Clickable, selective, and cell-permeable activity-based probe of human cathepsin B - Minimalistic approach for enhanced selectivity.

Human cathepsin B is a cysteine-dependent protease whose roles in both normal and diseased cellular states remain yet to be fully delineated. This is primarily due to overlapping substrate specificities and lack of unambiguously annotated physiological functions. In this work, a selective, cell-permeable, clickable and tagless small molecule cathepsin B probe, KDA-1, is developed and kinetically characterized. KDA-1 selectively targets active site Cys25 residue of cathepsin B for labeling and can detect active cellular cathepsin B in proteomes derived from live human MDA-MB-231 breast cancer cells and HEK293 cells. It is anticipated that KDA-1 probe will find suitable applications in functional proteomics involving human cathepsin B enzyme.

Alterations of elastin in female reproductive tissues arising from advancing parity.

Female reproductive tissues undergo significant alterations during pregnancy, which may compromise the structural integrity of extracellular matrix proteins. Here, we report on modifications of elastic fibers, which are primarily composed of elastin and believed to provide a scaffold to the reproductive tissues, due to parity and parturition. Elastic fibers from the upper vaginal wall of virgin Sprague Dawley rats were investigated and compared to rats having undergone one, three, or more than five pregnancies. Optical microscopy was used to study fiber level changes. Mass spectrometry, 13C and 2H NMR, was applied to study alterations of elastin from the uterine horns. Spectrophotometry was used to measure matrix metalloproteinases-2,9 and tissue inhibitor of metalloproteinase-1 concentration changes in the uterine horns. Elastic fibers were found to exhibit increase in tortuosity and fragmentation with increased pregnancies. Surprisingly, secondary structure, dynamics, and crosslinking of elastin from multiparous cohorts appear similar to healthy mammalian tissues, despite fragmentation observed at the fiber level. In contrast, elastic fibers from virgin and single pregnancy cohorts are less fragmented and comprised of elastin exhibiting structure and dynamics distinguishable from multiparous groups, with reduced crosslinking. These alterations were correlated to matrix metalloproteinases-2,9 and tissue inhibitor of metalloproteinase-1 concentrations. This work indicates that fiber level alterations resulting from pregnancy and/or parturition, such as fragmentation, rather than secondary structure (e.g. elastin crosslinking density), appear to govern scaffolding characteristics in the female reproductive tissues.

Cell penetrable, clickable and tagless activity-based probe of human cathepsin L.

Human cathepsin L is a ubiquitously expressed endopeptidase and is known to play critical roles in a wide variety of cellular signaling events. Its overexpression has been implicated in numerous human diseases, including highly invasive forms of cancer. Inhibition of cathepsin L is therefore considered a viable therapeutic strategy. Unfortunately, several redundant and even opposing roles of cathepsin L have recently emerged. Selective cathepsin L probes are therefore needed to dissect its function in context-specific manner before significant resources are directed into drug discovery efforts. Herein, the development of a clickable and tagless activity-based probe of cathepsin L is reported. The probe is highly efficient, active-site directed and activity-dependent, selective, cell penetrable, and non-toxic to human cells. Using zebrafish model, we demonstrate that the probe can inhibit cathepsin L function in vivo during the hatching process. It is anticipated that the probe will be a highly effective tool in dissecting cathepsin L biology at the proteome levels in both normal physiology and human diseases, thereby facilitating drug-discovery efforts targeting cathepsin L.

Quantification of desmosine and isodesmosine using MALDI-ion trap tandem mass spectrometry.

Desmosine (Des) and isodesmosine (Isodes), cross-linking amino acids in the biomolecule elastin, may be used as biomarkers for various pathological conditions associated with elastin degradation. The current study presents a novel approach to quantify Des and Isodes using matrix-assisted laser desorption ionization (MALDI)-tandem mass spectrometry (MS2) in a linear ion trap coupled to a vacuum MALDI source. MALDI-MS2 analyses of Des and Isodes are performed using stable-isotope-labeled desmosine d4 (labeled-Des) as an internal standard in different biological fluids, such as urine and serum. The method demonstrated linearity over two orders of magnitude with a detection limit of 0.02 ng/µL in both urine and serum without enrichment prior to mass spectrometry, and relative standard deviation of < 5%. The method is used to evaluate the time-dependent degradation of Des upon UV irradiation (254 nm) and found to be consistent with quantification by 1H NMR. This is the first characterized MALDI-MS2 method for quantification of Des and Isodes and illustrates the potential of MALDI-ion trap MS2 for effective quantification of biomolecules. The reported method represents improvement over current liquid chromatography-based methods with respect to analysis time and solvent consumption, while maintaining similar analytical characteristics. Graphical abstract ᅟ.

