Specific Protein Substrates Research Articles

Functional characterization of DnSIZ1, a SIZ/PIAS-type SUMO E3 ligase from Dendrobium.

BackgroundSUMOylation is an important post-translational modification of eukaryotic proteins that involves the reversible conjugation of a small ubiquitin-related modifier (SUMO) polypeptide to its specific protein substrates, thereby regulating numerous complex cellular processes. The PIAS (protein inhibitor of activated signal transducers and activators of transcription [STAT]) and SIZ (scaffold attachment factor A/B/acinus/PIAS [SAP] and MIZ) proteins are SUMO E3 ligases that modulate SUMO conjugation. The characteristic features and SUMOylation mechanisms of SIZ1 protein in monocotyledon are poorly understood. Here, we examined the functions of a homolog of Arabidopsis SIZ1, a functional SIZ/PIAS-type SUMO E3 ligase from Dendrobium.ResultsIn Dendrobium, the predicted DnSIZ1 protein has domains that are highly conserved among SIZ/PIAS-type proteins. DnSIZ1 is widely expressed in Dendrobium organs and has a up-regulated trend by treatment with cold, high temperature and wounding. The DnSIZ1 protein localizes to the nucleus and shows SUMO E3 ligase activity when expressed in an Escherichia coli reconstitution system. Moreover, ectopic expression of DnSIZ1 in the Arabidopsis siz1-2 mutant partially complements several phenotypes and results in enhanced levels of SUMO conjugates in plants exposed to heat shock conditions. We observed that DnSIZ1 acts as a negative regulator of flowering transition which may be via a vernalization-induced pathway. In addition, ABA-hypersensitivity of siz1-2 seed germination can be partially suppressed by DnSIZ1.ConclusionsOur results suggest that DnSIZ1 is a functional homolog of the Arabidopsis SIZ1 with SUMO E3 ligase activity and may play an important role in the regulation of Dendrobium stress responses, flowering and development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0613-3) contains supplementary material, which is available to authorized users.

Mistargeted mitochondrial proteins activate a proteostatic response in the cytosol.

Most of the mitochondrial proteome originates from nuclear genes and is transported into the mitochondria after synthesis in the cytosol. Complex machineries which maintain the specificity of protein import and sorting include the TIM23 translocase responsible for the transfer of precursor proteins into the matrix, and the mitochondrial intermembrane space import and assembly (MIA) machinery required for the biogenesis of intermembrane space proteins. Dysfunction of mitochondrial protein sorting pathways results in diminishing specific substrate proteins, followed by systemic pathology of the organelle and organismal death. The cellular responses caused by accumulation of mitochondrial precursor proteins in the cytosol are mainly unknown. Here we present a comprehensive picture of the changes in the cellular transcriptome and proteome in response to a mitochondrial import defect and precursor over-accumulation stress. Pathways were identified that protect the cell against mitochondrial biogenesis defects by inhibiting protein synthesis and by activation of the proteasome, a major machine for cellular protein clearance. Proteasomal activity is modulated in proportion to the quantity of mislocalized mitochondrial precursor proteins in the cytosol. We propose that this type of unfolded protein response activated by mistargeting of proteins (UPRam) is beneficial for the cells. UPRam provides a means for buffering the consequences of physiological slowdown in mitochondrial protein import and for counteracting pathologies that are caused or contributed by mitochondrial dysfunction.

Molecular dynamics investigations of BioH protein substrate specificity for biotin synthesis

