In Vivo Methylation of an Arginine in Chicken Myelin Basic Protein

Abstract: The amino acid sequence around the sole methylarginine residue in chicken myelin basic protein was determined and was found to be similar to that previously reported for mammalian myelin basic protein. The ratio N ^G, N '^G-dimethylarginine: N ^G-monomethylarginine:arginine was approximately 1.3:0.9:1.0. No N ^G, N ^G-dimethylarginine was detected in the protein. The in vivo incorporation of methyl groups from [methyl-³H]methionine into methylarginines in myelin was found to occur readily in 2-day-old chickens. Radioactively labelled N ^G, N '^G-dimeth-ylarginine and N ^G-monomethylarginine in myelin were derived solely from myelin basic protein. Radioactivity was also incorporated into N ^G, N ^G-dimeth-ylarginine, although this was not derived from myelin basic protein. As N ^G-monomethylarginine was easily separated from the dimethylarginines, and as it was derived from myelin basic protein, it may be a good marker for myelin basic protein turnover in vivo. A time course study of the incorporation showed that radioactivity was incorporated into N ^G-monomethylarginine up to 6 h after injection, and decayed slowly, with an apparent half-life of about 40 days.     

Translational Roles of Elongation Factor 2 Protein Lysine Methylation

Methylation of various components of the translational machinery has been shown to globally affect protein synthesis. Little is currently known about the role of lysine methylation on elongation factors. Here we show that in Saccharomyces cerevisiae, the product of the EFM3/YJR129C gene is responsible for the trimethylation of lysine 509 on elongation factor 2. Deletion of EFM3 or of the previously described EFM2 increases sensitivity to antibiotics that target translation and decreases translational fidelity. Furthermore, the amino acid sequences of Efm3 and Efm2, as well as their respective methylation sites on EF2, are conserved in other eukaryotes. These results suggest the importance of lysine methylation modification of EF2 in fine tuning the translational apparatus.     

Enhanced Arginine Methylation of Programmed Cell Death 4 Protein during Nutrient Deprivation Promotes Tumor Cell Viability

The role of programmed cell death 4 (PDCD4) in tumor biology is context-dependent. PDCD4 is described as a tumor suppressor, but its coexpression with protein arginine methyltransferase 5 (PRMT5) promotes accelerated tumor growth. Here, we report that PDCD4 is methylated during nutrient deprivation. Methylation occurs because of increased stability of PDCD4 protein as well as increased activity of PRMT5 toward PDCD4. During nutrient deprivation, levels of methylated PDCD4 promote cell viability, which is dependent on an enhanced interaction with eIF4A. Upon recovery from nutrient deprivation, levels of methylated PDCD4 are regulated by phosphorylation, which controls both the localization and stability of methylated PDCD4. This study reveals that, in response to particular environmental cues, the role of PDCD4 is up-regulated and is advantageous for cell viability. These findings suggest that the methylated form of PDCD4 promotes tumor viability during nutrient deprivation, ultimately allowing the tumor to grow more aggressively.     

Identification and Characterization of a Novel Human Methyltransferase Modulating Hsp70 Protein Function through Lysine Methylation

Hsp70 proteins constitute an evolutionarily conserved protein family of ATP-dependent molecular chaperones involved in a wide range of biological processes. Mammalian Hsp70 proteins are subject to various post-translational modifications, including methylation, but for most of these, a functional role has not been attributed. In this study, we identified the methyltransferase METTL21A as the enzyme responsible for trimethylation of a conserved lysine residue found in several human Hsp70 (HSPA) proteins. This enzyme, denoted by us as HSPA lysine (K) methyltransferase (HSPA-KMT), was found to catalyze trimethylation of various Hsp70 family members both in vitro and in vivo, and the reaction was stimulated by ATP. Furthermore, we show that HSPA-KMT exclusively methylates 70-kDa proteins in mammalian protein extracts, demonstrating that it is a highly specific enzyme. Finally, we show that trimethylation of HSPA8 (Hsc70) has functional consequences, as it alters the affinity of the chaperone for both the monomeric and fibrillar forms of the Parkinson disease-associated protein α-synuclein.     

