Ubiquitin recruiting chimera: more than just a PROTAC.

Ubiquitinylation of protein substrates results in various but distinct biological consequences, among which ubiquitin-mediated degradation is most well studied for its therapeutic application. Accordingly, artificially targeted ubiquitin-dependent degradation of various proteins has evolved into the therapeutically relevant PROTAC technology. This tethered ubiquitinylation of various targets coupled with a broad assortment of modifying E3 ubiquitin ligases has been made possible by rational design of bi-specific chimeric molecules that bring these proteins in proximity. However, forced ubiquitinylation inflicted by the binary warheads of a chimeric PROTAC molecule should not necessarily result in protein degradation but can be used to modulate other cellular functions. In this respect it should be noted that the ubiquitinylation of a diverse set of proteins is known to control their transport, transcriptional activity, and protein-protein interactions. This review provides examples of potential PROTAC usage based on non-degradable ubiquitinylation.

Discovery of a first-in-class protein degrader for the c-ros oncogene 1 (ROS1).

The c-ros oncogene 1 (ROS1), an oncogenic driver, is known to induce non-small cell lung cancer (NSCLC) when overactivated, particularly through the formation of fusion proteins. Traditional targeted therapies focus on inhibiting ROS1 activity with ROS 1 inhibitors to manage cancer progression. However, a new strategy involving the design of protein degraders offers a more potent approach by completely degrading ROS1 fusion oncoproteins, thereby effectively blocking their kinase activity and enhancing anti-tumour potential. Utilizing PROteolysis-TArgeting Chimera (PROTAC) technology and informed by molecular docking and rational design, we report the first ROS1-specific PROTAC, SIAIS039. This degrader effectively targets multiple ROS1 fusion oncoproteins (CD74-ROS1, SDC4-ROS1 and SLC34A2-ROS1) in engineered Ba/F3 cells and HCC78 cells, demonstrating anti-tumour effects against ROS1 fusion-driven cancer cells. It suppresses cell proliferation, induces cell cycle arrest, and apoptosis, and inhibits clonogenicity. The anti-tumour efficacy of SIAIS039 surpasses two approved drugs, crizotinib and entrectinib, and matches that of the top inhibitors, including lorlatinib and taletrectinib. Mechanistic studies confirm that the degradation induced by 039 requires the participation of ROS1 ligands and E3 ubiquitin ligases, and involves the proteasome and ubiquitination. In addition, 039 exhibited excellent oral bioavailability in a mouse xenograft model, highlighting its potential for clinical application. In conclusion, our study presents a promising and novel therapeutic strategy for ROS1 fusion-positive NSCLC by targeting ROS1 fusion oncoproteins for degradation, laying the foundation for the development of further PROTAC and offering hope for patients with ROS1 fusion-positive NSCLC.

A novel variant in GAS2 is associated with autosomal dominant nonsyndromic hearing impairment in a Chinese family.

Knockout of GAS2 (growth arrest-specific protein 2), causes disorganization and destabilization of microtubule bundles in supporting cells of the cochlear duct, leading to hearing loss in vivo. However, the molecular mechanism through which GAS2 variant results in hearing loss remains unknown. By Whole-exome sequencing, we identified a novel heterozygous splicing variant in GAS2 (c.616-2 A > G) as the only candidate mutation segregating with late-onset and progressive nonsyndromic hearing loss (NSHL) in a large dominant family. This splicing mutation causes an intron retention and produces a C-terminal truncated protein (named GAS2mu). Mechanistically, the degradation of GAS2mu via the ubiquitin-proteasome pathway is enhanced, and cells expressing GAS2mu exhibit disorganized microtubule bundles. Additionally, GAS2mu further promotes apoptosis by increasing the Bcl-xS/Bcl-xL ratio instead of through the p53-dependent pathway as wild-type GAS2 does, indicating that GAS2mu acts as a toxic molecule to exacerbate apoptosis. Our findings demonstrate that this novel variant of GAS2 promotes its own protein degradation, microtubule disorganization and cellular apoptosis, leading to hearing loss in carriers. This study expands the spectrum of GAS2 variants and elucidates the underlying pathogenic mechanisms, providing a foundation for future investigations of new therapeutic strategies to prevent GAS2-associated progressive hearing loss.

