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Cell Chemical Biology Jul 2021Induced protein degradation accomplishes elimination, rather than inhibition, of pathological proteins. Key to the success of this novel therapeutic modality is the... (Review)
Review
Induced protein degradation accomplishes elimination, rather than inhibition, of pathological proteins. Key to the success of this novel therapeutic modality is the modification of proteins with ubiquitin chains, which is brought about by molecular glues or bivalent compounds that induce proximity between the target protein and an E3 ligase. The human genome encodes ∼600 E3 ligases that differ widely in their structures, catalytic mechanisms, modes of regulation, and physiological roles. While many of these enzymes hold great promise for drug discovery, few have been successfully engaged by small-molecule degraders. Here, we review E3 ligases that are being used for induced protein degradation. Based on these prior successes and our growing understanding of the biology and biochemistry of E3 ligases, we propose new ubiquitylation enzymes that can be harnessed for drug discovery to firmly establish induced protein degradation as a specific and efficient therapeutic approach.
Topics: Enzyme Inhibitors; Humans; Proteolysis; Small Molecule Libraries; Ubiquitin-Protein Ligases
PubMed: 33891901
DOI: 10.1016/j.chembiol.2021.04.002 -
Methods in Molecular Biology (Clifton,... 2022N terminomics methods combine selective isolation of protein N-terminal peptides with mass spectrometry (MS)-based proteomics for global profiling of proteolytic...
N terminomics methods combine selective isolation of protein N-terminal peptides with mass spectrometry (MS)-based proteomics for global profiling of proteolytic cleavage sites. However, traditional N terminomics workflows require cell lysis before N-terminal enrichment and provide poor coverage of N termini derived from cell surface proteins. Here, we describe application of subtiligase-TM, a plasma membrane-targeted peptide ligase, for selective biotinylation of cell surface N termini, enabling their enrichment and analysis by liquid chromatography-tandem MS (LC-MS/MS). This method provides increased coverage of and specificity for cell surface N termini and is compatible with existing quantitative LC-MS/MS workflows.
Topics: Cell Membrane; Chromatography, Liquid; Peptide Synthases; Proteolysis; Subtilisins; Tandem Mass Spectrometry
PubMed: 35612736
DOI: 10.1007/978-1-0716-2124-0_6 -
Methods in Molecular Biology (Clifton,... 2023All cells contain proteases, which hydrolyze the peptide bonds between amino acids of a protein backbone. Typically, proteases are prevented from nonspecific proteolysis...
All cells contain proteases, which hydrolyze the peptide bonds between amino acids of a protein backbone. Typically, proteases are prevented from nonspecific proteolysis by regulation and by their physical separation into different subcellular compartments; however, this segregation is not retained during cell lysis, which is the initial step in any protein isolation procedure. Prevention of proteolysis during protein purification often takes the form of a two-pronged approach: first, inhibition of proteolysis in situ, followed by the early separation of the protease from the protein of interest via chromatographic purification. Protease inhibitors are routinely used to limit the effect of the proteases before they are physically separated from the protein of interest via column chromatography. In this chapter, commonly used approaches to reducing or avoiding proteolysis during protein expression and purification are reviewed.
Topics: Proteolysis; Peptide Hydrolases; Endopeptidases; Amino Acids; Cell Death
PubMed: 37646995
DOI: 10.1007/978-1-0716-3362-5_6 -
EBioMedicine Oct 2018There are several challenges towards the development and clinical use of small molecule inhibitors, which are currently the main type of targeted therapies towards... (Review)
Review
There are several challenges towards the development and clinical use of small molecule inhibitors, which are currently the main type of targeted therapies towards intracellular proteins. PROteolysis-TArgeting Chimeras (PROTACs) exploit the intracellular ubiquitin-proteasome system to selectively degrade target proteins. Recently, small-molecule PROTACs with high potency have been frequently reported. In this review, we summarize the emerging characteristics of small-molecule PROTACs, such as inducing a rapid, profound and sustained degradation, inducing a robust inhibition of downstream signals, displaying enhanced target selectivity, and overcoming resistance to small molecule inhibitors. In tumor xenografts, small-molecule PROTACs can significantly attenuate tumor progression. In addition, we also introduce recent developments of the PROTAC technology such as homo-PROTACs. The outstanding advantages over traditional small-molecule drugs and the promising preclinical data suggest that small-molecule PROTAC technology has the potential to greatly promote the development of targeted therapy drugs.
