[Deciphering cellular processes responding to lethality of 17ß-estradiol by quantitative phosphoproteomics].

17ß-Estradiol (E2), an important endocrine hormone in the mammalian body, participates in the regulation of the physiological functions of the reproductive system, mammary glands, bone, and cardiovascular system, among others. Paradoxically, despite the physiological actions of endogenous E2 (0.2-1.0 nmol/L), numerous clinical and experimental studies have demonstrated that high-dose E2 treatment can cause tumor regression and exert pro-apoptotic actions in multiple cell types; however, the underlying mechanism remains undescribed. In particular, little information of the cellular processes responding to the lethality of E2 is available. In the present study, we attempted to characterize the cellular processes responding to high-dose (µmol/L) E2 treatment using quantitative phosphoproteomics to obtain a better understanding of the regulatory mechanism of E2-induced cell death. First, the cell phenotype induced by high-dose E2 was determined by performing Cell Counting Kit-8 assay (CCK8), cell cytotoxicity analysis by trypan blue staining, and microscopic imaging on HeLa cells treated with 1-10 µmol/L E2 or dimethyl sulfoxide (DMSO) for 1-3 d. E2 inhibited cell proliferation and induced cell death in a dose- and time-dependent manner. Compared with the DMSO-treated HeLa cells, the cells treated with 5 µmol/L E2 for 2 d demonstrated >74% growth inhibition and approximately 50% cell death. Thus, these cells were used for quantitative phosphoproteomic analysis. Next, a solid-phase extraction (SPE)-based immobilized titanium ion affinity chromatography (Ti4+-IMAC) phosphopeptide-enrichment method coupled with data-independent acquisition (DIA)-based quantitative proteomics was employed for the in-depth screening of high-dose E2-regulated phosphorylation sites to investigate the intracellular processes responding to high-dose E2 treatment. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified over 10000 phosphorylation sites regulated by E2 and DMSO in HeLa cells. In comparison with the DMSO-treated cells, the cells treated with 5 µmol/L E2 showed 537 upregulated phosphorylation sites and 387 downregulated phosphorylation sites, with a threshold of p<0.01 and |log2(fold change)|≥1. A total of 924 phosphorylation sites on 599 proteins were significantly regulated by high-dose E2, and these sites were subjected to enrichment analysis. In addition, 453 differently regulated phosphorylation sites on 325 proteins were identified only in the E2- or DMSO-treated cell samples. These phosphorylation sites may be phosphorylated or dephosphorylated in response to high-dose E2 stimulation and were subjected to parallel enrichment analyses. Taken together, 1218 phosphorylation sites on 741 proteins were significantly regulated by high-dose E2 treatment. The functional phosphoproteins in these two groups were then analyzed using Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA) to determine the biological processes in which they participate and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. Consistent with the cell-phenotype data, cell cycle-related proteins were highly enriched in the two groups of E2-regulated phosphoproteins (p<0.05), indicating that high-dose E2 treatment can regulate cell proliferation. In addition, E2-regulated phosphoproteins were highly enriched in the cellular processes of ribosome biogenesis, nucleocytoplasmic transport, and messenger ribonucleic acid (mRNA) processing/splicing (p<0.05), indicating that the activation of these processes may contribute to high-dose E2-induced cell death. These results further confirm that high-dose E2 treatment inhibits protein translation and induces cell death. Furthermore, the significant upregulation of multiple phosphorylation sites associated with epidermal growth factor receptor (EGFR) and mitogen-activated protein kinases (MAPKs) MAPK1, MAPK4, and MAPK14 by high-dose E2 indicates that the EGFR and MAPK signaling pathways are likely involved in the regulation of E2-induced cell death. These phosphorylation sites likely play vital roles in E2-induced cell death in HeLa cells. Overall, our phosphoproteomic data could be a valuable resource for uncovering the regulatory mechanisms of E2 in the micromolar range.

Glycan-Imprinted Nanoparticle as Artificial Neutralizing Antibody for Efficient HIV-1 Recognition and Inhibition.

