SILAC-based quantitative proteomics and microscopy analysis of cancer cells treated with the N-glycolyl GM3-specific anti-tumor antibody 14F7.

Cancer immunotherapy represents a promising approach to specifically target and treat cancer. The most common mechanisms by which monoclonal antibodies kill cells include antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity and apoptosis, but also other mechanisms have been described. 14F7 is an antibody raised against the tumor-associated antigen NeuGc GM3, which was previously reported to kill cancer cells without inducing apoptotic pathways. The antibody was reported to induce giant membrane lesions in tumor cells, with apparent changes in the cytoskeleton. Here, we investigated the effect of humanized 14F7 on HeLa cells using stable isotope labeling with amino acids in cell culture (SILAC) in combination with LC-MS and live cell imaging. 14F7 did not kill the HeLa cells, however, it caused altered protein expression (MS data are available via ProteomeXchange with identifier PXD024320). Several cytoskeletal and nucleic-acid binding proteins were found to be strongly down-regulated in response to antibody treatment, suggesting how 14F7 may induce membrane lesions in cells that contain higher amounts of NeuGc GM3. The altered expression profile identified in this study thus contributes to an improved understanding of the unusual killing mechanism of 14F7.

Factor VII activating protease (FSAP) inhibits the outcome of ischemic stroke in mouse models.

The outcome of ischemic stroke can be improved by further refinements of thrombolysis and reperfusion strategies. Factor VII activating protease (FSAP) is a circulating serine protease that could be important in this context. Its levels are raised in patients as well as mice after stroke and a single nucleotide polymorphism (SNP) in the coding sequence, which results in an inactive enzyme, is linked to an increased risk of stroke. In vitro, FSAP cleaves fibrinogen to promote fibrinolysis, activates protease-activated receptors, and decreases the cellular cytotoxicity of histones. Based on these facts, we hypothesized that FSAP can be used as a treatment for ischemic stroke. A combination of tissue plasminogen activator (tPA), a thrombolytic drug, and recombinant serine protease domain of FSAP (FSAP-SPD) improved regional cerebral perfusion and neurological outcome and reduced infarct size in a mouse model of thromboembolic stroke. FSAP-SPD also improved stroke outcomes and diminished the negative consequences of co-treatment with tPA in the transient middle cerebral artery occlusion model of stroke without altering cerebral perfusion. The inactive MI-isoform of FSAP had no impact in either model. FSAP enhanced the lysis of blood clots in vitro, but in the tail transection model of hemostasis, FSAP-SPD treatment provoked a faster clotting time indicating that it also has pro-coagulant actions. Thus, apart from enhancing thrombolysis, FSAP has multiple effects on stroke progression and represents a promising novel therapeutic strategy in the treatment of ischemic stroke.

Crystal structure of MOA in complex with a peptide fragment: A protease caught in flagranti.

The Marasmius oreades agglutinin (MOA) is the holotype of an emerging family of fungal chimerolectins and an active Ca2+/Mn2+-dependent protease, which exhibits a unique papain-like fold with special active site features. Here we investigated the functional significance of the structural elements differentiating MOA from other papain-like cysteine proteases. X-ray crystal structures of MOA co-crystallized with two synthetic substrates reveal cleaved peptides bound to the catalytic site, corresponding to the final products of the proteolytic reaction. Anomalous diffraction data on crystals grown in the presence of calcium and manganese, cadmium or zinc resolve the calcium/manganese preference of MOA and elucidate the inhibitory roles of zinc and cadmium towards papain-like cysteine proteases in general. The reported structures, together with activity data of MOA active site variants, point to a conservation of the general proteolysis mechanism established for papain. Ultimately, the findings suggest that papain and the papain-like domain of MOA are the product of convergent evolution.

Characterization of the enzymatic activity of the serine protease domain of Factor VII activating protease (FSAP).

Factor VII (FVII) activating protease (FSAP) is a circulating serine protease. Human genetic studies, based on the Marburg I (MI) (Gly221Glu, chymotrypsin numbering system) polymorphism, implicate FSAP in the pathogenesis of many diseases. Here, we describe the molecular and functional changes caused by the Gly221Glu substitution in the 220 loop using recombinant proteins expressed in E. coli. The serine protease domain (SPD) of wild type (WT) FSAP displayed auto-catalytic activation whereas the MI isoform displayed very low autocatalytic activation and low proteolytic activity against the chromogenic substrate S-2288, Factor VII, tissue factor pathway inhibitor as well as pro-urokinase. Introduction of a thermolysin cleavage site in the activation position (Arg15Gln) led to cleavage of both WT- and MI-SPD and the resulting WT-SPD, but not the MI-SPD, was active. Mutating the Gly221 position to Asp, Gln and Leu led to a loss of activity whereas the Ala substitution was partially active. These results suggest a disturbance of the active site, or non-accessibility of the substrate to the active site in MI-SPD. With respect to regulation with metal ions, calcium, more than sodium, increased the enzymatic activity of WT-SPD. Thus, we describe a novel method for the production of recombinant FSAP-SPD to understand the role of the MI-single nucleotide polymorphism (SNP) in the regulation of its activity.