BioH, an enzyme of biotin synthesis, plays an important role in fatty acid synthesis which assembles the pimelate moiety. Pimeloyl-acyl carrier protein (ACP) methyl ester, which is long known to be a biotin precursor, is the physiological substrate of BioH. Azelayl methyl ester, which has a longer chain than pimeloyl methyl ester, conjugated to ACP is also indeed accepted by BioH with very low rate of hydrolysis. To date, the substrate specificity for BioH and the molecular origin for the experimentally observed rate changes of hydrolysis by the chain elongation have remained elusive. To this end, we have investigated chain elongation effects on the structures by using the fully atomistic molecular dynamics simulations combined with binding free energy calculations. The results indicate that the substrate specificity is determined by BioH together with ACP. The added two methylenes would increase the structural flexibility by protein motions at the interface of ACP and BioH, instead of making steric clashes with the side chains of the BioH hydrophobic cavity. On the other hand, the slower hydrolysis of azelayl substrate is suggested to be associated with the loose of contacts between BioH and ACP, and with the lost electrostatic interactions of two ionic/hydrogen bonding networks at the interface of the two proteins. The present study provides important insights into the structure–function relationships of the complex of BioH with pimeloyl-ACP methyl ester, which could contribute to further understanding about the mechanism of the biotin synthetic pathway, including the catalytic role of BioH.

In silico identification of AMPylating enzymes and study of their divergent evolution.

AMPylation is a novel post-translational modification (PTM) involving covalent attachment of an AMP moiety to threonine/tyrosine side chains of a protein. AMPylating enzymes belonging to three different families, namely Fic/Doc, GS-ATase and DrrA have been experimentally characterized. Involvement of these novel enzymes in a myriad of biological processes makes them interesting candidates for genome-wide search. We have used SVM and HMM to develop a computational protocol for identification of AMPylation domains and their classification into various functional subfamilies catalyzing AMPylation, deAMPylation, phosphorylation and phosphocholine transfer. Our analysis has not only identified novel PTM catalyzing enzymes among unannotated proteins, but has also revealed how this novel enzyme family has evolved to generate functional diversity by subtle changes in sequence/structures of the proteins. Phylogenetic analysis of Fic/Doc has revealed three new isofunctional subfamilies, thus adding to their functional divergence. Also, frequent occurrence of Fic/Doc proteins on highly mobile and unstable genomic islands indicated their evolution via extensive horizontal gene transfers. On the other hand phylogenetic analyses indicate lateral evolution of GS-ATase family and an early duplication event responsible for AMPylation and deAMPylation activity of GS-ATase. Our analysis also reveals molecular basis of substrate specificity of DrrA proteins.

O-GlcNAcylation of co-activator-associated arginine methyltransferase 1 regulates its protein substrate specificity.

Co-activator-associated arginine methyltransferase 1 (CARM1) asymmetrically di-methylates proteins on arginine residues. CARM1 was previously known to be modified through O-linked-β-N-acetylglucosaminidation (O-GlcNAcylation). However, the site(s) of O-GlcNAcylation were not mapped and the effects of O-GlcNAcylation on biological functions of CARM1 were undetermined. In the present study, we describe the comprehensive mapping of CARM1 post-translational modification (PTM) using top-down MS. We found that all detectable recombinant CARM1 expressed in human embryonic kidney (HEK293T) cells is automethylated as we previously reported and that about 50% of this automethylated CARM1 contains a single O-linked-β-N-acetylglucosamine (O-GlcNAc) moiety [31]. The O-GlcNAc moiety was mapped by MS to four possible sites (Ser595, Ser598, Thr601 and Thr603) in the C-terminus of CARM1. Mutation of all four sites [CARM1 quadruple mutant (CARM1QM)] markedly decreased O-GlcNAcylation, but did not affect protein stability, dimerization or cellular localization of CARM1. Moreover, CARM1QM elicits similar co-activator activity as CARM1 wild-type (CARM1WT) on a few transcription factors known to be activated by CARM1. However, O-GlcNAc-depleted CARM1 generated by wheat germ agglutinin (WGA) enrichment, O-GlcNAcase (OGA) treatment and mutation of putative O-GlcNAcylation sites displays different substrate specificity from that of CARM1WT. Our findings suggest that O-GlcNAcylation of CARM1 at its C-terminus is an important determinant for CARM1 substrate specificity.

Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins.

AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(ɛ)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ɛ-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ɛ-adducts, 1,N(6)-ethenoadenine (ɛA), 3,N(4)-ethenocytosine (ɛC) and 1,N(2)-ethenoguanine (1,N(2)-ɛG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ɛ-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed ɛA and ɛC from ds and ssDNA but were inactive toward 1,N(2)-ɛG. SC-1A repaired only ɛA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ɛ-adducts in dsDNA, while only ɛA and ɛC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only ɛC in ssDNA. Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N(2)-ɛG and that ALKBH3 removes only ɛC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins.

Prediction of substrate specificity and preliminary kinetic characterization of the hypothetical protein PVX_123945 from Plasmodium vivax

Members of the haloacid dehalogenase (HAD) superfamily are emerging as an important group of enzymes by virtue of their role in diverse chemical reactions. In different Plasmodium species their number varies from 16 to 21. One of the HAD superfamily members, PVX_123945, a hypothetical protein from Plasmodium vivax, was selected for examining its substrate specificity. Based on distant homology searches and structure comparisons, it was predicted to be a phosphatase. Thirty-eight metabolites were screened to identify potential substrates. Further, to validate the prediction, biochemical and kinetic studies were carried out that showed that the protein was a monomer with high catalytic efficiency for β-glycerophosphate followed by pyridoxal 5′-phosphate. The enzyme also exhibited moderate catalytic efficiencies for α-glycerophosphate, xanthosine 5′-monophosphate and adenosine 5′-monophosphate. It also hydrolyzed the artificial substrate p-nitrophenyl phosphate (pNPP). Mg2+ was the most preferred divalent cation and phosphate inhibited the enzyme activity. The study is the first attempt at understanding the substrate specificity of a hypothetical protein belonging to HAD superfamily from the malarial parasite P. vivax.

EF0176 and EF0177 from Enterococcus faecalis V583 are substrate-binding lipoproteins involved in ABC transporter mediated ribonucleoside uptake.

One of the ABC transporter systems in Enterococcus faecalis V583 is encoded by the ef0176-ef0180 gene cluster, which differs from orthologous operons in related bacteria in that it contains two genes putatively encoding substrate-binding proteins (SBPs). These SBPs, EF0176 and EF0177, have previously been identified on the surface of E. faecalis. By phenotypic studies of single and double knockout mutants, we show here that EF0176 and EF0177 are specific for ribonucleosides and, by inference, that the EF0176-EF0180 ABC transporter plays a role in nucleoside uptake. The specificity of the SBPs was mapped using growth experiments on a medium, RPMI 1640, that only supports growth of E. faecalis when supplemented with purine nucleosides or their corresponding bases. This analysis was complemented by studies with toxic fluorinated pyrimidine ribonucleoside analogues and competition experiments. The data show that EF0176 and EF0177 have broad and overlapping, but not identical, substrate specificities and that they, together, are likely to bind and facilitate the transport of all common ribonucleosides. Comparative sequence analysis and inspection of an available crystal structure of an orthologue, PnrA from Treponema pallidum, showed that the strongest binding interactions between the protein and the ligand involve the ribose moiety and that sequence variation in the binding site primarily affects interactions with the base. This explains both the broad substrate specificity of these binding proteins and the observed variations therein. The presence of two SBPs in this nucleoside ABC transporter system in E. faecalis may improve the bacterium's ability to scavenge nucleosides.

Characterization and identification of ubiquitin conjugation sites with E3 ligase recognition specificities.

