A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signalling.

Mechanisms that integrate the metabolic state of a cell with regulatory pathways are necessary to maintain cellular homeostasis. Endogenous, intrinsically reactive metabolites can form functional, covalent modifications on proteins without the aid of enzymes1,2, and regulate cellular functions such as metabolism3-5 and transcription6. An important 'sensor' protein that captures specific metabolic information and transforms it into an appropriate response is KEAP1, which contains reactive cysteine residues that collectively act as an electrophile sensor tuned to respond to reactive species resulting from endogenous and xenobiotic molecules. Covalent modification of KEAP1 results in reduced ubiquitination and the accumulation of NRF27,8, which then initiates the transcription of cytoprotective genes at antioxidant-response element loci. Here we identify a small-molecule inhibitor of the glycolytic enzyme PGK1, and reveal a direct link between glycolysis and NRF2 signalling. Inhibition of PGK1 results in accumulation of the reactive metabolite methylglyoxal, which selectively modifies KEAP1 to form a methylimidazole crosslink between proximal cysteine and arginine residues (MICA). This posttranslational modification results in the dimerization of KEAP1, the accumulation of NRF2 and activation of the NRF2 transcriptional program. These results demonstrate the existence of direct inter-pathway communication between glycolysis and the KEAP1-NRF2 transcriptional axis, provide insight into the metabolic regulation of the cellular stress response, and suggest a therapeutic strategy for controlling the cytoprotective antioxidant response in several human diseases

Metabolite-Induced Protein Modifications in Cell Signaling: A Direct Link between Metabolism and Regulatory Pathways of the Cell

Glycolysis is a primary and central metabolic pathway of the carbohydrates that supplies the energy source and building block precursors to the living system. A strong correlation between deregulated glucose metabolism and various human diseases has been studied, however mechanisms that integrate the metabolic state with regulatory pathways in cellular systems are largely unexplored. Here we report the direct link between glycolysis and KEAP1-NRF2 transcriptional program via a novel reactive metabolite-induced posttranslational modification. We demonstrate that reactive dicarbonyl metabolite methylglyoxal, mainly generated from glycolysis in cells, is a signaling messenger of the activation of NRF2 transcriptional program. Methylglyoxal induces the formation of a methylimidazole crosslink between cysteine electrophile sensor and proximal arginine residues posttranslational modification between two monomeric KEAP1 which was firstly characterized by our study. In-vivo experiment of NRF2-dependent UV-damage mouse model with the PGK1 inhibitor showed therapeutic efficacy, demonstrating the physiological relevance of regulating glucose metabolism for activating NRF2 signaling cascade. In summary, our work presented herein highlights the role of reactive metabolite-induced posttranslational modifications in cell signaling. Furthermore, we developed the novel chemical proteomic technologies to unveil the underlying protein communication networks and their dynamic and temporal interactions with other proteins or ligands in live cells. Our P3 profiling technology was designed to profile high resolution protein proximity maps by mild and fast proximity biotin labeling with light activation. Application of our approach to KEAP1 identified potential protein-protein interaction partners including well-characterized KEAP1 binding protein PGAM5, demonstrating that our novel chemical proteomic tool is applicable to study challenging research targets such as redox signaling networks of the cell. We also designed SILAC surface mapping assay to study the protein-ligand interactions in live cells, and we confirmed our strategy with photoaffinity analogue probe of GNF-2 and its binding site in c-ABL that is well-characterized. Indeed, the results of our quantitative proteomic workflow with PGK1 and its novel inhibitor CBR-470-1 suggest that CBR-470-1 may inhibit PGK1 activity by interfering its interaction with glycolytic intermediates 1,3-BPG and 3PG. Overall, these new chemical proteomic technologies with photoreactive functionalities and quantitative mass spectrometry may provide an unbiased, global insight into the nature of protein communications and regulations in complex biological systems that lead to elucidate the network of reactive metabolite-induced posttranslational modifications in cell signaling