Topics: Animals; Antineoplastic Agents; Biomarkers; Drug Discovery; Humans; Molecular Targeted Therapy; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proteolysis; Signal Transduction; Small Molecule Libraries; Ubiquitination
PubMed: 30224312
DOI: 10.1016/j.ebiom.2018.09.005 -
Journal of the American Chemical Society Oct 2021The targeted degradation of membrane proteins would afford an attractive and general strategy for treating various diseases that remain difficult with the current...
The targeted degradation of membrane proteins would afford an attractive and general strategy for treating various diseases that remain difficult with the current proteolysis-targeting chimera (PROTAC) methodology. We herein report a covalent nanobody-based PROTAC strategy, termed GlueTAC, for targeted membrane protein degradation with high specificity and efficiency. We first established a mass-spectrometry-based screening platform for the rapid development of a covalent nanobody (GlueBody) that allowed proximity-enabled cross-linking with surface antigens on cancer cells. By conjugation with a cell-penetrating peptide and a lysosomal-sorting sequence, the resulting GlueTAC chimera triggered the internalization and degradation of programmed death-ligand 1 (PD-L1), which provides a new avenue to target and degrade cell-surface proteins.
Topics: Proteolysis
PubMed: 34596400
DOI: 10.1021/jacs.1c08521 -
Molecular and Cellular Biochemistry Mar 2022Cadmium exhibits both toxic and carcinogenic effects, and its cytotoxicity is linked to various cellular pathways, such as oxidative stress, ubiquitin-proteasome, and...
Cadmium exhibits both toxic and carcinogenic effects, and its cytotoxicity is linked to various cellular pathways, such as oxidative stress, ubiquitin-proteasome, and p53-mediated response pathways. The molecular mechanism(s) underlying cadmium cytotoxicity appears to be complex, but remains largely unclear. Here, we examined the effects of cadmium on the protein catabolism using two surrogate markers, DNA topoisomerases I and II alpha and its contribution to cytotoxicity. We have found that cadmium exposure induced time- and concentration-dependent decreases in the protein level of surrogate markers and therefore suggest that cadmium may be involved in proteolysis system activation. A pharmacological study further revealed the novel role(s) of these proteolytic activities and reactive oxygen species (ROS) in the cadmium-induced acute toxicity: (i) Proteasome inhibition only partially relieved the cadmium-induced proteolysis of topoisomerases; (ii) Moreover, we report for the first time that the activation of metalloproteases, serine proteases, and cysteine proteases contributes to the acute cadmium cytotoxicity; (iii) Consistent with the notion that both ROS generation and proteolysis system activation contribute to the cadmium-induced proteolysis and cytotoxicity, the scavenger N-acetylcysteine and aforementioned protease inhibition not only reduced the cadmium-induced topoisomerase degradation but also alleviated the cadmium-induced cell killing. Taken together, acute cadmium exposure may activate multiple proteolytic systems and ROS formation, subsequently leading to intracellular damage and cytotoxicity. Thus, our results provide a novel insight into potential action mechanism(s) by which cadmium exerts its cytotoxic effect and suggest potential strategies to prevent cadmium-associated acute toxicity.
Topics: A549 Cells; Cadmium; Cytotoxins; DNA Topoisomerases; HeLa Cells; Humans; Proteolysis; Reactive Oxygen Species
PubMed: 35088369
DOI: 10.1007/s11010-021-04298-9 -
Methods in Molecular Biology (Clifton,... 2023Limited proteolysis coupled to mass spectrometry (LiP-MS) is a recent proteomics technique that allows structure-based target engagement profiling on a proteome-wide...
