Vibrational Circular Dichroism Sheds New Light on the Competitive Effects of Crowding and ß-Synuclein on the Fibrillation Process of α-Synuclein.

The effects of crowding, using the crowding agent Ficoll 70, and the presence of ß-synuclein on the fibrillation process of α-synuclein were studied by spectroscopic techniques, transmission electron microscopy, and thioflavin T assays. This combined approach, in which all techniques were applied to the same original sample, generated an unprecedented understanding of the effects of these modifying agents on the morphological properties of the fibrils. Separately, crowding gives rise to shorter mutually aligned fibrils, while ß-synuclein leads to branched, short fibrils. The combination of both effects leads to short, branched, mutually aligned fibrils. Moreover, it is shown that the nondestructive technique of vibrational circular dichroism is extremely sensitive to the length and the higher-order morphology of the fibrils.

Prolyl endopeptidase is involved in the degradation of neural cell adhesion molecules in vitro.

Membrane-associated glycoprotein neural cell adhesion molecule (NCAM) and its polysialylated form (PSA-NCAM) play an important role in brain plasticity by regulating cell-cell interactions. Here, we demonstrate that the cytosolic serine protease prolyl endopeptidase (PREP) is able to regulate NCAM and PSA-NCAM. Using a SH-SY5Y neuroblastoma cell line with stable overexpression of PREP, we found a remarkable loss of PSA-NCAM, reduced levels of NCAM180 and NCAM140 protein species, and a significant increase in the NCAM immunoreactive band migrating at an apparent molecular weight of 120 kDa in PREP-overexpressing cells. Moreover, increased levels of NCAM fragments were found in the concentrated medium derived from PREP-overexpressing cells. PREP overexpression selectively induced an activation of matrix metalloproteinase-9 (MMP-9), which could be involved in the observed degradation of NCAM, as MMP-9 neutralization reduced the levels of NCAM fragments in cell culture medium. We propose that increased PREP levels promote epidermal growth factor receptor (EGFR) signaling, which in turn activates MMP-9. In conclusion, our findings provide evidence for newly-discovered roles for PREP in mechanisms regulating cellular plasticity through NCAM and PSA-NCAM.

Study of the Conformational Dynamics of Prolyl Oligopeptidase by Mass Spectrometry: Lessons Learned.

Ion mobility mass spectrometry (IM-MS) can be used to analyze native proteins according to their size and shape. By sampling individual molecules, it allows us to study mixtures of conformations, as long as they have different collision cross sections and maintain their native conformation after dehydration and vaporization in the mass spectrometer. Even though conformational heterogeneity of prolyl oligopeptidase has been demonstrated in solution, it is not detectable in IM-MS. Factors that affect the conformation in solution, binding of an active site ligand, the stabilizing Ser554Ala mutation, and acidification do not qualitatively affect the collision-induced unfolding pattern. However, measuring the protection of accessible cysteines upon ligand binding provides a principle for the development of MS-based ligand screening methods.

In Vitro and In Situ Activity-Based Labeling of Fibroblast Activation Protein with UAMC1110-Derived Probes.

Fibroblast activation protein (FAP) is a proline-selective protease that belongs to the S9 family of serine proteases. It is typically highly expressed in the tumor microenvironment (TME) and especially in cancer-associated fibroblasts, the main cell components of the tumor stroma. The exact role of its enzymatic activity in the TME remains largely unknown. Hence, tools that enable selective, activity-based visualization of FAP within the TME can help to unravel FAP's function. We describe the synthesis, biochemical characterization, and application of three different activity-based probes (biotin-, Cy3-, and Cy5-labeled) based on the FAP-inhibitor UAMC1110, an in-house developed molecule considered to be the most potent and selective FAP inhibitor available. We demonstrate that the three probes have subnanomolar FAP affinity and pronounced selectivity with respect to the related S9 family members. Furthermore, we report that the fluorescent Cy3- and Cy5-labeled probes are capable of selectively detecting FAP in a cellular context, making these chemical probes highly suitable for further biological studies. Moreover, proof of concept is provided for in situ FAP activity staining in patient-derived cryosections of urothelial tumors.

