RESUMO
Neuropilin-2 (NRP2) is a cell surface receptor that plays key roles in lymphangiogenesis, but also in pathophysiological conditions such as cancer and inflammation. NRP2 targeting by efzofitimod, a novel immunomodulatory molecule, is currently being tested for the treatment of pulmonary sarcoidosis. To date, no anti-NRP2 antibodies are available for companion diagnostics. Here we describe the development and characterization of a novel NRP2 antibody. Using a variety of research techniques, that is, enzyme-linked immunoassay, Western blot, biolayer interferometry, and immunohistochemistry, we demonstrate that our antibody detects all major NRP2 isoforms and does not cross-react with NRP1. Using this antibody, we show high NRP2 expression in granulomas from sarcoidosis patient skin and lung biopsies. Our novel anti-NRP2 antibody could prove to be a useful clinical tool for sarcoidosis and other indications where NRP2 has been implicated. Clinical Trial Registration: clinicaltrials.gov NCT05415137.
Assuntos
Neoplasias , Sarcoidose , Humanos , Neuropilina-2/metabolismo , Anticorpos Monoclonais , Neoplasias/diagnóstico , Imuno-Histoquímica , Sarcoidose/diagnósticoRESUMO
Although blocking the binding of vascular endothelial growth factor (VEGF) to neuropilin-2 (NRP2) on tumor cells is a potential strategy to treat aggressive carcinomas, a lack of effective reagents that can be used clinically has hampered this potential therapy. Here, we describe the generation of a fully humanized, high-affinity monoclonal antibody (aNRP2-10) that specifically inhibits the binding of VEGF to NRP2, conferring antitumor activity without causing toxicity. Using triple-negative breast cancer as a model, we demonstrated that aNRP2-10 could be used to isolate cancer stem cells (CSCs) from heterogeneous tumor populations and inhibit CSC function and epithelial-to-mesenchymal transition. aNRP2-10 sensitized cell lines, organoids, and xenografts to chemotherapy and inhibited metastasis by promoting the differentiation of CSCs to a state that is more responsive to chemotherapy and less prone to metastasis. These data provide justification for the initiation of clinical trials designed to improve the response of patients with aggressive tumors to chemotherapy using this monoclonal antibody.
Assuntos
Neuropilina-2 , Neoplasias de Mama Triplo Negativas , Humanos , Neuropilina-2/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Ligação Proteica , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais/metabolismo , Linhagem Celular Tumoral , Neuropilina-1/metabolismoRESUMO
The autoimmune disease known as Jo-1 positive anti-synthetase syndrome (ASS) is characterized by circulating antibody titers to histidyl-tRNA synthetase (HARS), which may play a role in modulating the non-canonical functions of HARS. Monoclonal antibodies to HARS were isolated by single-cell screening and sequencing from three Jo-1 positive ASS patients and shown to be of high affinity, covering diverse epitope space. The immune response was further characterized by repertoire sequencing from the most productive of the donor samples. In line with previous studies of autoimmune repertoires, these antibodies tended to have long complementarity-determining region H3 sequences with more positive-charged residues than average. Clones of interest were clustered into groups with related sequences, allowing us to observe different somatic mutations in related clones. We postulated that these had found alternate structural solutions for high affinity binding, but that mutations might be transferable between clones to further enhance binding affinity. Transfer of somatic mutations between antibodies within the same clonal group was able to enhance binding affinity in a number of cases, including beneficial transfer of a mutation from a lower affinity clone into one of higher affinity. Affinity enhancement was seen with mutation transfer both between related single-cell clones, and directly from related repertoire sequences. To our knowledge, this is the first demonstration of somatic hypermutation transfer from repertoire sequences to further mature in vivo derived antibodies, and represents an additional tool to aid in affinity maturation for the development of antibodies.