BackgroundIn eukaryotes, ubiquitin-conjugation is an important mechanism underlying proteasome-mediated degradation of proteins, and as such, plays an essential role in the regulation of many cellular processes. In the ubiquitin-proteasome pathway, E3 ligases play important roles by recognizing a specific protein substrate and catalyzing the attachment of ubiquitin to a lysine (K) residue. As more and more experimental data on ubiquitin conjugation sites become available, it becomes possible to develop prediction models that can be scaled to big data. However, no development that focuses on the investigation of ubiquitinated substrate specificities has existed. Herein, we present an approach that exploits an iteratively statistical method to identify ubiquitin conjugation sites with substrate site specificities.ResultsIn this investigation, totally 6259 experimentally validated ubiquitinated proteins were obtained from dbPTM. After having filtered out homologous fragments with 40% sequence identity, the training data set contained 2658 ubiquitination sites (positive data) and 5532 non-ubiquitinated sites (negative data). Due to the difficulty in characterizing the substrate site specificities of E3 ligases by conventional sequence logo analysis, a recursively statistical method has been applied to obtain significant conserved motifs. The profile hidden Markov model (profile HMM) was adopted to construct the predictive models learned from the identified substrate motifs. A five-fold cross validation was then used to evaluate the predictive model, achieving sensitivity, specificity, and accuracy of 73.07%, 65.46%, and 67.93%, respectively. Additionally, an independent testing set, completely blind to the training data of the predictive model, was used to demonstrate that the proposed method could provide a promising accuracy (76.13%) and outperform other ubiquitination site prediction tool.ConclusionA case study demonstrated the effectiveness of the characterized substrate motifs for identifying ubiquitination sites. The proposed method presents a practical means of preliminary analysis and greatly diminishes the total number of potential targets required for further experimental confirmation. This method may help unravel their mechanisms and roles in E3 recognition and ubiquitin-mediated protein degradation.

Клонирование кДНК, экспрессия и определение субстратной специфичности селенсодержащего белка мыши SelV (Selenoprotein V)

To date various bioinformatics tools allowed to identify 25 selenocysteine-containing mammalian proteins. The name of these proteins assumes that they contain the amino acid selenocysteine (Sec). Functionally characterized selenocysteine-containing proteins are oxidoreductases with various functions, including glutathione peroxidases, thioredoxin reductases, deiodinases etc. However, the functions of more than half of identified proteins are still unclear, and mammalian selenoprotein SeIV is among them. We studied the selV in all stages of postnatal development with the maximum level of mRNA expression during puberty, whereas in adult mice (8-18 months) we observed a gradual decrease of expression. In order to get closer to the functional role of Selenoprotein V, we have carried out experiments on the substrate specificity and enzymatic activity measurement of this selenocysteine-containing protein. It was shown that SelV posseses glutathionperoxidase and thioredoxinreductase activities.

The specificity of thioredoxins and glutaredoxins is determined by electrostatic and geometric complementarity.

Thiol-disulfide oxidoreductases from the thioredoxin (Trx) family of proteins have a broad range of well documented functions and possess distinct substrate specificities. The mechanisms and characteristics that control these specificities are key to the understanding of both the reduction of catalytic disulfides as well as allosteric disulfides (thiol switches). Here, we have used the catalytic disulfide of E. coli 3'-phosphoadenosine 5'-phosphosulfate (PAPS) reductase (PR) that forms between the single active site thiols of two monomers during the reaction cycle as a model system to investigate the mechanisms of Trx and Grx protein specificity. Enzyme kinetics, ΔE'0 determination, and structural analysis of various Trx and Grx family members suggested that the redox potential does not determine specificity nor efficiency of the redoxins as reductant for PR. Instead, the efficiency of PR with various redoxins correlated strongly to the extent of a negative electric field of the redoxins reaching into the solvent outside the active site, and electrostatic and geometric complementary contact surfaces. These data suggest that, in contrast to common assumption, the composition of the active site motif is less important for substrate specificity than other amino acids in or even outside the immediate contact area.

Molecular insights into RNA and DNA helicase evolution from the determinants of specificity for a DEAD-box RNA helicase.

How different helicase families with a conserved catalytic 'helicase core' evolved to function on varied RNA and DNA substrates by diverse mechanisms remains unclear. In this study, we used Mss116, a yeast DEAD-box protein that utilizes ATP to locally unwind dsRNA, to investigate helicase specificity and mechanism. Our results define the molecular basis for the substrate specificity of a DEAD-box protein. Additionally, they show that Mss116 has ambiguous substrate-binding properties and interacts with all four NTPs and both RNA and DNA. The efficiency of unwinding correlates with the stability of the 'closed-state' helicase core, a complex with nucleotide and nucleic acid that forms as duplexes are unwound. Crystal structures reveal that core stability is modulated by family-specific interactions that favor certain substrates. This suggests how present-day helicases diversified from an ancestral core with broad specificity by retaining core closure as a common catalytic mechanism while optimizing substrate-binding interactions for different cellular functions.