Arabidopsis AtPARK13, which confers thermotolerance, targets misfolded proteins.

Mutations in HTRA2/Omi/PARK13 have been implicated in Parkinson disease (PD). PARK13 is a neuroprotective serine protease; however, little is known about how PARK13 confers stress protection and which protein targets are directly affected by PARK13. We have reported that Arabidopsis thaliana represents a complementary PD model, and here we demonstrate that AtPARK13, similar to human PARK13 (hPARK13), is a mitochondrial protease. We show that the expression/accumulation of AtPARK13 transcripts are induced by heat stress but not by other stress conditions, including oxidative stress and metals. Our data show that elevated levels of AtPARK13 confer thermotolerance in A. thaliana. Increased temperatures accelerate protein unfolding, and we demonstrate that although AtPARK13 can act on native protein substrates, unfolded proteins represent better AtPARK13 substrates. The results further show that AtPARK13 and hPARK13 can degrade the PD proteins α-synuclein (SNCA) and DJ-1/PARK7 directly, without autophagy involvement, and that misfolded SNCA and DJ-1 represent better substrates than their native counterparts. Comparative proteomic profiling revealed AtPARK13-mediated proteome changes, and we identified four proteins that show altered abundance in response to AtPARK13 overexpression and elevated temperatures. Our study not only suggests that AtPARK13 confers thermotolerance by degrading misfolded protein targets, but it also provides new insight into possible roles of this protease in neurodegeneration.

Studies on quantitative phosphopeptide analysis by matrix-assisted laser desorption/ionization mass spectrometry without label, chromatography or calibration curves.

RATIONALE: Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry combined with isotope labeling methods are effective for protein and peptide quantification, but limited in their multiplexing capacity, cost-effectiveness and dynamic range. This study investigates MALDI-MS-based quantification of peptide phosphorylation without labeling, and aims to overcome the shot-to-shot variability of MALDI using a mathematical transformation and extended data acquisition times. METHODS: A linear relationship between the reciprocal of phosphopeptide mole fraction and the reciprocal of phosphorylated-to-unphosphorylated signal ratio is derived, and evaluated experimentally using three separate phosphopeptide systems containing phosphorylated serine, threonine and tyrosine residues: mixtures of phosphopeptide and its des-phospho-analog with known stoichiometry measured by vacuum MALDI-linear ion trap mass spectrometry and fit to the linear model. The model is validated for quantifying in vitro phosphorylation assays with inhibition studies on Cdk2/cyclinA. RESULTS: Dynamic range of picomoles to femtomoles, good accuracy (deviations of 1.5-3.0% from expected values) and reproducibility (relative standard deviation (RSD) = 4.3-6.3%) are achieved. Inhibition of cyclin-dependent kinase phosphorylation by the classical inhibitors olomoucine and r-roscovitine was evaluated and IC50 values found to be in agreement with reported literature values. These results, achieved with single-point calibration, without isotope or chromatography, compare favorably to those arrived at using isotope dilution (p > 0.5 for accuracy). CONCLUSIONS: The mathematical relationship derived here can be applied to a method that we term Double Reciprocal Isotope-free Phosphopeptide Quantification (DRIP-Q), as a strategy for quantification of in vitro phosphorylation assays, the first MALDI-based, isotope- and calibration curve-free method of its type. These results also pave the way for further systematic studies investigating the effect of peptide composition and experimental conditions on quantitative, label-free MALDI.

Development of cell-active non-peptidyl inhibitors of cysteine cathepsins.

Cysteine cathepsins are an important class of enzymes that coordinate a variety of important cellular processes, and are implicated in various types of human diseases. However, small molecule inhibitors that are cell-permeable and non-peptidyl in nature are scarcely available. Herein the synthesis and development of sulfonyloxiranes as covalent inhibitors of cysteine cathepsins are reported. From a library of compounds, compound 5 is identified as a selective inhibitor of cysteine cathepsins. Live cell imaging and immunocytochemistry of metastatic human breast carcinoma MDA-MB-231 cells document the efficacy of compound 5 in inhibiting cysteine cathepsin activity in living cells. A cell-motility assay demonstrates that compound 5 is effective in mitigating the cell-migratory potential of highly metastatic breast carcinoma MDA-MB-231 cells.

