Introduction Cigar fermentation is crucial for developing its characteristic aroma, exogenous microorganisms can be used to enhance fermentation. It is reported that the citrus reticulata ‘Chachi’ (Chenpi, a traditional fermented ingredient) extract can improve the flavor of cigarette. However, there is no report on the influence of Chenpi-derived microorganisms on the fermentation process and flavor quality of cigar tobacco leaves (CTLs) till now. Methods A fermentation strain ( Enterobacter hoffmannii , G5Z-2) was isolated from Chenpi, and it was applied as a bioaugmentation agent in CTLs fermentation. A multi-omics approach, including metagenomics and metabolomics, was employed to investigate its impact. Results Inoculation with G5Z-2 significantly altered the microbial community structure, suppressing native Pseudomonas and reducing overall alpha diversity while enriching beneficial genera like Aspergillus and Staphylococcus . Metabolomic analysis revealed substantial restructuring of metabolic pathways, particularly the enrichment of amino acid metabolism (such as arginine biosynthesis and phenylalanine metabolism) and nicotinate/nicotinamide metabolism. This led to accelerated degradation of proteins and amino acids, providing precursors for Maillard reaction, and a marked increase (57.5%) in total volatile flavour compounds, including key aroma constituents from carotenoid and cembranoid degradation. Conclusion The Chenpi-derived E. hoffmannii G5Z-2 optimises the fermentation process by modulating the microbial consortium and driving metabolic shifts towards favourable flavour development, demonstrating significant potential for improving the quality of Chinese-style cigars.

ABSTRACT The rapid development of targeted protein degradation (TPD) has shown profound effects on disease treatment. Precise and effective targeted degradation tools that target endogenous proteins are essential to accelerate advances in treatment methods. Selective macroautophagy/autophagy relies on the activity of related receptors to achieve the degradation of specific intracellular components in lysosomes, but the methodology of selective autophagy for tumor therapy and chimeric antigen receptor (CAR)-T cell modification is yet unexplored. Here, we developed a peptide-based LC3-interacting region-targeting chimera (pLIRTAC) that accurately and efficiently targeted the degradation of AKT1 for glioma treatment. pLIRTAC could also inhibit the development of tumor cells by in vitro delivery after purification. For CAR-T cell therapy, pLIRTAC could significantly improve the efficacy of CAR-T cell-targeted lysis of tumor cells both in vitro and in vivo. pLIRTAC binds to autophagy-associated proteins through LC3-interacting region (LIR) motifs and to target proteins through protein-targeting short peptides, and targets the protein of interest (POI) based on the selective autophagy lysosomal pathway. pLIRTAC has been remarkably successful both in vivo and in vitro, providing a robust and effective tool for the control of endogenous abnormal proteins in cells, and can potentially further expand the therapeutic application of TPD technology. Abbreviation: ATG8s: mammalian Atg8 (autophagy related 8)-family proteins; Baf-A1: bafilomycin A1; CAR: chimeric antigen receptor; CQ: chloroquine; CRISPR: clustered regularly interspaced short palindromic repeats; EBSS: Earle’s balanced salt solution; LIR: LC3-interacting region; 3 MA: 3-methyladenine; MFI: mean fluorescence intensity; pLIRTAC: peptide-based LC3-interacting region-targeting chimera; POI: protein of interest; PROTAC: proteolysis-targeting chimera; SARS: selective autophagy receptors; TPD: targeted protein degradation.

The ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) directs substrates to the 26S proteasome, but its role in autophagic protein degradation remains unclear. FAT10 expression was analyzed by qRT-PCR and western blotting in colon cancer (CC) tissues and cells. Bioinformatics revealed FAT10-associated biological processes in CC. In vitro and in vivo assays examined CC cell invasion and metastasis. Autophagy was assessed by western blotting, mRFP-GFP-LC3 reporter (tfLC3), and electron microscopy. In vitro ubiquitination assays measured ubiquitination of zona occludens 1 (ZO-1) and autophagy-related gene 3 (ATG3). liquid chromatography‒tandem mass spectrometry (LC-MS/MS) identified FAT10-interacting proteins. Co-IP and GST pull-down confirmed FAT10-ATG3 binding. FAT10 was upregulated in CC tissues and cells. Bioinformatic analyses revealed that FAT10 expression was correlated with extracellular matrix binding and cell migration in CC. FAT10 overexpression enhanced CC invasion and metastasis. FAT10 promoted autophagic degradation of ZO-1, a key component of tight junctions, facilitating invasion and migration. Mechanistically, FAT10 stabilized ATG3 by competing with ubiquitin for binding, inhibiting ATG3 ubiquitination and activating autophagy, leading to ZO-1 degradation. The ATG3 inhibitor compound 189 reversed FAT10-induced ZO-1 loss and suppressed lung metastasis. Our findings demonstrate a novel pathway by which FAT10 activates autophagy by stabilizing ATG3, leading to ZO-1 degradation and promoting CC lung metastasis. This study provides new insights into the role of FAT10 in regulating protein homeostasis and offers a potential strategy for targeting the FAT10-ATG3-autophagy axis in the treatment of CC metastasis.

