Escherichia coli SecA truncated at its termini is functional and dimeric

Terminal residues in SecA, the dimeric ATPase motor of bacterial preprotein translocase, were proposed to be required for function and dimerization. To test this, we generated truncation mutants of the 901aa long SecA of Escherichia coli. We now show that deletions of carboxy-terminal domain (CTD), the extreme CTD of 70 residues, or of the N-terminal nonapeptide or of both, do not compromise protein translocation or viability. Deletion of additional C-terminal residues upstream of CTD compromised function. Functional truncation mutants like SecA9-861 are dimeric, conformationally similar to SecA, fully competent for nucleotide and SecYEG binding and for ATP catalysis. Our data demonstrate that extreme terminal SecA residues are not essential for SecA catalysis and dimerization.     

Characterising the Binding Specificities of the Subunits Associated with the KMT2/Set1 Histone Lysine Methyltransferase

KMT2/Set1 is the catalytic subunit of the complex of proteins associated with Set1 (COMPASS) that is responsible for the methylation of lysine 4 of histone H3 (H3K4) in Saccharomyces cerevisiae. Whereas monomethylated H3K4 (H3K4me1) is found throughout the genome, di- (H3K4me2) and tri- (H3K4me3) methylated H3K4 are enriched at specific loci, which correlates with the promoter and 5′-ends of actively transcribed genes in the case of H3K4me3. The COMPASS subunits contain a number of domains that are conserved in homologous complexes in higher eukaryotes and are reported to interact with modified histones. However, the exact organization of these subunits and their role within the complex have not been elucidated. In this study we showed that: (1) subunits Swd1 and Swd3 form a stable heterodimer that dissociates upon binding to a modified H3K4me2 tail peptide, suggesting a regulatory role in COMPASS; (2) the affinity of the subunit Spp1 for modified histone H3 substrates is much higher than that of Swd1 and Swd3; (3) Spp1 has a preference for H3K4me2/3 methylation state; and (4) Spp1 contains a high-affinity DNA-binding domain in the previously uncharacterised C-terminal region. These data allow us to suggest a mechanism for the regulation of COMPASS activity at an actively transcribed gene.     

Evidence for a pathway that facilitates nitric oxide diffusion in the brain

Nitric oxide (NO) is a diffusible messenger that conveys information based on its concentration dynamics, which is dictated by the interplay between its synthesis, inactivation and diffusion. Here, we characterized NO diffusion in the rat brain in vivo. By direct sub-second measurement of NO, we determined the diffusion coefficient of NO in the rat brain cortex. The value of 2.2 × 10⁻⁵ cm²/s obtained in vivo was only 14% lower than that obtained in agarose gel (used to evaluate NO free diffusion). These results reinforce the view of NO as a fast diffusing messenger but, noticeably, the data indicates that neither NO diffusion through the brain extracellular space nor homogeneous diffusion in the tissue through brain cells can account for the similarity between NO free diffusion coefficient and that obtained in the brain. Overall, the results support that NO diffusion in brain tissue is heterogeneous, pointing to the existence of a pathway that facilitates NO diffusion, such as cell membranes and other hydrophobic structures.     

Identification of a repeated domain within mammalian alpha-synemin that interacts directly with talin

The type VI intermediate filament (IF) protein synemin is a unique member of the IF protein superfamily. Synemin associates with the major type III IF protein desmin forming heteropolymeric intermediate filaments (IFs) within developed mammalian striated muscle cells. These IFs encircle and link all adjacent myofibrils together at their Z-lines, as well as link the Z-lines of the peripheral layer of cellular myofibrils to the costameres located periodically along and subjacent to the sarcolemma. Costameres are multi-protein assemblies enriched in the cytoskeletal proteins vinculin, alpha-actinin, and talin. We report herein a direct interaction of human alpha-synemin with the cytoskeletal protein talin by protein-protein interaction assays. The 312 amino acid insert (SNTIII) present only within alpha-synemin binds to the rod domain of talin in vitro and co-localizes with talin at focal adhesion sites within mammalian muscle cells. Confocal microscopy studies showed that synemin co-localizes with talin within the costameres of human skeletal muscle cells. Analysis of the primary sequences of human alpha- and beta-synemins revealed that SNTIII is composed of seven tandem repeats, each containing a specific Ser/Thr-X-Arg-His/Gln (S/T-X-R-H/Q) motif. Our results suggest human alpha-synemin plays an essential role in linking the heteropolymeric IFs to adherens-type junctions, such as the costameres within mammalian striated muscle cells, via its interaction with talin, thereby helping provide mechanical integration for the muscle cell cytoskeleton.     