Assuntos
Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos/imunologia , Autoanticorpos/imunologia , Técnicas Imunológicas/métodos , Miosite/imunologia , Anticorpos Monoclonais/isolamento & purificação , Autoanticorpos/isolamento & purificação , Histidina-tRNA Ligase/imunologia , Humanos , Hipermutação Somática de Imunoglobulina/imunologiaRESUMO
Throughout three domains of life, alanyl-tRNA synthetases (AlaRSs) recognize a G3:U70 base pair in the acceptor stem of tRNAAla as the major identity determinant of tRNAAla The crystal structure of the archaeon Archaeoglobus fulgidus AlaRS in complex with tRNAAla provided the basis for G3:U70 recognition with residues (Asp and Asn) that are conserved in the three domains [Naganuma M, et al. (2014) Nature 510:507-511]. The recognition mode is unprecedented, with specific accommodation of the dyad asymmetry of the G:U wobble pair and exclusion of the dyad symmetry of a Watson-Crick pair. With this conserved mode, specificity is based more on "fit" than on direct recognition of specific atomic groups. Here, we show that, in contrast to the archaeal complex, the Escherichia coli enzyme uses direct positive (energetically favorable) minor groove recognition of the unpaired 2-amino of G3 by Asp and repulsion of a competing base pair by Asn. Strikingly, mutations that disrupted positive recognition by the E. coli enzyme had little or no effect on G:U recognition by the human enzyme. Alternatively, Homo sapiens AlaRS selects G:U without positive recognition and uses Asp instead to repel a competitor. Thus, the widely conserved Asp-plus-Asn architecture of AlaRSs can select G:U in a straightforward (bacteria) or two different unconventional (eukarya/archaea) ways. The adoption of different modes for recognition of a widely conserved G:U pair in alanine tRNAs suggests an early and insistent role for G:U in the development of the genetic code.
Assuntos
Alanina-tRNA Ligase/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Modelos Moleculares , Motivos de Nucleotídeos , RNA de Transferência/química , Alanina-tRNA Ligase/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Humanos , Mutação , RNA de Transferência/genéticaRESUMO
The tRNA synthetases catalyze the first step of protein synthesis and have increasingly been studied for their nuclear and extra-cellular ex-translational activities. Human genetic conditions such as Charcot-Marie-Tooth have been attributed to dominant gain-of-function mutations in some tRNA synthetases. Unlike dominantly inherited gain-of-function mutations, recessive loss-of-function mutations can potentially elucidate ex-translational activities. We present here five individuals from four families with a multi-system disease associated with bi-allelic mutations in FARSB that encodes the beta chain of the alpha2beta2 phenylalanine-tRNA synthetase (FARS). Collectively, the mutant alleles encompass a 5'-splice junction non-coding variant (SJV) and six missense variants, one of which is shared by unrelated individuals. The clinical condition is characterized by interstitial lung disease, cerebral aneurysms and brain calcifications, and cirrhosis. For the SJV, we confirmed exon skipping leading to a frameshift associated with noncatalytic activity. While the bi-allelic combination of the SJV with a p.Arg305Gln missense mutation in two individuals led to severe disease, cells from neither the asymptomatic heterozygous carriers nor the compound heterozygous affected individual had any defect in protein synthesis. These results support a disease mechanism independent of tRNA synthetase activities in protein translation and suggest that this FARS activity is essential for normal function in multiple organs.
