Serine Hydrolase Activity Research Articles

Profiling Active Enzymes for Polysorbate Degradation in Biotherapeutics by Activity-Based Protein Profiling.

Polysorbate is widely used to maintain stability of biotherapeutic proteins in pharmaceutical formulation development. Degradation of polysorbate can lead to particle formation in drug products, which is a major quality concern and potential patient risk factor. Enzymatic activity from residual host cell enzymes such as lipases and esterases plays a major role for polysorbate degradation. Their high activity, often at very low concentration, constitutes a major analytical challenge in the biopharmaceutical industry. In this study, we evaluated and optimized the activity-based protein profiling (ABPP) approach to identify active enzymes responsible for polysorbate degradation. Using an optimized chemical probe, we established the first global profile of active serine hydrolases in harvested cell culture fluid (HCCF) for monoclonal antibodies (mAbs) production from two Chinese hamster ovary (CHO) cell lines. A total of eight known lipases were identified by ABPP with enzyme activity information, while only five lipases were identified by a traditional abundance-based proteomics (TABP) approach. Interestingly, phospholipase B-like 2 (PLBL2), a well-known problematic HCP was not found to be active in process-intermediates from two different mAbs. In a proof-of-concept study with downstream samples, phospholipase A2 group VII (PLA2G7) was only identified by ABPP and confirmed to contribute to polysorbate-80 degradation for the first time. The established ABBP approach is approved to be able to identify low-abundance host cell enzymes and fills the gap between lipase abundance and activity, which enables more meaningful polysorbate degradation investigations for biotherapeutic development.

Effects of Chlorpyrifos on Serine Hydrolase Activities, Lipid Mediators, and Immune Responses in Lungs of Neonatal and Adult Mice.

Chlorpyrifos (CPF) is an organophosphate (OP) pesticide that causes acute toxicity by inhibiting acetylcholinesterase (AChE) in the nervous system. However, endocannabinoid (eCB) metabolizing enzymes in brain of neonatal rats are more sensitive than AChE to inhibition by CPF, leading to increased levels of eCBs. Because eCBs are immunomodulatory molecules, we investigated the association between eCB metabolism, lipid mediators, and immune function in adult and neonatal mice exposed to CPF. We focused on lung effects because epidemiologic studies have linked pesticide exposures to respiratory diseases. CPF was hypothesized to disrupt lung eCB metabolism and alter lung immune responses to lipopolysaccharide (LPS), and these effects would be more pronounced in neonatal mice due to an immature immune system. We first assessed the biochemical effects of CPF in adult mice (≥8 weeks old) and neonatal mice after administering CPF (2.5 mg/kg, oral) or vehicle for 7 days. Tissues were harvested 4 h after the last CPF treatment and lung microsomes from both age groups demonstrated CPF-dependent inhibition of carboxylesterases (Ces), a family of xenobiotic and lipid metabolizing enzymes, whereas AChE activity was inhibited in adult lungs only. Activity-based protein profiling (ABPP)-mass spectrometry of lung microsomes identified 31 and 32 individual serine hydrolases in neonatal lung and adult lung, respectively. Of these, Ces1c/Ces1d/Ces1b isoforms were partially inactivated by CPF in neonatal lung, whereas Ces1c/Ces1b and Ces1c/BChE were partially inactivated in adult female and male lungs, respectively, suggesting age- and sex-related differences in their sensitivity to CPF. Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) activities in lung were unaffected by CPF. When LPS (1.25 mg/kg, i.p.) was administered following the 7-day CPF dosing period, little to no differences in lung immune responses (cytokines and immunophenotyping) were noted between the CPF and vehicle groups. However, a CPF-dependent increase in the amounts of dendritic cells and certain lipid mediators in female lung following LPS challenge was observed. Experiments in neonatal and adult Ces1d-/- mice yielded similar results as wild type mice (WT) following CPF treatment, except that CPF augmented LPS-induced Tnfa mRNA in adult Ces1d-/- mouse lungs. This effect was associated with decreased expression of Ces1c mRNA in Ces1d-/- mice versus WT mice in the setting of LPS exposure. We conclude that CPF exposure inactivates several Ces isoforms in mouse lung and, during an inflammatory response, increases certain lipid mediators in a female-dependent manner. However, it did not cause widespread altered lung immune effects in response to an LPS challenge.

A Superfamily-wide Activity Atlas of Serine Hydrolases in Drosophila melanogaster.