Crystallographic survey of active sites of an unclassified glutathione transferase from Bombyx mori

Background

Glutathione transferase (GST) catalyzes a major step in the xenobiotic detoxification pathway. We previously identified a novel, unclassified GST that is upregulated in an insecticide-resistant silkworm (Bombyx mori) upon insecticide exposure. Here, we sought to further characterize this GST, bmGSTu, by solving and refining its crystal structure and identifying its catalytic residues.

Methods

The structure of wild-type bmGSTu was determined with a resolution of 2.1 Å by synchrotron radiation and molecular modeling. Potential catalytic residues were mutated to alanine by means of site-directed mutagenesis, and kinetic data determined for wild-type and mutated bmGSTu.

Results

We found that bmGSTu occurred as a dimer, and that, like other GSTs, each subunit displayed a G-site and an H-site in the active center. Bound glutathione could be localized at the G-site. Kinetic data of the mutated forms of bmGSTu show that Val55, Glu67, and Ser68 in the G-site are important for catalysis. Furthermore, the H-site showed some unique features.

Conclusions

This is the first study to our knowledge to elucidate the molecular conformation of this B. mori GST. Our results indicate that residues Val55, Glu67, and Ser68, as well as Tyr7 and Ser12, in the glutathione-binding region of bmGSTu are critical for catalytic function.

General Significance

Our results, together with our previous finding that bmGSTu was preferentially induced in an insecticide-resistant strain, support the idea that bmGSTu functions in the transformation of exogenous chemical agents. Furthermore, the unique features observed in bmGSTu may shed light on mechanisms of insecticide resistance.     

Visualizing Compaction of Polysomes in Bacteria

During protein synthesis, many translating ribosomes are bound together with an mRNA molecule to form polysomes (or polyribosomes). While the spatial organization of bacterial polysomes has been well studied in vitro, little is known about how they cluster when cellular conditions are highly constrained. To better understand this, we used electron tomography, template matching, and three-dimensional modeling to analyze the supramolecular network of ribosomes after induction of translational pauses. In Escherichia coli, we overexpressed an mRNA carrying a polyproline motif known to induce pausing during translation. When working with a strain lacking transfer-messenger RNA, the principle actor in the “trans-translation” rescuing system, the cells survived the hijacking of the translation machinery but this resulted in a sharp modification of the ribosomal network. The results of our experiments demonstrate that single ribosomes are replaced with large amounts of compacted polysomes. These polysomes are highly organized, principally forming hairpins and dimers of hairpins that stack together. We propose that these spatial arrangements help maintain translation efficiency when the rescue systems are absent or overwhelmed.     

The mPlrp2 and mClps genes are involved in the hydrolysis of retinyl esters in the mouse liver

Retinyl esters are the major chemical forms of vitamin A stored in the liver, and can be delivered to peripheral tissues for conversion into biologically active forms. The function and regulation of the hepatic genes that are potentially involved in catalyzing the hydrolysis of retinyl esters remain unclear. Here we show that two lipid hydrolytic genes, pancreatic-related protein 2 (mPlrp2) and procolipase (mClps), expressed specifically in the mouse pancreas, are associated with the ratio of S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy). Light illumination deficiency or administration of 5'-AMP elevated the ratio of AdoMet to AdoHcy and induced the expression in the liver of mPlrp2 and mClps, which was blocked by all-trans retinoic acid. Mice fed a vitamin A-free diet exhibited increased activation of hepatic mPlrp2 and mClps expression, which was associated with increased methylation of histone H3K4 residues located near the mPlrp2 and mClps promoters. Inhibition of hepatic mPlrp2 and mClps expression by a methylase inhibitor, methylthioadenosine, markedly decreased plasma retinol levels in these mice. The activated hepatic stellate cell (HSC)-T6 cell line specifically expressed mClps and mPlrp2. Inhibition of mClps gene expressions by short hairpin RNA (shRNA) decreased hydrolysis of retinyl esters in the HSC-T6 cell line. These data suggest that the conditional expression of mPlrp2 and mClps is involved in the hydrolysis of retinyl esters in the mouse liver.     