The HIV-1 envelope is a heavily glycosylated class 1 trimeric fusion protein responsible for viral entry into CD4+ immune cells. Developing neutralizing antibodies against the specific envelope glycans is an alternative method for antiviral therapies. This work presents the first-ever development and characterization of artificial neutralizing antibodies using molecular imprinting technology to recognize and bind to the envelope protein of HIV-1. The prepared envelope glycan-imprinted nanoparticles (GINPs) can successfully prevent HIV-1 from infecting target cells by shielding the glycans on the envelope protein. In vitro experiments showed that GINPs have strong affinity toward HIV-1 (Kd = 36.7 ± 2.2 nM) and possess high anti-interference and specificity. GINPs demonstrate broad inhibition activity against both tier 1 and tier 2 HIV-1 strains with a pM-level IC50 and exhibit a significant inhibitory effect on long-term viral replication by more than 95%. The strategy provides a promising method for the inhibition and therapy of HIV-1 infection.

Kinome and phosphoproteome reprogramming underlies the aberrant immune responses in critically ill COVID-19 patients.

SARS-CoV-2 infection triggers extensive host immune reactions, leading to severe diseases in certain individuals. However, the molecular basis underlying the excessive yet non-productive immune responses in severe COVID-19 remains incompletely understood. In this study, we conducted a comprehensive analysis of the peripheral blood mononuclear cell (PBMC) proteome and phosphoproteome in sepsis patients positive or negative for SARS-CoV-2 infection, as well as healthy subjects, using quantitative mass spectrometry. Our findings demonstrate dynamic changes in the COVID-19 PBMC proteome and phosphoproteome during disease progression, with distinctive protein or phosphoprotein signatures capable of distinguishing longitudinal disease states. Furthermore, SARS-CoV-2 infection induces a global reprogramming of the kinome and phosphoproteome, resulting in defective adaptive immune response mediated by the B and T lymphocytes, compromised innate immune responses involving the SIGLEC and SLAM family of immunoreceptors, and excessive cytokine-JAK-STAT signaling. In addition to uncovering host proteome and phosphoproteome aberrations caused by SARS-CoV-2, our work recapitulates several reported therapeutic targets for COVID-19 and identified numerous new candidates, including the kinases PKG1, CK2, ROCK1/2, GRK2, SYK, JAK2/3, TYK2, DNA-PK, PKCδ, and the cytokine IL-12.

Individualizing Basketball-Specific Interval Training Using Anaerobic Speed Reserve: Effects on Physiological and Hormonal Adaptations.

PURPOSE: We compared the adaptive responses to supramaximal high-intensity interval training (HIIT) individualized according to anaerobic speed reserve (ASR), the 30-15 Intermittent Fitness Test (VIFT), and velocity associated with maximum oxygen uptake (MAS) to determine which approach facilitates more identical adaptations across athletes with different profiles. METHODS: Thirty national-level basketball players (age = 28.4 [5] y; body mass = 88.9 [6.3] kg; height = 190 [4.8] cm) were randomly assigned to 3 training groups performing 2 sets of 4, 6, 8, 6, 8, and 10-minute runs (from first to sixth week, respectively), consisting of 15-second running at Δ%20ASR (MAS + 0.2 × ASR), 95%VIFT, and 120%MAS, with 15 seconds recovery between efforts and a 3-minute relief between sets. RESULTS: All 3 interval interventions significantly (P < .05) enhanced maximum oxygen uptake (V˙O2max), oxygen pulse (V˙O2/HR), first and second ventilatory threshold (VT1 and VT2), cardiac output (Q˙max), stroke volume, peak and average power output, testosterone levels, and testosterone-to-cortisol ratio following the training period. Different values of interindividual variability (coefficient of variation) for the percentage changes of the measured variables were observed in response to HIITASR, HIITvIFT, and HIITMAS for V˙O2max (8.7%, 18.8%, 34.6%, respectively), V˙O2/HR (9.5%, 15.0%, 28.6%), VT1 (9.6%, 19.6%, 34.6%), VT2 (21.8%, 32.4%, 56.7%), Q˙max (8.2%, 16.9%, 28.8%), stroke volume (7.9%, 15.2%, 23.5%), peak power output (20%, 22%, 37.3%), average power output (21.1%, 21.3%, 32.5%), testosterone (52.9%, 61.6%, 59.9%), and testosterone-to-cortisol ratio (55.1%, 59.5%, 57.8%). CONCLUSIONS: Supramaximal HIIT performed at Δ%20ASR resulted in more uniform physiological adaptations than HIIT interventions prescribed using VIFT or MAS. Although hormonal changes do not follow this approach, all the approaches induced an anabolic effect.

