Synthesis of the Novel Covalent Cysteine Proteases Inhibitor with Iodoacetic Functional Group.

This work presents the synthesis of the novel covalent inhibitor of cysteine proteases where epoxide has been replaced by the iodoacetyl functional group. The molecule, similar in action to E-64 and DCG-04, the commonly applied inhibitors, is additionally biotinylated and contains tyrosyl iodination sites. The Fmoc solid phase synthesis has been applied. Conjugation of iodoacetic acid with the peptide was optimized by testing different conjugation agents. The purity of the final product was verified by mass spectrometry and its bioactivity was tested by incubation with a model cysteine protease-staphopain C. Finally, it was shown that the synthesized inhibitor binds to the protein at the ratio of 1:1. More detailed analysis by means of tandem mass spectrometry proved that the inhibitor binds to the cysteine present in the active site of the enzyme.

Changes in Protein Glycosylation as a Result of Aptamer Interactions with Cancer Cells.

PURPOSE: Based on the recent aptamer-related breast cancer studies, which indicate the therapeutic role of specific oligonucleotide sequences, experiments have been designed in an attempt to unravel the molecular targets of this mechanism. This article describes the study on glycoproteome changes in breast cancer cells as a result of their interactions with aptamers. EXPERIMENTAL DESIGN: Aberrations in protein glycosylation play an important role in tumorigenesis and influence cancer progression, metastasis, immunoresponse, and chemoresistance, therefore this study is focused on the identification of the alterations in glycan expression on the surface of proteins as a potential and innovative tool for biomedical applications of aptamers in cancer treatment. RESULTS: Two proteins, kinesin-like protein (KI13B) and proliferating cell nuclear antigen (PCNA), have been identified that carry N-glycan epitopes after conjugation with aptamer sequences. CONCLUSIONS AND CLINICAL RELEVANCE: Multiple features of aptamers as an alternative to protein antibodies are utilized for various biomedical applications ranging from biomarker discovery, bioimaging, targeted therapy, drug delivery, and drug pharmacokinetics and biodistribution. Frequently, aptamers bind to their target molecules and modulate their function. Such therapeutic aptamers can modify the biological pathways for treatment of many types of diseases, such as cancer.

IDE Degrades Nociceptin/Orphanin FQ through an Insulin Regulated Mechanism.

Insulin-degrading enzyme (IDE) was applied to catalyze hydrolysis of Nociceptin/Orphanin 1-16 (OFQ/N) to show the involvement of the enzyme in degradation of neuropeptides engaged in pain transmission. Moreover, IDE degradative action towards insulin (Ins) was inhibited by the OFQ/N fragments, suggesting a possible regulatory mechanism in the central nervous system. It has been found that OFQ/N and Ins affect each other degradation by IDE, although in a different manner. Indeed, while the digestion of OFQ/N is significantly affected by the presence of Ins, the kinetic profile of the Ins hydrolysis is not affected by the presence of OFQ/N. However, the main hydrolytic fragments of OFQ/N produced by IDE exert inhibitory activity towards the IDE-mediated Ins degradation. Here, we present the results indicating that, besides Ins, IDE cleaves neuropeptides and their released fragments act as inhibitors of IDE activity toward Ins. Having in mind that IDE is present in the brain, which also contains Ins receptors, it cannot be excluded that this enzyme indirectly participates in neural communication of pain signals and that neuropeptides involved in pain transmission may contribute to the regulation of IDE activity. Finally, preliminary results on the metabolism of OFQ/N, carried out in the rat spinal cord homogenate in the presence of various inhibitors specific for different classes of proteases, show that OFQ/N proteolysis in rat spinal cord could be due, besides IDE, also to a cysteine protease not yet identified.

The kisspeptin derivative kissorphin reduces the acquisition, expression, and reinstatement of ethanol-induced conditioned place preference in rats.

