Fluorogenic DNA ligase and base excision repair enzyme assays using substrates labeled with single fluorophores

Continuing our work on fluorogenic substrates labeled with single fluorophores for nucleic acid modifying enzymes, here we describe the development of such substrates for DNA ligases and some base excision repair enzymes. These substrates are hairpin-type synthetic DNA molecules with a single fluorophore located on a base close to the 3′ ends, an arrangement that results in strong fluorescence quenching. When such substrates are subjected to an enzymatic reaction, the position of the dyes relative to that end of the molecules is altered, resulting in significant fluorescence intensity changes. The ligase substrates described here were 5′ phosphorylated and either blunt-ended or carrying short, self-complementary single-stranded 5′ extensions. The ligation reactions resulted in the covalent joining of the ends of the molecules, decreasing the quenching effect of the terminal bases on the dyes. To generate fluorogenic substrates for the base excision repair enzymes formamido–pyrimidine–DNA glycosylase (FPG), human 8-oxo-G DNA glycosylase/AP lyase (hOGG1), endonuclease IV (EndoIV), and apurinic/apyrimidinic endonuclease (APE1), we introduced abasic sites or a modified nucleotide, 8-oxo-dG, at such positions that their enzymatic excision would result in the release of a short fluorescent fragment. This was also accompanied by strong fluorescence increases. Overall fluorescence changes ranged from approximately 4-fold (ligase reactions) to more than 20-fold (base excision repair reactions).     

In-gel protein phosphatase assay using fluorogenic substrates

We developed a method for the detection of phosphatase activity using fluorogenic substrates after polyacrylamide gel electrophoresis. When phosphatases such as Ca²⁺/calmodulin-dependent protein kinase phosphatase (CaMKP), protein phosphatase 2C (PP2C), protein phosphatase 5 (PP5), and alkaline phosphatase were resolved by polyacrylamide gel electrophoresis in the absence of SDS and the gel was incubated with a fluorogenic substrate such as 4-methylumbelliferyl phosphate (MUP), all of these phosphatase activities could be detected in situ. Although 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as well as MUP could be used as a fluorogenic substrate for an in-gel assay, MUP exhibited lower background fluorescence. Using this procedure, several fluorescent bands that correspond to endogenous phosphatases were observed after electrophoresis of various crude samples. The in-gel phosphatase assay could also be used to detect protein phosphatases resolved by SDS-polyacrylamide gel electrophoresis. In this case, however, the denaturation/renaturation process of resolved proteins was necessary for the detection of phosphatase activity. This procedure could be used for detection of renaturable protein phosphatases such as CaMKP and some other phosphatases expressed in cell extracts. The present fluorescent in-gel phosphatase assay is very useful, since no radioactive compounds or no special apparatus are required.     

Latent fluorophores based on a self-immolative linker strategy and suitable for protease sensing

The self-immolative spacer para-aminobenzyl alcohol (PABA) was used as a key component in the design of new protease-sensitive fluorogenic probes whose parent phenol-based fluorophore is released through an enzyme-initiated domino reaction. First, the conjugation of the phenylacetyl moiety to 7-hydroxycoumarin (umbelliferone) and 7-hydroxy-9 H-(9,9-dimethylacridin-2-one) (DAO) by means of the heterobifunctional PABA linker has led to pro-fluorophores 6a and 6d whose enzyme activation by penicillin amidase was demonstrated. The second part of this study was devoted to the extension of this latent fluorophore strategy to the caspase-3 protease, a key mediator of apoptosis in mammalian cells. Fluorogenic caspase-3 substrates 11 and 13 derived from umbelliferone and DAO, respectively, were prepared. It was demonstrated that pro-fluorophore 11 is a sensitive fluorimetric reagent for the detection of this cysteine protease. Furthermore, in vitro assays with fluorogenic probe 13 showed a deleterious effect of biological thiols on fluorescence of the released acridinone fluorophore DAO that, to our knowledge, had not been reported until now.     

The homogeneous fluorescence anisotropic sensing of salivary lysozyme using the 6-carboxyfluorescein-labeled DNA aptamer

A simple and sensitive fluorescence anisotropy method was developed for lysozyme, employing the coupling of fluorophore, 6-carboxyfluorescein (FAM), with lysozyme upon recognition between the target molecule and its DNA aptamer. It was found in this study that the rotational dynamic of the detecting system is crucial to obtain a high anisotropy signal that cannot always be achieved by simply increasing the molecular volume, because molecular volume increase may not be able to efficiently retard the rotational movement of the fluorophore. FAM was selected as the label of the ssDNA aptamer to effectively facilitate the change of the fluorophore from a primarily independent segmental movement to slow global rotation. The time-resolved measurements, including lifetime and dynamic fluorescence anisotropy, were conducted to study the recognition interaction and to better understand the methodology. The proposed method had a wide linear dynamic range of 12.5-300 nM and a high sensitivity with the limit of detection of 4.9 nM (3S/N). This proposed method was successfully applied to assay of human salivary lysozyme. The results based on the standard addition recovery and comparison with enzyme-linked immunosorbent assay (ELISA) demonstrated the feasibility of this method for biological samples. Using coupling between the fluorophore and the analyte can be one of the approaches working toward expanding the application of fluorescence anisotropy based on aptamer-target and antibody-antigen recognitions.     

