ABSTRACT
Protein-protein interactions (PPIs) are essentially fundamental to all cellular processes, so that developing small molecule inhibitors of PPIs have great significance despite representing a huge challenge. Studying PPIs with the help of peptide motifs could obtain the structural information and reference significance to reduce the difficulty in the development of small molecules. Computational methods are powerful tools to characterize peptide-protein interactions, especially molecular dynamics simulation and binding free energy calculation. Here, we established an affinity prediction model suitable for Casitas B lymphoma-b (Cbl-b) and phosphorylated motif system. According to the affinity data set of multiple truncated peptides, the force field, solvent model, and internal dielectric constant of molecular mechanics/generalized Born surface area (MM/GBSA) method were optimized. Further, we predicted the affinity of the rationally designed new sequences through this model and obtained a new 6-mer motif with a 7-fold increase in affinity and the comprehensive structure-activity relationship. Moreover, we proposed an insight of unexpected activity of the truncated 5-mer peptide and revealed the possible binding mode of the new highly active 6-mer motif by extended simulation. Our results showed that the activity enhancement of the truncated peptide was caused by the acetyl-mediated conformation change. The side chain of Arg and pTyr in the 6-mer motif co-occupied the site p1 to form numerous hydrogen bond interactions and increased hydrophobic interaction formed with Tyr266, leading to the higher affinity. The present work provided a reference to investigate the PPI of Cbl-b and phosphorylated substrates and guided the development of Cbl-b inhibitors.
Subject(s)
Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Peptides/pharmacology , Proto-Oncogene Proteins c-cbl/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Animals , Dose-Response Relationship, Drug , Ligands , Mice , Molecular Dynamics Simulation , Molecular Structure , Peptides/chemistry , Protein Binding/drug effects , Proto-Oncogene Proteins c-cbl/chemistry , Proto-Oncogene Proteins c-cbl/metabolism , Structure-Activity Relationship , Substrate SpecificityABSTRACT
The modulation of protein-protein interactions (PPIs) by small molecules represents a valuable strategy for pharmacological intervention in several human diseases. In this context, computer-aided drug discovery techniques offer useful resources to predict the network of interactions governing the recognition process between protein partners, thus furnishing relevant information for the design of novel PPI modulators. In this work, we focused our attention on the MUC1-CIN85 complex as a crucial PPI controlling cancer progression and metastasis. MUC1 is a transmembrane glycoprotein whose extracellular domain contains a variable number of tandem repeats (VNTRs) regions that are highly glycosylated in normal cells and under-glycosylated in cancer. The hypo-glycosylation fosters the exposure of the backbone to new interactions with other proteins, such as CIN85, that alter the intracellular signalling in tumour cells. Herein, different computational approaches were combined to investigate the molecular recognition pattern of MUC1-CIN85 PPI thus unveiling new structural information useful for the design of MUC1-CIN85 PPI inhibitors as potential anti-metastatic agents.
Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Mucin-1/chemistry , Mucin-1/metabolism , Binding Sites , Drug Design , Molecular Docking Simulation , Molecular Dynamics Simulation , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Interaction Domains and Motifs , Protein Multimerization , Proto-Oncogene Proteins c-cbl/chemistry , Proto-Oncogene Proteins c-cbl/metabolism , src Homology DomainsABSTRACT
CBL is a mammalian gene encoding the protein CBL, which is an E3 ubiquitin-protein ligase involved in cell signaling and protein ubiquitination. Pathogenic variants in this gene have been implicated in a number of human cancers, particularly acute myeloid leukemia (AML). Here, we present a 5-year-old male patient with a history of AML, diffuse midline glioma, and left brain lesion with histiocytic features. A variant of uncertain significance (VUS): p.L493F was detected in his CBL gene via clinical evaluation. Protein modeling predicts this variant to be pathogenic. Details of the clinical evaluation and modeling assay are discussed.
