Engineering of an industrial polyoxin producer for the rational production of hybrid peptidyl nucleoside antibiotics

Polyoxins and nikkomycins are potent antifungal peptidyl nucleoside antibiotics, which inhibit fungal cell wall biosynthesis. They consist of a nucleoside core and one or two independent peptidyl moieties attached to the core at different sites. Making mutations and introducing heterologous genes into an industrial Streptomyces aureochromogenes polyoxin producer, resulted in the production of four polyoxin-nikkomycin hybrid antibiotics designated as polyoxin N and nikkoxin B-D, whose structures were confirmed using high resolution MS and NMR. Two of the hybrid antibiotics, polyoxin N and nikkoxin D, were significantly more potent against some human or plant fungal pathogens than their parents. The data provides an example for rational generation of novel peptidyl nucleoside antibiotics in an industrial producer.     

Detection of endopeptidase activity and analysis of cleavage specificity using a radiometric solid-phase enzymatic assay

A radiometric procedure to detect the presence of proteolytic enzymes and analyze their substrate specificity is described. The enzymatic activity is first measured by the release into solution of a radiolabeled reporter group from an immobilized peptidyl substrate. Two peptidyl substrates encompassing multiple cleavage sites, a derivative of Leu-enkephalin and a peptide related to the bait region of human alpha 2-macroglobulin, are prepared and linked via a spacer molecule to an insoluble support. The labeled peptides released are then separated by high-performance liquid chromatography. The position of the released peptides upon chromatography allows direct identification of the sites of cleavage. The assay, using a radioactive iodinated tyrosine residue as reporter group, is extremely sensitive (less than 0.02 pg/ml of trypsin), reproducible, and easy to perform while yielding unambiguous identification of the sites of cleavage. This assay can be used to detect the presence of enzymatic activities and/or of enzyme inhibitors. Furthermore, it can be easily adapted to detect from a variety of sources all four classes of enzymes known by using appropriate peptidyl substrate sequences, buffer, pH, and incubation conditions.     

Bidirectional control of the inner dynamics of focal adhesions promotes cell migration

Focal adhesions (FA) are bidirectional mechanical biosensors that allow cells to integrate intracellular and extracellular cues. Their function is tightly regulated by changes in molecular composition and also by variation in the spatio-temporal dynamics of FA components within this structure. A closely regulated turnover of FA proteins within FA sites allows cells to respond appropriately to their environment, thereby impacting on cell shape and function. FA protein dynamics are linked to FA maturation and rates of assembly and disassembly, and have a significant influence on tumor cell migration. Using the FRAP technique to investigate the hidden internal dynamics of FA, we identified two new regulators of FA dynamics and cell migration: the Mgat5/galectin-3 lattice and tyrosine phosphorylated caveolin-1 (pY14Cav1). In this short review we first introduce FA and their complex dynamic behavior. We then present the Mgat5/galectin-3 lattice and caveolin-1 and discuss their concerted role in FA dynamics, which defines previously unknown, interdependent roles in tumor cell migration. We conclude with a discussion of interesting unexplored avenues that might lead to a better understanding of the complex mechanism of FA dynamics.Key words: focal adhesion, migration, caveolin-1, tyrosine 14, galectin-3, Mgat5, turnover, dynamics     

Triterpenoid carotenoids and related lipids. The triterpenoid carotenes of Streptococcus faecium UNH 564P

1. The occurrence of a novel series of triterpenoid carotenes in Streptococcus faecium UNH 564P is reported. 2. This series, which comprises the C(30) analogues of phytoene, phytofluene, zeta-carotene, 7,8,11,12-tetrahydrolycopene and neurosporene, appears to be analogous to the C(40) Porter-Lincoln series and a pathway of triterpenoid carotene dehydrogenation is proposed to account for the formation of these compounds. 3. Two cis isomers of the C(30) analogue of neurosporene are described. 4. An appropriate system of nomenclature for these novel compounds is proposed.     

Isodityrosine, a new cross-linking amino acid from plant cell-wall glycoprotein.

1. Cell-wall hydrolysates from calli of all higher plants tested contained a new phenolic amino acid for which the trivial name isodityrosine is proposed. Isodityrosine was shown to be an oxidatively coupled dimer of tyrosine with the two tyrosine units linked by a diphenyl ether bridge. 2. The amount of isodityrosine in sodium dodecyl sulphate-insoluble cell-wall preparations was proportional to the amount of hydroxyproline. 3. Acidified chlorite split the diphenyl ether bridge of isodityrosine, and concomitantly solubilized the cell-wall glycoprotein. 4. Dithiothreitol inhibited isodityrosine synthesis in vivo, and suppressed in parallel the covalent binding of newly synthesized protein in the cell wall. 5. It is suggested that isodityrosine is an inter-polypeptide cross-link responsible for the insolubility of plant cell-wall glycoprotein.     

