A novel nomogram to predict psoriatic arthritis in patients with plaque psoriasis.

OBJECTIVE: To construct a predictive model for Psoriatic Arthritis (PsA) based on clinical and ultrasonic characteristics in patients with plaque psoriasis (PsP). PATIENTS AND METHODS: Demographic, clinical, and ultrasound data were collected from patients with PsP and PsA between May 2019 and December 2022. RESULTS: A total of 212 patients with PsP and 123 with PsA in the training cohort, whereas the validation cohort comprised 91 patients with PsP and 49 with PsA. The multivariate logistic regression identified nail psoriasis (odds ratio [OR] 1.88, 95% CI: 1.07-3.29), synovitis (OR 18.23, 95% CI: 4.04-82.33), enthesitis (OR 3.71, 95% CI: 1.05-13.14), and bone erosion (OR 11.39, 95% CI: 3.05-42.63) as effective predictors for PsA. The area under the curve was 0.750 (95% CI, 0.691-0.806) and 0.804 (95% CI, 0.723-0.886) for the training and validation cohorts, respectively. The Hosmer-Lemeshow goodness-of-fit test showed good consistency for both the training cohort (p  =  0.970) and the validation cohort (p  =  0.967). Calibration curves also indicated good calibration for both cohorts. The DCA revealed that the predictive model had good clinical utility. CONCLUSIONS: We have developed a quantitative, intuitive, and convenient predictive model based on nail psoriasis, synovitis, enthesitis, and bone erosion to assess the risk of PsA in patients with plaque psoriasis.

P4HA2 hydroxylates SUFU to regulate the paracrine Hedgehog signaling and promote B-cell lymphoma progression.

Aberrations in the Hedgehog (Hh) signaling pathway are significantly prevailed in various cancers, including B-cell lymphoma. A critical facet of Hh signal transduction involves the dynamic regulation of the suppressor of fused homolog (SUFU)-glioma-associated oncogene homolog (GLI) complex within the kinesin family member 7 (KIF7)-supported ciliary tip compartment. However, the specific post-translational modifications of SUFU-GLI complex within this context have remained largely unexplored. Our study reveals a novel regulatory mechanism involving prolyl 4-hydroxylase 2 (P4HA2), which forms a complex with KIF7 and is essential for signal transduction of Hh pathway. We demonstrate that, upon Hh pathway activation, P4HA2 relocates alongside KIF7 to the ciliary tip. Here, it hydroxylates SUFU to inhibit its function, thus amplifying the Hh signaling. Moreover, the absence of P4HA2 significantly impedes B lymphoma progression. This effect can be attributed to the suppression of Hh signaling in stromal fibroblasts, resulting in decreased growth factors essential for malignant proliferation of B lymphoma cells. Our findings highlight the role of P4HA2-mediated hydroxylation in modulating Hh signaling and propose a novel stromal-targeted therapeutic strategy for B-cell lymphoma.

VAMP2 controls murine epidermal differentiation and carcinogenesis by regulation of nucleophagy.

Differentiation of murine epidermal stem/progenitor cells involves the permanent withdrawal from the cell cycle, the synthesis of various protein and lipid components for the cornified envelope, and the controlled dissolution of cellular organelles and nuclei. Deregulated epidermal differentiation contributes to the development of various skin diseases, including skin cancers. With a genome-wide shRNA screen, we identified vesicle-associated membrane protein 2 (VAMP2) as a critical factor involved in skin differentiation. Deletion of VAMP2 leads to aberrant skin stratification and enucleation in vivo. With quantitative proteomics, we further identified an autophagy protein, focal adhesion kinase family interacting protein of 200 kDa (FIP200), as a binding partner of VAMP2. Additionally, we showed that both VAMP2 and FIP200 are critical for murine keratinocyte enucleation and epidermal differentiation. Loss of VAMP2 or FIP200 enhances cutaneous carcinogenesis in vivo. Together, our findings identify important molecular mechanisms underlying epidermal differentiation and skin tumorigenesis.

Proteomics analysis of histone deacetylase inhibitor-resistant solid tumors reveals resistant signatures and potential drug combinations.

