Glucose-induced CRL4COP1-p53 axis amplifies glycometabolism to drive tumorigenesis.

The diabetes-cancer association remains underexplained. Here, we describe a glucose-signaling axis that reinforces glucose uptake and glycolysis to consolidate the Warburg effect and overcome tumor suppression. Specifically, glucose-dependent CK2 O-GlcNAcylation impedes its phosphorylation of CSN2, a modification required for the deneddylase CSN to sequester Cullin RING ligase 4 (CRL4). Glucose, therefore, elicits CSN-CRL4 dissociation to assemble the CRL4COP1 E3 ligase, which targets p53 to derepress glycolytic enzymes. A genetic or pharmacologic disruption of the O-GlcNAc-CK2-CSN2-CRL4COP1 axis abrogates glucose-induced p53 degradation and cancer cell proliferation. Diet-induced overnutrition upregulates the CRL4COP1-p53 axis to promote PyMT-induced mammary tumorigenesis in wild type but not in mammary-gland-specific p53 knockout mice. These effects of overnutrition are reversed by P28, an investigational peptide inhibitor of COP1-p53 interaction. Thus, glycometabolism self-amplifies via a glucose-induced post-translational modification cascade culminating in CRL4COP1-mediated p53 degradation. Such mutation-independent p53 checkpoint bypass may represent the carcinogenic origin and targetable vulnerability of hyperglycemia-driven cancer.

Cell type-specific cytonuclear coevolution in three allopolyploid plant species.

Cytonuclear disruption may accompany allopolyploid evolution as a consequence of the merger of different nuclear genomes in a cellular environment having only one set of progenitor organellar genomes. One path to reconcile potential cytonuclear mismatch is biased expression for maternal gene duplicates (homoeologs) encoding proteins that target to plastids and/or mitochondria. Assessment of this transcriptional form of cytonuclear coevolution at the level of individual cells or cell types remains unexplored. Using single-cell (sc-) and single-nucleus (sn-) RNAseq data from eight tissues in three allopolyploid species, we characterized cell type-specific variations of cytonuclear coevolutionary homoeologous expression and demonstrated the temporal dynamics of expression patterns across development stages during cotton fiber development. Our results provide unique insights into transcriptional cytonuclear coevolution in plant allopolyploids at the single-cell level.

O-GlcNAcylation promotes topoisomerase IIα catalytic activity in breast cancer chemoresistance.

DNA topoisomerase IIα (TOP2A) plays a vital role in replication and cell division by catalytically altering DNA topology. It is a prominent target for anticancer drugs, but clinical efficacy is often compromised due to chemoresistance. In this study, we investigate the role of TOP2A O-GlcNAcylation in breast cancer cells and patient tumor tissues. Our results demonstrate that elevated TOP2A, especially its O-GlcNAcylation, promotes breast cancer malignant progression and resistance to adriamycin (Adm). O-GlcNAcylation at Ser1469 enhances TOP2A chromatin DNA binding and catalytic activity, leading to resistance to Adm in breast cancer cells and xenograft models. Mechanistically, O-GlcNAcylation-modulated interactions between TOP2A and cell cycle regulators influence downstream gene expression and contribute to breast cancer drug resistance. These results reveal a previously unrecognized mechanistic role for TOP2A O-GlcNAcylation in breast cancer chemotherapy resistance and provide support for targeting TOP2A O-GlcNAcylation in cancer therapy.

Spatial Proteomic Profiling of Colorectal Cancer Revealed Its Tumor Microenvironment Heterogeneity.

Colorectal cancer is a predominant malignancy with a second mortality worldwide. Despite its prevalence, therapeutic options remain constrained and surgical operation is still the most useful therapy. In this regard, a comprehensive spatially resolved quantitative proteome atlas was constructed to explore the functional proteomic landscape of colorectal cancer. This strategy integrates histopathological analysis, laser capture microdissection, and proteomics. Spatial proteome profiling of 200 tissue section samples facilitated by the fully integrated sample preparation technology SISPROT enabled the identification of more than 4000 proteins on the Orbitrap Exploris 240 from 2 mm2 × 10 µm tissue sections. Compared with normal adjacent tissues, we identified a spectrum of cancer-associated proteins and dysregulated pathways across various regions of colorectal cancer including ascending colon, transverse colon, descending colon, sigmoid colon, and rectum. Additionally, we conducted proteomic analysis on tumoral epithelial cells and paracancerous epithelium from early to advanced stages in hallmark rectum cancer and sigmoid colon cancer. Bioinformatics analysis revealed functional proteins and cell-type signatures associated with different regions of colorectal tumors, suggesting potential clinical implications. Overall, this study provides a comprehensive spatially resolved functional proteome landscape of colorectal cancer, serving as a valuable resource for exploring potential biomarkers and therapeutic targets.

