake repeated cycles of deformation necessitates that both reticulocytes and erythrocytes regulate membrane-cytoskeletal protein interactions in order to maintain cellular stability. In the absence of transcriptional activity, modulation of these interactions in RBCs is likely to be achieved primarily through specific protein posttranslational modifications, which at present remain undefined. In this study, we use high-throughput methods to define the processes that underlie the response to deformation and shear stress in both reticulocytes and erythrocytes. Through combination of a bead-based microsphiltration assay with phosphoproteomics we describe posttranslational modification of RBC proteins associated with deformation. Using microsphiltration and microfluidic biochip-based assays, we explore the effect of inhibiting kinases identified using this dataset. We demonstrate roles for GSK3 and Lyn in capillary transit and maintenance of membrane stability following deformation and show that combined inhibition of these kinases significantly decreases reticulocyte capacity to undergo repeated deformation. Finally, we derive a comprehensive and integrative phosphoproteomic dataset that provides a valuable resource for further mechanistic dissection of the molecular pathways that underlie the RBC's response to mechanical stimuli and for the study of reticulocyte maturation.

Characterizing posttranslational modifications in prokaryotic metabolism using a multiscale workflow

Understanding the complex interactions of protein posttranslational modifications (PTMs) represents a major challenge in metabolic engineering, synthetic biology, and the biomedical sciences. Here, we present a workflow that integrates multiplex automated genome editing (MAGE), genome-scale metabolic modeling, and atomistic molecular dynamics to study the effects of PTMs on metabolic enzymes and microbial fitness. This workflow incorporates complementary approaches across scientific disciplines; provides molecular insight into how PTMs influence cellular fitness during nutrient shifts; and demonstrates how mechanistic details of PTMs can be explored at different biological scales. As a proof of concept, we present a global analysis of PTMs on enzymes in the metabolic network of Escherichia coli Based on our workflow results, we conduct a more detailed, mechanistic analysis of the PTMs in three proteins: enolase, serine hydroxymethyltransferase, and transaldolase. Application of this workflow identified the roles of specific PTMs in observed experimental phenomena and demonstrated how individual PTMs regulate enzymes, pathways, and, ultimately, cell phenotypes.

Shining a spotlight on intact proteins

Cells react to cues from their environment using various mechanisms that include changes in metabolites, gene expression, protein binding partners, protein localization, and protein posttranslational modifications (PTMs), all of which contribute to altered cellular signatures that enable appropriate cellular responses. Given the seemingly infinite number of mechanisms available to affect protein function and modulate biological processes, the question arises as to how cells manage to interpret protein readouts to accomplish the appropriate cell-type specific response to a particular stimulus.

Lamins in development, tissue maintenance and stress

Lamins are nuclear intermediate filament proteins. They provide mechanical stability, organize chromatin and regulate transcription, replication, nuclear assembly and nuclear positioning. Recent studies provide new insights into the role of lamins in development, differentiation and tissue response to mechanical, reactive oxygen species and thermal stresses. These studies also propose the existence of separate filament networks for A- and B-type lamins and identify new roles for the different networks. Furthermore, they show changes in lamin composition in different cell types, propose explanations for the more than 14 distinct human diseases caused by lamin A and lamin C mutations and propose a role for lamin B1 in these diseases.

Specific changes in total and mitochondrial proteomes are associated with higher levels of heterosis in maize hybrids

The phenomenon of hybrid vigor (heterosis) has long been harnessed by plant breeders to improve world food production. However, the changes that are essential for heterotic responses and the mechanisms responsible for heterosis remain undefined. Large increases in biomass and yield in high-heterosis hybrids suggest that alterations in bioenergetic processes may contribute to heterosis. Progeny from crosses between various inbred lines vary in the extent of vigor observed. Field-grown maize F₁ hybrids that consistently exhibited either low or high heterosis across a variety of environments were examined for changes in proteins that may be correlated with increased plant vigor and yield. Unpollinated ears at the time of flowering (ear shoots) were selected for the studies because they are metabolically active, rich in mitochondria, and the sizes of the ears are diagnostic of yield heterosis. Total protein and mitochondrial proteomes were compared among low- and higher-heterosis hybrids. Two-dimensional difference gel electrophoresis was used to identify allelic and/or isoform differences linked to heterosis. Identification of differentially regulated spots by mass spectrometry revealed proteins involved in stress responses as well as primary carbon and protein metabolism. Many of these proteins were identified in multiple spots, but analysis of their abundances by label-free mass spectrometry suggested that most of the expression differences were due to isoform variation rather than overall protein amount. Thus, our proteomics studies suggest that expression of specific alleles and/or post-translational modification of specific proteins correlate with higher levels of heterosis.