Synthesis and biological evaluation of thioglycosylated porphyrins for an application in photodynamic therapy.

The aim of this work is the synthesis of a new family of glycosylated porphyrins in which the sugar moieties are linked to the tetrapyrrole ring by a thioglycosidic bond. Two series have been designed. The first one corresponds to meso-aryl porphyrin derivatives. The second one has been obtained from protoporphyrin IX derivatization. Aryl-porphyrins were prepared from tristolyl o- and p-hydroxyporphyrins followed by bromoallylation and thioglycosylation with peracetylated S-glucose, mannose and galactose and deprotection. The other series has been synthesized from protoporphyrin IX dimethylester with a regioselective glycosylation of terminal alkenyl carbon. The UV-visible, NMR and MALDI mass spectra are presented. Photocytotoxicities of the synthesized compounds against K562 chronic leukaemia cell line has been evaluated.     

The Cpn10(1) Co-Chaperonin of A. thaliana Functions Only as a Hetero-Oligomer with Cpn20

The A. thaliana genome encodes five co-chaperonin homologs, three of which are destined to the chloroplast. Two of the proteins, Cpn10(2) and Cpn20, form functional homo-oligomers in vitro. In the current work, we present data on the structure and function of the third A. thaliana co-chaperonin, which exhibits unique properties. We found that purified recombinant Cpn10(1) forms inactive dimers in solution, in contrast to the active heptamers that are formed by canonical Cpn10s. Additionally, our data demonstrate that Cpn10(1) is capable of assembling into active hetero-oligomers together with Cpn20. This finding was reinforced by the formation of active co-chaperonin species upon mixing an inactive Cpn20 mutant with the inactive Cpn10(1). The present study constitutes the first report of a higher plant Cpn10 subunit that is able to function only upon formation of hetero-oligomers with other co-chaperonins.     

Structural and functional characterization of microcin C resistance peptidase MccF from Bacillus anthracis

Microcin C (McC) is heptapeptide adenylate antibiotic produced by Escherichia coli strains carrying the mccABCDEF gene cluster encoding enzymes, in addition to the heptapeptide structural gene mccA, necessary for McC biosynthesis and self-immunity of the producing cell. The heptapeptide facilitates McC transport into susceptible cells, where it is processed releasing a non-hydrolyzable aminoacyl adenylate that inhibits an essential aminoacyl-tRNA synthetase. The self-immunity gene mccF encodes a specialized serine peptidase that cleaves an amide bond connecting the peptidyl or aminoacyl moieties of, respectively, intact and processed McC with the nucleotidyl moiety. Most mccF orthologs from organisms other than E. coli are not linked to the McC biosynthesis gene cluster. Here, we show that a protein product of one such gene, MccF from Bacillus anthracis (BaMccF), is able to cleave intact and processed McC, and we present a series of structures of this protein. Structural analysis of apo-BaMccF and its adenosine monophosphate complex reveals specific features of MccF-like peptidases that allow them to interact with substrates containing nucleotidyl moieties. Sequence analyses and phylogenetic reconstructions suggest that several distinct subfamilies form the MccF clade of the large S66 family of bacterial serine peptidases. We show that various representatives of the MccF clade can specifically detoxify non-hydrolyzable aminoacyl adenylates differing in their aminoacyl moieties. We hypothesize that bacterial mccF genes serve as a source of bacterial antibiotic resistance.     

Bidirectional control of the inner dynamics of focal adhesions promotes cell migration

Focal adhesions (FA) are bidirectional mechanical biosensors that allow cells to integrate

intracellular and extracellular cues. Their function is tightly regulated by changes in

molecular composition and also by variation in the spatio-temporal dynamics of FA

components within this structure. A closely regulated turnover of FA proteins within FA

sites allows cells to respond appropriately to their environment, thereby impacting on cell

shape and function. FA protein dynamics are linked to FA maturation and rates of

assembly and disassembly, and have a significant influence on tumor cell migration.