Subject(s)
Germ-Line Mutation/genetics , Neoplasms/genetics , Proto-Oncogene Proteins c-cbl/chemistry , Proto-Oncogene Proteins c-cbl/genetics , Child , Child, Preschool , Female , Humans , Immunophenotyping , Infant , Leukemia, Myeloid, Acute/genetics , Male , Pedigree , Protein DomainsABSTRACT
CBL is a RING type E3 ubiquitin ligase that functions as a negative regulator of tyrosine kinase signaling and loss of CBL E3 function is implicated in several forms of leukemia. The Src-like adaptor proteins (SLAP/SLAP2) bind to CBL and are required for CBL-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling. Despite the established role of SLAP/SLAP2 in regulating CBL activity, the nature of the interaction and the mechanisms involved are not known. To understand the molecular basis of the interaction between SLAP/SLAP2 and CBL, we solved the crystal structure of CBL tyrosine kinase binding domain (TKBD) in complex with SLAP2. The carboxy-terminal region of SLAP2 adopts an α-helical structure which binds in a cleft between the 4H, EF-hand, and SH2 domains of the TKBD. This SLAP2 binding site is remote from the canonical TKBD phospho-tyrosine peptide binding site but overlaps with a region important for stabilizing CBL in its autoinhibited conformation. In addition, binding of SLAP2 to CBL in vitro activates the ubiquitin ligase function of autoinhibited CBL. Disruption of the CBL/SLAP2 interface through mutagenesis demonstrated a role for this protein-protein interaction in regulation of CBL E3 ligase activity in cells. Our results reveal that SLAP2 binding to a regulatory cleft of the TKBD provides an alternative mechanism for activation of CBL ubiquitin ligase function.
Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Proto-Oncogene Proteins c-cbl/chemistry , Proto-Oncogene Proteins c-cbl/metabolism , Proto-Oncogene Proteins pp60(c-src)/chemistry , Proto-Oncogene Proteins pp60(c-src)/metabolism , Ubiquitin/metabolism , Adaptor Proteins, Signal Transducing/genetics , Binding Sites , Down-Regulation , Humans , Molecular Conformation , Protein Binding , Protein Interaction Domains and Motifs , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-cbl/genetics , Proto-Oncogene Proteins pp60(c-src)/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Sequence Alignment , Signal Transduction , Ubiquitin-Protein Ligases/metabolism , src Homology DomainsABSTRACT
The Src-like adaptor proteins (SLAP/SLAP2) bind to CBL E3 ubiquitin ligase to downregulate antigen, cytokine and tyrosine kinase receptor signalling. In contrast to the phosphotyrosine-dependent binding of CBL substrates through its tyrosine kinase-binding domain (TKBD), CBL TKBD associates with the C-terminal tail of SLAP2 in a phospho-independent manner. To understand the distinct nature of this interaction, a purification protocol for SLAP2 in complex with CBL TKBD was established and the complex was crystallized. However, determination of the complex crystal structure was hindered by the apparent degradation of SLAP2 during the crystallization process, such that only the CBL TKBD residues could initially be modelled. Close examination of the CBL TKBD structure revealed a unique dimer interface that included two short segments of electron density of unknown origin. To elucidate which residues of SLAP2 to model into this unassigned density, a co-expression system was generated to test SLAP2 deletion mutants and define the minimal SLAP2 binding region. SLAP2 degradation products were also analysed by mass spectrometry. The model-building and map-generation features of the Phenix software package were employed, leading to successful modelling of the C-terminal tail of SLAP2 into the unassigned electron-density segments.
Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Proto-Oncogene Proteins c-cbl/chemistry , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Binding Sites , Crystallography, X-Ray , Electrons , Humans , Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/isolation & purification , Multiprotein Complexes/metabolism , Proto-Oncogene Proteins c-cbl/genetics , Proto-Oncogene Proteins c-cbl/metabolismABSTRACT
CblC is a chaperone that catalyzes removal of the ß-axial ligand of cobalamin (or B12), generating cob(II)alamin in an early step in the cofactor trafficking pathway. Cob(II)alamin is subsequently partitioned to support cellular needs for the synthesis of active cobalamin cofactor derivatives. In addition to the ß-ligand transferase activity, the Caenorhabdiitis elegans CblC (ceCblC) and clinical R161G/Q variants of the human protein exhibit robust thiol oxidase activity, converting glutathione to glutathione disulfide while concomitantly reducing O2 to H2O2. The chemical efficiency of the thiol oxidase side reaction during ceCblC-catalyzed dealkylation of alkylcobalamins is noteworthy in that it effectively scrubs ambient oxygen from the reaction mixture, leading to air stabilization of the highly reactive cob(I)alamin product. In this study, we report that the enhanced thiol oxidase activity of ceCblC requires the presence of KCl, which explains how the wasteful thiol oxidase activity is potentially curtailed inside cells where the chloride concentration is low. We have captured an unusual chlorocob(II)alamin intermediate that is formed in the presence of potassium chloride, a common component of the reaction buffer, and have characterized it by electron paramagnetic resonance, magnetic circular dichroism, and computational analyses. The ability to form a chlorocob(II)alamin intermediate could represent an evolutionary vestige in ceCblC, which is structurally related to bacterial B12-dependent reductive dehalogenases that have been proposed to form halogen cob(II)alamin intermediates in their catalytic cycle.