Search results for "Endopeptidase"
showing 10 items of 361 documents
Cholesterol as stabilizer of the oxytocin receptor
2002
AbstractThe function of the oxytocin receptor system is strongly dependent on steroids as demonstrated by several physiological studies. One key element of this dependence on steroids may be the interaction of cholesterol and the oxytocin receptor. In this study, we show that cholesterol stabilizes the solubilized human oxytocin receptor against thermal inactivation and proteolytic degradation. In the absence of additional cholesterol, the soluble receptor inactivates within minutes. Maximal stabilization of the oxytocin receptor requires a continuous supply with cholesterol from a cholesterol-rich environment. A structure–activity analysis of various cholesterol analogues and their effect …
A single mutation in the recombinant light chain of tetanus toxin abolishes its proteolytic activity and removes the toxicity seen after reconstituti…
1994
Specific proteolysis by the tetanus toxin light chain of a vesicle-associated membrane protein (VAMP) involved in exocytosis is thought to underlie its intracellular blockade of neurotransmitter release. To substantiate this mechanism, recombinant light chain was expressed as a maltose binding protein-light chain fusion product in Escherichia coli. After purification of affinity chromatography and cleavage with factor Xa, the resultant light chain was isolated and its identity confirmed by Western blotting and N-terminal sequencing. It exhibited activity similar to that of the native light chain in proteolyzing its target in isolated bovine small synaptic vesicles and in hydrolyzing a 62-re…
Production and characterization of the recombinant Sphingomonas chlorophenolica pentachlorophenol 4-monooxygenase.
2001
Abstract Pentachlorophenol 4-monooxygenase (PCP4MO) from Sphingomonas chlorophenolica is a flavoprotein that hydroxylates PCP in the presence of NADPH and oxygen. In order to investigate the structure and function of active site, recombinant PCP4MO (rePCP4MO) was produced in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Moreover, a tobacco etch virus (TEV) protease cleavage site (EKLYFQG) was introduced into GST-PCP4MO and a his-tagged TEV protease was employed. Hence, a two-step purification protocol was developed which allowed obtaining 15–20 mg of rePCP4MO from 1 L culture. The rePCP4MO revealed identity with native enzyme by SDS–PAGE and N-terminal sequence analy…
The E3 Ubiquitin Ligase MID1 Catalyzes Ubiquitination and Cleavage of Fu
2014
Sonic Hedgehog (SHH)-GLI signalling plays an important role during embryogenesis and in tumorigenesis. The survival and growth of several types of cancer depend on autonomously activated SHH-GLI signalling. A protein complex containing the ubiquitin-ligase MID1 and protein phosphatase 2A (PP2A) regulates the nuclear localization and transcriptional activity of GLI3, a transcriptional effector molecule of SHH, in cancer cell lines with autonomously activated SHH signalling. However, the exact molecular mechanisms that mediate the interaction between MID1 and GLI3 remained unknown. Here, we show that MID1 catalyses the ubiquitination and proteasomal cleavage of the GLI3-regulator Fu. Our data…
Amplicon-based high-throughput pooled sequencing identifies mutations in CYP7B1 and SPG7 in sporadic spastic paraplegia patients
2011
Hereditary spastic paraplegia (HSP) is a neurodegenerative disorder defined clinically by progressive lower limb spasticity and weakness. HSP is a genetically highly heterogeneous condition with at least 46 gene loci identified so far, involving X-linked, autosomal recessive (AR) and autosomal dominant inheritance. For correct diagnosis, molecular testing is essential because clinical parameters by themselves are not reliable to differentiate HSP forms. The purpose of this study was to establish amplicon-based high-throughput genotyping for AR-HSP. A sample of 187 index cases with apparently sporadic or recessive spastic paraplegia were analyzed by applying an array-based amplification stra…