Protease-Responsive Toolkit for Conditional Targeted Protein Degradation.

BioPROTACs are heterobifunctional proteins designed for targeted protein degradation. While they offer a potential therapeutic avenue for modulating disease-related proteins, the current strategies are static in nature and lack the ability to modulate protein degradation dynamically. Here, we introduce a synthetic framework for dynamic fine-tuning of target protein levels using protease control switches. The idea is to utilize proteases as an interfacing layer between exogenous inputs and protein degradation by modulating the recruitment of target proteins to E3 ligase by separating the two binding domains on bioPROTACs. By decoupling the external inputs from the primary protease layer, new conditional degradation phenotypes can be readily adapted with minimal modifications to the design. We demonstrate the adaptability of this approach using two highly efficient "bioPROTAC" systems: AdPROM and IpaH9.8-based Ubiquibodies. Using the TEV protease as the transducer, we can interface small-molecule and optogenetic inputs for conditional targeted protein degradation. Our findings highlight the potential of bioPROTACs with protease-responsive linkers as a versatile tool for conditional targeted protein degradation.

Material flow analysis of an upgraded anaerobic digestion treatment plant with separated utilization of carbon and nitrogen of food waste.

Anaerobic digestion of food waste can recover carbon in the form of biogas, while the high concentration of ammonia nitrogen in the digestion effluent becomes troublesome. Therefore, some new treatment plants use three-phase centrifugation to separate homogenized food waste into nitrogen-rich fine slag for insect cultivation and carbon-rich liquid for anaerobic digestion. To analyze the effects of the carbon-nitrogen separation, an upgraded plant's material and elementary flows were investigated. The three-phase separation process redistributed carbon and nitrogen, and the biogas slurry was the primary output. The principal endpoint for C was the crude oil, capturing 57.1 ±â€¯13.1 % of the total input; the find slag collected 48.3 ±â€¯6.9 % of the total N input, and the biogas slag accepted 52.9 ±â€¯4.4 % of the P input. The carbon-nitrogen separation strategy can improve digestion efficiency and increase treatment benefits significantly, marking a promising direction for future developments in food waste utilization.

Autophagy mediated targeting degradation, a promising strategy in drug development.

Targeted protein degradation (TPD) technologies have become promising therapeutic approaches through degrading disease-causing proteins via the protein degradation system. Autophagy is a fundamental biological process with a high relationship to protein degradation, which belongs to one of two main protein degradation pathways, the autophagy-lysosomal system. Recently, various autophagy-based TPD techniques ATTECs, AUTACs, and AUTOTACs, etc, have also been gradually developed, and they have achieved efficient degradation potency for the targeted protein, expanding the potential of degradation for large-size proteins or protein aggregates. Herein, we introduce the machinery of autophagy and its relation to protein degradation, and multiple methods for using autophagy to specifically degrade target proteins.

Identification of Proteolysis Targeting Chimeras (PROTACs) for Lysine Demethylase 5 and Their Neurite Outgrowth-Promoting Activity.

Lysine demethylase 5 (KDM5) proteins are involved in various neurological disorders, including Alzheimer's disease, and KDM5 inhibition is expected to be a therapeutic strategy for these diseases. However, the pharmacological effects of conventional KDM5 inhibitors are insufficient, as they only target the catalytic functionality of KDM5. To identify compounds that exhibit more potent pharmacological activity, we focused on proteolysis targeting chimeras (PROTACs), which degrade target proteins and thus inhibit their entire functionality. We designed and synthesized novel KDM5 PROTAC candidates based on previously identified KDM5 inhibitors. The results of cellular assays revealed that two compounds, 20b and 23b, exhibited significant neurite outgrowth-promoting activity through the degradation of KDM5A in neuroblastoma neuro 2a cells. These results suggest that KDM5 PROTACs are promising drug candidates for the treatment of neurological disorders.

Transcriptional Up-Regulation of FBXW7 by KCa1.1 K+ Channel Inhibition through the Nrf2 Signaling Pathway in Human Prostate Cancer LNCaP Cell Spheroid Model.