Non-label homogeneous protein detection based on laser interferometric photo-thermal displacement measurement using aptamers

Photo-thermal displacement measurement by laser interferometry involves the measurement of temperature change caused by illumination of the sample. To develop a system of detecting unlabeled homogeneous proteins based on laser interferometric measurement of photo-thermal displacement, we studied the interaction between aptamers and their target molecules by using thrombin and the thrombin aptamer as a model target and ligand, respectively. Because of the energy consumed by aptamer-thrombin interactions, the signals obtained from solutions containing aptamer-thrombin mixtures varied depending on the thrombin concentration. We propose that this method involving the use of aptamers and photo-thermal displacement measurement will provide a biomolecular detection system for rapid diagnosis.     

High-throughput kinase assays with protein substrates using fluorescent polymer superquenching

Background  

High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform.     

Reagentless optical sensing of glutamine using a dual-emitting glutamine-binding protein

Glutamine is a major source of nitrogen and carbon in cell culture media. Thus, glutamine monitoring is important in bioprocess control. Here we report a reagentless fluorescence sensing for glutamine based on the Escherichia coli glutamine-binding protein (GlnBP) that is sensitive in the submicromolar ranges. The S179C variant of GlnBP was labeled at the -SH and N-terminal positions with acrylodan and ruthenium bis-(2,2'-bipyridyl)-1,10-phenanthroline-9-isothiocyanate, respectively. The acrylodan emission is quenched in the presence of glutamine while the ruthenium acts as a nonresponsive long-lived reference. The apparent binding constant, K'(d), of 0.72 microM was calculated from the ratio of emission intensities of acrylodan and ruthenium (I(515)/I(610)). The presence of the long-lived ruthenium allowed for modulation sensing at lower frequencies (1-10 MHz) approaching an accuracy of +/-0.02 microM glutamine. Dual-frequency ratiometric sensing was also demonstrated. Finally, the extraordinary sensitivity of GlnBP allows for dilution of the sample, thereby eliminating the effects of background fluorescence from the culture media.     

Single cell protein from various chemically pretreated wood substrates using Chaetomium cellulolyticum

The growth behavior of Chaetomium cellulolyticum, a new cellulolytic fungus, has been examined in slurry fermentation systems using various chemically pretreated sawdusts from hardwoods as substrates. Both acid- and alkali-pretreatment methods were used and the fermentation media included the spent pretreatment liquor in an attempt to concurrently maximize substrate utilization and minimize the biological oxygen demand (BOD) level in the process effluent. Diauxic growth patterns were found in the three cases studied, suggesting an initial utilization of soluble hemicellulose sugars followed by utilization of the insoluble cellulose. This behavior patterns was supported by separate growth experiments using the major sugars of hemicellulose as carbon sources. The organism was found to be a good convertor of both cellulose and hemicelluloses into single cell protein (SCP). In terms of rate and extent of protein production in the insoluble biomass product, acid pretreatment appears to be better than alkali pretreatment if the product is intended as ruminant feed.     

Development of a protease activity assay using heat-sensitive Tus-GFP fusion protein substrates

Proteases are implicated in various diseases and several have been identified as potential drug targets or biomarkers. As a result, protease activity assays that can be performed in high throughput are essential for the screening of inhibitors in drug discovery programs. Here we describe the development of a simple, general method for the characterization of protease activity and its use for inhibitor screening. GFP was genetically fused to a comparatively unstable Tus protein through an interdomain linker containing a specially designed protease site, which can be proteolyzed. When this Tus–GFP fusion protein substrate is proteolyzed it releases GFP, which remains in solution after a short heat denaturation and centrifugation step used to eliminate uncleaved Tus–GFP. Thus, the increase in GFP fluorescence is directly proportional to protease activity. We validated the protease activity assay with three different proteases, i.e., trypsin, caspase 3, and neutrophil elastase, and demonstrated that it can be used to determine protease activity and the effect of inhibitors with small sample volumes in just a few simple steps using a fluorescence plate reader.     

