Investigation and identification of functional post-translational modification sites associated with drug binding and protein-protein interactions

Background

Protein post-translational modification (PTM) plays an essential role in various cellular processes that modulates the physical and chemical properties, folding, conformation, stability and activity of proteins, thereby modifying the functions of proteins. The improved throughput of mass spectrometry (MS) or MS/MS technology has not only brought about a surge in proteome-scale studies, but also contributed to a fruitful list of identified PTMs. However, with the increase in the number of identified PTMs, perhaps the more crucial question is what kind of biological mechanisms these PTMs are involved in. This is particularly important in light of the fact that most protein-based pharmaceuticals deliver their therapeutic effects through some form of PTM. Yet, our understanding is still limited with respect to the local effects and frequency of PTM sites near pharmaceutical binding sites and the interfaces of protein-protein interaction (PPI). Understanding PTM’s function is critical to our ability to manipulate the biological mechanisms of protein.

Results

In this study, to understand the regulation of protein functions by PTMs, we mapped 25,835 PTM sites to proteins with available three-dimensional (3D) structural information in the Protein Data Bank (PDB), including 1785 modified PTM sites on the 3D structure. Based on the acquired structural PTM sites, we proposed to use five properties for the structural characterization of PTM substrate sites: the spatial composition of amino acids, residues and side-chain orientations surrounding the PTM substrate sites, as well as the secondary structure, division of acidity and alkaline residues, and solvent-accessible surface area. We further mapped the structural PTM sites to the structures of drug binding and PPI sites, identifying a total of 1917 PTM sites that may affect PPI and 3951 PTM sites associated with drug-target binding. An integrated analytical platform (CruxPTM), with a variety of methods and online molecular docking tools for exploring the structural characteristics of PTMs, is presented. In addition, all tertiary structures of PTM sites on proteins can be visualized using the JSmol program.

Conclusion

Resolving the function of PTM sites is important for understanding the role that proteins play in biological mechanisms. Our work attempted to delineate the structural correlation between PTM sites and PPI or drug-target binding. CurxPTM could help scientists narrow the scope of their PTM research and enhance the efficiency of PTM identification in the face of big proteome data. CruxPTM is now available at http://csb.cse.yzu.edu.tw/CruxPTM/.

     

Computational approaches to predict protein functional families and functional sites

Understanding the mechanisms of protein function is indispensable for many biological applications, such as protein engineering and drug design. However, experimental annotations are sparse, and therefore, theoretical strategies are needed to fill the gap. Here, we present the latest developments in building functional subclassifications of protein superfamilies and using evolutionary conservation to detect functional determinants, for example, catalytic-, binding- and specificity-determining residues important for delineating the functional families. We also briefly review other features exploited for functional site detection and new machine learning strategies for combining multiple features.     

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.     

Redox proteomics: identification and functional role of glutathionylated proteins

Although radical oxygen and nitrogen species are harmful molecules that destroy cell functions, many operate as mediators of important cell signaling pathways when not in excess. Oxidants can modify protein function through the covalent, reversible addition of glutathione to cysteine. This review addresses different proteomic methods of identifying glutathionylation targets and emphasizes ways of defining their pattern of modification in response to oxidative stimuli in cells. Finally, the literature on nonproteomic studies that investigate the functional changes induced by glutathionylation are reviewed and future studies are commented on.     

Redox proteomics: identification and functional role of glutathionylated proteins

Although radical oxygen and nitrogen species are harmful molecules that destroy cell functions, many operate as mediators of important cell signaling pathways when not in excess. Oxidants can modify protein function through the covalent, reversible addition of glutathione to cysteine. This review addresses different proteomic methods of identifying glutathionylation targets and emphasizes ways of defining their pattern of modification in response to oxidative stimuli in cells. Finally, the literature on nonproteomic studies that investigate the functional changes induced by glutathionylation are reviewed and future studies are commented on.     

Poly ADP-ribosylation of proteins. Processivity of a post-translational modification

The nuclear enzyme poly(ADP-ribose) polymerase (EC 2.4.2.30) participates in DNA excision repair by post-translational selfmodification ("automodification") and the modification of other chromatin proteins ("heteromodification") with ADP-ribose polymers. We have studied the molecular mechanism of these reactions in a reconstituted in vitro system. After activation by DNA, poly(ADP-ribose) polymerase produces polymers with a distinct size pattern. These polymers are attached to a small subfraction of enzyme molecules. As the reaction progresses, more enzyme molecules are recruited for modification with an identical polymer size pattern. Likewise, the auto- and heteromodification reaction in nucleosomal core particles involves the consecutive addition of a highly conserved polymer size pattern to the acceptor proteins. Thus, a highly conserved polymer size pattern may constitute the molecular signal priming chromatin proteins for a role in DNA excision repair in vivo. The priming reaction is processive.     