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Glutathione Transferase/metabolism , Oxidoreductases/metabolism , Proto-Oncogene Proteins c-cbl/metabolism , Vitamin B 12/biosynthesis , Biocatalysis , Caenorhabditis elegans Proteins/chemistry , Glutathione Transferase/chemistry , Models, Molecular , Proto-Oncogene Proteins c-cbl/chemistry , Vitamin B 12/chemistryABSTRACT
Receptor Tyrosine Kinase (RTK) signaling is essential for normal biological processes and disruption of this regulation can lead to tumor initiation and progression. Cbl proteins (Cbl, Cbl-b and Cbl-c) are a family of RING finger (RF) ubiquitin ligases that negatively regulate a variety of RTKs, including EGFR, MET, and RET. Recent studies have identified Cbl mutations associated with human myeloid neoplasias in approximately 5% of the cases. Cbl-c is the most recently identified human Cbl protein and is expressed exclusively in epithelial cells. We identified a novel cDNA that was isolated from a mouse mammary cancer from the C3(1) Large T Antigen transgenic model. This mutant cDNA encodes a protein that has a deletion in the RF domain of Cbl-c, thereby resembling known Cbl family mutations associated with myeoloid neoplasias. Genomic analysis of both parental and transgenic lines shows no evidence of germline mutation indicating that this mutation is likely a somatic mutation. The mutant protein enhances transformation of NIH 3T3 cells when expressed in combination with SV40 Large T antigen. Together these data are consistent with a second hit mutation. In overexpression studies, this mutant Cbl-c protein fails to mediate ubiquitination of activated EGFR and acts in a dominant negative fashion to prevent ubiquitination and downregulation of the activated EGFR by wild type Cbl proteins. Mechanistically, the mutant Cbl-c binds to the EGFR and prevents recruitment of the wild type Cbl protein. Furthermore, data mining reveals Cbl-c mutations associated with solid tumors in humans. Subsequent cell-based analysis demonstrates a similar loss of E3 function and dominant negative effects for one of these human mutations. These data suggest that like Cbl mutations in myeloid neoplasms, loss of Cbl-c function may contribute to the pathogenesis of solid tumors in murine models and in humans.
Subject(s)
Loss of Function Mutation , Neoplasms/genetics , Proto-Oncogene Proteins c-cbl/genetics , Amino Acid Sequence , Animals , Antigens, Viral, Tumor/genetics , Base Sequence , Cell Transformation, Neoplastic/genetics , Female , HEK293 Cells , Humans , Male , Mammary Neoplasms, Experimental/genetics , Mice , Mice, Transgenic , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , NIH 3T3 Cells , Neoplasms/metabolism , Proto-Oncogene Proteins c-cbl/chemistry , Proto-Oncogene Proteins c-cbl/metabolism , RING Finger Domains/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Deletion , Signal TransductionABSTRACT
BACKGROUND: In its RING domain, tumor necrosis factor receptor-associated factor 6 (TRAF6) has ubiquitin E3 ligase activity that facilitates the formation of lysine 63-linked polyubiquitin chains. This activity is required to activate nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and plays an important role in the IκB kinase (IKK) complex. METHODS: An in vitro ubiquitination assay was used to establish whether c-Cbl could promote TRAF6 ubiquitination. We assessed direct binding and performed fine mapping between c-Cbl and TRAF6 based on the results of an immunoprecipitation assay with cultured 293 T cells. The luciferase reporter assay was applied to establish if c-Cbl-mediated ubiquitination affected NF-κB activation after stimulus from various TRAF-mediated signals: tumor necrosis factor-α (TNF-α), receptor activator of NF-κB ligand (RANKL), and interleukin-1ß (IL-1ß). An in vivo ubiquitination assay was performed using endogenous immunoprecipitation of TRAF6 in bone marrow macrophages (BMMs) and osteoclasts. RESULTS: Here, we report on a form of TRAF6 ubiquitination that is mediated by c-Cbl, leading to the formation of lysine 48-linked polyubiquitin chains. The NF-κB activity induced by RANKL and IL-1ß treatment is inhibited when c-Cbl is overexpressed, while the NF-κB activity induced by TNFα treatment is not. c-Cbl inhibits NF-κB activity mediated by TRAF6, but not by TRAF2. These findings show that c-Cbl ubiquitin ligase activity is essential for TRAF6 ubiquitination and negative regulation of NF-κB activity. Fine mapping revealed that the proline-rich domain of c-Cbl is critical for interaction with TRAF6. Stimulation with RANKL or interferon-γ (IFN-γ) caused c-Cbl to bind to polyubiquitinated TRAF6. CONCLUSIONS: These findings indicate that the interaction of TRAF6 with c-Cbl causes lysine 48-linked polyubiquitination for both negative feedback regulation and signaling cross-talk between RANKL and IFN-γ.
