Proteasome Pathway Research Articles

A hierarchical ubiquitination-mediated regulatory module controls bamboo lignin biosynthesis

Abstract The lignocellulosic feedstock of woody bamboo shows promising potential as an alternative to conventional wood, attributed to its excellent properties. The content and distribution of lignin serve as the foundation of these properties. While the regulation of lignin biosynthesis in bamboo has been extensively studied at the transcriptional level, its posttranslational control has remained poorly understood. This study provides a ubiquitinome dataset for moso bamboo (Phyllostachys edulis), identifying 13015 ubiquitinated sites in 4849 unique proteins. We further identified Kelch repeat F-boxprotein 9 (PeKFB9) that plays a negative role in lignin biosynthesis. Heterologous expression of PeKFB9 resulted in reduced accumulation of lignin and decreased phenylalanine ammonia-lyase (PAL) activities. Both in vitro and in vivo assays identified interaction between PeKFB9 and PePAL10. Further examination revealed that SCFPeKFB9 mediated the ubiquitination and degradation of PePAL10 via the 26S proteasome pathway. Moreover, PebZIP28667 could bind to the PePAL10 promoter to significantly inhibit its transcription, and ubiquitination of PebZIP28667 weakened this inhibition. Collectively, our findings reveal a PeKFB9-PePAL10/PebZIP28667-PePAL10 module that acts as a negative regulator of lignin biosynthesis. This study advances our understanding of posttranslational regulation in plant lignification, which will facilitate the improvement of the properties of bamboo wood and the breeding of varieties.

Temporal control of acute protein aggregate turnover by UBE3C and NRF1-dependent proteasomal pathways.

A hallmark of neurodegenerative diseases is the progressive loss of proteostasis, leading to the accumulation of misfolded proteins or protein aggregates, with subsequent cytotoxicity. To combat this toxicity, cells have evolved degradation pathways (ubiquitin-proteasome system and autophagy) that detect and degrade misfolded proteins. However, studying the underlying cellular pathways and mechanisms has remained a challenge, as formation of many types of protein aggregates is asynchronous, with individual cells displaying distinct kinetics, thereby hindering rigorous time-course studies. Here, we merge a kinetically tractable and synchronous agDD-GFP system for aggregate formation with targeted gene knockdowns, to uncover degradation mechanisms used in response to acute aggregate formation. We find that agDD-GFP forms amorphous aggregates by cryo-electron tomography at both early and late stages of aggregate formation. Aggregate turnover occurs in a proteasome-dependent mechanism in a manner that is dictated by cellular aggregate burden, with no evidence of the involvement of autophagy. Lower levels of misfolded agDD-GFP, enriched in oligomers, utilizes UBE3C-dependent proteasomal degradation in a pathway that is independent of RPN13 ubiquitylation by UBE3C. Higher aggregate burden activates the NRF1 transcription factor to increase proteasome subunit transcription, and subsequent degradation capacity of cells. Loss or gain of NRF1 function alters the turnover of agDD-GFP under conditions of high aggregate burden. Together, these results define the role of UBE3C in degradation of this class of misfolded aggregation-prone proteins and reveals a role for NRF1 in proteostasis control in response to widespread protein aggregation.

Up-regulated Lnc BTU promotes the production of duck plague virus DNA polymerase and inhibits the activation of JAK-STAT pathway to facilitate duck plague virus replication