The E3 ligase NEURL3 suppresses epithelial-mesenchymal transition and metastasis in nasopharyngeal carcinoma by promoting vimentin degradation.

BACKGROUND: Metastasis has emerged as the major reason of treatment failure and mortality in patients with nasopharyngeal carcinoma (NPC). Growing evidence links abnormal DNA methylation to the initiation and progression of NPC. However, the precise regulatory mechanism behind these processes remains poorly understood. METHODS: Bisulfite pyrosequencing, RT-qPCR, western blot, and immunohistochemistry were used to test the methylation and expression level of NEURL3 and its clinical significance. The biological function of NEURL3 was examined both in vitro and in vivo. Mass spectrometry, co-immunohistochemistry, immunofluorescence staining, and ubiquitin assays were performed to explore the regulatory mechanism of NEURL3. RESULTS: The promoter region of NEURL3, encoding an E3 ubiquitin ligase, was obviously hypermethylated, leading to its downregulated expression in NPC. Clinically, NPC patients with a low NEURL3 expression indicated an unfavorable prognosis and were prone to develop distant metastasis. Overexpression of NEURL3 could suppress the epithelial mesenchymal transition and metastasis of NPC cells in vitro and in vivo. Mechanistically, NEURL3 promoted Vimentin degradation by increasing its K48-linked polyubiquitination at lysine 97. Specifically, the restoration of Vimentin expression could fully reverse the tumor suppressive effect of NEURL3 overexpression in NPC cells. CONCLUSIONS: Collectively, our study uncovers a novel mechanism by which NEURL3 inhibits NPC metastasis, thereby providing a promising therapeutic target for NPC treatment.

Aurora B promotes the CENP-T-CENP-W interaction to guide accurate chromosome segregation in mitosis.

Accurate chromosome segregation in mitosis depends on kinetochores that connect centromeric chromatin to spindle microtubules. Centromeres are captured by individual microtubules via a kinetochore constitutive centromere-associated network (CCAN) during chromosome segregation. CCAN contains 16 subunits, including CENP-W and CENP-T. However, the molecular recognition and mitotic regulation of the CCAN assembly remain elusive. Here, we revealed that CENP-W binds to the histone fold domain and an uncharacterized N-terminal region of CENP-T. Aurora B phosphorylates CENP-W at Thr60, which enhances the interaction between CENP-W and CENP-T to ensure robust metaphase chromosome alignment and accurate chromosome segregation in mitosis. These findings delineate a conserved signaling cascade that integrates protein phosphorylation with CCAN integrity for the maintenance of genomic stability.

Robust Glycoproteomics Platform Reveals a Tetra-Antennary Site-Specific Glycan Capping with Sialyl-Lewis Antigen for Early Detection of Gastric Cancer.

The lack of efficient biomarkers for the early detection of gastric cancer (GC) contributes to its high mortality rate, so it is crucial to discover novel diagnostic targets for GC. Recent studies have implicated the potential of site-specific glycans in cancer diagnosis, yet it is challenging to perform highly reproducible and sensitive glycoproteomics analysis on large cohorts of samples. Here, a highly robust N-glycoproteomics (HRN) platform comprising an automated enrichment method, a stable microflow LC-MS/MS system, and a sensitive glycopeptide-spectra-deciphering tool is developed for large-scale quantitative N-glycoproteome analysis. The HRN platform is applied to analyze serum N-glycoproteomes of 278 subjects from three cohorts to investigate glycosylation changes of GC. It identifies over 20 000 unique site-specific glycans from discovery and validation cohorts, and determines four site-specific glycans as biomarker candidates. One candidate has branched tetra-antennary structure capping with sialyl-Lewis antigen, and it significantly outperforms serum CEA with AUC values > 0.89 compared against < 0.67 for diagnosing early-stage GC. The four-marker panel can provide improved diagnostic performances. Besides, discrimination powers of four candidates are also testified with a verification cohort using PRM strategy. This findings highlight the value of this strong tool in analyzing aberrant site-specific glycans for cancer detection.

