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1.
Anal Chem ; 91(24): 15818-15825, 2019 12 17.
Article in English | MEDLINE | ID: mdl-31743002

ABSTRACT

Activity-based chemical proteomics approaches used for identifying cellular targets of drugs are mainly dependent on the availability of probes derived from drugs. However, all chemical probes are structurally different from the drugs themselves and cannot fully mimic the real actions of drugs in cells. Here we present a concise and unbiased immunoaffinity-based strategy for identifying covalent drug targets in vivo. By using the specific antibody, we not only confirm the well-known ibrutinib-binding target BTK, but also identify some previously undescribed strongly binding proteins, such as CKAP4 in human cell lines and TAP1 in mouse organs. The observed target profiles between species may partially explain why certain drug candidates are very effective in mice but not in humans. This approach avoids the chemical modification of drugs, eliminates the nonspecific bindings of chemical probes, and allows to unbiasedly decode the underlying mechanisms of action of covalent drugs.


Subject(s)
Agammaglobulinaemia Tyrosine Kinase/metabolism , Peptides/chemistry , Proteomics , Pyrazoles/chemistry , Pyrimidines/chemistry , ATP Binding Cassette Transporter, Subfamily B, Member 2/chemistry , ATP Binding Cassette Transporter, Subfamily B, Member 2/metabolism , Adenine/analogs & derivatives , Agammaglobulinaemia Tyrosine Kinase/antagonists & inhibitors , Amino Acid Sequence , Animals , Antibodies/immunology , Cell Line , Humans , Liver/chemistry , Liver/metabolism , Liver/pathology , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence , Peptides/analysis , Piperidines , Protein Binding , Pyrazoles/immunology , Pyrazoles/metabolism , Pyrimidines/immunology , Pyrimidines/metabolism , Spleen/chemistry , Spleen/metabolism , Spleen/pathology
2.
Nature ; 551(7681): 525-528, 2017 11 23.
Article in English | MEDLINE | ID: mdl-29107940

ABSTRACT

The peptide-loading complex (PLC) is a transient, multisubunit membrane complex in the endoplasmic reticulum that is essential for establishing a hierarchical immune response. The PLC coordinates peptide translocation into the endoplasmic reticulum with loading and editing of major histocompatibility complex class I (MHC-I) molecules. After final proofreading in the PLC, stable peptide-MHC-I complexes are released to the cell surface to evoke a T-cell response against infected or malignant cells. Sampling of different MHC-I allomorphs requires the precise coordination of seven different subunits in a single macromolecular assembly, including the transporter associated with antigen processing (TAP1 and TAP2, jointly referred to as TAP), the oxidoreductase ERp57, the MHC-I heterodimer, and the chaperones tapasin and calreticulin. The molecular organization of and mechanistic events that take place in the PLC are unknown owing to the heterogeneous composition and intrinsically dynamic nature of the complex. Here, we isolate human PLC from Burkitt's lymphoma cells using an engineered viral inhibitor as bait and determine the structure of native PLC by electron cryo-microscopy. Two endoplasmic reticulum-resident editing modules composed of tapasin, calreticulin, ERp57, and MHC-I are centred around TAP in a pseudo-symmetric orientation. A multivalent chaperone network within and across the editing modules establishes the proofreading function at two lateral binding platforms for MHC-I molecules. The lectin-like domain of calreticulin senses the MHC-I glycan, whereas the P domain reaches over the MHC-I peptide-binding pocket towards ERp57. This arrangement allows tapasin to facilitate peptide editing by clamping MHC-I. The translocation pathway of TAP opens out into a large endoplasmic reticulum lumenal cavity, confined by the membrane entry points of tapasin and MHC-I. Two lateral windows channel the antigenic peptides to MHC-I. Structures of PLC captured at distinct assembly states provide mechanistic insight into the recruitment and release of MHC-I. Our work defines the molecular symbiosis of an ABC transporter and an endoplasmic reticulum chaperone network in MHC-I assembly and provides insight into the onset of the adaptive immune response.


