Optimizing the expression of chimeric genes in plant cells

Summary The Serratia marcescens chiA gene encodes a secreted chitinase activity which contributes to the fungal growth inhibition exhibited by this bacterium. The coding region from the chiA gene was fused to the promoter and 3 polyadenylation region of the Agrobacterium nopaline synthase gene. Site-directed mutagenesis of specific nucleotides surrounding the initiating AUG of the coding sequence of this chimeric gene resulted in up to an eight-fold increase in the amount of chitinase protein detected in transformed plant tissue. Analysis of the chiA mRNA indicated that these nucleotides also affected mRNA levels. At least 50% of the chitinase protein produced in transformed tobacco cells was the same molecular weight as the S. marcescen secreted protein.     

Combination of a Proteomics Approach and Reengineering of Meso Scale Network Models for Prediction of Mode-of-Action for Tyrosine Kinase Inhibitors

In drug discovery, the characterisation of the precise modes of action (MoA) and of unwanted off-target effects of novel molecularly targeted compounds is of highest relevance. Recent approaches for identification of MoA have employed various techniques for modeling of well defined signaling pathways including structural information, changes in phenotypic behavior of cells and gene expression patterns after drug treatment. However, efficient approaches focusing on proteome wide data for the identification of MoA including interference with mutations are underrepresented. As mutations are key drivers of drug resistance in molecularly targeted tumor therapies, efficient analysis and modeling of downstream effects of mutations on drug MoA is a key to efficient development of improved targeted anti-cancer drugs. Here we present a combination of a global proteome analysis, reengineering of network models and integration of apoptosis data used to infer the mode-of-action of various tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) cell lines expressing wild type as well as TKI resistance conferring mutants of BCR-ABL. The inferred network models provide a tool to predict the main MoA of drugs as well as to grouping of drugs with known similar kinase inhibitory activity patterns in comparison to drugs with an additional MoA. We believe that our direct network reconstruction approach, demonstrated on proteomics data, can provide a complementary method to the established network reconstruction approaches for the preclinical modeling of the MoA of various types of targeted drugs in cancer treatment. Hence it may contribute to the more precise prediction of clinically relevant on- and off-target effects of TKIs.     

Oxygen-independent coproporphyrinogen-III oxidase HemN from Escherichia coli

In bacteria the oxygen-independent coproporphyrinogen-III oxidase catalyzes the oxygen-independent conversion of coproporphyrinogen-III to protoporphyrinogen-IX. The Escherichia coli hemN gene encoding a putative part of this enzyme was overexpressed in E. coli. Anaerobically purified HemN is a monomeric protein with a native M(r) = 52,000 +/- 5,000. A newly established anaerobic enzyme assay was used to demonstrate for the first time in vitro coproporphyrinogen-III oxidase activity for recombinant purified HemN. The enzyme requires S-adenosyl-l-methionine (SAM), NAD(P)H, and additional cytoplasmatic components for catalysis. An oxygen-sensitive iron-sulfur cluster was identified by absorption spectroscopy and iron analysis. Cysteine residues Cys(62), Cys(66), and Cys(69), which are part of the conserved CXXXCXXC motif found in all HemN proteins, are essential for iron-sulfur cluster formation and enzyme function. Completely conserved residues Tyr(56) and His(58), localized closely to the cysteine-rich motif, were found to be important for iron-sulfur cluster integrity. Mutation of Gly(111) and Gly(113), which are part of the potential GGGTP S-adenosyl-l-methionine binding motif, completely abolished enzymatic function. Observed functional properties in combination with a recently published computer-based enzyme classification (Sofia, H. J., Chen, G., Hetzler, B. G., Reyes-Spindola, J. F., and Miller, N. E. (2001) Nucleic Acids Res. 29, 1097-1106) identifies HemN as "Radical SAM enzyme." An appropriate enzymatic mechanism is suggested.     

