Arginine methylation of the cellular nucleic acid binding protein does not affect its subcellular localization but impedes RNA binding

Cellular nucleic acid binding protein (CNBP) contains seven zinc finger (ZF) repeats and an arginine and glycine (RG) rich sequence between the first and the second ZF. CNBP interacts with protein arginine methyltransferase PRMT1. Full-length but not RG-deleted or mutated CNBP can be methylated. Treatment with a methylation inhibitor AdOx reduced CNBP methylation, but did not affect the concentrated nuclear localization of CNBP. Nevertheless, arginine methylation of CNBP appeared to interfere with its RNA binding activity. Our findings show that arginine methylation of CNBP in the RG motif did not change the subcellular localization, but regulated its RNA binding activity.Structured summary of protein interactionsPRMT1 binds to CNBP by pull down (View interaction)PRMT1 methylates CNBP by enzymatic study (View interaction)CNBP physically interacts with PRMT1 by anti tag coimmunoprecipitation (View interaction)     

Cellular nucleic acid binding protein binds G-rich single-stranded nucleic acids and may function as a nucleic acid chaperone

Cellular nucleic acid binding protein (CNBP) is a small single-stranded nucleic acid binding protein made of seven Zn knuckles and an Arg-Gly rich box. CNBP is strikingly conserved among vertebrates and was reported to play broad-spectrum functions in eukaryotic cells biology. Neither its biological function nor its mechanisms of action were elucidated yet. The main goal of this work was to gain further insights into the CNBP biochemical and molecular features. We studied Bufo arenarum CNBP (bCNBP) binding to single-stranded nucleic acid probes representing the main reported CNBP putative targets. We report that, although bCNBP is able to bind RNA and single-stranded DNA (ssDNA) probes in vitro, it binds RNA as a preformed dimer whereas both monomer and dimer are able to bind to ssDNA. A systematic analysis of variant probes shows that the preferred bCNBP targets contain unpaired guanosine-rich stretches. These data expand the knowledge about CNBP binding stoichiometry and begins to dissect the main features of CNBP nucleic acid targets. Besides, we show that bCNBP presents a highly disordered predicted structure and promotes the annealing and melting of nucleic acids in vitro. These features are typical of proteins that function as nucleic acid chaperones. Based on these data, we propose that CNBP may function as a nucleic acid chaperone through binding, remodeling, and stabilizing nucleic acids secondary structures. This novel CNBP biochemical activity broadens the field of study about its biological function and may be the basis to understand the diverse ways in which CNBP controls gene expression.     

Retinal isoforms of inosine 5'-monophosphate dehydrogenase type 1 are poor nucleic acid binding proteins

The RP 10 form of autosomal dominant retinitis pigmentosa (adRP) is caused by mutations in the widely expressed protein inosine 5′-monophosphate dehydrogenase type 1 (IMPDH1). These mutations have no effect on the enzymatic activity of IMPDH1, but do perturb the association of IMPDH1 with nucleic acids. Two newly discovered retinal-specific isoforms, IMPDH1(546) and IMPDH1(595), may provide the key to the photoreceptor specificity of disease [S.J. Bowne, Q. Liu, L.S. Sullivan, J. Zhu, C.J. Spellicy, C.B. Rickman, E.A. Pierce, S.P. Daiger, Invest. Ophthalmol. Vis. Sci. 47 (2006) 3754-3765]. Here we express and characterize the normal IMPDH1(546) and IMPDH1(595), together with their adRP-linked variants, D226N. The enzymatic activity of the purified IMPDH1(546), IMPDH1(595) and the D226N variants is indistinguishable from the canonical form. The intracellular distribution of IMPDH1(546) and IMPDH1(595) is also similar to the canonical IMPDH1 and unaffected by the D226N mutation. However, unlike the canonical IMPDH1, the retinal specific isoforms do not bind significant fractions of a random pool of oligonucleotides. This observation indicates that the C-terminal extension unique to the retinal isoforms blocks the nucleic acid binding site of IMPDH1, and thus uniquely regulates protein function within photoreceptors.     