Plasma proteomic characterization of colorectal cancer patients with FOLFOX chemotherapy by integrated proteomics technology.

BACKGROUND: Colorectal Cancer (CRC) is a prevalent form of cancer, and the effectiveness of the main postoperative chemotherapy treatment, FOLFOX, varies among patients. In this study, we aimed to identify potential biomarkers for predicting the prognosis of CRC patients treated with FOLFOX through plasma proteomic characterization. METHODS: Using a fully integrated sample preparation technology SISPROT-based proteomics workflow, we achieved deep proteome coverage and trained a machine learning model from a discovery cohort of 90 CRC patients to differentiate FOLFOX-sensitive and FOLFOX-resistant patients. The model was then validated by targeted proteomics on an independent test cohort of 26 patients. RESULTS: We achieved deep proteome coverage of 831 protein groups in total and 536 protein groups in average for non-depleted plasma from CRC patients by using a Orbitrap Exploris 240 with moderate sensitivity. Our results revealed distinct molecular changes in FOLFOX-sensitive and FOLFOX-resistant patients. We confidently identified known prognostic biomarkers for colorectal cancer, such as S100A4, LGALS1, and FABP5. The classifier based on the biomarker panel demonstrated a promised AUC value of 0.908 with 93% accuracy. Additionally, we established a protein panel to predict FOLFOX effectiveness, and several proteins within the panel were validated using targeted proteomic methods. CONCLUSIONS: Our study sheds light on the pathways affected in CRC patients treated with FOLFOX chemotherapy and identifies potential biomarkers that could be valuable for prognosis prediction. Our findings showed the potential of mass spectrometry-based proteomics and machine learning as an unbiased and systematic approach for discovering biomarkers in CRC.

Traffic-related air pollution and Parkinson's disease in central California.

BACKGROUND: Prior studies suggested that air pollution exposure may increase the risk of Parkinson's Disease (PD). We investigated the long-term impacts of traffic-related and multiple sources of particulate air pollution on PD in central California. METHODS: Our case-control analysis included 761 PD patients and 910 population controls. We assessed exposure at residential and occupational locations from 1981 to 2016, estimating annual average carbon monoxide (CO) concentrations - a traffic pollution marker - based on the California Line Source Dispersion Model, version 4. Additionally, particulate matter (PM2.5) concentrations were based on a nationwide geospatial chemical transport model. Exposures were assessed as 10-year averages with a 5-year lag time prior to a PD diagnosis for cases and an interview date for controls, subsequently categorized into tertiles. Logistic regression models were used, adjusting for various factors. RESULTS: Traffic-related CO was associated with an increased odds ratio for PD at residences (OR for T3 vs. T1: 1.58; 95% CI: 1.20, 2.10; p-trend = 0.02) and workplaces (OR for T3 vs. T1: 1.91; 95% CI: 1.22, 3.00; p-trend <0.01). PM2.5 was also positively associated with PD at residences (OR for T3 vs. T1: 1.62; 95% CI: 1.22, 2.15; p-trend <0.01) and workplaces (OR for T3 vs. T1: 1.85; 95% CI: 1.21, 2.85; p-trend <0.01). Associations remained robust after additional adjustments for smoking status and pesticide exposure and were consistent across different exposure periods. CONCLUSION: We found that long-term modeled exposure to local traffic-related air pollution (CO) and fine particulates from multiple sources (PM2.5) at homes and workplaces in central California was associated with an increased risk of PD.

Ambient long-term exposure to organophosphorus pesticides and the human gut microbiome: an observational study.