Family of Papain-Like Fungal Chimerolectins with Distinct Ca2+-Dependent Activation Mechanism.

An important function of fungal lectins is to protect their host. Marasmius oreades agglutinin (MOA) is toxic to nematodes and exerts its protective effect through protease activity. Its proteolytic function is associated with a papain-like dimerization domain. The closest homologue of MOA is Polyporus squamosus lectin 1a (PSL1a). Here, we probed PSL1a for catalytic activity and confirmed that it is a calcium-dependent cysteine protease, like MOA. The X-ray crystal structures of PSL1a (1.5 Å) and MOA (1.3 Å) in complex with calcium and the irreversible cysteine protease inhibitor E-64 elucidated the structural basis for their mechanism of action. The comparison with other calcium-dependent proteases (calpains, LapG) reveals a unique metal-dependent activation mechanism relying on a calcium-induced backbone shift and intradimer cooperation. Intriguingly, the enzymes appear to use a tyrosine-gating mechanism instead of pro-peptide processing. A search for potential MOA orthologues suggests the existence of a whole new family of fungal chimerolectins with these unique features.

Analysis of the substrate specificity of Factor VII activating protease (FSAP) and design of specific and sensitive peptide substrates.

Factor VII (FVII) activating protease (FSAP) is a circulating serine protease that is likely to be involved in a number of disease conditions such as stroke, atherosclerosis, liver fibrosis, thrombosis and cancer. To date, no systematic information is available about the substrate specificity of FSAP. Applying phage display and positional scanning substrate combinatorial library (PS-SCL) approaches we have characterised the specificity of FSAP towards small peptides. Results were evaluated in the context of known protein substrates as well as molecular modelling of the peptides in the active site of FSAP. The representative FSAP-cleaved sequence obtained from the phage display method was Val-Leu-Lys-Arg-Ser (P4-P1'). The sequence X-Lys/Arg-Nle-Lys/Arg (P4-P1) was derived from the PS-SCL method. These results show a predilection for cleavage at a cluster of basic amino acids on the nonprime side. Quenched fluorescent substrate (Ala-Lys-Nle-Arg-AMC) (amino methyl coumarin) and (Ala-Leu-Lys-Arg-AMC) had a higher selectivity for FSAP compared to other proteases from the hemostasis system. These substrates could be used to measure FSAP activity in a complex biological system such as plasma. In histone-treated plasma there was a specific activation of pro-FSAP as validated by the use of an FSAP inhibitory antibody, corn trypsin inhibitor to inhibit Factor XIIa and hirudin to inhibit thrombin, which may account for some of the haemostasis-related effects of histones. These results will aid the development of further selective FSAP activity probes as well as specific inhibitors that will help to increase the understanding of the functions of FSAP in vivo.

Polyporus squamosus Lectin 1a (PSL1a) Exhibits Cytotoxicity in Mammalian Cells by Disruption of Focal Adhesions, Inhibition of Protein Synthesis and Induction of Apoptosis.

PSL1a is a lectin from the mushroom Polyporus squamosus that binds to sialylated glycans and glycoconjugates with high specificity and selectivity. In addition to its N-terminal carbohydrate-binding domain, PSL1a possesses a Ca2+-dependent proteolytic activity in the C-terminal domain. In the present study, we demonstrate that PSL1a has cytotoxic effects on mammalian cancer cells, and we show that the cytotoxicity is dependent on the cysteine protease activity. PSL1a treatment leads to cell rounding and detachment from the substratum, concomitant with disruption of vinculin complexes in focal adhesions. We also demonstrate that PSL1a inhibits protein synthesis and induces apoptosis in HeLa cells, in a time- and concentration-dependent manner.

Surface micropattern limits bacterial contamination.