Using the FRAP technique to investigate the hidden internal dynamics of FA, we

identified two new regulators of FA dynamics and cell migration: the Mgat5/galectin-3

lattice and tyrosine phosphorylated caveolin-1 (pY14Cav1). In this short review we first

introduce FA and their complex dynamic behavior. We then present the Mgat5/galectin-3

lattice and caveolin-1 and discuss their concerted role in FA dynamics, which defines

previously unknown, interdependent roles in tumor cell migration. We conclude with a

discussion of interesting unexplored avenues that might lead to a better understanding of

the complex mechanism of FA dynamics.     

Cortactin as a Target for FAK in the Regulation of Focal Adhesion Dynamics

Background

Efficient cell movement requires the dynamic regulation of focal adhesion (FA) formation and turnover. FAs are integrin-associated sites of cell attachment and establish linkages to the cellular actin cytoskeleton. Cells without focal adhesion kinase (FAK), an integrin-activated tyrosine kinase, exhibit defects in FA turnover and cell motility. Cortactin is an actin binding adaptor protein that can influence FA dynamics. FAK and cortactin interact, but the cellular role of this complex remains unclear.

Principal Findings

Using FAK-null fibroblasts stably reconstituted with green fluorescent protein (GFP) tagged FAK constructs, we find that FAK activity and FAK C-terminal proline-rich region 2 (PRR2) and PRR3 are required for FA turnover and cell motility. Cortactin binds directly to FAK PRR2 and PRR3 sites via its SH3 domain and cortactin expression is important in promoting FA turnover and GFP-FAK release from FAs. FAK-cortactin binding is negatively-regulated by FAK activity and associated with cortactin tyrosine phosphorylation. FAK directly phosphorylates cortactin at Y421 and Y466 and over-expression of cortactin Y421, Y466, and Y482 mutated to phenylalanine (3YF) prevented FAK-enhanced FA turnover and cell motility. However, phospho-mimetic cortactin mutated to glutamic acid (3YE) did not affect FA dynamics and did not rescue FA turnover defects in cells with inhibited FAK activity or with PRR2-mutated FAK that does not bind cortactin.

Conclusions

Our results support a model whereby FAK-mediated FA remodeling may occur through the formation of a FAK-cortactin signaling complex. This involves a cycle of cortactin binding to FAK, cortactin tyrosine phosphorylation, and subsequent cortactin-FAK dissociation accompanied by FA turnover and cell movement.     

Structural Factors Enabling Successful GFP-Like Proteins with Alanine as the Third Chromophore-Forming Residue

GFP-like proteins from lancelets (lanFPs) is a new and least studied group that already generated several outstanding biomarkers (mNeonGreen is the brightest FP to date) and has some unique features. Here, we report the study of four homologous lanFPs with GYG and GYA chromophores. Until recently, it was accepted that the third chromophore-forming residue in GFP-like proteins should be glycine, and efforts to replace it were in vain. Now, we have the first structure of a fluorescent protein with a successfully matured chromophore that has alanine as the third chromophore-forming residue. Consideration of the protein structures revealed two alternative routes of posttranslational transformation, resulting in either chromophore maturation or hydrolysis of GYG/GYA tripeptide. Both transformations are catalyzed by the same set of catalytic residues, Arg88 and Glu35–Wat–Glu211 cluster, whereas the residues in positions 62 and 102 shift the equilibrium between chromophore maturation and hydrolysis.     

X-ray diffraction analysis of the inhibition of porcine pancreatic elastase by a peptidyl trifluoromethylketone

X-ray crystallographic data to 2.57 A resolution (1 A = 0.1 nm) have been measured for the complex of a peptidyl trifluoromethylketone inhibitor with porcine pancreatic elastase (PPE); R = 0.14. The inhibitor forms a stable complex with the enzyme by means of a covalent attachment to active site Ser195O gamma, resulting in a hemiketal moiety with tetrahedral geometry. The tripeptide protion binds as an antiparallel beta-sheet, with four hydrogen bonds augmenting the active-site covalent linkage, Ki = 9.5 microM. His57 exhibits a bifurcated H-bond to both Ser195O gamma and an F atom of the inhibitor. This study is one of a series which explores the binding geometry of a variety of small substrates and inhibitors to PPE. This peptidyl-PPE complex affords insight into the binding geometry of a novel trifluoromethylketone moiety to a serine proteinase.     

