Function and Localization Dynamics of Bifunctional Penicillin-Binding Proteins in Caulobacter crescentus

The peptidoglycan cell wall of bacteria is a complex macromolecule composed of glycan strands that are cross-linked by short peptide bridges. Its biosynthesis involves a conserved group of enzymes, the bifunctional penicillin-binding proteins (bPBPs), which contain both a transglycosylase and a transpeptidase domain, thus being able to elongate the glycan strands and, at the same time, generate the peptide cross-links. The stalked model bacterium Caulobacter crescentus possesses five bPBP paralogs, named Pbp1A, PbpC, PbpX, PbpY, and PbpZ, whose function is still incompletely understood. In this study, we show that any of these proteins except for PbpZ is sufficient for growth and normal morphogenesis when expressed at native or elevated levels, whereas inactivation of all five paralogs is lethal. Growth analyses indicate a central role of PbpX in the resistance of C. crescentus against the noncanonical amino acid d-alanine. Moreover, we show that PbpX and PbpY localize to the cell division site. Their recruitment to the divisome is dependent on the essential cell division protein FtsN and likely involves interactions with FtsL and the putative peptidoglycan hydrolase DipM. The same interaction pattern is observed for Pbp1A and PbpC, although these proteins do not accumulate at midcell. Our findings demonstrate that the bPBPs of C. crescentus are, to a large extent, redundant and have retained the ability to interact with the peptidoglycan biosynthetic machineries responsible for cell elongation, cytokinesis, and stalk growth. Nevertheless, they may preferentially act in specific peptidoglycan biosynthetic complexes, thereby facilitating the independent regulation of distinct growth processes.     

Visualization of Penicillin-Binding Proteins during Sporulation of Streptomyces griseus

We used fluorescein-tagged β-lactam antibiotics to visualize penicillin-binding proteins (PBPs) in sporulating cultures of Streptomyces griseus. Six PBPs were identified in membranes prepared from growing and sporulating cultures. The binding activity of an 85-kDa PBP increased fourfold by 10 to 12 h of sporulation, at which time the sporulation septa were formed. Cefoxitin inhibited the interaction of the fluorescein-tagged antibiotics with the 85-kDa PBP and also prevented septum formation during sporulation but not during vegetative growth. The 85-kDa PBP, which was the predominant PBP in membranes of cells that were undergoing septation, preferentially bound fluorescein-6-aminopenicillanic acid (Flu-APA). Fluorescence microscopy showed that the sporulation septa were specifically labeled by Flu-APA; this interaction was blocked by prior exposure of the cells to cefoxitin at a concentration that interfered with septation. We hypothesize that the 85-kDa PBP is involved in septum formation during sporulation of S. griseus.     

Penicillin-Binding Proteins in Selenomonas ruminantium

Diphenyl, o-phenylphenol and thiabendazole were analyzed in citrus fruits. The peel and edible parts were separately homogenized. These fungicides were extracted with dichloromethane from the homogenate, and they were fractionated with Sephadex LH-20 columns. Gas chromatography was used to determine the presence of these fungicides. The fungicides found in edible parts of citrus fruits were confirmed by gas chromatography-mass spectrometry.

Diphenyl, o-phenylphenol and thiabendazole were detected in imported grapefruits, lemons and oranges. Almost all fungicides were found in the peel. The concentrations of the three fungicides in the edible parts were very low. Some samples contained all three fungicides in the edible parts.     

Gene Disruption Studies of Penicillin-Binding Proteins 1a, 1b, and 2a in Streptococcus pneumoniae

The effects of inactivation of the genes encoding penicillin-binding protein 1a (PBP1a), PBP1b, and PBP2a in Streptococcus pneumoniae were examined. Insertional mutants did not exhibit detectable changes in growth rate or morphology, although a pbp1a pbp1b double-disruption mutant grew more slowly than its parent did. Attempts to generate a pbp1a pbp2a double-disruption mutant failed. The pbp2a mutants, but not the other mutants, were more sensitive to moenomycin, a transglycosylase inhibitor. These observations suggest that individually the pbp1a, pbp1b, and pbp2a genes are dispensable but that either pbp1a or pbp2a is required for growth in vitro. These results also suggest that PBP2a is a functional transglycosylase in S. pneumoniae.     

