Tailoring molecular sensing for peptide displaying engineered enzymes

Regulable enzymes displaying foreign peptides are valuable instruments for molecular targeting and fast analyte detection in homogeneous assays. Both the specificity and the intensity of the signal generated by the sensor are critical parameters that can be manipulated by trial-and-error protein engineering in the vicinity of the active site. An alternative approach is presented to enhance signal-background ratio in -galactosidase-based molecular sensors by an optimisation of the sensing conditions. The screening of the enzymatic response in a set of engineered enzymes has revealed an antibody-dependent increase in their specific activity up to 500% for the enzyme, HD72CA, that is reached with 0.25 pmol enzyme per reaction in presence of 1.75 mM substrate. This value, much higher than 200% enzyme activation achieved only by protein engineering, represents a step further in enhancing the enzyme's responsiveness. On the other hand, engineered -galactosidases are also highly dynamic without preliminary antibody incubation, rendering activation factors around 300% after global reaction times shorter than 15 min. Therefore, this enzymatic system has been revealed as extremely robust and suitable for efficient and fast molecular detection in the diagnosis of infectious diseases.     

Topography of prostaglandin H synthase. Antiinflammatory agents and the protease-sensitive arginine 253 region

Prostaglandin H synthase catalyzes two reactions: the bis-dioxygenation of arachidonic acid to form prostaglandin G2 (cyclooxygenase activity), and the reduction of hydroperoxides to the corresponding alcohols (peroxidase activity). The cyclooxygenase activity can be selectively inhibited by many nonsteroidal antiinflammatory agents including indomethacin. In the native synthase, there is a single prominent protease-sensitive region, located near Arg253; binding of the heme prosthetic group makes the synthase resistant to proteases. To investigate the spatial relationship between the area of the synthase which interacts with indomethacin and the protease-sensitive region, the effects of indomethacin and similar agents on the protease sensitivity of the two enzymatic activities and of the synthase polypeptide were examined. Incubation of the synthase apoenzyme with trypsin (3.6% w/w) resulted in the time-dependent coordinate loss (75% at 1 h) of both enzymatic activities and the cleavage (85% at 1 h) of the 70-kDa subunit into 38- and 33-kDa fragments, indicating that proteolytic cleavage of the polypeptide at Arg253, destroyed both activities of the synthase simultaneously. Indomethacin, (S)-flurbiprofen, or meclofenamate (each at 20 microM) rendered both activities and the synthase polypeptide (at 5 microM subunit) resistant to attack by trypsin or proteinase K; these agents also inhibited the cyclooxygenase activity of the intact synthase. Two reversible cyclooxygenase inhibitors, ibuprofen and flufenamate, also made both of the activities and the synthase polypeptide more resistant to trypsin. Titration of the apoenzyme with indomethacin (0-3 mol/mol of synthase dimer) resulted in proportional increases in the inhibition of the cyclooxygenase and in the resistance to attack by trypsin. (R)-Flurbiprofen did not increase the resistance to protease or appreciably inhibit the cyclooxygenase. These results suggest that the same stereospecific interaction of these agents with the synthase that produced inhibition of the cyclooxygenase led to a decreased accessibility of the Arg253 region to proteases. Aspirin treatment made the synthase less resistant to trypsin; aspirin-treated synthase became more resistant to trypsin when it was incubated with indomethacin before addition of the protease. The presence of 50 microM arachidonate during digestion of apoenzyme or aspirin-treated apoenzyme with trypsin did not decrease the cleavage of the synthase subunit.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tailoring the microenvironment of enzymes in water-poor systems.

Biocatalytic systems using enzymes in organic solvents open up the possibility of performing a whole range of reactions which would not normally occur under physiological conditions. The ability to perform reverse hydrolysis, or to convert substances relatively insoluble in aqueous environments on a scale of practical value in commercial applications are among those reactions for which water-poor systems are appropriate.     

Nanoparticles as vehicles for delivery of photodynamic therapy agents

Photodynamic therapy (PDT) in cancer treatment involves the uptake of a photosensitizer by cancer tissue followed by photoirradiation. The use of nanoparticles as carriers of photosensitizers is a very promising approach because these nanomaterials can satisfy all the requirements for an ideal PDT agent. This review describes and compares the different individual types of nanoparticles that are currently in use for PDT applications. Recent advances in the use of nanoparticles, including inorganic oxide-, metallic-, ceramic-, and biodegradable polymer-based nanomaterials as carriers of photosensitizing agents, are highlighted. We describe the nanoparticles in terms of stability, photocytotoxic efficiency, biodistribution and therapeutic efficiency. Finally, we summarize exciting new results concerning the improvement of the photophysical properties of nanoparticles by means of biphotonic absorption and upconversion.     

