Targeting Moonlighting Enzymes in Cancer.

Moonlighting enzymes are multifunctional proteins that perform multiple functions beyond their primary role as catalytic enzymes. Extensive research and clinical practice have demonstrated their pivotal roles in the development and progression of cancer, making them promising targets for drug development. This article delves into multiple notable moonlighting enzymes, including GSK-3, GAPDH, and ENO1, and with a particular emphasis on an enigmatic phosphatase, PTP4A3. We scrutinize their distinct roles in cancer and the mechanisms that dictate their ability to switch roles. Lastly, we discuss the potential of an innovative approach to develop drugs targeting these moonlighting enzymes: target protein degradation. This strategy holds promise for effectively tackling moonlighting enzymes in the context of cancer therapy.

Novel likely pathogenic variant in the EYA1 gene causing Branchio oto renal syndrome and the exploration of pathogenic mechanisms.

OBJECTIVE: Branchio-oto-renal syndrome (BOR, OMIM#113,650) is a rare autosomal dominant disorder that presents with a variety of symptoms, including hearing loss (sensorineural, conductive, or mixed), structural abnormalities affecting the outer, middle, and inner ear, branchial fistulas or cysts, as well as renal abnormalities.This study aims to identify the pathogenic variants by performing genetic testing on a family with Branchio-oto-renal /Branchio-otic (BO, OMIM#602,588) syndrome using whole-exome sequencing, and to explore possible pathogenic mechanisms. METHODS: The family spans 4 generations and consists of 9 individuals, including 4 affected by the BOR/BO syndrome. Phenotypic information, including ear malformation and branchial cleft, was collected from family members. Audiological, temporal bone imaging, and renal ultrasound examinations were also performed. Whole-exome sequencing was conducted to identify candidate pathogenic variants and explore the underlying molecular etiology of BOR/BO syndrome by minigene experiments. RESULTS: Intra-familial variability was observed in the clinical phenotypes of BOR/BO syndrome in this family. The severity and nature of hearing loss varied in family members, with mixed or sensorineural hearing loss. The proband, in particular, had profound sensorineural hearing loss on the left and moderate conductive hearing loss on the right. Additionally, the proband exhibited developmental delay, and her mother experienced renal failure during pregnancy and terminated the pregnancy prematurely. Genetic testing revealed a novel heterozygous variant NM_000503.6: c.639 + 3 A > C in the EYA1 gene in affected family members. In vitro minigene experiments demonstrated its effect on splicing. According to the American College of Medical Genetics (ACMG) guidelines, this variant was classified as likely pathogenic. CONCLUSION: This study highlights the phenotypic heterogeneity within the same family, reports the occurrence of renal failure and adverse pregnancy outcomes in a female patient at reproductive age with BOR syndrome, and enriches the mutational spectrum of pathogenic variants in the EYA1 gene.

The mechanism of covalent inhibition of LAR phosphatase by illudalic acid.

Leukocyte antigen-related (LAR) phosphatase is a receptor-type protein tyrosine phosphatase involved in cellular signaling and associated with human disease including cancer and metabolic disorders. Selective inhibition of LAR phosphatase activity by well characterized and well validated small molecules would provide key insights into the roles of LAR phosphatase in health and disease, but identifying selective inhibitors of LAR phosphatase activity has been challenging. Recently, we described potent and selective inhibition of LAR phosphatase activity by the fungal natural product illudalic acid. Here we provide a detailed biochemical characterization of the adduct formed between LAR phosphatase and illudalic acid. A mass spectrometric analysis indicates that two cysteine residues are covalently labeled by illudalic acid and a related analog. Mutational analysis supports the hypothesis that inhibition of LAR phosphatase activity is due primarily to the adduct with the catalytic cysteine residue. A computational study suggests potential interactions between the illudalic acid moiety and the enzyme active site. Taken together, these data offer novel insights into the mechanism of inhibition of LAR phosphatase activity by illudalic acid.

Association of TRAIL receptor with phosphatase SHP-1 enables repressing T cell receptor signaling and T cell activation through inactivating Lck.