Direct Quantitation of the Numbers of Individual Penicillin-Binding Proteins per Cell in Staphylococcus aureus

The penicillin-binding proteins (PBPs) are a set of enzymes that participate in bacterial peptidoglycan assembly. The absolute numbers of each PBP were determined by direct measurement and have been reported for two Staphylococcus aureus strains, RN4220 (methicillin-sensitive S. aureus) and RN450M (methicillin-resistant S. aureus). From the specific activity of the labeled penicillin and the absolute number of disintegrations per minute, and from the number of CFU per milliliter calculated from proteins and optical density, a determination of the number of PBPs per cell was made. These numbers ranged from approximately 150 to 825 PBPs/cell and represent the first direct determination of absolute numbers of PBPs in S. aureus.     

Crystal Structures of Penicillin-Binding Proteins 4 and 5 from Haemophilus influenzae

We have determined high-resolution apo crystal structures of two low molecular weight penicillin-binding proteins (PBPs), PBP4 and PBP5, from Haemophilus influenzae, one of the most frequently found pathogens in the upper respiratory tract of children. Novel β-lactams with notable antimicrobial activity have been designed, and crystal structures of PBP4 complexed with ampicillin and two of the novel molecules have also been determined. Comparing the apo form with those of the complexes, we find that the drugs disturb the PBP4 structure and weaken X-ray diffraction, to very different extents. PBP4 has recently been shown to act as a sensor of the presence of penicillins in Pseudomonas aeruginosa, and our models offer a clue to the structural basis for this effect. Covalently attached penicillins press against a phenylalanine residue near the active site and disturb the deacylation step. The ready inhibition of PBP4 by β-lactams compared to PBP5 also appears to be related to the weaker interactions holding key residues in a catalytically competent position.     

Temperature-Sensitive Cell Division Mutants of Escherichia coli with Thermolabile Penicillin-Binding Proteins

The thermostability of the penicillin-binding proteins (PBPs) of 31 temperature-sensitive cell division mutants of Escherichia coli has been examined. Two independent cell division mutants have been found that have highly thermolabile PBP3. Binding of [(14)C]benzylpenicillin to PBP3 (measured in envelopes prepared from cells grown at the permissive temperature) was about 30% of the normal level at 30 degrees C, and the ability to bind [(14)C]benzylpenicillin was rapidly lost on incubation at 42 degrees C. The other PBPs were normal in both mutants. At 30 degrees C both mutants were slightly longer than their parents and on shifting to 42 degrees C they ceased dividing, but cell mass and deoxyribonucleic acid synthesis continued and long filaments were formed. At 42 degrees C division slowly recommenced, but at 44 degrees C this did not occur. The inhibition of division at 42 degrees C was suppressed by 0.35 M sucrose, and in one of the mutants it was partially suppressed by 10 mM MgCl(2). PBP3 was not stabilized in vitro at 42 degrees C by these concentrations of sucrose or MgCl(2). Revertants that grew as normal rods at 42 degrees C regained both the normal level and the normal thermostability of PBP3. The results provide extremely strong evidence that the inactivation of PBP3 at 42 degrees C in the mutants is the cause of the inhibition of cell division at this temperature and identify PBP3 as an essential component of the process of cell division in E. coli. It is the inactivation of this protein by penicillins and cephalosporins that results in the inhibition of division characteristic of low concentrations of many of these antibiotics.     

Subfamily-Specific Adaptations in the Structures of Two Penicillin-Binding Proteins from Mycobacterium tuberculosis

Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows that Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis.     

Characterization of the Synechocystis Strain PCC 6803 Penicillin-Binding Proteins and Cytokinetic Proteins FtsQ and FtsW and Their Network of Interactions with ZipN