Atomistic Molecular-Dynamics Simulations Enable Prediction of the Arginine Permeation Pathway through OccD1/OprD from Pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram-negative bacterium that does not contain large, nonspecific porins in its outer membrane. Consequently, the outer membrane is highly impermeable to polar solutes and serves as a barrier against the penetration of antimicrobial agents. This is one of the reasons why such bacteria are intrinsically resistant to antibiotics. Polar molecules that permeate across the outer membrane do so through substrate-specific channels proteins. To design antibiotics that target substrate-channel proteins, it is essential to first identify the permeation pathways of their natural substrates. In P. aeruginosa, the largest family of substrate-specific proteins is the OccD (previously reported under the name OprD) family. Here, we employ equilibrium and steered molecular-dynamics simulations to study OccD1/OprD, the archetypical member of the OccD family. We study the permeation of arginine, one of the natural substrates of OccD1, through the protein. The combination of simulation methods allows us to predict the pathway taken by the amino acid, which is enabled by conformational rearrangements of the extracellular loops of the protein. Furthermore, we show that arginine adopts a specific orientation to form the molecular interactions that facilitate its passage through part of the protein. We predict a three-stage permeation process for arginine.

Evidence for substrate binding-induced zwitterion formation in the catalytic Cys-His dyad of the SARS-CoV main protease.

The coronavirus main protease (M(pro)) represents an attractive drug target for antiviral therapy of coronavirus (CoV) infections, including severe acute respiratory syndrome (SARS). The SARS-CoV M(pro) and related CoV proteases have several distinct features, such as an uncharged Cys-His catalytic dyad embedded in a chymotrypsin-like protease fold, that clearly separate these enzymes from archetypical cysteine proteases. To further characterize the catalytic system of CoV main proteases and to obtain information about improved inhibitors, we performed comprehensive simulations of the proton-transfer reactions in the SARS-CoV M(pro) active site that lead to the Cys(-)/His(+) zwitterionic state required for efficient proteolytic activity. Our simulations, comprising the free enzyme as well as substrate-enzyme and inhibitor-enzyme complexes, lead us to predict that zwitterion formation is fostered by substrate binding but not inhibitor binding. This indicates that M(pro) employs a substrate-induced catalytic mechanism that further enhances its substrate specificity. Our computational data are in line with available experimental results, such as X-ray geometries, measured pKa values, mutagenesis experiments, and the measured differences between the kinetic parameters of substrates and inhibitors. The data also provide an atomistic picture of the formerly postulated electrostatic trigger involved in SARS-CoV M(pro) activity. Finally, they provide information on how a specific microenvironment may finely tune the activity of M(pro) toward specific viral protein substrates, which is known to be required for efficient viral replication. Our simulations also indicate that the low inhibition potencies of known covalently interacting inhibitors may, at least in part, be attributed to insufficient fostering of the proton-transfer reaction. These findings suggest ways to achieve improved inhibitors.

Prediction of membrane transport proteins and their substrate specificities using primary sequence information.

