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1.
Blood ; 141(8): 917-929, 2023 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-36356299

RESUMO

Mutant calreticulin (CALR) proteins resulting from a -1/+2 frameshifting mutation of the CALR exon 9 carry a novel C-terminal amino acid sequence and drive the development of myeloproliferative neoplasms (MPNs). Mutant CALRs were shown to interact with and activate the thrombopoietin receptor (TpoR/MPL) in the same cell. We report that mutant CALR proteins are secreted and can be found in patient plasma at levels up to 160 ng/mL, with a mean of 25.64 ng/mL. Plasma mutant CALR is found in complex with soluble transferrin receptor 1 (sTFR1) that acts as a carrier protein and increases mutant CALR half-life. Recombinant mutant CALR proteins bound and activated the TpoR in cell lines and primary megakaryocytic progenitors from patients with mutated CALR in which they drive thrombopoietin-independent colony formation. Importantly, the CALR-sTFR1 complex remains functional for TpoR activation. By bioluminescence resonance energy transfer assay, we show that mutant CALR proteins produced in 1 cell can specifically interact in trans with the TpoR on a target cell. In comparison with cells that only carry TpoR, cells that carry both TpoR and mutant CALR are hypersensitive to exogenous mutant CALR proteins and respond to levels of mutant CALR proteins similar to those in patient plasma. This is consistent with CALR-mutated cells that expose TpoR carrying immature N-linked sugars at the cell surface. Thus, secreted mutant CALR proteins will act more specifically on the MPN clone. In conclusion, a chaperone, CALR, can turn into a rogue cytokine through somatic mutation of its encoding gene.


Assuntos
Transtornos Mieloproliferativos , Neoplasias , Humanos , Citocinas/metabolismo , Calreticulina/genética , Transtornos Mieloproliferativos/genética , Mutação , Fatores Imunológicos , Janus Quinase 2/genética
2.
Blood ; 137(14): 1920-1931, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33202418

RESUMO

Somatic mutations of calreticulin (CALR) have been identified as a main disease driver of myeloproliferative neoplasms, suggesting that development of drugs targeting mutant CALR is of great significance. Site-directed mutagenesis in the N-glycan binding domain (GBD) abolishes the ability of mutant CALR to oncogenically activate the thrombopoietin receptor (MPL). We therefore hypothesized that a small molecule targeting the GBD might inhibit the oncogenicity of the mutant CALR. Using an in silico molecular docking study, we identified candidate binders to the GBD of CALR. Further experimental validation of the hits identified a group of catechols inducing a selective growth inhibitory effect on cells that depend on oncogenic CALR for survival and proliferation. Apoptosis-inducing effects by the compound were significantly higher in the CALR-mutated cells than in CALR wild-type cells. Additionally, knockout or C-terminal truncation of CALR eliminated drug hypersensitivity in CALR-mutated cells. We experimentally confirmed the direct binding of the selected compound to CALR, disruption of the mutant CALR-MPL interaction, inhibition of the JAK2-STAT5 pathway, and reduction at the intracellular level of mutant CALR upon drug treatment. Our data indicate that small molecules targeting the GBD of CALR can selectively kill CALR-mutated cells by disrupting the CALR-MPL interaction and inhibiting oncogenic signaling.


Assuntos
Calreticulina/metabolismo , Hematoxilina/farmacologia , Mapas de Interação de Proteínas/efeitos dos fármacos , Receptores de Trombopoetina/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Calreticulina/química , Calreticulina/genética , Linhagem Celular , Descoberta de Drogas , Humanos , Camundongos , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Mutação , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/metabolismo , Ligação Proteica/efeitos dos fármacos , Receptores de Trombopoetina/química
3.
Blood ; 133(25): 2669-2681, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-30902807

RESUMO

Calreticulin (CALR) +1 frameshift mutations in exon 9 are prevalent in myeloproliferative neoplasms. Mutant CALRs possess a new C-terminal sequence rich in positively charged amino acids, leading to activation of the thrombopoietin receptor (TpoR/MPL). We show that the new sequence endows the mutant CALR with rogue chaperone activity, stabilizing a dimeric state and transporting TpoR and mutants thereof to the cell surface in states that would not pass quality control; this function is absolutely required for oncogenic transformation. Mutant CALRs determine traffic via the secretory pathway of partially immature TpoR, as they protect N117-linked glycans from further processing in the Golgi apparatus. A number of engineered or disease-associated TpoRs such as TpoR/MPL R102P, which causes congenital thrombocytopenia, are rescued for traffic and function by mutant CALRs, which can also overcome endoplasmic reticulum retention signals on TpoR. In addition to requiring N-glycosylation of TpoR, mutant CALRs require a hydrophobic patch located in the extracellular domain of TpoR to induce TpoR thermal stability and initial intracellular activation, whereas full activation requires cell surface localization of TpoR. Thus, mutant CALRs are rogue chaperones for TpoR and traffic-defective TpoR mutants, a function required for the oncogenic effects.