Assuntos
Aminoacil-tRNA Sintetases/genética , Pneumopatias/genética , Mutação/genética , Adolescente , Alelos , Doença de Charcot-Marie-Tooth/genética , Pré-Escolar , Feminino , Genes Recessivos/genética , Heterozigoto , Humanos , Lactente , Masculino , Biossíntese de Proteínas/genéticaRESUMO
The question of how dispersed mutations in one protein engender the same gain-of-function phenotype is of great interest. Here we focus on mutations in glycyl-tRNA synthetase (GlyRS) that cause an axonal form of Charcot-Marie-Tooth (CMT) diseases, the most common hereditary peripheral neuropathies. Because the disease phenotype is dominant, and not correlated with defects in the role of GlyRS in protein synthesis, the mutant proteins are considered to be neomorphs that gain new functions from altered protein structure. Given that previous crystal structures showed little conformational difference between dimeric wild-type and CMT-causing mutant GlyRSs, the mutant proteins were investigated in solution by hydrogen-deuterium exchange (monitored by mass spectrometry) and small-angle X-ray scattering to uncover structural changes that could be suppressed by crystal packing interactions. Significantly, each of five spatially dispersed mutations induced the same conformational opening of a consensus area that is mostly buried in the wild-type protein. The identified neomorphic surface is thus a candidate for making CMT-associated pathological interactions, and a target for disease correction. Additional result showed that a helix-turn-helix WHEP domain that was appended to GlyRS in metazoans can regulate the neomorphic structural change, and that the gain of function of the CMT mutants might be due to the loss of function of the WHEP domain as a regulator. Overall, the results demonstrate how spatially dispersed and seemingly unrelated mutations can perpetrate the same localized effect on a protein.
Assuntos
Proteínas Mutantes/química , Proteínas Mutantes/genética , Sequência de Aminoácidos , Fenômenos Biofísicos , Doença de Charcot-Marie-Tooth/enzimologia , Doença de Charcot-Marie-Tooth/genética , Medição da Troca de Deutério , Dimerização , Glicina-tRNA Ligase/química , Glicina-tRNA Ligase/genética , Humanos , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
A novel aminoacyl-tRNA synthetase that contains an iron-sulfur cluster in the tRNA anticodon-binding region and efficiently charges tRNA with tryptophan has been found in Thermotoga maritima. The crystal structure of TmTrpRS (tryptophanyl-tRNA synthetase; TrpRS; EC 6.1.1.2) reveals an iron-sulfur [4Fe-4S] cluster bound to the tRNA anticodon-binding (TAB) domain and an L-tryptophan ligand in the active site. None of the other T. maritima aminoacyl-tRNA synthetases (AARSs) contain this [4Fe-4S] cluster-binding motif (C-x22-C-x6-C-x2-C). It is speculated that the iron-sulfur cluster contributes to the stability of TmTrpRS and could play a role in the recognition of the anticodon.
Assuntos
Proteínas Ferro-Enxofre/química , Thermotoga maritima/enzimologia , Triptofano-tRNA Ligase/química , Sequência de Aminoácidos , Animais , Sequência Conservada , Cristalografia por Raios X , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Mistranslation arising from confusion of serine for alanine by alanyl-tRNA synthetases (AlaRSs) has profound functional consequences. Throughout evolution, two editing checkpoints prevent disease-causing mistranslation from confusing glycine or serine for alanine at the active site of AlaRS. In both bacteria and mice, Ser poses a bigger challenge than Gly. One checkpoint is the AlaRS editing centre, and the other is from widely distributed AlaXps-free-standing, genome-encoded editing proteins that clear Ser-tRNA(Ala). The paradox of misincorporating both a smaller (glycine) and a larger (serine) amino acid suggests a deep conflict for nature-designed AlaRS. Here we show the chemical basis for this conflict. Nine crystal structures, together with kinetic and mutational analysis, provided snapshots of adenylate formation for each amino acid. An inherent dilemma is posed by constraints of a structural design that pins down the alpha-amino group of the bound amino acid by using an acidic residue. This design, dating back more than 3 billion years, creates a serendipitous interaction with the serine OH that is difficult to avoid. Apparently because no better architecture for the recognition of alanine could be found, the serine misactivation problem was solved through free-standing AlaXps, which appeared contemporaneously with early AlaRSs. The results reveal unconventional problems and solutions arising from the historical design of the protein synthesis machinery.
