Activity-based Protein Profiling Approach Research Articles

Resveratrol, a novel inhibitor of fatty acid binding protein 5, inhibits cervical cancer metastasis by suppressing fatty acid transport into nucleus and downstream pathways.

Because of cervical cancer (CC) metastasis, the prognosis of diagnosed patients is poor. However, the molecular mechanisms and therapeutic approach for metastatic CC remain elusive. In this study, we first evaluated the effect of resveratrol (RSV) on CC cell migration and metastasis. Via an activity-based protein profiling (ABPP) approach, a photoaffinity probe of RSV (RSV-P) was synthesized, and the protein targets of RSV in HeLa cells were identified. Based on target information and subsequent in vivo and in vitro validation experiments, we finally elucidated the mechanism of RSV corresponding to its antimetastatic activity. The results showed that RSV concentration-dependently suppressed CC cell migration and metastasis. A list of proteins was identified as the targets of RSV, through the ABPP approach with RSV-P, among which fatty acid binding protein 5 (FABP5) attracted our attention based on The Cancer Genome Atlas (TCGA) database analysis. Subsequent knockout and overexpression experiments confirmed that RSV directly interacted with FABP5 to inhibit fatty acid transport into the nucleus, thereby suppressing downstream matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9) expression, thus inhibiting CC metastasis. Our study confirmed the key role of FABP5 in CC metastasis and provided important target information for the design of therapeutic lead compounds for metastatic CC.

Cover Picture: (Isr. J. Chem. 3‐4/2023)

Graphical Abstract The cover picture shows a graphic depiction of Benjamin Cravatt's face overlayed with and framed by some of the important components (chemical probes, biological systems, experimental results) of the activity-based protein profiling (ABPP) technologies that he has pioneered. As highlighted throughout this special issue, ABPP approaches allow studying a large variety of biological systems with many different applications in basic science and drug discovery. The cover picture was designed by Mary O'Reilly of O'Reilly Science Art, LLC.

Detection of Apoplastic Protease Inhibitors Using Convolution Activity-Based Protein Profiling.

Activity-based protein profiling (ABPP) is a powerful tool in biological chemistry to monitor protein activity using chemical probes that bind covalently and irreversible to active site of enzymes such as proteases. To date, there are three different ways to experimentally use ABPP: comparative, competitive, and convolution ABPP. Here we use and describe the convolution ABPP approach, a method used to detect changes in protease inhibitor abundance in different proteomes. We have applied this method to monitor the activity of Lolium perenne apoplastic cysteine proteases during the interaction with the fungal endophyte Epichloë festucae. We describe the method to isolate apoplastic fluids from infected and uninfected L. perenne ryegrass leaves and the protocol to perform a convolution ABPP experiment. Furthermore, we report how to quantify and analyze fluorescent gels obtained from the ABPP labeling.

Capture and Identification of Dual Specificity Mitogen-Activated Protein Kinase Kinase 7 as a Direct Proteomic Target of Berberine to Affect the c-JunN-Terminal Kinase Pathway

Capture and Identification of Dual Specificity Mitogen-Activated Protein Kinase Kinase 7 as a Direct Proteomic Target of Berberine to Affect the c-JunN-Terminal Kinase Pathway

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.

Rapid Polysorbate 80 Degradation by Liver Carboxylesterase in a Monoclonal Antibody Formulated Drug Substance at Early Stage Development

Polysorbates (PS) are surfactants commonly added in a therapeutic protein drug product to protect proteins from denaturation and aggregation during storage, transportation, and delivery. Significant degradation of PS in drug products could lead to particulate formation with shortened drug shelf life, and one of the major root causes of PS degradation is the host cell protein (HCP) derived lipase/esterase, which belong to the serine hydrolase family. Typically, PS degradation can only be observed in drug products after a long time of storage if very low levels of host cell protein impurity with PS degradation activities are present. In this study, PS80 degradation was observed in a monoclonal antibody (mAb) within 18 h at 5 °C with a low level of HCP presented (<20 ppm) based on ELISA quantitation. This observation suggested that a trace amount of unknown host cell protein(s) with strong enzymatic activity on polysorbate degradation was present in this drug substance. The activity-based protein profiling (ABPP) method with the ActivX FP serine hydrolase probe was employed to identify host cell proteins that can hydrolyze PS. Two hydrolases, liver carboxylesterase B-1-like protein (CES-B1L, A0A061I7X9) and liver carboxylesterase 1-like protein (CES-1L, A0A061IFE2) were identified with high confidence using the ABPP approach for the first time in a mAb drug substance during early stage development. PS80 became stable in the drug substance sample after the two hydrolases were depleted using the immobilized ActivX FP probe, confirming these two hydrolases were responsible for the rapid PS80 degradation. In addition, the PS80 degradation pattern was found to be equivalent to that generated by their human analog, human liver carboxylesterase-1 (hCES-1) and rabbit liver esterase (rLES). Overall, these results suggest that CES-B1L and CES-1L are the primary cause of PS80 degradation in this mAb drug.