Ligand Modification-Free Methods for the Profiling of Protein-Environmental Chemical Interactions.

Adverse health outcomes caused by environmental chemicals are often initiated via their interactions with proteins. Essentially, one environmental chemical may interact with a number of proteins and/or a protein may interact with a multitude of environmental chemicals, forming an intricate interaction network. Omics-wide protein-environmental chemical interaction profiling (PECI) is of prominent importance for comprehensive understanding of these interaction networks, including the toxicity mechanisms of action (MoA), and for providing systematic chemical safety assessment. However, such information remains unknown for most environmental chemicals, partly due to their vast chemical diversity. In recent years, with the continuous efforts afforded, especially in mass spectrometry (MS) based omics technologies, several ligand modification-free methods have been developed, and new attention for systematic PECI profiling was gained. In this Review, we provide a comprehensive overview on these methodologies for the identification of ligand-protein interactions, including affinity interaction-based methods of affinity-driven purification, covalent modification profiling, and activity-based protein profiling (ABPP) in a competitive mode, physicochemical property changes assessment methods of ligand-directed nuclear magnetic resonance (ligand-directed NMR), MS integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS), thermal proteome profiling (TPP), limited proteolysis-coupled mass spectrometry (LiP-MS), stability of proteins from rates of oxidation (SPROX), and several intracellular downstream response characterization methods. We expect that the applications of these ligand modification-free technologies will drive a considerable increase in the number of PECI identified, facilitate unveiling the toxicological mechanisms, and ultimately contribute to systematic health risk assessment of environmental chemicals.

CDK11 requires a critical activator SAP30BP to regulate pre-mRNA splicing.

CDK11 is an emerging druggable target for cancer therapy due to its prevalent roles in phosphorylating critical transcription and splicing factors and in facilitating cell cycle progression in cancer cells. Like other cyclin-dependent kinases, CDK11 requires its cognate cyclin, cyclin L1 or cyclin L2, for activation. However, little is known about how CDK11 activities might be modulated by other regulators. In this study, we show that CDK11 forms a tight complex with cyclins L1/L2 and SAP30BP, the latter of which is a poorly characterized factor. Acute degradation of SAP30BP mirrors that of CDK11 in causing widespread and strong defects in pre-mRNA splicing. Furthermore, we demonstrate that SAP30BP facilitates CDK11 kinase activities in vitro and in vivo, through ensuring the stabilities and the assembly of cyclins L1/L2 with CDK11. Together, these findings uncover SAP30BP as a critical CDK11 activator that regulates global pre-mRNA splicing.

miR-192 inhibits the activation of hepatic stellate cells by targeting Rictor.

The activation of hepatic stellate cells (HSCs) is regarded as the primary driving factor of liver fibrosis. miR-192, a miRNA associated with hepatocellular carcinoma and enriched in HSCs, has an undisclosed role in HSC activation and liver fibrosis. In this study, a CCl4-induced rat liver fibrosis model and transforming growth factor-beta 1 (TGF-ß1)-treated HSC lines (LX-2 and HSC-T6) were used to detect miR-192 and Rictor levels in vivo and in vitro. Bioinformatic analysis and a dual luciferase assay were used to predict and confirm the interaction of Rictor with miR-192. Gain- and/or loss-of-function methods evaluated molecular changes and HSC activation phenotypes, detected by quantitative real-time PCR, western blotting, and immunofluorescence. We observed a gradual downregulation of miR-192 and upregulation of Rictor during CCl4-induced liver fibrosis/cirrhosis in rats. Enriched miR-192 was downregulated, while Rictor was upregulated in TGF-ß1-activated HSCs. miR-192 inhibited the activation of HSCs by directly targeting Rictor. High miR-192/low Rictor expression attenuated the fibrotic-related gene expression by AKT/mTORC2 signaling. In conclusion, miR-192 could inhibit the activation of HSCs by directly targeting Rictor in the AKT/mTORC2 signaling pathway. This study provides insights into potential therapeutic targets for liver fibrosis and cirrhosis.