Duck plague virus (DPV) is the only herpes virus known to be transmissible among aquatic animals, leading to immunosuppression in ducks, geese and swans. Long noncoding RNAs (LncRNA) are known to participate in viral infections, acting as either immune defenders or viral targets to evade the host response, but their precise roles in waterfowl virus infections are yet to be fully understood. This study aimed to investigate the role of LncRNA in DPV-induced innate immune responses. Results showed that DPV infection greatly upregulated Lnc BTU expression in duck embryo fibroblasts (DEF) and Lnc BTU promoted DPV replication. Mechanically, 4 DPV proteins, namely UL46, UL42, VP22 and US10, interacted with Lnc BTU, leading to its upregulation. Specifically, Lnc BTU facilitated the production of DNA polymerase by enhancing UL42 expression, thereby promoting DPV replication. Additionally, Lnc BTU suppressed STAT1 expression by targeting the DNA binding domain (DBD) and promoting STAT1 degradation through the proteasome pathway. Furthermore, Lnc BTU inhibited the production of key antiviral factors such as IFN-α, IFN-β, MX and OASL during DPV infection. Treatment with 2 JAK-STAT pathway activators in DEFs resulted in the inhibition of Lnc BTU expression and DPV replication. Interestingly, DPV infection led to a decrease in STAT1 levels, which was reversed by Si-Lnc BTU. These findings suggest that DPV relies on Lnc BTU to inhibit the activation of the JAK-STAT pathway and limit the production of type 1 interferons (IFN) to complete immune evasion. Our study highlights the novel role of DPV proteins UL46, UL42, VP22, US10 as RNA-binding proteins in modulating the innate antiviral immune response, and discover the role of a new host factor, Lnc BTU, in DPV immune evasion, Lnc BTU and STAT1 can be used as a potential therapeutic target for DPV infection and immune evasion.

CRADD and cIAP1 antagonistically regulate caspase-9-mediated apoptosis in teleost

Caspase 9 (CASP9) is a well-known initiator caspase of intrinsic apoptosis. In humans, cIAP1 binds and induces degradation of the activated form of CASP9, but not pro-CASP9. In fish, the activity and regulation of CASP9 remain unknown. In this work, using flounder Paralichthys olivaceus as a representative species, we examined the regulatory mechanism of CASP9 in teleost. P. olivaceus CASP9 (PoCASP9) induced robust apoptosis, which was inhibited by P. olivaceus cIAP1 (PocIAP1). Unlike human cIAP1, PocIAP1 bound both pro- and active PoCASP9 and induced their degradation via the RING domain-involved proteasome pathway. In humans, the adaptor molecule CRADD cannot interact with CASP9. In contrast, P. olivaceus CRADD (PoCRADD) bound both pro- and active PoCASP9 via CARD-CARD interaction and enhanced apoptosis by promoting the cellular levels of pro- and active PoCASP9. Furthermore, PoCRADD abrogated the inhibition of PoCASP9 by PocIAP1 by preventing PocIAP1-PoCASP9 interaction. Together these results reveal a CASP9 regulation mechanism in teleost that differs from that in humans and demonstrate that teleost CASP9 is tightly and directly controlled by both negative and positive regulators that exert a regulation effect both before and after CASP9 activation. These findings advance our understanding of the regulation of CASP9-mediated apoptosis in vertebrates.

Deubiquitylase USP31 Induces Autophagy and Promotes the Progression in Lung Squamous Cell Carcinoma Cells by Stabilizing E2F1 Expression.

Autophagy exerts a vital role in the progression of lung squamous cell carcinoma (LUSC). Ubiquitin-specific peptidase 31 (USP31) has recently been found to be involved in the development of a variety of cancers. However, whether USP31 modulates autophagy in LUSC remains unclear. This study revealed that high levels of USP31 were discovered in LUSC tissue samples employing the Gene Expression Profiling Interactive Analysis (GEPIA) database, quantitative real- time PCR (qRT-PCR), and Western blot analysis. Cell proliferation was tested via cell counting kit 8 (CCK-8) as well as colony formation, demonstrating that USP31-stable knockdown reduced cell viability. Immunofluorescence analysis illustrated that USP31 knockdown blocked the occurrence of LUSC autophagy. Meanwhile, USP31 has been shown to stabilize the expression of E2F transcription factor 1 (E2F1) through the proteasome pathway. Furthermore, overexpressed E2F1 effectively eliminated the effect of USP31 knockdown on LUSC cell proliferation and autophagy. In summary, this investigation proved that USP31 promoted LUSC cell growth and autophagy, at least in part by stabilizing E2F1 expression, which provided a potential therapeutic gene for the treatment of LUSC.

