PAD2-mediated citrullination of MBP and early myelin destabilization in multiple sclerosis.

Myelin basic protein (MBP) represents a candidate autoantigen in multiple sclerosis (MS). We isolated MBP from normal and MS human white matter and purified six components (charge isomers) to compare the post-translational modifications on each. The sites and extent of methylation, deimination, and phosphorylation were documented for all tryptic peptides by mass spectrometry. We found that mono and dimethylated arginine 107 was increased in MS samples; deimination of arginine occurred at a number of sites and was elevated in MS; phosphorylation was observed in 10 peptides in normal samples but was greatly reduced or absent in most peptides from MS samples. Data obtained with MBP isolated from fresh brain obtained from a spontaneously demyelinating mouse model supported the view that the changes observed in human brain were probably related to pathogenesis of demyelination, i.e. we found decreased phosphorylation and decreased amounts of glycogen synthesis kinase in brain homogenates using specific antibodies. This study represents the first to define post-translational modifications in demyelinating disease and suggest an important role in pathogenesis.

Post-translational protein modifications can be recognized by B and T lymphocytes and can potentially make "self"-proteins appear foreign to the immune system. Such modifications may directly affect major histocompatibility complex-restricted T cell recognition of processed peptides or may perturb the processing events that generate such peptides. Using the tetanus toxin C fragment protein as a test case, we show that spontaneous deamidation of asparagine residues interferes with processing by the enzyme asparagine endopeptidase (AEP) and contributes to diminished antigen presentation. Deamidation inhibits AEP action either directly, when asparagine residues targeted by AEP are modified, or indirectly, when adjacent Asn residues are deamidated. Thus, deamidation of long-lived self-proteins may qualitatively or quantitatively affect the spectrum of self-peptides displayed to T cells and may thereby contribute to the onset or exacerbation of autoimmune disease.

Myelin basic protein (MBP) is an important component of the myelin sheath surrounding neurons, and it is directly affected in demyelinating diseases. MBP contains a relatively large number of post-translational modifications (PTMs), which have been reported to play a role in multiple sclerosis, while MBPs from lower vertebrates have been reported to be incapable of inducing multiple sclerosis or allergic encephalitis. This study reveals the extent of differences in PTM patterns for mammalian and nonmammalian MBPs. This included intact mass and de novo sequence analysis of approximately 85% of rattlesnake MBP, the first reptile MBP to be characterized, and of bovine MBP. We identified 12 PTMs at 11 sites in the five bovine MBP charge components, which include both previously reported and novel modifications. The most notable modification is an acetylation of lysine 121. Other modifications found in bovine MBP include N-terminal acetylation in components C1, C2, and C3; oxidation of methionine 19 in all five components; all charge isomers having both a mono- and dimethylated (symmetric) arginine at position 106; deimination in arginines 23 and 47 found only in component C8b; deimination of arginine 96 and deamidation in glutamine 102 found in components C2, C3, C8a, and C8b; phosphorylation in threonine 97 restricted to charge components C2 and C3; deimination in arginine 161 only found in component C3; deamidation of glutamine 120 was only observed in C3. All four deiminated arginines and one acetylated lysine were first experimentally revealed in this study for bovine MBP. Mascot database searching combined with de novo sequence analysis of rattlesnake MBP provided more than 85% sequence coverage. A few PTMs were also revealed in rattlesnake MBP: mono- and dimethylated Arg, protein N-terminal acetylation, and deiminated Arg. Overall, snake MBP was found to undergo less modification than bovine MBP on the basis of the mass heterogeneity of the intact protein, the bottom-up structure analysis, and the limited complexity of rattlesnake MBP chromatography. The combined data from this study and information from previous studies extend the known MBP PTMs, and PTMs unique to higher vertebrates are proposed.

With the rapid expansion of protein post-translational modification (PTM) research based on large-scale proteomic work, there is an increasing demand for a suitable repository to analyze PTM data. Here we present a curated, web-accessible PTM data base, SysPTM. SysPTM provides a systematic and sophisticated platform for proteomic PTM research equipped not only with a knowledge base of manually curated multi-type modification data but also with four fully developed, in-depth data mining tools. Currently, SysPTM contains data detailing 117,349 experimentally determined PTM sites on 33,421 proteins involving nearly 50 PTM types, curated from public resources including five data bases and four web servers and more than one hundred peer-reviewed mass spectrometry papers. Protein annotations including Pfam domains, KEGG pathways, GO functional classification, and ortholog groups are integrated into the data base. Four online tools have been developed and incorporated, including PTMBlast, to compare a user's PTM dataset with PTM data in SysPTM; PTMPathway, to map PTM proteins to KEGG pathways; PTMPhylog, to discover potentially conserved PTM sites; and PTMCluster, to find clusters of multi-site modifications. The workflow of SysPTM was demonstrated by analyzing an in-house phosphorylation dataset identified by MS/MS. It is shown that in SysPTM, the role of single-type and multi-type modifications can be systematically investigated in a full biological context. SysPTM could be an important contribution to modificomics research. SysPTM is freely available online at www.sysbio.ac.cn/SysPTM.