Identification of mRNA-binding proteins during development: characterization of Bufo arenarum cellular nucleic acid binding protein

Ultraviolet irradiation was used to covalently cross-link poly(A)+RNA and associated proteins in eggs and embryos of the toad Bufo arenarum. Four major proteins with apparent sizes of 60, 57, 45 and 30-24 kDa were identified. It was observed that the same mRNA-binding proteins were isolated from eggs to gastrula and neural stages of development. The 30 kDa polypeptide, p30, appeared as the main ultraviolet (UV) cross-linked protein in the developmental stages analyzed. By means of polyclonal antibodies, it was determined that this polypeptide has a cytoplasmic localization and it was detected in liver, eggs and embryos. The presence of p30 was also analyzed by western blot during oogenesis and development. The 30 kDa polypeptide was present in all stages analyzed but it could not be detected in stages I-II of oogenesis. At the neural stage, the relative amount of p30 began to decrease, reaching its lowest levels after stages 26-30 (tail-bud in Bufo arenarum). On the basis of purification, immunoprecipitation and western blot assays the 30 kDa protein was identified as the Bufo arenarum cellular nucleic acid binding protein.     

Predicting nucleic acid binding interfaces from structural models of proteins

The function of DNA‐ and RNA‐binding proteins can be inferred from the characterization and accurate prediction of their binding interfaces. However, the main pitfall of various structure‐based methods for predicting nucleic acid binding function is that they are all limited to a relatively small number of proteins for which high‐resolution three‐dimensional structures are available. In this study, we developed a pipeline for extracting functional electrostatic patches from surfaces of protein structural models, obtained using the I‐TASSER protein structure predictor. The largest positive patches are extracted from the protein surface using the patchfinder algorithm. We show that functional electrostatic patches extracted from an ensemble of structural models highly overlap the patches extracted from high‐resolution structures. Furthermore, by testing our pipeline on a set of 55 known nucleic acid binding proteins for which I‐TASSER produces high‐quality models, we show that the method accurately identifies the nucleic acids binding interface on structural models of proteins. Employing a combined patch approach we show that patches extracted from an ensemble of models better predicts the real nucleic acid binding interfaces compared with patches extracted from independent models. Overall, these results suggest that combining information from a collection of low‐resolution structural models could be a valuable approach for functional annotation. We suggest that our method will be further applicable for predicting other functional surfaces of proteins with unknown structure. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

Fatty acid interactions with native and mutant fatty acid binding proteins

The interactions of long chain fatty acids (FA) with wild type (WT) fatty acid binding proteins (FABP) and engineered FABP mutants have been monitored to determine the equilibrium binding constants as well as the rate constants for binding and dissociation. These measurements have been done using the fluorescent probes, ADIFAB and ADIFAB2, that allow the determination of the free fatty acid (FFA) concentration in the reaction of FA with proteins and membranes. The results of these studies indicate that for WT proteins from adipocyte, heart, intestine, and liver, Kd values are in the nM range and affinities decrease with increasing aqueous solubility of the FA. Binding affinities for heart and liver are generally greater than those for adipocyte and intestine. Moreover, measurements of the rate constants indicate that binding equilibrium at 37øC is achieved within seconds for all FA and FABPs. These results, together with the level of serum (unbound) FFA, suggests a buffering action of FABPs that helps to maintain the intracellular concentration of FFA so that the flux of FFA between serum and cells occurs down a concentration gradient. Measurements of the temperature dependence of binding reveal that the free energy is predominately enthalpic and that the enthalpy of the reaction results from FA-FABP interactions within the binding cavity. The nature of these interactions were investigated by determining the thermodynamics of binding to engineered point mutants of the intestinal FABP. These measurements showed that binding affinities did not report accurately the changes in protein-FA interactions because changes in the binding entropy and enthalpy tend to compensate. For example, an alanine substitution for arginine 106 yields a 30 fold increase in binding affinity, because the loss in enthalpy due to the elimination of the favorable interaction between the FA carboxylate and Arg106, is more than compensated for by an increase in entropy. Thus understanding the effects of amino acid replacements on FA-FABP interactions requires measurements of enthalpy and entropy, in addition to affinity.     

Carnitine palmitoyltransferase activities: Effects of serum albumin,acyl-CoA binding protein and fatty acid binding protein