BACKGROUND: T cell receptor (TCR) signaling and T cell activation are tightly regulated by gatekeepers to maintain immune tolerance and avoid autoimmunity. The TRAIL receptor (TRAIL-R) is a TNF-family death receptor that transduces apoptotic signals to induce cell death. Recent studies have indicated that TRAIL-R regulates T cell-mediated immune responses by directly inhibiting T cell activation without inducing apoptosis; however, the distinct signaling pathway that regulates T cell activation remains unclear. In this study, we screened for intracellular TRAIL-R-binding proteins within T cells to explore the novel signaling pathway transduced by TRAIL-R that directly inhibits T cell activation. METHODS: Whole-transcriptome RNA sequencing was used to identify gene expression signatures associated with TRAIL-R signaling during T cell activation. High-throughput screening with mass spectrometry was used to identify the novel TRAIL-R binding proteins within T cells. Co-immunoprecipitation, lipid raft isolation, and confocal microscopic analyses were conducted to verify the association between TRAIL-R and the identified binding proteins within T cells. RESULTS: TRAIL engagement downregulated gene signatures in TCR signaling pathways and profoundly suppressed phosphorylation of TCR proximal tyrosine kinases without inducing cell death. The tyrosine phosphatase SHP-1 was identified as the major TRAIL-R binding protein within T cells, using high throughput mass spectrometry-based proteomics analysis. Furthermore, Lck was co-immunoprecipitated with the TRAIL-R/SHP-1 complex in the activated T cells. TRAIL engagement profoundly inhibited phosphorylation of Lck (Y394) and suppressed the recruitment of Lck into lipid rafts in the activated T cells, leading to the interruption of proximal TCR signaling and subsequent T cell activation. CONCLUSIONS: TRAIL-R associates with phosphatase SHP-1 and transduces a unique and distinct immune gatekeeper signal to repress TCR signaling and T cell activation via inactivating Lck. Thus, our results define TRAIL-R as a new class of immune checkpoint receptors for restraining T cell activation, and TRAIL-R/SHP-1 axis can serve as a potential therapeutic target for immune-mediated diseases.

FLI1 induces erythroleukemia through opposing effects on UBASH3A and UBASH3B expression.

BACKGROUND: FLI1 is an oncogenic transcription factor that promotes diverse malignancies through mechanisms that are not fully understood. Herein, FLI1 is shown to regulate the expression of Ubiquitin Associated and SH3 Domain Containing A/B (UBASH3A/B) genes. UBASH3B and UBASH3A are found to act as an oncogene and tumor suppressor, respectively, and their combined effect determines erythroleukemia progression downstream of FLI1. METHODS: Promoter analysis combined with luciferase assays and chromatin immunoprecipitation (ChIP) analysis were applied on the UBASH3A/B promoters. RNAseq analysis combined with bioinformatic was used to determine the effect of knocking-down UBASH3A and UBASH3B in leukemic cells. Downstream targets of UBASH3A/B were inhibited in leukemic cells either via lentivirus-shRNAs or small molecule inhibitors. Western blotting and RT-qPCR were used to determine transcription levels, MTT assays to assess proliferation rate, and flow cytometry to examine apoptotic index. RESULTS: Knockdown of FLI1 in erythroleukemic cells identified the UBASH3A/B genes as potential downstream targets. Herein, we show that FLI1 directly binds to the UBASH3B promoter, leading to its activation and leukemic cell proliferation. In contrast, FLI1 indirectly inhibits UBASH3A transcription via GATA2, thereby antagonizing leukemic growth. These results suggest oncogenic and tumor suppressor roles for UBASH3B and UBASH3A in erythroleukemia, respectively. Mechanistically, we show that UBASH3B indirectly inhibits AP1 (FOS and JUN) expression, and that its loss leads to inhibition of apoptosis and acceleration of proliferation. UBASH3B also positively regulates the SYK gene expression and its inhibition suppresses leukemia progression. High expression of UBASH3B in diverse tumors was associated with worse prognosis. In contrast, UBASH3A knockdown in erythroleukemic cells increased proliferation; and this was associated with a dramatic induction of the HSP70 gene, HSPA1B. Accordingly, knockdown of HSPA1B in erythroleukemia cells significantly accelerated leukemic cell proliferation. Accordingly, overexpression of UBASH3A in different cancers was predominantly associated with good prognosis. These results suggest for the first time that UBASH3A plays a tumor suppressor role in part through activation of HSPA1B. CONCLUSIONS: FLI1 promotes erythroleukemia progression in part by modulating expression of the oncogenic UBASH3B and tumor suppressor UBASH3A.