Assuntos
Calreticulina/genética , Calreticulina/metabolismo , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Receptores de Trombopoetina/metabolismo , Animais , Humanos , Camundongos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Transporte Proteico/fisiologia
4.
J Allergy Clin Immunol ; 144(1): 224-235, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30707971

RESUMO

BACKGROUND: Janus kinase (JAK) 2 plays pivotal roles in signaling by several cytokine receptors. The mutant JAK2 V617F is the most common molecular event associated with myeloproliferative neoplasms. Selective targeting of the mutant would be ideal for treating these pathologies by sparing essential JAK2 functions. OBJECTIVE: We characterize inhibitory strategies for JAK2 V617F and assess their effect on physiologic signaling by distinct cytokine receptors. METHODS: Through structure-guided mutagenesis, we assessed the role of key residues around F617 and used a combination of cellular and biochemical assays to measure the activity of JAKs in reconstituted cells. We also assessed the effect of several specific JAK2 V617F inhibitory mutations on receptor dimerization using the NanoBiT protein complementation approach. RESULTS: We identified a novel Janus kinase homology 2 (JH2) αC mutation, A598F, which is suggested to inhibit the aromatic stacking between F617 with F594 and F595. Like other JAK2 V617F inhibitory mutations, A598F decreased oncogenic activation and spared cytokine activation while preventing JAK2 V617F-promoted erythropoietin receptor dimerization. Surprisingly, A598F and other V617F-inhibiting mutations (F595A, E596R, and F537A) significantly impaired IFN-γ signaling. This was specific for IFN-γ because the inhibitory mutations preserved responses to ligands of a series of receptor complexes. Similarly, homologous mutations in JAK1 prevented signaling by IFN-γ. CONCLUSIONS: The JH2 αC region, which is required for JAK2 V617F hyperactivation, is crucial for relaying cytokine-induced signaling of the IFN-γ receptor. We discuss how strategies aiming to inhibit JAK2 V617F could be used for identifying inhibitors of IFN-γ signaling.


Assuntos
Janus Quinase 2/genética , Receptores de Citocinas/metabolismo , Animais , Linhagem Celular , Humanos , Janus Quinase 2/metabolismo , Camundongos , Mutação , Transdução de Sinais
5.
Haematologica ; 103(4): 575-586, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29269524

RESUMO

Primary familial and congenital polycythemia is characterized by erythropoietin hypersensitivity of erythroid progenitors due to germline nonsense or frameshift mutations in the erythropoietin receptor gene. All mutations so far described lead to the truncation of the C-terminal receptor sequence that contains negative regulatory domains. Their removal is presented as sufficient to cause the erythropoietin hypersensitivity phenotype. Here we provide evidence for a new mechanism whereby the presence of novel sequences generated by frameshift mutations is required for the phenotype rather than just extensive truncation resulting from nonsense mutations. We show that the erythropoietin hypersensitivity induced by a new erythropoietin receptor mutant, p.Gln434Profs*11, could not be explained by the loss of negative signaling and of the internalization domains, but rather by the appearance of a new C-terminal tail. The latter, by increasing erythropoietin receptor dimerization, stability and cell-surface localization, causes pre-activation of erythropoietin receptor and JAK2, constitutive signaling and hypersensitivity to erythropoietin. Similar results were obtained with another mutant, p.Pro438Metfs*6, which shares the same last five amino acid residues (MDTVP) with erythropoietin receptor p.Gln434Profs*11, confirming the involvement of the new peptide sequence in the erythropoietin hypersensitivity phenotype. These results suggest a new mechanism that might be common to erythropoietin receptor frameshift mutations. In summary, we show that primary familial and congenital polycythemia is more complex than expected since distinct mechanisms are involved in the erythropoietin hypersensitivity phenotype, according to the type of erythropoietin receptor mutation.