Histone deacetylase inhibitors (HDACis) are important drugs for cancer therapy, but the indistinct resistant mechanisms of solid tumor therapy greatly limit their clinical application. In this study we conducted HDACi-perturbated proteomics and phosphoproteomics analyses in HDACi-sensitive and -resistant cell lines using a tandem mass tag (TMT)-based quantitative proteomic strategy. We found that the ribosome biogenesis proteins MRTO4, PES1, WDR74 and NOP16 vital to tumorigenesis might regulate the tumor sensitivity to HDACi. By integrating HDACi-perturbated protein signature with previously reported proteomics and drug sensitivity data, we predicted and validated a series of drug combination pairs potentially to enhance the sensitivity of HDACi in diverse solid tumor. Functional phosphoproteomic analysis further identified the kinase PDK1 and ROCK as potential HDACi-resistant signatures. Overall, this study reveals the potential HDACi-resistant signatures and may provide promising drug combination strategies to attenuate the resistance of solid tumor to HDACi.

A proteomic landscape of pharmacologic perturbations for functional relevance.

Pharmacological perturbation studies based on protein-level signatures are fundamental for drug discovery. In the present study, we used a mass spectrometry (MS)-based proteomic platform to profile the whole proteome of the breast cancer MCF7 cell line under stress induced by 78 bioactive compounds. The integrated analysis of perturbed signal abundance revealed the connectivity between phenotypic behaviors and molecular features in cancer cells. Our data showed functional relevance in exploring the novel pharmacological activity of phenolic xanthohumol, as well as the noncanonical targets of clinically approved tamoxifen, lovastatin, and their derivatives. Furthermore, the rational design of synergistic inhibition using a combination of histone methyltransferase and topoisomerase was identified based on their complementary drug fingerprints. This study provides rich resources for the proteomic landscape of drug responses for precision therapeutic medicine.

Prognostic biomarker discovery based on proteome landscape of Chinese lung adenocarcinoma.

Despite recent innovations in imaging and genomic screening promotes advance in diagnosis and treatment of lung adenocarcinoma (LUAD), there remains high mortality of LUAD and insufficient understanding of LUAD biology. Our previous study performed an integrative multi-omic analysis of LUAD, filling the gap between genomic alterations and their biological proteome effects. However, more detailed molecular characterization and biomarker resources at proteome level still need to be uncovered. In this study, a quantitative proteomic experiment of patient-derived benign lung disease samples was carried out. After that, we integrated the proteomic data with previous dataset of 103 paired LUAD samples. We depicted the proteomic differences between non-cancerous and tumor samples and among diverse pathological subtypes. We also found that up-regulated mitophagy was a significant characteristic of early-stage LUAD. Additionally, our integrative analysis filtered out 75 potential prognostic biomarkers and validated two of them in an independent LUAD serum cohort. This study provided insights for improved understanding proteome abnormalities of LUAD and the novel prognostic biomarker discovery offered an opportunity for LUAD precise management.

Metabolic regulation of homologous recombination repair by MRE11 lactylation.

Lactylation is a lactate-induced post-translational modification best known for its roles in epigenetic regulation. Herein, we demonstrate that MRE11, a crucial homologous recombination (HR) protein, is lactylated at K673 by the CBP acetyltransferase in response to DNA damage and dependent on ATM phosphorylation of the latter. MRE11 lactylation promotes its binding to DNA, facilitating DNA end resection and HR. Inhibition of CBP or LDH downregulated MRE11 lactylation, impaired HR, and enhanced chemosensitivity of tumor cells in patient-derived xenograft and organoid models. A cell-penetrating peptide that specifically blocks MRE11 lactylation inhibited HR and sensitized cancer cells to cisplatin and PARPi. These findings unveil lactylation as a key regulator of HR, providing fresh insights into the ways in which cellular metabolism is linked to DSB repair. They also imply that the Warburg effect can confer chemoresistance through enhancing HR and suggest a potential therapeutic strategy of targeting MRE11 lactylation to mitigate the effects.

ABPP-CoDEL: Activity-Based Proteome Profiling-Guided Discovery of Tyrosine-Targeting Covalent Inhibitors from DNA-Encoded Libraries.