Research has shown that opioids are involved in the rewarding effects of ethanol. Neuropeptide FF (NPFF) has been described as an anti-opioid peptide because, in many cases, it inhibits opioid and ethanol effects in rodents. Kissorphin (KSO) is a new peptide derived from kisspeptin-10 with structural similarities to NPFF. This peptide possesses NPFF-like biological activity in vitro. The aim of the current study was to investigate whether KSO (Tyr-Asn-Trp-Asn-Ser-Phe-NH2) influences the acquisition, expression, and reinstatement of ethanol-induced conditioned place preference (ethanol-CPP) in rats. The ethanol-CPP was established (conditioning for 5 days) by intraperitoneal (i.p.) administration of ethanol (1 g/kg, 20%, w/v) using an unbiased procedure. After that, one group of rats was used in final post-conditioning testing (expression of CPP) and the other group received a priming injection of ethanol after 10 days of extinction (reinstatement of CPP). Our experiments showed that KSO, given intravenously (i.v.) at the doses of 1, 3, and 10 nmol before every ethanol administration, inhibited the acquisition and, given acutely before the post-conditioning test or before the priming dose of ethanol, inhibited the expression and reinstatement of ethanol-CPP, respectively, in a dose-dependent manner. KSO given by itself neither induced place preference nor aversion and did not alter locomotor activity and coordination of rats. These results suggest that KSO can alter rewarding/motivational effects of ethanol. These data suggest this peptide possesses an anti-opioid character.

Advances in the Study of Aptamer-Protein Target Identification Using the Chromatographic Approach.

Ever since the development of the process known as the systematic evolution of ligands by exponential enrichment (SELEX), aptamers have been widely used in a variety of studies, including the exploration of new diagnostic tools and the discovery of new treatment methods. Aptamers' ability to bind to proteins with high affinity and specificity, often compared to that of antibodies, enables the search for potential cancer biomarkers and helps us understand the mechanisms of carcinogenesis. The blind spot of those investigations is usually the difficulty in the selective extraction of targets attached to the aptamer. There are many studies describing the cell SELEX for the prime choice of aptamers toward living cancer cells or even whole tumors in the animal models. However, a dilemma arises when a large number of proteins are being identified as potential targets, which is often the case. In this article, we present a new analytical approach designed to selectively target proteins bound to aptamers. During studies, we have focused on the unambiguous identification of the molecular targets of aptamers characterized by high specificity to the prostate cancer cells. We have compared four assay approaches using electrophoretic and chromatographic methods for "fishing out" aptamer protein targets followed by mass spectrometry identification. We have established a new methodology, based on the fluorescent-tagged oligonucleotides commonly used for flow-cytometry experiments or as optic aptasensors, that allowed the detection of specific aptamer-protein interactions by mass spectrometry. The use of atto488-labeled aptamers for the tracking of the formation of specific aptamer-target complexes provides the possibility of studying putative protein counterparts without needing to apply enrichment techniques. Significantly, changes in the hydrophobic properties of atto488-labeled aptamer-protein complexes facilitate their separation by reverse-phase chromatography combined with fluorescence detection followed by mass-spectrometry-based protein identification. These comparative results of several methodological approaches confirmed the universal applicability of this method to studying aptamer-protein interactions with high sensitivity, showing superior properties compared with pull-down techniques.

The new kisspeptin derivative - kissorphin (KSO) - attenuates acute hyperlocomotion and sensitization induced by ethanol and morphine in mice.

Kissorphin (KSO) is a new peptide derived from kisspeptin-10. This peptide possesses neuropeptide FF (NPFF)-like biological activity in vitro; NPFF, in many cases, inhibits opioid and ethanol effects in rodents. Therefore, the current study explored the influence of KSO on acute ethanol- and morphine-induced hyperactivity, and on the development and expression of locomotor sensitization induced by these drugs. In the present study, sensitization to locomotor effects was induced by repeated exposure to ethanol (2.4 g/kg, intraperitoneally [i.p.], 1 × 4 days) or morphine (10 mg/kg, subcutaneously [s.c.], 1 × 7 days). We found that KSO (1-10 nmol/300 µL, intravenously [i.v.]) did not have an impact on locomotor activity of naïve mice. However, it reduced both acute ethanol- (10 nmol/300 µL) and morphine-induced hyperactivity (3 and 10 nmol/300 µL). Pretreatment of animals with KSO (10 nmol/300 µL), before every ethanol or morphine injection during development of sensitization or before the ethanol or morphine challenge, attenuated the development, as well as the expression of locomotor sensitization to both substances. Moreover, prior administration of the NPFF receptor antagonist RF9 (10 nmol/300 µL, i.v.) inhibited the ability of KSO (10 nmol/300 µL) to reduce the expression of ethanol and morphine sensitization. KSO given alone, at all used doses, did not influence the motor coordination measured via the rotarod test. The results from this study show that KSO effectively attenuated acute and repeated effects of ethanol and morphine. Thus, KSO possesses NPFF-like anti-opioid activity in these behavioral studies.