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.

Activity-based protein profiling as a robust method for enzyme identification and screening in extremophilic Archaea

Archaea are characterized by a unique life style in often environmental extremes but their thorough investigation is currently hampered by a limited set of suitable in vivo research methodologies. Here, we demonstrate that in vivo activity-based protein profiling (ABPP) may be used to sensitively detect either native or heterogeneously expressed active enzymes in living archaea even under these extreme conditions. In combination with the development of a genetically engineered archaeal screening strain, ABPP can furthermore be used in functional enzyme screenings from (meta)genome samples. We anticipate that our ABPP approach may therefore find application in basic archaeal research but also in the discovery of novel enzymes from (meta)genome libraries.

Activity-Based Protein Profiling Shows Heterogeneous Signaling Adaptations to BRAF Inhibition.

Patients with BRAF V600E mutant melanoma are typically treated with targeted BRAF kinase inhibitors, such as vemurafenib and dabrafenib. Although these drugs are initially effective, they are not curative. Most of the focus to date has been upon genetic mechanisms of acquired resistance; therefore, we must better understand the global signaling adaptations that mediate escape from BRAF inhibition. In the current study, we have used activity-based protein profiling (ABPP) with ATP-analogue probes to enrich kinases and other enzyme classes that contribute to BRAF inhibitor (BRAFi) resistance in four paired isogenic BRAFi-naïve/resistant cell line models. Our analysis showed these cell line models, which also differ in their PTEN status, have considerable heterogeneity in their kinase ATP probe uptake in comparing both naïve cells and adaptations to chronic drug exposure. A number of kinases including FAK1, SLK, and TAOK2 had increased ATP probe uptake in BRAFi resistant cells, while KHS1 (M4K5) and BRAF had decreased ATP probe uptake in the BRAFi-resistant cells. Gene ontology (GO) enrichment analysis revealed BRAFi resistance is associated with a significant enhancement in ATP probe uptake in proteins implicated in cytoskeletal organization and adhesion, and decreases in ATP probe uptake in proteins associated with cell metabolic processes. The ABPP approach was able to identify key phenotypic mediators critical for each BRAFi resistant cell line. Together, these data show that common phenotypic adaptations to BRAF inhibition can be mediated through very different signaling networks, suggesting considerable redundancy within the signaling of BRAF mutant melanoma cells.

Substrate-Competitive Activity-Based Profiling of Ester Prodrug Activating Enzymes.

Understanding the mechanistic basis of prodrug delivery and activation is critical for establishing species-specific prodrug sensitivities necessary for evaluating preclinical animal models and potential drug-drug interactions. Despite significant adoption of prodrug methodologies for enhanced pharmacokinetics, functional annotation of prodrug activating enzymes is laborious and often unaddressed. Activity-based protein profiling (ABPP) describes an emerging chemoproteomic approach to assay active site occupancy within a mechanistically similar enzyme class in native proteomes. The serine hydrolase enzyme family is broadly reactive with reporter-linked fluorophosphonates, which have shown to provide a mechanism-based covalent labeling strategy to assay the activation state and active site occupancy of cellular serine amidases, esterases, and thioesterases. Here we describe a modified ABPP approach using direct substrate competition to identify activating enzymes for an ethyl ester prodrug, the influenza neuraminidase inhibitor oseltamivir. Substrate-competitive ABPP analysis identified carboxylesterase 1 (CES1) as an oseltamivir-activating enzyme in intestinal cell homogenates. Saturating concentrations of oseltamivir lead to a four-fold reduction in the observed rate constant for CES1 inactivation by fluorophosphonates. WWL50, a reported carbamate inhibitor of mouse CES1, blocked oseltamivir hydrolysis activity in human cell homogenates, confirming CES1 is the primary prodrug activating enzyme for oseltamivir in human liver and intestinal cell lines. The related carbamate inhibitor WWL79 inhibited mouse but not human CES1, providing a series of probes for analyzing prodrug activation mechanisms in different preclinical models. Overall, we present a substrate-competitive activity-based profiling approach for broadly surveying candidate prodrug hydrolyzing enzymes and outline the kinetic parameters for activating enzyme discovery, ester prodrug design, and preclinical development of ester prodrugs.