The serine hydrolase (SH) superfamily is, perhaps, one of the largest functional enzyme classes in all forms of life and consists of proteases, peptidases, lipases, and carboxylesterases as representative members. Consistent with the name of this superfamily, all members, without any exception to date, use a nucleophilic serine residue in the enzyme active site to perform hydrolytic-type reactions via a two-step ping-pong mechanism involving a covalent enzyme intermediate. Given the highly conserved catalytic mechanism, this superfamily has served as a classical prototype in the development of several platforms of chemical proteomics techniques, activity-based protein profiling (ABPP), to globally interrogate the functions of its different members in various native, yet complex, biological settings. While ABPP-based proteome-wide activity atlases for SH activities are available in numerous organisms, including humans, to the best of our knowledge, such an analysis for this superfamily is lacking in any insect model. To address this, we initially report a bioinformatics analysis toward the identification and categorization of nonredundant SHs in Drosophila melanogaster. Following up on this in silico analysis, leveraging discovery chemoproteomics, we identify and globally map the full complement of SH activities during various developmental stages and in different adult tissues of Drosophila. Finally, as a proof of concept of the utility of this activity atlas, we highlight sexual dimorphism in SH activities across different tissues in adult D. melanogaster, and we propose new research directions, resources, and tools that this study can provide to the fly community.

Application of the RBBP9 Serine Hydrolase Inhibitor, ML114, Decouples Human Pluripotent Stem Cell Proliferation and Differentiation.

Retinoblastoma binding protein 9 (RBBP9) is required for maintaining the expression of both pluripotency and cell cycle genes in human pluripotent stem cells (hPSCs). An siRNA-based study from our group showed it does so by influencing cell cycle progression through the RB/E2F pathway. In non-pluripotent cells, RBBP9 is also known to have serine hydrolase (SH) activity, acting on currently undefined target proteins. The role of RBBP9 SH activity in hPSCs, and during normal development, is currently unknown. To begin assessing whether RBBP9 SH activity might contribute to hPSC maintenance, hPSCs were treated with ML114—a selective chemical inhibitor of RBBP9 SH activity. Stem cells treated with ML114 showed significantly reduced population growth rate, colony size and progression through the cell cycle, with no observable change in cell morphology or decrease in pluripotency antigen expression—suggesting no initiation of hPSC differentiation. Consistent with this, hPSCs treated with ML114 retained the capacity for tri-lineage differentiation, as seen through teratoma formation. Subsequent microarray and Western blot analyses of ML114-treated hPSCs suggest the nuclear transcription factor Y subunit A (NFYA) may be a candidate effector of RBBP9 SH activity in hPSCs. These data support a role for RBBP9 in regulating hPSC proliferation independent of differentiation, whereby inhibition of RBBP9 SH activity de-couples decreased hPSC proliferation from initiation of differentiation.

Activity-based protein profiling of rice (Oryza sativa L.) bran serine hydrolases

Rice bran is an underutilized agricultural by-product with economic importance. The unique phytochemicals and fatty acid compositions of bran have been targeted for nutraceutical development. The endogenous lipases and hydrolases are responsible for the rapid deterioration of rice bran. Hence, we attempted to provide the first comprehensive profiling of active serine hydrolases (SHs) present in rice bran proteome by activity-based protein profiling (ABPP) strategy. The active site-directed fluorophosphonate probe (rhodamine and biotin-conjugated) was used for the detection and identification of active SHs. ABPP revealed 55 uncharacterized active-SHs and are representing five different known enzyme families. Based on motif and domain analyses, one of the uncharacterized and miss annotated SHs (Os12Ssp, storage protein) was selected for biochemical characterization by overexpressing in yeast. The purified recombinant protein authenticated the serine protease activity in time and protein-dependent studies. Os12Ssp exhibited the maximum activity at a pH between 7.0 and 8.0. The protease activity was inhibited by the covalent serine protease inhibitor, which suggests that the ABPP approach is indeed reliable than the sequence-based annotations. Collectively, the comprehensive knowledge generated from this study would be useful in expanding the current understanding of rice bran SHs and paves the way for better utilization/stabilization of rice bran.

Cyto-biological effects of microRNA-424-5p on human colorectal cancer cells.

