A permeabilized cell model for studying cytokinesis using mammalian tissue culture cells.

PtK1 cells lysed late in cell division in a medium containing the nonionic detergent Brij 58 and polyethylene glycol with continue to undergo cleavage after lysis. Maintenance of cleavage after lysis is dependent on the composition of the lysis medium; the pH must be around neutrality, MgATP must be present, and the free Ca++ concentration should be 1 microM for optimal constriction to occur. Cleavage can be stopped and reinitiated by raising and lowering the Ca++ levels in the lysis medium. Cleavage in the permeabilized cell is blocked by addition of phalloidin, cytochalasin B, and N-ethylmaleimide-modified myosin subfragment-1 to the lysis medium. This represents the first cell model system for studying cleavage since the pioneering studies of Hoffman-Berling in 1954

Ectodomain shedding of the amyloid precursor protein: Cellular control mechanisms and novel modifiers

Proteolytic cleavage in the ectodomain of the amyloid precursor protein (APP) is a key regulatory step in the generation of the Alzheimer's disease amyloid-beta (A beta) pepticle and occurs through two different protease activities termed alpha- and beta-secretase. Both proteases compete for APP cleavage, but have opposite effects on A beta generation. At present, little is known about the cellular pathways that control APP alpha- or beta-secretase cleavage and thus A beta generation. To explore the contributory pathways in more detail we have recently employed an expression cloning screen and identified several activators of APP cleavage by alpha- or beta-secretase. Among them were known activators of APP cleavage, for example protein kinase A, and novel activators, such as endophilin and the APP homolog amyloid precursor-like protein 1 (APLP1). Mechanistic analysis revealed that both endophilin and APLP1 reduce the rate of APP endocytosis and strongly increase APP cleavage by alpha-secretase. This review summarizes the results of the expression cloning screen in the context of recent developments in our understanding of the cellular regulation of APP alpha-secretase cleavage. Moreover, it highlights the particular importance of endocytic APP trafficking as a prime modulator of APP shedding. Copyright (c) 2006 S. Karger AG, Basel

Application of Peptides Containing the Cleavage Sequence of Pro-TNFα in Assessing TACE Activity of Whole Cells

Tumor necrosis factor-α (TNFα) is presumably shed from cell membranes by TNFα-cleaving enzyme (TACE). The peptides SPLAQAVRSSSR and Dabcyl-LAQAVRSSSR-Edans, each encompassing the cleavage sequence of pro-TNFα recognized by TACE, were applied to intact umbilical vein endothelium (HUVEC), peripheral blood leukocytes (PBL) and the mast cell line HMC-1, which express TACE, to homogenates of rat heart tissue and to membrane and cytoplasmic extracts of PBL. Formation of SPLAQA (specific cleavage) was determined by HPLC, while cleavage (specific plus non-specific) of Dabcyl-TNFα-Edans was followed over time by measuring fluorescence. Participation of TACE was assessed from inhibition due to the drug TAPI-2. Incubation with recombinant human TACE gave specific cleavage, fully inhibitable by TAPI-2 (IC50<0.1 μM). HUVEC rapidly degraded TNFα-peptide, but in a non-specific manner (no SPLAQA detectable) and 50 μM TAPI-2 was without effect. Fluorescence was evoked when Dabcyl- LAQAVRSSSR-Edans was incubated with HMC-1 or PBL and also with cytoplasmic and membrane fractions of lysed PBL, but in no case was there significant inhibition by TAPI-2. However, marginal (10%) inhibition of fluorescence by 50 μM TAPI-2 was observed with homogenized heart tissue. This contained TACE, about 75% of which was without the inhibitory cysteine switch (Western blot). In conclusion, simple peptide analogs of pro-TNFα cannot be employed as substrates for measuring membrane TACE activity, largely due to extensive non-specific proteolytic cleavage by whole cells and cell extracts

Rapid microwave-assisted CNBr cleavage of bead-bound peptides

Large libraries of peptides, cyclic peptides, and other molecules are standard tools for the discovery of drugs, molecular probes, and affinity reagents. In particular, one-bead-one-compound (OBOC) libraries,(1) prepared by the split-and-mix method,(2) provide access to a broad chemical space with a minimum of reagents. Once such a library has been screened against the target of interest, the chemical identity of the library elements on the hit beads is identified. For peptide libraries and their variants, mass spectrometry (MS) based peptide sequencing provides the most rapid method for such analysis. OBOC libraries are constructed in a number of ways to facilitate MS analysis,(3-5) but one common feature is that the peptide must be cleaved from the bead prior to being introduced into the mass spectrometer. While a number of chemical(6) and photochemical(7) cleavage strategies have been developed, the most common strategy is to incorporate a CNBr-cleavable methionine-linker group at the C-terminus of the peptide.(8) CNBr cleavage has also been widely used in proteomics to cleave proteins.(9) With such chemistry, up to 100 beads from an OBOC peptide library can be sequenced in a 24 h period.(10) A large fraction of that time, however, is devoted to the CNBr cleavage step. Standard literature protocols describe CNBr cleavage as requiring between 12 and 24 h, using 20−30 μL of 0.25 M CNBr in 70% aqueous formic acid at room temperature.(11) Although the CNBr cleavage time may be reduced to 2−4 h at elevated temperatures (47 °C), significant side-products may be generated.(12) All reports that we have found that describe CNBr cleavage chemistry from single beads have used the same conditions as for proteomics, although the two chemical processes are not necessarily equivalent