Mesothelial cells in suspension expose an enriched integrin repertoire capable of capturing soluble fibronectin and laminin

Pleural cavities are lined by a polarized monolayer of mesothelial cells (MC). During pleuritis, MC are shed into effusions, and pleural obstruction may occur. Integrins are cell surface receptors mediating interactions with extracellular matrix (ECM) proteins. The distribution of beta 1-, beta 3-, beta 4-integrins and fibronectin and laminin in normal and chronically inflamed pleura and in/on MC from pleural effusions was examined by immunomorphology and flow cytometry. Adhesion assays of MC to fibronectin and laminin were performed. In situ, resting MC expressed beta 1-, beta 3-, and beta 4-, and alpha v-subunits. Activated MC were beta 1- and alpha v-positive and also expressed alpha 3 and alpha 6; beta 4 was confined to the basal surface of MC; beta 3 was absent. Floating MC from effusions neoexpressed alpha 5 and reexpressed beta 3. In vitro, MC surface expressed beta 1, beta 3, alpha 3, alpha 5, alpha 6, alpha v, and also alpha 1 and alpha 2. In normal pleura, fibronectin and laminin were components of the basement membrane. In pleuritis, the basement membrane was desintegrated. Instead, newly formed fibronectin/laminin containing fibrils extended into the submesothelial connective tissue. Floating MC freshly isolated from effusions carried fibronectin and laminin on their surface and showed specific binding to these ECM proteins. Binding was blocked by anti-beta 1 or anti-alpha 5 and anti-alpha 6 antibodies, respectively. MC incubated with fibronectin showed a clear shift to the S phase, while laminin had no effect. In conclusion, activated and detached MC progressively enrich their integrin repertoire. By capturing soluble fibronectin and laminin and by matrix-mediated bridging, readhering MC may contribute to pleural obstruction. Further, soluble fibronectin bound to alpha 5 beta 1 might be life-sustaining for floating MC by driving cells into cell cycle.     

Crystal structure of coproporphyrinogen III oxidase reveals cofactor geometry of Radical SAM enzymes

'Radical SAM' enzymes generate catalytic radicals by combining a 4Fe-4S cluster and S-adenosylmethionine (SAM) in close proximity. We present the first crystal structure of a Radical SAM enzyme, that of HemN, the Escherichia coli oxygen-independent coproporphyrinogen III oxidase, at 2.07 A resolution. HemN catalyzes the essential conversion of coproporphyrinogen III to protoporphyrinogen IX during heme biosynthesis. HemN binds a 4Fe-4S cluster through three cysteine residues conserved in all Radical SAM enzymes. A juxtaposed SAM coordinates the fourth Fe ion through its amide nitrogen and carboxylate oxygen. The SAM sulfonium sulfur is near both the Fe (3.5 A) and a neighboring sulfur of the cluster (3.6 A), allowing single electron transfer from the 4Fe-4S cluster to the SAM sulfonium. SAM is cleaved yielding a highly oxidizing 5'-deoxyadenosyl radical. HemN, strikingly, binds a second SAM immediately adjacent to the first. It may thus successively catalyze two propionate decarboxylations. The structure of HemN reveals the cofactor geometry required for Radical SAM catalysis and sets the stage for the development of inhibitors with antibacterial function due to the uniquely bacterial occurrence of the enzyme.     

A novel human recombinant single-chain antibody targeting CD166/ALCAM inhibits cancer cell invasion in vitro and in vivo tumour growth

Screening a phage-display single-chain antibody library for binding to the breast cancer cell line PM-1 an antibody, scFv173, recognising activated leukocyte cell adhesion molecule (ALCAM, CD166) was isolated and its binding profile was characterized. Positive ALCAM immunohistochemical staining of frozen human tumour sections was observed. No ALCAM staining was observed in the majority of tested normal human tissues (nine of ten). Flow cytometry analyses revealed binding to 22 of 26 cancer cell lines of various origins and no binding to normal blood and bone marrow cells. Antibody binding inhibited invasion of the breast cancer cell line MDA-MB-231 by 50% in an in vitro Matrigel-coated membrane invasion assay. Reduced growth of tumours in nude mice was observed in an in vivo model in which the mice were injected subcutaneously with colorectal carcinoma HCT 116 cells and treated with scFv173 when compared to control. In summary, we have characterized a novel fully human scFv antibody recognising ALCAM on cancer cells and in tumour tissues that reduces cancer cell invasion and tumour growth in accordance with the hypothesised role for ALCAM in cell growth and migration control.     