Glycosylation Changes in Serum Proteins Identify Patients with Pancreatic Cancer.

After more than a decade of biomarker discovery using advanced proteomic and genomic approaches, very few biomarkers have been involved in clinical diagnostics. Most candidate biomarkers are focused on the protein component. Targeting post-translational modifications (PTMs) in combination with protein sequences will provide superior diagnostic information with regards to sensitivity and specificity. Glycosylation is one of the most common and functionally important PTMs. It plays a central role in many biological processes, including protein folding, host-pathogen interactions, immune response, and inflammation. Cancer-associated aberrant glycosylation has been identified in various types of cancer. Expression of cancer-specific glycan epitopes represents an excellent opportunity for diagnostics and potentially specific detection of tumors. Here, we report four proteins (LIFR, CE350, VP13A, HPT) found in sera from pancreatic cancer patients carrying aberrant glycan structures as compared to those of controls.

Flowing atmospheric pressure afterglow combined with laser ablation for direct analysis of compounds separated by thin-layer chromatography.

A thin-layer chromatography-mass spectrometry (TLC-MS) setup for characterization of low molecular weight compounds separated on standard TLC plates has been constructed. This new approach successfully combines TLC separation, laser ablation, and ionization using flowing atmospheric pressure afterglow (FAPA) source. For the laser ablation, a low-priced 445-nm continuous-wave diode laser pointer, with a power of 1 W, was used. The combination of the simple, low-budget laser pointer and the FAPA ion source has made this experimental arrangement broadly available, also for small laboratories. The approach was successfully applied for the characterization of low molecular weight compounds separated on TLC plates, such as a mixture of pyrazole derivatives, alkaloids (nicotine and sparteine), and an extract from a drug tablet consisting of paracetamol, propyphenazone, and caffeine. The laser pointer used was capable of ablating organic compounds without the need of application of any additional substances (matrices, staining, etc.) on the TLC spots. The detection limit of the proposed method was estimated to be 35 ng/cm(2) of a pyrazole derivative.

Insulin/IGF1-PI3K-dependent nucleolar localization of a glycolytic enzyme--phosphoglycerate mutase 2, is necessary for proper structure of nucleolus and RNA synthesis.

Phosphoglycerate mutase (PGAM), a conserved, glycolytic enzyme has been found in nucleoli of cancer cells. Here, we present evidence that accumulation of PGAM in the nucleolus is a universal phenomenon concerning not only neoplastically transformed but also non-malignant cells. Nucleolar localization of the enzyme is dependent on the presence of the PGAM2 (muscle) subunit and is regulated by insulin/IGF-1-PI3K signaling pathway as well as drugs influencing ribosomal biogenesis. We document that PGAM interacts with several 40S and 60S ribosomal proteins and that silencing of PGAM2 expression results in disturbance of nucleolar structure, inhibition of RNA synthesis and decrease of the mitotic index of squamous cell carcinoma cells. We conclude that presence of PGAM in the nucleolus is a prerequisite for synthesis and initial assembly of new pre-ribosome subunits.

Relevance of the poly(ethylene glycol) linkers in peptide surfaces for proteases assays.

Poly(ethylene glycol)s (PEGs) with different lengths were used as linkers during the preparation of peptide surfaces for protease detection. In the first approach, the PEG monolayers were prepared using a "grafting to" method on 3-aminopropyltrietoxysilane (APTES)-modified silicon wafers. Protected peptides with a fluorescent marker were synthesized by Fmoc solid phase synthesis. The protected peptide structures enabled their site-specific immobilization onto the PEG surfaces. Alternatively, the PEG-peptide surface was obtained by immobilizing a PEG-peptide conjugate directly onto the modified silicon wafer. The surfaces (composition, grafting density, hydrophilicity, and roughness) were characterized by time-of-flight-secondary ion mass spectrometry (ToF-SIMS), X-ray photoelectron spectroscopy (XPS), contact angle (CA), and atomic force microscopy (AFM). Introducing the PEG linker between the peptide and surface increased their resistance toward nonspecific protein adsorption. The peptide surfaces were examined as analytical platforms to study the action of trypsin as a representative protease. The products of the enzymatic hydrolysis were analyzed by fluorescence spectroscopy, electrospray ionization-mass spectrometry (ESI-MS), and ToF-SIMS. Conclusions about the optimal length of the PEG linker for the analytical application of PEG-peptide surfaces were drawn. This work demonstrates an effective synthetic procedure to obtain PEG-peptide surfaces as attractive platforms for the development of peptide microarrays.