Integrated proteomic analysis reveals a substantial enrichment of protein trafficking processes in hippocampus tissue after hypoxic stress.

Acute and chronic hypoxic episodes of the brain have been generally recognized as a common denominator of several neuropathologies of which cerebral ischemic stroke represents one of the leading causes of mortality and morbidity. In an attempt to clarify the plethora of molecular events elicited by ischemic or hypoxic stress, several studies have reported before on large-scale expression analysis; however, only a minority have put focus on proteome based changes. To further enrich our knowledge, we investigated the differences in protein levels following prolonged exposure of mice to global hypoxic stress in the hippocampus, one of the most susceptible regions of the brain. This was accomplished using the conventional 2-DE approach and peptide-centered quantitative methionyl-COFRADIC. Together both methods resulted in the identification of 110 unique hypoxia regulated proteins, and 34 posthypoxic reoxygenation regulated proteins based on 2-DE analysis alone. The integration and comparison of the implicated biological functions with other large-scale analyses of similar hypoxia and ischemic stroke models gave an overall resemblance of implicated biological processes apart from model specific alterations in distribution. Nevertheless, further examination of the data clearly depicted a substantial enrichment of protein trafficking and targeting processes in our data which could be related to synaptic plasticity and remodeling events.

Metal ions shape α-synuclein.

α-Synuclein is an intrinsically disordered protein that can self-aggregate and plays a major role in Parkinson's disease (PD). Elevated levels of certain metal ions are found in protein aggregates in neurons of people suffering from PD, and environmental exposure has also been linked with neurodegeneration. Importantly, cellular interactions with metal ions, particularly Ca2+, have recently been reported as key for α-synuclein's physiological function at the pre-synapse. Here we study effects of metal ion interaction with α-synuclein at the molecular level, observing changes in the conformational behaviour of monomers, with a possible link to aggregation pathways and toxicity. Using native nano-electrospray ionisation ion mobility-mass spectrometry (nESI-IM-MS), we characterize the heterogeneous interactions of alkali, alkaline earth, transition and other metal ions and their global structural effects on α-synuclein. Different binding stoichiometries found upon titration with metal ions correlate with their specific binding affinity and capacity. Subtle conformational effects seen for singly charged metals differ profoundly from binding of multiply charged ions, often leading to overall compaction of the protein depending on the preferred binding sites. This study illustrates specific effects of metal coordination, and the associated electrostatic charge patterns, on the complex structural space of the intrinsically disordered protein α-synuclein.

The proteome of the human neuroblastoma cell line SH-SY5Y: an enlarged proteome.

The human neuroblastoma cell line SH-SY5Y (ATCC: CRL-2266) is widely used as a neural cellular model system. The hitherto existing proteome data (115 proteins) are here extended. A total of 1103 unique proteins of this cell line were identified using 2D-LC combined with MALDI-TOF/TOF-MS, SDS-PAGE with nano-LC-MS/MS, N-terminal COFRADIC analysis with nano-LC-MS/MS and 2D-PAGE with MALDI-TOF/TOF-MS peptide mass fingerprinting. The obtained proteome profile of this cell line is discussed.

Spatiotemporal expression and inhibition of prolyl oligopeptidase contradict its involvement in key pathologic mechanisms of kainic acid-induced temporal lobe epilepsy in rats.