Abstract 2006: Kinases and adaptive signaling contribute to drug resistance in BRAF mutant melanoma

Abstract Melanoma, the most lethal form of skin cancer, is marked by numerous genetic modifications, including point mutations as well as overexpression and deletion of genes. Intrinsic and acquired resistance to BRAF V600E targeted therapies (BRAFi) in metastatic melanoma patients further underscores the need for global profiling of melanoma circuitry at the functional level. Therefore, activity-based protein profiling (ABPP) and phosphoproteomics was carried out to decipher steady state differences in global signaling mechanisms in naïve and BRAFi resistant melanoma cell lines with BRAF V600E mutations. Four cell lines (A375, 1205Lu, WM164 and WM793) were selected to evaluate different molecular backgrounds of BRAF mutation based on their PTEN status (either WT or null). For each cell type, both the naïve and BRAFi resistant lines were analyzed via ABPP as well as chemical labeling with tandem mass tags (TMT) prior to discovery phosphoproteomics. While the ABPP approach mined for kinases, phosphoproteomics identified STY phosphorylated peptides providing information on signaling via kinase substrates. LC-MS/MS discovery proteomics (RSLC and Q Exactive, Thermo) identified and relatively quantified all peptides observed in ABPP and TMT phosphoproteomics experiments. MaxQuant was used for data evaluation; preliminary statistical analyses were performed in Perseus to select significant differences for pathway mapping (GeneGO, Metacore) and follow up experiments using siRNA or pharmacological inhibition. Adaptive responses to combination treatment were also explored using the SysQuant workflow for quantitative expression analysis and phosphoproteomics. These experiments served as a basis for comparison for a pilot project of 12 metastatic tumors from BRAF mutant melanoma patients selected for comparison of good and poor survival outcomes. ABPP measurements on different cell line models (A375, 1205Lu, WM793 and WM164) reveal significant differences in ATP uptake of proteins in the resistant cell line model compared to its naïve counterpart. For example, in the 1205Lu cell line several proteins including EGFR, p38alpha, DNA-PK formed an interconnected pathway. Overall we identified between 2,000-2,800 proteins in each cell line with ∼150 kinases. Isobaric labeling coupled to phosphoproteomics identified ∼1,600 quantifiable proteins with ∼4,000 phosphorylation sites. Phosphoproteomics revealed concomitant increase in phosphorylation levels of the substrates acted upon by kinases showing higher ATP uptake in ABPP measurements. For example, in 1205Lu BRAFi resistant cells, CDK1, CDK2 and DNA-PK showed higher ATP uptake and their substrates SSK1, DPYSL3, and vimentin showed higher phosphorylation levels. The complementary nature of the two functional proteomics approaches provided holistic overview of signalling network in melanoma and enabled selection of targets for follow-up studies. Citation Format: Ritin Sharma, Manali Phadke, David Britton, Ian Pike, Keiran Smalley, John M. Koomen. Kinases and adaptive signaling contribute to drug resistance in BRAF mutant melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2006. doi:10.1158/1538-7445.AM2015-2006

Abstract A31: Activity-based protein profiling reveals adaptive response to pharmacological MEK inhibition in KRAS mutant non-small cell lung cancer

Abstract KRAS mutations account for development of approximately 30% of lung adenocarcinomas. While RAF-MEK-ERK signaling cascade is a key regulator of RAS-mediated proliferation and survival, the clinical efficacy of MEK inhibitors is still underwhelming in RAS-driven cancers. We treated KRAS mutant non-small cell lung cancer (NSCLC) cell lines with two MEK inhibitors (AZD6244 and MEK162) currently in clinical development. In all cell lines tested (N=11), we failed to observe significant effects on cell viability or induction of apoptosis. Because increasing evidence indicates that cancer cells undergo adaptive proteomic rewiring in response to kinase inhibitors, we performed activity-based protein profiling (ABPP) after treating 11 KRAS mutant NSCLC cell lines with MEK inhibitors in order to identify global enzymatic changes potentially involved in these adaptive processes. Our ABPP approach employs desthiobiotin-ATP probe, which enables selective labeling of active sites in ATP-binding proteins including kinases, chaperones and metabolic enzymes. Labeled proteins were subsequently purified and analyzed by mass spectrometry-based proteomics technique. Our ABPP results revealed that MEK inhibitors increased levels of multiple receptor tyrosine kinases (RTK) including EGFR and MET. MEK inhibitors induced MET phosphorylation and EGF-mediated AKT phosphorylation was markedly enhanced in the presence of MEK inhibitors, indicating increased RTK activity in adaptive response to MEK inhibition. We also found that MEK inhibition led to increased multiple heat shock proteins including HSP90 and HSP70, suggesting the possibility of chaperone-mediated adaptive resistance. Finally, MEK inhibitors globally increased enzymes involved in glucose utilization pathways implicating metabolic alterations in adaptive response to MEK inhibition. Collectively, our systematic activity-based proteomic profiling approach provides novel insights into adaptive resistance mechanisms to MEK inhibitors, suggesting rational combination treatment strategies for KRAS mutant NSCLC. Updated works will be presented. Citation Format: Jae-Young Kim, Bin Fang, Eric A. Welsh, Fumi Kinose, Jiannong Li, Steven A. Eschrich, John M. Koomen, Eric B. Haura. H. Activity-based protein profiling reveals adaptive response to pharmacological MEK inhibition in KRAS mutant non-small cell lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A31. doi: 10.1158/1557-3125.RASONC14-A31