Because very little is known about cell division in noncylindrical bacteria and cyanobacteria, we investigated 10 putative cytokinetic proteins in the unicellular spherical cyanobacterium Synechocystis strain PCC 6803. Concerning the eight penicillin-binding proteins (PBPs), which define three classes, we found that Synechocystis can survive in the absence of one but not two PBPs of either class A or class C, whereas the unique class B PBP (also termed FtsI) is indispensable. Furthermore, we showed that all three classes of PBPs are required for normal cell size. Similarly, the putative FtsQ and FtsW proteins appeared to be required for viability and normal cell size. We also used a suitable bacterial two-hybrid system to characterize the interaction web among the eight PBPs, FtsQ, and FtsW, as well as ZipN, the crucial FtsZ partner that occurs only in cyanobacteria and plant chloroplasts. We showed that FtsI, FtsQ, and ZipN are self-interacting proteins and that both FtsI and FtsQ interact with class A PBPs, as well as with ZipN. Collectively, these findings indicate that ZipN, in interacting with FtsZ and both FtsI and FtQ, plays a similar role to the Escherichia coli FtsA protein, which is missing in cyanobacteria and chloroplasts.The peptidoglycan layer (PG) of bacterial cell wall is a major determinant of cell shape, and the target of our best antibiotics. It is built from long glycan strands of repeating disaccharides cross-linked by short peptides (38). The resultant meshwork structure forms a strong and elastic exoskeleton essential for maintaining shape and withstanding intracellular pressure. Cell morphogenesis and division have been essentially studied in the rod-shaped organisms Escherichia coli and Bacillus subtilis, which divide through a single medial plane (8, 10, 21, 23). These organisms have two modes of cell wall synthesis: one involved in cell elongation and the second operating in septation (2). Each mode of synthesis is ensured by specific protein complexes involving factors implicated in the last step of PG synthesis (2). The complete assembly of PG requires a glycosyl transferase that polymerizes the glycan strands and a transpeptidase that cross-links them via their peptide side chains (35). Both activities are catalyzed by penicillin-binding proteins (PBPs), which can be divided into three classes: class A and class B high-molecular-weight (HMW) PBPs and class C low-molecular-weight (LMW) PBPs (35).Class A PBPs exhibit both transglycosylase and transpeptidase activities. In E. coli, they seem to be nonspecialized (2), as they operate in the synthesis of both cylindrical wall (cell elongation) and septal PG (cytokinesis). In B. subtilis, PBP1 (class A) is partially localized to septal sites and its depletion leads to cell division defects (31).Class B PBPs, which comprise two proteins in most bacteria, are monofunctional transpeptidases (35), each involved in longitudinal and septal growth of cell wall, respectively (36). In E. coli, this protein, PBP3, is also termed FtsI, because it belongs to the Fts group of cell division factors whose depletion leads to the filamentation phenotype (11). These at least 10 Fts proteins are recruited to the division site at mid-cell in the following sequential order: FtsZ, FtsA, ZipA, FtsK, FtsQ, FtsL/FtsB, FtsW, FtsI, and FtsN (11). The cytoplasmic protein FtsZ is the first recruited to the division site, where it polymerizes in a ring-like structure (1), which serves as a scaffold for the recruitment of the other Fts proteins and has been proposed to drive the division process (6). Together the Fts proteins form a complex machine coordinating nucleoid segregation, membrane constriction, septal PG synthesis, and possibly membrane fusion.Unlike the other PBPs, class C PBPs do not operate in PG synthesis but rather in maturation or recycling of PG during cell septation (35). They are subdivided into four types. Class C type 5 PBP removes the terminal d-alanine residue from pentapeptide side-chains (dd-carboxypeptidase activity). Types 4 and 7 are able to cleave the peptide cross-links (endopeptidase activity). Finally, type AmpH, which does not have a defined enzymatic activity, is believed to play a role in the normal course of PG synthesis, remodeling or recycling (for a review, see reference 35).In contrast to rod-shaped bacteria, less is known concerning PG synthesis, morphogenesis, and cytokinesis, and their relationships, in spherical-celled bacteria, even though a wealth of them have a strong impact on the environment and/or human health. Furthermore, unlike rod-shaped bacteria spherical-celled bacteria possess an infinite number of potential division planes at the point of greater cell diameter, and they divide through alternative perpendicular planes (26, 36, 37, 39). The spherical cells of Staphylococcus aureus seem to insert new PG strands only at the septum, and accordingly the unique class A PBP localizes at the septum during cell division (36). In contrast, the rugby-ball-shaped cells of Streptococcus pneumoniae synthesize cell wall at both the septum and the neighboring region called “equatorial rings” (36). Accordingly, class A PBP2a and PBP1a were found to operate in elongation and septation, respectively (29).In cyanobacteria, which are crucial to the biosphere in using solar energy to renew the oxygenic atmosphere and which make up the biomass for the food chain (7, 30, 40), cell division is currently investigated in two unicellular models with different morphologies: the rod-shaped Synechococcus elongatus strain PCC 7942 (19, 28) and the spherical-celled Synechocystis strain PCC 6803 (26), which both possess a small fully sequenced genome (http://genome.kazusa.or.jp/cyanobase/) that is easily manipulable (18). In both organisms FtsZ and ZipN/Arc6, a protein occurring only in cyanobacteria (ZipN) and plant chloroplasts (Arc6), were found to be crucial for cytokinesis (19, 26, 28) and to physically interact with each other (25, 26). Also, interestingly, recent studies of cell division in the filamentous cyanobacterium Anabaena (Nostoc) strain PCC 7120, showed that this process is connected with the differentiation of heterocysts, the cells dedicated to nitrogen fixation (34).In a continuous effort to study the cell division machine of the unicellular spherical cyanobacterium Synechocystis, we have presently characterized its eight presumptive PBPs (22) that define three classes and the putative cytokinetic proteins FtsQ and FtsW, as well as their network of interactions between each other and ZipN. Both FtsI and FtsQ were found to be key players in cell division in interacting with ZipN and class A PBPs. Consequently, ZipN in interacting with FtsZ (26), FtsI, and FtQ, like the FtsA protein of E. coli, could play a role similar to FtsA, which is absent in cyanobacteria and chloroplasts.     

