A blood-based multi-omic landscape for the molecular characterization of kidney stone disease.

Kidney stone disease (KSD, also named renal calculi, nephrolithiasis, or urolithiasis) is a common urological disease entailing the formation of minerals and salts that form inside the urinary tract, frequently caused by diabetes, high blood pressure, hypertension, and monogenetic components in most patients. 10% of adults worldwide are affected by KSD, which continues to be highly prevalent and with increasing incidence. For the identification of novel therapeutic targets in KSD, we adopted high-throughput sequencing and mass spectrometry (MS) techniques in this study and carried out an integrative analysis of exosome proteomic data and DNA methylation data from blood samples of normal and KSD individuals. Our research delineated the profiling of exosomal proteins and DNA methylation in both healthy individuals and those afflicted with KSD, finding that the overexpressed proteins and the demethylated genes in KSD samples are associated with immune responses. The consistency of the results in proteomics and epigenetics supports the feasibility of the comprehensive strategy. Our insights into the molecular landscape of KSD pave the way for a deeper understanding of its pathogenic mechanism, providing an opportunity for more precise diagnosis and targeted treatment strategies for KSD.

Integrative Analysis of Multi-Omic Data for the Characteristics of Endometrial Cancer.

Endometrial cancer (EC) is a frequently diagnosed gynecologic cancer. Identifying reliable prognostic genes for predicting EC onset is crucial for reducing patient morbidity and mortality. Here, a comprehensive strategy with transcriptomic and proteomic data was performed to measure EC's characteristics. Based on the publicly available RNA-seq data, death-associated protein kinase 3, recombination signal-binding protein for the immunoglobulin kappa J region, and myosin light chain 9 were screened out as potential biomarkers that affect the EC patients' prognosis. A linear model was further constructed by multivariate Cox regression for the prediction of the risk of being malignant. From further integrative analysis, exosomes were found to have a highly enriched role that might participate in EC occurrence. The findings were validated by qRT-polymerase chain reaction (PCR) and western blotting. Collectively, we constructed a prognostic-gene-based model for EC prediction and found that exosomes participate in EC incidents, revealing significantly promising support for the diagnosis of EC.

A pyridinium-based strategy for lysine-selective protein modification and chemoproteomic profiling in live cells.

Protein active states are dynamically regulated by various modifications; thus, endogenous protein modification is an important tool for understanding protein functions and networks in complicated biological systems. Here we developed a new pyridinium-based approach to label lysine residues under physiological conditions that is low-toxicity, efficient, and lysine-selective. Furthermore, we performed a large-scale analysis of the ∼70% lysine-selective proteome in MCF-7 cells using activity-based protein profiling (ABPP). We quantifically assessed 1216 lysine-labeled peptides in cell lysates and identified 386 modified lysine sites including 43 mitochondrial-localized proteins in live MCF-7 cells. Labeled proteins significantly preferred the mitochondria. This pyridinium-based methodology demonstrates the importance of analyzing endogenous proteins under native conditions and provides a robust chemical strategy utilizing either lysine-selective protein labeling or spatiotemporal profiling in a living system.

Sulfonium-Stapled Peptides-Based Neoantigen Delivery System for Personalized Tumor Immunotherapy and Prevention.

Neoantigen peptides hold great potential as vaccine candidates for tumor immunotherapy. However, due to the limitation of antigen cellular uptake and cross-presentation, the progress with neoantigen peptide-based vaccines has obviously lagged in clinical trials. Here, a stapling peptide-based nano-vaccine is developed, comprising a self-assembly nanoparticle driven by the nucleic acid adjuvant-antigen conjugate. This nano-vaccine stimulates a strong tumor-specific T cell response by activating antigen presentation and toll-like receptor signaling pathways. By markedly improving the efficiency of antigen/adjuvant co-delivery to the draining lymph nodes, the nano-vaccine leads to 100% tumor prevention for up to 11 months and without tumor recurrence, heralding the generation of long-term anti-tumor memory. Moreover, the injection of nano-vaccine with signal neoantigen eliminates the established MC-38 tumor (a cell line of murine carcinoma of the colon without exogenous OVA protein expression) in 40% of the mice by inducing potent cytotoxic T lymphocyte infiltration in the tumor microenvironment without substantial systemic toxicity. These findings represent that stapling peptide-based nano-vaccine may serve as a facile, general, and safe strategy to stimulate a strong anti-tumor immune response for the neoantigen peptide-based personalized tumor immunotherapy.

