Mechanisms of Relapse Following Targeted Therapy in An NRASG12V and Mll-AF9 Driven Mouse Model of AML

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2620-2620
Author(s):  
Craig E. Eckfeldt ◽  
Ernesto Diaz-Flores ◽  
Michaeleen D. Diers ◽  
Susan K. Rathe ◽  
Kevin M. Shannon ◽  
...  
Keyword(s):  
Mouse Model ◽  
Expression Analysis ◽  
Targeted Therapies ◽  
Expression Profiles ◽  
Critical Role ◽  
Negative Regulator ◽  
Erk Signaling ◽  
Genetic Mutations ◽  
Ras Signaling ◽  
Equity Ownership

Abstract Abstract 2620 Acute Myeloid Leukemia (AML) is driven by genetic mutations that promote proliferation and prevent maturation of myeloid progenitors. While the majority of adults with AML achieve a remission with aggressive cytotoxic chemotherapy, a large proportion will ultimately die of relapsed/refractory disease. Given the limited efficacy and significant toxicity associated with current AML treatment strategies, new therapies are needed. While many genetic mutations have been shown to contribute to the development AML, the genetic heterogeneity seen in AML presents a challenge for the development of targeted therapies. The NRAS proto-oncogene and other key mediators of RAS signaling are frequently mutated in AML, making RAS-targeted therapies an attractive strategy for the treatment of AML. We previously developed an in vivo genetically-engineered mouse (GEM) model of AML driven by a tetracycline-repressible, constitutively-active form of NRAS, NRASG12V, and the leukemogenic fusion gene Mll-AF9. The leukemia cells in this model are “addicted” to NRASG12V, and inhibiting its expression with doxycycline (dox) results in rapid, complete remission of the AML. However, we have found that some mice relapse with NRASG12V-independent “dox-resistant” disease after continued suppression of NRASG12V expression, a phenomenon we might expect to occur in human AML after Ras signal pathway inhibition. To investigate the mechanisms of relapse in this model we generated two NRASG12V-independent (NRI) AML clones from a single primary NRASG12V-dependent (NRD) AML. We performed Affymetrix-based global gene expression analysis to compare the expression profiles of the primary NRD AML with the relapsed NRI AMLs. We identified 79 genes that were expressed at ≥ 2-fold higher levels in the NRD AML compared to both NRI AMLs. Among these were the putative tumor suppressor Cav2, and a negative regulator of Ras/Raf/Mek/Erk signaling, Dusp6. Down regulation of Dusp6 could enhance Raf/Mek/Erk signaling in the absence of NRASG12V, and thereby circumvent “addiction” to NRASG12V. Expression analysis also identified 20 genes that were expressed at ≥ 2-fold higher levels in both relapsed NRI AMLs compared to the primary NRD AML. Interestingly a Myc oncogene family member, Mycn (N-Myc), was expressed at > 60-fold higher levels in relapsed NRI AMLs compared to the primary NRD AML. Enforced expression of Mycn is sufficient to give rise to AML in a mouse model, and MYCN is widely expressed in human AML (Kawagoe et al. Cancer Res. 2007;67:10677), suggesting a critical role for Mycn in the development of relapsed NRI AML in this model. We are in the process of investigating whether enforced expression of Mycn and/or loss of Dusp6 are sufficient for the development of resistant NRI AML, and conversely if loss of Mycn and/or enforced expression of Dusp6 restore NRASG12V dependence in this model. We are also performing multi-parameter phosho-flow cytometery to further elucidate the mechanisms of AML resistance and relapse and to identify potentially “druggable” targets for AML. This represents an important step toward understanding the genetic determinates of treatment response and disease relapse with Ras pathway targeted therapies for AML, and thus provides a foundation for developing more effective and less toxic therapies for AML. Disclosures: Largaespada: NeoClone Biotechnology, Inc.: Co-founder, Consultancy, Equity Ownership; Discovery Genomics Inc.: Co-founder, Consultancy, Equity Ownership.