Parathyroid hormone-related peptide and 8701-BC breast cancer cell growth and invasion in vitro: evidence for growth-inhibiting and invasion-promotin…
1995
It has been previously reported that 8701-BC cells, derived from a primary carcinoma of the breast, constitutively express parathyroid hormone-related peptide (PTHrP) gene and that N-terminal PTHrP immunoreactivity can be found in cell medium. Here we have firstly measured immunoreactive PTHrP in 8701-BC cell medium using antibodies raised against midregion and C-terminal fragments, and also demonstrated the expression of PTH/PTHrP receptor by 8701-BC cells. Secondly, we have examined the role, if any, elicited by diverse PTHrP domains on 8701-BC cell proliferation, and invasive behaviour in vitro related to production of extracellular proteolytic enzymes. Our data show that PTHrP [1-34], a…
Synthesis and Molecular Modeling Studies of Derivatives of a Highly Potent Peptidomimetic Vinyl Ester as Falcipain-2 Inhibitors
2012
Herein we report the synthesis of a set of constrained peptidomimetics endowed with an electrophilic vinyl ester warhead and structurally related to a previously identified lead compound, a potent and irreversible inhibitor of falcipain-2 (FP-2). FP-2 is the main hemoglobinase of the malaria parasite P. falciparum. The new compounds were evaluated for their inhibition against FP-2, and the results were rationalized on the basis of docking experiments. These studies underscore the pivotal role of both the ester function at the P1' site and the trifluoromethyl group of the P3 side chain in determining the correct orientation of the Michael acceptor warhead in the catalytic site, and as a cons…
Synthesis and biological evaluation of novel peptidomimetics as rhodesain inhibitors
2016
Novel rhodesain inhibitors were developed by combining an enantiomerically pure 3-bromoisoxazoline warhead with a 1,4-benzodiazepine scaffold as specific recognition moiety. All compounds were proven to inhibit rhodesain with Ki values in the low-micromolar range. Their activity towards rhodesain was found to be coupled to an in vitro antitrypanosomal activity, with IC50 values ranging from the mid-micromolar to a low-micromolar value for the most active rhodesain inhibitor (R,S,S)-3. All compounds showed a good selectivity against the target enzyme since all of them were proven to be poor inhibitors of human cathepsin L. Novel rhodesain inhibitors were developed by combining an enantiomeri…
Discovery of a new class of sortase a transpeptidase inhibitors to tackle gram-positive pathogens: 2-(2-phenylhydrazinylidene)alkanoic acids and rela…
2016
A FRET-based random screening assay was used to generate hit compounds as sortase A inhibitors that allowed us to identify ethyl 3-oxo-2-(2-phenylhydrazinylidene)butanoate as an example of a new class of sortase A inhibitors. Other analogues were generated by changing the ethoxycarbonyl function for a carboxy, cyano or amide group, or introducing substituents in the phenyl ring of the ester and acid derivatives. The most active derivative found was 3-oxo-2-(2-(3,4dichlorophenyl)hydrazinylidene)butanoic acid (2b), showing an IC50 value of 50 µM. For a preliminary assessment of their antivirulence properties the new derivatives were tested for their antibiofilm activity. The most active compo…
Dipeptidyl Enoates As Potent Rhodesain Inhibitors That Display a Dual Mode of Action
2015
Dipeptidyl enoates were prepared through a high-yielding two-step synthetic route. They have a dipeptidic structure with a 4-oxoenoate moiety as a warhead with multiple reactive sites. Dipeptidyl enoates were screened against rhodesain and human cathepsins B and L, and were found to be potent and selective inhibitors of rhodesain. Among them (S,E)-ethyl 5-((S)-2-{[(benzyloxy)carbonyl]amino}-3-phenylpropanamido)-7-methyl-4-oxooct-2-enoate (6) was the most potent, with an IC50 value of 16.4 nm and kinact/Ki=1.6×106 m−1 s−1 against rhodesain. These dipeptidyl enoates display a reversible mode of inhibition at very low concentrations and an irreversible mode at higher concentrations. Inhibition…