Interactions of Human Mismatch Repair Proteins MutS�� and MutL�� with Proteins of the ATR-Chk1 Pathway

At clinically relevant doses, chemotherapeutic S_N1 DNA methylating agents induce an ATR-mediated checkpoint response in human cells that is dependent on functional MutSα and MutLα. Deficiency of either mismatch repair activity renders cells highly resistant to this class of drug, but the mechanisms linking mismatch repair to checkpoint activation have remained elusive. In this study we have systematically examined the interactions of human MutSα and MutLα with proteins of the ATR-Chk1 pathway using both nuclear extracts and purified proteins. Using nuclear co-immunoprecipitation, we have detected interaction of MutSα with ATR, TopBP1, Claspin, and Chk1 and interaction of MutLα with TopBP1 and Claspin. We were unable to detect interaction of MutSα or MutLα with Rad17, Rad9, or replication protein A in the extract system. Use of purified proteins confirmed direct interaction of MutSα with ATR, TopBP1, and Chk1 and of MutLα with TopBP1. MutSα-Claspin and MutLα-Claspin interactions were not demonstrable with purified proteins, suggesting that extract interactions are indirect or depend on post-translational modification. Use of a modified chromatin immunoprecipitation assay showed that proliferating cell nuclear antigen, ATR, TopBP1, and Chk1 are recruited to chromatin in a MutLα- and MutSα-dependent fashion after N-methyl-N′-nitro-N-nitrosoguanidine treatment. However, chromatin enrichment of replication protein A, Claspin, Rad17-RFC, and Rad9-Rad1-Hus1 was not detected in these experiments. Although our failure to observe enrichment of the latter activities could be due to sensitivity limitations, these observations may indicate a novel mechanism for ATR activation.     

Globin-like蛋白质折叠类型识别

蛋白质折叠类型识别是蛋白质结构研究的重要内容.以SCOP中的Globin-like折叠为研究对象,选择其中序列同一性小于25%的17个代表性蛋白质为训练集,采用机器和人工结合的办法进行结构比对,产生序列排比,经过训练得到了适合Globin-like折叠的概形隐马尔科夫模型(profile HMM)用于该折叠类型的识别.以Astrall.65中的68057个结构域样本进行检验,识别敏感度为99.64%,特异性100%.在折叠类型水平上,与Pfam和SUPERFAMILY单纯使用序列比对构建的HMM相比,所用模型由多于100个归为一个,仍然保持了很高的识别效果.结果表明:对序列相似度很低但具有相同折叠类型的蛋白质,可以通过引入结构比对的方法建立统一的HMM模型,实现高准确率的折叠类型识别.     

