Human hepatoma cell line HuH-7 is an effective cellular system to produce recombinant factor IX with improved post-translational modifications.

Recombinant factor IX (rFIX) is increasingly used to treat patients with hemophilia B. CHO (Chinese Hamster Ovary) cells are commonly used for the production of rFIX but they have a limited capacity for introducing post-translational modifications (PTM) leading to incomplete γ-carboxylation, low phosphorylation and sulfation profiles as compared with plasma-derived preparations. Imperfect PTM might have an impact on the activity of Factor IX molecule. Several studies in animal models as well as clinical trials have previously reported a lower recovery of rFIX compared to plasma-derived FIX concentrates. In the present study, we aimed to produce a rFIX having a profile of PTM similar to plasma-derived FIX, using human hepatoma cell line HuH-7. We showed that rFIX produced by HuH-7 cells followed the classical intracellular pathway before secretion. In addition, improved PTM were associated with fully active molecule compared to plasma-derived and recombinant control FIX molecules. Secreted rFIX presented as a single band at the correct molecular weight. HuH-7 cellular clones were obtained and they secreted a biologically active human FIX. FIX was then purified for a detailed evaluation of PTM. Glycosylation and sialylation profiles were similar to plasma-derived and rFIX and mass spectrometry analysis demonstrated the presence of phosphorylated and sulfated forms of rFIX. These data strongly support that HuH-7 cells may represent an effective cellular system for production of rFIX exhibiting PTM similar to plasma-derived FIX.

Identification of two carbonic anhydrases in the mantle of the European Abalone Haliotis tuberculata (Gastropoda, Haliotidae): phylogenetic implications.

Carbonic anhydrases (CAs) represent a diversified family of metalloenzymes that reversibly catalyze the hydration of carbon dioxide. They are involved in a wide range of functions, among which is the formation of CaCO(3) skeletons in metazoans. In the shell-forming mantle tissues of mollusks, the location of the CA catalytic activity is elusive and gives birth to contradicting views. In the present paper, using the European abalone Haliotis tuberculata, a key model gastropod in biomineralization studies, we identified and characterized two CAs (htCA1 and htCA2) that are specific of the shell-forming mantle tissue. We analyzed them in a phylogenetic context. Combining various approaches, including proteomics, activity tests, and in silico analyses, we showed that htCA1 is secreted but is not incorporated in the organic matrix of the abalone shell and that htCA2 is transmembrane. Together with previous studies dealing with molluskan CAs, our findings suggest two possible modes of action for shell mineralization: the first mode applies to, for example, the bivalves Unio pictorum and Pinctada fucata, and involves a true CA activity in their shell matrix; the second mode corresponds to, for example, the European abalone, and does not include CA activity in the shell matrix. Our work provides new insight on the diversity of the extracellular macromolecular tools used for shell biomineralization study in mollusks.

The shell matrix of the pulmonate land snail Helix aspersa maxima.

In mollusks, the shell mineralization process is controlled by an array of proteins, glycoproteins and polysaccharides that collectively constitute the shell matrix. In spite of numerous researches, the shell protein content of a limited number of model species has been investigated. This paper presents biochemical data on the common edible land snail Helix aspersa maxima, a model organism for ecotoxicological purposes, which has however been poorly investigated from a biomineralization viewpoint. The shell matrix of this species was extracted and analyzed biochemically for functional in vitro inhibition assay, for amino acid and monosaccharides compositions. The matrix was further analyzed on 1 and 2D gels and short partial protein sequences were obtained from 2D gel spots. Serological comparisons were established with a set of heterologous antibodies, two of which were subsequently used for subsequent immunogold localization of matrix components. Our data suggest that the shell matrix of Helix aspersa maxima may differ widely from the shell secretory repertoire of the marine mollusks studied so far, such as the gastropod Haliotis or the pearl oyster Pinctada. In particular, most of the biochemical properties generally attributed to soluble shell matrices, such as calcium-binding capability, or the capacity to interfere in vitro with the precipitation of calcium carbonate or to inhibit the precipitation of calcium carbonate, were not recorded with this matrix. This drastic change in the biochemical properties of the landsnail shell matrix puts into question the existence of a unique molecular model for molluscan shell formation, and may be related to terrestrialisation.