Rictor mediates p53 deactivation to facilitate the malignant transformation of hepatocytes and promote hepatocarcinogenesis.

BACKGROUND: Mutations in TP53 gene is considered a main driver of hepatocellular carcinoma (HCC). While TP53 mutations are the leading cause of p53 dysfunction, their occurrence rates may drop to approximately 10% in cohorts without hepatitis B virus and aflatoxin exposure. This observation suggests that the deactivation of wild-type p53 (p53wt) may be a critical factor in the majority of HCC cases. However, the mechanism undermining p53wt activity in the liver remains unclear. METHODS: Microarray analysis and luciferase assay were utilized to confirm target associations. Gain- and/or loss-of-function methods were employed to assess alterations in signaling pathways. Protein interactions were analyzed by molecular immunological methods and further visualized by confocal microscopy. Bioinformatic analysis was performed to analyze clinical significance. Tumor xenograft nude mice were used to validate the findings in vivo. RESULTS: Our study highlights the oncogenic role of Rictor, a key component of the mammalian target of rapamycin complex 2 (mTORC2), in hepatocytes. Rictor exerts its oncogenic function by binding to p53wt and subsequently blocking p53wt activity based on p53 status, requiring the involvement of mTOR. Moreover, we observed a dynamic nucleocytoplasmic distribution pattern of Rictor, characterized by its translocation from the nucleus (in precancerous lesions) to the cytoplasm (in HCCs) during malignant transformation. Notably, Rictor is directly targeted by the liver-enriched microRNA miR-192, and the disruption of the miR-192-Rictor-p53-miR-192 signaling axis was consistently observed in both human and rat HCC models. Clinical analysis associated lower miR-192/higher Rictor with shorter overall survival and more advanced clinical stages (P < 0.05). In mice, xenograft tumors overexpressing miR-192 exhibited lower Rictor expression levels, leading to higher p53 activity, and these tumors displayed slower growth compared to untreated HCC cells. CONCLUSIONS: Rictor dynamically shuttles between the nucleus and cytoplasm during HCC development. Its pivotal oncogenic role involves binding and inhibiting p53wt activity within the nucleus in early hepatocarcinogenesis. Targeting Rictor presents a promising strategy for HCC based on p53 status.

The antipsychotic drug penfluridol inhibits N-linked glycoprotein processing and enhances T-cell-mediated tumor immunity.

Aberrant N-linked glycosylation is a prominent feature of cancers. Perturbance of oligosaccharide structure on cell surfaces directly affects key processes in tumor development and progression. In spite of the critical role played by N-linked glycans in tumor biology, the discovery of small molecules that specifically disturbs the N-linked glycans is still under investigation. To identify more saccharide-structure-perturbing compounds, a repurposed drug screen by using a library consisting of 1530 FDA-approved drugs was performed. Interestingly, an antipsychotic drug, penfluridol, was identified as being able to decrease cell surface Wheat germ agglutinin (WGA) staining. In the presence of penfluridol, cell membrane glycoproteins PD-L1 shifted to a lower molecular weight. Further studies demonstrated that penfluridol treatment caused an accumulation of high-mannose oligosaccharides, especially Man5-7GlcNAc2 glycan structures. Mechanistically, this effect is due to direct targeting of MAN1A1 mannosidase, a Golgi enzyme involved in N-glycan maturation. Moreover, we found that altered glycosylation of PD-L1 caused by penfluridol disrupted interactions between PD-1 and PD-L1, resulting in activation of T-cell tumor immunity. In a mouse xenograft and glioma model, penfluridol enhanced the anti-tumor effect of the anti-PD-L1 antibody in vivo. Overall, these findings revealed an important biological activity of the antipsychotic drug penfluridol as an inhibitor of glycan processing and proposed a repurposed use of penfluridol in anti-tumor therapy through activation of T-cell immunity.

DNAJA4 suppresses epithelial-mesenchymal transition and metastasis in nasopharyngeal carcinoma via PSMD2-mediated MYH9 degradation.