The tumor suppressor gene F-box and WD repeat domain-containing (FBXW) 7 reduces cancer stemness properties by promoting the protein degradation of pluripotent stem cell markers. We recently demonstrated the transcriptional repression of FBXW7 by the three-dimensional (3D) spheroid formation of several cancer cells. In the present study, we found that the transcriptional activity of FBXW7 was promoted by the inhibition of the Ca2+-activated K+ channel, KCa1.1, in a 3D spheroid model of human prostate cancer LNCaP cells through the Akt-Nrf2 signaling pathway. The transcriptional activity of FBXW7 was reduced by the siRNA-mediated inhibition of the CCAAT-enhancer-binding protein C/EBP δ (CEBPD) after the transfection of miR223 mimics in the LNCaP spheroid model, suggesting the transcriptional regulation of FBXW7 through the Akt-Nrf2-CEBPD-miR223 transcriptional axis in the LNCaP spheroid model. Furthermore, the KCa1.1 inhibition-induced activation of FBXW7 reduced (1) KCa1.1 activity and protein levels in the plasma membrane and (2) the protein level of the cancer stem cell (CSC) markers, c-Myc, which is a molecule degraded by FBXW7, in the LNCaP spheroid model, indicating that KCa1.1 inhibition-induced FBXW7 activation suppressed CSC conversion in KCa1.1-positive cancer cells.

Dynamic modulation of nodulation factor receptor levels by phosphorylation-mediated functional switch of a RING-type E3 ligase during legume nodulation.

The precise control of receptor levels is crucial for initiating cellular signaling transduction in response to specific ligands; however, such mechanisms regulating nodulation factor (NF) receptor (NFR)-mediated perception of NFs to establish symbiosis remain unclear. In this study, we unveil the pivotal role of the NFR-interacting RING-type E3 ligase 1 (NIRE1) in regulating NFR1/NFR5 homeostasis to optimize rhizobial infection and nodule development in Lotus japonicus. We demonstrated that NIRE1 has a dual function in this regulatory process. It associates with both NFR1 and NFR5, facilitating their degradation through K48-linked polyubiquitination before rhizobial inoculation. However, following rhizobial inoculation, NFR1 phosphorylates NIRE1 at a conserved residue, Tyr-109, inducing a functional switch in NIRE1, which enables NIRE1 to mediate K63-linked polyubiquitination, thereby stabilizing NFR1/NFR5 in infected root cells. The introduction of phospho-dead NIRE1Y109F leads to delayed nodule development, underscoring the significance of phosphorylation at Tyr-109 in orchestrating symbiotic processes. Conversely, expression of the phospho-mimic NIRE1Y109E results in the formation of spontaneous nodules in L. japonicus, further emphasizing the critical role of the phosphorylation-dependent functional switch in NIRE1. In summary, these findings uncover a fine-tuned symbiotic mechanism that a single E3 ligase could undergo a phosphorylation-dependent functional switch to dynamically and precisely regulate NF receptor protein levels.

USP36 inhibits apoptosis by deubiquitinating cIAP1 and survivin in colorectal cancer cells.

Chemotherapeutic agents for treating colorectal cancer (CRC) primarily induce apoptosis in tumor cells. The ubiquitin-proteasome system is critical for apoptosis regulation. Deubiquitinating enzymes (DUBs) remove ubiquitin from substrates to reverse ubiquitination. Although over 100 DUB members have been discovered, the biological functions of only a small proportion of DUBs have been characterized. Here, we aimed to systematically identify the DUBs that contribute to the development of CRC. Among the DUBs, ubiquitin-specific protease 36 (USP36) is upregulated in CRC. We showed that the knockdown of USP36 induces intrinsic and extrinsic apoptosis. Through gene silencing and coimmunoprecipitation techniques, we identified survivin and cIAP1 as USP36 targets. Mechanistically, USP36 binds and removes lysine-11-linked ubiquitin chains from cIAP1 and lysine-48-linked ubiquitin chains from survivin to abolish protein degradation. Overexpression of USP36 disrupts the formation of the XIAP-second mitochondria-derived activator of caspase complex and promotes receptor-interacting protein kinase 1 ubiquitination, validating USP36 as an inhibitor to intrinsic and extrinsic apoptosis through deubiquitinating survivin and cIAP1. Therefore, our results suggest that USP36 is involved in CRC progression and is a potential therapeutic target.

ProteoCure : an European network to fine-tune the proteome.