Subject(s)
Lysine/metabolism , NF-kappa B/metabolism , Polyubiquitin/metabolism , Proto-Oncogene Proteins c-cbl/metabolism , TNF Receptor-Associated Factor 6/metabolism , Ubiquitination , HEK293 Cells , Humans , Interferon-gamma/pharmacology , Protein Binding , Proto-Oncogene Proteins c-cbl/chemistry , RANK Ligand/pharmacology , RING Finger Domains , TNF Receptor-Associated Factor 6/chemistry , Ubiquitination/drug effectsABSTRACT
Casitas B lineage lymphoma (c-Cbl) is a multifunctional protein with a ubiquitin E3 ligase activity capable of degrading diverse sets of proteins. Although previous work had focused mainly on c-Cbl mutations in humans with hematological malignancies, recent emerging evidence suggests a critical role of c-Cbl in angiogenesis and human solid organ tumors. The combination of its unique structure, modular function, and ability to channelize cues from a rich network of signaling cascades, empowers c-Cbl to assume a central role in these disease models. This review consolidates the structural and functional insights based on recent studies that highlight c-Cbl as a target with tantalizing therapeutic potential in various models of angiogenesis and tumorigenesis.
Subject(s)
Carcinogenesis/metabolism , Neovascularization, Pathologic/enzymology , Proto-Oncogene Proteins c-cbl/metabolism , Animals , Humans , Mice , Molecular Targeted Therapy , Phospholipase C gamma/metabolism , Proto-Oncogene Proteins c-cbl/chemistry , Ubiquitination , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism , Wnt Signaling Pathway , beta Catenin/metabolismABSTRACT
Arf-GAP with Rho-GAP domain, ANK repeat and PH domain-containing protein 1 (ARAP1), Cbl-interacting protein of 85 kDa (CIN85), and casitas B-lineage lymphoma (Cbl) play important roles in epidermal growth factor receptor (EGFR) internalization and recycling. In previous studies, ARAP1 was found to interact with CIN85, and their interaction attenuated the ubiquitination of EGFR. However, the molecular mechanism was still unclear. In this study, we first biochemically and structurally characterized the interaction between ARAP1 and CIN85, and found that the CIN85 SH3B domain bound to the ARAP1 PXPXXRX (except P) XXR/H/K motif with high affinity and specificity. Based on this binding model, we further predicted other potential CIN85 binding partners and tested their interactions biochemically. Moreover, our swapping data and structure alignment analysis suggested that the ß2-ß3 loops of the CIN85 SH3 domains and the H87ARAP1/E132CIN85 interaction were critical for ARAP1 binding specificity. Finally, our competitive analytical gel-filtration chromatography and isothermal titration calorimetry (ITC) results showed that ARAP1 could compete with Cbl for CIN85 binding, which provides a biochemical basis for the regulatory roles of ARAP1 in the CIN85-mediated EGFR internalizing process.
Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Models, Molecular , Multiprotein Complexes/chemistry , Neoplasm Proteins/chemistry , Nerve Tissue Proteins/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Animals , Mice , Multiprotein Complexes/metabolism , Neoplasm Proteins/metabolism , Nerve Tissue Proteins/metabolism , Protein Binding , Protein Domains , Protein Structure, Quaternary , Protein Structure, Secondary , Proto-Oncogene Proteins c-cbl/chemistry , Proto-Oncogene Proteins c-cbl/metabolism , Structure-Activity RelationshipABSTRACT
Cbl proteins are E3 ubiquitin ligases specialized for the regulation of tyrosine kinases by ubiquitylation. Human Cbl proteins are activated by tyrosine phosphorylation, thus setting up a feedback loop whereby the activation of tyrosine kinases triggers their own degradation. Cbl proteins are targeted to their substrates by a phosphotyrosine-binding SH2 domain. Choanoflagellates, unicellular eukaryotes that are closely related to metazoans, also contain Cbl. The tyrosine kinase complement of choanoflagellates is distinct from that of metazoans, and it is unclear if choanoflagellate Cbl is regulated similarly to metazoan Cbl. Here, we performed structure-function studies on Cbl from the choanoflagellate species Salpingoeca rosetta and found that it undergoes phosphorylation-dependent activation. We show that S. rosetta Cbl can be phosphorylated by S. rosetta Src kinase, and that it can ubiquitylate S. rosetta Src. We also compared the substrate selectivity of human and S. rosetta Cbl by measuring ubiquitylation of Src constructs in which Cbl-recruitment sites are placed in different contexts with respect to the kinase domain. Our results indicate that for both human and S. rosetta Cbl, ubiquitylation depends on proximity and accessibility, rather than being targeted toward specific lysine residues. Our results point to an ancient interplay between phosphotyrosine and ubiquitin signaling in the metazoan lineage.