Cutaneous human papillomavirus type 38 E7 regulates actin cytoskeleton structure for increasing cell proliferation through CK2 and the eukaryotic elongation factor 1A.

We previously reported that the oncoproteins E6 and E7 from cutaneous human papillomavirus type 38 (HPV38) can immortalize primary human keratinocytes in vitro and sensitize transgenic mice to develop skin cancer in vivo. Immunofluorescence staining revealed that human keratinocytes immortalized by HPV38 E6 and E7 display fewer actin stress fibers than do control primary keratinocyte cells, raising the possibility of a role of the viral oncoproteins in the remodeling of the actin cytoskeleton. In this study, we show that HPV38 E7 induces actin stress fiber disruption and that this phenomenon correlates with its ability to downregulate Rho activity. The downregulation of Rho activity by HPV38 E7 is mediated through the activation of the CK2-MEK-extracellular signal-regulated kinase (ERK) pathway. In addition, HPV38 E7 is able to induce actin fiber disruption by binding directly to eukaryotic elongation factor 1A (eEF1A) and abolishing its effects on actin fiber formation. Finally, we found that the downregulation of Rho activity by HPV38 E7 through the CK2-MEK-ERK pathway facilitates cell growth proliferation. Taken together, our data support the conclusion that HPV38 E7 promotes keratinocyte proliferation in part by negatively regulating actin cytoskeleton fiber formation through the CK2-MEK-ERK-Rho pathway and by binding to eEF1A and inhibiting its effects on actin cytoskeleton remodeling.

Molecular evolution of mollusc shell proteins: insights from proteomic analysis of the edible mussel Mytilus.

Shell matrix proteins (SMPs) that are embedded within calcified layers of mollusc shells are believed to play an essential role in controlling the biomineral synthesis and in increasing its mechanical properties. Among the wide diversity of mollusc shell textures, nacro-prismatic shells represent a tremendous opportunity for the investigation of the SMP evolution. Indeed, nacro-prismatic texture appears early in Cambrian molluscs and is still present in the shell of some bivalves, gastropods, cephalopods and very likely also, of some monoplacophorans. One key question is to know whether these shells are constructed from similar matrix protein assemblages, i.e. whether they share a common origin. Most of the molecular data published so far are restricted to two genera, the bivalve Pinctada and the gastropod Haliotis. The shell protein content of these two genera are clearly different, suggesting independent origins or considerable genetic drift from a common ancestor. In order to describe putatively conserved mollusc shell proteins, here we have investigated the SMP set of a new bivalve model belonging to another genera, the edible mussel Mytilus, using an up-to-date proteomic approach based on the interrogation of more than 70,000 EST sequences, recently available from NCBI public databases. We describe nine novel SMPs, among which three are completely novel, four are homologues of Pinctada SMPs and two are very likely homologues of Haliotis SMPs. This latter result constitutes the first report of conserved SMPs between bivalves and gastropods. More generally, our data suggest that mollusc SMP set may follow a mosaic pattern within the different mollusc models (Mytilus, Pinctada, Haliotis). We discuss the function of such proteins in calcifying matrices, the molecular evolution of SMP genes and the origin of mollusc nacro-prismatic SMPs.

Novel proteins from the calcifying shell matrix of the Pacific oyster Crassostrea gigas.

The shell of the Pacific oyster Crassostrea gigas is composed of more than 99% CaCO3 and of around 0.5% of occluded organic matrix. According to classical views, this matrix is supposed to regulate the shell mineral deposition. In this study, we developed one of the first proteomic approaches applied to mollusk shell in order to characterise the calcifying matrix proteins. The insoluble organic matrix, purified after demineralisation of the shell powder, was digested with trypsin enzyme, and separated on nano-LC, prior to nanospray quadrupole/time-of-flight analysis. MS/MS spectra were searched against the above 220,000 EST sequences available in the public database for Crassostrea. Using this approach, we were able to identify partial or full-length sequence transcripts that encode eight novel shell matrix proteins.