Rapid identification of fluorochrome modification sites in proteins by LC ESI-Q-TOF mass spectrometry

Conjugation of either a fluorescent dye or a drug molecule to the ε-amino groups of lysine residues of proteins has many applications in biology and medicine. However, this type of conjugation produces a heterogeneous population of protein conjugates. Because conjugation of fluorochrome or drug molecule to a protein may have deleterious effects on protein function, the identification of conjugation sites is necessary. Unfortunately, the identification process can be time-consuming and laborious; therefore, there is a need to develop a rapid and reliable way to determine the conjugation sites of the fluorescent label or drug molecule. In this study, the sites of conjugation of fluorescein-5'-isothiocyanate and rhodamine-B-isothiocyanate to free amino groups on the insert-domain (I-domain) protein derived from the α-subunit of lymphocyte function-associated antigen-1 (LFA-1) were determined by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS) along with peptide mapping using trypsin digestion. A reporter fragment of the fluorochrome moiety that is generated in the collision cell of the Q-TOF without explicit MS/MS precursor selection was used to identify the conjugation site. Selected ion plots of the reporter ion readily mark modified peptides in chromatograms of the complex digest. Interrogation of theses spectra reveals a neutral loss/precursor pair that identifies the modified peptide. The results show that one to seven fluorescein molecules or one to four rhodamine molecules were attached to the lysine residue(s) of the I-domain protein. No modifications were found in the metal ion-dependent adhesion site (MIDAS), which is an important binding region of the I-domain.     

Oxidative post-translational modification of tryptophan residues in cardiac mitochondrial proteins

We examined the distribution of N-formylkynurenine, a product of the dioxidation of tryptophan residues in proteins, throughout the human heart mitochondrial proteome. This oxidized amino acid is associated with a distinct subset of proteins, including an over-representation of complex I subunits as well as complex V subunits and enzymes involved in redox metabolism. No relationship was observed between the tryptophan modification and methionine oxidation, a known artifact of sample handling. As the mitochondria were isolated from normal human heart tissue and not subject to any artificially induced oxidative stress, we suggest that the susceptible tryptophan residues in this group of proteins are "hot spots" for oxidation in close proximity to a source of reactive oxygen species in respiring mitochondria.     

Structural insights of post-translational modification sites in the proteome of Thermus thermophilus

Phosphorylation and acetylation are the most prevalent post-translational modifications (PTMs) detected in not only eukaryotes but also bacteria. We performed phosphoproteome and acetylome analyses of proteins from an extremely thermophilic eubacterium Thermus thermophilus HB8, and identified numerous phosphorylation and acetylation sites. To facilitate the elucidation of the structural aspects of these PTM events, we mapped the PTM sites on the known tertiary structures for the respective proteins and their homologs. Wu et al. (Mol Cell Proteomics 12:2701–2713, 2013) recently reported phosphoproteome analysis of proteins from T. thermophilus HB27. Therefore, we assessed the structural characteristics of these phosphorylation and acetylation sites on the tertiary structures of the identified proteins or their homologs. Our study revealed that many of the identified phosphosites are in close proximity to bound ligands, i.e., the numbers of ‘nearby’ and ‘peripheral’ phosphorylation sites represent 56 % (48/86 sites) of total identified phosphorylation sites. In addition, approximately 60 % of all phosphosites exhibited <10 % accessible surface area of their side chains, suggesting some structural rearrangement is required for phosphoryl transfer by kinases. Our findings also indicate that phosphorylation of a residue occurs more frequently at a flexible region of the protein, whereas lysine acetylation occurs more frequently in an ordered structure.     

A general user interface for prediction servers of proteins' post-translational modification sites

Post-translational modifications (PTMs) of proteins play essential roles in governing the functions and dynamics of proteins and are implicated in many cellular processes. Several types of PTMs have been investigated through computational approaches, including phosphorylation, sumoylation, palmitoylation, and lysine and arginine methylation, among others. Because the large diversity in the user interfaces (UIs) of different prediction servers for PTMs could possibly hinder experimental biologists in using these servers, we propose to develop a protocol for a unified UI for PTM prediction servers, based on our own work and that of other groups on PTM site prediction. By following this protocol, tool developers can provide a uniform UI regardless of the PTM types and the underlying computational algorithms. With such uniformity in the UI, experimental biologists would be able to use any PTM prediction server compliant with this protocol once they had learned to use one of them. It takes a typical PTM prediction server compliant with this unified UI several minutes to calculate the prediction results for a protein 1,000 amino acids in length.     