Limited proteolysis coupled to mass spectrometry (LiP-MS) is a recent proteomics technique that allows structure-based target engagement profiling on a proteome-wide level. To achieve this, native lysates are first incubated with a compound, followed by a short incubation with a nonspecific protease. Binding of a compound can change accessibility at the binding site or induce other structural changes in the target. This leads to treatment-specific proteolytic fingerprints upon limited proteolysis, which can be analyzed by standard bottom-up MS-based proteomics. Here, we describe a basic LiP-MS protocol using the natural product rapamycin as an example compound. Along with the provided LiP-MS reference data available via ProteomeXchange with identifier PXD035183, this enables the straightforward implementation of the method by scientists with a basic biochemistry and mass spectrometry background. We describe how the procedure can easily be adapted to other protein samples and small molecules.
Topics: Proteolysis; Mass Spectrometry; Proteome; Binding Sites; Peptide Hydrolases
PubMed: 37558949
DOI: 10.1007/978-1-0716-3397-7_13 -
Analytical Chemistry Aug 2021Monitoring the dynamic alterations of protein structures within an aqueous solution remains enormously challenging. In this study, we describe a size-selective VAILase...
Monitoring the dynamic alterations of protein structures within an aqueous solution remains enormously challenging. In this study, we describe a size-selective VAILase proteolysis (SVP)-mass spectrometry (MS) strategy to probe the protein structure changes without strict control of the proteolysis kinetics. The unique conformation selectivity of SVP depends on the uniform nano-sized entrance pores of the VAILase hexameric cage as well as the six inherent molecular rulers in the VAILase-substrate recognition and cleavage. The dynamic insights into subtle conformation alterations of both myoglobin unfolding transition and Aurora kinase A-inhibitor binding are successfully captured using the SVP strategy, which matches well with the results in the molecular dynamics simulation. Our work provides a new paradigm of size-selective native proteolysis for exploring the aqueous protein structure-function relationships.
Topics: Kinetics; Mass Spectrometry; Protein Conformation; Proteins; Proteolysis
PubMed: 34291915
DOI: 10.1021/acs.analchem.1c02042 -
Nature Chemical Biology Jul 2019Ligand-dependent protein degradation has emerged as a compelling strategy to pharmacologically control the protein content of cells. So far, however, only a limited...
Ligand-dependent protein degradation has emerged as a compelling strategy to pharmacologically control the protein content of cells. So far, however, only a limited number of E3 ligases have been found to support this process. Here, we use a chemical proteomic strategy that leverages broadly reactive, cysteine-directed electrophilic fragments coupled to selective ligands for intracellular proteins (for example, SLF for FKBP12, JQ1 for BRD4) to screen for heterobifunctional degrader compounds (or proteolysis targeting chimeras, PROTACs) that operate by covalent adduction of E3 ligases. This approach identified DCAF16-a poorly characterized substrate recognition component of CUL4-DDB1 E3 ubiquitin ligases-as a target of electrophilic PROTACs that promote the nuclear-restricted degradation of proteins. We find that only a modest fraction (~10-40%) of DCAF16 needs to be modified to support protein degradation, pointing to the potential for electrophilic PROTACs to induce neosubstrate degradation without substantially perturbing the function of the participating E3 ligase.
Topics: Dose-Response Relationship, Drug; HEK293 Cells; Humans; Ligands; Molecular Structure; Nuclear Proteins; Protein Kinase Inhibitors; Proteolysis; Structure-Activity Relationship
PubMed: 31209349
DOI: 10.1038/s41589-019-0279-5 -
Current Opinion in Structural Biology Dec 2013
Topics: Internationality; Peptide Hydrolases; Proteolysis; Research Design
PubMed: 24210951
DOI: 10.1016/j.sbi.2013.10.004