NrasQ61R/+ and Kras-/- cooperate to downregulate Rasgrp1 and promote lympho-myeloid leukemia in early T-cell precursors

Blood ◽  
2021 ◽  
Author(s):  
Zhi Wen ◽  
Grant Yun ◽  
Alexander Hebert ◽  
Guangyao Kong ◽  
Erik A. Ranheim ◽  
...  
Keyword(s):  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Transcript Level ◽  
Negative Regulator ◽  
Erk Signaling ◽  
Ras Signaling ◽  
Nucleotide Exchange ◽  
Promote Cell Proliferation ◽  
Oncogenic Ras ◽  
Molecular Features

Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) is an aggressive subtype of T-ALL. Although genetic mutations hyperactivating cytokine receptor/Ras signaling are prevalent in ETP-ALL, it remains unknown how activated Ras signaling contributes to ETP-ALL. Here, we find that in addition to the frequent oncogenic RAS mutations, wild-type (WT) KRAS transcript level was significantly downregulated in human ETP-ALL cells. Similarly, loss of WT Kras in NrasQ61R/+ mice promoted hyperactivation of ERK signaling, thymocyte hyperproliferation, and expansion of ETP compartment. Kras-/-;NrasQ61R/+ mice developed early onset of T-cell malignancy that recapitulates many biological and molecular features of human ETP-ALL. Mechanistically, RNA-Seq analysis and quantitative proteomics study identified that Rasgrp1, a Ras guanine nucleotide exchange factor, was greatly downregulated in mouse and human ETP-ALL. Unexpectedly, hyperactivated Nras/ERK signaling suppressed Rasgrp1 expression and reduced Rasgrp1 level led to increased ERK signaling, thereby establishing a positive feedback loop to augment Nras/ERK signaling and promote cell proliferation. Corroborating our cell line data, Rasgrp1 haploinsufficiency induced Rasgrp1 downregulation, increased pERK level, and ETP expansion in NrasQ61R/+ mice. Our study identifies Rasgrp1 as a negative regulator of Ras/ERK signaling in oncogenic Nras-driven ETP-like leukemia.

Activated NRAS Mediates Self-Renewal Capacity in AML by Facilitating the Mll/AF9-Specified Gene Expression Signature

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5116-5116
Author(s):  
Zohar Sachs ◽  
Hanh Nguyen ◽  
Nurul Azyan Mohd Hassan ◽  
Susan K Rathe ◽  
Karen Sachs ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mouse Model ◽  
Gene Expression Signature ◽  
Mtor Pathway ◽  
Ras Signaling ◽  
Therapeutic Targeting ◽  
Self Renewal ◽  
Expression Signature ◽  
Renewal Program ◽  
Equity Ownership

Abstract Abstract 5116 RAS is a known oncogene in AML (Schubbert et al. Nat Rev Cancer 2007, Bowen et al. Blood 2006), however the specific effects of targeting RAS-activated pathways on AML physiology are unclear. NRASG12V transgene repression in an NRASG12V/Mll/AF9 transgenic murine AML model leads to apoptosis and disease remission (Kim et al. Blood 2009). To better understand possibilities for and implications of therapeutic targeting of RAS-activated pathways, we inactivate NRASG12V in this mouse model and characterize the subsequent signaling and transcriptional response. We profiled signaling intermediates and markers of apoptosis and cell cycle using mass cytometry, a next-generation flow cytometry technology, which simultaneously measured the levels of 32 antibody-labeled proteins in single cells. These analyses revealed specific signaling changes that varied with the surface immunophenotype of the AML cells and highlighted relevant RAS-directed signaling pathways. Parallel RNA sequencing and gene expression microarrays revealed that NRASG12V-expressing cells express hematopoietic self-renewal genes and repression of NRASG12V leads to loss of this program. Importantly, the NRASG12V-dependent gene expression program mimics the Mll/AF9-determined, Myb-mediated self-renewal program reported by Zuber et al. (Zuber et al. Genes Dev 2011). These data suggest a novel role for RAS in AML self-renewal capacity by maintaining the Mll/AF9-mediated self-renewal program. Furthermore, inhibition of the PI3K-mTOR pathway decreases viability, in vitro colony formation, and recapitulates the effects of NRASG12V inactivation on self renewal-associated genes, implicating this pathway as the mediator of RAS-directed leukemia self renewal capacity. These data provide rationale for therapeutic targeting of leukemia stem cells via PI3K-mTOR pathway inhibition and for using this mouse model as a tool to test such therapeutic approaches. Since a variety of AML-specific genetic mutations lead to perturbations in RAS signaling, these results may be generalizable to AML with a broader range of mutations. Figure: NRAS-dependent gene expression signature recapitulates Mll/AF9 gene expression signature. Figure:. NRAS-dependent gene expression signature recapitulates Mll/AF9 gene expression signature. Disclosures: Largaespada: Discovery Genomics, Inc. : Consultancy, Equity Ownership; NeoClone Biotechnology, Inc. : Consultancy, Equity Ownership.