Green fluorescent protein rendered susceptible to proteolysis: positions for protease-sensitive insertions

The green fluorescent protein (GFP) is highly resistant to proteolysis and remains uncleaved after prolonged incubation with trypsin or pronase despite several putative tryptic and chymotryptic sites in exposed loops. We have rendered GFP sensitive to proteolysis by inserting five amino acids, IEGRS, in loops at position 157, 172, or 189. Excitation and emission maxima of the three insertion mutants were similar to those of wild type, but quantum yields of mutants Omega172 and Omega189 were lower, indicating increased freedom of the fluorophore. Trypsin cleaved the native (folded) form of each mutant at a unique site defined by the insert. Pronase also yields similar digestion patterns in these variants, but further proteolysis was also observed, suggesting that the primary cleavage relaxes GFP structure and reveals previously inaccessible sites. Fluorescence of Omega189 changed little upon digestion with trypsin but decreased progressively by as much as 40% upon digestion with increasing amounts of pronase. Fluorescence of other variants was not affected significantly by the proteases, further confirming the remarkable stabilities of GFP variants. These constructs define a new conformation-sensitive site around residue 189 of GFP and show that GFP may be useful for design of protease-susceptible molecules for monitoring of specific proteolytic activities in vivo.     

Ribosomes specifically bind to mammalian mitochondria via protease-sensitive proteins on the outer membrane

The interaction of ribosomes with specific components of membranes is one of the central themes to the co-translational targeting and import of proteins. To examine ribosome binding to mammalian mitochondria, we used ribosome-nascent chain complexes (RNCs) to follow the in vitro binding of ribosomes that correspond to the initial targeting stage of proteins. Mitochondria were found to contain a limited number of RNC binding sites on the outer membrane. It required more than twice the amount of non-translating ribosomes to inhibit RNC binding by one-half, indicating that RNCs have a competitive binding advantage. In addition, we found that RNCs bind mainly through the ribosomal component and not the nascent chain. RNCs bind via protease-sensitive proteins on the outer membrane, as well as by protease-insensitive components suggesting that two classes of receptors exist. We also show that binding is sensitive to cation conditions. Nearly all of the binding was inhibited in 0.5 m KCl, indicating that they interact with the membrane primarily through electrostatic interactions. In addition, disruption of RNC structure by removing magnesium causes the complete inhibition of binding under normal binding conditions indicating that it is the intact ribosome that is crucial for binding and not the nascent chain. These findings support the hypothesis that the outer mitochondrial membrane contains receptors specific for ribosomes, which would support the conditions necessary for co-translational import.     

Metallobacteriochlorophylls as potential dual agents for photodynamic therapy and chemotherapy

A theoretical analysis of bacteriochlorophyll a containing its non-native divalent metal ions: Co, Ni, Cu, Zn, Ru, Rh, Pd, and Pt, has been carried out by means of density functional theory (DFT) calculations. The main stress was put on the derivatives with metals, which already found applications as coordination compounds in anti-tumor therapy (Ru, Pt, Pd, and Rh). The idea was to combine their cytotoxic properties with the known suitability of bacteriochlorophylls macrocycle for photodynamic therapy. The geometries of the studied systems are compared and reveal a number of similarities. The cores of the modified bacteriochlorophylls are flat, and the introduced metal ions lie in plane of the macrocycle, showing its large ability to accommodate metal ions of different sizes. However, four metal–nitrogen bonds, linking the central ions with the macrocycle ligand, are not equivalent. Metals are the strongest attached to nitrogens, which come from the pyrrole, which is fused with isocyclic ring. Based on the known spectroscopic data, the absorption properties of the proposed systems are predicted. Finally, it is found that all studied metal–macrocycle adducts are stable in aqueous media. The only exceptions are Mg-BChla (the finding is reflected by experimental facts) and Zn-BChla. The predicted high stability of Ru-, Rh-, Pt- and Pd-bacteriochlorophylls might turn out beneficial for therapeutic purposes.     