Protein Tyrosine Phosphatase PRL-3: A Key Player in Cancer Signaling.

Protein phosphatases are primarily responsible for dephosphorylation modification within signal transduction pathways. Phosphatase of regenerating liver-3 (PRL-3) is a dual-specific phosphatase implicated in cancer pathogenesis. Understanding PRL-3's intricate functions and developing targeted therapies is crucial for advancing cancer treatment. This review highlights its regulatory mechanisms, expression patterns, and multifaceted roles in cancer progression. PRL-3's involvement in proliferation, migration, invasion, metastasis, angiogenesis, and drug resistance is discussed. Regulatory mechanisms encompass transcriptional control, alternative splicing, and post-translational modifications. PRL-3 exhibits selective expressions in specific cancer types, making it a potential target for therapy. Despite advances in small molecule inhibitors, further research is needed for clinical application. PRL-3-zumab, a humanized antibody, shows promise in preclinical studies and clinical trials. Our review summarizes the current understanding of the cancer-related cellular function of PRL-3, its prognostic value, and the research progress of therapeutic inhibitors.

Exploring the Anti-Diabetic Potential of Quercetagitrin through Dual Inhibition of PTPN6 and PTPN9.

Protein tyrosine phosphatases (PTPs) are pivotal contributors to the development of type 2 diabetes (T2DM). Hence, directing interventions towards PTPs emerges as a valuable therapeutic approach for managing type 2 diabetes. In particular, PTPN6 and PTPN9 are targets for anti-diabetic effects. Through high-throughput drug screening, quercetagitrin (QG) was recognized as a dual-target inhibitor of PTPN6 and PTPN9. We observed that QG suppressed the catalytic activity of PTPN6 (IC50 = 1 µM) and PTPN9 (IC50 = 1.7 µM) in vitro and enhanced glucose uptake by mature C2C12 myoblasts. Additionally, QG increased the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and insulin-dependent phosphorylation of Akt in mature C2C12 myoblasts. It further promoted the phosphorylation of Akt in the presence of palmitic acid, suggesting the attenuation of insulin resistance. In summary, our results indicate QG's role as a potent inhibitor targeting both PTPN6 and PTPN9, showcasing its potential as a promising treatment avenue for T2DM.

Structural analysis of PTPN21 reveals a dominant-negative effect of the FERM domain on its phosphatase activity.

PTPN21 belongs to the four-point-one, ezrin, radixin, moesin (FERM) domain-containing protein tyrosine phosphatases (PTP) and plays important roles in cytoskeleton-associated cellular processes like cell adhesion, motility, and cargo transport. Because of the presence of a WPE loop instead of a WPD loop in the phosphatase domain, it is often considered to lack phosphatase activity. However, many of PTPN21's biological functions require its catalytic activity. To reconcile these findings, we have determined the structures of individual PTPN21 FERM, PTP domains, and a complex between FERM-PTP. Combined with biochemical analysis, we have found that PTPN21 PTP is weakly active and is autoinhibited by association with its FERM domain. Disruption of FERM-PTP interaction results in enhanced ERK activation. The oncogenic HPV18 E7 protein binds to PTP at the same location as PTPN21 FERM, indicating that it may act by displacing the FERM domain from PTP. Our results provide mechanistic insight into PTPN21 and benefit functional studies of PTPN21-mediated processes.

The protein tyrosine phosphatase PPH-7 is required for fertility and embryonic development in C. elegans at elevated temperatures.

