An infectivity-enhancing site on the SARS-CoV-2 spike protein targeted by antibodies.

Antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein prevent SARS-CoV-2 infection. However, the effects of antibodies against other spike protein domains are largely unknown. Here, we screened a series of anti-spike monoclonal antibodies from coronavirus disease 2019 (COVID-19) patients and found that some of antibodies against the N-terminal domain (NTD) induced the open conformation of RBD and thus enhanced the binding capacity of the spike protein to ACE2 and infectivity of SARS-CoV-2. Mutational analysis revealed that all of the infectivity-enhancing antibodies recognized a specific site on the NTD. Structural analysis demonstrated that all infectivity-enhancing antibodies bound to NTD in a similar manner. The antibodies against this infectivity-enhancing site were detected at high levels in severe patients. Moreover, we identified antibodies against the infectivity-enhancing site in uninfected donors, albeit at a lower frequency. These findings demonstrate that not only neutralizing antibodies but also enhancing antibodies are produced during SARS-CoV-2 infection.

Comprehensive characterization of the phosphoproteome of gastric cancer from endoscopic biopsy specimens.

Rationale: Cancer phosphoproteomics can provide insights regarding kinases that can be targeted for therapeutic applications. Monitoring the phosphoproteomics in cancer is expected to play a key role in optimizing treatments with kinase inhibitors. Clinical phosphoproteomics in surgical tissues and patient-derived models has been studied intensively. However, the reported data may not accurately reflect the phosphosignaling status in patients due to the effect of ischemia occurring during surgery or changes in the characteristics of cancer cells when establishing the models. In contrast, endoscopic biopsies have an advantage for clinical phosphoproteomics because they can be rapidly cryo-preserved. We aimed to develop a highly sensitive method for phosphoproteomics in endoscopic biopsies of gastric cancer. Methods: Three tumor biopsies and three normal gastric biopsies were obtained by endoscopy at one time, and subjected to our optimized phosphoproteomics. Phosphopeptides were enriched with an immobilized metal affinity chromatography, and labeled with Tandem Mass Tag reagent. Quantified phosphosites were compared between the pairs of tumor/normal biopsies within same patient. Cancer-specific activated pathways and kinases were identified by pathway enrichment analysis and kinase-substrate enrichment analysis. Results: Our protocol enabled the identification of more than 10,000 class 1 phosphosites from endoscopic biopsies. A comparison between samples from cancer tissue and normal mucosa demonstrated differences in the phosphosignaling, including biomarkers of response to DNA damage. Finally, cancer-specific activation of DNA damage response signaling was validated by additional phosphoproteomics of other patients and western blotting of gastric cancer/normal cells. Conclusion: In summary, our pioneering approach will facilitate more accurate clinical phosphoproteomics in endoscopic biopsies, which can be applied to monitor the activities of therapeutic kinases and, ultimately, can be a useful tool to precision medicine.

Improved phosphoproteomic analysis for phosphosignaling and active-kinome profiling in Matrigel-embedded spheroids and patient-derived organoids.

Many attempts have been made to reproduce the three-dimensional (3D) cancer behavior. For that purpose, Matrigel, an extracellular matrix from Engelbreth-Holm-Swarm mouse sarcoma cell, is widely used in 3D cancer models such as scaffold-based spheroids and patient-derived organoids. However, severe ion suppression caused by contaminants from Matrigel hampers large-scale phosphoproteomics. In the present study, we successfully performed global phosphoproteomics from Matrigel-embedded spheroids and organoids. Using acetone precipitations of tryptic peptides, we identified more than 20,000 class 1 phosphosites from HCT116 spheroids. Bioinformatic analysis revealed that phosphoproteomic status are significantly affected by the method used for the recovery from the Matrigel, i.e., Dispase or Cell Recovery Solution. Furthermore, we observed the activation of several phosphosignalings only in spheroids and not in adherent cells which are coincident with previous study using 3D culture. Finally, we demonstrated that our protocol enabled us to identify more than 20,000 and nearly 3,000 class 1 phosphosites from 1.4 mg and 150 µg of patient-derived organoid, respectively. Additionally, we were able to quantify phosphosites with high reproducibility (r = 0.93 to 0.95). Our phosphoproteomics protocol is useful for analyzing the phosphosignalings of 3D cancer behavior and would be applied for precision medicine with patient-derived organoids.

Deep Phospho- and Phosphotyrosine Proteomics Identified Active Kinases and Phosphorylation Networks in Colorectal Cancer Cell Lines Resistant to Cetuximab.

Abnormality in cellular phosphorylation is closely related to oncogenesis. Thus, kinase inhibitors, especially tyrosine kinase inhibitors (TKIs), have been developed as anti-cancer drugs. Genomic analyses have been used in research on TKI sensitivity, but some types of TKI resistance have been unclassifiable by genomic data. Therefore, global proteomic analysis, especially phosphotyrosine (pY) proteomic analysis, could contribute to predict TKI sensitivity and overcome TKI-resistant cancer. In this study, we conducted deep phosphoproteomic analysis to select active kinase candidates in colorectal cancer intrinsically resistant to Cetuximab. The deep phosphoproteomic data were obtained by performing immobilized metal-ion affinity chromatography-based phosphoproteomic and highly sensitive pY proteomic analyses. Comparison between sensitive (LIM1215 and DLD1) and resistant cell lines (HCT116 and HT29) revealed active kinase candidates in the latter, most of which were identified by pY proteomic analysis. Remarkably, genomic mutations were not assigned in most of these kinases. Phosphorylation-based signaling network analysis of the active kinase candidates indicated that SRC-PRKCD cascade was constitutively activated in HCT116 cells. Treatment with an SRC inhibitor significantly inhibited proliferation of HCT116 cells. In summary, our results based on deep phosphoproteomic data led us to propose novel therapeutic targets against cetuximab resistance and showed the potential for anti-cancer therapy.

Deep Phosphotyrosine Proteomics by Optimization of Phosphotyrosine Enrichment and MS/MS Parameters.

Phosphorylation is a major post-translational modification that regulates protein function, with phosphotyrosine (pY) modifications being implicated in oncogenesis. However, global profiling of pY statuses without treatment with a tyrosine phosphatase inhibitor such as pervanadate is still challenging due to the low occupancy of pY sites. In this study, we greatly improved the identification of pY sites by liquid chromatography-tandem mass spectrometry (LC-MS/MS) by optimization of both the pY-immunoprecipitation (pY-IP) protocol and the LC-MS/MS parameters. Our highly sensitive method reproducibly identified more than 1000 pY sites from 8 mg of protein lysate without the need for tyrosine phosphatase inhibitor treatment. Furthermore, >30% of the identified pY sites were not assigned in the PhosphositePlus database. We further applied our method to the comparison of pY status between PC3 cells with and without treatment using the epidermal growth factor receptor (EGFR) inhibitor Erlotinib. Under Erlotinib treatment, we observed not only a decrease in well-known modes of EGFR downstream signaling but also modulations of kinases that are not relevant to the EGFR cascade, such as PTK6 and MAPK13. Our newly developed method for pY proteomics has the potential to reveal unknown pY signaling modes and to identify novel kinase anticancer targets.