Enhanced Methylarginine Characterization by Post-Translational Modification-Specific Targeted Data Acquisition and Electron-Transfer Dissociation Mass Spectrometry

When localizing protein post-translational modifications (PTMs) using liquid-chromatography (LC)-tandem mass spectrometry (MS/MS), existing implementations are limited by inefficient selection of PTM-carrying peptides for MS/MS, particularly when PTM site occupancy is sub-stoichiometric. The present contribution describes a method by which peptides carrying specific PTMs of interest-in this study, methylarginines-may be selectively targeted for MS/MS: peptide features are extracted from high mass accuracy single-stage MS data, searched against theoretical PTM-carrying peptide masses, and matching features are subjected to targeted data acquisition LC-MS/MS. Using trypsin digested Saccharomyces cerevisiae Npl3, in which evidence is presented for 18 methylarginine sites-17 of which fall within a glycine-arginine-rich (GAR) domain spanning <120 amino acids-it is shown that this approach outperforms conventional data dependent acquisition (DDA): when applied to a complex protein mixture featuring in vivo methylated Npl3, 95% more (P=0.030) methylarginine-carrying peptides are selected for MS/MS than DDA, leading to an 86% increase (P=0.044) in the number of methylated peptides producing Mascot ion scores ≥20 following electron-transfer dissociation (ETD). Notably, significantly more low abundance arginine methylated peptides (maximum ion intensities <6×10(4) cps) are selected for MS/MS using this approach relative to DDA (50% more in a digest of purified in vitro methylated Npl3). It is also demonstrated that relative to collision-induced dissociation (CID), ETD facilitates a 586% increase (P=0.016) in average Mascot ion scores of methylarginine-carrying peptides. The present PTM-specific targeted data acquisition approach, though described using methylarginine, is applicable to any ionizable PTM of known mass.

Statins for treatment of CNS diseases. Status report from research and clinical practice

3-Hydroxy-3-methyglutaryl coenzyme A (HMG-CoA) reductase inhibitors, "statins," are widely used oral cholesterol-lowering drugs. Statins competitively inhibit HMG-CoA reductase, the enzyme that catalyzes conversion of HMG-CoA to L-mevalonate, a key intermediate in cholesterol synthesis. Certain metabolites of L-mevalonate are also involved in posttranslational modifications of specific proteins with cell proliferation and differentiation properties. Thus, statins have important biologic effects beyond their cholesterol-reducing properties. Here we discuss recent experimental and clinical data that may support a potential role for statins in the treatment of three central nervous system (CNS) neurological diseases: Multiple sclerosis (MS), Alzheimer's disease (AD), and ischemic stroke. Despite their considerable pathogenic differences, in animal models of these disorders statins have shown beneficial effects. In both stroke and AD cohort studies suggest a beneficial treatment effect in humans; in MS, results from small open-label studies look encouraging. Multicenter, randomized, placebo-controlled clinical trials are in the planning or recruiting stage to evaluate the therapeutic effects of statins in all three disorders.

Statins as potential therapeutic agents in multiple sclerosis.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (ie, statins) are oral cholesterol-lowering drugs. Statins are well tolerated and have an excellent safety record. These agents competitively inhibit HMG CoA reductase, which is the enzyme that catalyzes the conversion of HMG CoA to L-mevalonate. Although L-mevalonate is a key intermediate in cholesterol synthesis, several of its metabolites are involved in post-translational modification of specific proteins involved in cell proliferation and differentiation. Thus, independent of their cholesterol-reducing properties, statins have important pleiotropic biologic effects. Recent reports indicate that statins have anti-inflammatory and neuroprotective properties. Whether statins will be of clinical benefit for patients with multiple sclerosis and other neurodegenerative diseases of the central nervous system will only be known after they are evaluated in prospective randomized clinical trials.

The potential therapeutic role of statins in central nervous system autoimmune disorders.

3-hydroxy-3-methyglutaryl coenzyme A (HMG-CoA) reductase inhibitors, 'statins' are widely used oral cholesterol-lowering drugs. Statins competitively inhibit HMG-CoA reductase, the enzyme that catalyzes conversion of HMG-CoA to L-mevalonate, a key intermediate in cholesterol synthesis. Certain metabolites of mevalonate are also involved in posttranslational modification of specific proteins involved in cell proliferation and differentiation. Thus, statins have important biologic effects that may be independent of their cholesterol-reducing properties. Recent studies indicate that statins have antiinflammatory and neuroprotective properties which may be beneficial in the treatment of multiple sclerosis as well as other central nervous system (CNS) neurodegenerative diseases. This article will outline current experimental evidence that may suggest potential clinical benefits for patients with CNS autoimmune disorders. Ultimately, clinical trials will have to determine the safety and efficacy of statins in this patient population.