Structures of human N-Acetylglucosamine kinase in two complexes with N-Acetylglucosamine and with ADP/glucose: insights into substrate specificity and regulation

N-Acetylglucosamine (GlcNAc), a major component of complex carbohydrates, is synthesized de novo or salvaged from lysosomally degraded glycoconjugates and from nutritional sources. The salvage pathway requires that GlcNAc kinase converts GlcNAc to GlcNAc-6-phosphate, a component utilized in UDP-GlcNAc biosynthesis or energy metabolism. GlcNAc kinase belongs to the sugar kinase/Hsp70/actin superfamily that catalyze phosphoryl transfer from ATP to their respective substrates, and in most cases catalysis is associated with a large conformational change in which the N-terminal small and C-terminal large domains enclose the substrates. Here we report two crystal structures of homodimeric human GlcNAc kinase, one in complex with GlcNAc and the other in complex with ADP and glucose. The active site of GlcNAc kinase is located in a deep cleft between the two domains of the V-shaped monomer. The enzyme adopts a "closed" configuration in the GlcNAc-bound complex and GlcNAc interacts with residues of both domains. In addition, the N-acetyl methyl group contacts residues of the other monomer in the homodimer, a unique feature compared to other members of the sugar kinase/Hsp70/actin superfamily. This contrasts an "open" configuration in the ADP/glucose-bound structure, where glucose cannot form these interactions, explaining its low binding affinity for GlcNAc kinase. Our results support functional implications derived from apo crystal structures of GlcNAc kinases from Chromobacter violaceum and Porphyromonas gingivalis and show that Tyr205, which is phosphorylated in thrombin-activated platelets, lines the GlcNAc binding pocket. This suggests that phosphorylation of Tyr205 may modulate GlcNAc kinase activity and/or specificity.     

S100A6 Competes with the TAZ2 Domain of p300 for Binding to p53 and Attenuates p53 Acetylation

S100A6 is a calcium binding protein that, like some other members of the S100 protein family, is able to bind p53. This interaction may be physiologically relevant considering the numerous connotations of S100 proteins and of S100A6, in particular, with cancer and metastasis. In this work, we show that the interaction with S100A6 is limited to unmodified or phosphorylated p53 and is inhibited by p53 acetylation. Using in vitro acetylation assay, we show that the presence of S100A6 attenuates p53 acetylation by p300. Furthermore, using ELISA, we show that S100A6 and the TAZ2 domain of p300 bind p53 with similar affinities and that S100A6 effectively competes with TAZ2 for binding to p53. Our results add another element to the complicated scheme of p53 activation.     

Identification and clarification of the role of key active site residues in bacterial glutathione S-transferase zeta/maleylpyruvate isomerase

The maleylpyruvate isomerase NagL from Ralstonia sp. strain U2, which has been structurally characterized previously, catalyzes the isomerization of maleylpyruvate to fumarylpyruvate. It belongs to the class zeta glutathione S-transferases (GSTZs), part of the cytosolic GST family (cGSTs). In this study, site-directed mutagenesis was conducted to probe the functions of 13 putative active site residues. Steady-state kinetic information for mutants in the reduced glutathione (GSH) binding site, suggested that (a) Gln64 and Asp102 interact directly with the glutamyl moiety of glutathione, (b) Gln49 and Gln64 are involved in a potential electron-sharing network that influences the ionization of the GSH thiol. The information also suggests that (c) His38, Asn108 and Arg109 interact with the GSH glycine moiety, (d) His104 has a role in the ionization of the GSH sulfur and the stabilization of the maleyl terminal carboxyl group in the reaction intermediate and (e) Arg110 influences the electron distribution in the active site and therefore the ionization of the GSH thiolate. Kinetic data for mutants altered in the substrate-binding site imply that (a) Arg8 and Arg176 are critical for maleylpyruvate orientation and enolization, and (b) Arg109 (exclusive to NagL) participates in k_cat regulation. Surprisingly, the T11A mutant had a decreased GSH K_m value, whereas little impact on maleylpyruvate kinetics was observed, suggesting that this residue plays an important role in GSH binding. An evolutionary trend in this residue appears to have developed not only in prokaryotic and eukaryotic GSTZs, but also among the wider class of cGSTs.     

RCC1 Uses a Conformationally Diverse Loop Region to Interact with the Nucleosome: A Model for the RCC1-Nucleosome Complex

The binding of RCC1 (regulator of chromosome condensation 1) to chromatin is critical for cellular processes such as mitosis, nucleocytoplasmic transport, and nuclear envelope formation because RCC1 recruits the small GTPase Ran (Ras-related nuclear protein) to chromatin and sets up a Ran-GTP gradient around the chromosomes. However, the molecular mechanism by which RCC1 binds to nucleosomes, the repeating unit of chromatin, is not known. We have used biochemical approaches to test structural models for how the RCC1 β-propeller protein could bind to the nucleosome. In contrast to the prevailing model, RCC1 does not appear to use the β-propeller face opposite to its Ran-binding face to interact with nucleosomes. Instead, we find that RCC1 uses a conformationally flexible loop region we have termed the switchback loop in addition to its N-terminal tail to bind to the nucleosome. The juxtaposition of the RCC1 switchback loop to its Ran binding surface suggests a novel mechanism for how nucleosome-bound RCC1 recruits Ran to chromatin. Furthermore, this model accounts for previously unexplained observations for how Ran can interact with the nucleosome both dependent and independent of RCC1 and how binding of the nucleosome can enhance RCC1's Ran nucleotide exchange activity.     