Roles of Low-Molecular-Weight Penicillin-Binding Proteins in Bacillus subtilis Spore Peptidoglycan Synthesis and Spore Properties

The peptidoglycan cortex of endospores of Bacillus species is required for maintenance of spore dehydration and dormancy, and the structure of the cortex may also allow it to function in attainment of spore core dehydration. A significant difference between spore and growing cell peptidoglycan structure is the low degree of peptide cross-linking in cortical peptidoglycan; regulation of the degree of this cross-linking is exerted by d,d-carboxypeptidases. We report here the construction of mutant B. subtilis strains lacking all combinations of two and three of the four apparent d,d-carboxypeptidases encoded within the genome and the analysis of spore phenotypic properties and peptidoglycan structure for these strains. The data indicate that while the dacA and dacC products have no significant role in spore peptidoglycan formation, the dacB and dacF products both function in regulating the degree of cross-linking of spore peptidoglycan. The spore peptidoglycan of a dacB dacF double mutant was very highly cross-linked, and this structural modification resulted in a failure to achieve normal spore core dehydration and a decrease in spore heat resistance. A model for the specific roles of DacB and DacF in spore peptidoglycan synthesis is proposed.Peptidoglycan (PG) is the structural element of the bacterial cell wall which determines cell shape and which resists the turgor pressure within the cell. The bacterial endospores produced by species of Bacillus, Clostridium, and several other bacterial genera are modified cells that are able to survive long periods and extreme conditions in a dormant, relatively dehydrated state. The PG wall within the endospore is required for maintenance of the dehydrated state (10, 11), which is the major determinant of spore heat resistance (2, 17, 22). Spore PG appears to be comprised of two distinct though contiguous layers. The thin inner layer, the germ cell wall, appears to have a structure similar to that of the vegetative wall and serves as the initial cell wall of the germinated spore (1, 20, 21, 31). The thicker outer layer, the spore cortex, has a modified structure which may determine its ability to carry out roles specific to the spore, and is rapidly degraded during spore germination (1, 20, 35, 37). The most dramatic of the cortex structural modifications results in partial cleavage or complete removal of ∼75% of the peptide side chains from the glycan strands. Loss of these peptides limits the cross-linking potential of the PG and results in the formation of only one peptide cross-link per 35 disaccharide units in the spore PG, compared to one peptide cross-link per 2.3 to 2.9 disaccharide units in the vegetative PG (1, 20, 36). This low degree of cross-linking has been predicted to give spore PG a flexibility that allows it to have a role in attainment of spore core dehydration (14, 34) in addition to its clear role in maintenance of dehydration. We are studying the structure and mechanism of synthesis of spore PG in an attempt to discern the roles of this structure and its individual components in determining spore properties.A family of proteins called the penicillin-binding proteins (PBPs) polymerizes PG on the external surface of the cell membrane (reviewed in reference 7). The high-molecular-weight (high-MW) members of this family (generally ≥60 kDa) carry the transglycosylase and transpeptidase activities involved in polymerization and cross-linking of the glycan strands. The low-MW PBPs have commonly been found to possess d,d-carboxypeptidase activity. This activity can remove the terminal d-alanine of the peptide side chains and thereby prevent the side chain from serving as a donor in the formation of a peptide cross-link. Analysis of the B. subtilis genome reveals six low-MW PBP-encoding genes: dacA (33), dacB (4), dacC (19), dacF (38), pbpE (23), and pbpX (accession no. {"type":"entrez-nucleotide","attrs":{"text":"Z99112","term_id":"32468745","term_text":"Z99112"}}Z99112). The four dac gene products exhibit very high sequence similarity to proven d,d-carboxypeptidases, and this activity has been demonstrated in vitro for the dacA and dacB products, PBP5 (12) and PBP5* (32), respectively. The sequences of the pbpE and pbpX products are more distantly related, and no activity has yet been established or ruled out for them.PBP5 is the major penicillin-binding and d,d-carboxypeptidase activity found in vegetative cells (12). Although dacA expression declines significantly during sporulation, a significant amount of PBP5 remains during the time of spore PG synthesis (29). A dacA-null mutation results in no obvious effects on vegetative growth, sporulation, spore characteristics, or spore germination (3, 33). However, loss of PBP5 does result in a reduction of cleavage of peptide side chains from the tetrapeptide to the tripeptide form in the spore PG (20). PBP5* is expressed only during sporulation and only in the mother cell compartment of the sporangium, under the control of the RNA polymerase ς^E subunit (4, 5, 28, 29). A dacB-null mutation leading to loss of this d,d-carboxypeptidase results in a fourfold increase in the effective cross-linking of the spore PG (1, 20, 22). This structural change is accompanied by only slight decreases in spore core dehydration and heat resistance (3, 22). The suspected d,d-carboxypeptidase activities of the products of the dacC and dacF genes have not been demonstrated. The latter two genes are expressed only during the postexponential growth phase: dacC is expressed during early stationary phase under the control of ς^H (19) and dacF is expressed only within the forespore under the control of ς^F (27, 38). Null mutations effecting either gene result in no obvious phenotype and no change in spore PG structure (19, 38).The multiplicity of these proteins in sporulating cells and the lack of effect of loss of some of them suggested redundancy of function among these proteins, a situation observed previously with PBPs of a high-MW class (25, 30, 39). In order to examine this possibility we have constructed mutants lacking multiple low-MW PBPs and have examined their sporulation efficiency, spore PG structure, spore heat resistance and wet density, and spore germination and outgrowth. The present study demonstrates a role for the dacF gene product in synthesis of spore PG, and we also present a model for the roles of the dacB and dacF gene products in spore PG formation.     