Assuntos
Mutação em Linhagem Germinativa , Policitemia/etiologia , Receptores da Eritropoetina/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Eritropoetina/farmacologia , Humanos , Camundongos , Proteínas Mutantes , Policitemia/genética , Multimerização Proteica/genética , Estabilidade Proteica , Receptores da Eritropoetina/metabolismo
6.
PNAS Nexus ; 3(5): pgae184, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38756234

RESUMO

The glycoproteins MICA and MICB are upregulated on the surface of cells undergoing stress, for instance due to (viral) infection or malignant transformation. MICA/B are the ligands for the activating receptor NKG2D, found on cytotoxic immune cells like NK cells, CD8+ T cells, and γδ T cells. Upon engagement of NKG2D, these cells are activated to eradicate the MICA/B-positive targets, assisted by the secretion of cytokines. Nanobodies, or VHHs, are derived from the variable regions of camelid heavy-chain only immunoglobulins. Nanobodies are characterized by their small size, ease of production, stability, and specificity of recognition. We generated nanobodies that recognize membrane-bound MICA with high affinity. Here, we use these nanobodies as building blocks for a chimeric antigen receptor (CAR) to establish VHH-based CAR NK cells. These anti-MICA nanobody-based CAR NK cells recognize and selectively kill MICA-positive tumor cells in vitro and in vivo. We track localization of the VHH-based CAR NK cells to MICA-positive lung metastases by immuno-positron emission tomography imaging.

7.
Res Sq ; 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-39149504

RESUMO

Bispecific antibody engagers are fusion proteins composed of a nanobody that recognizes immunoglobulin kappa light chains ( VHH kappa ) and a nanobody that recognizes either CTLA-4 or PD-L1. These fusions show strong antitumor activity in mice through recruitment of polyclonal immunoglobulins independently of specificity or isotype. In the MC38 mouse model of colorectal carcinoma, the anti-CTLA-4 VHH-VHH kappa conjugate eradicates tumors and reduces the number of intratumoral regulatory T cells. The anti-PD-L1 VHH-VHH kappa conjugate is less effective in the MC38 model, whilst still outperforming an antibody of similar specificity. The potency of the anti-PD-L1 VHH-VHH kappa conjugate was strongly enhanced by installation of the cytotoxic drug maytansine or a STING agonist. The ability of such fusions to engage the Fc-mediated functions of all immunoglobulin isotypes is an appealing strategy to further improve on the efficacy of immune checkpoint blockade, commonly delivered as a monoclonal immunoglobulin of a single defined isotype.

8.
J Biol Chem ; 287(10): 7246-55, 2012 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-22241472

RESUMO

The purpose of the present work was to identify the catalytic activity of AGXT2L1 and AGXT2L2, two closely related, putative pyridoxal-phosphate-dependent enzymes encoded by vertebrate genomes. The existence of bacterial homologues (40-50% identity with AGXT2L1 and AGXT2L2) forming bi- or tri-functional proteins with a putative kinase belonging to the family of aminoglycoside phosphotransferases suggested that AGXT2L1 and AGXT2L2 acted on phosphorylated and aminated compounds. Vertebrate genomes were found to encode a homologue (AGPHD1) of these putative bacterial kinases, which was therefore likely to phosphorylate an amino compound bearing a hydroxyl group. These and other considerations led us to hypothesize that AGPHD1 corresponded to 5-hydroxy-L-lysine kinase and that AGXT2L1 and AGXT2L2 catalyzed the pyridoxal-phosphate-dependent breakdown of phosphoethanolamine and 5-phosphohydroxy-L-lysine. The three recombinant human proteins were produced and purified to homogeneity. AGPHD1 was indeed found to catalyze the GTP-dependent phosphorylation of 5-hydroxy-L-lysine. The phosphorylation product made by this enzyme was metabolized by AGXT2L2, which converted it to ammonia, inorganic phosphate, and 2-aminoadipate semialdehyde. AGXT2L1 catalyzed a similar reaction on phosphoethanolamine, converting it to ammonia, inorganic phosphate, and acetaldehyde. AGPHD1 and AGXT2L2 are likely to be the mutated enzymes in 5-hydroxylysinuria and 5-phosphohydroxylysinuria, respectively. The high level of expression of AGXT2L1 in human brain, as well as data in the literature linking AGXT2L1 to schizophrenia and bipolar disorders, suggest that these diseases may involve a perturbation of brain phosphoethanolamine metabolism. AGXT2L1 and AGXT2L2, the first ammoniophospholyases to be identified, belong to a family of aminotransferases acting on ω-amines.