Post-translational modifications are key in the regulation of activity, structure, localization, and stability of most proteins in eukaryotes. Phosphorylation is potentially the most studied post-translational modification, also due to its reversibility and thereby the regulatory role this modification often plays. While most research attention was focused on kinases in the past, phosphatases remain understudied, most probably because the addition and presence of the modification is more easily studied than its removal and absence. Here, we report the identification of an uncharacterized protein tyrosine phosphatase PPH-7 in C. elegans, a member of the evolutionary conserved PTPN family of phosphatases. Lack of PPH-7 function led to reduction of fertility and embryonic lethality at elevated temperatures. Proteomics revealed changes in the regulation of targets of the von Hippel-Lindau (VHL) E3 ligase, suggesting a potential role for PPH-7 in the regulation of VHL.

The Eyes Absent family members EYA4 and EYA1 promote PLK1 activation and successful mitosis through tyrosine dephosphorylation.

The Eyes Absent proteins (EYA1-4) are a biochemically unique group of tyrosine phosphatases known to be tumour-promoting across a range of cancer types. To date, the targets of EYA phosphatase activity remain largely uncharacterised. Here, we identify Polo-like kinase 1 (PLK1) as an interactor and phosphatase substrate of EYA4 and EYA1, with pY445 on PLK1 being the primary target site. Dephosphorylation of pY445 in the G2 phase of the cell cycle is required for centrosome maturation, PLK1 localization to centrosomes, and polo-box domain (PBD) dependent interactions between PLK1 and PLK1-activation complexes. Molecular dynamics simulations support the rationale that pY445 confers a structural impairment to PBD-substrate interactions that is relieved by EYA-mediated dephosphorylation. Depletion of EYA4 or EYA1, or chemical inhibition of EYA phosphatase activity, dramatically reduces PLK1 activation, causing mitotic defects and cell death. Overall, we have characterized a phosphotyrosine signalling network governing PLK1 and mitosis.

Antibody agonists trigger immune receptor signaling through local exclusion of receptor-type protein tyrosine phosphatases.

Antibodies can block immune receptor engagement or trigger the receptor machinery to initiate signaling. We hypothesized that antibody agonists trigger signaling by sterically excluding large receptor-type protein tyrosine phosphatases (RPTPs) such as CD45 from sites of receptor engagement. An agonist targeting the costimulatory receptor CD28 produced signals that depended on antibody immobilization and were sensitive to the sizes of the receptor, the RPTPs, and the antibody itself. Although both the agonist and a non-agonistic anti-CD28 antibody locally excluded CD45, the agonistic antibody was more effective. An anti-PD-1 antibody that bound membrane proximally excluded CD45, triggered Src homology 2 domain-containing phosphatase 2 recruitment, and suppressed systemic lupus erythematosus and delayed-type hypersensitivity in experimental models. Paradoxically, nivolumab and pembrolizumab, anti-PD-1-blocking antibodies used clinically, also excluded CD45 and were agonistic in certain settings. Reducing these agonistic effects using antibody engineering improved PD-1 blockade. These findings establish a framework for developing new and improved therapies for autoimmunity and cancer.

Targeting Protein Tyrosine Phosphatases to Improve Cancer Immunotherapies.

Advances in immunotherapy have brought significant therapeutic benefits to many cancer patients. Nonetheless, many cancer types are refractory to current immunotherapeutic approaches, meaning that further targets are required to increase the number of patients who benefit from these technologies. Protein tyrosine phosphatases (PTPs) have long been recognised to play a vital role in the regulation of cancer cell biology and the immune response. In this review, we summarize the evidence for both the pro-tumorigenic and tumour-suppressor function of non-receptor PTPs in cancer cells and discuss recent data showing that several of these enzymes act as intracellular immune checkpoints that suppress effective tumour immunity. We highlight new data showing that the deletion of inhibitory PTPs is a rational approach to improve the outcomes of adoptive T cell-based cancer immunotherapies and describe recent progress in the development of PTP inhibitors as anti-cancer drugs.