scholarly journals Whole Exome Sequencing of Pediatric Acute Lymphoblastic Leukemia Patients Identify Mutations in 11 Pathways: A Report from the Children's Oncology Group

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 455-455
Author(s):  
Mignon L. Loh ◽  
Jinghui Zhang ◽  
Deqing Pei ◽  
Yunfeng Dai ◽  
Xiaotu Ma ◽  
...  
Keyword(s):  
Lymphoblastic Leukemia ◽  
B Cell Development ◽  
Genetic Mutations ◽  
Ras Signaling ◽  
Childhood All ◽  
Cytoplasmic Transport ◽  
Whole Exome ◽  
Increased Risk ◽  
Equity Ownership ◽  
Risk Of Relapse

Abstract Survival for childhood acute lymphoblastic leukemia (ALL) now approaches 90% with risk adapted therapy based on National Cancer Institute risk group (NCI RG) at diagnosis, somatic lymphoblast genetics, and early response to therapy as measured by minimal residual disease (MRD). Recent studies have identified multiple somatic genetic mutations in ALL, some of which confer increased risk of relapse or identify opportunities for additional targeted therapies. However, there are few genome sequencing analyses of representative cohorts of childhood ALL treated on contemporary regimens. To define the mutational landscape of childhood B-ALL, we performed whole exome sequencing (WES) on diagnostic tumor and remission samples of 192 patients with B-ALL consecutively enrolled between 4/1/06-9/8/06 on the Children's Oncology Group AALL03B1 classification trial that enrolled 11,145 patients up to age 30, which included patients enrolled on clinical trials for standard-risk (SR) B-ALL (N=5226, age 1-10 years and white blood cell count (WBC) < 50,000/uL) and high-risk (HR) B-ALL (N=2907; age ≥10 years or WBC ≥50,000/uL). An 8-gene expression low density array card identified Ph-like patients, and single nucleotide polymorphism arrays and multiplex ligation assays were used to determine DNA copy number alterations. Approximately 2/3 NCI SR and 1/3 NCI HR patients with sufficient banked samples were selected to reflect a population-based cohort (Table 1). Comparison of those selected vs. those not revealed significantly more NCI SR patients with a higher WBC and MRD, and fewer with double trisomy 4 and 10. Selected NCI HR patients were younger, had a higher WBC, and had more ETV6/RUNX1. There were a total of 3576 non-silent mutations with a median of 15 mutations/case (range 1-134). One case had 102 non-silent mutations and a germline mutation in MSH3. The additional mutations clustered in 11 pathways (Table 2), several of which were novel including cell-matrix interaction (e.g. SSPO, FAT1) and intracellular trafficking/cytoplasmic transport (e.g. DYNC2H1, ANK3, UNC13C). Most commonly altered were B-cell development (49.4%), transcription factors (45.8%), tumor suppressor genes (32.7%), cytoplasmic transport (27.3%), and Ras signaling (26.7%). Several pathways Ras signaling, Jak/STAT, and transcription factor mutations/deletions were associated with genetic lesions commonly used for risk stratification. Other pathways (epigenetic, B-cell development, or tumor suppressor) occurred in all subtypes. The most commonly identified genetic mutations were NRAS (n=33), KRAS (n=26), FLT3 (n=13), PAX5 (n=10), CREBBP (n=10), XBP1 (n=9), WHSC1 (n=7), and UBA2 (n=7). XBP1 and UBA2 mutations were novel. XBP1 (X-Box binding Protein 1) encodes a transcription factor that regulates the unfolded protein response. UBA2 (ubiquitin like modifier activating enzyme 2) encodes a protein involved in sumoylation to regulate protein structure and intracellular localization. Univariable analysis revealed no significant associations with any of these pathways or mutations with an increased risk of relapse with the exception of IKZF1 mutations or deletions (n=36; p=0.0087). Multivariable analysis modeling including IKZF1, age, presenting WBC, gender, NCI RG, ETV6/RUNX1, BCR/ABL1, Ph-like, white race, and MRD revealed only BCR/ABL1 and MRD positivity being significantly predictive of relapse. However, the risk of relapse was significantly increased based on the number of mutations identified in any one single patient (HR 1.02, 95% CI 1.01-1.023, p < 0.0004). Of note, only 18 patients (NCI SR, n=8, NCI HR, n=10) were Ph-like in this cohort, likely explaining the lack of significance between Ph-like status and an increased risk of relapse in this analysis. In summary, WES of a consecutively enrolled cohort of NCI SR and HR patients revealed a large number of novel genetic mutations that could be broadly assigned to 11 classes. Outcomes for patients overall were not influenced by any one of these classes, demonstrating that sentinel genetic alterations currently used in risk stratification are of paramount importance in directing therapy intensification for ALL. These data provide important information about pathways commonly mutated in childhood ALL, identifying classes of drugs that can be considered for clinical testing to further improve outcome. Disclosures Loh: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding. Hunger:Amgen: Equity Ownership; Pfizer: Equity Ownership; Merck: Equity Ownership; Jazz Pharmaceuticals: Honoraria; Sigma Tau Pharmaceuticals: Honoraria; Erytech: Honoraria; Patent: Patents & Royalties: Dr. Hunger is a co-inventor of a patent (#8658,964) for the identification of novel subgroups in high risk B-ALL and outcome correlations and diagnostic methods related to the same; Spectrum Pharmaceuticals: Honoraria.