BACKGROUND: Organophosphorus pesticides (OP) have been associated with various human health conditions. Animal experiments and in-vitro models suggested that OP may also affect the gut microbiota. We examined associations between ambient chronic exposure to OP and gut microbial changes in humans. METHODS: We recruited 190 participants from a community-based epidemiologic study of Parkinson's disease living in a region known for heavy agricultural pesticide use in California. Of these, 61% of participants had Parkinson's disease and their mean age was 72 years. Microbiome and predicted metagenome data were generated by 16S rRNA gene sequencing of fecal samples. Ambient long-term OP exposures were assessed using pesticide application records combined with residential addresses in a geographic information system. We examined gut microbiome differences due to OP exposures, specifically differences in microbial diversity based on the Shannon index and Bray-Curtis dissimilarities, and differential taxa abundance and predicted Metacyc pathway expression relying on regression models and adjusting for potential confounders. RESULTS: OP exposure was not associated with alpha or beta diversity of the gut microbiome. However, the predicted metagenome was sparser and less evenly expressed among those highly exposed to OP (p = 0.04). Additionally, we found that the abundance of two bacterial families, 22 genera, and the predicted expression of 34 Metacyc pathways were associated with long-term OP exposure. These pathways included perturbed processes related to cellular respiration, increased biosynthesis and degradation of compounds related to bacterial wall structure, increased biosynthesis of RNA/DNA precursors, and decreased synthesis of Vitamin B1 and B6. CONCLUSION: In support of previous animal studies and in-vitro findings, our results suggest that ambient chronic OP pesticide exposure alters gut microbiome composition and its predicted metabolism in humans.

A phosphoglycerate mutase 1 allosteric inhibitor overcomes drug resistance to EGFR-targeted therapy via disrupting IL-6/JAK2/STAT3 signaling pathway in lung adenocarcinoma.

Resistance to epidermal growth factor receptor (EGFR) inhibitors, from the first-generation erlotinib to the third generation osimertinib, is a clinical challenge in the treatment of patients with EGFR-mutant lung adenocarcinoma. Our previous work found that a novel allosteric inhibitor of phosphoglycerate mutase 1 (PGAM1), HKB99, restrains erlotinib resistance in lung adenocarcinoma cells. However, the role of HKB99 in osimertinib resistance and its underlying molecular mechanism remains to be elucidated. Herein, we found that IL-6/JAK2/STAT3 signaling pathway is aberrantly activated in both erlotinib and osimertinib resistant cells. Importantly, HKB99 significantly blocks the interaction of PGAM1 with JAK2 and STAT3 via the allosteric sites of PGAM1, which leads to inactivation of JAK2/STAT3 and thereby disrupts IL-6/JAK2/STAT3 signaling pathway. Consequently, HKB99 remarkably restores EGFR inhibitor sensitivity and exerts synergistic tumoricidal effect. Additionally, HKB99 alone or in combination with osimertinib down-regulated the level of p-STAT3 in xenograft tumor models. Collectively, this study identifies PGAM1 as a key regulator in IL-6/JAK2/STAT3 axis in the development of resistance to EGFR inhibitors, which could serve as a therapeutic target in lung adenocarcinoma with acquired resistance to EGFR inhibitors.

Compensatory Genetic and Transcriptional Cytonuclear Coordination in Allopolyploid Lager Yeast (Saccharomyces pastorianus).

Cytonuclear coordination between biparental-nuclear genomes and uniparental-cytoplasmic organellar genomes in plants is often resolved by genetic and transcriptional cytonuclear responses. Whether this mechanism also acts in allopolyploid members of other kingdoms is not clear. Additionally, cytonuclear coordination of interleaved allopolyploid cells/individuals within the same population is underexplored. The yeast Saccharomyces pastorianus provides the opportunity to explore cytonuclear coevolution during different growth stages and from novel dimensions. Using S. pastorianus cells from multiple growth stages in the same environment, we show that nuclear mitochondria-targeted genes have undergone both asymmetric gene conversion and growth stage-specific biased expression favoring genes from the mitochondrial genome donor (Saccharomyces eubayanus). Our results suggest that cytonuclear coordination in allopolyploid lager yeast species entails an orchestrated and compensatory genetic and transcriptional evolutionary regulatory shift. The common as well as unique properties of cytonuclear coordination underlying allopolyploidy between unicellular yeasts and higher plants offers novel insights into mechanisms of cytonuclear evolution associated with allopolyploid speciation.

ppGalNAc-T4-catalyzed O-Glycosylation of TGF-ß type â ¡ receptor regulates breast cancer cells metastasis potential.