Tailoring microbes to upgrade lignin

Lignin depolymerization generates a mixture of numerous compounds that are difficult to separate cost-effectively. To address this heterogeneity issue, microbes have been employed to ‘biologically funnel’ a broad range of compounds present in depolymerized lignin into common central metabolites that can be converted into a single desirable product. Because the composition of depolymerized lignin varies significantly with the type of biomass and the depolymerization method, microbes should be selected and engineered by considering this compositional variation. An ideal microbe must efficiently metabolize all relevant lignin-derived compounds regardless of the compositional variation of feedstocks, but discovering or developing such a perfect microbe is very challenging. Instead, developing multiple tailored microbes to tolerate a given mixture of lignin-derived compounds and to convert most of these into a target product is more practical. This review summarizes recent progress toward the development of such microbes for lignin valorization and offers future directions.     

Easily available enzymes as natural retting agents

Easily available commercial enzymes currently have great potential in bast fibre processing and can be modified for different end uses. There are several new technologies using enzymes that are able to modify fibre parameters, achieve requested properties, improve processing results and are more beneficial to the ecology in the area of bast fibre processing and fabrics finishing. Enzymatic methods for retting of flax, "cottonisation" of bast fibres, hemp separation, and processing of flax rovings before wet spinning, etc., fall into this group of new technologies. Such enzymatic biotechnologies can provide benefits in textile, composite, reinforced plastic and other technical applications. Laboratory, pilot and industrial scale results and experiences have demonstrated the ability of selected enzymes to decompose interfibre-bonding layers based on pectin, lignin and hemicelluloses. Texazym SER spray is able to increase flax long fibre yields by more than 40%. Other enzymes in combination with mild mechanical treatment can replace aggressive and energy-intensive processing like Laroche "cottonisation". Texazym SCW and DLG pretreatments of flax rovings are presented.     

Dansylcadaverine specific staining for transamidating enzymes.

Versatile fluorescent staining methodologies, based on the incorporation of dansylcadaverine[N-(5-aminopentyl)-5-dimethylamino-l-naphthalenesulfonamide] into N,N-dimethylcasein, are described for the detection of transamidating enzymes of the endo-γ-glutamine:ε-lysine transferase type. Activity staining was employed for comparing the electrophoretic behaviors of such transamidating enzymes derived from human and guinea pig tissues. Two enzymatically active forms of guinea pig liver transglutaminase were found.     

Functional representation of enzymes by specific peptides

Predicting the function of a protein from its sequence is a long-standing goal of bioinformatic research. While sequence similarity is the most popular tool used for this purpose, sequence motifs may also subserve this goal. Here we develop a motif-based method consisting of applying an unsupervised motif extraction algorithm (MEX) to all enzyme sequences, and filtering the results by the four-level classification hierarchy of the Enzyme Commission (EC). The resulting motifs serve as specific peptides (SPs), appearing on single branches of the EC. In contrast to previous motif-based methods, the new method does not require any preprocessing by multiple sequence alignment, nor does it rely on over-representation of motifs within EC branches. The SPs obtained comprise on average 8.4 +/- 4.5 amino acids, and specify the functions of 93% of all enzymes, which is much higher than the coverage of 63% provided by ProSite motifs. The SP classification thus compares favorably with previous function annotation methods and successfully demonstrates an added value in extreme cases where sequence similarity fails. Interestingly, SPs cover most of the annotated active and binding site amino acids, and occur in active-site neighboring 3-D pockets in a highly statistically significant manner. The latter are assumed to have strong biological relevance to the activity of the enzyme. Further filtering of SPs by biological functional annotations results in reduced small subsets of SPs that possess very large enzyme coverage. Overall, SPs both form a very useful tool for enzyme functional classification and bear responsibility for the catalytic biological function carried out by enzymes.     

Data mining of enzymes using specific peptides

Background  

Predicting the function of a protein from its sequence is a long-standing challenge of bioinformatic research, typically addressed using either sequence-similarity or sequence-motifs. We employ the novel motif method that consists of Specific Peptides (SPs) that are unique to specific branches of the Enzyme Commission (EC) functional classification. We devise the Data Mining of Enzymes (DME) methodology that allows for searching SPs on arbitrary proteins, determining from its sequence whether a protein is an enzyme and what the enzyme's EC classification is.     