DNA-encoded chemical library (DEL) has been extensively used for lead compound discovery for decades in academia and industry. Incorporating an electrophile warhead into DNA-encoded compounds recently permitted the discovery of covalent ligands that selectively react with a particular cysteine residue. However, noncysteine residues remain underexplored as modification sites of covalent DELs. Herein, we report the design and utility of tyrosine-targeting DELs of 67 million compounds. Proteome-wide reactivity analysis of tyrosine-reactive sulfonyl fluoride (SF) covalent probes suggested three enzymes (phosphoglycerate mutase 1, glutathione s-transferase 1, and dipeptidyl peptidase 3) as models of tyrosine-targetable proteins. Enrichment with SF-functionalized DELs led to the identification of a series of tyrosine-targeting covalent inhibitors of the model enzymes. In-depth mechanistic investigation revealed their novel modes of action and reactive ligand-accessible hotspots of the enzymes. Our strategy of combining activity-based proteome profiling and covalent DEL enrichment (ABPP-CoDEL), which generated selective covalent binders against a variety of target proteins, illustrates the potential use of this methodology in further covalent drug discovery.

Antiandrogen treatment induces stromal cell reprogramming to promote castration resistance in prostate cancer.

Lineage plasticity causes therapeutic resistance; however, it remains unclear how the fate conversion and phenotype switching of cancer-associated fibroblasts (CAFs) are implicated in disease relapse. Here, we show that androgen deprivation therapy (ADT)-induced SPP1+ myofibroblastic CAFs (myCAFs) are critical stromal constituents that drive the development of castration-resistant prostate cancer (CRPC). Our results reveal that SPP1+ myCAFs arise from the inflammatory CAFs in hormone-sensitive PCa; therefore, they represent two functional states of an otherwise ontogenically identical cell type. Antiandrogen treatment unleashes TGF-ß signaling, resulting in SOX4-SWI/SNF-dependent CAF phenotype switching. SPP1+ myCAFs in turn render PCa refractory to ADT via an SPP1-ERK paracrine mechanism. Importantly, these sub-myCAFs are associated with inferior therapeutic outcomes, providing the rationale for inhibiting polarization or paracrine mechanisms to circumvent castration resistance. Collectively, our results highlight that therapy-induced phenotypic switching of CAFs is coupled with disease progression and that targeting this stromal component may restrain CRPC.

Integrative multi-omics and drug-response characterization of patient-derived prostate cancer primary cells.

Prostate cancer (PCa) is the second most prevalent malignancy in males across the world. A greater knowledge of the relationship between protein abundance and drug responses would benefit precision treatment for PCa. Herein, we establish 35 Chinese PCa primary cell models to capture specific characteristics among PCa patients, including gene mutations, mRNA/protein/surface protein distributions, and pharmaceutical responses. The multi-omics analyses identify Anterior Gradient 2 (AGR2) as a pre-operative prognostic biomarker in PCa. Through the drug library screening, we describe crizotinib as a selective compound for malignant PCa primary cells. We further perform the pharmacoproteome analysis and identify 14,372 significant protein-drug correlations. Surprisingly, the diminished AGR2 enhances the inhibition activity of crizotinib via ALK/c-MET-AKT axis activation which is validated by PC3 and xenograft model. Our integrated multi-omics approach yields a comprehensive understanding of PCa biomarkers and pharmacological responses, allowing for more precise diagnosis and therapies.

Phosphorylation of PHF2 by AMPK releases the repressive H3K9me2 and inhibits cancer metastasis.