A comparative study of glycoproteomes in androgen-sensitive and -independent prostate cancer cell lines.

Prostate cancer is one of the most common malignancies in men and is predicted to be the second leading cause of cancer-related deaths. After 6-18 months, hormone ablation treatment results in androgen-independent growth of cancer cells, metastasis and progression. The mechanism of androgen-independent growth of prostatic carcinoma cells is still unknown. Identification of factors that facilitate the transition from androgen-dependent to independent states is crucial in designing future diagnostics and medication strategies. To understand the biochemical meaning of hormone dependency deprivation, glycoproteins enriched profiles were compared between DU145 (hormone non-responding) and LNCaP (hormone responding) prostate cancer cells. These results allow for anticipation on the important role of glycosylation in malignant transformation. Both Tn antigen and complex antennary N-oligosaccharides were recognized. Their occurrence might be involved in the development and progression of tumor, and failure of hormone ablation therapy. Among identified proteins in androgen-sensitive cells nucleolin (P19338) was found that is widely described as apoptosis inhibitor, and also transporter of molecules from the membrane to the cytoplasm or nucleus. In addition, 14-3-3 protein family (P27348, P31946, P61981, P63104, P62258, Q04917, and P31947) was investigated across available databases as it forms stable complexes with glycoproteins. Our studies indicate that isoforms: sigma and eta were found in androgen-dependent prostate cancer cells, while other isoforms were present in androgen non-responding cells. 14-3-3 binding partners are involved in cancer pathogenesis. These findings may contribute to a better understanding of prostate cancer tumorigenesis and to a more efficient prognosis and individual therapy in a future. However, it still remains to be revealed how important those changes are for androgen dependency loss in prostate cancer patients carried out on clinically relevant populations.

Synthesis and characterisation of PEG-peptide surfaces for proteolytic enzyme detection.

Peptide surfaces were obtained by the covalent immobilisation of fluorescently labelled pentapeptides carboxyfluorescein-glycine-arginine-methionine-leucine-glycine, either directly or through a poly(ethylene glycol) (PEG) linker on modified silicon wafers. Each step during the preparation of the peptide surfaces was confirmed by several surface characterisation techniques. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy were used to determine the surface composition, the wafers philicity was measured by contact angle and atomic force microscopy was used to investigate the surface morphology. Exposure of the peptide surfaces to trypsin resulted in the release of a fluorescently labelled peptide product, which allowed the kinetics of the enzymatic reaction to be followed with the aid of fluorescence spectroscopy. The electrospray ionisation mass spectrometry analysis of the post-digestion solution confirmed that the pentapeptides attached to the solid support undergo specific trypsin hydrolysis at the C-terminus of the arginine residues. Detailed surface analyses before and after the enzyme action was performed using ToF-SIMS. Because of the limited accessibility of the short peptide directly attached to the surface, a quantitative yield of enzymatic hydrolysis was observed only in case when the peptide was bound through the PEG linker. The insertion of the PEG linker increased the number of immobilised peptides and the rate of enzymatic digestion which consequently improved the quality of the enzyme assays. The described approach may be used for different peptide sequences designed for other proteases.

Phenylmethanesulfonyl fluoride, a serine protease inhibitor, suppresses naloxone-precipitated withdrawal jumping in morphine-dependent mice.