MicroRNA (miR)-424-5p is overexpressed in colorectal cancer (CRC); however, its role, clinical significance and underlying molecular mechanism have remained to be fully elucidated. The aim of the present study was to investigate the roles of miR-424-5p in CRC and the underlying mechanisms. It was demonstrated that miR-424-5p is overexpressed in CRC, based on bioinformatics analysis using The Cancer Genome Atlas TCGA and analysis of tissue samples from patients with CRC from The First Hospital of Hebei Medical University, and the expression of miR-424-5p was associated with the depth of invasion and Dukes' staging. In CRC cells, the oncogenic roles of miR-424-5p were also verified by Cell Counting Kit-8, wound healing and Transwell assays. To identify target genes, all transcripts were compared between miR-424-5p mimic-transfected SW480 cells and mimic control cells by transcriptome sequencing. Subsequently, the differentially expressed genes (DEGs) were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The DEGs were revealed to be significantly enriched in the GO terms ‘serine hydrolase activity,’ ‘serine-type peptidase activity’ and ‘serine-type endopeptidase activity’. KEGG signaling pathway analysis indicated that the DEGs were significantly enriched in ‘endocytosis’, ‘regulation of actin cytoskeleton’, ‘Wnt signaling pathway’ and ‘ubiquitin-mediated proteolysis signaling pathway’. These results suggested that miR-424-5p is a potential target in the treatment of CRC.

Abstract 6482: Activity based protein profiling by SWATH/DIA-MS mode (ABPP-SWATH/DIA-MS) from OCT-embedded tissues biopsies reveals lung tumor-specific protease profiles

Abstract (a) Proteomics relies on protein digestion into smaller fragments/peptides and converge to proteome sequence information, classification, and expression levels without a screen of the cellular protein activities. Activity-based proteome profiling (ABPP) is emerging as a tool for functional proteomics based on mass spectrometry. We established an ABPP-SWATH/DIA MS platform compatible with OCT-embedded biopsies for differential global serine hydrolase (SHs) activity profiling and determine the Relative Depletion-Detected Activity (RDDA) index that reflects the relative activity of SHs in type IIIA lung adenocarcinoma biopsies. (b) To prevent digestion on streptavidin beads and MS spectra contamination, we used SWATH/DIA-MS2 signal intensities for the assessment of the ratio between the biopsy-extract solution either treated with DMSO or which had earlier been depleted with chemical probe for active enzyme molecules. We processed 24 lung biopsies (having a solution of depleted and non-depleted proteome) of long and short survivors with type IIIA lung adenocarcinoma tumor and their normal tissue counterparts and included peptides of 65 known endogenous inhibitors of SH. In parallel experiments, we analyzed the catalytically depleted part of the molecule by conventional ABPP-MS approach integrated on bead sample digestion. (c) We quantified 117 proteins annotated to the serine hydrolase family, of which as much as 60% was depleted with a CI of 90% in the biopsy-extract solution incubated with chemical probe. Among 133 enzymes monitored over the entire clinical cohort, we found as many as 89 enzymes with significant depletion that we further used to generate the activity SH profiles of each biopsy. We found 36 enzymes exclusively depleted in tumor tissue, both in short and long survivals, and with depletion absent from normal counterparts. By quantification of the catalytically active part of the molecule on the streptavidin bead sample pull-down digest, we found only 9 proteins that contained quality data for spectral counting comparisons. Partial Least Squares Discriminant Analysis (PLS-DA) analysis revealed the activity profiles of 9 enzymes perfectly discriminating the tumor tissues of short survivals, and these enzyme signatures were enhanced by the elevated activity of 2 enzymes - ADH1C and IAH1- in short-term survivors. (d) We have demonstrated that this approach can substantially increase the differential detection of protease activity between oncogenic lung tissue and their normal tissue counterparts. Citation Format: Tatjana Sajic, Stephan Arni, Rudolf Aebersold, Sven Hillinger. Activity based protein profiling by SWATH/DIA-MS mode (ABPP-SWATH/DIA-MS) from OCT-embedded tissues biopsies reveals lung tumor-specific protease profiles [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6482.