The carnitine palmitoyltransferase activity of various subcellular preparations measured with octanoyl-CoA as substrate was markedly increased by bovine serum albumin at low M concentrations of octanoyl-CoA. However, even a large excess (500 M) of this acyl-CoA did not inhibit the activity of the mitochondrial outer carnitine palmitoyltransferase, a carnitine palmitoyltransferase isoform that is particularly sensitive to inhibition by low M concentrations of palmitoyl-CoA. This bovine serum albumin stimulation was independent of the salt activation of the carnitine palmitoyltransferase activity. The effects of acyl-CoA binding protein (ACBP) and the fatty acid binding protein were also examined with palmitoyl-CoA as substrate. The results were in line with the findings of stronger binding of acyl-CoA to ACBP but showed that fatty acid binding protein also binds acyl-CoA esters. Although the effects of these proteins on the outer mitochondrial carnitine palmitoyltransferase activity and its malonyl-CoA inhibition varied with the experimental conditions, they showed that the various carnitine palmitoyltransferase preparations are effectively able to use palmitoyl-CoA bound to ACBP in a near physiological molar ratio of 1:1 as well as that bound to the fatty acid binding protein. It is suggested that the three proteins mentioned above effect the carnitine palmitoyltransferase activities not only by binding of acyl-CoAs, preventing acyl-CoA inhibition, but also by facilitating the removal of the acylcarnitine product from carnitine palmitoyltransferase. These results support the possibility that the acyl-CoA binding ability of acyl-CoA binding protein and of fatty acid binding protein have a role in acyl-CoA metabolismin vivo.Abbreviations ACBP acyl-CoA binding protein - BSA bovine serum albumin - CPT carnitine palmitoyltransferase - CPT₀ malonyl-CoA sensitive CPT of the outer mitochondrial membrane - CPT malonyl-CoA insensitive CPT of the inner mitochondrial membrane - OG octylglucoside - OMV outer membrane vesicles - IMV inner membrane vesicles Affiliated to the Department of Experimental Medicine, University of Montreal     

Arginine methylation of SARS-Cov-2 nucleocapsid protein regulates RNA binding,its ability to suppress stress granule formation,and viral replication

Viral proteins are known to be methylated by host protein arginine methyltransferases (PRMTs) necessary for the viral life cycle, but it remains unknown whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins are methylated. Herein, we show that PRMT1 methylates SARS-CoV-2 nucleocapsid (N) protein at residues R95 and R177 within RGG/RG motifs, preferred PRMT target sequences. We confirmed arginine methylation of N protein by immunoblotting viral proteins extracted from SARS-CoV-2 virions isolated from cell culture. Type I PRMT inhibitor (MS023) or substitution of R95 or R177 with lysine inhibited interaction of N protein with the 5’-UTR of SARS-CoV-2 genomic RNA, a property required for viral packaging. We also defined the N protein interactome in HEK293 cells, which identified PRMT1 and many of its RGG/RG substrates, including the known interacting protein G3BP1 as well as other components of stress granules (SGs), which are part of the host antiviral response. Methylation of R95 regulated the ability of N protein to suppress the formation of SGs, as R95K substitution or MS023 treatment blocked N-mediated suppression of SGs. Also, the coexpression of methylarginine reader Tudor domain-containing protein 3 quenched N protein–mediated suppression of SGs in a dose-dependent manner. Finally, pretreatment of VeroE6 cells with MS023 significantly reduced SARS-CoV-2 replication. Because type I PRMT inhibitors are already undergoing clinical trials for cancer treatment, inhibiting arginine methylation to target the later stages of the viral life cycle such as viral genome packaging and assembly of virions may represent an additional therapeutic application of these drugs.     

Molecular dynamics simulations of apo and holo forms of fatty acid binding protein 5 and cellular retinoic acid binding protein II reveal highly mobile protein,retinoic acid ligand,and water molecules

Structural and dynamic properties from a series of 300 ns molecular dynamics, MD, simulations of two intracellular lipid binding proteins, iLBPs, (Fatty Acid Binding Protein 5, FABP5, and Cellular Retinoic Acid Binding Protein II, CRABP-II) in both the apo form and when bound with retinoic acid reveal a high degree of protein and ligand flexibility. The ratio of FABP5 to CRABP-II in a cell may determine whether it undergoes natural apoptosis or unrestricted cell growth in the presence of retinoic acid. As a result, FABP5 is a promising target for cancer therapy. The MD simulations presented here reveal distinct differences in the two proteins and provide insight into the binding mechanism. CRABP-II is a much larger, more flexible protein that closes upon ligand binding, where FABP5 transitions to an open state in the holo form. The traditional understanding obtained from crystal structures of the gap between two β-sheets of the β-barrel common to iLBPs and the α-helix cap that forms the portal to the binding pocket is insufficient for describing protein conformation (open vs. closed) or ligand entry and exit. When the high degree of mobility between multiple conformations of both the ligand and protein are examined via MD simulation, a new mode of ligand motion that improves understanding of binding dynamics is revealed.     