Development of glutathione-coupled cantilever for the single-molecule force measurement by scanning force microscopy

The accuracy and the fidelity of a single-molecule force measurement largely rely on how the molecule of interest is attached to the solid substrate surface (bead, cantilever, cover glass and etc.). A site-specific attachment of a protein without affecting its structure and enzymatic function has been a major concern. Here, we established a glutathione-coupled cantilever to which any glutathione S-transferase (GST)-fused proteins can be attached in a desired direction. The rupture force between glutathione and GST was approximately 100 pN on average. By using this cantilever, we succeeded in measuring the interaction force between importin alpha and importin beta.     

Different requirements of the kinase and UHM domains of KIS for its nuclear localization and binding to splicing factors

The protein kinase KIS is made by the juxtaposition of a unique kinase domain and a C-terminal domain with a U2AF homology motif (UHM), a sequence motif for protein interaction initially identified in the heterodimeric pre-mRNA splicing factor U2AF. This domain of KIS is closely related to the C-terminal UHM domain of the U2AF large subunit, U2AF⁶⁵. KIS phosphorylates the splicing factor SF1, which in turn enhances SF1 binding to U2AF⁶⁵ and the 3′ splice site, an event known to take place at an early step of spliceosome assembly. Here, the analysis of the subcellular localization of mutated forms of KIS indicates that the kinase domain of KIS is the necessary domain for its nuclear localization. As in the case of U2AF⁶⁵, the UHM-containing C-terminal domain of KIS is required for binding to the splicing factors SF1 and SF3b155. The efficiency of KIS binding to SF1 and SF3b155 is similar to that of U2AF⁶⁵ in pull-down assays. These results further support the functional link of KIS with splicing factors. Interestingly, when compared to other UHM-containing proteins, KIS presents a different specificity for the UHM docking sites that are present in the N-terminal region of SF3b155, thus providing a new insight into the variety of interactions mediated by UHM domains.     

Mapping of two O-GlcNAc modification sites in the capsid protein of the potyvirus Plum pox virus

A large number of O-linked N-acetylglucosamine (O-GlcNAc) residues have been mapped in vertebrate proteins, however targets of O-GlcNAcylation in plants still have not been characterized. We show here that O-GlcNAcylation of the N-terminal region of the capsid protein of Plum pox virus resembles that of animal proteins in introducing O-GlcNAc monomers. Thr-19 and Thr-24 were specifically O-GlcNAcylated. These residues are surrounded by amino acids typical of animal O-GlcNAc acceptor sites, suggesting that the specificity of O-GlcNAc transferases is conserved among plants and animals. In laboratory conditions, mutations preventing O-GlcNAcylation of Thr-19 and Thr-24 did not have noticeable effects on PPV competence to infect Prunus persicae or Nicotiana clevelandii. However, the fact that Thr-19 and Thr-24 are highly conserved among different PPV strains suggests that their O-GlcNAc modification could be relevant for efficient competitiveness in natural conditions.     

Charged Amino Acids in a Preprotein Inhibit SecA-Dependent Protein Translocation

Sec translocase catalyzes membrane protein insertion and translocation. We have introduced stretches of charged amino acid residues into the preprotein proOmpA and have analyzed their effect on in vitro protein translocation into Escherichia coli inner membrane vesicles. Both negatively and positively charged amino acid residues inhibit translocation of proOmpA, yielding a partially translocated polypeptide chain that blocks the translocation site and no longer activates preprotein-stimulated SecA ATPase activity. Stretches of positively charged residues are much stronger translocation inhibitors and suppressors of the preprotein-stimulated SecA ATPase activity than negatively charged residues. These results indicate that both clusters of positively and negatively charged amino acids are poor substrates for the Sec translocase and that this is reflected by their inability to stimulate the ATPase activity of SecA.     