GalNAc-type O-glycosylation, initially catalyzed by polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts), is one of the most abundant and complex posttranslational modifications of proteins. Emerging evidence has proven that aberrant ppGalNAc-Ts are involved in malignant tumor transformation. However, the exact molecular functions of ppGalNAc-Ts are still unclear. Here, the role of one isoform, ppGalNAc-T4, in breast cancer cell lines was investigated. The expression of ppGalNAc-T4 was found to be negatively associated with migration of breast cancer cells. Loss-of-function studies revealed that ppGalNAc-T4 attenuated the migration and invasion of breast cancer cells by inhibiting the epithelial-mesenchymal transition (EMT) process. Correspondingly, transforming growth factor beta (TGF-ß) signaling, which is the upstream pathway of EMT, was impaired by ppGalNAc-T4 expression. ppGalNAc-T4 knockout decreased O-GalNAc modification of TGF-ß type Ⅰ and Ⅱ receptor (TßR Ⅰ and Ⅱ) and led to the elevation of TGF-ß receptor dimerization and activity. Importantly, a peptide from TßR Ⅱ was identified as a naked peptide substrate of ppGalNAc-T4 with a higher affinity than ppGalNAc-T2. Further, Ser31, corresponding to the extracellular domain of TßR Ⅱ, was identified as the O-GalNAcylation site upon in vitro glycosylation by ppGalNAc-T4. The O-GalNAc-deficient S31 A mutation enhanced TGF-ß signaling activity and EMT in breast cancer cells. Together, these results identified a novel mechanism of ppGalNAc-T4-catalyzed TGF-ß receptors O-GalNAcylation that suppresses breast cancer cell migration and invasion via the EMT process. Targeting ppGalNAc-T4 may be a potential therapeutic strategy for breast cancer treatment.

High-Resolution Metabolomic Assessment of Pesticide Exposure in Central Valley, California.

Pesticides are widely used in the agricultural Central Valley region of California. Historically, this has included organophosphates (OPs), organochlorines (OCs), and pyrethroids (PYRs). This study aimed to identify perturbations of the serum metabolome in response to each class of pesticide and mutual associations between groups of metabolites and multiple pesticides. We conducted high-resolution metabolomic profiling of serum samples from 176 older adults living in the California Central Valley using liquid chromatography with high-resolution mass spectrometry. We estimated chronic pesticide exposure (from 1974 to year of blood draw) to OPs, OCs, and PYRs from ambient sources at homes and workplaces with a geographic information system (GIS)-based model. Based on partial least-squares regression and pathway enrichment analysis, we identified metabolites and metabolic pathways associated with one or multiple pesticide classes, including mitochondrial energy metabolism, fatty acid and lipid metabolism, and amino acid metabolism. Utilizing an integrative network approach, we found that the fatty acid ß-oxidation pathway is a common pathway shared across all three pesticide classes. The disruptions of the serum metabolome suggested that chronic pesticide exposure might result in oxidative stress, inflammatory reactions, and mitochondrial dysfunction, all of which have been previously implicated in a wide variety of diseases. Overall, our findings provided a comprehensive view of the molecular mechanisms of chronic pesticide toxicity, and, for the first time, our approach informs exposome research by moving from macrolevel population exposures to microlevel biologic responses.

Reduced expression of ppGalNAc-T4 promotes proliferation of human breast cancer cells.