Activity-based protein profiling guided identification of urine proteinase 3 activity in subclinical rejection after renal transplantation

BackgroundThe pathophysiology of subclinical versus clinical rejection remains incompletely understood given their equivalent histological severity but discordant graft function. The goal was to evaluate serine hydrolase enzyme activities to explore if there were any underlying differences in activities during subclinical versus clinical rejection.MethodsSerine hydrolase activity-based protein profiling (ABPP) was performed on the urines of a case control cohort of patients with biopsy confirmed subclinical or clinical transplant rejection. In-gel analysis and affinity purification with mass spectrometry were used to demonstrate and identify active serine hydrolase activity. An assay for proteinase 3 (PR3/PRTN3) was adapted for the quantitation of activity in urine.ResultsIn-gel ABPP profiles suggested increased intensity and diversity of serine hydrolase activities in urine from patients undergoing subclinical versus clinical rejection. Serine hydrolases (n = 30) were identified by mass spectrometry in subclinical and clinical rejection patients with 4 non-overlapping candidates between the two groups (i.e. ABHD14B, LTF, PR3/PRTN3 and PRSS12). Western blot and the use of a specific inhibitor confirmed the presence of active PR3/PRTN3 in samples from patients undergoing subclinical rejection. Analysis of samples from normal donors or from several serial post-transplant urines indicated that although PR3/PRTN3 activity may be highly associated with low-grade subclinical inflammation, the enzyme activity was not restricted to this patient group.ConclusionsThere appear to be limited qualitative and quantitative differences in serine hydrolase activity in patients with subclinical versus clinical renal transplant rejection. The majority of enzymes identified were present in samples from both groups implying that in-gel quantitative differences may largely relate to the activity status of shared enzymes. However qualitative compositional differences were also observed indicating differential activities. The PR3/PRTN3 analyses indicate that the activity status of urine in transplant patients is dynamic possibly reflecting changes in the underlying processes in the transplant. These data suggest that differential serine hydrolase pathways may be active in subclinical versus clinical rejection which requires further exploration in larger patient cohorts. Although this study focused on PR3/PRTN3, this does not preclude the possibility that other enzymes may play critical roles in the rejection process.

Characterization of a monoacylglycerol lipase in the medicinal leech, Hirudo verbana.

Endocannabinoids are a class of lipid neuromodulators found throughout the animal kingdom. Among the endocannabinoids, 2-arachydonoyl glycerol (2-AG) is the most prevalent endocannabinoid and monoacylglycerol lipase (MAGL) is a serine hydrolase primarily responsible for metabolizing 2-AG in mammals. In the medicinal leech, Hirudo verbana, 2-AG has been found to be an important and multi-functional modulator of synaptic transmission and behavior. However, very little is known about the molecular components of its synthesis and degradation. In this study we have identified cDNA in Hirudo that encodes a putative MAGL (HirMAGL). The encoded protein exhibits considerable sequence and structural conservation with mammalian forms of MAGL, especially in the catalytic triad that mediates 2-AG metabolism. Additionally, HirMAGL transcripts are detected in the Hirudo central nervous system. When expressed in HEK 293 cells HirMAGL segregates to the plasma membrane as expected. It also exhibits serine hydrolase activity that is blocked when a critical active site residue is mutated. HirMAGL also demonstrates the capacity to metabolize 2-AG and this capacity is also prevented when the active site is mutated. Finally, HirMAGL activity is inhibited by JZL184 and MJN110, specific inhibitors of mammalian MAGL. To our knowledge these findings represent the first characterization of an invertebrate form of MAGL and show that HirMAGL exhibits many of the same properties as mammalian MAGL's that are responsible for 2-AG metabolism.

High-Resolution Confocal Fluorescence Imaging of Serine Hydrolase Activity in Cryosections \u2013 Application to Glioma Brain Unveils Activity Hotspots Originating from Tumor-Associated Neutrophils

BackgroundSerine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. ABPP utilizes cell/tissue proteomes and features the FP-warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Existing ABPP approaches characterize global SH activity based on mobility in gel or MS-based target identification and cannot reveal the identity of the cell-type responsible for an individual SH activity originating from complex proteomes.ResultsHere, by using an activity probe with broad reactivity towards the SH family, we advance the ABPP methodology to glioma brain cryosections, enabling for the first time high-resolution confocal fluorescence imaging of global SH activity in the tumor microenvironment. Tumor-associated cell types were identified by extensive immunohistochemistry on activity probe-labeled sections. Tissue-ABPP indicated heightened SH activity in glioma vs. normal brain and unveiled activity hotspots originating from tumor-associated neutrophils (TANs), rather than tumor-associated macrophages (TAMs). Thorough optimization and validation was provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification.ConclusionsOur study advances the ABPP methodology to tissue sections, enabling high-resolution confocal fluorescence imaging of global SH activity in anatomically preserved complex native cellular environment. To achieve global portrait of SH activity throughout the section, a probe with broad reactivity towards the SH family members was employed. As ABPP requires no a priori knowledge of the identity of the target, we envisage no imaginable reason why the presently described approach would not work for sections regardless of species and tissue source.

