Eukaryotic-like gephyrin and cognate membrane receptor coordinate corynebacterial cell division and polar elongation.

The order Corynebacteriales includes major industrial and pathogenic actinobacteria such as Corynebacterium glutamicum or Mycobacterium tuberculosis . Their elaborate multi-layered cell wall, composed primarily of the mycolyl-arabinogalactan-peptidoglycan complex, and their polar growth mode impose a stringent coordination between the septal divisome, organized around the tubulin-like protein FtsZ, and the polar elongasome, assembled around the tropomyosin-like protein Wag31. Here, we report the identification of two new divisome members, a gephyrin-like repurposed molybdotransferase (GLP) and its membrane receptor (GLPR). We show that the interplay between the GLPR/GLP module, FtsZ and Wag31 is crucial for orchestrating cell cycle progression. Our results provide a detailed molecular understanding of the crosstalk between two essential machineries, the divisome and elongasome, and reveal that Corynebacteriales have evolved a protein scaffold to control cell division and morphogenesis similar to the gephyrin/GlyR system that in higher eukaryotes mediates synaptic signaling through network organization of membrane receptors and the microtubule cytoskeleton.

Glypican-2 levels in cerebrospinal fluid predict the status of adult hippocampal neurogenesis.

Adult hippocampal neurogenesis is a remarkable form of brain plasticity through which new neurons are generated throughout life. Despite its important roles in cognition and emotion and its modulation in various preclinical disease models, the functional importance of adult hippocampal neurogenesis in human health has not been revealed because of a lack of tools for monitoring adult neurogenesis in vivo. Therefore, we performed an unbiased proteomics screen to identify novel proteins expressed during neuronal differentiation using a human neural stem cell model, and we identified the proteoglycan Glypican-2 (Gpc2) as a putative secreted marker of immature neurons. Exogenous Gpc2 binds to FGF2 and inhibits FGF2-induced neural progenitor cell proliferation. Gpc2 is enriched in neurogenic regions of the adult brain. Its expression is increased by physiological stimuli that increase hippocampal neurogenesis and decreased in transgenic models in which neurogenesis is selectively ablated. Changes in neurogenesis also result in changes in Gpc2 protein level in cerebrospinal fluid (CSF). Gpc2 is detectable in adult human CSF, and first pilot experiments with a longitudinal cohort indicate a decrease over time. Thus, Gpc2 may serve as a potential marker to monitor adult neurogenesis in both animal and human physiology and disease, warranting future studies.

Potency and selectivity of inhibition of cathepsin K, L and S by their respective propeptides.

The prodomains of several cysteine proteases of the papain family have been shown to be potent inhibitors of their parent enzymes. An increased interest in cysteine proteases inhibitors has been generated with potential therapeutic targets such as cathepsin K for osteoporosis and cathepsin S for immune modulation. The propeptides of cathepsin S, L and K were expressed as glutathione S-transferase-fusion proteins in Escherichia coli. The proteins were purified on glutathione affinity columns and the glutathione S-transferase was removed by thrombin cleavage. All three propeptides were tested for inhibitor potency and found to be selective within the cathepsin L subfamily (cathepsins K, L and S) compared with cathepsin B or papain. Inhibition of cathepsin K by either procathepsin K, L or S was time-dependent and occurred by an apparent one-step mechanism. The cathepsin K propeptide had a Ki of 3.6-6.3 nM for each of the three cathepsins K, L and S. The cathepsin L propeptide was at least a 240-fold selective inhibitor of cathepsin K (Ki = 0.27 nM) and cathepsin L (Ki = 0.12 nM) compared with cathepsin S (Ki = 65 nM). Interestingly, the cathepsin S propeptide was more selective for inhibition of cathepsin L (Ki = 0.46 nM) than cathepsin S (Ki = 7.6 nM) itself or cathepsin K (Ki = 7.0 nM). This is in sharp contrast to previously published data demonstrating that the cathepsin S propeptide is equipotent for inhibition of human cathepsin S and rat and paramecium cathepsin L [Maubach, G., Schilling, K., Rommerskirch, W., Wenz, I., Schultz, J. E., Weber, E. & Wiederanders, B. (1997), Eur J. Biochem. 250, 745-750]. These results demonstrate that limited selectivity of inhibition can be measured for the procathepsins K, L and S vs. the parent enzymes, but selective inhibition vs. cathepsin B and papain was obtained.