Breast cancer, one of the most frequently diagnosed and aggressive malignancies, is the major cause of cancer-related death greatly threatening women health. Polypeptide N-acetylgalactosaminyltransferase 4 (ppGalNAc-T4), responsible for the initial step of mucin-type O-glycosylation, has been reported to be implicated in diverse types of human tumors. However, the biological role of ppGalNAc-T4 in breast cancer is still undetermined. In this study, we investigate the effects and mechanism of ppGalNAc-T4 to breast cancer cell proliferation. From analysis of high throughput RNA sequencing datasets of Gene Expression Omnibus and ArrayExpress, a positive correlation between ppGalNAc-T4 and the recurrence-free survival was observed in multigroup of human breast cancer datasets. Moreover, transcriptomes analysis using RNA-sequencing in MCF7 cells revealed that cell cycle-related genes induced the effects of ppGalNAc-T4 on breast cancer cell proliferation. Additionally, investigations showed that ppGalNAc-T4 impaired cell proliferation in MCF-7 and MDA-MB-231 breast cells. Furthermore, our results suggested that the ppGalNAc-T4 knockout activated Notch signaling pathway and enhanced cell proliferation. Collectively, our data indicated that ppGalNAc-T4 affected the proliferation of human breast cancer cells, which appears to be a novel target for understanding the underlying molecular mechanism of breast cancer.

NRF2-GPX4/SOD2 axis imparts resistance to EGFR-tyrosine kinase inhibitors in non-small-cell lung cancer cells.

Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) have achieved satisfactory clinical effects in the therapy of non-small cell lung cancer (NSCLC), but acquired resistance limits their clinical application. NRF2 has been shown to enhance the resistance to apoptosis induced by radiotherapy and some chemotherapy. In this study, we investigated the role of NRF2 in resistance to EGFR-TKIs. We showed that NRF2 protein levels were markedly increased in a panel of EGFR-TKI-resistant NSCLC cell lines due to slow degradation of NRF2 protein. NRF2 knockdown overcame the resistance to EGFR-TKIs in HCC827ER and HCC827GR cells. Furthermore, we demonstrated that NRF2 imparted EGFR-TKIs resistance in HCC827 cells via upregulation of GPX4 and SOD2, and suppression of GPX4 and SOD2 reversed resistance to EGFR-TKIs. Thus, we conclude that targeting NRF2-GPX4/SOD2 pathway is a potential strategy for overcoming resistance to EGFR-TKIs.

Epidemiology, Clinical Characteristics, and Visual Outcomes of Patients with Intraocular Foreign Bodies in Southwest China: A 10-Year Review.

INTRODUCTION: Intraocular foreign bodies (IOFBs) are a serious subset of open-globe injury that can result in visual loss. This study analyzed the epidemiology, clinical characteristics, and visual outcomes of patients with IOFBs in Southwest China. METHODS: This retrospective study comprised 1,176 patients with the primary diagnosis of IOFBs who resided in Sichuan Province over a 10-year period. All data were collected from medical records and analyzed statistically. RESULTS: The annual incidence for IOFBs was 0.14 per 100,000 (95% confidence interval 0.12-0.16 per 100,000) people in Southwest China. In that period, IOFBs accounted for 22.3% of all open-globe injuries. Working-age male patients accounted for 79.1% of all IOFBs patients and there had significant differences in age distributions between genders (p < 0.001). Metallic IOFBs were the most common (74.6%) IOFB, but there were significant differences in the materials of IOFBs between adults and children of different age-groups (p < 0.001). At discharge, 277 (23.6%) patients had increased visual acuity (VA) and 95 (8.0%) had no light perception. Initial VA <20/200 (odds ratio [OR], 5.5; p < 0.001), increasing wound size (OR, 1.3; p = 0.004), IOFBs in the posterior segment (OR, 2.6; p = 0.002) and existing complications (traumatic cataract, endophthalmitis, retinal detachment, or retinal break) were independent risk factors for final VA <20/200. CONCLUSION: The incidence of IOFBs in Southwest China differed from global statistics. Adults and children had different clinical characteristics. Thus, their prevention strategies should be different.

D283 Med, a Cell Line Derived from Peritoneal Metastatic Medulloblastoma: A Good Choice for Missing Protein Discovery.