Nautilin-63, a novel acidic glycoprotein from the shell nacre of Nautilus macromphalus.

UNLABELLED: In molluscs, and more generally in metazoan organisms, the production of a calcified skeleton is a complex molecular process that is regulated by the secretion of an extracellular organic matrix. This matrix constitutes a cohesive and functional macromolecular assemblage, containing mainly proteins, glycoproteins and polysaccharides that, together, control the biomineral formation. These macromolecules interact with the extruded precursor mineral ions, mainly calcium and bicarbonate, to form complex organo-mineral composites of well-defined microstructures. For several reasons related to its remarkable mechanical properties and to its high value in jewelry, nacre is by far the most studied molluscan shell microstructure and constitutes a key model in biomineralization research. To understand the molecular mechanism that controls the formation of the shell nacreous layer, we have investigated the biochemistry of Nautilin-63, one of the main nacre matrix proteins of the cephalopod Nautilus macromphalus. After purification of Nautilin-63 by preparative electrophoresis, we demonstrate that this soluble protein is glycine-aspartate-rich, that it is highly glycosylated, that its sugar moieties are acidic, and that it is able to bind chitin in vitro. Interestingly, Nautilin-63 strongly interacts with the morphology of CaCO(3) crystals precipitated in vitro but, unexpectedly, it exhibits an extremely weak ability to inhibit in vitro the precipitation of CaCO(3) . The partial resolution of its amino acid sequence by de novo sequencing of its tryptic peptides indicates that Nautilin-63 exhibits short collagenous-like domains. Owing to specific polyclonal antibodies raised against the purified protein, Nautilin-63 was immunolocalized mainly in the intertabular nacre matrix. In conclusion, Nautilin-63 exhibits 'hybrid' biochemical properties that are found both in the soluble and insoluble proteins, rendering it difficult to classify according to the standard view on nacre proteins. DATABASE: The protein sequences of N63 appear on the UniProt Knowledgebase under accession number P86702.

Structural and functional changes in human insulin induced by the lipid peroxidation byproducts 4-hydroxy-2-nonenal and 4-hydroxy-2-hexenal.

Lipid peroxidation produces many reactive byproducts including 4-hydroxy-2-hexenal (HHE) and 4-hydroxy-2-nonenal (HNE) derived from the peroxidation of n-3 and n-6 polyunsaturated fatty acids, respectively. HNE and HHE can modify circulating biomolecules through the formation of covalent adducts. It remains, however, unknown whether HHE and HNE could induce functional and structural changes in the insulin molecule, which may in turn be pivotal in the development of insulin resistance and diabetes. Recombinant human insulin was incubated in the presence of HHE or HNE, and the formation of covalent adducts on insulin was analyzed by mass spectrometry analysis. Insulin tolerance test in mice and stimulation of glucose uptake by 3T3 adipocytes and L6 muscle cells were used to evaluate the biological efficiency of adducted insulin compared with the native one. One to 5 adducts were formed on insulin through Michael adduction, involving histidine residues. Glucose uptake in 3T3-L1 and L6C5 cells as well as the hypoglycemic effect in mice was significantly reduced after treatment with adducted insulin compared to native insulin. The formation of HNE- and HHE-Michael adducts significantly disrupts the biological activity of insulin. These structural and functional abnormalities of the insulin molecule might contribute to the pathogenesis of insulin resistance.

Proteomic identification of novel proteins from the calcifying shell matrix of the Manila clam Venerupis philippinarum.