Linker Histones and HMG1 Proteins of Higher Plants

Abstract: Linker histones and the proteins belonging to the high mobility group 1 (HMG1) family are the most abundant proteins associated with the internucleosomal linker DNA in eukaryotic chromatin. Despite their relative abundance, there are indications that they may be involved in specific developmental pathways. In higher plants, a variety of these chromosomal proteins has been identified and characterized. In this report, we give an overview of the present knowledge about these two protein families, and discuss their likely functions in plants.     

Histochemical Tests for Proteins and Amino Acids; The Characterization of Basic Proteins

For cytophysiological work it is important to have ways of demonstrating proteins and amino acids and especially of characterizing basic and non-basic proteins. The author presents a review of the more usually employed histochemical reactions for amino acids and proteic compounds in general, with several modifications which increase their sensitivity, or specificity and localization. The author describes the histochemical arginine reaction, recently introduced by him, by means of which the characterization of basic and non-basic proteins can be easily accomplished in every laboratory without costly apparatus; this reaction serves also for the demonstration of proteins in general. The application of protein histochemical tests for quantitative purposes is discussed in connection with the characterization of the basic proteins and the determination of the relative concentration and the active metabolic changes of proteic compounds.     

Coronin1 Proteins Dictate Rac1 Intracellular Dynamics and Cytoskeletal Output

Rac1 regulates lamellipodium formation, myosin II-dependent contractility, and focal adhesions during cell migration. While the spatiotemporal assembly of those processes is well characterized, the signaling mechanisms involved remain obscure. We report here that the cytoskeleton-related Coronin1A and -1B proteins control a myosin II inactivation-dependent step that dictates the intracellular dynamics and cytoskeletal output of active Rac1. This step is signaling-branch specific, since it affects the functional competence of active Rac1 only when forming complexes with downstream ArhGEF7 and Pak proteins in actomyosin-rich structures. The pathway is used by default unless Rac1 is actively rerouted away from the structures by upstream activators and signals from other Rho GTPases. These results indicate that Coronin1 proteins are at the center of a regulatory hub that coordinates Rac1 activation, effector exchange, and the F-actin organization state during cell signaling. Targeting this route could be useful to hamper migration of cancer cells harboring oncogenic RAC1 mutations.     

Human Cytidine Triphosphate Synthetase 1 Interacting Proteins

We investigated the interacting proteins and intracellular localization of CTP synthetase 1 (CTPS1) in mammalian cells. CTPS1 interacted with a GST- peptidyl prolyl isomerase, Pin1 fusion (GST-Pin1) in a Ser 575 (S575) phosphorylation-dependent manner. Immunoprecipitation experiments demonstrated that CTPS1 also bound tubulin, and thirteen additional coimmunoprecipitating proteins were identified by mass spectrometry. Immunolocalization experiments showed that tubulin and CTPS1 colocalized subcellularly. Taxol treatment enhanced this but cotreatment of cells with the CTPS inhibitor, cyclopentenyl cytosine (CPEC), and taxol failed to disrupt the colocalization. Thus, these studies provide novel information on the potential interacting proteins that may regulate CTPS1 function or intracellular localization.     