Assuntos
Alanina-tRNA Ligase/metabolismo , Alanina/metabolismo , Escherichia coli/enzimologia , Biossíntese de Proteínas , Serina/metabolismo , Alanina-tRNA Ligase/química , Alanina-tRNA Ligase/genética , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Domínio Catalítico , Cristalização , Cinética , Modelos Moleculares , Mutação , Conformação Proteica , RNA de Transferência de Alanina/metabolismo , Relação Estrutura-AtividadeRESUMO
Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs for protein synthesis. However, the aminoacylation reaction can be diverted to produce diadenosine tetraphosphate (Ap4A), a universal pleiotropic signaling molecule needed for cell regulation pathways. The only known mechanism for Ap4A production by a tRNA synthetase is through the aminoacylation reaction intermediate aminoacyl-AMP, thus making Ap4A synthesis amino acid-dependent. Here, we demonstrate a new mechanism for Ap4A synthesis. Crystal structures and biochemical analyses show that human glycyl-tRNA synthetase (GlyRS) produces Ap4A by direct condensation of two ATPs, independent of glycine concentration. Interestingly, whereas the first ATP-binding pocket is conserved for all class II tRNA synthetases, the second ATP pocket is formed by an insertion domain that is unique to GlyRS, suggesting that GlyRS is the only tRNA synthetase catalyzing direct Ap4A synthesis. A special role for GlyRS in Ap4A homeostasis is proposed.
Assuntos
Fosfatos de Dinucleosídeos/química , Fosfatos de Dinucleosídeos/fisiologia , Glicina-tRNA Ligase/química , Glicina-tRNA Ligase/fisiologia , Trifosfato de Adenosina/química , Sítios de Ligação , Catálise , Cromatografia em Camada Fina/métodos , Cristalografia por Raios X/métodos , Glicina/química , Homeostase , Humanos , Modelos Biológicos , Modelos Moleculares , Conformação Molecular , Ligação ProteicaRESUMO
Protein synthesis involves the accurate attachment of amino acids to their matching transfer RNA (tRNA) molecules. Mistranslating the amino acids serine or glycine for alanine is prevented by the function of independent but collaborative aminoacylation and editing domains of alanyl-tRNA synthetases (AlaRSs). We show that the C-Ala domain plays a key role in AlaRS function. The C-Ala domain is universally tethered to the editing domain both in AlaRS and in many homologous free-standing editing proteins. Crystal structure and functional analyses showed that C-Ala forms an ancient single-stranded nucleic acid binding motif that promotes cooperative binding of both aminoacylation and editing domains to tRNA(Ala). In addition, C-Ala may have played an essential role in the evolution of AlaRSs by coupling aminoacylation to editing to prevent mistranslation.
Assuntos
Alanina-tRNA Ligase/química , Alanina-tRNA Ligase/metabolismo , RNA de Transferência de Alanina/química , RNA de Transferência de Alanina/metabolismo , Aminoacilação de RNA de Transferência , Motivos de Aminoácidos , Sequência de Aminoácidos , Bactérias/enzimologia , Sequência de Bases , Cristalografia por Raios X , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Evolução Molecular , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Filogenia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/metabolismoRESUMO
AlaXp is a widely distributed (from bacteria to humans) genome-encoded homolog of the editing domain of alanyl-tRNA synthetases. Editing repairs the confusion of serine and glycine for alanine through clearance of mischarged (with Ser or Gly) tRNA(Ala). Because genome-encoded fragments of editing domains of other synthetases are scarce, the AlaXp redundancy of the editing domain of alanyl-tRNA synthetase is thought to reflect an unusual sensitivity of cells to mistranslation at codons for Ala. Indeed, a small defect in the editing activity of alanyl-tRNA synthetase is causally linked to neurodegeneration in the mouse. Although limited earlier studies demonstrated that AlaXp deacylated mischarged tRNA(Ala) in vitro, the significance of this activity in vivo has not been clear. Here we describe a bacterial system specifically designed to investigate activity of AlaXp in vivo. Serine toxicity, experienced by a strain harboring an editing-defective alanyl-tRNA synthetase, was rescued by an AlaXp-encoding transgene. Rescue was dependent on amino acid residues in AlaXp that are needed for its in vitro catalytic activity. Thus, the editing activity per se of AlaXp was essential for suppressing mistranslation. The results support the idea that the unique widespread distribution of AlaXp arises from the singular difficulties, for translation, poised by alanine.