Novel Role of Vitamin K in Preventing Oxidative Injury to Developing Oligodendrocytes and Neurons

Oxidative stress is believed to be the cause of cell death in multiple disorders of the brain, including perinatal hypoxia/ischemia. Glutamate, cystine deprivation, homocysteic acid, and the glutathione synthesis inhibitor buthionine sulfoximine all cause oxidative injury to immature neurons and oligodendrocytes by depleting intracellular glutathione. Although vitamin K is not a classical antioxidant, we report here the novel finding that vitamin K1 and K2 (menaquinone-4) potently inhibit glutathione depletion-mediated oxidative cell death in primary cultures of oligodendrocyte precursors and immature fetal cortical neurons with EC50 values of 30 nm and 2 nm, respectively. The mechanism by which vitamin K blocks oxidative injury is independent of its only known biological function as a cofactor for gamma-glutamylcarboxylase, an enzyme responsible for posttranslational modification of specific proteins. Neither oligodendrocytes nor neurons possess significant vitamin K-dependent carboxylase or epoxidase activity. Furthermore, the vitamin K antagonists warfarin and dicoumarol and the direct carboxylase inhibitor 2-chloro-vitamin K1 have no effect on the protective function of vitamin K against oxidative injury. Vitamin K does not prevent the depletion of intracellular glutathione caused by cystine deprivation but completely blocks free radical accumulation and cell death. The protective and potent efficacy of this naturally occurring vitamin, with no established clinical side effects, suggests a potential therapeutic application in preventing oxidative damage to undifferentiated oligodendrocytes in perinatal hypoxic/ischemic brain injury.

Monitoring post-translational modification of proteins with allosteric ribozymes

An allosteric hammerhead ribozyme activated specifically by the unphosphorylated form of the protein kinase ERK2 was created through a rational design strategy that relies on molecular recognition of ERK2 to decrease the formation of an alternate, inactive ribozyme conformer. Neither closely related mitogen-activated protein kinases (MAPKs) nor the phosphorylated form of ERK2 induced ribozyme activity. The ribozyme quantitatively detected ERK2 added to mammalian cell lysates and also functioned quantitatively in a multiplexed solution-phase assay. This same strategy was used to construct a second ribozyme selectively activated by the phosphorylated (active) form of ERK2. This approach is generally applicable to the development of ribozymes capable of monitoring post-translational modification of specific proteins.

Identification of α1-Antitrypsin Variants in Plasma with the Use of Proteomic Technology

Proteomic technology permits the investigation of genetic metabolic diseases at the level of protein expression. Changes in the expression, polypeptide structure, and posttranslational modification of individual proteins can be detected in complex mixtures of proteins. We used high-resolution two-dimensional polyacrylamide gel electrophoresis to separate isoforms of plasma proteins and detect abnormalities of mass and/or charge. We confirmed the identity of the separated proteins by in-gel digestion with proteases and N-glycanases and then analyzed the released peptides and glycans by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry. Complete characterization of the polypeptide sequences and glycosylation of alpha(1)-antitrypsin isoforms was achieved in plasma from controls and from patients with three different known alpha(1)-antitrypsin deficiencies and congenital disorder of glycosylation type Ia. This study shows that proteomic techniques are a powerful and sensitive means of detecting changes in the amino acid sequence and abnormal posttranslational modifications of specific proteins in a complex biologic matrix.

Profiling Changes in Gene Expression during Differentiation and Maturation of Monocyte-derived Dendritic Cells Using Both Oligonucleotide Microarrays and Proteomics

Dendritic cells (DCs) are antigen-presenting cells that play a major role in initiating primary immune responses. We have utilized two independent approaches, DNA microarrays and proteomics, to analyze the expression profile of human CD14(+) blood monocytes and their derived DCs. Analysis of gene expression changes at the RNA level using oligonucleotide microarrays complementary to 6300 human genes showed that approximately 40% of the genes were expressed in DCs. A total of 255 genes (4%) were found to be regulated during DC differentiation or maturation. Most of these genes were not previously associated with DCs and included genes encoding secreted proteins as well as genes involved in cell adhesion, signaling, and lipid metabolism. Protein analysis of the same cell populations was done using two-dimensional gel electrophoresis. A total of 900 distinct protein spots were included, and 4% of them exhibited quantitative changes during DC differentiation and maturation. Differentially expressed proteins were identified by mass spectrometry and found to represent proteins with Ca(2+) binding, fatty acid binding, or chaperone activities as well as proteins involved in cell motility. In addition, proteomic analysis provided an assessment of post-translational modifications. The chaperone protein, calreticulin, was found to undergo cleavage, yielding a novel form. The combined oligonucleotide microarray and proteomic approaches have uncovered novel genes associated with DC differentiation and maturation and has allowed analysis of post-translational modifications of specific proteins as part of these processes.