Epithelial to mesenchymal transition (EMT) plays a crucial role in cancer metastasis, accompanied with vast epigenetic changes. AMP-activated protein kinase (AMPK), a cellular energy sensor, plays regulatory roles in multiple biological processes. Although a few studies have shed light on AMPK regulating cancer metastasis, the inside epigenetic mechanisms remain unknown. Herein we show that AMPK activation by metformin relieves the repressive H3K9me2-mediated silencing of epithelial genes (e.g., CDH1) during EMT processes and inhibits lung cancer metastasis. PHF2, a H3K9me2 demethylase, was identified to interact with AMPKα2. Genetic deletion of PHF2 aggravates lung cancer metastasis and abolishes the H3K9me2 downregulation and anti-metastasis effect of metformin. Mechanistically, AMPK phosphorylates PHF2 at S655 site, enhancing PHF2 demethylation activity and triggering the transcription of CDH1. Furthermore, the PHF2-S655E mutant that mimics AMPK-mediated phosphorylation status further reduces H3K9me2 and suppresses lung cancer metastasis, while PHF2-S655A mutant presents opposite phenotype and reverses the anti-metastasis effect of metformin. PHF2-S655 phosphorylation strikingly reduces in lung cancer patients and the higher phosphorylation level predicts better survival. Altogether, we reveal the mechanism of AMPK inhibiting lung cancer metastasis via PHF2 mediated H3K9me2 demethylation, thereby promoting the clinical application of metformin and highlighting PHF2 as the potential epigenetic target in cancer metastasis.

Investigation of targets and anticancer mechanisms of covalently acting natural products by functional proteomics.

Eriocalyxin B (EB), 17-hydroxy-jolkinolide B (HJB), parthenolide (PN), xanthatin (XT) and andrographolide (AG) are terpenoid natural products with a variety of promising antitumor activities, which commonly bear electrophilic groups (α,ß-unsaturated carbonyl groups and/or epoxides) capable of covalently modifying protein cysteine residues. However, their direct targets and underlying molecular mechanisms are still largely unclear, which limits the development of these compounds. In this study, we integrated activity-based protein profiling (ABPP) and quantitative proteomics approach to systematically characterize the covalent targets of these natural products and their involved cellular pathways. We first demonstrated the anti-proliferation activities of these five compounds in triple-negative breast cancer cell MDA-MB-231. Tandem mass tag (TMT)-based quantitative proteomics showed all five compounds commonly affected the ubiquitin mediated proteolysis pathways. ABPP platform identified the preferentially modified targets of EB and PN, two natural products with high anti-proliferation activity. Biochemical experiments showed that PN inhibited the cell proliferation through targeting ubiquitin carboxyl-terminal hydrolase 10 (USP10). Together, this study uncovered the covalently modified targets of these natural products and potential molecular mechanisms of their antitumor activities.

Discovery of toxoflavin, a potent IRE1α inhibitor acting through structure-dependent oxidative inhibition.

Inositol-requiring enzyme 1α (IRE1α) is the most conserved endoplasmic reticulum (ER) stress sensor with two catalytic domains, kinase and RNase, in its cytosolic portion. IRE1α inhibitors have been used to improve existing clinical treatments against various cancers. In this study we identified toxoflavin (TXF) as a new-type potent small molecule IRE1α inhibitor. We used luciferase reporter systems to screen compounds that inhibited the IRE1α-XBP1s signaling pathway. As a result, TXF was found to be the most potent IRE1α RNase inhibitor with an IC50 value of 0.226 µM. Its inhibitory potencies on IRE1α kinase and RNase were confirmed in a series of cellular and in vitro biochemical assays. Kinetic analysis showed that TXF caused time- and reducing reagent-dependent irreversible inhibition on IRE1α, implying that ROS might participate in the inhibition process. ROS scavengers decreased the inhibition of IRE1α by TXF, confirming that ROS mediated the inhibition process. Mass spectrometry analysis revealed that the thiol groups of four conserved cysteine residues (CYS-605, CYS-630, CYS-715 and CYS-951) in IRE1α were oxidized to sulfonic groups by ROS. In molecular docking experiments we affirmed the binding of TXF with IRE1α, and predicted its binding site, suggesting that the structure of TXF itself participates in the inhibition of IRE1α. Interestingly, CYS-951 was just near the docked site. In addition, the RNase IC50 and ROS production in vitro induced by TXF and its derivatives were negative correlated (r = -0.872). In conclusion, this study discovers a new type of IRE1α inhibitor that targets a predicted new alternative site located in the junction between RNase domain and kinase domain, and oxidizes conserved cysteine residues of IRE1α active sites to inhibit IRE1α. TXF could be used as a small molecule tool to study IRE1α's role in ER stress.

ARID1A loss induces polymorphonuclear myeloid-derived suppressor cell chemotaxis and promotes prostate cancer progression.