Development of homogeneous immunoassays based on protein fragment complementation

We demonstrate a functional in vitro proof-of-principle homogeneous assay capable of detecting small (<1 kDa) to large (150 kDa) analytes using TEM-1 β-lactamase protein fragment complementation. In the assays reported here, complementary components are added together in the presence of analyte and substrate resulting in colorimetric detection within 10-min. We demonstrate the use of functional mutations leading to either increased enzymatic activity, reduced fragment self-association or increased inhibitor resistance upon analyte driven fragment complementation. Kinetic characterization of the resulting reconstituted enzyme illustrates the importance of balancing increased enzyme activity with fragment self-association, producing diagnostically relevant signal-to-noise ratios. Complementation can be utilized as a homogeneous immunoassay platform for the potential detection of a range of analytes including, antibodies, antigens and biomarkers.     

Label-free detection of protein interactions using deep UV fluorescence lifetime microscopy

We present a label-free detection of protein interaction between beta-galactosidase from Escherichia coli (Ecbeta-Gal) and monoclonal anti-Ecbeta-Gal using deep UV laser-based fluorescence lifetime microscopy. The native fluorescence from intrinsic tryptophan emission was observed after one-photon excitation at 266 nm. Applying the time-correlated single-photon counting (TCSPC) method, we investigated the mean fluorescence lifetime and lifetime distributions from tryptophan residues in Ecbeta-Gal protein, monoclonal anti-Ecbeta-Gal, and corresponding complex. The results demonstrate that deep UV laser-based fluorescence lifetime microscopy is useful for sensitive identification of biological macromolecules interaction using intrinsic fluorescence.     

Large-scale prediction of protein ubiquitination sites using a multimodal deep architecture

Background

Ubiquitination, which is also called “lysine ubiquitination”, occurs when an ubiquitin is attached to lysine (K) residues in targeting proteins. As one of the most important post translational modifications (PTMs), it plays the significant role not only in protein degradation, but also in other cellular functions. Thus, systematic anatomy of the ubiquitination proteome is an appealing and challenging research topic. The existing methods for identifying protein ubiquitination sites can be divided into two kinds: mass spectrometry and computational methods. Mass spectrometry-based experimental methods can discover ubiquitination sites from eukaryotes, but are time-consuming and expensive. Therefore, it is priority to develop computational approaches that can effectively and accurately identify protein ubiquitination sites.

Results

The existing computational methods usually require feature engineering, which may lead to redundancy and biased representations. While deep learning is able to excavate underlying characteristics from large-scale training data via multiple-layer networks and non-linear mapping operations. In this paper, we proposed a deep architecture within multiple modalities to identify the ubiquitination sites. First, according to prior knowledge and biological knowledge, we encoded protein sequence fragments around candidate ubiquitination sites into three modalities, namely raw protein sequence fragments, physico-chemical properties and sequence profiles, and designed different deep network layers to extract the hidden representations from them. Then, the generative deep representations corresponding to three modalities were merged to build the final model. We performed our algorithm on the available largest scale protein ubiquitination sites database PLMD, and achieved 66.4% specificity, 66.7% sensitivity, 66.43% accuracy, and 0.221 MCC value. A number of comparative experiments also indicated that our multimodal deep architecture outperformed several popular protein ubiquitination site prediction tools.

Conclusion

The results of comparative experiments validated the effectiveness of our deep network and also displayed that our method outperformed several popular protein ubiquitination site prediction tools. The source codes of our proposed method are available at https://github.com/jiagenlee/deepUbiquitylation.

     

Effect of parathyroid hormone on rat renal cAMP-dependent protein kinase and protein kinase C activity measured using synthetic peptide substrates

The actions of parathyroid hormone (PTH) on the renal cortex are thought to be mediated primarily by cAMP-dependent protein kinase (PKA) with some suggestion of a role for protein kinase C (PKC). However, present methods for assaying PKA and PKC in subcellular fractions are insensitive and require large amounts of protein. Recently, a sensitive method for measuring the activity of protein kinases has been reported. This method uses synthetic peptides as substrates and a tandem chromatographic procedure for isolating the phosphorylated peptides. We have adapted this method to study the effect of PTH on PKA and PKC activity using thin slices of rat renal cortex. PTH (250 nM) stimulated cytosolic PKA activity four- to fivefold within 30 s, and PKA activity was sustained for at least 5 min. PTH also rapidly stimulated PKC activity in the membrane fraction and decreased PKC activity in the cytosol. These changes were maximal at 30 s, but unlike changes in PKA, they declined rapidly thereafter. PTH significantly activated PKC only at concentrations of 10 nM or greater. This study demonstrates that PTH does activate PKC in renal tissue, although the duration of activation is much less than for PKA. It also demonstrates that a combination of synthetic peptides with tandem chromatography can be used as a sensitive assay procedure for protein kinase activity in biological samples.     