Methylation of Ribosomal Protein L42 Regulates Ribosomal Function and Stress-adapted Cell Growth

Lysine methylation is one of the most common protein modifications. Although lysine methylation of histones has been extensively studied and linked to gene regulation, that of non-histone proteins remains incompletely understood. Here, we show a novel regulatory role of ribosomal protein methylation. Using an in vitro methyltransferase assay, we found that Schizosaccharomyces pombe Set13, a SET domain protein encoded by SPAC688.14, specifically methylates lysine 55 of ribosomal protein L42 (Rpl42). Mass spectrometric analysis revealed that endogenous Rpl42 is monomethylated at lysine 55 in wild-type S. pombe cells and that the methylation is lost in Δset13 mutant cells. Δset13 and Rpl42 methylation-deficient mutant S. pombe cells showed higher cycloheximide sensitivity and defects in stress-responsive growth control compared with wild type. Genetic analyses suggested that the abnormal growth phenotype was distinct from the conserved stress-responsive pathway that modulates translation initiation. Furthermore, the Rpl42 methylation-deficient mutant cells showed a reduced ability to survive after entering stationary phase. These results suggest that Rpl42 methylation plays direct roles in ribosomal function and cell proliferation control independently of the general stress-response pathway.     

Automethylation of Protein Arginine Methyltransferase 8 (PRMT8) Regulates Activity by Impeding S-Adenosylmethionine Sensitivity

Protein arginine methyltransferase (PRMT) 8 is unique among the PRMTs, as it has a highly restricted tissue expression pattern and an N terminus that contains two automethylation sites and a myristoylation site. PRMTs catalyze the transfer of a methyl group from S-adenosylmethionine (AdoMet) to a peptidylarginine on a protein substrate. Currently, the physiological roles, regulation, and cellular substrates of PRMT8 are poorly understood. However, a thorough understanding of PRMT8 kinetics should provide insights into each of these areas, thereby enhancing our understanding of this unique enzyme. In this study, we determined how automethylation regulates the enzymatic activity of PRMT8. We found that preventing automethylation with lysine mutations (preserving the positive charge of the residue) increased the turnover rate and decreased the K_m of AdoMet but did not affect the K_m of the protein substrate. In contrast, mimicking automethylation with phenylalanine (i.e. mimicking the increased hydrophobicity) decreased the turnover rate. The inhibitory effect of the PRMT8 N terminus could be transferred to PRMT1 by creating a chimeric protein containing the N terminus of PRMT8 fused to PRMT1. Thus, automethylation of the N terminus likely regulates PRMT8 activity by decreasing the affinity of the enzyme for AdoMet.     

The JmjN Domain of Jhd2 Is Important for Its Protein Stability,and the Plant Homeodomain (PHD) Finger Mediates Its Chromatin Association Independent of H3K4 Methylation

Histone lysine methylation is a dynamic process that plays an important role in regulating chromatin structure and gene expression. Recent studies have identified Jhd2, a JmjC domain-containing protein, as an H3K4-specific demethylase in budding yeast. However, important questions regarding the regulation and functions of Jhd2 remain unanswered. In this study, we show that Jhd2 has intrinsic activity to remove all three states of H3K4 methylation in vivo and can dynamically associate with chromatin to modulate H3K4 methylation levels on both active and repressed genes and at the telomeric regions. We found that the plant homeodomain (PHD) finger of Jhd2 is important for its chromatin association in vivo. However, this association is not dependent on H3K4 methylation and the H3 N-terminal tail, suggesting the presence of an alternative mechanism by which Jhd2 binds nucleosomes. We also provide evidence that the JmjN domain and its interaction with the JmjC catalytic domain are important for Jhd2 function and that Not4 (an E3 ligase) monitors the structural integrity of this interdomain interaction to maintain the overall protein levels of Jhd2. We show that the S451R mutation in human SMCX (a homolog of Jhd2), which has been linked to mental retardation, and the homologous T359R mutation in Jhd2 affect the protein stability of both of these proteins. Therefore, our findings provide a mechanistic explanation for the observed defects in patients harboring this SMCX mutant and suggest the presence of a conserved pathway involving Not4 that modulates the protein stability of both yeast Jhd2 and human SMCX.     

Functional Cross-talk between Distant Domains of Chikungunya Virus Non-structural Protein 2 Is Decisive for Its RNA-modulating Activity

Chikungunya virus (CHIKV) non-structural protein 2 (nsP2) is a multifunctional protein that is considered a master regulator of the viral life cycle and a main viral factor responsible for cytopathic effects and subversion of antiviral defense. The C-terminal part of nsP2 possesses protease activity, whereas the N-terminal part exhibits NTPase and RNA triphosphatase activity and is proposed to have helicase activity. Bioinformatics analysis classified CHIKV nsP2 into helicase superfamily 1. However, the biochemical significance of a coexistence of two functionally unrelated modules in this single protein remains unknown. In this study, recombinant nsP2 demonstrated unwinding of double-stranded RNA in a 5′–3′ directionally biased manner and RNA strand annealing activity. Comparative analysis of NTPase and helicase activities of wild type nsP2 with enzymatic capabilities of different truncated or N-terminally extended variants of nsP2 revealed that the C-terminal part of the protein is indispensable for helicase functionality and presumably provides a platform for RNA binding, whereas the N-terminal-most region is apparently involved in obtaining a conformation of nsP2 that allows for its maximal enzymatic activities. The establishment of the protocols for the production of biochemically active CHIKV nsP2 and optimization of the parameters for helicase and NTPase assays are expected to provide the starting point for a further search of possibilities for therapeutic interventions to suppress alphaviral infections.     