Reversible post-translational modification of proteins by nitrated fatty acids in vivo

Nitric oxide ((*)NO)-derived reactive species nitrate unsaturated fatty acids, yielding nitroalkene derivatives, including the clinically abundant nitrated oleic and linoleic acids. The olefinic nitro group renders these derivatives electrophilic at the carbon beta to the nitro group, thus competent for Michael addition reactions with cysteine and histidine. By using chromatographic and mass spectrometric approaches, we characterized this reactivity by using in vitro reaction systems, and we demonstrated that nitroalkene-protein and GSH adducts are present in vivo under basal conditions in healthy human red cells. Nitro-linoleic acid (9-, 10-, 12-, and 13-nitro-9,12-octadecadienoic acids) (m/z 324.2) and nitro-oleic acid (9- and 10-nitro-9-octadecaenoic acids) (m/z 326.2) reacted with GSH (m/z 306.1), yielding adducts with m/z of 631.3 and 633.3, respectively. At physiological concentrations, nitroalkenes inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which contains a critical catalytic Cys (Cys-149). GAPDH inhibition displayed an IC(50) of approximately 3 microM for both nitroalkenes, an IC(50) equivalent to the potent thiol oxidant peroxynitrite (ONOO(-)) and an IC(50) 30-fold less than H(2)O(2), indicating that nitroalkenes are potent thiol-reactive species. Liquid chromatography-mass spectrometry analysis revealed covalent adducts between fatty acid nitroalkene derivatives and GAPDH, including at the catalytic Cys-149. Liquid chromatography-mass spectrometry-based proteomic analysis of human red cells confirmed that nitroalkenes readily undergo covalent, thiol-reversible post-translational modification of nucleophilic amino acids in GSH and GAPDH in vivo. The adduction of GAPDH and GSH by nitroalkenes significantly increased the hydrophobicity of these molecules, both inducing translocation to membranes and suggesting why these abundant derivatives had not been detected previously via traditional high pressure liquid chromatography analysis. The occurrence of these electrophilic nitroalkylation reactions in vivo indicates that this reversible post-translational protein modification represents a new pathway for redox regulation of enzyme function, cell signaling, and protein trafficking.     

The role of posttranslational modification in moonlighting glyceraldehyde-3-phosphate dehydrogenase structure and function

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a moonlighting protein exhibiting distinct activities apart from its classical role in glycolysis. Regulation of its moonlighting functions and its subcellular localization may be dependent on its posttranslational modification (PTM). The latter include its phosphorylation, which is required for its role in intermembrane trafficking, synaptic transmission and cancer survival; nitrosylation, which is required for its function in apoptosis, heme metabolism and the immune response; acetylation which is necessary for its modulation of apoptotic gene regulation; and N-acetylglucosamine modification which may induce changes in GAPDH oligomeric structure. These findings suggest a structure function relationship between GAPDH posttranslational modification and its diverse moonlighting activities.

     

Granzyme M: characterization with sites of post-translational modification and specific sites of interaction with substrates and inhibitors

Granzymes kill cells in a variety of ways. They induce mitochondrial dysfunction through caspase dependent and caspase-independent pathways and destroy DNA and the integrity of the nucleus. For gaining a better understanding of the molecular function of granzyme M and its NK cell specificity, structural characterization of this enzyme by molecular modeling as well as its detailed comparison with other granzymes is presented in this study. The study includes mode of action of granzyme M using cationic binding sites, substrate specificity, post-translational structural modification and its functional relationship and interaction of the enzyme with inhibitor in an attempt to explore how the activity of human granzyme M is controlled under physiological conditions. It is concluded from the present study that the post-translational modification, including Oglycosylation of serine, phosphorylation of serine and threonine and myristoylation of glycine, play an important role in the interaction of enzyme with the cell surface membrane and regulate protein trafficking and stability. Phosphorylated serine and threonine also plays a role in tumor elimination, viral clearance and tissue repair. In Gzm M there are cationic sites, cs1 and cs2 that may participate in binding of Gzm M to the cell surface, thereby promoting its uptake and eventual release into the cytoplasm. Gzm M shows apoptotic activity both by caspase dependent and independent pathways. Modeling of inhibitors bound to the granzyme active site shows that the dimer also contributes to substrate specificity in a unique manner by extending the active-site cleft.     