Caspase-resistant BAP31 inhibits fas-mediated apoptotic membrane fragmentation and release of cytochrome c from mitochondria.

BAP31 is a 28-kDa integral membrane protein of the endoplasmic reticulum whose cytosolic domain contains two identical caspase recognition sites (AAVD.G) that are preferentially cleaved by initiator caspases, including caspase 8. Cleavage of BAP31 during apoptosis generates a p20 fragment that remains integrated in the membrane and, when expressed ectopically, is a potent inducer of cell death. To examine the consequences of maintaining the structural integrity of BAP31 during apoptosis, the caspase recognition aspartate residues were mutated to alanine residues, and Fas-mediated activation of caspase 8 and cell death were examined in human KB epithelial cells stably expressing the caspase-resistant mutant crBAP31. crBAP31 only modestly slowed the time course for activation of caspases, as assayed by the processing of procaspases 8 and 3 and the measurement of total DEVDase activity. As a result, cleavage of the caspase targets poly(ADP-ribosyl) polymerase and endogenous BAP31, as well as the redistribution of phosphatidylserine and fragmentation of DNA, was observed. In contrast, cytoplasmic membrane blebbing and fragmentation and apoptotic redistribution of actin were strongly inhibited, cell morphology was retained near normal, and the irreversible loss of cell growth potential following removal of the Fas stimulus was delayed. Of note, crBAP31-expressing cells also resisted Fas-mediated release of cytochrome c from mitochondria, and the mitochondrial electrochemical potential was only partly reduced. These results argue that BAP31 cleavage is important for manifesting cytoplasmic apoptotic events associated with membrane fragmentation and reveal an unexpected cross talk between mitochondria and the endoplasmic reticulum during Fas-mediated apoptosis in vivo.

A general method for the rapid characterization of tyrosine-phosphorylated proteins by mini two-dimensional gel electrophoresis.

Our preliminary results are reported in the investigation of the tyrosine phosphorylation cascade triggered by the stimulation of the insulin receptor in the adipocyte cell line 3T3-L1 using a mini two-dimensional gel electrophoresis approach. The minigel format, 8 x 10 cm, was found sufficiently resolving and reproducible to study complex biological samples while considerably increasing throughput and lowering costs compared to larger gel formats. Consequently, we used the minigel format to rapidly screen a large number of samples, of which only the most relevant were then analyzed by optimized, preparative two-dimensional gels. The accurate localization and relative quantification of tyrosine-phosphorylated proteins was performed using a nonradioactive triple labeling method. After transfer onto polyvinylidene difluoride (PVDF) membranes, proteins were stained with Sypro Ruby to verify the separation quality and to localize the general region of interest for immunostaining. The membranes were subsequently blocked with polyvinylpyrrolidone-40 and probed with the relevant antibodies for visualization of the phosphorylated proteins by chemiluminescence. Finally, membranes were stained with colloidal gold to obtain a pattern reminiscent of the silver staining of a polyacrylamide gel. We believe that the presented strategy can be generalized for any gel application in which a protein has to be detected and identified based on its immunoreactivity.

Data-dependent modulation of solid-phase extraction capillary electrophoresis for the analysis of complex peptide and phosphopeptide mixtures by tandem mass spectrometry: application to endothelial nitric oxide synthase.