Overcoming breast cancer resistance through targeted protein degradation and next generation chimeras.

Histone deacetylases (HDACs) are key epigenetic regulators involved in a variety of cancers, rendering them attractive therapeutic targets. Although several HDAC inhibitors have achieved clinical success, challenges such as poor isoform selectivity, acquired resistance, and off-target toxicity limit their broader application. Proteolysis-targeting chimeras (PROTACs) represent an innovative therapeutic strategy that enables ubiquitin-proteasome-mediated degradation of HDACs. This approach enhances specificity, overcomes resistance mechanisms, including those resulting from point mutations or persistent target activity, and enables sustained suppression at low concentrations, owing to its catalytic and event-driven mode of action. This review summarizes the structural classification and biological functions of HDACs and surveys recent advances in the design of HDAC-directed PROTACs. Key emphasis is placed on rational warhead selection, linker optimization, and the strategic choice of E3 ligase recruiters to guide degradation efficiency and isoform specificity. Representative degraders are evaluated for their pharmacological characteristics and antitumor efficacy across diverse malignancies. Current challenges and future directions for the development of HDAC degraders as clinically viable agents are also discussed.

The egg cytoplasm undergoes large-scale remodeling after fertilization. Here, we reveal that the germ granule component, HERD-1, is involved in selective degradation of maternal plasma membrane proteins after fertilization in Caenorhabditis elegans. HERD-1 is specifically expressed in the germline and mainly localized in a subtype of germ granules, Z granules. HERD-1 loss caused maternal plasma membrane protein accumulation in ubiquitin-positive early and late endosomal assemblies in the early embryos. The proteomic analysis showed that HERD-1 loss substantially reduced the protein levels of a subset of endolysosomal regulators such as ESCRT-0 components regulating the multivesicular body pathway. Defects in maternal membrane protein degradation in herd-1-deficient embryos were suppressed with the loss of DEPS-1 or PRG-1, which are required for germ granule organization and small-RNA biogenesis. These results suggest that several germ granule components maintain appropriate endolysosomal component levels via small RNA-mediated regulation during the oocyte-to-embryo transition, promoting endosomal switching toward embryogenesis.

Cellular responses to amino acid fluctuations often hinge on ubiquitin-mediated control of metabolic enzymes, yet the underlying E3 ligase pathways remain poorly defined. Using quantitative proteomics and active cullin-RING ligase (CRL) profiling, we identify LRRC58 as a cysteine-responsive substrate receptor whose stability increases sharply under cysteine starvation. Proteomics reveals an inverse relationship between LRRC58 and the metabolic enzyme cysteine dioxygenase 1 (CDO1), suggesting a cysteine-linked regulatory axis. Biochemical reconstitution and cryo-EM structures show that LRRC58 forms an active CUL2- or CUL5-based CRL that selectively positions CDO1 for ubiquitylation at Lys8. Disease mutant versions of CDO1 mapping to the LRRC58 interface and impaired for the endogenous ubiquitylation pathway were degraded through orthogonal targeting by a VHL-based degrader. Together, our proteomics-guided discovery pipeline, cellular stability studies, and structural analyses uncover a metabolically-tuned LRRC58-CDO1 pathway that links cysteine availability to selective proteasomal turnover, reveals principles of metabolite-regulated CRL activity, and showcases mechanisms distinguishing endogenous and targeted protein degradation.