BACKGROUND: Bacterial surface contamination contributes to transmission of nosocomial infections. Chemical cleansers used to control surface contamination are often toxic and incorrectly implemented. Additional non-toxic strategies should be combined with regular cleanings to mitigate risks of human error and further decrease rates of nosocomial infections. The Sharklet micropattern (MP), inspired by shark skin, is an effective tool for reducing bacterial load on surfaces without toxic additives. The studies presented here were carried out to investigate the MP surfaces capability to reduce colonization of methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) compared to smooth control surfaces. METHODS: The MP and smooth surfaces produced in acrylic film were compared for remaining bacterial contamination and colonization following inoculation. Direct sampling of surfaces was carried out after inoculation by immersion, spray, and/or touch methods. Ultimately, a combination assay was developed to assess bacterial contamination after touch transfer inoculation combined with drying (persistence) to mimic common environmental contamination scenarios in the clinic or hospital environment. The combination transfer and persistence assay was then used to test antimicrobial copper beside the MP for the ability to reduce MSSA and MRSA challenge. RESULTS: The MP reduced bacterial contamination with log reductions ranging from 87-99% (LR = 0.90-2.18; p < 0.05) compared to smooth control surfaces. The MP was more effective than the 99.9% pure copper alloy C11000 at reducing surface contamination of S. aureus (MSSA and MRSA) through transfer and persistence of bacteria. The MP reduced MSSA by as much as 97% (LR = 1.54; p < 0.01) and MRSA by as much as 94% (LR = 1.26; p < 0.005) compared to smooth controls. Antimicrobial copper had no significant effect on MSSA contamination, but reduced MRSA contamination by 80% (LR = 0.70; p < 0.005). CONCLUSION: The assays developed in this study mimic hospital environmental contamination events to demonstrate the performance of a MP to limit contamination under multiple conditions. Antimicrobial copper has been implemented in hospital room studies to evaluate its impact on nosocomial infections and a decrease in HAI rate was shown. Similar implementation of the MP has potential to reduce the incidence of HAIs although future clinical studies will be necessary to validate the MP's true impact.

Microarray analysis of Mu transposition in Salmonella enterica, serovar Typhimurium: transposon exclusion by high-density DNA binding proteins.

All organisms contain transposons with the potential to disrupt and rearrange genes. Despite the presence of these destabilizing sequences, some genomes show remarkable stability over evolutionary time. Do bacteria defend the genome against disruption by transposons? Phage Mu replicates by transposition and virtually all genes are potential insertion targets. To test whether bacteria limit Mu transposition to specific parts of the chromosome, DNA arrays of Salmonella enterica were used to quantitatively measure target site preference and compare the data with Escherichia coli. Essential genes were as susceptible to transposon disruption as non-essential ones in both organisms, but the correlation of transposition hot spots among homologous genes was poor. Genes in highly transcribed operons were insulated from transposon mutagenesis in both organisms. A 10 kb cold spot on the pSLT plasmid was near parS, a site to which the ParB protein binds and spreads along DNA. Deleting ParB erased the plasmid cold spot, and an ectopic parS site placed in the Salmonella chromosome created a new cold spot in the presence of ParB. Our data show that competition between cellular proteins and transposition proteins on plasmids and the chromosome is a dominant factor controlling the genetic footprint of transposons in living cells.

A gyrase mutant with low activity disrupts supercoiling at the replication terminus.

When a mutation in an essential gene shows a temperature-sensitive phenotype, one usually assumes that the protein is inactive at nonpermissive temperature. DNA gyrase is an essential bacterial enzyme composed of two subunits, GyrA and GyrB. The gyrB652 mutation results from a single base change that substitutes a serine residue for arginine 436 (R436-S) in the GyrB protein. At 42 degrees C, strains with the gyrB652 allele stop DNA replication, and at 37 degrees C, such strains grow but have RecA-dependent SOS induction and show constitutive RecBCD-dependent DNA degradation. Surprisingly, the GyrB652 protein is not inactive at 42 degrees C in vivo or in vitro and it doesn't directly produce breaks in chromosomal DNA. Rather, this mutant has a low k(cat) compared to wild-type GyrB subunit. With more than twice the normal mean number of supercoil domains, this gyrase hypomorph is prone to fork collapse and topological chaos near the terminus of DNA replication.

Bacteriophage Mu targets the trinucleotide sequence CGG.

Target specificity for bacteriophage Mu was studied using a new phage derivative that enabled cloning of Mu-host junctions from phage DNA. Insertions distributed throughout the chromosome showed no orientation bias with respect to transcription or replication polarity. Genes with a high frequency of the triplet CGG were preferred targets.

Microarray analysis of transposition targets in Escherichia coli: the impact of transcription.

Transposable elements have influenced the genetic and physical composition of all modern organisms. Defining how different transposons select target sites is critical for understanding the biochemical mechanism of this type of recombination and the impact of mobile genes on chromosome structure and function. Phage Mu replicates in Gram-negative bacteria using an extremely efficient transposition reaction. Replicated copies are excised from the chromosome and packaged into virus particles. Each viral genome plus several hundred base pairs of host DNA covalently attached to the prophage right end is packed into a virion. To study Mu transposition preferences, we used DNA microarray technology to measure the abundance of >4,000 Escherichia coli genes in purified Mu phage DNA. Insertion hot- and cold-spot genes were found throughout the genome, reflecting >1,000-fold variation in utilization frequency. A moderate preference was observed for genes near the origin compared to terminus of replication. Large biases were found at hot and cold spots, which often include several consecutive genes. Efficient transcription of genes had a strong negative influence on transposition. Our results indicate that local chromosome structure is more important than DNA sequence in determining Mu target-site selection.