OBJECTIVE: Prolyl oligopeptidase (PREP) has been implicated in neuroinflammatory processes and neuroplasticity and has been suggested as a target for the treatment of neurodegenerative disease. The aim of this investigation was to explore the involvement of PREP in the neuropathologic mechanisms relevant to temporal lobe epilepsy (TLE) using a PREP inhibitor in a well-established rat model. METHODS: PREP activity and expression was studied in Sprague-Dawley rats 2 and 12 weeks following kainic acid-induced status epilepticus (KASE). Continuous video-electroencephalography monitoring was performed for 2 weeks in the 12-week cohort to identify a relationship of PREP expression/activity with epileptic seizures. In addition, the animals included in the 2-week time point were treated with a specific inhibitor of PREP, KYP-2047, or saline continuously, starting immediately after SE. PREP activity and its expression were analyzed in rat brain by using enzyme kinetics and western blot. In addition, markers for microglial activation, astrogliosis, cell loss, and cell proliferation were evaluated. RESULTS: Enzymatic activity of PREP was unchanged following induction of SE after 2 and 12 weeks in rats. PREP activity in epileptic rats did not relate to the number of seizures/day at the 12-week time point. Moreover, continuous inhibition of PREP for 2 weeks after KASE did not alter the SE-mediated neuroinflammatory response, cell loss, or cell proliferation in the hippocampal subgranule zone measured at the 2-week time point. SIGNIFICANCE: PREP inhibition does not affect key pathologic mechanisms, including activation of glial cells, cell loss, and neural progenitor cell proliferation, in this KASE model of TLE. The results do not support a direct role of PREP in seizure burden during the chronic epilepsy period in this model.

The development and validation of a combined kinetic fluorometric activity assay for fibroblast activation protein alpha and prolyl oligopeptidase in plasma.

BACKGROUND: Fibroblast activiation protein alpha (FAP) is considered a diagnostic and prognostic biomarker for various types of cancer. FAP shares substrate specificity with prolyl oligopeptidase (PREP), studied in (neuro)inflammation and neurodegeneration as well as cancer. Current assays inadequately discriminate between FAP and PREP and there is need for an assay that reliably quantitates the FAP/PREP activity ratio in plasma. METHODS: FAP and PREP activities were measured in human EDTA-plasma in presence of well characterized PREP and FAP inhibitors. RESULTS: A combined kinetic assay was developed in conditions to optimally measure FAP as well as PREP activity with Z-Gly-Pro-AMC as substrate. Limit of detection was 0.009 U/L and limit of quantitation was 0.027 U/L for the combined FAP-PREP assay. Within-run coefficient of variation was 3% and 4% and between-run precision was 7% and 12% for PREP and FAP, respectively. Accuracy was demonstrated by comparison with established end-point assays. Hemolysis interferes with the assay with 1.5 g/L hemoglobin as cut-off value. PREP (but not FAP) activity can increase upon lysis of platelets and red blood cells during sample preparation. CONCLUSION: With this new assay, on average 67% of the Z-Gly-Pro-AMC converting activity in plasma can be attributed to FAP.

Expression profiling of the cerebral ischemic and hypoxic response.

In mammals, the major component in energy production is molecular oxygen. This has led to the development of several elaborate strategies that tightly regulate oxygen homeostasis in order to allow appropriate cell function and survival. However, a sustained drop in oxygen supply to neuronal tissue has detrimental consequences to cell functioning and survival. Disturbances in oxygen supply have been implicated in a number of CNS disorders that can be related to hypoxia or ischemia. On a cellular level, oxygen-deprived stress induces a multitude of spatially and temporally regulated responses, ranging from adapted channel activity to altered gene expression. Global analysis of expression changes over several time points and tissue regions or cells has already shed light on previous known and unknown biological processes and molecules. By combining knowledge from several different expression-profiling studies into one database, the first steps are made in unifying and categorizing the molecular response to oxygen-deprived conditions, such as stroke. In this review, some of the queries that can be extracted from the database are discussed in regard to the biological context.

Crystal structure of Porphyromonas gingivalis dipeptidyl peptidase 4 and structure-activity relationships based on inhibitor profiling.

The Gram-negative anaerobe Porphyromonas gingivalis is associated with chronic periodontitis. Clinical isolates of P. gingivalis strains with high dipeptidyl peptidase 4 (DPP4) expression also had a high capacity for biofilm formation and were more infective. The X-ray crystal structure of P. gingivalis DPP4 was solved at 2.2 Å resolution. Despite a sequence identity of 32%, the overall structure of the dimer was conserved between P. gingivalis DPP4 and mammalian orthologues. The structures of the substrate binding sites were also conserved, except for the region called S2-extensive, which is exploited by specific human DPP4 inhibitors currently used as antidiabetic drugs. Screening of a collection of 450 compounds as inhibitors revealed a structure-activity relationship that mimics in part that of mammalian DPP9. The functional similarity between human and bacterial DPP4 was confirmed using 124 potential peptide substrates.