Electrospray ionization (ESI) tandem mass spectrometry (MS/MS) of peptides in conjunction with automated sequence database searching of the resulting collision-induced dissociation (CID) spectra has become a powerful method for the identification of purified proteins or the components of protein mixtures. The success of the method is critically dependent on the manner by which the peptides are introduced into the mass spectrometer. In this report, we describe a capillary electrophoresis-based system for the automated, sensitive analysis of complex peptide mixtures. The system consists of an ESI-MS/MS instrument, a solid-phase extraction (SPE)-capillary zone electrophoresis (CZE) device for peptide concentration and separation, and an algorithm written in Instrument Control Language (ICL) which modulates the electrophoretic conditions in a data-dependent manner to optimize available time for the generation of high-quality CID spectra of peptides in complex samples. We demonstrate that the data-dependent modulation of the electric field significantly expands the analytical window for each peptide analyzed and that the sensitivity of the SPE-CZE technique is not noticeably altered by the procedure. By applying the technique to the analysis of in vivo phosphorylation sites of endothelial nitric oxide synthase (eNOS), we demonstrate the power of this system for the MS/MS analysis of minor peptide species in complex samples such as phosphopeptides generated by the proteolytic digestion of a large protein, eNOS, phosphorylated at low stoichiometry.

A simplified gradient solvent delivery system for capillary liquid chromatography-electrospray ionization mass spectrometry.

We describe a simple solvent delivery system for gradient capillary HPLC at nanoliter per minute flow rates. The novel aspect of the system is that solvents are delivered one at a time, using a switching valve, into a relatively large-volume mixing chamber. Efficient mixing in the chamber causes the formation of a sigmoidal gradient from the initial solvent to the subsequent solvent, which is then delivered to a capillary column. The shape of the gradients formed can be predicted from a simple theoretical model. Gradients of different slope can be formed by varying either the size of the chamber or the system flow rate. The system is robust, reproducible, and simple to operate. We provide a detailed protocol of how to construct a low-cost capillary HPLC system consisting of two syringe pumps, a capillary mixing chamber, a capillary column, and a zero dead-volume microelectrospray interface. We demonstrate that the coupling of this HPLC system to a mass spectrometer enabled us to identify proteins at the low femtomole level in solution-phase digests and at the picomole level in digests of samples separated on SDS-PAGE gels. We believe that the strategy presented will be useful as a general method for the characterization of proteins and peptides by capillary HPLC-electrospray mass spectrometry.

RGSZ1, a Gz-selective RGS protein in brain. Structure, membrane association, regulation by Galphaz phosphorylation, and relationship to a Gz gtpase-activating protein subfamily.

We cloned the cDNA for human RGSZ1, the major Gz-selective GTPase-activating protein (GAP) in brain (Wang, J., Tu, Y., Woodson, J., Song, X., and Ross, E. M. (1997) J. Biol. Chem. 272, 5732-5740) and a member of the RGS family of G protein GAPs. Its sequence is 83% identical to RET-RGS1 (except its N-terminal extension) and 56% identical to GAIP. Purified, recombinant RGSZ1, RET-RGS1, and GAIP each accelerated the hydrolysis of Galphaz-GTP over 400-fold with Km values of approximately 2 nM. RGSZ1 was 100-fold selective for Galphaz over Galphai, unusually specific among RGS proteins. Other enzymological properties of RGSZ1, brain Gz GAP, and RET-RGS1 were identical; GAIP differed only in Mg2+ dependence and in its slightly lower selectivity for Galphaz. RGSZ1, RET-RGS1, and GAIP thus define a subfamily of Gz GAPs within the RGS proteins. RGSZ1 has no obvious membrane-spanning region but is tightly membrane-bound in brain. Its regulatory activity in membranes depends on stable bilayer association. When co-reconstituted into phospholipid vesicles with Gz and m2 muscarinic receptors, RGSZ1 increased agonist-stimulated GTPase >15-fold with EC50 <12 nM, but RGSZ1 added to the vesicle suspension was <0.1% as active. RGSZ1, RET-RGS1, and GAIP share a cysteine string sequence, perhaps targeting them to secretory vesicles and allowing them to participate in the proposed control of secretion by Gz. Phosphorylation of Galphaz by protein kinase C inhibited the GAP activity of RGSZ1 and other RGS proteins, providing a mechanism for potentiation of Gz signaling by protein kinase C.

Reconstitution, characterisation and mass analysis of the pentacylindrical allophycocyanin core complex from the cyanobacterium Anabaena sp. PCC 7120.