Mycoparasitism illuminated by genome and transcriptome sequencing of Coniothyrium minitans, an important biocontrol fungus of the plant pathogen Sclerotinia sclerotiorum.

Coniothyrium minitans is a mycoparasite of the notorious plant pathogen Sclerotinia sclerotiorum. To further understand the parasitism of C. minitans, we assembled and analysed its genome and performed transcriptome analyses. The genome of C. minitans strain ZS-1 was assembled into 350 scaffolds and had a size of 39.8 Mb. A total of 11 437 predicted genes and proteins were annotated, and 30.8 % of the blast hits matched proteins encoded by another member of the Pleosporales, Paraphaeosphaeria sporulosa, a worldwide soilborne fungus with biocontrol ability. The transcriptome of strain ZS-1 during the early interaction with S. sclerotiorum at 0, 4 and 12 h was analysed. The detected expressed genes were involved in responses to host defenses, including cell-wall-degrading enzymes, transporters, secretory proteins and secondary metabolite productions. Seventeen differentially expressed genes (DEGs) of fungal cell-wall-degrading enzymes (FCWDs) were up-regulated during parasitism, with only one down-regulated. Most of the monocarboxylate transporter genes of the major facilitator superfamily and all the detected ABC transporters, especially the heavy metal transporters, were significantly up-regulated. Approximately 8 % of the 11 437 proteins in C. minitans were predicted to be secretory proteins with catalytic activity. In the molecular function category, hydrolase activity, peptidase activity and serine hydrolase activity were enriched. Most genes involved in serine hydrolase activity were significantly up-regulated. This genomic analysis and genome-wide expression study demonstrates that the mycoparasitism process of C. minitans is complex and a broad range of proteins are deployed by C. minitans to successfully invade its host. Our study provides insights into the mechanisms of the mycoparasitism between C. minitans and S. sclerotiorum and identifies potential secondary metabolites from C. minitans for application as a biocontrol agent.

Structure Dependent Determination of Organophosphate Targets in Mammalian Tissues Using Activity-Based Protein Profiling.

Acute and chronic exposures to organophosphates (OPs), including agricultural pesticides, industrial chemicals, and chemical warfare agents, remain a significant worldwide health risk. The mechanisms by which OPs alter development and cognition in exposed individuals remain poorly understood, in part due to the large number of structurally diverse OPs and the wide range of affected proteins and signaling pathways. To investigate the influence of structure on OP targets in mammalian systems, we have developed a series of probes for activity-based protein profiling (ABPP) featuring two distinct reactive groups that mimic OP chemical reactivity. FOP features a fluorophosphonate moiety, and PODA and CODA utilize a dialkynyl phosphate ester; both reactive group types target serine hydrolase activity. As the oxon represents the highly reactive and toxic functional group of many OPs, the new probes described herein enhance our understanding of tissue-specific reactivity of OPs. Chemoproteomic analysis of mouse tissues treated with the probes revealed divergent protein profiles, demonstrating the influence of probe structure on protein targeting. These targets also vary in sensitivity toward different OPs. The simultaneous use of multiple probes in ABPP experiments may therefore offer more comprehensive coverage of OP targets; FOP consistently labeled more targets in both brain and liver than PODA or CODA, suggesting the dialkyne warhead is more selective for enzymes in major signaling pathways than the more reactive fluorophosphonate warhead. Additionally, the probes can be used to assess reactivation of OP-inhibited enzymes by N-oximes and may serve as diagnostic tools for screening of therapeutic candidates in a panel of protein targets. These applications will help clarify the short- and long-term effects of OP toxicity beyond acetylcholinesterase inhibition, investigate potential points of convergence for broad spectrum therapeutic development, and support future efforts to screen candidate molecules for efficacy in various model systems.