The shell of the Manila clam Venerupis philippinarum is composed of more than 99% calcium carbonate and of a small amount of organic matrix (around 0.2%). In this study, we developed one of the first proteomic approaches applied to mollusc shell in order to characterise the matrix proteins that are believed to be essential for the formation of the biomineral. The insoluble organic matrix, purified after demineralisation of the shell powder with cold acetic acid (5%), was digested with trypsin enzyme and then separated on nano-LC prior to nanospray/quadrupole time-of-flight analysis. MS/MS spectra were searched against the above 11,000 EST sequences available on the NCBI public database for Venerupis. Using this approach, we were able to identify partial or full-length sequence transcripts that encode for shell matrix proteins. These include three novel shell proteins whose sequences do not present any homologous proteins or already described domains, two putative protease inhibitor proteins containing Kazal-type domains, and a putative Ca(2+)-binding protein containing two EF-hand domains. Biomineral formation and evolutionary implications are discussed.

An improved method to unravel phosphoacceptors in Ser/Thr protein kinase-phosphorylated substrates.

Identification of the phosphorylated residues of bacterial Ser/Thr protein kinase (STPK) substrates still represents a challenging task. Herein, we present a new strategy allowing the rapid determination of phosphoacceptors in kinase substrates, essentially based on the dual expression of the kinase with its substrate in the surrogate E. coli, followed by MS analysis in a single-step procedure. The performance of this strategy is illustrated using two distinct proteins from Mycobacterium tuberculosis as model substrates, the GroEL2 and HspX chaperones. A comparative analysis with a standard method that includes mass spectrometry analysis of in vitro phosphorylated substrates is also addressed.

Proteomic analysis of the acid-soluble nacre matrix of the bivalve Unio pictorum: detection of novel carbonic anhydrase and putative protease inhibitor proteins.

The matrix extracted from mollusc shell nacre is a mixture of proteins and glycoproteins that is thought to play a major role in controlling biomineral synthesis and in increasing its mechanical properties. We investigated the nacreous shell of the freshwater mussel Unio pictorum, to which we applied a proteomics approach adapted to mollusc shell proteins. On one hand, the acid-soluble nacre matrix was fractionated by SDS-PAGE and the five main protein bands (P95, P50, P29, P16, and P12) were digested with trypsin and analyzed by nanoLC-MS/MS followed by de novo sequencing. On the other hand, the acid-soluble nacre matrix was analyzed in a similar manner, without any preliminary fractionation. In total, we obtained about 140 peptides, of between 9 and 21 residues, as well as several shorter peptides. Interestingly, it appears that the different protein bands share several identical peptides; this has implications for the underlying genetic machinery that synthesizes nacre proteins. Homology searches against sequences in the Swiss-Prot protein database and the 800,000 mollusc expressed sequence tag database were performed, but surprisingly, only a few obvious homologies were established. Among the peptides that match with known sequences, some from P50 and P16/P12 proteins align with carbonic anhydrase (CA) and with the protease inhibitor, respectively. The evolutionary implications of our findings are discussed.

Proteomic analysis of Parietaria judaica pollen and allergen profiling by an immunoproteomic approach.

Parietaria judaica pollen is a common cause of airway allergic disease in the Mediterranean area. Proteome analysis of mature Parietaria judaica pollen by two-dimensional gel electrophoresis (2-DE) and mass spectrometry has established the first reference proteome map of this weed. Proteins involved in a variety of cellular functions as well as the occurrence of allergens were detected. By using 2-DE and immunoblotting with sera from Parietaria judaica allergic patients we obtained a more detailed characterization of Parietaria judaica allergen profile so to improve our comprehension of the pathogenesis of pollen-induced allergic reaction.

Strategy for identification by mass spectrometry of a new human hemoglobin variant with two mutations in Cis in the beta-globin chain: Hb S-Clichy [beta6(A3)Glu-->Val; beta8(A5)Lys-->Thr].