Proteins are essential molecular actors in every cellular process. From their synthesis to their degradation, they are subject to continuous quality control mechanisms to ensure that they fulfil cellular needs in proper and timely fashion. Proteostasis is a key process allowing cells or organisms to maintain an appropriate but dynamic equilibrium of their proteome (the ensemble of all their proteins). It relies on multiple mechanisms that together control the level, fate and function of individual proteins, and ensure elimination of abnormal ones. The proteostasis network is essential for development and adaptation to environmental changes or challenges. Its dysfunctions can lead to accumulation of deleterious proteins or, conversely, to excessive degradation of beneficial ones, and are implicated in many diseases such as cancers, neurodegeneration, or developmental and aging disorders. Manipulating this network to control abundance of selected target proteins is therefore a strategy with enormous therapeutic or biotechnological potential. The ProteoCure COST Action gathers more than 350 researchers and their teams (31 countries represented) from the academic, clinical, and industrial sectors, who share the conviction that our understanding of proteostasis is mature enough to develop novel and highly specific therapies based on selective tunning of protein levels. Towards this objective, the Action organizes community-building activities to foster synergies among its participants and reinforce training of the next generation of European researchers. Its ambition is to function as a knowledge-based network and a creative exchange hub on normal and pathologic proteostasis, focusing on developing innovative tools modulating the level of specific protein(s).

The U-Box E3 Ubiquitin Ligase PUB35 Negatively Regulates ABA Signaling through AFP1-mediated Degradation of ABI5.

Abscisic acid (ABA) signaling is crucial for plant responses to various abiotic stresses. The Arabidopsis (Arabidopsis thaliana) transcription factor ABA INSENSITIVE 5 (ABI5) is a central regulator of ABA signaling. ABI5 BINDING PROTEIN 1 (AFP1) interacts with ABI5 and facilitates its 26S-proteasome-mediated degradation, although the detailed mechanism has remained unclear. Here, we report that an ABA-responsive U-box E3 ubiquitin ligase, PLANT U-BOX 35 (PUB35), physically interacts with AFP1 and ABI5. PUB35 directly ubiquitinated ABI5 in a bacterially reconstituted ubiquitination system and promoted ABI5 protein degradation in vivo. ABI5 degradation was enhanced by AFP1 in response to ABA treatment. Phosphorylation of the T201 and T206 residues in ABI5 disrupted the ABI5-AFP1 interaction and affected the ABI5-PUB35 interaction and PUB35-mediated degradation of ABI5 in vivo. Genetic analysis of seed germination and seedling growth showed that pub35 mutants were hypersensitive to ABA as well as to salinity and osmotic stresses, whereas PUB35 overexpression lines were hyposensitive. Moreover, abi5 was epistatic to pub35, whereas the pub35-2 afp1-1 double mutant showed a similar ABA response to the two single mutants. Together, our results reveal a PUB35-AFP1 module involved in fine-tuning ABA signaling through ubiquitination and 26S-proteasome-mediated degradation of ABI5 during seed germination and seedling growth.

Omeprazole Induces CYP3A4 mRNA Expression but Not CYP3A4 Protein Expression in HepaRG Cells.

Unknown interactions between drugs remain the limiting factor for clinical application of drugs, and the induction and inhibition of drug-metabolizing CYP enzymes are considered the key to examining the drug-drug interaction (DDI). In this study, using human HepaRG cells as an in vitro model system, we analyzed the potential DDI based on the expression levels of CYP3A4 and CYP1A2. Rifampicin and omeprazole, the potent inducers for CYP3A4 and CYP1A2, respectively, induce expression of the corresponding CYP enzymes at both the mRNA and protein levels. We noticed that, in addition to inducing CYP1A2, omeprazole induced CYP3A4 mRNA expression in HepaRG cells. However, unexpectedly, CYP3A4 protein expression levels were not increased after omeprazole treatment. Concurrent administration of rifampicin and omeprazole showed an inhibitory effect of omeprazole on the CYP3A4 protein expression induced by rifampicin, while its mRNA induction remained intact. Cycloheximide chase assay revealed increased CYP3A4 protein degradation in the cells exposed to omeprazole. The data presented here suggest the potential importance of broadening the current DDI examination beyond conventional transcriptional induction and enzyme-activity inhibition tests to include post-translational regulation analysis of CYP enzyme expression.