Pathogenic CARD11 mutations affect B cell development and differentiation through a noncanonical pathway

2019 ◽  
Vol 4 (41) ◽  
pp. eaaw5618 ◽  
Author(s):  
Zheng Wei ◽  
Yan Zhang ◽  
Jingjing Chen ◽  
Yu Hu ◽  
Pan Jia ◽  
...  
Keyword(s):  
B Cells ◽  
Mouse Model ◽  
B Cell ◽  
Critical Role ◽  
Cell Development ◽  
B Cell Development ◽  
Negative Regulator ◽  
Regulatory Function ◽  
Akt Activation ◽  
Development And Differentiation

Pathogenic CARD11 mutations cause aberrant nuclear factor κB (NF-κB) activation, which is presumably responsible for multiple immunological disorders. However, whether there is an NF-κB–independent regulatory mechanism contributing to CARD11 mutations related to pathogenesis remains undefined. Using three distinct genetic mouse models, the Card11 knockout (KO) mouse model mimicking primary immunodeficiency, the CARD11 E134G point mutation mouse model representing BENTA (B cell expansion with NF-κB and T cell anergy) disease, and the mouse model bearing oncogenic K215M mutation, we show that CARD11 has a noncanonical function as a negative regulator of the AKT-FOXO1 signal axis, independent of NF-κB activation. Although BENTA disease–related E134G mutant elevates NF-κB activation, we find that E134G mutant mice phenotypically copy Card11 KO mice, in which NF-κB activation is disrupted. Mechanistically, the E134G mutant causes exacerbated AKT activation and reduced FOXO1 protein in B cells similar to that in Card11 KO cells. Moreover, the oncogenic CARD11 mutant K215M reinforces the importance of the noncanonical function of CARD11. In contrast to the E134G mutant, K215M shows a stronger inhibitory effect on AKT activation and more stabilized FOXO1. Likewise, E134G and K215M mutants have converse impacts on B cell development and differentiation. Our results demonstrate that, besides NF-κB, CARD11 also governs the AKT/FOXO1 signaling pathway in B cells. The critical role of CARD11 is further revealed by the effects of pathogenic CARD11 mutants on this noncanonical regulatory function on the AKT-FOXO1 signaling axis.

scholarly journals Characterization of somatic mutation-associated microenvironment signatures in acute myeloid leukemia patients based on TCGA analysis

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jun Wang ◽  
Feng-Ting Dao ◽  
Lu Yang ◽  
Ya-Zhen Qin
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Risk Stratification ◽  
Myeloid Leukemia ◽  
Expression Profiles ◽  
Critical Role ◽  
Genetic Mutation ◽  
Functional Enrichment ◽  
Genetic Mutations ◽  
Acute Myeloid