Signal peptide peptidases (SPPs) play a critical role in intramembrane proteolysis of signal peptides in mammals. However, their function in plants remains poorly understood. Here, we uncover the critical role of two rice SPP-like proteins, OsSPPL1/ 2, in ER-associated degradation (ERAD). Their expression is directly upregulated by OsbZIP50 under ER stress conditions. Mutations in OsSPPL1/2 result in increased ER stress sensitivity, whereas their overexpression enhance ER stress tolerance. We further demonstrate that OsSPPL1/2 localize in ER, and physically interact with the ERAD components OsDER1/2, indicating their involvement in ERAD. Using a GFP protein fused with a segment of maize floury-2 protein defective in signal peptide cleavage (ZmFL2m-GFP), we show that OsSPPL1/2 interact with ZmFL2m-GFP in ER and facilitate its degradation in tobacco leaves and rice plants. Additionally, OsSPPL1/2 double mutants exhibit exaggerated thermal sensitivity, while OsSPPL1/2-overexpressing plants display improved thermotolerance. Together, our findings identify OsSPPL1/2 as components of ERAD and highlight the importance of ERAD in plant thermotolerance.

The rapid construction of chiral pharmacophores through stepwise and modular synthesis approaches presents a significant opportunity to accelerate lead compound discovery in drug development. While carbon-based chiral cores currently dominate drug molecule structures, the stepwise functionalization of sp³ carbon centers via C-H or C-X bond manipulations remains challenging. In contrast, silicon atoms offer superior functionalization capabilities with reactive Si-H or Si-X bonds, making them ideal central atoms for the modular and stepwise construction of pharmacophores. In this study, we constructed a total of 64 silicon-containing pharmacophores through efficient modular and stepwise synthesis, starting from Si-H or Si-X bonds. Screening these sila-pharmacophores for their ability to recruit protein degradation systems led to the identification of several potent sila-protein degraders (SiDs). The versatility of these SiDs was demonstrated by their successful conjugation to various small-molecule binders/inhibitors, resulting in efficient degradation of multiple target proteins and thus expanding their potential across diverse drug targets. Notably, we observed significant tumor-suppressive activity of a SiD-conjugated ALK degrader in a xenograft model using the H3122 cell line (ALK-positive), highlighting the therapeutic potential of sila-pharmacophores. These findings underscore that silicon-centered drug motifs not only offer synthetic accessibility and convenience but also enhance drug-like properties. The silicon-carbon switch strategy introduced herein provides an innovative approach to new drug motif development, highlighting its extensive potential in drug discovery and opening new avenues for chemical biology research.

A novel FAK-targeted degrader: Design, synthesis, and therapeutic potential against colorectal cancer.

The Bowenoid subtype of cutaneous squamous cell carcinoma (SCC) has been distinguished from conventional SCC for over a century based on its different histopathologic appearance, clinical features, and biologic potential. While there has been extensive molecular analysis of conventional SCC, there has been comparatively little investigation of Bowenoid SCC. Here we show that loss of RB1 protein expression is a defining feature of Bowenoid SCC and reflects biallelic genetic inactivation which most commonly proceeds through mutation of one RB1 allele and copy number loss of the other allele. Neither RB1 protein loss nor RB1 mutations were seen in conventional SCC. A third, but much less common, form of cutaneous SCC is caused by human papillomavirus (HPV). This subtype showed similar morphologic features to Bowenoid SCC and also showed loss of RB1 protein expression. However, these tumors lacked RB1 mutation and likely inactivate RB1 through HPV's known capacity to promote post-translational degradation of RB1 protein. These data suggest that the two most common subtypes of cutaneous SCC proceed through distinct pathways of molecular pathogenesis and highlight an unexpected relationship between Bowenoid and HPV-associated cutaneous SCC.

Design, synthesis, and biological evaluation of ALK5 PROTAC degraders for pulmonary fibrosis.

The E1A-associated protein p300 (EP300) is a key regulator of oncogenic transcription factors, making it a promising target for cancer therapy. However, its high sequence similarity to its paralog, CREB-binding protein (CBP), has hindered the development of selective inhibitors, leading to dose-limiting toxicities. Here, we describe the discovery of a highly potent and selective p300 degrader. Unlike dual p300/CBP degraders, this compound forms a more stable ternary complex with p300, driving enhanced proteasomal recruitment and ubiquitination. Notably, our data uncover a previously unrecognized mechanism of paralog selectivity mediated by regioselective ubiquitination of a unique lysine residue on p300. Hematological malignancies, including multiple myeloma, non-Hodgkin lymphoma, and acute myeloid leukemia, exhibit marked sensitivity to selective p300 degradation, resulting in cell lethality and robust antitumor activity in xenograft models. These findings establish selective p300 degradation as a mechanistically distinct and promising therapeutic strategy in hematological malignancies.