DIA-based quantitative proteomic analysis on porcine meat quality at different chilling rates

The objective of this study was to evaluate the effects of chilling rate on porcine meat quality from the perspective of proteome using data independent acquisition (DIA)-based quantitative proteomic strategy. M. longissimus thoracis et lumborum (n = 9) was assigned randomly to the control group (3.72 °C/h), very fast chilling-I group (VFC-I, 9.31 °C/h) and VFC-II group (14.43 °C/h). The DIA was used to analyze the difference in proteins under different chilling rates. Results showed that tenderness was improved significantly in meat at the chilling rate of 14.43 °C/h. Seventy-nine differential abundant proteins (fold change > 1.5, P < 0.05), including 46 up-regulated and 33 down-regulated proteins, were identified and mainly involved in carbon metabolism, pyruvate metabolism and proteasome pathways. These pathways indicated that VFC delayed cell metabolism and glycolysis by down-regulating the expression of metabolic enzymes. The tenderness was improved by up-regulating the expression of proteasome and m-calpain.

MdSINA2-MdNAC104 Module Regulates Apple Alkaline Resistance by Affecting γ-Aminobutyric Acid Synthesis and Transport.

Soil alkalization is an adverse factor limiting plant growth and yield. As a signaling molecule and secondary metabolite, γ-aminobutyric acid (GABA) responds rapidly to alkaline stress and enhances the alkaline resistance of plants. However, the molecular mechanisms by which the GABA pathway adapts to alkaline stress remain unclear. In this study, a transcription factor, MdNAC104 is identified, from the transcriptome of the alkaline-stressed roots of apple, which effectively reduces GABA levels and negatively regulates alkaline resistance. Nevertheless, applying exogenous GABA compensates the negative regulatory mechanism of overexpressed MdNAC104 on alkaline resistance. Further research confirms that MdNAC104 repressed the GABA biosynthetic gene MdGAD1/3 and the GABA transporter gene MdALMT13 by binding to their promoters. Here, MdGAD1/3 actively regulates alkaline resistance by increasing GABA synthesis, while MdALMT13 promotes GABA accumulation and efflux in roots, resulting in an improved resistance to alkaline stress. This subsequent assays reveal that MdSINA2 interacts with MdNAC104 and positively regulates root GABA content and alkaline resistance by ubiquitinating and degrading MdNAC104 via the 26S proteasome pathway. Thus, the study reveals the regulation of alkaline resistance and GABA homeostasis via the MdSINA2-MdNAC104-MdGAD1/3/MdALMT13 module in apple. These findings provide novel insight into the molecular mechanisms of alkaline resistance in plants.

20(S)-Ginsenoside Rh2 overcomes gemcitabine resistance in pancreatic cancer by inhibiting LAMC2-Modulated ABC transporters

IntroductionGemcitabine (GEM) is the first-line drug for pancreatic ductal adenocarcinoma (PDAC), but drug resistance severely restricts its chemotherapeutic efficacy. Laminin subunit γ2 (LAMC2) plays a crucial role in extracellular matrix formation in the development of GEM-resistance. However, the biological function of LAMC2 in GEM resistance and its molecular mechanisms are still unclear. 20(S)-Ginsenoside Rh2 (Rh2), one of the principal active components isolated from Ginseng Radix et Rhizoma, possesses strong anti-tumor effects. However, the effects of Rh2 on overcoming GEM resistance and its action mechanisms remain to be elucidated. ObjectivesThis study aimed to determine the efficacy of Rh2 on overcoming GEM resistance and to explore its underlying molecular mechanisms. MethodsClinical study, Western blotting, publicly available databasesand bioinformatic analyses were performed to investigate the protein expression of LAMC2 in the GEM-resistant PDAC patients and the acquired GEM-resistant PDAC cells. Then, the effects of Rh2 on overcoming the GEM resistance in PDAC were evaluated both in vitro and in vivo. Stable silencing or overexpression of LAMC2 in the GEM-resistant PDAC cells were established for validating the role of LAMC2 on Rh2 overcoming the GEM resistance in PDAC. ResultsThe protein expression of LAMC2 was markedly increased in the GEM-resistant PDAC patient biopsies compared to the sensitive cases. The protein expression of LAMC2 was significantly higher in the acquired GEM-resistant PDAC cells than that in their parental cells. Rh2 enhanced the chemosensitivity of GEM in the GEM-resistant PDAC cells, and inhibited the tumor growth of Miapaca-2-GR cell-bearing mice and Krastm4TyjTrp53tm1BrnTg (Pdx1-cre/Esr1*) #Dam/J (KPC) mice. Rh2 effectively reversed the GEM resistance in Miapaca-2-GR and Capan-2-GR cells by inhibiting LAMC2 expression through regulating the ubiquitin–proteasome pathway. Knockdown of LAMC2 enhanced the chemosensitivity of GEM and the effects of Rh2 on overcoming the GEM resistance in PDAC cells and the orthotopic PDAC mouse model. Conversely, LAMC2 overexpression aggravated the chemoresistance of GEM and abolished the effects of Rh2 on overcoming GEM resistance via modulating ATP-binding cassette (ABC) transporters leading to the active GEM efflux. ConclusionsLAMC2 plays an important role in the GEM resistance in PDAC, and Rh2 is a potential adjuvant for overcoming the chemoresistance of GEM in PDAC.