Hemoglobinopathies are the most frequent genetic diseases in the world. Among them, the Hb S variant [beta6(A3)Glu-->Val], which, in the homozygous state, produces a severe disease known as sickle cell anemia with polymerization of Hb S inside red blood cells under hypoxic conditions. Additional mutations, in cis or in trans of the beta(S)-globin chain, may inhibit or enhance the polymerization process. We describe here a new hemoglobin (Hb) variant (Hb S-Clichy) which carries the beta(S)-globin chain and an additional mutation beta8(A5)Lys-->Thr. The variant was detected by routine electrophoretic techniques and cation exchange liquid chromatography (CE-LC). Globin chain separation by reversed phase LC (RP-LC) showed normal and abnormal beta chains, confirming that the additional abnormality was located in cis to Hb S. Electrospray ionization mass spectrometry (ESI-MS) gave a 57 Da mass decrease for the abnormal globin chain. The abnormal chain was isolated and submitted to trypsin digestion. Normal peptides betaT-1 and betaT-2 were not observed on the matrix-assisted laser desorption-time of flight (MALDI-TOF) mass spectrum but a new peptide betaT-1,2 was detected. Nano LC-ESI-MS/MS of the new peptide showed that the glutamic acid at codon 6 was replaced by a valine residue, and the lysine at codon 8 was replaced by a threonine residue, as confirmed by DNA sequencing. This example demonstrates that in a population where Hb S is present, every unidentified Hb needs to be clearly characterized to prevent major sickle cell syndromes. In addition, the identification of these variants must be considered in newborn screening for sickle cell disease, using either classical biochemical methods or MS techniques.

Description of two new alpha variants: Hb Canuts [alpha85(F6)Asp-->His (alpha1)] and Hb Ambroise Pare [alpha117(GH5)Phe-->Ile (alpha2)]; two new beta variants: Hb Beaujolais [beta84(EF8)Thr-->Asn] and Hb Monplaisir [beta147 (Tyr-Lys-Leu-Ala-Phe-Phe-Leu-Leu-Ser-Asn-Phe-Tyr-158-COOH)] and one new delta variant: Hb (A2)North Africa [delta59(E3)Lys-->Met].

We present here five new hemoglobin (Hb) variants which have been identified during routine Hb analysis before their genotypic characterization. Four of these result from a classical missense mutation: Hb Canuts [alpha85(F6)Asp-->His (alpha1)], Hb Ambroise Pare [alpha117(GH5)Phe-->Ile (alpha2)], Hb Beaujolais [beta84(EF8)Thr-->Asn] and HbA(2)-North Africa [delta59(E3)Lys-->Met]. The last one, Hb Monplaisir [beta147 (Tyr-Lys-Leu-Ala-Phe-Phe-Leu-Leu-Ser-Asn-Phe-Tyr-158-COOH)], results from a frameshift mutation at the stop codon of the beta-globin gene which leads to a modified C-terminal sequence in the beta-globin chain. None of these variants seem to have a particular clinical expression in the heterozygous state. The circumstances of the discovery of these five new Hb variants emphasize the fact that an association of techniques is necessary for a complete screening of Hb variants during routine Hb analysis. Globin chain separation by reversed phase liquid chromatography (RP-LC) appears to be the most relevant method.

Probing the conformation of the resting state of a bacterial multidrug ABC transporter, BmrA, by a site-directed spin labeling approach.

Previously published 3-D structures of a prototypic ATP-binding cassette (ABC) transporter, MsbA, have been recently corrected revealing large rigid-body motions possibly linked to its catalytic cycle. Here, a closely related multidrug bacterial ABC transporter, BmrA, was studied using site-directed spin labeling by focusing on a region connecting the transmembrane domain and the nucleotide-binding domain (NBD). Electron paramagnetic resonance (EPR) spectra of single spin-labeled cysteine mutants suggests that, in the resting state, this sub-domain essentially adopts a partially extended conformation, which is consistent with the crystal structures of MsbA and Sav1866. Interestingly, one of the single point mutants (Q333C) yielded an immobilized EPR spectrum that could arise from a direct interaction with a vicinal tyrosine residue. Inspection of different BmrA models pointed to Y408, within the NBD, as the putative interacting partner, and its mutation to a Phe residue indeed dramatically modified the EPR spectra of the spin labeled Q333C. Moreover, unlike the Y408F mutation, the Y408A mutation abolished both ATPase activity and drug transport of BmrA, suggesting that a nonpolar bulky residue is required at this position. The spatial proximity of Q333 and Y408 was also confirmed by formation of a disulfide bond when both Q333 and T407 (or S409) were replaced jointly by a cysteine residue. Overall, these results indicate that the two regions surrounding Q333 and Y408 are close together in the 3-D structure of BmrA and that residues within these two sub-domains are essential for proper functioning of this transporter.