Proteasomal Serine Hydrolases Are Up-Regulated by and Required for Influenza Virus Infection

Interactions between viruses and their host cells are important determinants of virus replication and of immune responses to the virus. However, these interactions and resulting consequences of these interactions remain poorly defined. Numerous recent quantitative proteomic approaches have measured host proteins affected by virus infection. Here, we used activity-based protein profiling (ABPP) to measure functional alterations in host serine hydrolases after influenza A virus infection of Madin-Darby canine kidney and human A549 lung cells. We identified 62 serine proteases. We then combined the ABPP approach with stable isotope labeling to directly measure how serine hydrolase activities were affected by virus infection. Differentially regulated SHs mapped into a few key cellular pathway systems, most notably the proteasomal system. The specific serine protease inhibitors Aprotinin and Pefablock and specific proteasomal inhibitors Bortezomib and MG132 significantly inhibited influenza virus growth. Some inhibitors also down-regulated activities of several proteasomal proteins, including PSMA1, PSMA2, and PMSB3. Genetic knockdown of PMSA2 also attenuated influenza virus replication. These findings further our understanding of enzymatic cellular processes affected by influenza virus and may be beneficial in the search for additional antiviral therapeutic targets.

Multiplexed Activity-based Protein Profiling of the Human Pathogen Aspergillus fumigatus Reveals Large Functional Changes upon Exposure to Human Serum

Environmental adaptability is critical for survival of the fungal human pathogen Aspergillus fumigatus in the immunocompromised host lung. We hypothesized that exposure of the fungal pathogen to human serum would lead to significant alterations to the organism's physiology, including metabolic activity and stress response. Shifts in functional pathway and corresponding enzyme reactivity of A. fumigatus upon exposure to the human host may represent much needed prognostic indicators of fungal infection. To address this, we employed a multiplexed activity-based protein profiling (ABPP) approach coupled to quantitative mass spectrometry-based proteomics to measure broad enzyme reactivity of the fungus cultured with and without human serum. ABPP showed a shift from aerobic respiration to ethanol fermentation and utilization over time in the presence of human serum, which was not observed in serum-free culture. Our approach provides direct insight into this pathogen's ability to survive, adapt, and proliferate. Additionally, our multiplexed ABPP approach captured a broad swath of enzyme reactivity and functional pathways and provides a method for rapid assessment of the A. fumigatus response to external stimuli.

Activity-based protein profiling of the hepatitis C virus replication in Huh-7 hepatoma cells using a non-directed active site probe

BackgroundHepatitis C virus (HCV) poses a growing threat to global health as it often leads to serious liver diseases and is one of the primary causes for liver transplantation. Currently, no vaccines are available to prevent HCV infection and clinical treatments have limited success. Since HCV has a small proteome, it relies on many host cell proteins to complete its life cycle. In this study, we used a non-directed phenyl sulfonate ester probe (PS4≡) to selectively target a broad range of enzyme families that show differential activity during HCV replication in Huh-7 cells.ResultsThe PS4≡ probe successfully targeted 19 active proteins in nine distinct protein families, some that were predominantly labeled in situ compared to the in vitro labeled cell homogenate. Nine proteins revealed altered activity levels during HCV replication. Some candidates identified, such as heat shock 70 kDa protein 8 (or HSP70 cognate), have been shown to influence viral release and abundance of cellular lipid droplets. Other differentially active PS4≡ targets, such as electron transfer flavoprotein alpha, protein disulfide isomerase A5, and nuclear distribution gene C homolog, constitute novel proteins that potentially mediate HCV propagation.ConclusionsThese findings demonstrate the practicality and versatility of non-directed activity-based protein profiling (ABPP) to complement directed methods and accelerate the discovery of altered protein activities associated with pathological states such as HCV replication. Collectively, these results highlight the ability of in situ ABPP approaches to facilitate the identification of enzymes that are either predominantly or exclusively labeled in living cells. Several of these differentially active enzymes represent possible HCV-host interactions that could be targeted for diagnostic or therapeutic purposes.