In situ prolyl oligopeptidase activity assay in neural cell cultures.

Prolyl oligopeptidase (PREP, E.C.3.4.21.26) is a cytosolic serine protease that hydrolyzes small (<3 kDa), proline-containing peptides on the carboxyl terminal side of proline residues, and is widely distributed in the brain. High PREP activity, due to aging or neurodegenerative disease, has been hypothesised to lead to an increased breakdown of neuropeptides, resulting in a decline of cognitive functions and an acceleration of neurodegeneration. Recent data have suggested that PREP involvement in neurodegeneration cannot be explained by its extracellular space proteolytic activity alone, but may involve intracellular PREP activities as well. In order to test this, appropriate methods for measuring PREP intracellular activity must first be developed. In the present study, we developed and validated an in situ PREP intracellular activity assay in primary rat cortical neurons, using nitroblue tetrazolium chloride salt (NBT) and a PREP specific substrate (S)-benzyl 2-(2-(4-hydroxynaphthalen-l-ylcarbanoyl)pyrrolidin-l-yl)-2-oxoethylcarbamate (UAMC-00682). This novel in situ PREP activity assay was further validated in neuroblastoma SH-SY5Y cells, under conditions of PREP overexpression and inhibited PREP expression. Using this assay, we demonstrated that PREP inhibitors, Z-Pro-Pro-aldehyde-dimethylacetal, Boc-Asn-Phe-Pro-aldehyde, and (S)-1-((S)-1-(4-phenylbutanoyl)-pyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile (KYP-2047), were able to inhibit intracellular PREP activity in primary rat cortical neurons. KYP-2047 was the most potent PREP inhibitor in all assay systems tested. The validated assay enables localization and quantification of in situ PREP activity in primary rat cortical neurons and neuroblastoma SH-SY5Y cells, as well allows testing cell permeability and efficiency of novel PREP inhibitors.

P2-substituted N-acylprolylpyrrolidine inhibitors of prolyl oligopeptidase: biochemical evaluation, binding mode determination, and assessment in a cellular model of synucleinopathy.

We have investigated the effect of regiospecifically introducing substituents in the P2 part of the typical dipeptide derived basic structure of PREP inhibitors. This hitherto unexplored modification type can be used to improve target affinity, selectivity, and physicochemical parameters in drug discovery programs focusing on PREP inhibitors. Biochemical evaluation of the produced inhibitors identified several substituent types that significantly increase target affinity, thereby reducing the need for an electrophilic "warhead" functionality. Pronounced PREP specificity within the group of Clan SC proteases was generally observed. Omission of the P1 electrophilic function did not affect the overall binding mode of three representative compounds, as studied by X-ray crystallography, while the P2 substituents were demonstrated to be accommodated in a cavity of PREP that, to date, has not been probed by inhibitors. Finally, we report on results of selected inhibitors in a SH-SY5Y cellular model of synucleinopathy and demonstrate a significant antiaggregation effect on α-synuclein.

Structure and function relationship in prolyl oligopeptidase.

Prolyl oligopeptidase (POP) belongs to a unique class of serine proteases. Based on extensive enzyme kinetic measurements it has become clear that POP acts in a multifaceted way. This is reflected in the complex behavior in different reaction conditions with different substrates. Also the typical structural architecture of POP, with the active site located at the interface of the catalytic domain and the ß-propeller domain, has instigated many researchers to speculate about the mechanism of functioning. The latest developments support the idea of extended conformational changes upon substrate binding. In this review the plethora of available information is assembled into a coherent and stepwise description of the structural composition of POP. In one aspect the composition and contribution of structural boundaries at the active site are described. Attention is focused on the catalytic components and the features that are presumed to confine the substrate specificity. Complementary to this, the specificity of POP towards the residues surrounding the scissile bond is described by means of a consecutive evaluation of the preferred physico-chemical properties. Together, these two approaches may facilitate a better understanding of the concepts that determine catalytic behavior of POP in physiological conditions.