Post-translational modifications in proteins involved in blood coagulation

Citrullination of arginine residues is a post-translational modification (PTM) found on myelin basic protein (MBP), which neutralizes MBPs positive charge, and is implicated in myelin damage and multiple sclerosis (MS). Here we identify lysine acetylation as another neutralizing PTM to MBP that may be involved in myelin damage. We quantify changes in lysine and arginine PTMs on MBP derived from mice induced with an experimental autoimmune encephalomyelitis (EAE) model of MS using liquid chromatography tandem mass spectrometry. The changes in PTMs are correlated to changes in neurological disability scoring (NDS), as a marker of myelin damage. We found that lysine acetylation increased by 2-fold on MBP during peak NDS post-EAE induction. We also found that mono- and dimethyl-lysine, as well as asymmetric dimethyl-arginine residues on MBP were elevated at peak EAE disability. These findings suggest that the acetylation and methylation of lysine on MBP are PTMs associated with the neurological disability produced by EAE. Since histone deacetylase (HDAC) inhibitors have been previously shown to improve neurological disability, we also show that treatment with trichostatin A (a HDAC inhibitor) improves the NDS of EAE mice but does not change MBP acetylation.

Molecular disturbances of neurotransmitter systems have long been held to be a core feature of the pathophysiology of schizophrenia. Despite years of study of neurotransmitter associated protein expression at multiple levels of gene expression, reports of abnormal neurotransmitter receptor transcript, protein, and signaling complex expression in schizophrenia brain have often been conflicting. These inconsistencies led us to reconsider neurotransmitter-based hypotheses of schizophrenia not as a problem of receptor number, or as a defect of neurotransmitter systems, but rather as a dysregulation of central cellular processes regulating the intracellular distribution of signaling proteins. Our working hypothesis is that a fundamental dysregulation of intracellular processes exists in schizophrenia, resulting in abnormal assembly, trafficking, and intracellular targeting of many key proteins involved in neurotransmission and other critical cellular functions. Previous studies have shown the important roles posttranslational lipid and carbohydrate modifications play in targeting receptors, transporters, and other proteins between intracellular compartments and the synapse, and in the lateral translocation of such molecules between lipid microdomains at the distal end of forward trafficking pathways. Accordingly, we have predicted that abnormal posttranslational lipid and/or sugar modification of proteins by occurs in schizophrenia. We have previously reported changes in extent of N-linked glycosylation as well as of the lipid modifications palmitoylation and N-myristoylation on target proteins in schizophrenia brain. In an ongoing project to elucidate mechanisms of these changes, we have studied expression patterns of key enzymes associated with these posttranslational modifications. Using well characterized samples of postmortem brain from schizophrenia and matched comparison subjects, we assayed transcript expression of enzymes associated with posttranslational protein modifications by lipids and carbohydrates using microarrays and qPCR. Next, we assayed protein expression of a subset of enzymes using western blot analyses. To determine the brain cell specificity of protein changes, we used laser capture microdissection (LCM) of neuronal and glial cells to harvest specific cell populations from postmortem brains, and developed and validated a capillary electrophoresis system for ultra-low quantity protein concentration (the ProteinSimple WES system) to measure protein expression within LCM harvested cells. Using microarray and qPCR, multiple transcript changes were found in schizophrenia cortex. The most substantial number of altered transcripts were found for those encoding enzymes associated with multiple aspects of posttranslational carbohydrate modifications of proteins, including N-acetylglucosaminyltransferases (GlcNAcTs), glucosyltransferases, glucosidases, N-acetylgalactosaminyltransferases (GalNAcTs), galactosyltransferases, mannosidases, fucosyltransferases, fucosidases, sialyltransferases, and sialidases. Fewer changes were found for lipid modification enzymes. Subsequently, protein expression of candidate proteins associated with these posttranslational modifications were determined by western blot analyses. Changes in protein expression of mutlple enzymes associated with glycan modification of proteins were found in schizophrenia. These include significant decreases in expression of the N-acetylglucosaminyltransferases B3GNT8 and MGAT4A, and the fucosyltransferase FUT8. Increased protein expression was found for the fucosyltransferase POFUT2, the sialyltransferase ST8SIA2, the glucosyltransferase UGGT2, and the mannosidase EDEM2. Numerous protein changes were also found in enzymes associated with lipid and glycolipid modifications. These include decreased expression of the prenylation associate denzyme subunits farnesyl-protein transferase α-subunit (FNTA), geranylgeranyltransferase type I β-subunit (PGGT1B), and rab geranylgeranyltransferase β-subunit (RABGGTB). Glycophosphatidylinositol (GPI)-anchor attachment 1 protein (GPAA1) is increased in these subjects. To determine the cell-specific pattern of these protein changes, we have developed an LCM-capillary electrophoresis assay to isolate protein from LCM harvested cells to allow multiplex protein quantification in 500 ng of protein obtained from these cells. We have validated that we can reliably harvest cortical neuronal subtypes and astroglia, are able to measure 4 proteins simultaneously in samples from these cells lines, and are currently collecting cells to extend these findings into cell-specific studies to determine if the changes we have found in posttranslational modification proteins are widely specific or specific to given subpopulations of brain cells. These data support our earlier findings of altered patterns of the posttranslational modifications of both glycosylation and lipid modification of proteins in the cortex of schizophrenia. By identifying changes in both mRNA and protein expression of key enzymes associated with these posttranslational modifications, we have begun to elucidate potential mechanisms of these earlier observations. One of the challenges that has plagued schizophrenia research for decades is that many different neurotransmitter and neurochemical systems have been implicated and studied in this illness, and reconciling this large literature is challenging. These many changes in numerous different systems suggest, however, that rather than schizophrenia being a disorder of a given neurotransmitter system, it is rather a disturbance of core intracellular processes that underlie regulation of multiple neurochemical systems. The machinery associated with posttranslational modifications of proteins is a possible substrate that could reconcile prior abnormalities identified in myriad systems. We have proposed that a fundamental defect in the brains of those affected with this illness is abnormal assembly, trafficking and receptor dynamics of many different proteins in schizophrenia that is due mechanistically to abnormal posttranslational modifications that influence intracellular targeting and trafficking of proteins between subcellular compartments. Dysregulation of lipid and glycan modification of proteins are likely candidates for such a process, and these present data begin to elucidate the mechanisms from which these abnormalities occur.