Monoclonal antibodies to an artificial immunogen, specifically demonstrating phosphotyrosine-containing proteins

The importance of protein phosphorylation at tyrosyl hydroxy groups in the control of cell proliferation has recently been established. For identification of tyrosine-phosphorylated proteins, monoclonal antibodies (Mabs) against artificial immunogens containing O-phosphotyrosine (pTyr) or tripeptide pTyr-Gly-Gly as haptens were generated; the haptens were coupled to carrier proteins (bovine serum albumin, human immunoglobulin, keyhole limpet hemocyanin). After immunization of mice with pTyr coupled to keyhole limpet hemocyanin, Mabs were generated which were highly specific for pTyr and did not cross-react with O-phosphoserine, O-phosphothreonine, tyrosine or nucleoside-5'-monophosphates. The Mabs specifically react with tyrosinephosphorylated proteins in the Rous sarcoma virus-transformed rat XC-cell.     

cDNA nucleotide sequence and expression of a tobacco cytoplasmic ribosomal protein L2 gene.

The ribosomal protein L2 is an essential component of the ribosomal large subunit by its relation to the peptidyl transferase reaction, subunit association and elongation factor G-GTP binding. We have isolated a 937 nucleotide long cDNA encoding a cytoplasmic ribosomal L2 protein. Its deduced protein contains 260 amino acid residues and shows 65% identity with eucaryotic RL2 but only 32% identity with the chloroplast homologue. In addition, the protein presents the PROSITE signature which matches all the 50S and 60S L2 proteins and the two residues involved in the peptidyl transferase activity. The corresponding mRNA is accumulated in young plant tissues, in growing cell suspension and in germinating seeds but is not detectable in mature plant tissues, stationary cell suspension and in dry seeds. The mRNA accumulation is correlated with the growth process. Southern blot hybridization shows that cytoplasmic ribosomal protein L2 is encoded by two types of gene which could originate from each parent. highly homologous L2 genes were also detected by Southern blots in the genomes of several monocot and dicot plant species.     

Solution structure of the human parvulin-like peptidyl prolyl cis/trans isomerase, hPar14

The hPar14 protein is a peptidyl prolyl cis/trans isomerase and is a human parvulin homologue. The hPar14 protein shows about 30 % sequence identity with the other human parvulin homologue, hPin1. Here, the solution structure of hPar14 was determined by nuclear magnetic resonance spectroscopy. The N-terminal 35 residues preceding the peptidyl prolyl isomerase domain of hPar14 are unstructured, whereas hPin1 possesses the WW domain at its N terminus. The fold of residues 36-131 of hPar14, which comprises a four-stranded beta-sheet and three alpha-helices, is superimposable onto that of the peptidyl prolyl isomerase domain of hPin1. To investigate the interaction of hPar14 with a substrate, the backbone chemical-shift changes of hPar14 were monitored during titration with a tetra peptide. Met90, Val91, and Phe94 around the N terminus of alpha3 showed large chemical-shift changes. These residues form a hydrophobic patch on the molecular surface of hPar14. Two of these residues are conserved and have been shown to interact with the proline residue of the substrate in hPin1. On the other hand, hPar14 lacks the hPin1 positively charged residues (Lys63, Arg68, and Arg69), which determine the substrate specificity of hPin1 by interacting with phosphorylated Ser or Thr preceding the substrate Pro, and exhibits a different structure in the corresponding region. Therefore, the mechanism determining the substrate specificity seems to be different between hPar14 and hPin1.     

Engineering a thermostable human prolyl endopeptidase for antibody-directed enzyme prodrug therapy

We present a new antibody-directed enzyme prodrug therapy strategy (ADEPT) based on a post-proline cleaving endopeptidase and prodrugs, in which cytotoxic moieties are linked to a proline-containing peptide. Human prolyl endopeptidase was expressed in Escherichia coli and purified to homogeneity. The enzyme was active in buffer and in human serum but was rapidly thermally inactivated by incubation at 37 degrees C, thus preventing applications in vivo. While prolyl endopeptidase display on filamentous phage abolished viral infectivity and prevented directed evolution strategies based on phage display, we robotically screened 10752 individual colonies of mutant enzymes using a fluorogenic assay to improve enzyme stability. A single amino acid mutation (Glu289 --> Gly) improved protein stability, resulting in a half-life of 16 h at 37 degrees C in phosphate buffer. Two prodrugs were synthesized, in which an N-protected glycine-proline dipeptide was covalently coupled to doxorubicin and melphalan. (Benzyloxycarbonyl)glycylprolylmelphalan, but not the more sterically hindered doxorubicin prodrug, could be efficiently activated by prolyl endopeptidase [specific activity = 813.3 nmol min(-1) (mg of enzyme)(-1) at 25 degrees C]. The melphalan prodrug was essentially nontoxic to CHO, F9 teratocarcinoma, MCF7 breast adenocarcinoma, and p3U1 mouse myeloma cells up to millimolar concentrations, while prodrug incubation with the engineered prolyl endopeptidase mutant led to a cell killing profile superimposable to the one of melphalan. The prolyl endopeptidase mutant was then chemically coupled to the human antibody L19, specific to the EDB domain of fibronectin, a marker of angiogenesis. The resulting immunoconjugate retains antigen binding and enzymatic activity, thus opening the way to anticancer ADEPT applications.     