Abstract P224: RbFox2 is a Rho-related BTB Domain Containing 1 (RhoBTB1)-Cullin 3 (CUL3) Pathway Target that Regulates the Actin Cytoskeleton

Blood pressure (BP) is controlled by an array of cellular pathways. Our prior research revealed that endothelial nitric oxide (NO)-dependent vessel tone and BP are regulated through the RhoBTB1-CUL3 mediated proteasomal degradation of Phosphodiesterase 5 (PDE5). RhoBTB1 is an adaptor protein which delivers protein substrates to the CUL3 E3 ubiquitin ligase for proteasomal degradation. However, the range of protein targets of RhoBTB1 remains unknown. We employed the ascorbate peroxidase (APEX2) proximity labelling system coupled with mass spectroscopy to identify RhoBTB1 targets in A7r5 smooth muscle cells. This analysis identified nearly 75 proteins which putatively bind to the C terminal half of RhoBTB1 (known as B1B2C), the region we previously identified was essential for adaptor function. One of these proteins was RNA binding motif protein 9, known as RBM9 and RbFox2, a key regulator of splicing events. Subsequent co-immunoprecipitation (Co-IP) assays validated the interaction of RbFox2 with RhoBTB1 and its B1B2C domains. Expression of Rbfox2 was increased in response to treatment of A7r5 cells with either MG132, an inhibitor of the proteasome, and MLN4924, an inhibitor of the CUL family. Similarly, RbFox2 expression was increased in CRISPR-Cas9 generated CUL3-deficient HEK293 cells, in aorta isolated from SMC-specific CUL3-deficient mice, and in A7r5 cells expressing an siRNA targeting RhoBTB1. Ang-II treatment of mice, which raised blood pressure by 40 mmHg, also increased RbFox2 expression, presumably through down regulation of RhoBTB1. Targeting RbFox2 by siRNA in A7r5 cells resulted in a decrease in actin fiber formation detected by phalloidin staining. In summary, our findings suggest that RbFox2 regulation may occur through the RhoBTB1-CUL3 proteasomal pathway and may potentially play a mechanistic role in actin cytoskeleton organization and thus arterial stiffness associated with hypertension.

ANKRD1 aggravates renal ischaemia‒reperfusion injury via promoting TRIM25-mediated ubiquitination of ACSL3.