Histone H2A and H4 N-terminal Tails Are Positioned by the MEP50 WD Repeat Protein for Efficient Methylation by the PRMT5 Arginine Methyltransferase

The protein arginine methyltransferase PRMT5 is complexed with the WD repeat protein MEP50 (also known as Wdr77 or androgen coactivator p44) in vertebrates in a tetramer of heterodimers. MEP50 is hypothesized to be required for protein substrate recruitment to the catalytic domain of PRMT5. Here we demonstrate that the cross-dimer MEP50 is paired with its cognate PRMT5 molecule to promote histone methylation. We employed qualitative methylation assays and a novel ultrasensitive continuous assay to measure enzyme kinetics. We demonstrate that neither full-length human PRMT5 nor the Xenopus laevis PRMT5 catalytic domain has appreciable protein methyltransferase activity. We show that histones H4 and H3 bind PRMT5-MEP50 more efficiently compared with histone H2A(1–20) and H4(1–20) peptides. Histone binding is mediated through histone fold interactions as determined by competition experiments and by high density histone peptide array interaction studies. Nucleosomes are not a substrate for PRMT5-MEP50, consistent with the primary mode of interaction via the histone fold of H3-H4, obscured by DNA in the nucleosome. Mutation of a conserved arginine (Arg-42) on the MEP50 insertion loop impaired the PRMT5-MEP50 enzymatic efficiency by increasing its histone substrate K_m, comparable with that of Caenorhabditis elegans PRMT5. We show that PRMT5-MEP50 prefers unmethylated substrates, consistent with a distributive model for dimethylation and suggesting discrete biological roles for mono- and dimethylarginine-modified proteins. We propose a model in which MEP50 and PRMT5 simultaneously engage the protein substrate, orienting its targeted arginine to the catalytic site.     

Leucine Carboxyl Methyltransferase 1 (LCMT1)-dependent Methylation Regulates the Association of Protein Phosphatase 2A and Tau Protein with Plasma Membrane Microdomains in Neuroblastoma Cells

Down-regulation of protein phosphatase 2A (PP2A) methylation occurs in Alzheimer disease (AD). However, the regulation of PP2A methylation remains poorly understood. We have reported that altered leucine carboxyl methyltransferase (LCMT1)-dependent PP2A methylation is associated with down-regulation of PP2A holoenzymes containing the Bα subunit (PP2A/Bα) and subsequent accumulation of phosphorylated Tau in N2a cells, in vivo and in AD. Here, we show that pools of LCMT1, methylated PP2A, and PP2A/Bα are co-enriched in cholesterol-rich plasma membrane microdomains/rafts purified from N2a cells. In contrast, demethylated PP2A is preferentially distributed in non-rafts wherein small amounts of the PP2A methylesterase PME-1 are exclusively present. A methylation-incompetent PP2A mutant is excluded from rafts. Enhanced methylation of PP2A promotes the association of PP2A and Tau with the plasma membrane. Altered PP2A methylation following expression of a catalytically inactive LCMT1 mutant, knockdown of LCMT1, or alterations in one-carbon metabolism all result in a loss of plasma membrane-associated PP2A and Tau in N2a cells. This correlates with accumulation of soluble phosphorylated Tau, a hallmark of AD and other tauopathies. Thus, our findings reveal a distinct compartmentalization of PP2A and PP2A regulatory enzymes in plasma membrane microdomains and identify a novel methylation-dependent mechanism involved in modulating the targeting of PP2A, and its substrate Tau, to the plasma membrane. We propose that alterations in the membrane localization of PP2A and Tau following down-regulation of LCMT1 may lead to PP2A and Tau dysfunction in AD.     