Subject(s)
Antigen Presentation , Cryoelectron Microscopy , Histocompatibility Antigens Class I/metabolism , Multiprotein Complexes/metabolism , Multiprotein Complexes/ultrastructure , ATP Binding Cassette Transporter, Subfamily B, Member 2/chemistry , ATP Binding Cassette Transporter, Subfamily B, Member 2/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 2/ultrastructure , ATP Binding Cassette Transporter, Subfamily B, Member 3/chemistry , ATP Binding Cassette Transporter, Subfamily B, Member 3/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 3/ultrastructure , Binding Sites , Burkitt Lymphoma/chemistry , Calreticulin/chemistry , Calreticulin/metabolism , Calreticulin/ultrastructure , Cytosol/immunology , Cytosol/metabolism , Disease Progression , Endoplasmic Reticulum/chemistry , Endoplasmic Reticulum/metabolism , Histocompatibility Antigens Class I/chemistry , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class I/ultrastructure , Humans , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/metabolism , Membrane Transport Proteins/ultrastructure , Models, Biological , Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/immunology , Protein Disulfide-Isomerases/chemistry , Protein Disulfide-Isomerases/metabolism , Protein Disulfide-Isomerases/ultrastructure , Protein Domains
3.
Sci Rep ; 7(1): 11137, 2017 09 11.
Article in English | MEDLINE | ID: mdl-28894200

ABSTRACT

Partner of sld five 1 (PSF1) is an evolutionary conserved DNA replication factor involved in DNA replication in lower species, which is strongly expressed in normal stem cell populations and progenitor cell populations. Recently, we have investigated PSF1 functions in cancer cells and found that PSF1 plays a significant role in tumour growth. These findings provide initial evidence for the potential of PSF1 as a therapeutic target. Here, we reveal that PSF1 contains an immunogenic epitope suitable for an antitumour vaccine. We analysed PSF1 peptides eluted from affinity-purified human leukocyte antigen (HLA) by mass spectrometry and identified PSF179-87 peptide (YLYDRLLRI) that has the highest prediction score using an in silico algorithm. PSF179-87 peptide induced PSF1-specific cytotoxic T lymphocyte responses such as the production of interferon-γ and cytotoxicity. Because PSF1 is expressed in cancer cell populations and highly expressed in cancer stem cell populations, these data suggest that vaccination with PSF179-87 peptide may be a novel therapeutic strategy for cancer treatment.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 2/immunology , Cancer Vaccines/immunology , HLA Antigens/immunology , Peptides/immunology , ATP Binding Cassette Transporter, Subfamily B, Member 2/chemistry , Animals , Antigen Presentation , Cell Line, Tumor , Chromatography, Liquid , Cytotoxicity, Immunologic , Disease Models, Animal , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/immunology , Female , Histocompatibility Antigens Class I/immunology , Humans , Immunogenicity, Vaccine , Mice , Neoplasms/immunology , Peptides/blood , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism , Tandem Mass Spectrometry
4.
PLoS One ; 12(5): e0178238, 2017.
Article in English | MEDLINE | ID: mdl-28542489

ABSTRACT

ATP-binding cassette (ABC) transporters form a large family of transmembrane importers and exporters. Using two nucleotide-binding domains (NBDs), which form a canonical ATP-sandwich dimer at some point within the transport cycle, the transporters harness the energy from ATP binding and hydrolysis to drive substrate transport. However the structural elements that enable and tune the dimerization propensity of the NBDs have not been fully elucidated. Here we compared the biochemical properties of the NBDs of human and rat TAP1, a subunit of the heterodimeric transporter associated with antigen processing (TAP). The isolated human TAP1 NBD was monomeric in solution, in contrast to the previously observed ATP-mediated homodimerization of the isolated rat TAP1 NBD. Using a series of human-rat chimeric constructs, we identified the D-helix, an α-helix N-terminal to the conserved D-loop motif, as an important determinant of NBD dimerization. The ATPase activity of our panel of TAP1 NBD constructs largely correlated with dimerization ability, indicating that the observed dimerization uses the canonical ATP-sandwich interface. The N-terminus of the D-helix from one protomer interacts with the ATP-binding Walker A motif of the second protomer at the ATP-sandwich interface. However, our mutational analysis indicated that residues farther from the interface, within the second and third turn of the D-helix, also influence dimerization. Overall, our data suggest that although the D-helix sequence is not conserved in ABC transporters, its precise positioning within the NBD structure has a critical role in NBD dimerization.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 2/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 2/chemistry , ATP Binding Cassette Transporter, Subfamily B, Member 2/genetics , Adenosine Triphosphate/metabolism , Animals , Binding Sites , Escherichia coli , Humans , Hydrolysis , Models, Molecular , Mutation , Protein Domains , Protein Multimerization , Protein Structure, Secondary , Rats , Solutions , Ultracentrifugation
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