Assuntos
Bactérias , Proteínas de Bactérias , Etanolaminas , Genoma Bacteriano/fisiologia , Genoma Humano/fisiologia , Hidroxilisina/análogos & derivados , Transaminases , Animais , Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transtorno Bipolar/enzimologia , Transtorno Bipolar/genética , Etanolaminas/química , Etanolaminas/metabolismo , Humanos , Hidroxilisina/química , Hidroxilisina/metabolismo , Mutação , Esquizofrenia/enzimologia , Esquizofrenia/genética , Homologia de Sequência de Aminoácidos , Transaminases/química , Transaminases/genética , Transaminases/metabolismo
9.
Sci Immunol ; 8(84): eadg9459, 2023 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-37352373

RESUMO

The immune system eliminates pathogen intruders such as viruses and bacteria. To recruit immune effectors to virus-infected cells, we conjugated a small molecule, the influenza neuraminidase inhibitor zanamivir, to a nanobody that recognizes the kappa light chains of mouse immunoglobulins. This adduct was designed to achieve half-life extension of zanamivir through complex formation with the much-larger immunoglobulins in the circulation. The zanamivir moiety targets the adduct to virus-infected cells, whereas the anti-kappa component simultaneously delivers polyclonal immunoglobulins of indeterminate specificity and all isotypes. Activation of antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity promoted elimination of influenza neuraminidase-positive cells. A single dose of the conjugate protected mice against influenza A or B viruses and was effective even when given several days after infection with a lethal dose of virus. In the absence of circulating immunoglobulins, we observed no in vivo protection from the adduct. The type of conjugates described here may thus find application for both anti-influenza prophylaxis and therapy.


Assuntos
Influenza Humana , Zanamivir , Camundongos , Animais , Humanos , Zanamivir/farmacologia , Zanamivir/uso terapêutico , Oseltamivir/farmacologia , Oseltamivir/uso terapêutico , Cadeias Leves de Imunoglobulina/uso terapêutico , Neuraminidase/uso terapêutico , Influenza Humana/prevenção & controle , Camundongos Endogâmicos BALB C
10.
Elife ; 122023 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-37338955

RESUMO

Dimerization of the thrombopoietin receptor (TpoR) is necessary for receptor activation and downstream signaling through activated Janus kinase 2. We have shown previously that different orientations of the transmembrane (TM) helices within a receptor dimer can lead to different signaling outputs. Here we addressed the structural basis of activation for receptor mutations S505N and W515K that induce myeloproliferative neoplasms. We show using in vivo bone marrow reconstitution experiments that ligand-independent activation of TpoR by TM asparagine (Asn) substitutions is proportional to the proximity of the Asn mutation to the intracellular membrane surface. Solid-state NMR experiments on TM peptides indicate a progressive loss of helical structure in the juxtamembrane (JM) R/KWQFP motif with proximity of Asn substitutions to the cytosolic boundary. Mutational studies in the TpoR cytosolic JM region show that loss of the helical structure in the JM motif by itself can induce activation, but only when localized to a maximum of six amino acids downstream of W515, the helicity of the remaining region until Box 1 being required for receptor function. The constitutive activation of TpoR mutants S505N and W515K can be inhibited by rotation of TM helices within the TpoR dimer, which also restores helicity around W515. Together, these data allow us to develop a general model for activation of TpoR and explain the critical role of the JM W515 residue in the regulation of the activity of the receptor.


Assuntos
Receptores de Trombopoetina , Transdução de Sinais , Receptores de Trombopoetina/genética , Receptores de Trombopoetina/metabolismo , Linhagem Celular , Mutação , Estrutura Secundária de Proteína , Transdução de Sinais/genética
11.
Leukemia ; 34(2): 510-521, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31471561

RESUMO

Frameshifting mutations (-1/+2) of the calreticulin (CALR) gene are responsible for the development of essential thrombocythemia (ET) and primary myelofibrosis (PMF). The mutant CALR proteins activate the thrombopoietin receptor (TpoR) inducing cytokine-independent megakaryocyte progenitor proliferation. Here, we generated via CRISPR/Cas9 technology two knock-in mouse models that are heterozygous for a type-I murine Calr mutation. These mice exhibit an ET phenotype with elevated circulating platelets compared with wild-type controls, consistent with our previous results showing that murine CALR mutants activate TpoR. We also show that the mutant CALR proteins can be detected in plasma. The phenotype of Calr del52 is transplantable, and the Calr mutated hematopoietic cells have a slow-rising advantage over wild-type hematopoiesis. Importantly, a homozygous state of a type-1 Calr mutation is lethal at a late embryonic development stage, showing narrowed ventricular myocardium walls, similar to the murine Calr knockout phenotype, pointing to the C terminus of CALR as crucial for heart development.


Assuntos
Calreticulina/genética , Éxons/genética , Coração/fisiologia , Trombocitemia Essencial/genética , Animais , Sistemas CRISPR-Cas/genética , Feminino , Mutação da Fase de Leitura/genética , Hematopoese/genética , Homozigoto , Masculino , Camundongos , Mielofibrose Primária/genética , Receptores de Trombopoetina/genética , Trombocitose/genética
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