The phycobilisome (PBS) of Anabaena sp. PCC 7120 was allowed to dissociate into its constituents and the resulting allophycocyanin (AP) fraction was purified. Its reconstitution yielded a complex which according to negative stain electron microscopy and spectral analysis was identical to the native pentacylindrical PBS core domain. Each cylinder of the central tricylindric unit was comprised of four AP (alphabeta)3 disks. Mass analysis using the scanning transmission electron microscope (STEM) showed the presence of 16 AP trimers in the intact reconstitute, which had a total mass of 1966(+/-66) kDa. Composition analysis indicated an AP trimer distribution of (AP-II):(AP-LCM):(AP-B):(AP-I)=6:2:2:6, i.e. an addition of two AP-I and two AP-II complexes compared to a tricylindrical PBS core domain. Therefore, we suggest that each supplementary half-core cylinder found in pentacylindrical AP core domains is comprised of one AP-I and one AP-II trimer, in agreement with the current model. The structural significance of the 127 kDa core membrane linker polypeptide was further investigated by subjecting the AP core reconstitute to mild chymotryptic degradation. After isolation, the digested complex exhibited a tricylindrical appearance while STEM mass analysis confirmed the presence of only 12 AP complexes. Polypeptide analysis by SDS-PAGE and Edman degradation related the half-cylinder loss to cleavage of the Rep4 domain of the core membrane linker polypeptide. On the basis of these data, a general model for the assembly of the three hemidiscoidal PBS types known to date is discussed.

Expression and conservation of apolipoprotein AIV in an avian species.

In birds, intestinally derived lipoproteins are thought to be secreted directly into the portal vein rather than to enter the circulation via the lymphatic system as in mammals. Hepatic clearance of these so-called portomicrons must be rapid, but the protein(s) mediating their catabolism, presumably analogues of the 36-kDa mammalian apolipoprotein E, have not been identified. In searching for such a mediator(s), we have isolated a hitherto unknown 38-kDa protein from chicken serum, which we identified by microsequencing and molecular cloning as a counterpart to mammalian apolipoprotein AIV (apoAIV). Mature chicken apoAIV consists of 347 amino acids, lacks cysteine residues, and displays 57% sequence identity with human apoAIV and, to a significantly lesser extent, with apoAIVs of rodents. This first nonmammalian apoAIV characterized is the smallest homologue reported so far, because of the lack of repeated motifs at the carboxyl terminus with the consensus sequence Glu-Gln-Glu/Ala-Gln, a hallmark of mammalian apoAIVs. Chicken apoAIV (isoelectric point, 4.65) is also considerably more acidic than its human counterpart. Agarose gel electrophoresis revealed that unlike human apoAIV, which migrates to a pre-alpha-position, chicken apoAIV shows fast alpha migration. Functional characterization demonstrated that the avian protein is able to activate the enzyme lecithin:cholesterol acyltransferase. Roosters and hens express apoAIV predominantly in the gut, one-fifth as much in the liver, and no other sites of expression are identifiable by Northern blot analysis. Although pronounced intestinal synthesis is common to apoAIVs, the features of the avian protein support the notion that it represents a prototype of an apoprotein that evolved to acquire possibly distinct functions in mammals and birds.

Chiral high performance liquid chromatography resolution of ibuprofen esters.

Two cellulose-based chiral stationary phases (Chiralcel OD and Chiralcel OJ) were compared on their ability to resolve various aliphatic ibuprofen esters. Chiralcel OJ with hexane as the mobile phase allows for the separation of most of the esters. Observed changes in resolution depending on the solute nature (basicity of the solute, esterified alcohol chain length, presence of a double bond) are discussed. An example of the application of this method for following the kinetic resolution of racemic ibuprofen is presented.

High throughput protein characterization by automated reverse-phase chromatography/electrospray tandem mass spectrometry.