Abstract Recurrent genetic mutations occur in acute myeloid leukemia (AML) and have been incorporated into risk stratification to predict the prognoses of AML patients. The bone marrow microenvironment plays a critical role in the development and progression of AML. However, the characteristics of the genetic mutation-associated microenvironment have not been comprehensively identified to date. In this study, we obtained the gene expression profiles of 173 AML patients from The Cancer Genome Atlas (TCGA) database and calculated their immune and stromal scores by applying the ESTIMATE algorithm. Immune scores were significantly associated with OS and cytogenetic risk. Next, we categorized the intermediate and poor cytogenetic risk patients into individual-mutation and wild-type groups according to RUNX1, ASXL1, TP53, FLT3-ITD, NPM1 and biallelic CEBPA mutation status. The relationships between the immune microenvironment and each genetic mutation were investigated by identifying differentially expressed genes (DEGs) and conducting functional enrichment analyses of them. Significant immune- and stromal-relevant DEGs associated with each mutation were identified, and most of the DEGs (from the FLT3-ITD, NPM1 and biallelic CEBPA mutation groups) were validated in the GSE14468 cohort downloaded from the Gene Expression Omnibus (GEO) database. In summary, we identified key immune- and stromal-relevant gene signatures associated with genetic mutations in AML, which may provide new biomarkers for risk stratification and personalized immunotherapy.

scholarly journals Comparative Transcriptome Analysis of the Expression of Antioxidant and Immunity Genes in the Spleen of a Cyanidin 3-O-Glucoside-Treated Alzheimer’s Mouse Model

Antioxidants ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1435
Author(s):  
Varun Jaiswal ◽  
Miey Park ◽  
Hae-Jeung Lee
Keyword(s):  
Gene Expression ◽  
Mouse Model ◽  
Transcriptome Analysis ◽  
Immune Modulation ◽  
Expression Profiles ◽  
Critical Role ◽  
Therapeutic Effects ◽  
Comparative Transcriptome ◽  
Antioxidant Genes ◽  
Comparative Transcriptome Analysis

Cyanidin 3-O-glucoside (C3G) is a well-known antioxidant found as a dietary anthocyanin in different fruits and vegetables. It has protective and therapeutic effects on various diseases. It can reduce neuronal death from amyloid-beta (Aβ)-induced toxicity and promote the inhibition of Aβ fibrillization. Antioxidant and immune modulation might play a critical role in the properties of C3G against Alzheimer’s disease (AD) and other diseases. However, limited studies have been performed on the mechanism involved in the effect of C3G through transcriptome analysis. Thus, the objective of this study was to perform comparative transcriptome analysis of the spleen to determine gene expression profiles of wild-type mice (C57BL/6J Jms), an Alzheimer’s mouse model (APPswe/PS1dE9 mice), and a C3G-treated Alzheimer’s mouse model. Differentially expressed antioxidant, immune-related, and AD pathways genes were identified in the treated group. The validation of gene expression data via RT-PCR studies further supported the current findings. Six important antioxidant genes (S100a8, S100a9, Prdx2, Hp, Mpst, and Prxl2a) and a high number of immune-related genes were found to be upregulated in the treatment groups, suggesting the possible antioxidant and immunomodulatory mechanisms of C3G, respectively. Further studies are strongly recommended to elucidate the precise role of these essential genes and optimize the therapeutic function of C3G in AD and other disease conditions.

scholarly journals MAP kinase phosphatase MKP-1 regulates p-ERK1/2 signaling pathway with fluoride treatment

2020 ◽  
Author(s):  
Lin Zhao ◽  
Jiali Su ◽  
Sijia Liu ◽  
Yang Li ◽  
Tao Li ◽  
...  
Keyword(s):  
Map Kinase ◽  
Molecular Mechanisms ◽  
Tooth Enamel ◽  
Dental Fluorosis ◽  
Critical Role ◽  
Negative Regulator ◽  
Erk Signaling ◽  
High Dose ◽  
Dependent Manner ◽  
Map Kinase Phosphatase

Abstract Background Dental fluorosis is characterized by hypomineralization of tooth enamel caused by ingestion of excessive fluoride during enamel formation. Excess fluoride could have effects on the ERK signaling, which is essential for the ameloblasts differentiation and tooth development. MAP kinase phosphatase-1 (MKP-1) plays a critical role in regulating ERK related kinases. However, the role of MKP-1 in ameloblast and the mechanisms of MKP-1/ERK signaling in the pathogenesis of dental fluorosis are incompletely understood. Results Here, we adopted an in vitro fluorosis cell model using murine ameloblasts-like LS8 cells by employing sodium fluoride (NaF) as inducer. Using this system, we demonstrated that fluoride exposure led to an inhibition of p- MEK and p-ERK1/2 with a subsequent increase in MKP-1 expression in a dose-dependent manner. We further identified, under high dose fluoride, MKP-1 acted as a negative regulator of the fluoride-induced p-ERK1/2 signaling, leading to downregulation of CREB, c-myc, and Elk-1. Conclusion Our results identify a novel MKP-1/ERK signaling mechanism that regulates dental fluorosis and provide a framework for studying the molecular mechanisms of intervention and fluorosis remodeling under normal and pathological conditions. MKP-1 inhibitors may prove to be a benefit therapeutic strategy for dental fluorosis treatment.