Phenotype determination of hemoglobinopathies by mass spectrometry.

OBJECTIVES: Nowadays, nearly 1000 hemoglobin (Hb) variants are known. The standard biochemical techniques used in Hb analysis are mainly: isoelectric focusing, cation-exchange liquid chromatography (LC) and reversed-phase LC. In addition to this approach, a protein analysis is achieved by mass spectrometry (MS) and additional DNA studies are performed. The aim of this review is to emphasize the significance of MS methods applied to Hb variants analysis. RESULTS AND PERSPECTIVES: To perform Hb studies, different MS techniques are currently used such as electrospray ionization (ESI), matrix-assisted laser desorption ionization (MALDI) and tandem mass spectrometry (MS/MS). As shown here, MS is an efficient tool for identification of all types of variants (substitution of a single amino acid residue, several substitutions in the same globin chain, insertions/deletions, fusion Hbs). The use of MS in neonatal screening of Hb variants is also presented. CONCLUSIONS: MS is a powerful technique for Hb analysis. It appears as being an important additional method in the set of biochemical techniques.

The MurC ligase essential for peptidoglycan biosynthesis is regulated by the serine/threonine protein kinase PknA in Corynebacterium glutamicum.

The Mur ligases play an essential role in the biosynthesis of bacterial cell-wall peptidoglycan and thus represent attractive targets for the design of novel antibacterials. These enzymes catalyze the stepwise formation of the peptide moiety of the peptidoglycan disaccharide peptide monomer unit. MurC is responsible of the addition of the first residue (L-alanine) onto the nucleotide precursor UDP-MurNAc. Phosphorylation of proteins by Ser/Thr protein kinases has recently emerged as a major physiological mechanism of regulation in prokaryotes. Herein, the hypothesis of a phosphorylation-dependent mechanism of regulation of the MurC activity was investigated in Corynebacterium glutamicum. We showed that MurC was phosphorylated in vitro by the PknA protein kinase. An analysis of the phosphoamino acid content indicated that phosphorylation exclusively occurred on threonine residues. Six phosphoacceptor residues were identified by mass spectrometry analysis, and we confirmed that mutagenesis to alanine residues totally abolished PknA-dependent phosphorylation of MurC. In vitro and in vivo ligase activity assays showed that the catalytic activity of MurC was impaired following mutation of these threonine residues. Further in vitro assays revealed that the activity of the MurC-phosphorylated isoform was severely decreased compared with the non-phosphorylated protein. To our knowledge, this is the first demonstration of a MurC ligase phosphorylation in vitro. The finding that phosphorylation is correlated with a decrease in MurC enzymatic activity could have significant consequences in the regulation of peptidoglycan biosynthesis.

Detection of a thalassemic alpha-chain variant (Hemoglobin Groene Hart) by reversed-phase liquid chromatography.

BACKGROUND: Hemoglobin (Hb) Groene Hart [alpha119 (H2)Pro-->Ser (alpha1)], also known as Hb Bernalda, is a nondeletional alpha-thalassemic Hb variant that is frequent in southern Italy and North Africa. This variant is not supposed to be produced in the erythrocytes of carriers. The alpha-thalassemic behavior of this variant has been explained as an impaired interaction between the alpha-globin chain and the alpha-Hb-stabilizing protein. METHODS: To separate globin chains, we developed a modified reversed-phase liquid chromatography (RPLC) procedure that uses acetonitrile-water solvents containing up to 3 mL/L trifluoroacetic acid. After RPLC, we characterized the isolated globin chains by electrospray ionization (ESI) mass spectrometry (MS) and analyzed their tryptic peptides with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and nano-LC-ESI-MS/MS. RESULTS: RPLC detected an abnormal peak with a retention time substantially greater than that of the wild-type alpha(A)-globin chain. We identified this variant as Hb Groene Hart and found it in the hemolysates of 11 unrelated patients (1 homozygote, 9 heterozygotes, and 1 heterozygote associated with the -alpha(3.7) deletion). These patients possessed abnormal hematologic features suggesting an alpha-thalassemia phenotype. Molecular modeling suggested that the increase in hydrophobicity was due to opening of the GH interhelical segment following replacement of amino acid residue 119 with a nonhelix breaker residue. CONCLUSIONS: This method allows the detection of Hb variants at low concentrations, and adjusting the composition of the organic solvents enables the method to identify Hb variants with large changes in hydrophobicity.