BackgroundMembrane transport proteins (transporters) move hydrophilic substrates across hydrophobic membranes and play vital roles in most cellular functions. Transporters represent a diverse group of proteins that differ in topology, energy coupling mechanism, and substrate specificity as well as sequence similarity. Among the functional annotations of transporters, information about their transporting substrates is especially important. The experimental identification and characterization of transporters is currently costly and time-consuming. The development of robust bioinformatics-based methods for the prediction of membrane transport proteins and their substrate specificities is therefore an important and urgent task.ResultsSupport vector machine (SVM)-based computational models, which comprehensively utilize integrative protein sequence features such as amino acid composition, dipeptide composition, physico-chemical composition, biochemical composition, and position-specific scoring matrices (PSSM), were developed to predict the substrate specificity of seven transporter classes: amino acid, anion, cation, electron, protein/mRNA, sugar, and other transporters. An additional model to differentiate transporters from non-transporters was also developed. Among the developed models, the biochemical composition and PSSM hybrid model outperformed other models and achieved an overall average prediction accuracy of 76.69% with a Mathews correlation coefficient (MCC) of 0.49 and a receiver operating characteristic area under the curve (AUC) of 0.833 on our main dataset. This model also achieved an overall average prediction accuracy of 78.88% and MCC of 0.41 on an independent dataset.ConclusionsOur analyses suggest that evolutionary information (i.e., the PSSM) and the AAIndex are key features for the substrate specificity prediction of transport proteins. In comparison, similarity-based methods such as BLAST, PSI-BLAST, and hidden Markov models do not provide accurate predictions for the substrate specificity of membrane transport proteins. TrSSP: The Transporter Substrate Specificity Prediction Server, a web server that implements the SVM models developed in this paper, is freely available at http://bioinfo.noble.org/TrSSP.

Ada response - a strategy for repair of alkylated DNA in bacteria.

Alkylating agents are widespread in the environment and also occur endogenously. They can be cytotoxic or mutagenic to the cells introducing alkylated bases to DNA or RNA. All organisms have evolved multiple DNA repair mechanisms to counteract the effects of DNA alkylation: the most cytotoxic lesion, N3-methyladenine (3meA), is excised by AlkA glycosylase initiating base excision repair (BER); toxic N1-methyladenine (1meA) and N3-methylcytosine (3meC), induced in DNA and RNA, are removed by AlkB dioxygenase; and mutagenic and cytotoxic O6-methylguanine (O6meG) is repaired by Ada methyltransferase. In Escherichia coli, Ada response involves the expression of four genes, ada, alkA, alkB, and aidB, encoding respective proteins Ada, AlkA, AlkB, and AidB. The Ada response is conserved among many bacterial species; however, it can be organized differently, with diverse substrate specificity of the particular proteins. Here, an overview of the organization of the Ada regulon and function of individual proteins is presented. We put special effort into the characterization of AlkB dioxygenases, their substrate specificity, and function in the repair of alkylation lesions in DNA/RNA.

Substrate recognition and specificity of double-stranded RNA binding proteins.

Recognition of double-stranded (ds) RNA is an important part of many cellular pathways, including RNA silencing, viral recognition, RNA editing, processing, and transport. dsRNA recognition is often achieved by dsRNA binding domains (dsRBDs). We use atomistic molecular dynamics simulations to examine the binding interface of the transactivation response RNA binding protein (TRBP) dsRBDs to dsRNA substrates. Our results explain the exclusive selectivity of dsRBDs toward dsRNA and against DNA–RNA hybrid and dsDNA duplexes. We also provide corresponding experimental evidence. The dsRNA duplex is recognized by dsRBDs through the A-form of three duplex grooves and by the chemical properties of RNA bases, which have 2′-hydroxyl groups on their sugar rings. Our simulations show that TRBP dsRBD discriminates dsRNA- from DNA-containing duplexes primarily through interactions at two duplex grooves. The simulations also reveal that the conformation of the DNA–RNA duplex can be altered by dsRBD proteins, resulting in a weak binding of dsRBDs to DNA–RNA hybrids. Our study reveals the structural and molecular basis of protein–RNA interaction that gives rise to the observed substrate specificity of dsRNA binding proteins.