Renal ischaemia‒reperfusion injury (IRI) is the primary cause of acute kidney injury (AKI). To date, effective therapies for delaying renal IRI and postponing patient survival remain absent. Ankyrin repeat domain 1 (ANKRD1) has been implicated in some pathophysiologic processes, but its role in renal IRI has not been explored. The mouse model of IRI-AKI and in vitro model were utilised to investigate the role of ANKRD1. Immunoprecipitation-mass spectrometry was performed to identify potential ANKRD1-interacting proteins. Protein‒protein interactions and protein ubiquitination were examined using immunoprecipitation and proximity ligation assay and immunoblotting, respectively. Cell viability, damage and lipid peroxidation were evaluated using biochemical and cellular techniques. First, we unveiled that ANKRD1 were significantly elevated in renal IRI models. Global knockdown of ANKRD1 in all cell types of mouse kidney by recombinant adeno-associated virus (rAAV9)-mitigated ischaemia/reperfusion-induced renal damage and failure. Silencing ANKRD1 enhanced cell viability and alleviated cell damage in human renal proximal tubule cells exposed to hypoxia reoxygenation or hydrogen peroxide, while ANKRD1 overexpression had the opposite effect. Second, we discovered that ANKRD1's detrimental function during renal IRI involves promoting lipid peroxidation and ferroptosis by directly binding to and decreasing levels of acyl-coenzyme A synthetase long-chain family member 3 (ACSL3), a key protein in lipid metabolism. Furthermore, attenuating ACSL3 in vivo through pharmaceutical approach and in vitro via RNA interference mitigated the anti-ferroptotic effect of ANKRD1 knockdown. Finally, we showed ANKRD1 facilitated post-translational degradation of ACSL3 by modulating E3 ligase tripartite motif containing 25 (TRIM25) to catalyse K63-linked ubiquitination of ACSL3, thereby amplifying lipid peroxidation and ferroptosis, exacerbating renal injury. Our study revealed a previously unknown function of ANKRD1 in renal IRI. By driving ACSL3 ubiquitination and degradation, ANKRD1 aggravates ferroptosis and ultimately exacerbates IRI-AKI, underlining ANKRD1's potential as a therapeutic target for kidney IRI. Ankyrin repeat domain 1 (ANKRD1) is rapidly activated in renal ischaemia‒reperfusion injury (IRI) models in vivo and in vitro. ANKRD1 knockdown mitigates kidney damage and preserves renal function. Ferroptosis contributes to the deteriorating function of ANKRD1 in renal IRI. ANKRD1 promotes acyl-coenzyme A synthetase long-chain family member 3 (ACSL3) degradation via the ubiquitin‒proteasome pathway. The E3 ligase tripartite motif containing 25 (TRIM25) is responsible for ANKRD1-mediated ubiquitination of ACSL3.

Discovery of a proteolysis targeting chimera (PROTAC) as a potent regulator of FOXP3

Regulatory T (Treg) cells play a central role in immune homeostasis. Forkhead box P3 (Foxp3), a hallmark molecule in Treg cells, is a vital transcription factor for their development and function. Studies have shown that degradation of the Foxp3 could provide therapeutic benefits in achieving effective anti-tumor immunity. In this study, we designed three PROTAC molecules, P60-L1-VHL, P60-L2-VHL, and P60-L3-VHL, based on a 15-mer peptide inhibitor of Foxp3 (P60), and explored their potential in regulating Foxp3 expression and function. Our data show that, among these molecules, P60-L3-VHL can inhibit the expression and nuclear localization of Foxp3 in HEK 293 T and HeLa cells, respectively. Meanwhile, use of proteasome inhibitor in P60-L3-VHL treated cells revealed an increased Foxp3 expression, indicating that P60-L3-VHL mediates the inhibition of Foxp3 through its degradation in the proteasome pathway. We further substantiate that P60-L3-VHL reduces the differentiation and Foxp3 expression in the in-vitro activated Treg cells. Overall, our findings suggest that P60-L3-VHL inhibits the differentiation of Treg cells by degrading the Foxp3, which may have potential implications in cancer immunotherapy.

Environmentally relevant concentration PFNA promotes degradation of SMAD7 to drive progression of ovarian cancer via TGF-β/SMADs signaling pathway

Perfluorononanoic acid (PFNA), an acknowledged environmental endocrine disruptor, is increasingly utilized as a substitute for perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA). Despite its growing use, limited research has been conducted to investigate its potential impact on tumorigenesis and progression, and the potential molecular mechanisms. Earlier studies linked perfluoroalkyl and polyfluoroalkyl substances (PFAS) exposure to breast and gynecological cancer progression in humans, lacking a clear understanding of the underlying mechanisms, notably in ovarian cancer. Our investigation into PFNA's effects at environmental concentrations (0.25–2 mM) showed no significant impact on cell proliferation but a notable increase in invasion and migration of ovarian cancer cells. This led to alterations in epithelial-mesenchymal transition (EMT) markers, including Claudin1, Vimentin, and Snail. Notably, PFNA exposure activated the TGF-β/SMADs signaling pathway. Crucially, SMAD7 degradation through the ubiquitin-proteasome system emerged as PFNA's pivotal molecular target for inducing EMT, corroborated in mouse models. In summary, this study presented evidence that environmentally relevant concentrations of PFNA could induce SMAD7 degradation via the proteasome pathway, subsequently activating the TGF-β/SMADs signaling pathway, and promoting EMT in ovarian cancer. These results illuminated the association between PFNA exposure and metastasis of ovarian cancer.