Combined Effects of Optimized Heat Treatment and Nickel Coating for the Improvement of Interfacial Bonding in Aluminum–Iron Alloys Hybrid Structures

The effects of nickel coating and heat treatment on the interfacial bonds of aluminum–iron (Al/Fe) alloys hybrid structures were investigated using microstructural analysis. The application of a nickel coating successfully suppressed the formation of defects such as gaps and oxide scale, improving the physical bonding of the interface. Optimizing the heat treatment conditions generated superior chemical bonding at the interface and facilitated the formation of a nickel-bearing phase in the Al matrix. Also, the types of nickel-bearing phase were influenced by solution treatment and proximity to the interface. By analyzing the isopleth phase diagram of the aluminum system for the ranges of nickel present in the Al, it was confirmed that the Ni:Cu ratio affected the precipitation characteristics of the system. However, when heated under conditions that were optimized for chemical bonding, the Al matrix decreased by approximately 40% (from 100 HV to 60 HV), due to grain growth. The effect of artificial aging increased the hardness of the Al matrix away from the interface by 35% (from 63 HV to 90 HV). On the other hand, this did not occur in the Al matrix near the interface. These results indicate that the nickel that diffused into the Al matrix interfered with the precipitation hardening effect

Bispecific Antibodies: A Smart Arsenal for Cancer Immunotherapies

Following the clinical success of cancer immunotherapies such as immune checkpoint inhibitors blocking B7/CTLA-4 or PD-1/PD-L1 signaling and ongoing numerous combination therapies in the clinic,3 bispecific antibodies (BsAbs) are now emerging as a growing class of immunotherapies with the potential to improve clinical efficacy and safety further. Here, we describe four classes of BsAbs: (a) immune effector cell redirectors; (b) tumor-targeted immunomodulators; (c) dual immunomodulators; and (d) dual tumor-targeting BsAbs. This review describes each of these classes of BsAbs and presents examples of BsAbs in development. We reviewed the biological rationales and characteristics of BsAbs and summarized the current status and limitations of clinical development of BsAbs and strategies to overcome limitations. The field of BsAb-based cancer immunotherapy is growing, and more data from clinical trials are accumulating. Thus, BsAbs could be the next generation of new treatment options for cancer patients

Kiatomab is a Novel Antitumor Monoclonal Antibody Targeting Cancer Stem Cells

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B7-H3-targeted 4-1BB activation potentiates CD8 T cell-dependent antitumor immunity without systemic toxicity

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Profiling Reactive Metabolites via Chemical Trapping and Targeted Mass Spectrometry

Metabolomic profiling studies aim to provide a comprehensive, quantitative, and dynamic portrait of the endogenous metabolites in a biological system. While contemporary technologies permit routine profiling of many metabolites, intrinsically labile metabolites are often improperly measured or omitted from studies due to unwanted chemical transformations that occur during sample preparation or mass spectrometric analysis. The primary glycolytic metabolite 1,3-bisphosphoglyceric acid (1,3-BPG) typifies this class of metabolites, and, despite its central position in metabolism, has largely eluded analysis in profiling studies. Here we take advantage of the reactive acylphosphate group in 1,3-BPG to chemically trap the metabolite with hydroxylamine during metabolite isolation, enabling quantitative analysis by targeted LC–MS/MS. This approach is compatible with complex cellular metabolome, permits specific detection of the reactive (1,3-) instead of nonreactive (2,3-) BPG isomer, and has enabled direct analysis of dynamic 1,3-BPG levels resulting from perturbations to glucose processing. These studies confirmed that standard metabolomic methods misrepresent cellular 1,3-BPG levels in response to altered glucose metabolism and underscore the potential for chemical trapping to be used for other classes of reactive metabolites

Identification of Lactobacillus strains for development of vegan probiotic products with anti-obesity and well-aging effects

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