Chemical Epigenetic Regulation Secondary Metabolites Derived from Aspergillus sydowii DL1045 with Inhibitory Activities for Protein Tyrosine Phosphatases.

Protein tyrosine phosphatases (PTPs) are ubiquitous in living organisms and are promising drug targets for cancer, diabetes/obesity, and autoimmune disorders. In this study, a histone deacetylase inhibitor called suberoylanilide hydroxamic acid (SAHA) was added to a culture of marine fungi (Aspergillus sydowii DL1045) to identify potential drug candidates related to PTP inhibition. Then, the profile of the induced metabolites was characterized using an integrated metabolomics strategy. In total, 46% of the total SMs were regulated secondary metabolites (SMs), among which 20 newly biosynthesized metabolites (10% of the total SMs) were identified only in chemical epigenetic regulation (CER) broth. One was identified as a novel compound, and fourteen compounds were identified from Aspergillus sydowii first. SAHA derivatives were also biotransformed by A. sydowii DL1045, and five of these derivatives were identified. Based on the bioassay, some of the newly synthesized metabolites exhibited inhibitory effects on PTPs. The novel compound sydowimide A (A11) inhibited Src homology region 2 domain-containing phosphatase-1 (SHP1), T-cell protein tyrosine phosphatase (TCPTP) and leukocyte common antigen (CD45), with IC50 values of 1.5, 2.4 and 18.83 µM, respectively. Diorcinol (A3) displayed the strongest inhibitory effect on SHP1, with an IC50 value of 0.96 µM. The structure-activity relationship analysis and docking studies of A3 analogs indicated that the substitution of the carboxyl group reduced the activity of A3. Research has demonstrated that CER positively impacts changes in the secondary metabolic patterns of A. sydowii DL1045. The compounds produced through this approach will provide valuable insights for the creation and advancement of novel drug candidates related to PTP inhibition.

A syndromic neurodevelopmental disorder caused by rare variants in PPFIA3.

PPFIA3 encodes the protein-tyrosine phosphatase, receptor-type, F-polypeptide-interacting-protein-alpha-3 (PPFIA3), which is a member of the LAR-protein-tyrosine phosphatase-interacting-protein (liprin) family involved in synapse formation and function, synaptic vesicle transport, and presynaptic active zone assembly. The protein structure and function are evolutionarily well conserved, but human diseases related to PPFIA3 dysfunction are not yet reported in OMIM. Here, we report 20 individuals with rare PPFIA3 variants (19 heterozygous and 1 compound heterozygous) presenting with developmental delay, intellectual disability, hypotonia, dysmorphisms, microcephaly or macrocephaly, autistic features, and epilepsy with reduced penetrance. Seventeen unique PPFIA3 variants were detected in 18 families. To determine the pathogenicity of PPFIA3 variants in vivo, we generated transgenic fruit flies producing either human wild-type (WT) PPFIA3 or five missense variants using GAL4-UAS targeted gene expression systems. In the fly overexpression assays, we found that the PPFIA3 variants in the region encoding the N-terminal coiled-coil domain exhibited stronger phenotypes compared to those affecting the C-terminal region. In the loss-of-function fly assay, we show that the homozygous loss of fly Liprin-α leads to embryonic lethality. This lethality is partially rescued by the expression of human PPFIA3 WT, suggesting human PPFIA3 function is partially conserved in the fly. However, two of the tested variants failed to rescue the lethality at the larval stage and one variant failed to rescue lethality at the adult stage. Altogether, the human and fruit fly data reveal that the rare PPFIA3 variants are dominant-negative loss-of-function alleles that perturb multiple developmental processes and synapse formation.

Drugging Protein Tyrosine Phosphatases through Targeted Protein Degradation.