We have previously shown that intracerebroventricular (i.c.v.) administration of cysteine protease inhibitors suppresses naloxone-precipitated withdrawal jumping in morphine-dependent mice, presumably through the inhibition of dynorphin degradation (see (Tan-No, K., Sato, T., Shimoda, M., Nakagawasai, O., Niijima, F., Kawamura, S., Furuta, S., Sato, T., Satoh, S., Silberring, J., Terenius, L., Tadano, T., 2010. Suppressive effects by cysteine protease inhibitors on naloxone-precipitated withdrawal jumping in morphine-dependent mice. Neuropeptides 44, 279-283)). In the present study, we examined the effect of phenylmethanesulfonyl fluoride (PMSF), a serine protease inhibitor, on naloxone-precipitated withdrawal jumping in morphine-dependent mice. The doses of morphine (mg/kg per injection) were subcutaneously given twice daily for 2 days [day 1 (30) and day 2 (60)]. On day 3, naloxone (8 mg/kg) was intraperitoneally administered 3h after the final injection of morphine (60 mg/kg), and the number of jumps was immediately recorded for 20 min. Naloxone-precipitated withdrawal jumping was significantly suppressed by i.c.v. administration of PMSF (4 nmol), given 5 min before each morphine treatment during the induction phase, with none given on the test day. The expression of tissue plasminogen activator (tPA), a serine protease that converts plasminogen to plasmin, in the prefrontal cortex was significantly increased in morphine-dependent and -withdrawal mice, as compared with saline-treated mice. Moreover, trans-4-(aminomethyl)-cyclohexanecarboxylic acid (300 pmol), an antiplasmin agent, and (Tyr(1))-thrombin receptor activating peptide 7 (0.45 and 2 nmol), an antagonist of protease activated receptor-1 (PAR-1), significantly suppressed naloxone-precipitated withdrawal jumping. The present results suggest that PMSF suppresses naloxone-precipitated withdrawal jumping in morphine-dependent mice, presumably through the inhibition of activities of tPA and plasmin belonging to the serine proteases family, which subsequently activates PAR-1.

Dynorphin convertases and their functions in CNS.

Neuropeptides play crucial, mediatory roles in many processes that occur in both CNS and PNS. The commonly accepted dogma for the release of bioactive peptides involves cleavage of inactive precursor by sequential action of proteinases recognizing dibasic stretches, followed by truncation of C-terminal Arg/Lys by the carboxypeptidase-like enzyme(s). Dynorphin convertases play an important role in CNS by regulating dynorphins level and also releasing enkephalins, thus maintaining a balance between these neuropeptides and their distinct functions (dynorphins are preferentially bound to kappa receptors and enkephalins are directed toward delta receptors). Knowledge on the cleavage fragments of dynorphins is important for understanding the pharmacological activity of these peptides. As some new data emerged in the literature, we would like to update recent achievements and progress concerning functions of such convertases, inhibitors, and their potential role in future pharmacotherapy.

Myeloperoxidase-catalyzed oxidative inactivation of human kininogens: the impairment of kinin-precursor and prekallikrein-binding functions.

Bradykinin-related vasoactive peptides (kinins) are important mediators of local and systemic inflammatory reactions. However, at local inflammatory foci, the production of kinins from proteinaceous precursors (kininogens) can be affected by reactive oxygen species released by phagocyte cells. One of the predominant oxidants at these places is hypochlorous acid which is formed from hydrogen peroxide and chloride ions by neutrophil myeloperoxidase. In this study, inactivation of human kininogens after oxidation with the myeloperoxidase-H2O2-chloride system was observed and analyzed by protein chemistry methods. The kinin release from oxidized kininogens by major kinin-producing enzymes, plasma and tissue kallikreins, proceed with a very low rate. This effect was assigned to apparent inability of kallikreins to process the kinin N-terminus owing to the conversion of the adjacent Met-361 residue to methionine sulfoxide. Additionally, the oxidized high-molecular mass kininogen lost its natural ability to bind plasma prekallikrein. This effect was assigned to the oxidation of Trp-569 residue within the prekallikrein-binding region which is subsequently destructed owing to cleavage of the peptide bond after that residue. One possible pathophysiological consequence of the described effects on kininogens could be the impairment of the normal assembly and triggering of the kinin-forming system on defense cell surfaces.

Suppressive effects by cysteine protease inhibitors on naloxone-precipitated withdrawal jumping in morphine-dependent mice.