Since the Chromosome-Centric Human Proteome Project (C-HPP) was launched in 2010, many techniques have been adopted for the discovery of missing proteins (MPs). Because of these efforts, only 1481 MPs remained as of July 2020; however, by relying only on technique optimization, researchers have reached a bottleneck in MP discovery. Protein expression is tissue- or cell-type-dependent. The tissues of the human testis and brain have been reported to harbor a large number of tissue-specific genes and proteins; however, few studies have been performed on human brain tissue or cells to identify MPs. Herein a metastatic cell line derived from brain cancer, D283 Med, was used to search for MPs. With a traditional and simple shotgun workflow to separate the peptides into 20 fractions, 12 MPs containing at least two unique non-nested peptides (amino acid length ≥9) were identified in this cell line with a protein false discovery rate of <1%. Following the same experimental protocol, only one MP was found in a nonmetastatic brain cancer cell line, U-118 MG. Furthermore, 12 MPs were verified as having two non-nested unique peptides by matching them with corresponding chemically synthesized peptides through parallel reaction monitoring. These results clearly demonstrate that the appropriate selection of experimental materials, either tissues or cell lines, is imperative for MP discovery. The data obtained in this study are available via ProteomeXchange (PXD021482) and PeptideAtlas (PASS01627).

Exploration of Missing Proteins by a Combination Approach to Enrich the Low-Abundance Hydrophobic Proteins from Four Cancer Cell Lines.

The mission of the Chromosome-Centric Human Proteome Project (C-HPP) to discover missing proteins (MPs) has become increasingly difficult due to the remaining low-abundance, high-hydrophobicity, or low-molecular-weight MPs. We have reported two approaches to resolve these identification problems for the low-abundance and high-hydrophobicity MPs, respectively. In this study, to improve the identification of low-abundance MPs with high hydrophobicity, we combined two approaches and obtained MPs from several different cancer cell lines. Their membrane fractions were isolated by ultracentrifugation, and the low-abundance proteins were enriched at the protein level with the ProteoMiner kit. After that, the peptides from the enriched proteins were separated by high concentrations of organic solvents according to their hydrophobicity as the first dimension of separation at the peptide level, and the second and third dimensions of separation involved a high pH reversed-phase and an acid reversed-phase column, respectively. In total, 16 MPs (at least two non-nested unique peptides with ≥9 amino acids) with 61 unique peptides were identified from four human cancer cell lines, including 2, 8, 2, and 7 MPs from HeLa, HCT116, SNU-1, and HepG2 cells, respectively. Furthermore, all MPs were verified with two non-nested unique peptides through parallel reaction monitoring (PRM) by matching the peptides with their chemically synthesized peptides. Interestingly, two additional MPs were verified from the same cell line by PRM assay, although the two non-nested unique peptides with ≥9 amino acids for each MP were identified from different MS injections or cell lines by data-dependent acquisition (DDA). Thus, a total of 18 MPs were dug out in this study. The data are available via ProteomeXchange (PXD014058) and PeptideAtlas (PASS01388).

Evaluation and minimization of nonspecific tryptic cleavages in proteomic sample preparation.

High specificity of trypsin is a prerequisite for accurate identification and quantification of proteins in shotgun proteomics. It is important to minimize nonspecific enzymatic cleavages during proteomic sample preparation. METHODS: In this study, protein extraction and trypsin digestion conditions were extensively evaluated using the less-complex Escherichia coli lysates to improve the sensitivity of detecting low-abundance nonspecific peptides by liquid chromatography/tandem mass spectrometry. RESULTS: Trypsin digestion buffers and digestion times were proved to have a significant effect on nonspecific cleavages. The triethylammonium bicarbonate buffer induces significantly lower nonspecific cleavages than the other two buffers, but a freshly prepared urea solution does not induce more than sodium dodecyl sulfate. Because prolonged trypsin digestion resulted in a considerable number of nonspecific cleavages, an optimized 2-h protocol was developed with 45.2% less semispecific tryptic peptides but 18.5% more unmodified peptides identified than the commonly used 16-h protocol. CONCLUSIONS: The significant decrease in nonspecific cleavages and artificial modifications improves the accuracy of protein quantification and the identification of low-abundance proteins, and it is especially useful for studying protein posttranslational modifications. For trypsin digestion, the proposed 2-h protocol can potentially be a replacement for the traditional 16-h protocol.

Identification of the O-GalNAcylation site(s) on FOXA1 catalyzed by ppGalNAc-T2 enzyme in vitro.