Functional annotation of serine hydrolases in the asexual erythrocytic stage of Plasmodium falciparum

Enzymes of the serine hydrolase superfamily are ubiquitous, highly versatile catalysts that mediate a wide variety of metabolic reactions in eukaryotic cells, while also being amenable to selective inhibition. We have employed a fluorophosphonate-based affinity capture probe and mass spectrometry to explore the expression profile and metabolic roles of the 56-member P. falciparum serine hydrolase superfamily in the asexual erythrocytic stage of P. falciparum. This approach provided a detailed census of active serine hydrolases in the asexual parasite, with identification of 21 active serine hydrolases from α/β hydrolase, patatin, and rhomboid protease families. To gain insight into their functional roles and substrates, the pan-lipase inhibitor isopropyl dodecylfluorophosphonate was employed for competitive activity-based protein profiling, leading to the identification of seven serine hydrolases with potential lipolytic activity. We demonstrated how a chemoproteomic approach can provide clues to the specificity of serine hydrolases by using a panel of neutral lipase inhibitors to identify an enzyme that reacts potently with a covalent monoacylglycerol lipase inhibitor. In combination with existing phenotypic data, our studies define a set of serine hydrolases that likely mediate critical metabolic reactions in asexual parasites and enable rational prioritization of future functional characterization and inhibitor development efforts.

Abstract 4934: Modified SWATH MS analysis of serine hydrolase activity in lung adenocarcinoma

Abstract Lung cancer, especially adenocarcinoma as the most common subtype, is still the most deadly human cancers. Therefore biomarker discovery for implementation of individualised therapies remains of utmost importance. Serine hydrolases (SHs), one of the largest enzyme families, have previously been shown to be implicated in the development of lung cancers. Activity-based protein profiling (ABPP) is a proteomic method that uses active site-directed chemical probes to selectively target the active form of the subsets of the enzymes in question, and then by a combination of a streptavidin-biotin enrichment step and mass spectrometry quantifies the catalytically active amount of the enzyme molecule. In this project, we monitored both forms of serine hydrolase, the catalytically active and inactive, in a patient cohort of lung adenocarcinoma biopsies. For this purpose, we combined the activity-based proteomics for serine hydrolase and SWATH mass spectrometry (MS), which ensures highly reproducible protein quantification in a large panel of clinical samples. Twenty four lung biopsies of long- and short surviving patients with stage IIIA adenocarcinomas and their normal tissue counterparts were available as OCT-embedded tissue. Although OCT represents a non-reactive compound used to preserve tissue morphology in long-term sample storage, this substance is incompatible with mass spectrometry measurements. We optimized and developed an OCT clean-up protocol compatible with enzyme-substrate binding of activity-based chemical probes and the targeted portion of the active enzyme. To prevent digestion on streptavidin beads and MS spectra contamination, we used the reproducible and accurate SWATH MS to indirectly measure the active enzyme form in the biopsy-extract solution which had earlier been depleted for all active enzyme molecules by precipitation with chemical probes attached to the beads. We identified over 4000 lung tissue proteins from a few milligrams of OCT-embedded biopsies, and confirmed good data quality for further SH enzyme quantification. In addition to the analysis of total proteome, 278 distinct proteins were identified on the parallel streptavidin bead samples, with chemical probes being used to deplete the active enzyme from the biopsy-extract. Each SWATH experiment reported the percentage of active enzyme form through the indirectly measured ratio between the inactive SHs (sample depleted for active SHs) and the total SHs (non-depleted sample). We detected around 80 enzymes with the active form comparably measured in all three independent experiments, which amount generally accounted for between 5-55% of the total enzyme concentration. We conclude that with the modified protocol compatible with OCT-embedded tissue biopsies the combination of ABPP and SWATH-MS enables highly reproducible protein quantification in biomarker discovery. Citation Format: Tatjana Sajic, Stephan Arni, Walter Weder, Rudolf Aebersold, Sven Hillinger. Modified SWATH MS analysis of serine hydrolase activity in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4934.

Functional characterization of a subtilisin-like serine protease from Vibrio cholerae