Intracellular Delivery of Stabilized Peptide Blocking MTDH-SND1 Interaction for Breast Cancer Suppression.

Triple-negative breast cancer is one of the most prevalent malignant cancers worldwide. Disrupting the MTDH-SND1 protein-protein interaction has recently been shown to be a promising strategy for breast cancer therapy. In this work, a novel potent stabilized peptide with a stronger binding affinity was obtained through rational structure-based optimization. Furthermore, a sulfonium-based peptide delivery system was established to improve the cell penetration and antitumor effects of stabilized peptides in metastatic breast cancer. Our study further broadens the in vivo applications of the stabilized peptides for blocking MTDH-SND1 interaction and provides promising opportunities for breast cancer therapy.

Pyridinium-Based Strategy for a Bioorthogonal Conjugation-Assisted Purification Method for Profiling Cell Surface Proteome.

Cell surface proteins (CSPs) are valuable targets for therapeutic agents, but achieving highly selective CSP enrichment in cellular physiology remains a technical challenge. To address this challenge, we propose a newly developed sulfo-pyridinium ester (SPE) cross-linking probe, followed by two-step imaging and enrichment. The SPE probe showed higher efficiency in labeling proteins than similar NHS esters at the level of cell lysates and demonstrated specificity for Lys in competitive experiments. More importantly, this probe could selectively label the cell membranes in cell imaging with only negligible labeling of the intracellular compartment. Moreover, we successfully performed this strategy on MCF-7 live cells to label 425 unique CSPs from 1162 labeled proteins. Finally, we employed our probe to label the CSPs of insulin-cultured MCF-7, revealing several cell surface targets of key functional biomarkers and insulin-associated pathogenesis. The above results demonstrate that the SPE method provides a promising tool for the selective labeling of cell surface proteins and monitoring transient cell surface events.

Selective Protein of Interest Degradation through the Split-and-Mix Liposome Proteolysis Targeting Chimera Approach.

Proteolysis Targeting Chimera (PROTAC) technology represents a promising new approach for target protein degradation using a cellular ubiquitin-proteasome system. Recently, we developed a split-and-mix nanoplatform based on peptide self-assembly, which could serve as a self-adjustable platform for multifunctional applications. However, the lower drug efficacy limits further biomedical applications of peptide-based SM-PROTAC. In this study, we develop a novel split-and-mix PROTAC system based on liposome self-assembly (LipoSM-PROTAC), concurrent with modification of FA (folate) to enhance its tumor-targeting capabilities. Estrogen receptors (ERα) were chosen as the protein of interest (POI) to validate the efficacy of Lipo degraders. Results demonstrate that this PROTAC can be efficiently and selectively taken up into the cells by FA receptor-positive cells (FR+) and degrade the POI with significantly reduced concentration. Compared to the peptide-based SM-PROTACs, our designed LipoSM-PROTAC system could achieve therapeutic efficacy with a lower concentration and provide opportunities for clinical translational potential. Overall, the LipoSM-based platform shows a higher drug efficacy, which offers promising potential applications for PROTAC and other biomolecule regulations.

Development of Lysine Crotonyl-Mimic Probe to Covalently Identify H3K27Cr Interacting Proteins.

Histone lysine crotonylation (Kcr) is one newly discovered acylation modification and regulates numerous pathophysiological processes. The binding affinity between Kcr and its interacting proteins is generally weak, which makes it difficult to effectively identify Kcr-interacting partners. Changing the amide of crotonyl to an ester increased reactivity with proximal cysteines and retained specificity for Kcr antibody. The probe "H3g27Cr" was designed by incorporating the ester functionality into a H3K27 peptide. Using this probe, multiple Kcr-interacting partners including STAT3 were successfully identified, and this has not been reported previously. Further experiments suggested that STAT3 possibly could form complexes with Histone deacetylase HDACs to downregulate the acetylation and crotonylation of Histone H3K27. Our unique design provided intriguing tools to further explore Kcr-interacting proteins and elucidate their working mechanisms.

Histidine-specific bioconjugation via visible-light-promoted thioacetal activation.

Histidine (His, H) undergoes various post-translational modifications (PTMs) and plays multiple roles in protein interactions and enzyme catalyzed reactions. However, compared with other amino acids such as Lys or Cys, His modification is much less explored. Herein we describe a novel visible-light-driven thioacetal activation reaction which enables facile modification on histidine residues. An efficient addition to histidine imidazole N3 under biocompatible conditions was achieved with an electrophilic thionium intermediate. This method allows chemo-selective modification on peptides and proteins with good conversions and efficient histidine-proteome profiling with cell lysates. 78 histidine containing proteins were for the first time found with significant enrichment, most functioning in metal accumulation in brain related diseases. This facile His modification method greatly expands the chemo-selective toolbox for histidine-targeted protein conjugation and helps to reveal histidine's role in protein functions.