Biological roles of specific peptides in enzymes

It has recently been shown (Kunik et al., PLOS Comput Biol 2007;3(8):e167) that the occurrence of specific peptides (SPs) on sequences of enzymes allows for accurate EC classification of enzymes. We inquire whether these SPs play important roles in bringing about the enzymatic function. This is assessed by cross-checking the occurrence of SPs on enzymes with Swiss-Prot annotations and PDB spatial structures of enzymes. Analyzing the coverage of functional annotations of enzymes, we demonstrate that SPs contain major fractions of all annotated features. This result is statistically highly significant and associates over 10% of all SPs with important biological markers. Concentrating on DNA binding regions, relevant to LexA repressor enzymes, we find interesting coverage patterns. Moreover, for the same data, we demonstrate that SPs allow for subclassification of the relevant bacteria into phylogenetic classes. An analysis of mutagen annotations on SPs appearing on all enzymes leads to the conclusion that mutations on SPs tend to damage the enzymatic function much more than expected from a background model, hence SPs are of high importance to enzymatic functions. SPs that lie in 3D pockets that are shared by active and binding sites, are shown to be significantly enriched by glycine, leading to the hypothesis that they are responsible for conformational plasticity. Finally we show that SPs can partially resolve outstanding difficult problems of convergent evolution by representing correctly enzyme functions in spite of remote homologies in sequence and in structure.     

Stabilization of enzymes by their specific antibodies.

In nature, increased stability of enzymes has often been found to be associated with noncovalent protein-protein interactions. Specific antibodies should be suitable for this purpose. To test this hypothesis, we used a number of model enzymes, complexed them with their specific antibodies, and exposed them and the free enzymes to low and high temperature, lyophilization, oxidation, and alcohol. The retained activity of the antibody-complexed enzymes was substantially, and in some cases dramatically, higher. In general mechanistic terms, stabilization may have been accomplished either by noncovalent antibody crosslinking of discontinuous oligopeptide chains on the surface of the enzyme, thereby increasing resistance to unfolding of the enzyme, or by physical shielding by the antibodies of vulnerable sites on the surface of the enzyme.     

Elucidation of a protease-sensitive site involved in the binding of calcium to C-reactive protein

C-reactive protein (CRP) is a Ca2+-binding protein composed of five identical 23-kDa subunits arranged as a cyclic pentamer, present in greatly elevated concentration in the blood during the acute phase of processes involving tissue injury and necrosis. In the present work, it was found that treatment of human CRP with Pronase or Nagarse protease produces two major fragments which remain associated in physiological buffers but are separable under denaturing conditions. To localize the cleavage site(s), the fragments were characterized according to molecular mass, amino acid composition, partial amino acid sequence, and reactivity with monoclonal antibodies specific for the fragments and for defined CRP epitopes including residues 147-152 and 199-206. Nagarse protease cleaves the CRP subunit between residues 145 and 146, producing two fragments, 16 and 6.5 kDa (calculated molecular mass). Pronase cleaves the CRP subunit between residues 146 and 147, producing a 16-kDa fragment (A1) and a 6.5-kDa fragment (B); an additional fragment (A2) approximately 1 kDa smaller than fragment A1 is also apparently produced due to a secondary cleavage site in fragment A1. Cleavage appears to be completely inhibited in the presence of 1 mM CaCl2. Ca2+ does not protect cleaved CRP from heat-induced aggregation (i.e., precipitation) as it does the intact protein. Protease-cleaved CRP loses the ability to bind to the Ca2+-dependent ligand phosphorylcholine but remains the ability to bind to the Ca2+-independent ligand arginine-rich histone. Equilibrium dialysis indicates that intact CRP binds 2 mol of Ca2+/mol of subunit with a Kd of 6 X 10(-5) M.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro hybridization and separation of hybrids of human adenylosuccinate lyase from wild-type and disease-associated mutant enzymes

Human adenylosuccinate lyase (ASL) deficiency is an inherited metabolic disease in which the majority of the patients are compound heterozygotes for the mutations that occur in the ASL gene. Starting with purified wild-type (WT) and single-mutant human ASL, we generated in vitro hybrids that mimic compound heterozygote ASL. For this study, we used His-tagged WT/non-His-tagged WT, His-tagged WT/non-His-tagged R396C, His-tagged WT/non-His-tagged R396H, His-tagged R194C/non-His-tagged R396C, and His-tagged L311V/non-His-tagged R396H enzyme pairs. We generated various hybrids by denaturing pairs of enzymes in 1 M guanidinium chloride and renaturing them by removing the denaturant. The hybrids were separated on a nickel-nitrilotriacetic acid-agarose column based on the number of His tags present in the enzyme tetramer. Analytical ultracentrifuge data indicate that the hybrids have predominant amounts of heterotetramers. Analysis of the V(max) values of the hybrids indicates that most of the subunits behave independently; however, the hybrid tetramers retain weak positive cooperativity, indicating that there is some interaction between the different subunit types. The interactions between WT and mutant subunits may be advantageous to the parents of ASL deficient patients, while the interactions between some mutant subunits may assist heterozygote ASL deficient patients.