Vibrio cholerae, the causative agent of the human diarrheal disease cholera, exports numerous enzymes that facilitate its adaptation to both intestinal and aquatic niches. These secreted enzymes can mediate nutrient acquisition, biofilm assembly, and V. cholerae interactions with its host. We recently identified a V. cholerae-secreted serine protease, IvaP, that is active in V. cholerae-infected rabbits and human choleric stool. IvaP alters the activity of several host and pathogen enzymes in the gut and, along with other secreted V. cholerae proteases, decreases binding of intelectin, an intestinal carbohydrate-binding protein, to V. cholerae in vivo IvaP bears homology to subtilisin-like enzymes, a large family of serine proteases primarily comprised of secreted endopeptidases. Following secretion, IvaP is cleaved at least three times to yield a truncated enzyme with serine hydrolase activity, yet little is known about the mechanism of extracellular maturation. Here, we show that IvaP maturation requires a series of sequential N- and C-terminal cleavage events congruent with the enzyme's mosaic protein domain structure. Using a catalytically inactive reporter protein, we determined that IvaP can be partially processed in trans, but intramolecular proteolysis is most likely required to generate the mature enzyme. Unlike many other subtilisin-like enzymes, the IvaP cleavage pattern is consistent with stepwise processing of the N-terminal propeptide, which could temporarily inhibit, and be cleaved by, the purified enzyme. Furthermore, IvaP was able to cleave purified intelectin, which inhibited intelectin binding to V. cholerae These results suggest that IvaP plays a role in modulating intelectin-V. cholerae interactions.

The effect of a LysR-type transcriptional regulator gene of Pseudomonas plecoglossicida on the immune responses of Epinephelus coioides

As an important pathogen in aquaculture, Pseudomonas plecoglossicida has caused heavy losses. It was determined with RNA-seq that the expression of a LysR-type transcriptional regulator gene (L321_20267) of P. plecoglossicida at 18 °C was significantly higher than that at 28 °C, which was verified by quantitative real-time PCR (qRT-PCR). RNAi significantly reduced the content of L321_20267 mRNA in P. plecoglossicida, with a maximal decrease of 90.63%. Compared with the wild-type strain, infection with the L321_20267-RNAi strain resulted in a 50% reduction in mortality and an onset time delay of Epinephelus coioides, as well as alleviation of the symptoms in E. coioides spleens. Compared with the wild-type strain of P. plecoglossicida, the L321_20267-RNAi strain resulted in a significant change in the spleen transcriptome of infected E. coioides. The results of GO and KEGG analysis showed that genes of serine hydrolase activity, the antigen processing and presentation pathway, the B cell receptor signalling pathway and the chemokine signalling pathway were most affected by the L321_20267 gene of P. plecoglossicida. Meanwhile, the immune genes were related to different numbers of miRNAs and lncRNAs, and some miRNAs were related to more than one gene. The results indicated that 1. L321_20267 is a virulence gene of P. plecoglossicida; 2. the upregulation of the immune pathways facilitated E. coioides to remove the L321_20267-RNAi strain compared with the wild-type strain of P. plecoglossicida; and 3. the immune genes were regulated by miRNA and lncRNA in a complex manner.

Activity-based proteomics reveals nine target proteases for the recombinant protein-stabilizing inhibitor SlCYS8 in Nicotiana benthamiana.

SummaryCo‐expression of protease inhibitors like the tomato cystatin Sl CYS8 is useful to increase recombinant protein production in plants, but key proteases involved in protein proteolysis are still unknown. Here, we performed activity‐based protein profiling to identify proteases that are inhibited by Sl CYS8 in agroinfiltrated Nicotiana benthamiana. We discovered that Sl CYS8 selectively suppresses papain‐like cysteine protease (PLCP) activity in both apoplastic fluids and total leaf extracts, while not affecting vacuolar‐processing enzyme and serine hydrolase activity. A robust concentration‐dependent inhibition of PLCPs occurred in vitro when purified Sl CYS8 was added to leaf extracts, indicating direct cystatin–PLCP interactions. Activity‐based proteomics revealed that nine different Cathepsin‐L/‐F‐like PLCPs are strongly inhibited by Sl CYS8 in leaves. By contrast, the activity of five other Cathepsin‐B/‐H‐like PLCPs, as well as 87 Ser hydrolases, was unaffected by Sl CYS8. Sl CYS8 expression prevented protein degradation by inhibiting intermediate and mature isoforms of granulin‐containing proteases from the Resistant‐to‐Desiccation‐21 (RD21) PLCP subfamily. Our data underline the key role of endogenous PLCPs on recombinant protein degradation and reveal candidate proteases for depletion strategies.

Activity-Based Protein Profiling of Intraoperative Serine Hydrolase Activities during Cardiac Surgery.