Detection of a calcium-activated protein kinase in mougeotia by using synthetic Peptide substrates

By using a synthetic peptide, KM-14, a protein kinase was detected and partially purified from Mougeotia sp. The peptide contains the sequence of the regulatory light chain of smooth muscle myosin that is phosphorylated by calcium-calmodulin-dependent myosin light chain kinase (MLCK). The Mougeotia kinase was stimulated 40-fold by calcium with half-maximal stimulation occurring at 1.5 micromolar. The enzyme was fractionated from calmodulin and was depleted of calmodulin based on enzyme activator analysis. The calmodulin-depleted enzyme was fully active and calcium dependent, and was not stimulated further by exogenous calmodulin nor by the calcium effectors phosphatidylserine and diacylglycerol. The enzyme phosphorylated intact chicken gizzard myosin light chain as well as the KM-14 substrate. KM-13, a peptide analog of KM-14 with a deletion of a glutamine at position 5, was a poor substrate with a V_max/K_m ratio 200-fold lower than KM-14. Thus, similarly to vertebrate MLCK, the Mougeotia enzyme is very sensitive to changes in sequence surrounding the phosphorylation site. Calcium-dependent KM-14 kinase activity also was detected in two other algae, Mesotaenium caldariorum and Spirogyra sp., as well as in pea seedlings. The data suggest that plant and algal tissues possess an enzyme with a substrate specificity similar to MLCK, but unlike MLCK, does not appear to require calmodulin for activity.     

Recognition and fluorescence sensing of specific amino acid residue on protein surface using designed molecules

Many biological processes are mediated by surface recognition between proteins. Small molecules that recognize and bind a specific region of a protein surface may be promising agents for disrupting certain protein-protein surface interactions, which consequently leads to regulation of cellar functions. This article describes our recent efforts toward the development of the designed small molecules, which can recognize histidine or phosphorylated amino acid residues on peptide surfaces in a sequence-selective manner. These results demonstrate that cooperative metal-ligand interaction is powerful for tight and selective binding to the specific amino acid residues of proteins in aqueous medium.     

Ultrasensitive staining-free protein detection after PAA gel electrophoresis using deep UV fluorescence

We present the observation of separated protein bands after polyacrylamide (PAA) gel electrophoresis based on the staining-free detection of their ultra violet (UV)-induced fluorescence employing deep UV confocal fluorescence microscopy. Mixtures of the three biological compounds beta-Galactosidase (from Escherichia coli), apo-Transferrin (bovine) and bovine serum albumin (BSA) have been separated and a staining free detection limit below 80 pg (7.0 x 10(8) molecules) per band has been achieved. This corresponds to approximately 270 molecules in the detection volume for confocal microscopy.     

Analysis of protein tyrosine phosphatase interactions with microarrayed phosphopeptide substrates using imaging mass spectrometry

Microarrays of peptide and recombinant protein libraries are routinely used for high-throughput studies of protein–protein interactions and enzymatic activities. Imaging mass spectrometry (IMS) is currently applied as a method to localize analytes on thin tissue sections and other surfaces. Here, we have applied IMS as a label-free means to analyze protein–peptide interactions in a microarray-based phosphatase assay. This IMS strategy visualizes the entire microarray in one composite image by collecting a predefined raster of matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectrometry spectra over the surface of the chip. Examining the bacterial tyrosine phosphatase YopH, we used IMS as a label-free means to visualize enzyme binding and activity with a microarrayed phosphopeptide library printed on chips coated with either gold or indium–tin oxide. Furthermore, we demonstrate that microarray-based IMS can be coupled with surface plasmon resonance imaging to add kinetic analyses to measured binding interactions. The method described here is within the capabilities of many modern MALDI–TOF instruments and has general utility for the label-free analysis of microarray assays.     

A protease inhibitor discovery method using fluorescence correlation spectroscopy with position-specific labeled protein substrates

We developed novel substrates for protease activity evaluation by fluorescence correlation spectroscopy (FCS). Substrates were labeled in a position-specific manner with a fluorophore near the N terminus and included a C-terminal, 30 kDa, highly soluble protein (elongation factor Ts [EF-Ts]). The C-terminal protein enhanced the substrate peptide solubility and increased the molecular weight, enabling sensitive detection by FCS. Using the labeled substrates, caspase-3 and matrix metalloproteinase-9 (MMP-9) activities were confirmed by FCS. To demonstrate the suitability of this FCS-based assay for high-throughput screening, we screened various chemical compounds for MMP-9 inhibitors. The screening results confirmed the inhibitory activity of one compound and also revealed another potential MMP-9 inhibitor. Thus, this combination of position-specific labeled protein substrates and FCS may serve as a useful tool for evaluating activities of various proteases and for protease inhibitor screening.