Electrophoresis combined with novel mass spectrometry techniques: powerful tools for the analysis of proteins and proteomes.

Analytical and preparative electrophoresis separation techniques have been essential tools in protein biochemistry and the biological sciences in general. The combination of high resolution electrophoresis techniques with high performance analytical procedures has dramatically enhanced analytical protein biochemistry. In this report we describe the combination of electrophoretic separation techniques with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). A series of different techniques, consisting of automated high performance liquid chromatography (HPLC)-MS/MS, capillary-HPLC-MS/MS, and solid phase extraction (SPE)-capillary zone electrophoresis (CZE)-MS/MS, are described in the context of the identification of high pmol to the low fmol amounts of proteins. Application of these powerful new tools for the analysis of proteins on a large proteome-wide scale is presented. Furthermore, the combination of orthogonal separation techniques, such as immobilized metal affinity chromatography (IMAC) with SPE-CZE-MS/MS, and IMAC followed by HPLC, and by SPE-CZE-MS/MS, are presented for the detailed investigation of post-translational modifications of specific proteins.

ADP-ribosylation and gene expression.

Gene expression can be defined as the conversion of information existing in a molecule of DNA into a mature RNA or protein product and each step in the process, which requires the concerted action of several macromolecules for completion, may be perturbed by the post-translational modification of specific proteins with ADP-ribose. The participation of poly(ADP-ribose) in the regulation of transcription initiation was examined using cell-free systems for both ribosomal RNA and ribosomal proteins. The presence or absence of poly(ADP-ribose) polymerase did not influence the transcription process. Similarly, under conditions optimal for poly(ADP-ribose) polymerase activity, no change in transcription was observed. A direct contribution of poly(ADP-ribosyl)ation to gene transcription thus could not be detected. In contrast, the addition of 3-aminobenzamide to quiescent hepatoma cells treated with insulin inhibited the stimulation of rRNA synthesis. The high concentrations necessary for this effect suggest that a mono(ADP-ribosyl)ation event participates in the cellular action of insulin. A role in the signal transduction pathway leading to activation of rRNA gene expression has been proposed.

Adaptation of cyanobacteria to environmental stimuli: new steps towards molecular mechanisms

Dating from the Pre-Cambrian era, cyanobacteria have a long history of adaptation to the Earth's environment. By evolving oxygen via photosynthetic reactions similar to those of plants and green algae, these prokaryotes were essential to the evolution of the present biosphere. They continue to make a large contribution to the equilibrium of the Earth's atmosphere by production oxygen and removing carbon dioxide. To survive in extreme or variable environments, cyanobacteria have developed specific regulatory systems, in addition to more general mechanisms equivalent to those of other prokaryotes or photosynthesis eukaryotes. Specific regulatory systems control the differentiation of specialized nitrogen-fixing cells and of cell types facilitating the dispersion of species. In the past decade, considerable progress has been made towards understanding the expression of the cyanobacterial genome in response to variations in the intensity and spectral quality of incident light and in response to nutritional conditions, especially carbon, nitrogen and sulphur sources. These studies have provided insights into the relationships between carbon and nitrogen intermediary metabolism, and a start towards understanding of the interconnected pathways which lead from the perception of environmental signals to the regulation of enzyme activities and gene expression. Cyanobacterial regulatory mechanisms share common features with those of other prokaryotes, but are unique since these essentially photo-autotrophic organisms must maintain a proper cellular C/N balance, in spite of dailty variations in incident light. Thus an appropriate coordination between photosynthesis and other metabolic processes must be achieved through control of the catalytic activity of key enzymes by reducing equivalents and ATP produced by photosynthetic or respiratory electron transport. Recently discovered kinases/phosphatases act by post-translational modification of specific proteins which probably act as signal transducers or modulators of gene expression in a manner similar to the well-known two-component regulatory systems described in other bacteria. In this overview, we present our current knowledge on the molecular aspects of the biology of cyanobacteria, as well as on their mechanisms of resistance to metal ions and their responses to metabolic stress.