Emerging evidence indicates that DNA methylation plays an important role in the initiation and progression of nasopharyngeal carcinoma (NPC). DNAJA4 is hypermethylated in NPC, while its role in regulating NPC progression remains unclear. Here, we revealed that the promoter of DNAJA4 was hypermethylated and its expression was downregulated in NPC tissues and cells. Overexpression of DNAJA4 significantly suppressed NPC cell migration, invasion, and EMT in vitro, and markedly inhibited the inguinal lymph node metastasis and lung metastatic colonization in vivo, while it did not affect NPC cell viability and proliferation capability. Mechanistically, DNAJA4 facilitated MYH9 protein degradation via the ubiquitin-proteasome pathway by recruiting PSMD2. Furthermore, the suppressive effects of DNAJA4 on NPC cell migration, invasion, and EMT were reversed by overexpression of MYH9 in NPC cells. Clinically, a low level of DNAJA4 indicated poor prognosis and an increased probability of distant metastasis in NPC patients. Collectively, DNAJA4 serves as a crucial driver for NPC invasion and metastasis, and the DNAJA4-PSMD2-MYH9 axis might contain potential targets for NPC treatments.

Integrated Protein Solubility Shift Assays for Comprehensive Drug Target Identification on a Proteome-Wide Scale.

Target proteins are often stabilized after binding with a ligand and thereby typically become more resistant to denaturation. Based on this phenomenon, several methods without the need to covalently modify the ligand have been developed to identify target proteins for a specific ligand. These methods usually employ complicated workflows with high cost and limited throughput. Here, we develop an iso-pH shift assay (ipHSA) method, a proteome-wide target identification method that detects ligand-induced protein solubility shifts by precipitating proteins with a single concentration of acidic agent followed by protein quantification via data-independent acquisition (DIA). Using a pan-kinase inhibitor, staurosporine, we demonstrated that ipHSA increased throughput compared to the previously developed pH-dependent protein precipitation (pHDPP) method. ipHSA was found to have high complementarity in staurosporine target identification compared with the improved isothermal shift assay (iTSA) and isosolvent shift assay (iSSA) using DIA instead of tandem mass tags (TMTs) for quantification. To further improve target identification sensitivity, we developed an integrated protein solubility shift assay (IPSSA) by pooling the supernatants yielded from ipHSA, iTSA, and iSSA methods. IPSSA exhibited increased sensitivity in screening staurosporine targets by 38, 29, and 38% compared to individual methods. Increasing the number of replicate experiments further enhanced the sensitivity of target identification. Meanwhile, IPSSA also improved the throughput and reduced the cost compared with previous methods. As a fast and efficient tool for drug target identification, IPSSA is expected to have broad applications in the study of the mechanism of action.

Hydrophobic Derivatization Strategy Facilitates Comprehensive Profiling of Protein Methylation.

Protein methylation is receiving more and more attention due to its essential role in diverse biological processes. Large-scale analysis of protein methylation requires the efficient identification of methylated peptides at the proteome level; unfortunately, a significant number of methylated peptides are highly hydrophilic and hardly retained during reversed-phase chromatography, making it difficult to be identified by conventional approaches. Herein, we report the development of a novel strategy by combining hydrophobic derivatization and high pH strong cation exchange enrichment, which significantly expands the identification coverage of the methylproteome. Noteworthily, the total number of identified methylated short peptides was improved by more than 2-fold. By this strategy, we identified 492 methylation sites from NCI-H460 cells compared to only 356 sites identified in native forms. The identification of methylation sites before and after derivatization was highly complementary. Approximately 2-fold the methylation sites were obtained by combining the results identified in both approaches (native and derivatized) as compared with the only analysis in native forms. Therefore, this novel chemical derivatization strategy is a promising approach for the comprehensive identification of protein methylation by improving the identification of methylated short peptides.

Photoreactive Probe-Based Strategy Enables the Specific Identification of the Transient Substrates of Methyltransferase at the Proteome Scale.

To decipher the biological function of protein arginine methyltransferases (PRMTs), the identification of their substrate proteins is crucial. However, this is not a trivial task as the stable and strong interacting proteins always prevail over the weak and transient substrate proteins. Herein, we report the development of a novel photoreactive probe-based strategy to identify the substrate proteins of methyltransferases. By applying it to PRMT1, we demonstrate that this strategy can effectively distinguish substrate proteins from other interacting proteins and allows the identification of highly confident substrate proteins. Noteworthily, we found for the first time that hypomethylation of proteins is a prerequisite for efficient capturing of substrate proteins. This study describes the development of a robust chemical proteomics tool for profiling the transient substrates and can be adapted for broad biomedical applications.