Protein tyrosine phosphatases (PTPs) are an important class of enzymes that regulate protein tyrosine phosphorylation levels of a large variety of proteins in cells. Anomalies in protein tyrosine phosphorylation have been associated with the development of numerous human diseases, leading to a heightened interest in PTPs as promising targets for drug development. However, therapeutic targeting of PTPs has faced skepticism about their druggability. Besides the conventional small molecule inhibitors, proteolysis-targeting chimera (PROTAC) technology offers an alternative approach to target PTPs. PROTAC molecules utilize the ubiquitin-proteasome system to degrade specific proteins and have unique advantages compared with inhibitors: 1) PROTACs are highly efficient and can work at much lower concentrations than that expected based on their biophysical binding affinity; 2) PROTACs may achieve higher selectivity for the targeted protein than that dictated by their binding affinity alone; and 3) PROTACs may engage any region of the target protein in addition to the functional site. This review focuses on the latest advancement in the development of targeted PTP degraders and deliberates on the obstacles and prospective paths of harnessing this technology for therapeutic targeting of the PTPs.

Targeting prostate tumor low-molecular weight tyrosine phosphatase for oxidation-sensitizing therapy.

Protein tyrosine phosphatases (PTPs) play major roles in cancer and are emerging as therapeutic targets. Recent reports suggest low-molecular weight PTP (LMPTP)-encoded by the ACP1 gene-is overexpressed in prostate tumors. We found ACP1 up-regulated in human prostate tumors and ACP1 expression inversely correlated with overall survival. Using CRISPR-Cas9-generated LMPTP knockout C4-2B and MyC-CaP cells, we identified LMPTP as a critical promoter of prostate cancer (PCa) growth and bone metastasis. Through metabolomics, we found that LMPTP promotes PCa cell glutathione synthesis by dephosphorylating glutathione synthetase on inhibitory Tyr270. PCa cells lacking LMPTP showed reduced glutathione, enhanced activation of eukaryotic initiation factor 2-mediated stress response, and enhanced reactive oxygen species after exposure to taxane drugs. LMPTP inhibition slowed primary and bone metastatic prostate tumor growth in mice. These findings reveal a role for LMPTP as a critical promoter of PCa growth and metastasis and validate LMPTP inhibition as a therapeutic strategy for treating PCa through sensitization to oxidative stress.

Druggable targets of protein tyrosine phosphatase Family, viz. PTP1B, SHP2, Cdc25, and LMW-PTP: Current scenario on medicinal Attributes, and SAR insights.

Protein tyrosine phosphatases (PTPs) are the class of dephosphorylation enzymes that catalyze the removal of phosphate groups from tyrosine residues on proteins responsible for various cellular processes. Any disbalance in signal pathways mediated by PTPs leads to various disease conditions like diabetes, obesity, cancers, and autoimmune disorders. Amongst the PTP superfamily, PTP1B, SHP2, Cdc25, and LMW-PTP have been prioritized as druggable targets for developing medicinal agents. PTP1B is an intracellular PTP enzyme that downregulates insulin and leptin signaling pathways and is involved in insulin resistance and glucose homeostasis. SHP2 is involved in the RAS-MAPK pathway and T cell immunity. Cdk-cyclin complex activation occurs by Cdc25-PTPs involved in cell cycle regulation. LMW-PTPs are involved in PDGF/PDGFR, Eph/ephrin, and insulin signaling pathways, resulting in certain diseases like diabetes mellitus, obesity, and cancer. The signaling cascades of PTP1B, SHP2, Cdc25, and LMW-PTPs have been described to rationalize their medicinal importance in the pathophysiology of diabetes, obesity, and cancer. Their binding sites have been explored to overcome the hurdles in discovering target selective molecules with optimum potency. Recent developments in the synthetic molecules bearing heterocyclic moieties against these targets have been explored to gain insight into structural features. The elaborated SAR investigation revealed the effect of substituents on the potency and target selectivity, which can be implicated in the further discovery of newer medicinal agents targeting the druggable members of the PTP superfamily.

Developmental roles of glomerular epithelial protein-1 in mice molar morphogenesis.