The processes involved in the initiation of acute kidney injury (AKI) following cardiopulmonary bypass (CPB) are thought to occur during the intraoperative period. Such a rapid development might indicate that some of the inductive events are not dependent on de novo protein synthesis, raising the possibility that changes in activities of pre-existing enzymes could contribute to the development of AKI. Activity-based protein profiling (ABPP) was used to compare the serine hydrolase enzyme activities present in the urines of CPB patients who subsequently developed AKI versus those who did not (non-AKI) during the intra- and immediate postoperative periods. Sequential urines collected from a nested case-control cohort of AKI and non-AKI patients were reacted with a serine hydrolase activity probe, fluorophosphonate-TAMRA, and separated by SDS-PAGE. The patterns and levels of probe-labeled proteins in the two groups were initially comparable. However, within 1 h of CPB there were significant pattern changes in the AKI group. Affinity purification and mass spectrometry-based analysis of probe-labeled enzymes in AKI urines at 1 h CPB and arrival to the intensive care unit (ICU) identified 28 enzymes. Quantitative analysis of the activity of one of the identified enzymes, kallikrein-1, revealed some trends suggesting differences in the levels and temporal patterns of enzyme activity between a subset of patients who developed AKI and those who did not. A comparative analysis of affinity-purified probe reacted urinary proteins from these patient groups during the intraoperative period suggested the presence of both shared and unique enzyme patterns. These results indicate that there are intraoperative changes in the levels and types of serine hydrolase activities in patients who subsequently develop AKI. However, the role of these activity differences in the development of AKI remains to be determined.

Chemical Proteomic Analysis of Serine Hydrolase Activity in Niemann-Pick Type C Mouse Brain.

The endocannabinoid system (ECS) is considered to be an endogenous protective system in various neurodegenerative diseases. Niemann-Pick type C (NPC) is a neurodegenerative disease in which the role of the ECS has not been studied yet. Most of the endocannabinoid enzymes are serine hydrolases, which can be studied using activity-based protein profiling (ABPP). Here, we report the serine hydrolase activity in brain proteomes of a NPC mouse model as measured by ABPP. Two ABPP methods are used: a gel-based method and a chemical proteomics method. The activities of the following endocannabinoid enzymes were quantified: diacylglycerol lipase (DAGL) α, α/β-hydrolase domain-containing protein 4, α/β-hydrolase domain-containing protein 6, α/β-hydrolase domain-containing protein 12, fatty acid amide hydrolase, and monoacylglycerol lipase. Using the gel-based method, two bands were observed for DAGL α. Only the upper band corresponding to this enzyme was significantly decreased in the NPC mouse model. Chemical proteomics showed that three lysosomal serine hydrolase activities (retinoid-inducible serine carboxypeptidase, cathepsin A, and palmitoyl-protein thioesterase 1) were increased in Niemann-Pick C1 protein knockout mouse brain compared to wild-type brain, whereas no difference in endocannabinoid hydrolase activity was observed. We conclude that these targets might be interesting therapeutic targets for future validation studies.

Molecular cloning of adipose triglyceride lipase (ATGL) gene from blunt snout bream and its expression after LPS-induced TNF-α factor.

The aims of the present study were to clone the full-length cDNA of adipose triglyceridelipase (ATGL) and to analyze its expression after lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-α). The cDNA obtained covered 1801bp with an open reading frame of 1500bp encoding 499 amino acids. Sequence alignment and phylogenetic analysis show the best identity with Cyprinus carpio (86%). The ATGL protein shared a highly conserved 169-amino acid patatin domain, containing a glycine-rich motif, an active serine hydrolase motif, and an aspartic active site. The highest ATGL expression was observed in the liver followed by muscle, whereas relatively low values were detected in the brain and adipose. TNF-α is regarded as an important factor in regulating fat metabolism. Here, LPS was used to induce TNF-α in vivo to verify whether TNF-α can affect ATGL expression. TNF-α expression in liver and muscle is increased and remains unchanged in adipose tissue and brain. The variation of ATGL activity is consistent with that of TNF-α gene expression. Next, we explored the mechanism by which LPS-induced TNF-α mediates the mRNA expression of ATGL in the liver and muscle. For liver, the mRNA levels of c-Jun N-terminal kinase (JNK), nuclear factor kappa B (NF-κB), Sirtuin 1 (SIRT1), and AMP-activated protein kinase (AMPK) were increased by LPS-induced TNF-α. Differencing from the situation in the liver, there was a near-significant decrease trend in the expression of SIRT1 in muscle. Those results indicated that the ATGL gene of blunt snout bream shared a high similarity with the other vertebrates. The expression level of ATGL in tissues with high-fat content was intended to be high. LPS can induce ATGL expression perhaps related to TNF-α.