The effects of various protease inhibitors on naloxone-precipitated withdrawal jumping were examined in morphine-dependent mice. The doses of morphine were subcutaneously given twice daily for 2 days (day 1, 30 mg/kg; day 2, 60 mg/kg). On day 3, naloxone (8 mg/kg) was intraperitoneally administered 3h after final injection of morphine (60 mg/kg), and the number of jumping was immediately recorded for 20 min. Naloxone-precipitated withdrawal jumping was significantly suppressed by the intracerebroventricular administration of N-ethylmaleimide (0.5 nmol) and Boc-Tyr-Gly-NHO-Bz (0.4 nmol), inhibitors of cysteine proteases involved in dynorphin degradation, 5 min before each morphine treatment during the induction phase, with none given on the test day, as well as by dynorphin A (62.5 pmol) and dynorphin B (250 pmol). However, amastatin, an aminopeptidase inhibitor, phosphoramidon, an endopeptidase 24.11 inhibitor, and captopril, an angiotensin-converting enzyme inhibitor, caused no changes. The present results suggest that cysteine protease inhibitors suppress naloxone-precipitated withdrawal jumping in morphine-dependent mice, presumably through the inhibition of dynorphin degradation.

The new face of nucleolin in human melanoma.

Nucleolin is multifunctional protein mainly present in nucleoli but also detected in cytoplasm and plasma membranes. Extranuclear nucleolin differs from the nuclear form by its glycosylation. Studies on expression of nucleolin in breast cancer suggest a possible association to the metastatic cascade. In the present study, Vicia villosa lectin (VVL) precipitation followed by subsequent polyacrylamide gel electrophoresis and mass spectrometry analysis demonstrates nucleolin as a VVL-positive glycoprotein expressed in melanoma. The presence of VVL-positive nucleolin in the melanoma cell membrane and cytoplasm was confirmed by confocal microscopy. Using bioinformatic peptide prediction programs, nucleolin was shown to contain multiple possible MHC class-I binding peptides in its sequence which makes nucleolin an interesting melanoma marker and target for immunodiagnostic and possibly therapeutic purposes.

Cysteine protease inhibitors suppress the development of tolerance to morphine antinociception.

The effects of various protease inhibitors on the development of antinociceptive tolerance to morphine were examined in mice. Intrathecal (i.t.) administration of morphine (0.01-1 nmol) produced a dose-dependent and significant antinociceptive effect in the 0.5% formalin test. When the doses of morphine (mg/kg, s.c. per injection) were given as pretreatment twice daily for two days [first day (30) and second day (60)], i.t. administration of morphine (0.1 nmol) was inactive due to antinociceptive tolerance on the third day. Tolerance to i.t. morphine was significantly suppressed by the i.t. injection of N-ethylmaleimide or Boc-Tyr-Gly-NHO-Bz, inhibitors of cysteine proteases involved in dynorphin degradation, as well as by dynorphin A, dynorphin B and (-) U-50,488, a selective kappa-opioid receptor agonist. On the other hand, amastatin, an aminopeptidase inhibitor, phosphoramidon, an endopeptidase 24.11 inhibitor, lisinopril, an angiotensin-converting enzyme inhibitor, and phenylmethanesulfonyl fluoride, a serine protease inhibitor, were inactive. These results suggest that cysteine protease inhibitors suppress the development of morphine tolerance presumably through the inhibition of dynorphin degradation.

A novel cryptic peptide derived from the rat neuropeptide FF precursor reverses antinociception and conditioned place preference induced by morphine.

Neuropeptide FF (NPFF) precursors from different species contain at least three known neuropeptides, i.e. FF (FLFQPQRF-NH(2)), AF (AGEGLSSPFWSLAAPQR-NH(2)) and SF (SLAAPQRF-NH(2)). We demonstrate that the rat NPFF precursor contains another bioactive sequence, NAWGPWSKEQLSPQA, spanning between positions 85 and 99. Synthetic NPFF precursor (85-99) (10 and 20 nmol, i.c.v.) blocked the expression of conditioned place preference induced by morphine (5 mg/kg, s.c.). This peptide alone (10 and 20 nmol, i.c.v.) had no influence on the baseline latency of a nociceptive reaction but reversed the antinociceptive activity of morphine (5 mg/kg, s.c.) in the tail-immersion test in rats. These data suggest the existence of a novel bioactive cryptic peptide within an already known NPFF precursor.