Genetic characterization of DDX11 variants identified in a Chinese family with Warsaw breakage syndrome.

Colorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide. The classic development of CRC is a process from normal colonic mucosa to polyp to eventually adenocarcinoma. However, the critical genes regulating this process and the underlying molecular mechanisms remain elusive. Here, we identified ZNF473 as an upregulated and key functional gene in CRC progression. Specifically, comprehensive bioinformatics analyses were performed to explore the expression of ZNF473 in CRC samples and to investigate its correlation with clinicopathological characteristics, prognosis, and potential biological functions. In vitro experiments were performed to elucidate the potential role and molecular mechanisms of ZNF473 in CRC progression. Results demonstrate that ZNF473 is highly expressed in CRC and correlates with poor prognosis. Functionally, ZNF473 knockdown significantly inhibits cell viability and proliferation. Furthermore, gene function enrichment analyses reveal an association between ZNF473 and pathways related to drug metabolism (Cytochrome P450) and chemotherapy resistance. Mechanistically, ZNF473 physically interact with p53 to promote its protein degradation, consequently upregulates the Survivin expression. In summary, this study reveals the role and molecular function of ZNF473 in CRC progression, uncovering a potential novel ZNF473/p53/Survivin axis and providing a hint for targeting ZNF473 to suppress tumor growth and potential chemoresistance.

To investigate the prognostic value of a model based on ferroptosis-related long non-coding RNAs (lncRNAs) for patients with diffuse large B-cell lymphoma (DLBCL). Ferroptosis-related lncRNA expression matrices and corresponding clinical information for DLBCL patients were downloaded from the GEO public database (datasets GSE10846 and GSE11318). A prognostic risk model was constructed using the GSE10846 dataset through the following steps: identification of ferroptosis-related genes, Pearson correlation analysis with lncRNAs, Kaplan-Meier survival analysis, univariate Cox regression analysis, LASSO regression analysis, and multivariate Cox regression analysis. The accuracy of the model was evaluated using Receiver Operating Characteristic (ROC) curves. The model was subsequently validated using the GSE11318 dataset. Further systematic assessments of the model's biological significance and clinical translational potential were conducted via immune infiltration analysis and drug sensitivity prediction. A prognostic model comprising 9 lncRNAs was established. Time-dependent ROC curve analysis showed that the Area Under the Curve (AUC) values for 3-year and 5-year survival predictions were both greater than 0.75. Consistent results were obtained in the validation set, indicating good model robustness. Immune microenvironment analysis revealed that the high-risk group exhibited an immunosuppressive "cold tumor" phenotype, characterized by a significant reduction in the infiltration of effector immune cells such as cytotoxic cells and CD8⁺ T cells (all P < 0.001). Drug sensitivity analysis indicated that the high-risk group was more sensitive to PI3K/mTOR inhibitors (e.g., Dactolisib) but showed resistance to certain histone deacetylase inhibitors. Enrichment analysis suggested that these lncRNAs might influence DLBCL progression by regulating pathways such as protein degradation and RNA processing. The prognostic model constructed based on ferroptosis-related lncRNAs demonstrates considerable reliability. It not only effectively predicts patient survival but also reflects tumor immune status and drug response characteristics, showing potential as an auxiliary tool for prognosis assessment and personalized treatment in DLBCL.

Targeted protein degradation (TPD) is a therapeutic strategy that utilizes small molecules to induce the proximity-driven degradation of disease-causing proteins. Because the efficacy and selectivity of TPD compounds must be validated across thousands of proteins, high-throughput proteomics is essential for the rapid screening and characterization of these novel degraders. Here, we developed a 300 samples per day (SPD) LC-MS/MS method using the Orbitrap Astral mass spectrometer for ultrahigh-throughput TPD compound screening. We identified close to 8000 protein groups from a single cell line with a coefficient of variation (CV) of less than 10%, highlighting the deep proteome coverage and method reproducibility even at 300 SPD. This high degree of precision provides the statistical confidence to detect subtle, yet significant, changes in protein abundance that were previously challenging to quantify in high-throughput workflows. To evaluate the quantitation accuracy of this method, we further mixed the digests from two or three species at different ratios. Our three-proteome mixture results demonstrated highly accurate quantitation for proteins with both small and large fold changes. Moreover, our two-proteome mixture experiment, where 20 to 160 ng of yeast digest was spiked into 200 ng of HeLa digest, showed an R2 of 0.999 for the yeast proteome, underscoring the quantitation accuracy of the method. Utilizing this workflow, we studied dose-dependent protein degradation patterns induced by pomalidomide, iberdomide, and mezigdomide. Our results indicate that mezigdomide may possess enhanced efficacy in T cells by degrading additional proteins such as IKZF2, thereby boosting anticancer immunity. Together, we developed an ultrahigh-throughput LC-MS/MS method with excellent proteome coverage and quantitation accuracy that is highly suitable for chemoproteomics screening of drug libraries.