Cys Palmitoylation of the β Subunit Modulates Gating of the Epithelial Sodium Channel

The epithelial Na⁺ channel (ENaC) is comprised of three homologous subunits (α, β, and γ) that have a similar topology with two transmembrane domains, a large extracellular region, and cytoplasmic N and C termini. Although ENaC activity is regulated by a number of factors, palmitoylation of its cytoplasmic Cys residues has not been previously described. Fatty acid-exchange chemistry was used to determine whether channel subunits were Cys-palmitoylated. We observed that only the β and γ subunits were modified by Cys palmitoylation. Analyses of ENaCs with mutant β subunits revealed that Cys-43 and Cys-557 were palmitoylated. Xenopus oocytes expressing ENaC with a β C43A,C557A mutant had significantly reduced amiloride-sensitive whole cell currents, enhanced Na⁺ self-inhibition, and reduced single channel P_o when compared with wild-type ENaC, while membrane trafficking and levels of surface expression were unchanged. Computer modeling of cytoplasmic domains indicated that β Cys-43 is in proximity to the first transmembrane α helix, whereas β Cys-557 is within an amphipathic α-helix contiguous with the second transmembrane domain. We propose that β subunit palmitoylation modulates channel gating by facilitating interactions between cytoplasmic domains and the plasma membrane.     

TMPRSS4-dependent activation of the epithelial sodium channel requires cleavage of the γ-subunit distal to the furin cleavage site

The epithelial sodium channel (ENaC) is activated by a unique mechanism, whereby inhibitory tracts are released by proteolytic cleavage within the extracellular loops of two of its three homologous subunits. While cleavage by furin within the biosynthetic pathway releases one inhibitory tract from the α-subunit and moderately activates the channel, full activation through release of a second inhibitory tract from the γ-subunit requires cleavage once by furin and then at a distal site by a second protease, such as prostasin, plasmin, or elastase. We now report that coexpression of mouse transmembrane protease serine 4 (TMPRSS4) with mouse ENaC in Xenopus oocytes was associated with a two- to threefold increase in channel activity and production of a unique ～70-kDa carboxyl-terminal fragment of the γ-subunit, similar to the ～70-kDa γ-subunit fragment that we previously observed with prostasin-dependent channel activation. TMPRSS4-dependent channel activation and production of the ～70-kDa fragment were partially blocked by mutation of the prostasin-dependent cleavage site (γRKRK186QQQQ). Complete inhibition of TMPRSS4-dependent activation of ENaC and γ-subunit cleavage was observed when three basic residues between the furin and prostasin cleavage sites were mutated (γK173Q, γK175Q, and γR177Q), in addition to γRKRK186QQQQ. Mutation of the four basic residues associated with the furin cleavage site (γRKRR143QQQQ) also prevented TMPRSS4-dependent channel activation. We conclude that TMPRSS4 primarily activates ENaC by cleaving basic residues within the tract γK173-K186 distal to the furin cleavage site, thereby releasing a previously defined key inhibitory tract encompassing γR158-F168 from the γ-subunit.     

Intra- and extracellular regulation of activity and processing of legumain by cystatin E/M

Legumain, an asparaginyl endopeptidase, is up-regulated in tumour and tumour-associated cells, and is linked to the processing of cathepsin B, L, and proMMP-2. Although legumain is mainly localized to the endosomal/lysosomal compartments, legumain has been reported to be localized extracellularly in the tumour microenvironment and associated with extracellular matrix and cell surfaces. The most potent endogenous inhibitor of legumain is cystatin E/M, which is a secreted protein synthesised with an export signal. Therefore, we investigated the cellular interplay between legumain and cystatin E/M. As a cell model, HEK293 cells were transfected with legumain cDNA, cystatin E/M cDNA, or both, and over-expressing monoclonal cell lines were selected (termed M38L, M4C, and M3CL, respectively). Secretion of prolegumain from M38L cells was inhibited by treatment with brefeldin A, whereas bafilomycin A1 enhanced the secretion. Cellular processing of prolegumain to the 46 and 36 kDa enzymatically active forms was reduced by treatment with either substance alone. M38L cells showed increased, but M4C cells decreased, cathepsin L processing suggesting a crucial involvement of legumain activity. Furthermore, we observed internalization of cystatin E/M and subsequently decreased intracellular legumain activity. Also, prolegumain was shown to internalize followed by increased intracellular legumain processing and activation. In addition, in M4C cells incomplete processing of the internalized prolegumain was observed, as well as nuclear localized cystatin E/M. Furthermore, auto-activation of secreted prolegumain was inhibited by cystatin E/M, which for the first time shows a regulatory role of cystatin E/M in controlling both intra- and extracellular legumain activity.