Ultraviolet radiation reduces desmosine cross-links in elastin.

Elastic fibers, a major component of the extracellular matrix of the skin, are often exposed to ultraviolet (UV) radiation throughout mammalian life. We report on an in vitro study of the alterations in bovine nuchal ligament elastic fibers resulting from continuous UV-A exposure by the use of transmission electron microscopy (TEM), histology, mass spectrometry, and solid state 13C NMR methodologies. TEM images reveal distinct cracks in elastic fibers as a result of UV-A irradiation and histological measurements show a disruption in the regular array of elastic fibers present in unirradiated samples; elastic fibers appear shorter, highly fragmented, and thinner after UV-A treatment. Magic angle spinning 13C NMR was applied to investigate possible secondary structural changes or dynamics in the irradiated samples; our spectra reveal no differences between UV-A irradiated and non-irradiated samples. Lastly, MALDI mass spectrometry indicates that the concentration of desmosine, which forms cross-links in elastin, is observed to decrease by 11 [Formula: see text] following 9 days of continuous UV-A irradiation, in comparison to unirradiated samples. These alterations presumably play a significant role in the loss of elasticity observed in UV exposed skin.

Conformational Differentiation of α-Cyanohydroxycinnamic Acid Isomers: A Raman Spectroscopic Study.

Two α-cyanohydroxycinnamic acid positional isomers, α-cyano-4-hydroxycinnamic acid (CHCA4) and α-cyano-3-hydroxycinnamic acid (CHCA3), were characterized using Raman spectroscopy. We analyzed the implications of the collected Raman spectral shifts, and verified them through other spectroscopic techniques, to arrive at plausible three dimensional structures of CHCA3 and CHCA4. The positions of these groups were mapped by systematically analyzing the orientation and type of interactions functional groups make in each CHCA isomer. We determined whether or not the carboxylic moieties are forming dimeric links and ascertained the existence of ring-ring π-stacking interactions. We also assessed the nature of the hydrogen bonding between -CN and -OH groups. The results were then taken together to model plausible three dimensional structures for each compound. The data revealed a structure for CHCA4 that matches the published x-ray crystallographic structure. We then applied the same spectral analysis to CHCA3 to reveal its plausible three dimensional structure. The structural details revealed may account for the functional properties of the two α-cyanohydroxycinnamic acid positional isomers.

A Proteomics Approach to Investigate miR-153-3p and miR-205-5p Targets in Neuroblastoma Cells.

MicroRNAs are key regulators associated with numerous diseases. In HEK293 cells, miR-153-3p and miR-205-5p down-regulate alpha-synuclein (SNCA) and Leucine-rich repeat kinase 2 (LRRK2), two key proteins involved in Parkinson's disease (PD). We have used two-dimensional gel electrophoresis (2D-PAGE) coupled to mass spectrometry (MS) to identify a spectrum of miR-153-3p and miR-205-5p targets in neuronal SH-SY5Y cells. We overexpressed and inhibited both microRNAs in SH-SY5Y cells and through comparative proteomics profiling we quantified ~240 protein spots from each analysis. Combined, thirty-three protein spots were identified showing significant (p-value < 0.05) changes in abundance. Modulation of miR-153-3p resulted in seven up-regulated proteins and eight down-regulated proteins. miR-205 modulation resulted in twelve up-regulated proteins and six down-regulated proteins. Several of the proteins are associated with neuronal processes, including peroxiredoxin-2 and -4, cofilin-1, prefoldin 2, alpha-enolase, human nucleoside diphosphate kinase B (Nm23) and 14-3-3 protein epsilon. Many of the differentially expressed proteins are involved in diverse pathways including metabolism, neurotrophin signaling, actin cytoskeletal regulation, HIF-1 signaling and the proteasome indicating that miR-153-3p and miR-205-5p are involved in the regulation of a wide variety of biological processes in neuroblastoma cells.