Subject(s)
Choanoflagellata/enzymology , Choanoflagellata/metabolism , Proto-Oncogene Proteins c-cbl/metabolism , Animals , Caenorhabditis elegans/enzymology , Crystallography, X-Ray , Drosophila melanogaster/enzymology , Mass Spectrometry , Models, Molecular , Phosphorylation , Proto-Oncogene Proteins c-cbl/chemistryABSTRACT
The synthesis and structural characterization of Co-(dN)25 -Cbl (Cbl: cobalamin; dN: deoxynucleotide) and Co-(dN)39 -Cbl, which are organometallic DNA-B12 conjugates with single DNA strands consisting of 25 and 39 deoxynucleotides, respectively, and binding studies of these two DNA-Cbl conjugates to three homologous human Cbl transporting proteins, transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are reported. This investigation tests the suitability of such DNA-Cbls for the task of eventual in vivo oligonucleotide delivery. The binding of DNA-Cbl to TC, IF, and HC was investigated in competition with either a fluorescent Cbl derivative and Co-(dN)25 -Cbl, or radiolabeled vitaminâ B12 (57 Co-CNCbl) and Co-(dN)25 -Cbl or Co-(dN)39 -Cbl. Binding of the new DNA-Cbl conjugates was fast and tight with TC, but poorer with HC and IF, which extends a similar original finding with the simpler DNA-Cbl, Co-(dN)18 -Cbl. The contrasting affinities of TC versus IF and HC for the DNA-Cbl conjugates are rationalized herein by a stepwise mechanism of Cbl binding. Critical contributions to overall affinity result from gradual conformational adaptations of the Cbl-binding proteins to the DNA-Cbl, which is first bound to the respective ß domains. This transition is fast with TC, but slow with IF and HC, with which weaker binding results. The invariably tight interaction of the DNA-Cbl conjugates with TC makes the Cbl moiety a potential natural vector for the specific delivery of oligonucleotide loads from the blood into cells.
Subject(s)
DNA/chemistry , Genetic Vectors/chemistry , Genetic Vectors/chemical synthesis , Oligonucleotides/chemistry , Organometallic Compounds/chemistry , Proto-Oncogene Proteins c-cbl/chemistry , Vitamin B 12/chemistry , Binding Sites , Humans , Oligonucleotides/chemical synthesisABSTRACT
Mutations of the tyrosine kinase-directed ubiquitin ligase CBL cause myeloid leukemias, but the molecular determinants of the dominant leukemogenic activity of mutant CBL oncogenes are unclear. Here, we first define a gain-of-function attribute of the most common leukemia-associated CBL mutant, Y371H, by demonstrating its ability to increase proliferation of hematopoietic stem/progenitor cells (HSPCs) derived from CBL-null and CBL/CBL-B-null mice. Next, we express second-site point/deletion mutants of CBL-Y371H in CBL/CBL-B-null HSPCs or the cytokine-dependent human leukemic cell line TF-1 to show that individual or combined Tyr â Phe mutations of established phosphotyrosine residues (Tyr-700, Tyr-731, and Tyr-774) had little impact on the activity of the CBL-Y371H mutant in HSPCs, and the triple Tyr â Phe mutant was only modestly impaired in TF-1 cells. In contrast, intact tyrosine kinase-binding (TKB) domain and proline-rich region (PRR) were critical in both cell models. PRR deletion reduced the stem cell factor (SCF)-induced hyper-phosphorylation of the CBL-Y371H mutant and the c-KIT receptor and eliminated the sustained p-ERK1/2 and p-AKT induction by SCF. GST fusion protein pulldowns followed by phospho-specific antibody array analysis identified distinct CBL TKB domains or PRR-binding proteins that are phosphorylated in CBL-Y371H-expressing TF-1 cells. Our results support a model of mutant CBL gain-of-function in which mutant CBL proteins effectively compete with the remaining wild type CBL-B and juxtapose TKB domain-associated PTKs with PRR-associated signaling proteins to hyper-activate signaling downstream of hematopoietic growth factor receptors. Elucidation of mutant CBL domains required for leukemogenesis should facilitate targeted therapy approaches for patients with mutant CBL-driven leukemias.