Molecular basis for protein histidine N1-specific methylation of the "His-x-His" motifs by METTL9.

Histidine methylation serves as an intriguing strategy to introduce altered traits of target proteins, including metal ion chelation, histidine-based catalysis, molecular assembly, and translation regulation. As a newly identified histidine methyltransferase, METTL9 catalyzes N1-methylation of protein substrates containing the "His-x-His" motif (HxH, x denotes small side chain residue). Here our structural and biochemical studies revealed that METTL9 specifically methylates the second histidine of the "HxH" motif, while exploiting the first one as a recognition signature. We observed an intimate engagement between METTL9 and a pentapeptide motif, where the small "x" residue is embedded and confined within the substrate pocket. Upon complex formation, the N3 atom of histidine imidazole ring is stabilized by an aspartate residue such that the N1 atom is presented to S-adenosylmethionine for methylation. Moreover, METTL9 displayed a feature in preferred consecutive and "C-to-N" directional methylation of tandem "HxH" repeats that exist in many METTL9 substrates. Collectively, our work illustrates the molecular design of METTL9 in N1-specific methylation of the broadly existing "HxH" motifs, highlighting its importance in histidine methylation biology.

O-Glycopeptide Truncation Strategy for Heterogeneous O-GalNAc Glycoproteomics Characterization.

Mucin-type O-glycosylation (or O-GalNAcylation) takes place on most membrane and secretory proteins and is vital in regulating protein functions and many biological processes. O-GalNAcylation generally exhibits highly diverse and dense O-glycans linked to carrier proteins, which challenges the analysis of O-GalNAc glycoproteome using conventional methodologies. Here, we report an O-glycopeptide truncation strategy for the characterization of protein O-GalNAcylation in biological samples. The O-glycopeptide truncation strategy utilizes proteases or O-glycopeptidases for targeted cleavage of the enriched tryptic O-glycopeptides. It simplifies the O-glycopeptide backbones, O-glycans, or both, and has been shown to aid the improvement of the analytical coverage of O-GalNAc glycopeptides and glycoproteins. Tryptic O-glycopeptides covered with O-glycan clusters and terminal sialic acids could be well isolated by the hydrophilic-based enrichment approaches. The enriched O-glycopeptides are then enzymatically truncated into shorter or less multiply O-glycosylated peptides, which are more favorable for mass spectrometry detection and database search in general bottom-up glycoproteomics. We also investigate different proteolysis which could be well integrated into the O-glycopeptide truncation strategy. For large-scale analysis, we exploit different truncation schemes and identify nearly 2000 O-glycopeptides corresponding to 391 glycoproteins from 75 µL human serum, achieving the deepest-scale coverage of O-glycoproteins compared to other plasma/serum O-glycoproteomic studies. Together, the O-glycopeptide truncation strategy has great potential to facilitate the in-depth study of O-GalNAc glycoproteomics in biological samples.

Temporal Tracking of Insulin Action on the Cell Surface of Proteins at a Resolution of Ten Seconds.

The ligand-receptor signaling occurring on the cell surface governs cell growth, proliferation, and survival via rapidly triggering a cascade of events. Here, we for the first time report an in situ perturbation-free and rapid surface proteomic profiling at a temporal resolution of ten seconds. By this innovation, about 1022 cell surface-associated proteins were reproducibly identified and quantified. It is noteworthy that, upon a model ligand insulin stimulus, a few rapid-responding proteins at 10 s to 2 min were identified, e.g., CNNM3. Moreover, temporal response patterns were established for the members of GLUT4 storage vesicles (GSVs; responsible for glucose transportation) and confirmed with five known GSV proteins. This pattern was then exploited to uncover seven new regulatory proteins (LDLR, HFE, ECE1, MRC2, CORO1C, CPD, and BST2). Collectively, we showed a powerful surface proteomic tool to decipher rapid signaling of cell-surface proteins and to uncover new subunits involved in rapidly trafficking vesicles.