We describe an integrated workstation for the automated, high-throughput, and conclusive identification of proteins by reverse-phase chromatography electrospray ionization tandem mass spectrometry. The instrumentation consists of a refrigerated autosampler, a submicrobore reverse-phase liquid chromatograph, and an electrospray triple quadrupole mass spectrometer. For protein identification, enzymatic digests of either homogeneous polypeptides or simple protein mixtures were generated and loaded into the autosampler. Samples were sequentially injected every 32 min. Ions of eluting peptides were automatically selected by the mass spectrometer and subjected to collision-induced dissociation. Following each run, the resulting tandem mass spectra were automatically analyzed by SEQUEST, a program that correlates uninterpreted peptide fragmentation patterns with amino acid sequences contained in databases. Protein identification was established by SEQUEST_SUMMARY a program that combines the SEQUEST scores of peptides originating from the same protein and ranks the cumulative results in a short summary. The workstation's performance was demonstrated by the unattended identification of 90 proteins from the yeast Saccharomyces cerevisiae, which were separated by high-resolution two-dimensional PAGE. The system was found to be very robust and identification was reliably and conclusively established for proteins if quantities exceeding 1-5 pmol were applied to the gel. The level of automation, the throughput, and the reliability of the results suggest that this system will be useful for the many projects that require the characterization of large numbers of proteins.

High-sensitivity detection of 4-(3-pyridinylmethylaminocarboxypropyl) phenylthiohydantoins by capillary liquid chromatography-microelectrospray ion trap mass spectrometry.

We describe the separation and detection at the low-femtomole level of 4-(3-pyridinylmethylaminocarboxypropyl) phenylthiohydantoins (311-PTHs) by capillary liquid chromatography-microelectrospray ion trap mass spectrometry. Highest sensitivity was obtained in the multiple-ion monitoring operating mode in which we detected 311-PTHs at the 5-fmol level with a signal-to-noise ratio of approximately 10. We investigated the fragmentation patterns of the isobaric 311-PTH isoleucine and 311-PTH leucine by electrospray ionization ion trap tandem mass spectrometry. The compounds could be differentiated by a fragment ion of mass m/z = 366.1 which was specific for the breakdown of 311-PTH leucine, thus allowing for the unambiguous identification of the 311-PTH derivatives of all 20 naturally occurring amino acids by their masses and fragmentation patterns.

Colloidal silver staining of electroblotted proteins for high sensitivity peptide mapping by liquid chromatography-electrospray ionization tandem mass spectrometry.

Mass spectrometric techniques for the identification of proteins either by amino acid sequencing or by correlation of mass spectral data with sequence databases are becoming increasingly sensitive and are rapidly approaching the limit of detection achieved by the staining of proteins in gels or, after electroblotting, on membranes. Here we present a technique for the sensitive staining of proteins electroblotted onto nitrocellulose or polyvinylidene difluoride membranes and enzymatic cleavage conditions for such proteins to achieve optimal recovery of peptides. The technique is based on the deposition of colloidal silver on the membrane-bound proteins. Peptide mixtures generated by proteolysis on the membrane were recovered at high yields and were compatible with analysis by reverse-phase chromatography and on-line electrospray ionization mass spectrometry. This simple and rapid colloidal silver staining procedure allowed the visualization of less than 5 ng of protein in a band and thus approached the sensitivity of silver staining in gels. We demonstrate that this method allows the detection of subpicomole amounts of electroblotted proteins and their identification by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry.

Membrane amine oxidase cloning and identification as a major protein in the adipocyte plasma membrane.

A 97-kDa protein present in the glucose transporter (GLUT4 isotype)-containing vesicles from rat adipocytes has been isolated, the sequences of two tryptic peptides were obtained, and on the basis of these its cDNA partially cloned. The 97-kDa protein is almost certainly identical to a major integral glycoprotein of this size in the rat adipocyte plasma membrane, since its predicted N-terminal sequence is the same as that recently determined for this glycoprotein by amino acid sequencing. Moreover, the predicted partial sequence (322 amino acids) of the 97-kDa protein is highly homologous to the corresponding region of a human placental amine oxidase, which was cloned simultaneously and proposed to be a secreted protein. The amino acid sequence of the 97-kDa rat/human amine oxidase indicates that the protein consists of a very short N-terminal cytoplasmic domain followed by a single transmembrane segment and a large extracellular domain containing the catalytic site. Thus this study establishes the 97-kDa rat/human amine oxidase as the first integral membrane amine oxidase to be cloned. The membrane amine oxidase was more abundant in the plasma membranes than the low density microsomes of the adipocyte, and in contrast to some other proteins found in GLUT4 vesicles, it did not redistribute to the plasma membrane in response to treatment of the cells with insulin.