PERK Activation Preserves the Viability and Function of Remyelinating Oligodendrocytes in Immune-Mediated Demyelinating Diseases

The myelin basic protein (MBP) family comprises a variety of developmentally-regulated members arising from different transcription start sites, differential splicing, and post-translational modifications. The “classic” isoforms of MBP include the 18.5-kDa form, which predominates in adult human myelin and facilitates compaction of the mature myelin sheath in the central nervous system, thereby maintaining its structural integrity. In addition to membrane-association, the 18.5-kDa and all other classic isoforms are able to interact with a multitude of proteins, including Ca<sup>2+</sup>-calmodulin, actin, tubulin, and SH3-domain-containing proteins, and thus may be signalling linkers during myelin development and remodelling. All proteins in this family are intrinsically disordered, creating a large effective surface to facilitate multiple protein associations, and are post-translationally modified to various degrees by methylation, phosphorylation, and deimination. We have used spectroscopic (fluorescence, circular dichroism, electron paramagnetic resonance, and nuclear magnetic resonance) and computational (molecular dynamics) approaches to study MBP’s conformational adaptability. A highly-conserved central domain consists of an amphipathic α-helix that associates with a phospholipid membrane. In multiple sclerosis, this segment represents a primary immunodominant epitope. This α-helical structure is adjacent to a proline-rich region that contains a classic SH3-ligand along with two threonyl MAP-kinase phosphorylation sites, and forms a poly-proline type II (PPII) structure. This α-helical segment of the protein is thus essential to proper positioning of the PPII protein-interaction motif, with the local conformation and accessibility being modulated by MAP-kinases, and may represent an important molecular switch. Aberrant post-translational or other modifications in this segment of the protein may participate in the onset and pathogenesis of the human demyelinating disease multiple sclerosis.