Clerocidin selectively modifies the gyrase-DNA gate to induce irreversible and reversible DNA damage

Clerocidin (CL), a microbial diterpenoid, reacts with DNA via its epoxide group and stimulates DNA cleavage by type II DNA topoisomerases. The molecular basis of CL action is poorly understood. We establish by genetic means that CL targets DNA gyrase in the gram-positive bacterium Streptococcus pneumoniae, and promotes gyrase-dependent single- and double-stranded DNA cleavage in vitro. CL-stimulated DNA breakage exhibited a strong preference for guanine preceding the scission site (-1 position). Mutagenesis of -1 guanines to A, C or T abrogated CL cleavage at a strong pBR322 site. Surprisingly, for double-strand breaks, scission on one strand consistently involved a modified (piperidine-labile) guanine and was not reversed by heat, salt or EDTA, whereas complementary strand scission occurred at a piperidine-stable -1 nt and was reversed by EDTA. CL did not induce cleavage by a mutant gyrase (GyrA G79A) identified here in CL-resistant pneumococci. Indeed, mutations at G79 and at the neighbouring S81 residue in the GyrA breakage-reunion domain discriminated poisoning by CL from that of antibacterial quinolones. The results suggest a novel mechanism of enzyme inhibition in which the -1 nt at the gyrase-DNA gate exhibit different CL reactivities to produce both irreversible and reversible DNA damage

The hyaluronan-binding serine protease from human plasma cleaves HMW and LMW kininogen and releases bradykinin

The influence of the hyaluronanbinding protease (PHBSP), a plasma enzyme with FVII- and pro-urokinase-activating potency, on components of the contact phase (kallikrein/kinin) system was investigated. No activation or cleavage of the proenzymes involved in the contact phase system was observed. The procofactor high molecular weight kininogen (HK), however, was cleaved in vitro by PHBSP in the absence of any charged surface, releasing the activated cofactor and the vasoactive nonapeptide bradykinin. Glycosoaminoglycans strongly enhanced the reaction. The cleavage was comparable to that of plasma kallikrein, but clearly different from that of coagulation factor FXIa. Upon extended incubation with PHBSP, the light chain was further processed, partially removing about 60 amino acid residues from the Nterminus of domain D5 of the light chain. These cleavage site(s) were distinct from plasma kallikrein or FXIa cleavage sites. PHBSP and, more interestingly, also plasma kallikrein could cleave low molecular weight kininogen in vitro, indicating that domains D5(H) and D6(H) are no prerequisite for kininogen cleavage. PHBSP was also able to release bradykinin from HK in plasma where the pro-cofactor circulates predominantly in complex with plasma kallikrein or FXI. In conclusion, PHBSP represents a novel kininogen-cleaving and bradykinin-releasing enzyme in plasma that shares significant catalytic similarities with plasma kallikrein. Since they are structurally unrelated in their heavy chains (propeptide), their similar in vivo catalytic activities might be directed at distinct sites where PHBSP could induce processes that are related to the kallikrein/kinin system

Cloning and characterisation of a maize carotenoid cleavage dioxygenase (ZmCCD1) and its involvement in the biosynthesis of apocarotenoids with various roles in mutualistic and parasitic interactions

Colonisation of maize roots by arbuscular mycorrhizal (AM) fungi leads to the accumulation of apocarotenoids (cyclohexenone and mycorradicin derivatives). Other root apocarotenoids (strigolactones) are involved in signalling during early steps of the AM symbiosis but also in stimulation of germination of parasitic plant seeds. Both apocarotenoid classes are predicted to originate from cleavage of a carotenoid substrate by a carotenoid cleavage dioxygenase (CCD), but the precursors and cleavage enzymes are unknown. A Zea mays CCD (ZmCCD1) was cloned by RT-PCR and characterised by expression in carotenoid accumulating E. coli strains and analysis of cleavage products using GC¿MS. ZmCCD1 efficiently cleaves carotenoids at the 9, 10 position and displays 78% amino acid identity to Arabidopsis thaliana CCD1 having similar properties. ZmCCD1 transcript levels were shown to be elevated upon root colonisation by AM fungi. Mycorrhization led to a decrease in seed germination of the parasitic plant Striga hermonthica as examined in a bioassay. ZmCCD1 is proposed to be involved in cyclohexenone and mycorradicin formation in mycorrhizal maize roots but not in strigolactone formatio

Truncated Chicken Interleukin-1β with Increased Biologic Activity

Chicken interleukin-1β (ChIL-1β) is synthesized as a precursor molecule that unlike its mammalian counterpart, lacks a typical caspase-1 cleavage site. Therefore, it was unclear if proteolytic cleavage of ChIL-1β can occur and if cleavage might modulate the biologic activity of this cytokine. Using an avian indicator cell line that carries an NF-κB-regulated luciferase reporter gene, we established a sensitive and highly specific bioassay for ChIL-1β. Experiments with a rabbit antiserum indicated that the NF-κB-stimulating activity in supernatants of lipopolysaccharide (LPS)-treated chicken HD-11 macrophages is largely due to IL-1β and that proteolytic processing of natural and recombinant ChIL-1β is not very efficient. Functional analyses further revealed that cDNAs for either full-length or N-terminally truncated chicken ChIL-1β yielded active cytokine. A truncated molecule that closely resembled putative mature ChIL-1β exhibited more than 100-fold enhanced biologic activity after expression in mammalian cells, indicating that precursor cleavage is indeed of critical importance for maximal activity