Low-Toxicity Sulfonium-Based Probes for Cysteine-Specific Profiling in Live Cells.

Despite being a low-abundance amino acid, cysteine plays an essential role in regulating protein function and serves as a satisfactory target of post-translational modifications and drug developments. To comprehensively assess reactive-cysteine-containing proteins, the development of chemical proteomic probes to label cysteine residues in human cells is an important objective. Cysteine modification using sulfonium-based probes is a novel method to identify reactive cysteine residues in proteins. Herein, we reported a set of "cysteine-reactive sulfonium-based (C-Sul)" probes to label the reactive cysteine sites in cellular proteins. Notably, water-soluble C-Sul probes have a significantly enhanced stability and cellular uptakes, displaying a high specificity toward reactive cysteines and compatibility with quantitative proteomic profiling. In comparison to the conventional iodoacetamide-based probe, C-Sul particularly has no inhibitory effects on cell viability, enabling its application in proteomic profiling of reactive cysteine residues under biorelevant conditions. We propose C-Sul probes as optimal tools of cysteine profiling for further broadly basic research.

Design and Evaluation of a Novel Peptide-Drug Conjugate Covalently Targeting SARS-CoV-2 Papain-like Protease.

Coronavirus disease 2019 (COVID-19) pandemic, a global health threat, was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 papain-like cysteine protease (PLpro) was recognized as a promising drug target because of multiple functions in virus maturation and antiviral immune responses. Inhibitor GRL0617 occupied the interferon-stimulated gene 15 (ISG15) C-terminus-binding pocket and showed an effective antiviral inhibition. Here, we described a novel peptide-drug conjugate (PDC), in which GRL0617 was linked to a sulfonium-tethered peptide derived from PLpro-specific substrate LRGG. The EM-C and EC-M PDCs showed a promising in vitro IC50 of 7.40 ± 0.37 and 8.63 ± 0.55 µM, respectively. EC-M could covalently label PLpro active site C111 and display anti-ISGylation activities in cellular assays. The results represent the first attempt to design PDCs composed of stabilized peptide inhibitors and GRL0617 to inhibit PLpro. These novel PDCs provide promising opportunities for antiviral drug design.

Electrophilic Sulfonium-Promoted Peptide and Protein Amidation in Aqueous Media.

A novel amidation strategy using electrophilic sulfonium, which is soluble and stable in aqueous conditions, was developed. The sulfoniums could activate thioacid and carboxyl acid to efficiently react with amines to afford amides. This method enables applications in amidation in both aqueous media and solid-phase peptide synthesis, peptide/protein modifications, and reactive lysines of a proteome at pH 10 with activity-based protein profiling. A peptide ligand-directed labeling of the USP7-UBL2 domain was also performed using this method.

B,N-Doped PdRu Aerogels as High-Performance Peroxidase Mimics for Sensitive Detection of Glucose.

Among plentiful porous nanomaterials, noble metal aerogels taken as nanozymes attract broad attention in sensing applications with their distinct enzyme mimic functions. In the catalytic field, the heteroatom doping strategy is a kind of way with great promise in improving the enzyme mimic activity of noble metal aerogels. In this experiment, we find a type of creative materials that were prepared by the fast and simple method. Due to the unique porous structure and synergetic effect from doped atoms, PdRu aerogels co-doped with boron and nitrogen (B, N-PdRu aerogels) were prepared using NH3BH3 as a reductant, which present improved peroxidase mimicking activity. With the existence of H2O2, the oxidation of 3,3',5,5'-tetramethylbenzidine was catalyzed by B, N-PdRu aerogels fairly efficiently, whose solution would be a blue appearance at optimum absorption wavelength 652 nm. Thus, by the tandem reaction bound to the enzyme glucose oxidase, the B, N-PdRu aerogels can be used for the sensitive determination of glucose. The new method has a good linear detection effect for glucose in the range of 10 µM to 2 mM. The minimum limit of detection can reach as low as 6 µM. This work will contribute to research on the rational design of metal aerogels based on the heteroatomic doping strategy and enhance the corresponding performance for a variety of applications.