Subject(s)
Mutant Proteins/chemistry , Mutation , Oncogenes , Proto-Oncogene Proteins c-cbl/chemistry , Animals , Binding Sites , Cell Line, Tumor , Cell Separation , Cytokines/metabolism , Flow Cytometry , Gene Expression Regulation, Leukemic , Glutathione Transferase/metabolism , Hematopoietic Stem Cells/cytology , Humans , Mice , Mice, Knockout , Mutagenesis , Mutant Proteins/genetics , Phenotype , Phenylalanine/chemistry , Phosphorylation , Proline/chemistry , Protein Domains , Proto-Oncogene Proteins c-cbl/genetics , Recombinant Fusion Proteins/chemistry , Signal Transduction , Tyrosine/chemistryABSTRACT
Cbl-b is a RING-type ubiquitin ligase. Previously, we showed that Cbl-b-mediated ubiquitination and proteosomal degradation of IRS-1 contribute to muscle atrophy caused by unloading stress. The phospho-pentapeptide DGpYMP (Cblin) mimics Tyr612-phosphorylated IRS-1 and inhibits the Cbl-b-mediated ubiquitination and degradation of IRS-1 in vitro and in vivo. In this study, we confirmed the direct interaction between Cblin and the TKB domain of Cbl-b using NMR. Moreover, we showed that the shortened tripeptide GpYM also binds to the TKB domain. To elucidate the inhibitory mechanism of Cblin, we solved the crystal structure of the TKB-Cblin complex at a resolution of 2.5 Å. The pY in Cblin inserts into a positively charged pocket in the TKB domain via hydrogen-bond networks and hydrophobic interactions. Within this complex, the Cblin structure closely resembles the TKB-bound form of another substrate-derived phosphopeptide, Zap-70-derived phosphopeptide. These peptides lack the conserved intrapeptidyl hydrogen bond between pY and a conserved residue involved in TKB-domain binding. Instead of the conserved interaction, these peptides specifically interact with the TKB domain. Based on this binding mode of Cblin to the TKB domain, we can design drugs against unloading-mediated muscle atrophy.
Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Oligopeptides/metabolism , Proto-Oncogene Proteins c-cbl/chemistry , Proto-Oncogene Proteins c-cbl/metabolism , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , HEK293 Cells , Humans , Insulin Receptor Substrate Proteins/metabolism , Models, Molecular , Oligopeptides/pharmacology , Protein Binding , Protein Structure, Tertiary , Proto-Oncogene Proteins c-cbl/antagonists & inhibitors , Ubiquitination/drug effectsABSTRACT
Oncogenic mutations in the monomeric Casitas B-lineage lymphoma (Cbl) gene have been found in many tumors, but their significance remains largely unknown. Several human c-Cbl (CBL) structures have recently been solved, depicting the protein at different stages of its activation cycle and thus providing mechanistic insight underlying how stability-activity tradeoffs in cancer-related proteins-may influence disease onset and progression. In this study, we computationally modeled the effects of missense cancer mutations on structures representing four stages of the CBL activation cycle to identify driver mutations that affect CBL stability, binding, and activity. We found that recurrent, homozygous, and leukemia-specific mutations had greater destabilizing effects on CBL states than random noncancer mutations. We further tested the ability of these computational models, assessing the changes in CBL stability and its binding to ubiquitin-conjugating enzyme E2, by performing blind CBL-mediated EGFR ubiquitination assays in cells. Experimental CBL ubiquitin ligase activity was in agreement with the predicted changes in CBL stability and, to a lesser extent, with CBL-E2 binding affinity. Two thirds of all experimentally tested mutations affected the ubiquitin ligase activity by either destabilizing CBL or disrupting CBL-E2 binding, whereas about one-third of tested mutations were found to be neutral. Collectively, our findings demonstrate that computational methods incorporating multiple protein conformations and stability and binding affinity evaluations can successfully predict the functional consequences of cancer mutations on protein activity, and provide a proof of concept for mutations in CBL.
