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PubMed1977

Membrane and intracellular hydrolysis of peptides: differentiation, role and interrelations with transport.

The possible relative importance of the membrane and intracellular peptidases of the enterocytes in splitting dietary peptides to amino acids has been analysed. On the anoxic criterion, membrane hydrolysis was found to be predominant. Model experiments revealed cooperative interactions between the membrane enzyme and the coupled transport system. This cooperativity allows the main characteristics of oligomer transport to be described in terms of membrane digestion. Comparison of the behaviour of membrane and intracellular peptidase under different conditions has shown that the former are largely involved in digestion and the latter in intracellular metabolism. It is suggested that the efficiency of the membrane system is high enough to account for the hydrolysis of protein, especially taking into account the stimulation of enzyme processes in the brush border that occurs after ingestion of protein and carbohydrate. A sequential model based on the concept of three interacting enzyme layers (glycocalyx, lipoprotein membrane and cytosol) is presented.

Allosteric Regulation↗
PubMed1977

Transport and hydrolysis of peptides by microorganisms.

The structural specificities of the dipeptide and oligopeptide permeases of E. coli are briefly reviewed and related to the requirements found for other microorganisms. New, quick, sensitive methods for studying peptide transport are described, based on the following: (i) peptide-dependent incorporation of free radioactive amino acid into newly synthesized protein by a double amino acid auxotroph, (ii) colorimetric assay of peptide-dependent enzyme synthesis by an amino acid auxotroph, (iii) dansyl fingerprint technique. These approaches provide information on peptide binding affinity to a permease and rates of peptide uptake and amino acid efflux. Among current and future research areas considered are: the influence of the pKb of the N-terminal amino group on transport, generality of peptide transport in microorganisms, energy coupling and regulation, involvement of binding proteins, and the 'smugglin' concept. Peptide hydrolysis, and nutritional ultilization of peptides, by microorganisms are briefly discussed.

Bacteria↗
PubMed2005

Generation of peptides by human erythrocytes: facts and artifacts.

Previously reported data on peptide composition of human erythrocyte lysate were obtained under conditions that did not exclude proteolytic degradation of hemoglobin in the process of peptide isolation. Comparative chromatographic analysis of the diluted erythrocyte lysate incubated in acidic conditions with or without proteolytic enzyme inhibitors showed that several peptides earlier identified as intraerythrocyte ones in fact result from hemoglobin degradation by erythrocyte acidic protease(s) during incubation of the lysate. A rational scheme excluding postlysis proteolysis was developed for isolation of peptide fraction. Further analysis resulted in determination of structure and content of about 50 endogenous intraerythrocyte hemoglobin fragments. A primary endopeptidase splitting of alpha- and beta-globin chains followed by consecutive exopeptidase trimming of primary fragments is suggested as a degradation mechanism. The intraerythrocyte peptides were shown to differ from peptides excreted by the erythrocytes to the extracellular medium in the primary culture. It was also found that intraerythrocyte peptides cannot play the role of precursors of hemoglobin fragments present in tissue extracts.

Amino Acid Sequence↗
PubMed2007

Quantum dots and peptides: a bright future together.

Nanocrystalline semi-conductor materials, called quantum dots (QDs), exhibit unique optical and spectroscopic properties, which include, broad absorption, narrow and tunable emission, resistance to photobleaching, strong luminescence, and long luminescent lifetimes. These remarkable properties of QDs have resulted in their use as an alternative to both small-molecule and protein fluorophores in innumerable biological applications. The overlap of QDs with the rich chemistry and biology that is characteristic of the peptide arena is an emerging research area. Peptides engineered with appropriate cysteines or histidines have served as ligands for producing water soluble QDs as well as for tagging protein ligands and biosensors to QD surfaces. Incorporation of cell-penetrating peptides on QD surfaces has allowed for the translocation of functionalized QDs into cells for intracellular imaging applications. QDs containing fluorescently labeled peptide substrates have shown utility in the development of novel protease assays. Moreover, QDs-labeled peptides that serve as ligands for cellular receptors provide an alternative to antibody mediated imaging at the whole-cell and single molecule level to study receptor distribution and trafficking. This review highlights the overlap between QD and peptide chemistry and speculates on future research directions.

Biocompatible Materials↗
PubMed2007

Activity based fingerprinting of proteases using FRET peptides.