Chronic inflammation and an immunosuppressive microenvironment promote prostate cancer (PCa) progression and diminish the response to immune checkpoint blockade (ICB) therapies. However, it remains unclear how and to what extent these two events are coordinated. Here, we show that ARID1A, a subunit of the SWI/SNF chromatin remodeling complex, functions downstream of inflammation-induced IKKß activation to shape the immunosuppressive tumor microenvironment (TME). Prostate-specific deletion of Arid1a cooperates with Pten loss to accelerate prostate tumorigenesis. We identify polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) as the major infiltrating immune cell type that causes immune evasion and reveal that neutralization of PMN-MDSCs restricts the progression of Arid1a-deficient tumors. Mechanistically, inflammatory cues activate IKKß to phosphorylate ARID1A, leading to its degradation via ß-TRCP. ARID1A downregulation in turn silences the enhancer of A20 deubiquitinase, a critical negative regulator of NF-κB signaling, and thereby unleashes CXCR2 ligand-mediated MDSC chemotaxis. Importantly, our results support the therapeutic strategy of anti-NF-κB antibody or targeting CXCR2 combined with ICB for advanced PCa. Together, our findings highlight that the IKKß/ARID1A/NF-κB feedback axis integrates inflammation and immunosuppression to promote PCa progression.

The length of stay and inpatient burden in inpatients with different psoriasis subtypes.

Background: Heavy disease burden of psoriasis has been indicated by previous studies. However, the cost of care and length of stay (LOS) in inpatients with different psoriasis subtypes were rarely addressed. This study aimed to investigate the cost of care and LOS in Chinese patients with different psoriasis types and to clarify the independent factors affecting LOS. Methods: We conducted a cross-sectional study by enrolling patients with psoriasis who were hospitalized between 13 Feb 2017 and 29 Mar 2021. Demographic and clinical characteristics of the patients were collected by reviewing their Electronic Medical Records. Multivariate linear regression was used to estimate the associations with adjustments. Results: A total of 310 adult patients with psoriasis were included (mean cost of care: 13.0±22.3 kCNY; mean LOS: 7.9±4.3 days). Statistically significant differences were found among patients with different psoriasis subtypes in LOS (P<0.001) but not in the cost of care (P=0.530). Relative to psoriasis vulgaris, pustular psoriasis (Adjusted coefficient: 2.37, 95% confidence interval (CI): 0.87-3.87) and erythrodermic psoriasis (Adjusted coefficient: 2.92, 95%CI: 1.38-4.47) were significantly associated with an increased LOS. Meanwhile, respiratory tract infections (Adjusted coefficient: 1.60, 95%CI: 0.11-3.10) also significantly increased the LOS. On the contrary, a decreased LOS was found in psoriatic arthritis patients treated with TNF-alpha inhibitors (Adjusted coefficient: -2.21, 95%CI: -4.37 to -0.05). Conclusions: LOS differed significantly among different psoriasis subtypes while the inpatient burden for a single hospitalization was alike. Infection is an important factor associated with a longer LOS. TNF-alpha inhibitors evidently reduced the total hospital stay period for patients with psoriatic arthritis.

SLC1A1-mediated cellular and mitochondrial influx of R-2-hydroxyglutarate in vascular endothelial cells promotes tumor angiogenesis in IDH1-mutant solid tumors.