Subject(s)
Lung Neoplasms/enzymology , Lung Neoplasms/genetics , Mutation, Missense , Proto-Oncogene Proteins c-cbl/genetics , Proto-Oncogene Proteins c-cbl/metabolism , Uterine Cervical Neoplasms/enzymology , Uterine Cervical Neoplasms/genetics , Carcinoma, Non-Small-Cell Lung/enzymology , Carcinoma, Non-Small-Cell Lung/genetics , Enzyme Activation , ErbB Receptors/chemistry , ErbB Receptors/metabolism , Female , HEK293 Cells , HeLa Cells , Humans , Models, Molecular , Phosphorylation , Protein Stability , Proto-Oncogene Proteins c-cbl/chemistry , Signal Transduction , Thermodynamics , Transfection , UbiquitinationABSTRACT
The casitas b-lineage lymphoma (c-Cbl) is an important adaptor protein with an intrinsic E3 ubiquitin ligase activity that interacts with E2 proteins such as UbCH7. c-Cbl plays a vital role in regulating receptor tyrosine kinase signaling. c-Cbl involves in whole-body energy homeostasis, which makes it a potential target for the treatment of type 2 diabetes and obesity. In the present study, we have designed two parental peptides and 55 modified peptides based on the structure of UbCH7 loop L1 and L2. Thirteen of the modified peptides showed increased inhibitory activity in a fluorescence polarization-based assay. In the in vivo proof of study principle, mice treated with peptides 10, 34, 49 and 51 were protected against high-fat diet-induced obesity and insulin resistant. These inhibitors may potentially lead to new therapeutic alternatives for obesity and type 2 diabetes.
Subject(s)
Anti-Obesity Agents/pharmacology , Diabetes Mellitus, Experimental/drug therapy , Hypoglycemic Agents/pharmacology , Obesity/drug therapy , Peptides/pharmacology , Proto-Oncogene Proteins c-cbl/antagonists & inhibitors , Animals , Anti-Obesity Agents/chemical synthesis , Blood Glucose/metabolism , Diabetes Mellitus, Experimental/etiology , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Experimental/pathology , Diet, High-Fat/adverse effects , Energy Metabolism/drug effects , Gene Expression , Hypoglycemic Agents/chemical synthesis , Injections, Intraperitoneal , Insulin/metabolism , Insulin Resistance , Insulin Secretion , Male , Mice , Mice, Inbred C57BL , Obesity/etiology , Obesity/genetics , Obesity/pathology , Peptides/chemical synthesis , Protein Structure, Secondary , Protein Structure, Tertiary , Proto-Oncogene Proteins c-cbl/chemistry , Proto-Oncogene Proteins c-cbl/genetics , Proto-Oncogene Proteins c-cbl/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Controlled self-renewal and differentiation of hematopoietic stem/progenitor cells (HSPCs) are critical for vertebrate development and survival. These processes are tightly regulated by the transcription factors, signaling molecules and epigenetic factors. Impaired regulations of their function could result in hematological malignancies. Using a large-scale zebrafish N-ethyl-N-nitrosourea mutagenesis screening, we identified a line named LDD731, which presented significantly increased HSPCs in hematopoietic organs. Further analysis revealed that the cells of erythroid/myeloid lineages in definitive hematopoiesis were increased while the primitive hematopoiesis was not affected. The homozygous mutation was lethal with a median survival time around 14-15 days post fertilization. The causal mutation was located by positional cloning in the c-cbl gene, the human ortholog of which, c-CBL, is found frequently mutated in myeloproliferative neoplasms (MPN) or acute leukemia. Sequence analysis showed the mutation in LDD731 caused a histidine-to-tyrosine substitution of the amino acid codon 382 within the RING finger domain of c-Cbl. Moreover, the myeloproliferative phenotype in zebrafish seemed dependent on the Flt3 (fms-like tyrosine kinase 3) signaling, consistent with that observed in both mice and humans. Our study may shed new light on the pathogenesis of MPN and provide a useful in vivo vertebrate model of this syndrome for screening drugs.
Subject(s)
Myeloproliferative Disorders/genetics , Point Mutation , Proto-Oncogene Proteins c-cbl/genetics , Animals , Cell Proliferation , Hematopoiesis , Hematopoietic Stem Cells/physiology , Humans , Myeloproliferative Disorders/etiology , Phenotype , Protein Structure, Tertiary , Proto-Oncogene Proteins c-cbl/chemistry , ZebrafishABSTRACT
Juvenile myelomonocytic leukemia (JMML) is a pediatric myeloproliferative neoplasm that arises from malignant transformation of the stem cell compartment and results in increased production of myeloid cells. Somatic and germline variants in CBL (Casitas B-lineage lymphoma proto-oncogene) have been associated with JMML. We report an incompletely penetrant CBL Y371C mutation discovered by whole-exome sequencing in three individuals with JMML in a large pedigree with 35 years of follow-up. The Y371 residue is highly evolutionarily conserved among CBL orthologs and paralogs. In silico bioinformatics prediction programs suggested that the Y371C mutation is highly deleterious. Protein structural modeling revealed that the Y371C mutation abrogated the ability of the CBL protein to adopt a conformation that is required for ubiquitination. Clinically, the three mutation-positive JMML individuals exhibited variable clinical courses; in two out of three, primary hematologic abnormalities persisted into adulthood with minimal clinical symptoms. The penetrance of the CBL Y371C mutation was 30% for JMML and 40% for all leukemia. Of the 8 mutation carriers in the family with available photographs, only one had significant dysmorphic features; we found no evidence of a clinical phenotype consistent with a "CBL syndrome". Although CBL Y371C has been previously reported in familial JMML, we are the first group to follow a complete pedigree harboring this mutation for an extended period, revealing additional information about this variant's penetrance, function and natural history.