Targeted protein degradation (TPD) by PROteolysis TArgeting Chimeras (PROTACs) has emerged as a powerful chemical biology and therapeutic modality, yet many degraders exhibit incomplete target clearance and characteristic rebound kinetics despite continuous exposure. The mechanistic basis for this behavior remains poorly understood. Here we uncover protein age as a previously unrecognized determinant of PROTAC efficacy. Using CG □SLENP, a chemical genetics strategy that selectively labels newly synthesized and pre □existing proteins within the same living cell, we directly resolve PROTAC□induced degradation of distinct intracellular protein populations. Applying this approach to the bromodomain protein BRD4, we show that two mechanistically and structurally distinct PROTACs, dBET6 and MZ□1, preferentially degrade pre □existing BRD4, while newly synthesized BRD4 is degraded substantially more slowly and incompletely. This age□dependent degradation bias is observed in live□cell imaging, across compound concentrations and time scales, and for both reporter and endogenous BRD4. These findings reveal that PROTAC□mediated degradation is governed not only by target engagement and ternary complex formation, but also by the dynamic balance between protein synthesis and degradation. By identifying temporal proteostasis as a critical parameter in TPD, this work provides a mechanistic framework for incomplete degradation and rebound kinetics and establishes protein maturation state as an important consideration for degrader design and evaluation.

p62/SQSTM1 self-assembles with polyubiquitin into liquid-like condensates ("p62 bodies") that function as stress-signaling hubs and selective autophagy cargo. We show that TBK1-dependent phosphorylation at Ser403 acts as a threshold-dependent modulator of a condensate's physical properties and promotes their rapid autophagic clearance. Phosphorylation within p62 bodies drives a transition from large, fluid droplets to compact, gel-like condensates that efficiently capture LC3-positive isolation membranes and accelerate the autophagic removal of ubiquitinated proteins. PP2A holoenzymes containing PPP2R5A/B/E, recruited via a KEAP1 bridge, counteract TBK1 by dephosphorylating Ser403. Homozygous p62S403E/S403E knock-in embryonic stem cells differentiate into post-mitotic neurons enriched in miniaturized, gel-like p62 bodies. Consistently, phosphorylation-mimetic knock-in mice show similar remodeling of p62 condensates in vivo, demonstrating that this phosphorylation-driven mechanism maintains proteostasis across scales. We propose that Ser403 phosphorylation functions as a molecular switch that couples the material state of p62 condensates to their stability and serves as a central control point for p62-mediated protein degradation.

CEP152 is essential for centriole function and neurodevelopment, and pathogenic recessive variants in CEP152 cause primary microcephaly. We identified new compound heterozygous CEP152 variants, c.314 G > A,p.(W105*) and c.2689 A > T,p.(K897*), in a microcephalic patient and analyzed them alongside a homozygous variant c.95 A > C,p.(Q32P) associated with severe microcephaly with marked gyral simplification. In vitro assays revealed distinct effects: p.K897* prevented centrosomal localization, p.W105* led to protein degradation, and p.Q32P retained centrosomal targeting but disrupted binding to Polo-like kinase 4, a key centriole biogenesis kinase and CEP152 partner. In vivo, both Cep152W105*/K897* and Cep152Q32P/Q32P knock-in mice displayed microcephaly; notably, Cep152Q32P/Q32P mice also exhibited severe cortical defects during brain development. Cellular analyses revealed centrosome dysfunction, mitotic errors, and increased apoptosis, which were exacerbated in Cep152Q32P/Q32P brains. Morphological examination, including electron microscopy, further demonstrated structural abnormalities of the centrosomes and centrioles in Cep152Q32P/Q32P brains. Electrophysiological and gene expression analyses confirmed variant-specific neuronal impairments, which correlate with clinical severity. Collectively, these findings demonstrate that distinct CEP152 variants disrupt neurodevelopment through different mechanisms, thereby explaining the spectrum of microcephaly severity and associated phenotypes.