Selective degradation of hyperphosphorylated tau by proteolysis-targeting chimeras ameliorates cognitive function in Alzheimer's disease model mice.

Alzheimer's disease (AD) is one of the most common chronic neurodegenerative diseases. Hyperphosphorylated tau plays an indispensable role in neuronal dysfunction and synaptic damage in AD. Proteolysis-targeting chimeras (PROTACs) are a novel type of chimeric molecule that can degrade target proteins by inducing their polyubiquitination. This approach has shown promise for reducing tau protein levels, which is a potential therapeutic target for AD. Compared with traditional drug therapies, the use of PROTACs to reduce tau levels may offer a more specific and efficient strategy for treating AD, with fewer side effects. In the present study, we designed and synthesized a series of small-molecule PROTACs to knock down tau protein. Of these, compound C8 was able to lower both total and phosphorylated tau levels in HEK293 cells with stable expression of wild-type full-length human tau (termed HEK293-htau) and htau-overexpressed mice. Western blot findings indicated that C8 degraded tau protein through the ubiquitin-proteasome system in a time-dependent manner. In htau-overexpressed mice, the results of both the novel object recognition and Morris water maze tests revealed that C8 markedly improved cognitive function. Together, our findings suggest that the use of the small-molecule PROTAC C8 to degrade phosphorylated tau may be a promising therapeutic strategy for AD.

PA200-Mediated Proteasomal Protein Degradation and Regulation of Cellular Senescence.

Cellular senescence is closely related to DNA damage, proteasome inactivity, histone loss, epigenetic alterations, and tumorigenesis. The mammalian proteasome activator PA200 (also referred to as PSME4) or its yeast ortholog Blm10 promotes the acetylation-dependent degradation of the core histones during transcription, DNA repair, and spermatogenesis. According to recent studies, PA200 plays an important role in senescence, probably because of its role in promoting the degradation of the core histones. Loss of PA200 or Blm10 is a major cause of the decrease in proteasome activity during senescence. In this paper, recent research progress on the association of PA200 with cellular senescence is summarized, and the potential of PA200 to serve as a therapeutic target in age-related diseases is discussed.

Targeted protein degradation combined with PET imaging reveals the role of host PD-L1 in determining anti-PD-1 therapy efficacy.

PURPOSE: Immunohistochemical staining of programmed death-ligand 1 (PD-L1) in tumor biopsies acquired through invasive procedures is routinely employed in clinical practice to identify patients who are most likely to benefit from anti-programmed cell death protein 1 (PD-1) therapy. Nevertheless, PD-L1 expression is observed in various cellular subsets within tumors and their microenvironments, including tumor cells, dendritic cells, and macrophages. The impact of PD-L1 expression across these different cell types on the responsiveness to anti-PD-1 treatment is yet to be fully understood. METHODS: We synthesized polymer-based lysosome-targeting chimeras (LYTACs) that incorporate both PD-L1-targeting motifs and liver cell-specific asialoglycoprotein receptor (ASGPR) recognition elements. Small-animal positron emission tomography (PET) imaging of PD-L1 expression was also conducted using a PD-L1-specific radiotracer 89Zr-αPD-L1/Fab. RESULTS: The PD-L1 LYTAC platform was capable of specifically degrading PD-L1 expressed on liver cancer cells through the lysosomal degradation pathway via ASGPR without impacting the PD-L1 expression on host cells. When coupled with whole-body PD-L1 PET imaging, our studies revealed that host cell PD-L1, rather than tumor cell PD-L1, is pivotal in the antitumor response to anti-PD-1 therapy in a mouse model of liver cancer. CONCLUSION: The LYTAC strategy, enhanced by PET imaging, has the potential to surmount the limitations of knockout mouse models and to provide a versatile approach for the selective degradation of target proteins in vivo. This could significantly aid in the investigation of the roles and mechanisms of protein functions associated with specific cell subsets in living subjects.

Molecular insights and clinical implications for the tumor suppressor role of SCFFBXW7 E3 ubiquitin ligase.