The liver fatty acid binding protein - comparison of cavity properties of intracellular lipid-binding proteins

The crystal and solution structures of all of the intracellular lipid binding proteins (iLBPs) reveal a common -barrel framework with only small local perturbations. All existing evidence points to the binding cavity and a poorly delimited portal region as defining the function of each family member. The importance of local structure within the cavity appears to be its influence on binding affinity and specificity for the lipid. The portal region appears to be involved in the regulation of ligand exchange. Within the iLBP family, liver fatty acid binding protein or LFABP, has the unique property of binding two fatty acids within its internalized binding cavity rather than the commonly observed stoichiometry of one. Furthermore, LFABP will bind hydrophobic molecules larger than the ligands which will associate with other iLBPs. The crystal structure of LFABP contains two bound oleate molecules and provides the explanation for its unusual stoichiometry. One of the bound fatty acids is completely internalized and has its carboxylate interacting with an arginine and two serines. The second oleate represents an entirely new binding mode with the carboxylate on the surface of LFABP. The two oleates also interact with each other. Because of this interaction and its inner location, it appears the first oleate must be present before the second more external molecule is bound.     

Modern tools for identification of nucleic acid-binding proteins

Numerous biological mechanisms depend on nucleic acid--protein interactions. The first step to the understanding of these mechanisms is to identify interacting molecules. Knowing one partner, the identification of other associated molecular species can be carried out using affinity-based purification procedures. When the nucleic acid-binding protein is known, the nucleic acid can be isolated and identified by sensitive techniques such as polymerase chain reaction followed by DNA sequencing or hybridization on chips. The reverse identification procedure is less straightforward in part because interesting nucleic acid-binding proteins are generally of low abundance and there are no methods to amplify amino acid sequences. In this article, we will review the strategies that have been developed to identify nucleic acid-binding proteins. We will focus on methods permitting the identification of these proteins without a priori knowledge of protein candidates.     

Fatty acid binding proteins from developing human fetal brain: Characterization and binding properties

Two fatty acid binding proteins (FABPs) of identicalM _r, 13 kDa, have been isolated from developing human fetal brain. A delipidated 105,000 g supernatant was incubated with [1 -¹⁴C]oleate and subjected to a Sephacryl S-200 column followed by gel filtration chromatography on a Sephadex G-75 column and ion-exchange chromatography using a DEAE-Sephacel column. Purity was checked by UV spectroscopy, SDS-PAGE, isoelectric focusing and immunological cross-reactivity. The two FABPs designated as DE-I (pI 5.4) and DE-II (pI 6.9) showed cross-reactivity with each other and no alteration at the antigenic site during intrauterine development. Anti-human fetal brain FABP does not cross-react with purified human fetal heart, gut, lung or liver FABPs. The molecular mass of DE-I and DE-II is lower than those of fetal lung and liver FABPs. Like liver FABP, these proteins bind organic anions, fatty acids and acyl CoAs but differ in their binding affinities. Both DE-I and DE-II have been found to exhibit higher affinity for oleate (K _d = 0.23 μM) than palmitate (K _d = 0.9μM) or palmitoyl-CoA (K _d = 0.96 μM), with DE-I binding less fatty acids than DE-II. DE-II is more efficient in transferring fatty acid from phospholipid vesjcles than DE-I indicating that human fetal brain FABPs may play a significant role in fatty acid transport in developing fetal brain.     

Epigenetic regulation: methylation of histone and non-histone proteins

Histone methylation is believed to play important roles in epigenetic memory in various biological processes. However, questions like whether the methylation marks themselves are faithfully transmitted into daughter cells and through what mechanisms are currently under active investigation. Previously, methylation was considered to be irreversible, but the recent discovery of histone lysine demethylases revealed a dynamic nature of histone methylation regulation on four of the main sites of methylation on histone H3 and H4 tails (H3K4, H3K9, H3K27 and H3K36). Even so, it is still unclear whether demethylases specific for the remaining two sites, H3K79 and H4K20, exist. Furthermore, besides histone proteins, the lysine methylation and demethylation also occur on non-histone proteins, which are probably subjected to similar regulation as histones. This review discusses recent progresses in protein lysine methylation regulation focusing on the above topics, while referring readers to a number of recent reviews for the biochemistry and biology of these enzymes     

A method for detecting distant evolutionary relationships between protein or nucleic acid sequences in the presence of deletions or insertions