We have successfully developed a protease assay using fluorescence resonance energy transfer based peptide libraries, which allows not only general detection of enzymatic activities, but more importantly substrate fingerprinting of proteases from different classes. The method allows the generation of substrate fingerprints of a protease from both the nonprime and prime sites. Therefore, it is well suited for profiling of major metalloproteases such as thermolysin and MMPs. We envisage that this method will provide a useful tool in the emerging field of Catalomics for high-throughput studies of proteases.

Fluorescence Resonance Energy Transfer↗
PubMed2004

An optimized protocol for nano-LC-MALDI-TOF-MS coupling for the analysis of proteolytic digests of glycoproteins.

Matrix-assissted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analyses of complete proteolytic digests are often hampered by contaminations and the complexity of the sample. This results in suppression effects and the formation of adducts which are difficult to assign, thus leading to low scores in database searches. In particular, signals of post-translationally modified peptides such as glycopeptides are often of low intensity or completely suppressed. Online liquid chromatography electrospray ionization mass spectrometry (ESI-MS) can, in part, overcome this problem, but the analytes are completely consumed during the run. Coupling of nano-flow HPLC (nano-LC), microfractionation and MALDI-TOF-MS combines separation and high-sensitivity UV detection with the possibility of collecting fractionated peptides and preserving the sample for detailed mass spectrometric analyses. Here we report on an optimized protocol for nano-LC-MALDI-TOF-MS analyses of glycoproteins. This protocol improves spectral quality, resulting in better protein identification scores in database searches. Furthermore, post-translationally modified peptides could be detected with higher sensitivity by changing the experimental conditions, allowing assignment, localization and characterization of the respective carbohydrate substituents.

Asialoglycoproteins↗
PubMed2006

Mapping protease substrates by using a biotinylated phage substrate library.

We describe a bacteriophage M13 substrate library encoding the AviTag (BirA substrate) and combinatorial heptamer peptides displayed at the N terminus of the mature form of capsid protein III. Phages are biotinylated efficiently (> or = 50%) when grown in E. coli cells coexpressing BirA, and such viral particles can be immobilized on a streptavidin-coated support and released by protease cleavage within the combinatorial peptide. We have used this library to map the specificity of human Factor Xa and a neuropeptidase, neurolysin (EC3.4.24.16). Validation by analysis of isolated peptide substrates has revealed that neurolysin recognizes the motif hydrophobic-X-Pro-Arg-hydrophobic, where Arg-hydrophobic is the scissile bond.

Bacteriophage M13↗
PubMed2006

Modulation of infectivity in phage display as a tool to determine the substrate specificity of proteases.

Proteases play an important role in human and animal diseases. Rapid determination of substrate specificity is possible through the use of substrate phage display; however, current methods possess several drawbacks. They require phage-immobilization and cannot be used for infectivity-destroying or affinity tag-destroying proteases; this can make entire libraries useless. To overcome these limitations, here we introduce infectivity-modulated phage display (IMOP). IMOP uses a protease-resistant and infectivity-reducing tag fused to substrate-displaying polyvalent phages, and the specific cleavage of the substrate increases the infectivity of the phages by releasing the infectivity-reducing tag. The resulting phages were first tested with the infectivity-destroying detergent protease subtilisin; this resulted in a highly specific substrate at a 200-fold enrichment. In a second example, the protease ompT was used and led to an enrichment of the known double-arginine motif. The IMOP system thus substantially improves and simplifies previous systems.

Peptide Hydrolases↗
PubMed2006

A targeted protease substrate for a quantitative determination of protease activities in the endolysosomal pathway.

Inside the cell, proteases act in concert in the degradation of proteins and peptides. In order to understand the significance of an individual proteolytic activity within an ensemble of proteases, protocols and probes are required that enable a quantitative determination of the contribution of a protease to the break-down of a given substrate. Here we present a fluorescence resonance energy transfer-based probe and protocols for a quantitative determination of proteolytic activities inside the endolysosomal compartment. A peptide substrate that is readily cleaved by different cathepsins is flanked by fluorescein and tetramethylrhodamine-labeled lysine residues. Efficient endolysosomal targeting of the substrate is achieved by N-terminal elongation with the cell-penetrating peptide nona-arginine. The proteasome inhibitor lactacystin has a small, but significant effect on the break-down of the substrate, thus demonstrating that only a minor fraction of the peptide reaches the cytoplasm in its intact form. Nona-arginine therefore constitutes a highly efficient low-molecular-weight moiety for targeting the endolysosomal compartment.

Catalysis↗