Protein kinase C-mediated phosphorylation of the two polybasic regions of synaptotagmin VI regulates their function in acrosomal exocytosis

We have previously reported that synaptotagmin VI is present in human sperm cells and that a recombinant protein containing the C2A and C2B domains abrogates acrosomal exocytosis in permeabilized spermatozoa, an effect that was regulated by phosphorylation. In this report, we show that each individual C2 domain blocks acrosomal exocytosis. The inhibitory effect was completely abrogated by phosphorylation of the domains with purified PKCbetaII. We found by site-directed mutagenesis that Thr418 and/or Thr419 in the polybasic region (KKKTTIK) of the C2B domain--a key region for the function of synaptotagmins--are the PKC target that regulates its inhibitory effect on acrosomal exocytosis. Similarly, we showed that Thr284 in the polybasic region of C2A (KCKLQTR) is the target for PKC-mediated phosphorylation in this domain. An antibody that specifically binds to the phosphorylated polybasic region of the C2B domain recognized endogenous phosphorylated synaptotagmin in the sperm acrosomal region. The antibody was inhibitory only at early stages of exocytosis in sperm acrosome reaction assays, and the immunolabeling decreased upon sperm stimulation, indicating that the protein is dephosphorylated during acrosomal exocytosis. Our results indicate that acrosomal exocytosis is regulated through the PKC-mediated phosphorylation of conserved threonines in the polybasic regions of synaptotagmin VI.     

The Tudor Domain of the PHD Finger Protein 1 Is a Dual Reader of Lysine Trimethylation at Lysine 36 of Histone H3 and Lysine 27 of Histone Variant H3t

PHF1 associates with the Polycomb repressive complex 2 and it was demonstrated to stimulate its H3K27-trimethylation activity. We studied the interaction of the PHF1 Tudor domain with modified histone peptides and found that it recognizes H3K36me3 and H3tK27me3 (on the histone variant H3t) and that it uses the same trimethyllysine binding pocket for the interaction with both peptides. Since both peptide sequences are very different, this result indicates that reading domains can have dual specificities. Sub-nuclear localization studies of full-length PHF1 in human HEK293 cells revealed that it co-localizes with K27me3, but not with K36me3, and that this co-localization depends on the trimethyllysine binding pocket indicating that K27me3 is an in vivo target for the PHF1 Tudor domain. Our data suggest that PHF1 binds to H3tK27me3 in human chromatin, and H3t has a more general role in Polycomb regulation.     

The effect of leader peptide mutations on the biological function of bovine myostatin gene

The growth of muscle fibers can be negatively regulated by bovine myostatin. The first two exons of myostatin gene code for the N-propeptide and its third exon codes for the C-polypeptide. Myostatin is secreted as a latent configuration formed by dimerization of two matured C peptides non-covalently linked with the N terminal pro-peptide. Pro-peptide has two distinct functions in guiding protein folding and regulating biological activity of myostatin. When the structure of the leader peptide is altered via mutations resulting in more tight binding with the mature peptide, myostatin function is inhibited, resulting in the changes of P21 and CDK2 expression levels which are relatedto the regulation of cell cycle. In the present study, the coding region of bMSTN (bovine myostatin) gene was amplified and mutated (A224C and G938A) through fusion PCR, and the mutated bMSTN gene (bMSTN-mut) was inserted in frame into the pEF1a-IRES-DsRed-Express2 vector and transfected into bovine fibroblast cells. The expression levels of bMSTN-mut, P21 and CDK2 (cyclin dependent kinase 2) were examined with qPCR and Western-blotting. Changes in cell cycle after transfection were also analyzed with flow cytometry. The results indicated that leader peptide mutation resulted in down-regulation of P21 expression levels and up-regulation of CDK2 expression levels. The flow cytometry results showed that the proportion of cells in the G0/G1-phase was lower and that of cells in the S-phase was higher in bMSTN-mut transfected group than that in the control group. The proliferation rate of bMSTN-mut transfected cells was also significantly higher than that of the control cells. In conclusion, the studies have shown that the pEF1a-IRES-DsRed-Express2–bMSTN-mut recombinant plasmid could effectively promote the proliferation of bovine fibroblast cells. The site-directed mutagenesis of bMSTN gene leader peptide and in vitro expression in bovine fibroblast cells could be helpful to further the studies of bMSTN in regulating bovine muscle cell growth and development.     

Requirement of the transmembrane semaphorin Sema4C for myogenic differentiation

Semaphorins constitute a large family of signaling proteins that contribute to axonal guidance. Here we demonstrate that the transmembrane semaphorin Sema4C is up-regulated both in the early stage of differentiation of C2C12 mouse skeletal myoblasts into myotubes and during injury-induced muscle regeneration in vivo. Depletion of Sema4C in C2C12 cells resulted in marked attenuation of myotube formation. A fusion protein containing the extracellular Sema domain and a peptide corresponding to the intracellular COOH-terminal region of Sema4C each inhibited the differentiation of C2C12 cells. These findings indicate that Sema4C-mediated interaction among myoblasts plays an important role in terminal myogenic differentiation.