Acute-phase plasma proteomics of rabbit lung VX2 tumors treated by image-guided microwave ablation.

To explore the plasma proteomic changes of rabbit lung VX2 tumors treated by microwave ablation, and to explore the molecular pathway mechanisms that may be involved. New Zealand white rabbits were inoculated with VX2 tumor cell suspension in the right lower lung and treated with microwave ablation after 2-3 weeks of tumor formation. Blood was collected at 5 time points (TP1~TP5) before and after ablation by cardiac blood sampling and pre-treated before proteomic analysis. The plasma proteome was analyzed by Data-Independent Acquisition (DIA). Different molecular pathways were activated at different time points:(i) TP1vsTP2: more proteins were down-regulated and enrichment analysis showed that the proteasome pathway was activated. The abnormal protein folding process involved in this pathway is closely related to the process of tumor development. (ii) TP2vsTP3: more proteins were up-regulated although the number of differentially differentiated proteins was lower and enrichment analysis showed that the phagosome pathway was activated. After microwave ablation inactivates tumor cells, it activates the phagosomal pathway for immune clearance of necrotic tumor tissue. (iii) TP3vsTP4: more down-regulated proteins, enrichment analysis showed that cysteine and methionine metabolism pathway was activated. Decreased metabolism of these amino acids suggests that cancer progression may be blocked after microwave ablation therapy. (iv) TP4vsTP5: the number of differential proteins was less and more down-regulated proteins, enrichment analysis showed that glutathione metabolism and metabolism of xenobiotics by cytochrome P450 pathway were activated. The down-regulated proteins in this pathway may suggest that microwave ablation may have reduced resistance to certain chemotherapeutic agents following. In the process of lung cancer treatment by microwave ablation, the changes of proteins on the possible molecular pathways at each time point are related to lung cancer, and not only involve some simple inflammatory reactions, and some of the proteins released by destroying the tumor cells can be used as possible drug binding sites and reduce drug resistance.

Rice A20/AN1 protein, OsSAP10, confers water-deficit stress tolerance via proteasome pathway and positive regulation of ABA signaling in Arabidopsis.

Overexpression of rice A20/AN1 zinc-finger protein, OsSAP10, improves water-deficit stress tolerance in Arabidopsis via interactionwith multiple proteins. Stress-associated proteins (SAPs) constitute a class of A20/AN1 zinc-finger domain containing proteins and their genes are induced in response to multiple abiotic stresses. The role of certain SAP genes in conferring abiotic stress tolerance is well established, but their mechanism of action is poorly understood. To improve our understanding of SAP gene functions, OsSAP10, a stress-inducible rice gene, was chosen for the functional and molecular characterization. To elucidate its role in water-deficit stress (WDS) response, we aimed to functionally characterize its roles in transgenic Arabidopsis, overexpressing OsSAP10. OsSAP10 transgenics showed improved tolerance to water-deficit stress at seed germination, seedling and mature plant stages. At physiological and biochemical levels, OsSAP10 transgenics exhibited a higher survival rate, increased relative water content, high osmolyte accumulation (proline and soluble sugar), reduced water loss, low ROS production, low MDA content and protected yield loss under WDS relative to wild type (WT). Moreover, transgenics were hypersensitive to ABA treatment with enhanced ABA signaling and stress-responsive genes expression. The protein-protein interaction studies revealed that OsSAP10 interacts with proteins involved in proteasomal pathway, such as OsRAD23, polyubiquitin and with negative and positive regulators of stress signaling, i.e., OsMBP1.2, OsDRIP2, OsSCP and OsAMTR1. The A20 domain was found to be crucial for most interactions but insufficient for all interactions tested. Overall, our investigations suggest that OsSAP10 is an important candidate for improving water-deficit stress tolerance in plants, and positively regulates ABA and WDS signaling via protein-protein interactions and modulation of endogenous genes expression in ABA-dependent manner.