Subject(s)
Germ-Line Mutation , Leukemia, Myelomonocytic, Juvenile/genetics , Mutation, Missense , Pedigree , Proto-Oncogene Proteins c-cbl/genetics , Ubiquitination/genetics , Adolescent , Adult , Child , Child, Preschool , Exome , Female , Follow-Up Studies , Humans , Infant , Male , Models, Molecular , Penetrance , Protein Structure, Tertiary , Proto-Oncogene Mas , Proto-Oncogene Proteins c-cbl/chemistryABSTRACT
Human CblC catalyzes the elimination of the upper axial ligand in cobalamin or B12 derivatives entering the cell from circulation. This processing step is critical for assimilation of dietary cobalamin into the active cofactor forms that support the B12-dependent enzymes, methionine synthase and methylmalonyl-CoA mutase. Using a modified nitroreductase scaffold tailored to bind cobalamin and glutathione, CblC exhibits versatility in the mechanism by which it removes cyano versus alkyl ligands in cobalamin. In this study, we have characterized the effects of two pathogenic missense mutations at the same residue, R161G and R161Q, which are associated with early and late onset of the CblC disorder, respectively. We find that the R161Q and R161G CblC mutants display lower protein stability and decreased dealkylation but not decyanation activity, suggesting that cyanocobalamin might be therapeutically useful for patients carrying mutations at Arg-161. The mutant proteins also exhibit impaired glutathione binding. In the presence of physiologically relevant glutathione concentrations, stabilization of the cob(II)alamin derivative is observed, which occurs at the expense of increased oxidation of glutathione. Futile redox cycling, which is suppressed in wild-type human CblC, explains the reported increase in oxidative stress levels associated with the CblC disorder.
Subject(s)
Biocatalysis , Metabolism, Inborn Errors/genetics , Mutation, Missense , Proto-Oncogene Proteins c-cbl/genetics , Proto-Oncogene Proteins c-cbl/metabolism , Vitamin B 12/metabolism , Alkylation , Arginine/metabolism , Glutathione/pharmacology , Humans , Models, Molecular , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Nitriles/metabolism , Oxidation-Reduction/drug effects , Protein Stability , Protein Structure, Tertiary , Proto-Oncogene Proteins c-cbl/chemistry , Reactive Oxygen Species/metabolism , Vitamin B 12/analogs & derivatives , Vitamin B 12/chemistryABSTRACT
The human B12trafficking chaperone protein hCblC is responsible for escorted delivery and early processing of B12in intracellular B12 metabolism. In this study, we characterized a putative B12trafficking chaperone of Caenorhabditis elegans (cCblC), which shows 26% amino acid sequence identity with hCblC. cCblC was shown to bind B12with a broad specificity for the upper axial ligand, as previously observed with other homologous proteins. In addition, cCblC catalyzed glutathione (GSH)-dependent elimination of alkyl and GSH upper axial ligands from alkylcobalamins and glutathionylcobalamin (GSCbl), respectively. Dealkylation of methylcobalamin (MeCbl) generated cob(II)alamin with S-methylglutathione. Cob(I)alamin was detected as the intermediate for cob(II)alamin generation, indicating that the reaction is a nucleophilic displacement using the thiolate of GSH. Deglutathionylation of GSCbl also generated cob(II)alamin, via cob(I)alamin intermediate, with glutathione disulfide, indicating the reaction is chemically analogous with dealkylation. Cob(II)alamin generated by dealkylation and deglutathionylation was bound to cCblC in the base-off state and stable under aerobic conditions, which would be favorable for subsequent enzyme cofactor synthesis. These results demonstrate that cCblC is a B12trafficking chaperone of C. elegans catalyzing dealkylation and deglutathionylation via a nucleophilic displacement using the thiolate of GSH.