From the characterization of the four serine/threonine protein kinases (PknA/B/G/L) of Corynebacterium glutamicum toward the role of PknA and PknB in cell division.

Corynebacterium glutamicum contains four serine/threonine protein kinases (STPKs) named PknA, PknB, PknG, and PknL. Here we present the first biochemical and comparative analysis of all four C. glutamicum STPKs and investigate their potential role in cell shape control and peptidoglycan synthesis during cell division. In vitro assays demonstrated that, except for PknG, all STPKs exhibited autokinase activity. We provide evidence that activation of PknG is part of a phosphorylation cascade mechanism that relies on PknA activity. Following phosphorylation by PknA, PknG could transphosphorylate its specific substrate OdhI in vitro. A mass spectrometry profiling approach was also used to identify the phosphoresidues in all four STPKs. The results indicate that the nature, number, and localization of the phosphoacceptors varies from one kinase to the other. Disruption of either pknL or pknG in C. glutamicum resulted in viable mutants presenting a typical cell morphology and growth rate. In contrast, we failed to obtain null mutants of pknA or pknB, supporting the notion that these genes are essential. Conditional mutants of pknA or pknB were therefore created, leading to partial depletion of PknA or PknB. This resulted in elongated cells, indicative of a cell division defect. Moreover, overexpression of PknA or PknB in C. glutamicum resulted in a lack of apical growth and therefore a coccoid-like morphology. These findings indicate that pknA and pknB are key players in signal transduction pathways for the regulation of the cell shape and both are essential for sustaining corynebacterial growth.

Processing and nuclear localization of CRMP2 during brain development induce neurite outgrowth inhibition.

Collapsin response mediator proteins (CRMPs) are believed to play a crucial role in neuronal differentiation and axonal outgrowth. Among them, CRMP2 mediates axonal guidance by collapsing growth cones during development. This activity is correlated with the reorganization of cytoskeletal proteins. CRMP2 is implicated in the regulation of several intracellular signaling pathways. Two subtypes, A and B, and multiple cytosolic isoforms of CRMP2B with apparent masses between 62 and 66 kDa have previously been reported. Here, we show a new short isoform of 58 kDa, expressed during brain development, derived from C-terminal processing of the CRMP2B subtype. Although full-length CRMP2 is restricted to the cytoplasm, using transfection experiments, we demonstrate that a part of the short isoform is found in the nucleus. Interestingly, at the tissue level, this short CRMP2 is also found in a nuclear fraction of brain extract. By mutational analysis, we demonstrate, for the first time, that nuclear translocation occurs via nuclear localization signal (NLS) within residues Arg(471)-Lys(472) in CRMP2 sequence. The NLS may be unmasked after C-terminal processing; thereby, this motif may be surface-exposed. This short CRMP2 induces neurite outgrowth inhibition in neuroblastoma cells and suppressed axonal growth in cultured cortical neurons, whereas full-length CRMP2 promotes neurite elongation. The NLS-mutated short isoform, restricted to the cytoplasm, abrogates both neurite outgrowth and axon growth inhibition, indicating that short nuclear CRMP2 acts as a dominant signal. Therefore, post-transcriptional processing of CRMP2 together with its nuclear localization may be an important key in the regulation of neurite outgrowth in brain development.