Host-specific SRSF7 regulates polymerase activity and replication of influenza A virus

Avian influenza viruses crossing the host barrier to infect humans have caused great panic in human society and seriously threatened public health. Herein, we revealed that knockdown of SRSF7 significantly down-regulated influenza virus titers and viral protein expression. We further observed for the first time that human SRSF7, but not avian SRSF7, significantly inhibited polymerase activity (PB2627E). Molecular mapping demonstrated that amino acids 206 to 228 of human SRSF7 play a decisive role in regulating the polymerase activity, which contains the amino acid motif absent in avian SRSF7. Importantly, our results illustrated that the PB2627K-encoding influenza virus induces SRSF7 protein degradation more strongly via the lysosome pathway and not via the proteasome pathway. Functional enrichment analysis of SRSF7-related KEGG pathways indicated that SRSF7 is closely related to cell growth and death. Lastly, our results showed that knocking down SRSF7 interferes with normal polymerase activity. Taken together, our results advance our understanding of interspecies transmission and our findings point out new targets for the development of drugs preventing or treating influenza virus infection.

Glutathione-dependent degradation of SMARCA2/4 for targeted lung cancer therapy with improved selectivity

SMARCA2 and SMARCA4 are the mutually exclusive catalytic subunits of the mammalian Switch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complex, and have recently been considered as attractive synthetic lethal targets for PROTAC-based cancer therapy. However, the potential off-tissue toxicity towards normal tissues remains a concern. Here, we optimize a GSH-inducible SMARCA2/4-based PROTAC precursor with selective antitumor activity towards lung cancer cells and negligible cytotoxicity towards normal cells in both in vitro and in vivo studies. The precursor is not bioactive or cytotoxic, but preferentially responds to endogenous GSH in GSH-rich lung cancer cells, releasing active PROTAC to degrade SMARCA2/4 via PROTAC-mediated proteasome pathway. Subsequent xenograft model study reveals that selective SMARCA2/4 degradation in lung tumors triggers DNA damage and apoptosis, which significantly inhibits lung cancer cell proliferation without obvious adverse events towards normal tissues. This study exemplifies the targeted degradation of SMARCA2/4 in lung cancer cells by the GSH-responsive PROTAC precursor, highlighting its potential as an encouraging cancer therapeutic strategy.

Aberrantly Expressed tRNA-Val Fragments Can Distinguish Canine Hepatocellular Carcinoma from Canine Hepatocellular Adenoma.

Hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC) can be difficult to differentiate but must be diagnosed correctly as treatment and prognosis for these tumors differ markedly. Relevant diagnostic biomarkers are thus needed, and those identified in dogs may have utility in human medicine because of the similarities between human and canine HCA and HCC. A tRNA-derived fragment (tRF), tRNA-Val, is a promising potential biomarker for canine mammary gland tumors but has not previously been investigated in hepatic tumors. Accordingly, we aimed to elucidate the potential utility of tRNA-Val as a biomarker for canine HCA and HCC using clinical samples (tumor tissue and plasma extracellular vesicles [EVs]) and tumor cell lines with qRT-PCR assays. We also investigated relevant functions and signaling pathways with bioinformatic analyses (Gene Ontology and Kyoto Encyclopedia of Genes and Genomes). tRNA-Val was markedly downregulated in HCC tumor tissue versus HCA tumor tissue and normal liver tissue, and a similar trend was shown in plasma EVs and HCC cell lines versus healthy controls. Based on areas under the receiver operating characteristic curves (AUCs), tRNA-Val significantly distinguished HCC (AUC = 1.00, p = 0.001) from healthy controls in plasma EVs and HCC from HCA (AUC = 0.950, p = 0.01). Bioinformatics analysis revealed that tRNA-Val may be primarily involved in DNA repair, mRNA processing, and splicing and may be linked to the N-glycan and ubiquitin-mediated proteasome pathways. This is the first report on the expression of tRNA-Val in canine HCC and HCA and its possible functions and signaling pathways. We suggest that tRNA-Val could be a promising novel biomarker to distinguish canine HCC from HCA. This study provides evidence for a greater understanding of the role played by tRNA-Val in the development of canine HCC.