Glomerular epithelial protein-1 (Glepp1), a R3 subtype family of receptor-type protein tyrosine phosphatases, plays important role in the activation of Src family kinases and regulates cellular processes such as cell proliferation, differentiation, and apoptosis. In this study, we firstly examined the functional evaluation of Glepp1 in tooth development and morphogenesis. The precise expression level and developmental function of Glepp1 were examined by RT-qPCR, in situ hybridization, and loss and gain of functional study using a range of in vitro organ cultivation methods. Expression of Glepp1 was detected in the developing tooth germs in cap and bell stage of tooth development. Knocking down Glepp1 at E13 for 2 days showed the altered expression levels of tooth development-related signaling molecules, including Bmps, Dspp, Fgf4, Lef1, and Shh. Moreover, transient knock down of Glepp1 revealed alterations in cellular physiology, examined by the localization patterns of Ki67 and E-cadherin. Similarly, knocking down of Glepp1 showed disrupted enamel rod and interrod formation in 3-week renal transplanted teeth. In addition, due to attrition of odontoblastic layers, the expression signals of Dspp and the localization of NESTIN were almost not detected after knock down of Glepp1; however, their expressions were increased after Glepp1 overexpression. Thus, our results suggested that Glepp1 plays modulating roles during odontogenesis by regulating the expression levels of signaling molecules and cellular events to achieve the proper structural formation of hard tissue matrices in mice molar development.

Nasal polyp antibody-secreting cells display proliferation signature in aspirin-exacerbated respiratory disease.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) causes nasal obstruction and olfactory dysfunction. Aspirin-exacerbated respiratory disease (AERD) is the triad of CRSwNP, asthma, and respiratory reactions to COX-1 inhibitors. Patients with AERD have elevated nasal IL-5 levels and high numbers of antibody-secreting cells (ASCs), including plasma cells and plasmablasts, in their polyp tissue; in addition, their nasal polyp (NP) IgE levels are correlated with disease severity and recurrence of nasal polyposis. OBJECTIVE: We sought to explore differences in the transcriptomic profile, activation markers, and IL-5Rα expression and function of NP ASCs from patients with AERD and CRSwNP. METHODS: NP tissue was collected from patients with AERD and CRSwNP and digested into single-cell suspensions. NP cells were analyzed for protein expression by mass cytometry. For IL-5Rα functional studies, plasma cells were purified and cultured in vitro with or without IL-5 and analyzed by bulk RNA sequencing. RESULTS: Compared with polyp tissue from patients with CRSwNP, polyp tissue from patients with AERD contained significantly more ASCs and had increased ASC expression of IL-5Rα. ASCs from patients with AERD expressed higher protein levels of B-cell activation and regulatory markers (CD40, CD19, CD32, and CD38) and the proliferation marker Ki-67. ASCs from patients with AERD also expressed more IL5RA, IGHE, and cell cycle- and proliferation-related transcripts (CCND2, MKI67, CDC25A, and CDC25B) than did ASCs from patients with CRSwNP. Stimulation of plasma cells from patients with AERD with IL-5 induced key cell cycle genes (CCND2 and PTP4A3), whereas IL-5 stimulation of ASCs from patients with CRSwNP induced few transcriptomic changes. CONCLUSION: NP tissue ASCs from patients with AERD express higher levels of functional IL-5Rα and markers associated with cell cycling and proliferation than do ASCs from patients with aspirin-tolerant CRSwNP.

In Vitro Phosphatase Assays for the Eya2 Tyrosine Phosphatase.

Protein tyrosine phosphatases (PTP), such as the Eyes Absent (Eya) family of proteins, play important roles in diverse biological processes. In vitro phosphatase assays are essential tools for characterizing the enzymatic activity as well as discovering inhibitors and regulators of these phosphatases. Two common types of in vitro phosphatase assays use either a small molecule substrate that produces a fluorescent or colored product, or a peptide substrate that produces a colorimetric product in a malachite green assay. In this chapter, we describe detailed protocols of a phosphatase assay using small molecule 3-O-methylfluorescein phosphate (OMFP) as a substrate and a malachite green assay using the pH2AX peptide as a substrate to evaluate the phosphatase activity of EYA2 and the effect of small molecule inhibitors of EYA2. These protocols can be easily adapted to study other protein tyrosine phosphatases.