Mutant isocitrate dehydrogenase 1 (mIDH1) drives tumorigenesis via producing oncometabolite R-2-hydroxyglutarate (R-2-HG) across various tumor types. However, mIDH1 inhibitors appear only effective in hematological tumors. The therapeutic benefit in solid tumors remains elusive, likely due to the complex tumor microenvironment. In this study, we discover that R-2-HG produced by IDH1-mutant tumor cells is preferentially imported into vascular endothelial cells and remodels mitochondrial respiration to promote tumor angiogenesis, conferring a therapeutic vulnerability in IDH1-mutant solid tumors. Mechanistically, SLC1A1, a Na+-dependent glutamate transporter that is preferentially expressed in endothelial cells, facilitates the influx of R-2-HG from the tumor microenvironment into the endothelial cells as well as the intracellular trafficking of R-2-HG from cytoplasm to mitochondria. R-2-HG hijacks SLC1A1 to promote mitochondrial Na+/Ca2+ exchange, which activates the mitochondrial respiratory chain and fuels vascular endothelial cell migration in tumor angiogenesis. SLC1A1 deficiency in mice abolishes mIDH1-promoted tumor angiogenesis as well as the therapeutic benefit of mIDH1 inhibitor in solid tumors. Moreover, we report that HH2301, a newly discovered mIDH1 inhibitor, shows promising efficacy in treating IDH1-mutant cholangiocarcinoma in preclinical models. Together, we identify a new role of SLC1A1 as a gatekeeper of R-2-HG-mediated crosstalk between IDH1-mutant tumor cells and vascular endothelial cells, and demonstrate the therapeutic potential of mIDH1 inhibitors in treating IDH1-mutant solid tumors via disrupting R-2-HG-promoted tumor angiogenesis.

Histone methyltransferase WHSC1 loss dampens MHC-I antigen presentation pathway to impair IFN-γ-stimulated antitumor immunity.

IFN-γ-stimulated MHC class I (MHC-I) antigen presentation underlies the core of antitumor immunity. However, sustained IFN-γ signaling also enhances the programmed death ligand 1 (PD-L1) checkpoint pathway to dampen antitumor immunity. It remains unclear how these opposing effects of IFN-γ are regulated. Here, we report that loss of the histone dimethyltransferase WHSC1 impaired the antitumor effect of IFN-γ signaling by transcriptional downregulation of the MHC-I machinery without affecting PD-L1 expression in colorectal cancer (CRC) cells. Whsc1 loss promoted tumorigenesis via a non-cell-autonomous mechanism in an Apcmin/+ mouse model, CRC organoids, and xenografts. Mechanistically, we found that the IFN-γ/STAT1 signaling axis stimulated WHSC1 expression and, in turn, that WHSC1 directly interacted with NLRC5 to promote MHC-I gene expression, but not that of PD-L1. Concordantly, silencing Whsc1 diminished MHC-I levels, impaired antitumor immunity, and blunted the effect of immune checkpoint blockade. Patient cohort analysis revealed that WHSC1 expression positively correlated with enhanced MHC-I expression, tumor-infiltrating T cells, and favorable disease outcomes. Together, our findings establish a tumor-suppressive function of WHSC1 that relays IFN-γ signaling to promote antigen presentation on CRC cells and provide a rationale for boosting WHSC1 activity in immunotherapy.

An Integrative Proteome-Based Pharmacologic Characterization and Therapeutic Strategy Exploration of SAHA in Solid Malignancies.

Targeting histone epigenetic modification is an important strategy for anticancer therapy. Histone deacetylase inhibitors (HDACis) have been clinically approved in the treatment of diverse hematological cancers, but mechanisms of drug resistance and poor therapeutic efficacy in solid malignancies remain largely unknown. In this study, we applied a mass spectrometry-based quantitative proteomic strategy to investigate the molecular differences in HDACi vorinostat (SAHA) sensitive and resistant cell lines. The proteomic results revealed that the glycolysis pathway was highly enriched after vorinostat treatment in the resistant cell line, leading to the prediction of a new drug combination, SAHA and hexokinase inhibitor (2-deoxyglucose). The efficacy of this combination was further verified in several solid tumor cell lines. Quantitative proteomics revealed that alterations in the transcription process and protein homeostasis could play roles in the synergetic utilization of these two compounds. Our study showed the application of proteomics in elucidating the drug mechanism and predicting drug combination and the potential of expanding the utilization of HDACi.

[Optimization and evaluation of protein C-terminal peptide enrichment strategy based on arginine cleavage].