Design, synthesis, anti-tumor activity and mechanism of novel PROTACs as degraders of PD-L1 and inhibitors of PD-1/PD-L1 interaction

Currently, antibody drugs targeting programmed cell death ligand 1 (PD-L1) have achieved promising results in cancer treatment, while the development of small-molecule drugs lags behind. In this study, we designed and synthesized a series of PD-L1-degrading agents based on the PROTAC design principle, utilizing the PD-L1 inhibitor A56. Through systematic screening of ligands and linkers and investigating the structure–activity relationship of the degraders, we identified two highly active compounds, 9i and 9j. These compounds enhance levels of CD4+, CD8+, granzyme B, and perforin, demonstrating significant in vivo antitumor effects with a tumor growth inhibition (TGI) of up to 57.35 %. Both compounds facilitate the internalization of PD-L1 from the cell surface and promote its degradation through proteasomal and lysosomal pathways, while also maintaining inhibition of the PD-1/PD-L1 interaction. In summary, our findings provide a novel strategy and mechanism for developing biphenyl-based PROTAC antitumor drugs targeting and degrading PD-L1.

O-GlcNAcylation determines the function of the key O-GalNAc glycosyltransferase C1GalT1 in bladder cancer.

Protein glycosylation is a type of protein post-translational modification. One specific example is the modification of proteins with O-linked β-N-acetylglucosamine (O-GlcNAc) and O-linked α-N-acetylgalactosamine (O-GalNAc). Enhanced levels of both O-GalNAc and O-GlcNAc in bladder cancer (BlCa) have been reported previously. However, the interplay between O-GalNAc and O-GlcNAc has yet to be explored. Herein, we find that the expression level of core1 β-1,3-galactosyltransferase (C1GalT1), which is responsible for extending and maturing mucin-type O-glycans, is increased in BlCa. This increase is accompanied by O-GlcNAc modification of C1GalT1. This modification stabilizes C1GalT1 expression and strengthens its interaction with its chaperone Cosmc. Mutation at Thr229 or Thr233 attenuates C1GalT1 stability and facilitates its degradation via the proteasome pathway. Furthermore, a decrease in C1GalT1 inhibits the pro-tumorigenic effect on bladder cancer cells by suppressing glycolysis.

Scenedesmus acuminatus as a potential phycoremediator: Enrichment and detoxification of cadmium/lead

Heavy metal pollution in water can cause severe environmental problems, harm the ecosystem, and ultimately threaten human health. The traditional methods used to remove heavy metals from water are expensive, inefficient, and can cause secondary pollution. However, microalgae have potential applications in the removal of toxic heavy metal pollutants from water. In this study, six green algae were screened in terms of their ability to adsorb cadmium (Cd) and lead (Pb) from a liquid medium. Ion content analyses confirmed that Scenedesmus acuminatus had the strongest Cd- and Pb-absorption ability. S. acuminatus was subjected to Cd and Pb treatments, and then transcriptome sequencing analysis was conducted. The results of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that the “proteasome” pathway was enriched with differentially expressed genes (DEGs) under Cd treatment, and the “photosynthesis” pathway was enriched with DEGs under Pb treatment. These results imply that the proteasome pathway maintained protein homeostasis and cellular activity by degrading damaged proteins under Cd toxicity, and that photosynthesis was affected by Pb toxicity in S. acuminatus. In conclusion, S. acuminatus showed a strong ability to accumulate Cd/Pb and might alleviate heavy metal toxicity and reduce heavy metal efflux using the ubiquitin-proteasome system. The results of this study identify a novel microalgal strain with potential applications in phytoextraction and phytoremediation, and provide information about the molecular mechanisms of Cd/Pb accumulation in this alga. These data provide a theoretical basis for reducing Cd/Pb toxicity and will be useful for devising strategies to protect aquatic animals and plants.