scholarly journals Genome-wide identification of cyclin-dependent kinase (CDK) genes affecting adipocyte differentiation in cattle

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Cuili Pan ◽  
Zhaoxiong Lei ◽  
Shuzhe Wang ◽  
Xingping Wang ◽  
Dawei Wei ◽  
...  
Keyword(s):  
Gene Family ◽  
Expression Analysis ◽  
Adipocyte Differentiation ◽  
Bos Taurus ◽  
Adipogenic Differentiation ◽  
Critical Role ◽  
Bos Indicus ◽  
Specific Expression ◽  
Genome Wide ◽  
A Genome

Abstract Background Cyclin-dependent kinases (CDKs) are protein kinases regulating important cellular processes such as cell cycle and transcription. Many CDK genes also play a critical role during adipogenic differentiation, but the role of CDK gene family in regulating bovine adipocyte differentiation has not been studied. Therefore, the present study aims to characterize the CDK gene family in bovine and study their expression pattern during adipocyte differentiation. Results We performed a genome-wide analysis and identified a number of CDK genes in several bovine species. The CDK genes were classified into 8 subfamilies through phylogenetic analysis. We found that 25 bovine CDK genes were distributed in 16 different chromosomes. Collinearity analysis revealed that the CDK gene family in Bos taurus is homologous with Bos indicus, Hybrid-Bos taurus, Hybrid Bos indicus, Bos grunniens and Bubalus bubalis. Several CDK genes had higher expression levels in preadipocytes than in differentiated adipocytes, as shown by RNA-seq analysis and qPCR, suggesting a role in the growth of emerging lipid droplets. Conclusion In this research, 185 CDK genes were identified and grouped into eight distinct clades in Bovidae, showing extensively homology. Global expression analysis of different bovine tissues and specific expression analysis during adipocytes differentiation revealed CDK4, CDK7, CDK8, CDK9 and CDK14 may be involved in bovine adipocyte differentiation. The results provide a basis for further study to determine the roles of CDK gene family in regulating adipocyte differentiation, which is beneficial for beef quality improvement.

scholarly journals A Novel Cysteine Protease Inhibitor of Naegleria fowleri That Is Specifically Expressed during Encystation and at Mature Cysts

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 388
Author(s):  
Hương Giang Lê ◽  
A-Jeong Ham ◽  
Jung-Mi Kang ◽  
Tuấn Cường Võ ◽  
Haung Naw ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Cysteine Protease ◽  
Expression Profiles ◽  
Critical Role ◽  
Cyst Formation ◽  
Cysteine Proteases ◽  
Cysteine Protease Inhibitor ◽  
Naegleria Fowleri ◽  
Nægleria Fowleri ◽  
Cystatin Family

Naegleria fowleri is a free-living amoeba that is ubiquitous in diverse natural environments. It causes a fatal brain infection in humans known as primary amoebic meningoencephalitis. Despite the medical importance of the parasitic disease, there is a great lack of knowledge about the biology and pathogenicity of N. fowleri. In this study, we identified and characterized a novel cysteine protease inhibitor of N. fowleri (NfCPI). NfCPI is a typical cysteine protease inhibitor belonging to the cystatin family with a Gln-Val-Val-Ala-Gly (QVVAG) motif, a characteristic motif conserved in the cystatin family of proteins. Bacterially expressed recombinant NfCPI has a dimeric structure and exhibits inhibitory activity against several cysteine proteases including cathespin Bs of N. fowleri at a broad range of pH values. Expression profiles of nfcpi revealed that the gene was highly expressed during encystation and cyst of the amoeba. Western blot and immunofluorescence assays also support its high level of expression in cysts. These findings collectively suggest that NfCPI may play a critical role in encystation or cyst formation of N. fowleri by regulating cysteine proteases that may mediate encystation or mature cyst formation of the amoeba. More comprehensive studies to investigate the roles of NfCPI in encystation and its target proteases are necessary to elucidate the regulatory mechanism and the biological significance of NfCPI.

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