GABAAergic stimulation modulates intracellular protein arginine methylation

Changes in cytoplasmic pH are known to regulate diverse cellular processes and influence neuronal activities. In neurons, the intracellular alkalization is shown to occur after stimulating several channels and receptors. For example, it has previously demonstrated in P19 neurons that a sustained intracellular alkalinization can be mediated by the Na+/H+ antiporter. In addition, the benzodiazepine binding subtypes of the γ-amino butyric acid type A (GABAA) receptor mediate a transient intracellular alkalinization when they are stimulated. Because the activities of many enzymes are sensitive to pH shift, here we investigate the effects of intracellular pH modulation resulted from stimulating GABAA receptor on the protein arginine methyltransferases (PRMT) activities. We show that the major benzodiazepine subtype (2α1, 2β2, 1γ2) is constitutively expressed in both undifferentiated P19 cells and retinoic acid (RA) differentiated P19 neurons. Furthermore stimulation with diazepam and, diazepam plus muscimol produce an intracellular alkalinization that can be detected ex vivo with the fluorescence dye. The alkalinization results in significant perturbation in protein arginine methylation activity as measured in methylation assays with specific protein substrates. Altered protein arginine methylation is also observed when cells are treated with the GABAA agonist muscimol but not an antagonist, bicuculline. These data suggest that pH-dependent and pH-independent methylation pathways can be activated by GABAAergic stimulation, which we verified using hippocampal slice preparations from a mouse model of fragile X syndrome.

Understanding the influence of codon translation rates on cotranslational protein folding.

Protein domains can fold into stable tertiary structures while they are synthesized by the ribosome in a process known as cotranslational folding. If a protein does not fold cotranslationally, however, it has the opportunity to do so post-translationally, that is, after the nascent chain has been fully synthesized and released from the ribosome. The rate at which a ribosome adds an amino acid encoded by a particular codon to the elongating nascent chain can vary significantly and is called the codon translation rate. Recent experiments have illustrated the profound impact that codon translation rates can have on the cotranslational folding process and the acquisition of function by nascent proteins. Synonymous codon mutations in an mRNA molecule change the chemical identity of a codon and its translation rate without changing the sequence of the synthesized protein. This change in codon translation rate can, however, cause a nascent protein to malfunction as a result of cotranslational misfolding. In some situations, such dysfunction can have profound implications; for example, it can alter the substrate specificity of an ABC transporter protein, resulting in patients who are nonresponsive to chemotherapy treatment. Thus, codon translation rates are crucial in coordinating protein folding in a cellular environment and can affect downstream cellular processes that depend on the proper functioning of newly synthesized proteins. As the importance of codon translation rates makes clear, a necessary aspect of fully understanding cotranslational folding lies in considering the kinetics of the process in addition to its thermodynamics. In this Account, we examine the contributions that have been made to elucidating the mechanisms of cotranslational folding by using the theoretical and computational tools of chemical kinetics, molecular simulations, and systems biology. These efforts have extended our ability to understand, model, and predict the influence of codon translation rates on cotranslational protein folding and misfolding. The application of such approaches to this important problem is creating a framework for making quantitative predictions of the impact of synonymous codon substitutions on cotranslational folding that has led to a novel hypothesis regarding the role of fast-translating codons in coordinating cotranslational folding. In addition, it is providing new insights into proteome-wide cotranslational folding behavior and making it possible to identify potential molecular mechanisms by which molecular chaperones can influence such behavior during protein synthesis. As we discuss in this Account, bringing together these theoretical developments with experimental approaches is increasingly helping answer fundamental questions about the nature of nascent protein folding on the ribosome.

Cover Picture: Porous Protein Crystals as Catalytic Vessels for Organometallic Complexes (Chem. Asian J. 5/2014)

Biohybrid catalysts have been developed by incorporating organometallic complexes into cross-linked porous protein crystals. As reported by Takafumi Ueno, Susumu Kitagawa et al. in their Full Paper on page 1373 ff., organoruthenium complexes in cross-linked porous hen egg white lysozyme (HEWL) crystals had an improved catalytic activity of hydrogen transfer reduction of acetophenone derivatives as compared to that of a buffer solution containing the organoruthenium complexes and HEWL. The reaction catalyzed in the tetragonal and orthorhombic HEWL crystals gave different enantiomers by specific protein–substrate interactions. This method represents a new approach for the construction of reaction nanovessels using protein crystals.