Computational identification of putative programmed translational frameshift sites

MOTIVATION: In an effort to identify potential programmed frameshift sites by statistical analysis, we explore the hypothesis that selective pressure would have rendered such sites underabundant and underrepresented in protein-coding sequences. We developed a computer program to compare the frequencies of k-length subsequences of nucleotides with the frequencies predicted by a zero order Markov chain determined by the codon bias of the same set of sequences. The program was used to calculate and evaluate the distribution of 7-base oligonucleotides in the 6000+ putative protein-coding sequences of S. cerevisiae preliminary to the laboratory testing of the most highly underrepresented oligos for frameshifting efficiency. RESULTS: Among the most significant results is the finding that the heptanucleotides CUU-AGG-C and CUU-AGU-U, sites of the programmed +1 translational frameshifts required for the production in yeast of actin filament-binding protein ABP140 and telomerase subunit EST3, respectively, rank among the least represented of phase I heptanucleotides in the coding sequences of S. cerevisiae. Laboratory experiments demonstrated that other underrepresented heptanucleotides identified by the program, for example GGU-CAG-A, are also prone to significant translational frameshifting, suggesting the possibility that genes containing other underrepresented heptamers may also encode transframe products. AVAILABILITY: The program is available for download from http://www.gesteland.genetics.utah.edu/freqAnalysis SUPPLEMENTARY INFORMATION: Complete results from the analysis of S. cerevisiae are available on http://www.gesteland.genetics.utah.edu/freqAnalysis     

Computational identification of proteins for selectivity assays

At the stage of optimization of a chemical series the compounds are normally assayed for binding or inhibition on the target protein as well as on several proteins from a selectivity panel. These proteins are normally identified on the basis of sequence homology to the target protein. Experimental selectivity data are also taken into account if available. Cases when a nonhomologous protein has a significant affinity to the compound series are going to be missed if the selectivity panel is identified by homology. Experimental data is usually either unavailable or limited to a small fraction of proteins that should be considered. We have developed a computational method of identification of selectivity panel proteins. It is based on the evaluation of binding site similarity to the target protein using docking scores of target-selected molecular probes. These probes are obtained by docking a large library of drug-like compounds to the target protein followed by selecting a diverse subset from the best virtual binders. Docking scores of these probes to other proteins measure binding site similarity to the target. Because the method does not require prior knowledge of either affinities or structures of inhibitors for the target, it can be applied to any protein with known 3D structure. Validation of the method includes rediscovery of nonhomologous proteins that bind common ligands: estradiol, tamoxifen, and riboflavin. Given 3D structures, the method can effectively discriminate proteins with similar binding sites from random proteins independent of sequence homology.     

Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications

Identifying sites of post-translational modifications on proteins is a major challenge in proteomics. O-Linked beta-N-acetylglucosamine (O-GlcNAc) is a dynamic nucleocytoplasmic modification more analogous to phosphorylation than to classical complex O-glycosylation. We describe a mass spectrometry-based method for the identification of sites modified by O-GlcNAc that relies on mild beta-elimination followed by Michael addition with dithiothreitol (BEMAD). Using synthetic peptides, we also show that biotin pentylamine can replace dithiothreitol as the nucleophile. The modified peptides can be efficiently enriched by affinity chromatography, and the sites can be mapped using tandem mass spectrometry. This same methodology can be applied to mapping sites of serine and threonine phosphorylation, and we provide a strategy that uses modification-specific antibodies and enzymes to discriminate between the two post-translational modifications. The BEMAD methodology was validated by mapping three previously identified O-GlcNAc sites, as well as three novel sites, on Synapsin I purified from rat brain. BEMAD was then used on a purified nuclear pore complex preparation to map novel sites of O-GlcNAc modification on the Lamin B receptor and the nucleoporin Nup155. This method is amenable for performing quantitative mass spectrometry and can also be adapted to quantify cysteine residues. In addition, our studies emphasize the importance of distinguishing between O-phosphate versus O-GlcNAc when mapping sites of serine and threonine post-translational modification using beta-elimination/Michael addition methods.     

The procyclic acidic repetitive proteins of Trypanosoma brucei. Purification and post-translational modification

The procyclic acidic repetitive protein (PARP) of Trypanosoma brucei was purified by cell fractionation followed by ion-exchange and concanavalin A-Sepharose affinity chromatography. PARP is membrane-bound and comprises about 1% of the total procyclic trypanosome protein or 6 x 10(6) molecules per parasite. The results of NH2-terminal sequencing and amino acid analysis indicate that PARP is processed by removal of an N-terminal signal sequence and the hydrophobic COOH terminus. Metabolic labeling of PARP with [3H] ethanolamine is consistent with attachment of the protein to the membrane via a glycosylphosphatidylinositol anchor. The glycolipid can be removed by base hydrolysis or nitrous acid deamination but is not susceptible to bacterial phosphatidylinositol-specific phospholipase C.