A Conserved Mechanism of GABA Binding and Antagonism Is Revealed by Structure-Function Analysis of the Periplasmic Binding Protein Atu2422 in Agrobacterium tumefaciens

Bacterial periplasmic binding proteins (PBPs) and eukaryotic PBP-like domains (also called as Venus flytrap modules) of G-protein-coupled receptors are involved in extracellular GABA perception. We investigated the structural and functional basis of ligand specificity of the PBP Atu2422, which is implicated in virulence and transport of GABA in the plant pathogen Agrobacterium tumefaciens. Five high-resolution x-ray structures of Atu2422 liganded to GABA, Pro, Ala, and Val and of point mutant Atu2422-F77A liganded to Leu were determined. Structural analysis of the ligand-binding site revealed two essential residues, Phe⁷⁷ and Tyr²⁷⁵, the implication of which in GABA signaling and virulence was confirmed using A. tumefaciens cells expressing corresponding Atu2422 mutants. Phe⁷⁷ restricts ligand specificity to α-amino acids with a short lateral chain, which act as antagonists of GABA signaling in A. tumefaciens. Tyr²⁷⁵ specifically interacts with the GABA γ-amino group. Conservation of these two key residues in proteins phylogenetically related to Atu2422 brought to light a subfamily of PBPs in which all members could bind GABA and short α-amino acids. This work led to the identification of a fingerprint sequence and structural features for defining PBPs that bind GABA and its competitors and revealed their occurrence among host-interacting proteobacteria.     

The Gos28 SNARE Protein Mediates Intra-Golgi Transport of Rhodopsin and Is Required for Photoreceptor Survival

SNARE proteins play indispensable roles in membrane fusion events in many cellular processes, including synaptic transmission and protein trafficking. Here, we characterize the Golgi SNARE protein, Gos28, and its role in rhodopsin (Rh1) transport through Drosophila photoreceptors. Mutations in gos28 lead to defective Rh1 trafficking and retinal degeneration. We have pinpointed a role for Gos28 in the intra-Golgi transport of Rh1, downstream from α-mannosidase-II in the medial- Golgi. We have confirmed the necessity of key residues in Gos28''s SNARE motif and demonstrate that its transmembrane domain is not required for vesicle fusion, consistent with Gos28 functioning as a t-SNARE for Rh1 transport. Finally, we show that human Gos28 rescues both the Rh1 trafficking defects and retinal degeneration in Drosophila gos28 mutants, demonstrating the functional conservation of these proteins. Our results identify Gos28 as an essential SNARE protein in Drosophila photoreceptors and provide mechanistic insights into the role of SNAREs in neurodegenerative disease.     

A Novel 3-Methylhistidine Modification of Yeast Ribosomal Protein Rpl3 Is Dependent upon the YIL110W Methyltransferase

We have shown that Rpl3, a protein of the large ribosomal subunit from baker''s yeast (Saccharomyces cerevisiae), is stoichiometrically monomethylated at position 243, producing a 3-methylhistidine residue. This conclusion is supported by top-down and bottom-up mass spectrometry of Rpl3, as well as by biochemical analysis of Rpl3 radiolabeled in vivo with S-adenosyl-l-[methyl-³H]methionine. The results show that a +14-Da modification occurs within the GTKKLPRKTHRGLRKVAC sequence of Rpl3. Using high-resolution cation-exchange chromatography and thin layer chromatography, we demonstrate that neither lysine nor arginine residues are methylated and that a 3-methylhistidine residue is present. Analysis of 37 deletion strains of known and putative methyltransferases revealed that only the deletion of the YIL110W gene, encoding a seven β-strand methyltransferase, results in the loss of the +14-Da modification of Rpl3. We suggest that YIL110W encodes a protein histidine methyltransferase responsible for the modification of Rpl3 and potentially other yeast proteins, and now designate it Hpm1 (Histidine protein methyltransferase 1). Deletion of the YIL110W/HPM1 gene results in numerous phenotypes including some that may result from abnormal interactions between Rpl3 and the 25 S ribosomal RNA. This is the first report of a methylated histidine residue in yeast cells, and the first example of a gene required for protein histidine methylation in nature.     

Protein Polymorphism Is Negatively Correlated with Conservation of Intronic Sequences and Complexity of Expression Patterns in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

We report a significant negative correlation between nonsynonymous polymorphism and intron length in Drosophila melanogaster. This correlation is similar to that between protein divergence and intron length previously reported in Drosophila. We show that the relationship can be explained by the content of conserved noncoding sequences (CNS) within introns. In addition, genes with a high regulatory complexity and many genetic interactions also exhibit larger amounts of CNS within their introns and lower values of nonsynonymous polymorphism. The present study provides relevant evidence on the importance of intron content and expression patterns on the levels of coding polymorphism. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. [Reviewing Editor: Dr. Dmitri Petrov]