Identification of a Novel Penicillin-Binding Protein from Helicobacter pylori

The Helicobacter pylori genome encodes four penicillin-binding proteins (PBPs). PBPs 1, 2, and 3 exhibit similarities to known PBPs. The sequence of PBP 4 is unique in that it displays a novel combination of two highly conserved PBP motifs and an absence of a third motif. Expression of PBP 4, but not PBP 1, 2, or 3, is significantly increased during mid- to late-log-phase growth.     

鼠肝炎冠状病毒非结构蛋白1及其突变体的原核表达

目的:构建鼠肝炎冠状病毒(MHV)非结构蛋白1(NSP1)及其突变体(NSP1 mu)的原核重组表达质粒,在大肠杆菌中分别融合表达重组NSP1及NSP1 mu。方法:以现有质粒载体为模板,扩增编码NSP1及NSP1 mu的基因片段,并克隆至pMD18-T克隆载体;菌落PCR鉴定阳性克隆并测序分析;将阳性克隆的目的片段亚克隆至表达载体pET-28a,并转化大肠杆菌TOP10感受态细胞,PCR和双酶切鉴定转化菌落;将阳性质粒转化大肠杆菌BL21(DE3)感受态细胞并加入IPTG诱导表达,SDS-PAGE和免疫印迹分析目的蛋白的表达。结果:PCR扩增得到表达NSP1及NSP1 mu的特异片段,并克隆到pMD18-T载体,测序结果正确无误;构建了NSP1和NSP1 mu的重组表达质粒,并在大肠杆菌BL21(DE3)中分别融合表达了重组NSP1及NSP1 mu,表达的目的蛋白均能与His单克隆抗体特异结合;用Ni-NTA琼脂糖试剂盒纯化重组蛋白,获得可溶性的NSP1及NSP1 mu,相对分子质量分别为27×103和28×103。结论:在大肠杆菌中分别表达并纯化获得了大量可溶性重组NSP1及NSP1 mu。     

Screening for Differentially Expressed Proteins Relevant to the Differential Diagnosis of Sarcoidosis and Tuberculosis

Background

In this study, we sought to identify differentially expressed proteins in the serum of patients with sarcoidosis or tuberculosis and to evaluate these proteins as markers for the differential diagnosis of sarcoidosis and sputum-negative tuberculosis.

Methods

Using protein microarrays, we identified 3 proteins exhibiting differential expression between patients with sarcoidosis and tuberculosis. Elevated expression of these proteins was verified using the enzyme-linked immunosorbent assay (ELISA) and was further confirmed by immunohistochemistry. Receiver operating characteristic (ROC) curve, logistic regression analysis, parallel, and serial tests were used to evaluate the diagnostic efficacy of the proteins.

Results

Intercellular Adhesion Molecule 1(ICAM-1) and leptin were screened for differentially expressed proteins relevant to sarcoidosis and tuberculosis. Using ROC curves, we found that ICAM-1 (cutoff value: 57740 pg/mL) had an area under the curve (AUC), sensitivity, and specificity of 0.718, 62.3%, and 79.5% respectively, while leptin (cutoff value: 1193.186 pg/mL) had an AUC, sensitivity, and specificity of 0.763, 88.3%, and 65.8%, respectively. Logistic regression analysis revealed that the AUC, sensitivity, and specificity of combined leptin and ICAM-1 were 0.787, 89.6%, and 65.8%, respectively, while those of combined leptin, ICAM-1, and body mass index (BMI) were 0.837, 90.9%, and 64.4%, respectively, which had the greatest diagnostic value. Parallel and serial tests indicated that the BMI-leptin parallel with the ICAM-1 serial was the best diagnostic method, achieving a sensitivity and specificity of 86.5% and 73.1%, respectively. Thus, our results identified elevated expression of ICAM-1 and leptin in serum and granulomas of sarcoidosis patients.

Conclusions

ICAM-1 and leptin were found to be potential markers for the diagnosis of sarcoidosis and differential diagnosis of sarcoidosis and sputum-negative tuberculosis.