Summary A method for detecting homology between two protein or nucleic acid sequences which require insertions or deletions for optimum alignment has been devised for use with a computer. Sequences are assessed for possible relationship by Monte Carlo methods involving comparisons between the alignment of the real sequences and alignments of randomly scrambled sequences of the Same composition as the real sequences, each alignment having the optimum number of gaps. As each gap is successively introduced into a comparison (real or random) a maximum score is determined from the similarity of the aligned residues. From the distribution of the maximum alignment scores of randomly scrambled sequences having the same number of gaps, the percentage of random comparisons having higher scores is determined, and the smallest of these percentage levels for each pair of sequences (real or random) indicates the optimum alignment. The fraction of the comparisons of random sequences having percentage levels at their optimum alignment below that of the real sequence comparison at its optimum estimates the probability that such an alignment might have arisen by chance. Related sequences are detected since their optimum alignment score, by virtue of a contribution from ancestral homology in addition to optimised random considerations, occupies a more extreme position in the appropriate frequency distribution of scores than do the majority of optimum scores of randomly scrambled sequences in their appropriate distributions.Application of this optimum match method of sequence comparison shows that the sensitivity of the maximum match method of Needleman and Wunsch (1970) decreases quite dramatically with sequence comparisons which require only a few gaps for a reasonable alignment, or when sequences differ greatly in length. The maximum match method as applied by Barker and Dayhoff (1972) has the additional disadvantage that deletions which have occurred in the longer of two homologous protein sequences further decrease the sensitivity of detection of relationship. The constrained match method of Sankoff and Cedergren (1973) is seen to be misleading since large increments in the alignment score from added gaps do not necessarily result in a high total alignment score required to demonstrate sequence homology.     

肝型脂肪酸结合蛋白研究进展

肝型脂肪酸结合蛋白（liver fatty acid binding protein,L-FABP）是脂肪酸结合蛋白（fatty acid binding proteins,FABPs）家族重要的成员,在肝脏、小肠、肾脏等组织中均有表达。L-FABP在不饱和脂肪酸、饱和脂肪酸、胆固醇、胆汁酸等转运过程中扮演重要角色。目前研究显示L-FABP在脂肪肝、肝硬化以及肝癌发生发展中起到重要作用,并有望作为肝损伤的早期检测指标。此外,新近研究发现尿中L-FABP水平还可以用于预测1型糖尿病患者的临床结局。在2型糖尿病中,尿中L-FABP与糖尿病性肾病的病程有密切关系。主要就L-FABP的特性、结构及其与疾病的关系做一综述。     

Protein and ligand adaptation in a retinoic acid binding protein.

A retinoic acid binding protein isolated from the lumen of the rat epididymis (ERABP) is a member of the lipocalin superfamily. ERABP binds both the all-trans and 9-cis isomers of retinoic acid, as well as the synthetic retinoid (E)-4-[2-(5,6,7,8)-tetrahydro-5,5,8,8-tetramethyl-2 napthalenyl-1 propenyl]-benzoic acid (TTNPB), a structural analog of all-trans retinoic acid. The structure of ERABP with a mixture of all-trans and 9-cis retinoic acid has previously been reported. To elucidate any structural differences in the protein when bound to the all-trans and 9-cis isomers, the structures of all-trans retinoic acid-ERABP and 9-cis retinoic acid ERABP were determined. Our results indicate that the all-trans isomer of retinoic acid adopts an 8-cis structure in the binding cavity with no concomitant conformational change in the protein. The structure of TTNPB-ERABP is also reported herein. To accommodate this all-trans analog, which cannot readily adopt a cis-like structure, alternative positioning of critical binding site side chains is required. Consequently, both protein and ligand adaption are observed in the formation of the various holo-proteins.     

Effect of Centchroman (67/20) and 78/224 on nucleic acid and protein biosynthesis during implantation in rats

The biosynthesis of nucleic acids and proteins was studied in rat uterus by following the incorporation of [³H]-thymidine, [³H]-uridineand[¹⁴C]-leucinein control and pregnant rats in the presence and absence of two anti-implantation drugs. One of the drugs, 78/224 caused a significant increase in incorporation whereas the other drug, Centchroman, caused an inhibition in incorporation of all the three precursors. The implications of these changes in the light of estrogenicity, agonist and antagonist actions of anti-estrogens have been analysed. The importance of homeostatic mechanisms involved in nucleic acids and proteins for the maintenance of constant internal milieu for blastocyst attachment has been discussed.     

羧酸还原酶的研究进展

羧酸还原酶(carboxylic acid reductases,CARs)可以催化羧酸还原为相应的醛,反应条件温和、拥有广阔的底物范围且副反应较少。本文旨在综述近年来羧酸还原酶系统发育、结构与催化机理、蛋白质工程和固定化工程等方面的研究成果,揭示其作为重要工具酶在生物转化及合成生物学中的应用前景。