FBXW7 is one of the most well-characterized F-box proteins, serving as substrate receptor subunit of SKP1-CUL1-F-box (SCF) E3 ligase complexes. SCFFBXW7 is responsible for the degradation of various oncogenic proteins such as cyclin E, c-MYC, c-JUN, NOTCH, and MCL1. Therefore, FBXW7 functions largely as a major tumor suppressor. In keeping with this notion, FBXW7 gene mutations or downregulations have been found and reported in many types of malignant tumors, such as endometrial, colorectal, lung, and breast cancers, which facilitate the proliferation, invasion, migration, and drug resistance of cancer cells. Therefore, it is critical to review newly identified FBXW7 regulation and tumor suppressor function under physiological and pathological conditions to develop effective strategies for the treatment of FBXW7-altered cancers. Since a growing body of evidence has revealed the tumor-suppressive activity and role of FBXW7, here, we updated FBXW7 upstream and downstream signaling including FBXW7 ubiquitin substrates, the multi-level FBXW7 regulatory mechanisms, and dysregulation of FBXW7 in cancer, and discussed promising cancer therapies targeting FBXW7 regulators and downstream effectors, to provide a comprehensive picture of FBXW7 and facilitate the study in this field.

Unveiling a new strategy for PDIA1 inhibition: Integration of activity-based probes profiling and targeted degradation.

The overexpression of PDIA1 in cancer has spurred the quest for effective inhibitors. However, existing inhibitors often bind to only one active site, limiting their efficacy. In our study, we developed a PROTAC-mimetic probe dPA by combining PACMA31 (PA) analogs with cereblon-directed pomalidomide. Through protein profiling and analysis, we confirmed dPA's specific interaction with PDIA1's active site cysteines. We further synthesized PROTAC variants with a thiophene ring and various linkers to enhance degradation efficiency. Notably, H4, featuring a PEG linker, induced significant PDIA1 degradation and inhibited cancer cell proliferation similarly to PA. The biosafety profile of H4 is comparable to that of PA, highlighting its potential for further development in cancer therapy. Our findings highlight a novel strategy for PDIA1 inhibition via targeted degradation, offering promising prospects in cancer therapeutics. This approach may overcome limitations of conventional inhibitors, presenting new avenues for advancing anti-cancer interventions.

A small-molecule degron with a phenylpropionic acid scaffold.

Targeted protein degradation (TPD), employing proteolysis-targeting chimeras (PROTACs) composed of ligands for both a target protein and ubiquitin ligase (E3) to redirect the ubiquitin-proteasome system (UPS) to the target protein, has emerged as a promising strategy in drug discovery. However, despite the vast number of E3 ligases, the repertoire of E3 ligands utilized in PROTACs remains limited. Here, we report the discovery of a small-molecule degron with a phenylpropionic acid skeleton, derived from a known ligand of S-phase kinase-interacting protein 2 (Skp2), an E3 ligase. We used this degron to design PROTACs inducing proteasomal degradation of HaloTag-fused proteins, and identified key structural relationships. Surprisingly, our mechanistic studies excluded the involvement of Skp2, suggesting that this degron recruits other protein(s) within the UPS.

XBAT31 regulates reproductive thermotolerance through controlling the accumulation of HSFB2a/B2b under heat stress conditions.

Heat shock transcription factors (HSFs) play a crucial role in heat stress tolerance in vegetative tissues. However, their involvement in reproductive tissues and their post-translational modifications are not well understood. In this study, we identify the E3 ligase XB3 ORTHOLOG 1 IN ARABIDOPSIS THALIANA (XBAT31) as a key player in the ubiquitination and degradation of HSFB2a/B2b. Our results show that the xbat31 mutant exhibits a higher percentage of unfertile siliques and decreased expression of HSPs in flowers under heat stress conditions compared to the wild type. Conversely, the hsfb2a hsfb2b double mutant displays improved reproductive thermotolerance. We find that XBAT31 interacts with HSFB2a/B2b and mediates their ubiquitination. Furthermore, HSFB2a/B2b ubiquitination is reduced in the xbat31-1 mutant, resulting in higher accumulation of HSFB2a/B2b in flowers under heat stress conditions. Overexpression of HSFB2a or HSFB2b leads to an increase in unfertile siliques under heat stress conditions. Thus, our results dissect the important role of the XBAT31-HSFB2a/B2b module in conferring reproductive thermotolerance in plants.