A cell-intrinsic inhibitor of HIV-1 reverse transcription in CD4(+) T cells from elite controllers.

HIV-1 reverse transcription represents the predominant target for pharmacological inhibition of viral replication, but cell-intrinsic mechanisms that can block HIV-1 reverse transcription in a clinically significant way are poorly defined. We find that effective HIV-1 reverse transcription depends on the phosphorylation of viral reverse transcriptase by host cyclin-dependent kinase (CDK) 2 at a highly conserved Threonine residue. CDK2-dependent phosphorylation increased the efficacy and stability of viral reverse transcriptase and enhanced viral fitness. Interestingly, p21, a cell-intrinsic CDK inhibitor that is upregulated in CD4(+) T cells from "elite controllers," potently inhibited CDK2-dependent phosphorylation of HIV-1 reverse transcriptase and significantly reduced the efficacy of viral reverse transcription. These data suggest that p21 can indirectly block HIV-1 reverse transcription by inhibiting host cofactors supporting HIV-1 replication and identify sites of viral vulnerability that are effectively targeted in persons with natural control of HIV-1 replication.

Development of a highly potent, selective, and cell-active inhibitor of cysteine cathepsin L-A hybrid design approach.

A hybrid-design approach is undertaken to develop a highly potent and selective inhibitor of human cathepsin L. Studies involving human breast carcinoma MDA-MB-231 cells establish that this inhibitor can successfully block intracellular cathepsin L activity, and retard the cell-migratory potential of these highly metastatic cells.

Tethered domains and flexible regions in tRNase Z(L), the long form of tRNase Z.

tRNase Z, a member of the metallo-ß-lactamase family, endonucleolytically removes the pre-tRNA 3' trailer in a step central to tRNA maturation. The short form (tRNase Z(S)) is the only one found in bacteria and archaebacteria and is also present in some eukaryotes. The homologous long form (tRNase Z(L)), exclusively found in eukaryotes, consists of related amino- and carboxy-domains, suggesting that tRNase Z(L) arose from a tandem duplication of tRNase Z(S) followed by interdependent divergence of the domains. X-ray crystallographic structures of tRNase Z(S) reveal a flexible arm (FA) extruded from the body of tRNase Z remote from the active site that binds tRNA far from the scissile bond. No tRNase Z(L) structures have been solved; alternative biophysical studies are therefore needed to illuminate its functional characteristics. Structural analyses of tRNase Z(L) performed by limited proteolysis, two dimensional gel electrophoresis and mass spectrometry establish stability of the amino and carboxy domains and flexibility of the FA and inter-domain tether, with implications for tRNase Z(L) function.

Design, synthesis, and evaluation of 2-(arylsulfonyl)oxiranes as cell-permeable covalent inhibitors of protein tyrosine phosphatases.

A structure-based design approach has been applied to develop 2-(arylsulfonyl)oxiranes as potential covalent inhibitors of protein tyrosine phosphatases. A detailed kinetic analysis of inactivation by these covalent inhibitors reveals that this class of compounds inhibits a panel of protein tyrosine phosphatases in a time- and dose-dependent manner, consistent with the covalent modification of the enzyme active site. An inactivation experiment in the presence of sodium arsenate, a known competitive inhibitor of protein tyrosine phosphatase, indicated that these inhibitors were active site bound. This finding is consistent with the mass spectrometric analysis of the covalently modified protein tyrosine phosphatase enzyme. Additional experiments indicated that these compounds remained inert toward other classes of arylphosphate-hydrolyzing enzymes, and alkaline and acid phosphatases. Cell-based experiments with human A549 lung cancer cell lines indicated that 2-(phenylsulfonyl)oxirane (1) caused an increase in intracellular pTyr levels in a dose-dependent manner thereby suggesting its cell-permeable nature. Taken together, the newly identified 2-(arylsulfonyl)oxiranyl moiety could serve as a novel chemotype for the development of activity-based probes and therapeutic agents against protein tyrosine phosphatase superfamily of enzymes.

Rapid isolation and identification of bacteriophage T4-encoded modifications of Escherichia coli RNA polymerase: a generic method to study bacteriophage/host interactions.