Structural and functional characterization of a promiscuous feruloyl esterase (Est1E) from the rumen bacterium Butyrivibrio proteoclasticus

The release of polysaccharide from the plant cell wall is a key process to release the stored energy from plant biomass. Within the ruminant digestive system, a host of commensal microorganisms speed the breakdown of plant cell matter releasing fermentable sugars. The presence of phenolic compounds, most notably ferulic acid (FA), esterified within the cell wall is thought to pose a significant impediment to the degradation of the plant cell wall. The structure of a FA esterase from the ruminant bacterium Butyrivibrio proteoclasticus has been determined in two different space groups, in both the apo‐form, and the ligand bound form with FA located in the active site. The structure reveals a new lid domain that has no structural homologues in the PDB. The flexibility of the lid domain is evident by the presence of three different conformations adopted by different molecules in the crystals. In the FA‐bound structures, these conformations show sequential binding and closing of the lid domain over the substrate. Enzymatic activity assays demonstrate a broad activity against plant‐derived hemicellulose, releasing at least four aromatic compounds including FA, coumaric acid, coumarin‐3‐carboxylic acid, and cinnamic acid. The rumen is a complex ecosystem that efficiently degrades plant biomass and the genome of B. proteoclasticus contains greater than 130 enzymes, which are potentially involved in this process of which Est1E is the first to be well characterized. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

XIAP reverses various functional activities of FRNK in endothelial cells

In endothelial cells, focal adhesion kinase (FAK) regulates cell proliferation, migration, adhesion, and shear-stimulated activation of MAPK. We recently found that FAK is recruited into focal adhesion (FA) sites through interactions with XIAP (X-chromosome linked inhibitor of apoptosis protein) and activated by Src kinase in response to shear stress. In this study, we examined which domain(s) of FAK is(are) important for various vascular functions such as FA recruiting, XIAP-binding and shear stress-stimulated ERK activation. Through a series of experiments, we determined that the FRNK domain is recruited into FA sites and promotes endothelial cell adhesion. Interestingly, XIAP knockdown was shown to reduce FA recruitment of FRNK and the cell adhesive effect of FRNK. In addition, we found that XIAP interacts with FRNK, suggesting cross-talk between XIAP and FRNK. We also demonstrated that FRNK inhibits endothelial cell migration and shear-stimulated ERK activation. These inhibitory effects of FRNK were reversed by XIAP knockdown. Taken together, we can conclude that XIAP plays a key role in vascular functions of FRNK or FRNK domain-mediated vascular functions of FAK.     

Ferulate–coniferyl alcohol cross-coupled products formed by radical coupling reactions

Radical coupling reactions between ethyl ferulate (Et-FA), a simple model for feruloyl polysaccharides in planta, and coniferyl alcohol (CA), a monolignol, were studied in order to better understand the polymer cross-coupling interactions among polysaccharides and monolignols or lignin, mediated by ferulate (FA), in plant cell walls. Cross-coupled FA/CA dimers produced in an aqueous buffer (pH 5.0) containing peroxidase/hydrogen peroxide were isolated and characterized by NMR. The total coupling products were characterized by 2D ¹³C–¹H correlation (HSQC) NMR spectroscopy and GC–MS. Results from this study showed that ferulate readily cross-couples with coniferyl alcohol through free radical coupling mechanisms producing a series of cross-coupled FA/CA dimers with β-O-4-, β-5-/8-5-, and 8-β-linkages; the syntheses and isolation of β-5- and 8-5-cross-coupled dimers are reported here. The transformation from 8-β-coupled FA/CA hydroxyl esters into lactones through intramolecular transesterification is demonstrated for the first time and mechanisms behind these transformations are discussed. The finding of both β-5- and 8-5-cross-coupled dimers in this study suggests that analogs of both may be present in plant cell walls. Finally it is suggested that ferulates in plants indeed react with monolignols through free radical mechanisms producing a more diverse array of cross-coupled dimers than previously reported.