As unique biomarkers, protein C-termini are involved in various biological processes such as protein trafficking, subcellular relocation, and signal transduction. Dysregulation of protein C-terminal status is critical during the development of various diseases, including cardiovascular, neurodegenerative, and metabolic diseases and cancer. Thus, global profiling of protein C-termini is of great value in providing mechanistic insight into biological or pathological processes, as well as for identifying potential new targets for therapeutic treatment. Polymer-based negative enrichment is a prominent C-terminomics strategy with advantages of universal applicability and parallel sample preparation. Compared with other methods of such a strategy, the profiling depth of the approaches based on enzymatic cleavage of Arg residues still needs to be improved. This greatly limits our understanding of the physiological functions and molecular mechanisms of C-termini. To add a more powerful tool for C-terminomics, Arg cleavage-based negative enrichment C-terminomics was optimized and evaluated. First, the sample preparation process was optimized. A one-pot enrichment platform based on a V-shaped filter was established, which reduced sample loss, avoided cross-contamination between reactions, and shortened sample preparation time. In addition, the protein-level acetylation conditions were investigated with the optimal labeling conditions as follows: triple coupling using 5 mmol/L Ac-NHS at pH 7.0 and 500 mmol/L ammonium for 15 min provided minimized acetylation rates (acetylation labeling efficiencies of Ser, Thr, and Tyr were lower than 4%, 2%, and 1%, respectively), along with the highest peptide-spectrum match number and satisfactory Lys labeling efficiency (up to 98%). These optimized conditions would not only minimize acetylation, but also facilitate the identification of C-terminal peptides. Second, it was speculated that the unexpected low identification rate was primarily caused by the interference of the large number of organic compounds accumulated during the peptide-level reactions, including reagents, organic buffering agents, and their complex side-reaction products. Therefore, the conditions for StageTip-based fractionation, including pH, the amount of Empore C18 beads, and the number of fractions, were optimized. As a result, by separating the sample enriched from 300 µg proteome into seven fractions, sample complexity was largely decreased and a total of 696 C-termini were identified in duplicates from strict data filtration, that is, percolator false discovery rate (FDR)<0.01, ion score≥20, and C-terminal amidation by ethanolamine. If only peptide FDR<0.01 was considered, the identified C-termini further increased to 933, which was among the largest C-terminome datasets obtained from the polymer-based strategy. Furthermore, compared with the results of a previous study, the optimized method would be a practical strategy for broader C-terminome coverage. Finally, to further broaden the coverage of the sub-C-terminome generated by Arg-specific cleavage, this study explored a new method in which ArgN-specific cleavage (cleavage at the N-terminal of Arg by LysargiNase) was combined with different N-terminal protections (dimethylation and acetylation). Among all the combinations, the additional use of the "LysargiNase+N-terminal acetylation" method increased 47% more identifications of unique C-termini on the basis of "trypsin+N-terminal demethylation" and the two covered 87% of the total C-termini. Therefore, the parallel use of the two methods would further expand the coverage of Arg-cleaved C-terminal peptides. With the analysis of the physicochemical properties of the peptides identified by the two methods, the reason why the C-terminal peptides identified by different strategies are complementary was explained. In conclusion, in this study, the optimized C-terminomics platform can deeply profile Arg cleavage-generated C-terminal peptides using a polymer-based approach. This method provides a powerful tool for the global characterization of protein C-termini.

Global identification of phospho-dependent SCF substrates reveals a FBXO22 phosphodegron and an ERK-FBXO22-BAG3 axis in tumorigenesis.

SKP1-CUL1-F-box (SCF) ubiquitin ligases play fundamental roles in cellular functions. Typically, substrate phosphorylation is required for SCF recognition and subsequent degradation. However, phospho-dependent substrates remain largely unidentified. Here, using quantitative phoshoproteome approach, we performed a system-wide investigation of phospho-dependent SCF substrates. This strategy identified diverse phospho-dependent candidates. Biochemical verification revealed a mechanism by which SCFFBXO22 recognizes the motif XXPpSPXPXX as a conserved phosphodegron to target substrates for destruction. We further demonstrated BAG3, a HSP70 co-chaperone, is a bona fide substrate of SCFFBXO22. FBXO22 mediates BAG3 ubiquitination and degradation that requires ERK-dependent BAG3 phosphorylation at S377. FBXO22 depletion or expression of a stable BAG3 S377A mutant promotes tumor growth via defects in apoptosis and cell cycle progression in vitro and in vivo. In conclusion, our study identified broad phosphorylation-dependent SCF substrates and demonstrated a phosphodegron recognized by FBXO22 and a novel ERK-FBXO22-BAG3 axis involved in tumorigenesis.