The post-translational modification citrullination has been proposed to play a role in the pathogenesis of multiple sclerosis (MS). Myelin basic protein (MBP) is a candidate autoantigen which is citrullinated to a minor extent under physiological conditions and hypercitrullinated in MS. We examined immune cell responses elicited by hypercitrullinated MBP (citMBP) in cultures of mononuclear cells from 18 patients with MS and 42 healthy donors (HDs). The immunodominant peptide of MBP, MBP85-99, containing citrulline in position 99, outcompeted the binding of native MBP85-99 to HLA-DR15, which is strongly linked to MS. Moreover, using the monoclonal antibody MK16 as probe, we observed that B cells and monocytes from HLA-DR15+ patients with MS presented MBP85-99 more efficiently after challenge with citMBP than with native MBP. Both citMBP and native MBP induced proliferation of CD4+ T cells from patients with MS as well as TNF-α production by their B cells and CD4+ T cells, and citrullination of MBP tended to enhance TNF-α secretion by CD4+ T cells from HLA-DR15+ patients. Unlike native MBP, citMBP induced differentiation into Th17 cells in cultures from HDs, while neither form of MBP induced Th17-cell differentiation in cultures from patients with MS. These data suggest a role for citrullination in the breach of tolerance to MBP in healthy individuals and in maintenance of the autoimmune response to MBP in patients with MS.

Posttranslational Protein Modifications: Current Implications for Cancer Detection, Prevention, and Therapeutics

Progress in Multiple Sclerosis Research

The post-translational modifications of proteins by mono- and poly-ADP-ribosylation involve the cleavage of βNAD⁺, with the release of its nicotinamide moiety, accompanied by the transfer of a single (mono) or several (poly) ADP-ribose molecules from βNAD⁺ to a specific amino-acid residue of various cellular proteins. Thus, both mono- and poly-ADP-ribosylation are NAD⁺-consuming reactions. ADP-ribosylation reactions have been reported to have important roles in the nucleus, and in mitochondrial activity. Distinct subcellular NAD⁺ pools have been identified, not only in the nucleus and the mitochondria, but also in the endoplasmic reticulum and peroxisomes. Recent reports have shed new light on the correlation between NAD⁺-dependent ADP-ribosylation reactions and the endoplasmic reticulum. We have demonstrated that ARTD15/PARP16 is a novel mono-ADP-ribosyltransferase with a new intracellular location, as it is associated with the endoplasmic reticulum. The endoplasmic reticulum is a membranous network of tubules, vesicles, and cisternae that are interconnected in the cytoplasm of eukaryotic cells. This intracellular compartment is responsible for many cellular functions, including facilitation of protein folding and assembly, biosynthesis of lipids, storage of intracellular Ca²⁺, and transport of proteins. ARTD15 might have a role in both the nucleo-cytoplasmic shuttling, through importinβ1 mono-ADP-ribosylation, and in the unfolded protein response through its ability to ADP-ribosylate two components of this pathway: PERK and IRE1. This review summarizes our present knowledge of the enzymes and targets involved in ADP-ribosylation reactions, with special regard to the novel regulatory reactions that occurs at the level of the endoplasmic reticulum, and that can affect the function of this organelle.

Post-translational modifications of pancreatic fluid proteins collected via the endoscopic pancreatic function test (ePFT)

Dysregulated peptidyl deiminase 2 (PAD2) and aberrant protein citrullination (PC), a posttranslational modification (PTM), are involved in various inflammatory and neurodegenerative diseases. We previously showed in transgenic mice and postmortem human tissues that PC and PAD2 are altered in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by motor neurons loss, paralysis, and death. Herein, we investigated the role of PAD2 in ALS by PAD2 knockout in a SOD1-ALS mouse model. To investigate the role of PAD2-induced citrullination in ALS pathogenesis, we generated PAD2 knockout (PAD2KO) in SOD1 G93A ALS mouse model and investigated the consequent modulation on the neuropathology and clinical symptoms, using molecular biology techniques such as qPCR, Western blotting, confocal microscopy, and electron microscopy. Additionally, we identified C3 as being citrullinated in human ALS using ionFinder. Our results show that PAD2KO blocked the increased PC and reduced myelin basic protein (MBP) aggregates in the ALS model. PAD2KO also improved motor neuron survival and the integrity of myelin, axons, and neuromuscular junctions, and reduced microgliosis in the white matter and C3 protein levels in astrocytes. Clinically, data from monitoring the body weight changes suggests that PAD2KO modulates the course of the disease in the ALS mouse model, accelerating the onset while slowing the progression after the onset, and modestly extending the survival of male mice. These results show that PAD2 is responsible for the increased PC in ALS and PC contributes to neuroinflammation and degeneration of motor neurons and myelinated axons. The modest modulation of the disease phenotype suggests that the role of PC in ALS is complex, involving altered PC in numerous proteins and in multiple cell types. Future studies are needed to investigate how PC modulates individual protein functions in various cell types to understand the contribution of PC to ALS pathogenesis.