FOXA1 functions as a pioneer factor of transcriptional regulation that binds to specific sites in the chromatin and recruits other transcription factors, promoting the initiation of gene transcription and mediating the regulation of downstream target gene expression. FOXA1 was reported to facilitate or reprogram ERα binding, thus playing a key function in breast cancer progression. Our previous results indicated that the O-linked N-acetylgalactosamine (O-GalNAc) modification of FOXA1 plays a potentially significant role in the ERα transcription network. However, further investigations are needed to identify the specific mechanism of modification and the specific glycosylation sites on FOXA1. In this study, we first suggested that FOXA1 could be O-GalNAcylated by ppGalNAc-T2 in vitro. By dividing and expressing recombinant FOXA1 as three segments, two O-GalNAcylation sites were found on FOXA1, both located at the C-terminal of the protein. Then, synthesized peptides, including the predicted O-GalNAc sites in the C-terminus of FOXA1, were used in a vitro reaction, and peptides mutated at the predicted O-GalNAc sites were employed as controls. Through an ESI-MS assay, S354 and S355 were identified as probable O-GalNAcylation sites on FOXA1. Additionally, we performed ESI-ETD-MS/MS analysis of the full-length O-GalNAcylated FOXA1 protein and identified S355 as the O-GalNAc modification site on FOXA1, consistent with the peptide reaction. In conclusion, our results demonstrated that FOXA1 can be O-GalNAcylated by ppGalNAc-T2 at S355 in vitro. These results will provide new insights for studying the role of O-GalNAcylation in the development of breast cancer.

Improvement of Peptide Separation for Exploring the Missing Proteins Localized on Membranes.

Following an enormous effort by the global scientific community coordinated by HUPO's Human Proteome Project, the number of proteins without high-quality MS or other evidence (colloquially termed missing proteins) has substantially decreased; however, some highly hydrophobic MPs remain on the list. We believe that efficient peptide separation is an approach that can be used to improve the identification of these hydrophobic MPs. We propose that peptides prepared from the membrane fractions of human cell lines and placental tissue can be well separated from hydrophilic peptides in organic solvents at high concentrations due to the precipitation of hydrophilic peptides with lower solubility. Using a combination strategy of peptide separation in 98% acetonitrile prior to traditional 2D reverse-phase liquid chromatography, more hydrophobic peptides were detected in the supernatants of the organic solvent extractions than were found in the pellets. When this strategy was adopted, 30 MPs (≥2 non-nested unique peptides with ≥9 amino acids) with 114 unique peptides were identified at protein false discovery rate (FDR) < 1%, including 7, 12, and 13 MPs obtained from membrane preparations derived from K562, HeLa cells, and human placenta, respectively. Of the 30 MPs identified in this study, 19 were categorized as membrane proteins or extracellular matrix proteins. Furthermore, 20 were verified to possess two non-nested unique peptides through parallel reaction monitoring with the corresponding chemically synthesized peptides. The use of organic solvents at high concentrations was shown to be an efficient way to improve the exploration of hydrophobic MPs. The data obtained in this study are available via ProteomeXchange (PXD010630) and PeptideAtlas (PASS01218).

Self-Assembly of Supramolecular Fractals from Generation 1 to 5.

In the seeking of molecular expression of fractal geometry, chemists have endeavored in the construction of molecules and supramolecules during the past few years, while only a few examples were reported, especially for the discrete architectures. We herein designed and constructed five generations of supramolecular fractals (G1-G5) based on the coordination-driven self-assembly of terpyridine ligands. All the ligands were synthesized from triphenylamine motif, which played a central role in geometry control. Different approaches based on direct Sonogashira coupling and/or ⟨tpy-Ru(II)-tpy⟩ connectivity were employed to prepare complex Ru(II)-organic building blocks. Fractals G1-G5 were obtained in high yields by precise coordination of organic or Ru(II)-organic building blocks with Zn(II) ions. Characterization of those architectures were accomplished by 1D and 2D NMR spectroscopy, electrospray ionization mass spectrometry (ESI-MS), traveling-wave ion mobility mass spectrometry (TWIM-MS), and transmission electron microscopy (TEM). Furthermore, the two largest fractals also hierarchically self-assemble into ordered supramolecular nanostructures either at solid/liquid interface or in solution on the basis of their well-defined scaffolds.