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1.
Sci China Life Sci ; 66(8): 1869-1887, 2023 08.
Article in English | MEDLINE | ID: mdl-37059927

ABSTRACT

Protein-biomolecule interactions play pivotal roles in almost all biological processes. For a biomolecule of interest, the identification of the interacting protein(s) is essential. For this need, although many assays are available, highly robust and reliable methods are always desired. By combining a substrate-based proximity labeling activity from the pupylation pathway of Mycobacterium tuberculosis and the streptavidin (SA)-biotin system, we developed the Specific Pupylation as IDEntity Reporter (SPIDER) method for identifying protein-biomolecule interactions. Using SPIDER, we validated the interactions between the known binding proteins of protein, DNA, RNA, and small molecule. We successfully applied SPIDER to construct the global protein interactome for m6A and mRNA, identified a variety of uncharacterized m6A binding proteins, and validated SRSF7 as a potential m6A reader. We globally identified the binding proteins for lenalidomide and CobB. Moreover, we identified SARS-CoV-2-specific receptors on the cell membrane. Overall, SPIDER is powerful and highly accessible for the study of protein-biomolecule interactions.


Subject(s)
COVID-19 , Humans , SARS-CoV-2 , Proteins , Protein Binding
2.
Acta Biochim Biophys Sin (Shanghai) ; 54(10): 1453-1463, 2022 Oct 25.
Article in English | MEDLINE | ID: mdl-36239351

ABSTRACT

Type 2 diabetes mellitus (T2DM) is recognized as a serious public health concern with increasing incidence. The dipeptidyl peptidase-4 (DPP-4) inhibitor sitagliptin has been used for the treatment of T2DM worldwide. Although sitagliptin has excellent therapeutic outcome, adverse effects are observed. In addition, previous studies have suggested that sitagliptin may have pleiotropic effects other than treating T2DM. These pieces of evidence point to the importance of further investigation of the molecular mechanisms of sitagliptin, starting from the identification of sitagliptin-binding proteins. In this study, by combining affinity purification mass spectrometry (AP-MS) and stable isotope labeling by amino acids in cell culture (SILAC), we discover seven high-confidence targets that can interact with sitagliptin. Surface plasmon resonance (SPR) assay confirms the binding of sitagliptin to three proteins, i. e., LYPLAL1, TCP1, and CCAR2, with binding affinities (K D) ranging from 50.1 µM to 1490 µM. Molecular docking followed by molecular dynamic (MD) simulation reveals hydrogen binding between sitagliptin and the catalytic triad of LYPLAL1, and also between sitagliptin and the P-loop of ATP-binding pocket of TCP1. Molecular mechanics Poisson-Boltzmann Surface Area (MMPBSA) analysis indicates that sitagliptin can stably bind to LYPLAL1 and TCP1 in active sites, which may have an impact on the functions of these proteins. SPR analysis validates the binding affinity of sitagliptin to TCP1 mutant D88A is ~10 times lower than that to the wild-type TCP1. Our findings provide insights into the sitagliptin-targets interplay and demonstrate the potential of sitagliptin in regulating gluconeogenesis and in anti-tumor drug development.


Subject(s)
Diabetes Mellitus, Type 2 , Dipeptidyl-Peptidase IV Inhibitors , Sitagliptin Phosphate , Humans , Adaptor Proteins, Signal Transducing , Carrier Proteins , Diabetes Mellitus, Type 2/chemically induced , Dipeptidyl-Peptidase IV Inhibitors/pharmacology , Hypoglycemic Agents/pharmacology , Molecular Docking Simulation , Sitagliptin Phosphate/pharmacology
3.
Acta Biochim Biophys Sin (Shanghai) ; 53(9): 1134-1141, 2021 Aug 31.
Article in English | MEDLINE | ID: mdl-34159380

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global health threat since December 2019, and there is still no highly effective drug to control the pandemic. To facilitate drug target identification for drug development, studies on molecular mechanisms, such as SARS-CoV-2 protein interactions, are urgently needed. In this study, we focused on Nsp2, a non-structural protein with largely unknown function and mechanism. The interactome of Nsp2 was revealed through the combination of affinity purification mass spectrometry (AP-MS) and stable isotope labeling by amino acids in cell culture (SILAC), and 84 proteins of high-confidence were identified. Gene ontology analysis demonstrated that Nsp2-interacting proteins are involved in several biological processes such as endosome transport and translation. Network analysis generated two clusters, including ribosome assembly and vesicular transport. Bio-layer interferometry (BLI) assay confirmed the bindings between Nsp2- and 4-interacting proteins, i.e. STAU2 (Staufen2), HNRNPLL, ATP6V1B2, and RAP1GDS1 (SmgGDS), which were randomly selected from the list of 84 proteins. Our findings provide insights into the Nsp2-host interplay and indicate that Nsp2 may play important roles in SARS-CoV-2 infection and serve as a potential drug target for anti-SARS-CoV-2 drug development.


Subject(s)
COVID-19 Drug Treatment , SARS-CoV-2/chemistry , Viral Nonstructural Proteins/chemistry , Drug Delivery Systems , Guanine Nucleotide Exchange Factors/chemistry , Guanine Nucleotide Exchange Factors/metabolism , HEK293 Cells , Heterogeneous-Nuclear Ribonucleoproteins/chemistry , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Humans , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , Protein Binding , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , SARS-CoV-2/metabolism , Vacuolar Proton-Translocating ATPases/chemistry , Vacuolar Proton-Translocating ATPases/metabolism , Viral Nonstructural Proteins/metabolism
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