Bacteriophages are bacterial viruses that infect bacterial cells, and they have developed ingenious mechanisms to modify the bacterial RNA polymerase. Using a rapid, specific, single-step affinity isolation procedure to purify Escherichia coli RNA polymerase from bacteriophage T4-infected cells, we have identified bacteriophage T4-dependent modifications of the host RNA polymerase. We suggest that this methodology is broadly applicable for the identification of bacteriophage-dependent alterations of the host synthesis machinery.

Prediction of cyclin-dependent kinase phosphorylation substrates.

Protein phosphorylation, mediated by a family of enzymes called cyclin-dependent kinases (Cdks), plays a central role in the cell-division cycle of eukaryotes. Phosphorylation by Cdks directs the cell cycle by modifying the function of regulators of key processes such as DNA replication and mitotic progression. Here, we present a novel computational procedure to predict substrates of the cyclin-dependent kinase Cdc28 (Cdk1) in the Saccharomyces cerevisiae. Currently, most computational phosphorylation site prediction procedures focus solely on local sequence characteristics. In the present procedure, we model Cdk substrates based on both local and global characteristics of the substrates. Thus, we define the local sequence motifs that represent the Cdc28 phosphorylation sites and subsequently model clustering of these motifs within the protein sequences. This restraint reflects the observation that many known Cdk substrates contain multiple clustered phosphorylation sites. The present strategy defines a subset of the proteome that is highly enriched for Cdk substrates, as validated by comparing it to a set of bona fide, published, experimentally characterized Cdk substrates which was to our knowledge, comprehensive at the time of writing. To corroborate our model, we compared its predictions with three experimentally independent Cdk proteomic datasets and found significant overlap. Finally, we directly detected in vivo phosphorylation at Cdk motifs for selected putative substrates using mass spectrometry.

Analysis of protein phosphorylation by hypothesis-driven multiple-stage mass spectrometry.

We describe a strategy, which we term hypothesis-driven multiple-stage mass spectrometry (HMS-MS), for the sensitive detection and identification of phosphopeptides derived from enzymatic digests of phosphoproteins. In this strategy, we postulate that any or all of the potential sites of phosphorylation in a given protein may be phosphorylated. Using this assumption, we calculate the m/z values of all the corresponding singly charged phosphopeptide ions that could, in theory, be produced by the enzyme employed for proteolysis. We test ions at these m/z values for the presence of phosphoserine or phosphothreonine residues using tandem mass spectrometry (MS(2)) in a vacuum MALDI ion trap mass spectrometer, where the neutral loss of the elements of H(3)PO(4) (98 Da) provides a sensitive assay for the presence of phosphopeptides. Subsequent MS(3) analysis of the (M + H - 98)(+) peaks allows us to confirm or reject the hypotheses that the putative phosphopeptides are present in the sample. HMS-MS was successfully applied to the detection and identification of phosphopeptides from substrates of the Saccharomyces cerevisiae cyclin-dependent kinase (Cdk) Cdc28, phosphorylated in vitro (Ipl1) and in vivo (Orc6), basing hypothesis formation on the minimal Cdk consensus phosphorylation motif Ser/Thr-Pro. The method was also used to find in vitro phosphopeptides from a domain of the Drosophila melanogaster protein PERIOD, hypothesizing possible phosphorylations of all Ser/Thr residues without assuming a consensus motif. Our results demonstrate that HMS-MS is a sensitive, highly specific tool for systematically surveying proteins for Ser/Thr phosphorylation, and represents a significant step toward our goal of comprehensive phosphorylation mapping.

Targeted proteomic study of the cyclin-Cdk module.

The cell division cycle of the yeast S. cerevisiae is driven by one Cdk (cyclin-dependent kinase), which becomes active when bound to one of nine cyclin subunits. Elucidation of Cdk substrates and other Cdk-associated proteins is essential for a full understanding of the cell cycle. Here, we report the results of a targeted proteomics study using affinity purification coupled to mass spectrometry. Our study identified numerous proteins in association with particular cyclin-Cdk complexes. These included phosphorylation substrates, ubiquitination-degradation proteins, adaptors, and inhibitors. Some associations were previously known, and for others, we confirmed their specificity and biological relevance. Using a hypothesis-driven mass spectrometric approach, we also mapped in vivo phosphorylation at Cdk consensus motif-containing peptides within several cyclin-associated candidate Cdk substrates. Our results demonstrate that this approach can be used to detect a host of transient and dynamic protein associations within a biological module.