Identification of proteins by capillary electrophoresis-tandem mass spectrometry. Evaluation of an on-line solid-phase extraction device.

Capillary electrophoresis-tandem mass spectrometry has been used successfully for the analysis of complex peptide mixtures. The method is limited by a relatively high concentration limit of detection and by matrix effects. Here we describe on-line coupling of a solid-phase microextraction device to a capillary electrophoresis-tandem mass spectrometry system. The performance of the integrated instrument was evaluated for the identification of proteins by their amino acid sequence. We report that the concentration limit of detection was improved at least 1000 fold to the low attomole/microliter range and that matrix effects were minimized by extensive sample clean-up during solid-phase extraction. We demonstrate that the implementation of a solid-phase extraction device significantly enhances capillary electrophoresis-tandem mass spectrometry as a method for the identification of low abundance proteins isolated from high-resolution two-dimensional polyacrylamide gels.

Axonal proteins involved in myelination: characterization of a collagen-like protein.

An anti-axolemma monoclonal antibody, designated G21.3, has been isolated in order to understand molecular mechanisms involved in myelination. Both biochemical and morphological studies showed that the monoclonal antibody inhibits myelin production by oligodendrocytes in cerebellar slice cultures. On Western blots of axolemma preparations, the antibody recognized 140- and 120-kD proteins. The present study involves the isolation and characterization of the G21.3 antigen. The G21.3-immunoreactive proteins of 140 and 120 kD were purified from the adult rat sciatic nerve and amino acid sequencing of these proteins revealed significant homology to alpha I and alpha II chains of collagen type I. Biochemical and Western blot analysis using pure collagen, collagen I antibody and collagenase D suggest that the antigen isolated from sciatic nerve is collagen. However, immunofluorescence studies using the G21.3 antibody, collagen I antibody, collagenase D and Northern blot analysis using a collagen probe do not fully support the view that the G21.3 antigen in the CNS is also a collagen. We conclude that the G21.3 antigen is a collagen-like protein involved in CNS myelination.

Independent evolution of the prochlorophyte and green plant chlorophyll a/b light-harvesting proteins.

The prochlorophytes are oxygenic prokaryotes differing from other cyanobacteria by the presence of a light-harvesting system containing both chlorophylls (Chls) a and b and by the absence of phycobilins. We demonstrate here that the Chl a/b binding proteins from all three known prochlorophyte genera are closely related to IsiA, a cyanobacterial Chl a-binding protein induced by iron starvation, and to CP43, a constitutively expressed Chl a antenna protein of photosystem II. The prochlorophyte Chl a/b protein (pcb) genes do not belong to the extended gene family encoding eukaryotic Chl a/b and Chl a/c light-harvesting proteins. Although higher plants and prochlorophytes share common pigment complements, their light-harvesting systems have evolved independently.

Protein identification by solid phase microextraction-capillary zone electrophoresis-microelectrospray-tandem mass spectrometry.

We describe an analytical system for the rapid identification of proteins by correlation of tandem mass spectra with protein sequence databases. The system consists of an integrated solid phase microextraction/capillary zone electrophoresis peptide separation device that is connected through a microelectrospray ion source to a tandem mass spectrometer. The limits of detection are 660 amol of sample at a concentration limit of < 33 amol/microliters for peptide mass measurement, and < 10 fmol of sample, at a concentration limit of < 300 amol/microliters for peptide analysis by collision-induced dissociation. Using this system, we have identified low nanogram amounts of yeast proteins separated by high-resolution two-dimensional gel electrophoresis.