Recruitment Of Effectors Research Articles

The beta 2 adrenergic receptor cross-linked interactome identifies 14-3-3 proteins as regulating the availability of signaling-competent receptors.

The emerging picture of G protein-coupled receptor function suggests that the global signaling response is an integrated sum of a multitude of individual receptor responses, each regulated by their local protein environment. The beta 2 adrenergic receptor (B2AR) has long served as an example receptor in the development of this model. But the mechanism and the identity of the protein-protein interactions that govern the availability of receptors competent for signaling remains incompletely characterized. To address this question, we characterized the interactome of agonist-stimulated B2AR in HEK293 cells using FLAG co-immunoprecipitation coupled to SILAC labeling and mass spectrometry. Our B2AR cross-linked interactome identified 190 high-confidence proteins, including almost all known interacting proteins and six out of seven isoforms of the 14-3-3 family of scaffolding proteins. Inhibiting 14-3-3 proteins with the peptide difopein enhanced isoproterenol-stimulated adrenergic signaling via cAMP approximately three-fold, and increased both miniGs and arrestin recruitment to B2AR more than two fold each, without noticeably changing EC50 with respect to cAMP signaling or effector recruitment upon stimulation. Our results show that 14-3-3 proteins negatively regulate downstream signaling by inhibiting access of B2AR to effector proteins. We propose that 14-3-3 proteins maintain a dynamic pool of B2AR that has reduced signaling efficacy in response to acute agonist stimulation, limiting the amount of signaling-competent receptors at the plasma membrane. Significance Statement This study presents a new interactome of the agonist-stimulated beta 2 adrenergic receptor (B2AR), a paradigmatic GPCR that is both a model system for members of this class and an important signaling protein in respiratory, cardiovascular, and metabolic regulation. We identify 14-3-3 proteins as responsible for restricting B2AR access to signaling effectors and maintaining a receptor population that is insensitive to acute stimulation by agonists.

Progress on the development of Class A GPCR-biased ligands.

Class A G protein-coupled receptors (GPCRs) continue to garner interest for their essential roles in cell signalling and their importance as drug targets. Although numerous drugs in the clinic target these receptors, over 60% GPCRs remain unexploited. Moreover, the adverse effects triggered by the available unbiased GPCR modulators, limit their use and therapeutic value. In this context, the elucidation of biased signalling has opened up new pharmacological avenues holding promise for safer therapeutics. Functionally selective ligands favour receptor conformations facilitating the recruitment of specific effectors and the modulation of the associated pathways. This review surveys the current drug discovery landscape of GPCR-biased modulators with a focus on recent advances. Understanding the biological effects of this preferential coupling is at different stages depending on the Class A GPCR family. Therefore, with a focus on individual GPCR families, we present a compilation of the functionally selective modulators reported over the past few years. In doing so, we dissect their therapeutic relevance, molecular determinants and potential clinical applications.

Epigenetic memory is governed by an effector recruitment specificity toggle in Heterochromatin Protein 1

HP1 proteins are essential for establishing and maintaining transcriptionally silent heterochromatin. They dimerize, forming a binding interface to recruit diverse chromatin-associated factors. Although HP1 proteins are known to rapidly evolve, the extent of variation required to achieve functional specialization is unknown. To investigate how changes in amino acid sequence impacts heterochromatin formation, we performed a targeted mutagenesis screen of the S. pombe HP1 homolog, Swi6. Substitutions within an auxiliary surface adjacent to the HP1 dimerization interface produce Swi6 variants with divergent maintenance properties. Remarkably, substitutions at a single amino acid position lead to the persistent gain or loss of epigenetic inheritance. These substitutions increase Swi6 chromatin occupancy in vivo and altered Swi6-protein interactions that reprogram H3K9me maintenance. We show how relatively minor changes in Swi6 amino acid composition in an auxiliary surface can lead to profound changes in epigenetic inheritance providing a redundant mechanism to evolve HP1-effector specificity.

The lipid flippase ATP8A1 regulates the recruitment of ARF effectors to the trans-Golgi Network

Formation of transport vesicles requires the coordinate activity of the coating machinery that selects cargo into the nascent vesicle and the membrane bending machinery that imparts curvature to the forming bud. Vesicle coating at the trans-Golgi Network (TGN) involves AP1, GGA2 and clathrin, which are recruited to membranes by activated ARF GTPases. The ARF activation at the TGN is mediated by the BIG1 and BIG2 guanine nucleotide exchange factors (GEFs). Membrane deformation at the TGN has been shown to be mediated by lipid flippases, including ATP8A1, that moves phospholipids from the inner to the outer leaflet of the TGN membrane. We probed a possible coupling between the coating and deformation machineries by testing for an interaction between BIG1, BIG2 and ATP8A1, and by assessing whether such an interaction may influence coating efficiency. Herein, we document that BIG1 and BIG2 co-localize with ATP8A1 in both, static and highly mobile TGN elements, and that BIG1 and BIG2 bind ATP8A1. We show that the interaction involves the catalytic Sec7 domain of the GEFs and the cytosolic C-terminal tail of ATP8A1. Moreover, we report that the expression of ATP8A1, but not ATP8A1 lacking the GEF-binding cytosolic tail, increases the generation of activated ARFs at the TGN and increases the selective recruitment of AP1, GGA2 and clathrin to TGN membranes. This occurs without increasing BIG1 or BIG2 levels at the TGN, suggesting that the binding of the ATP8A1 flippase tail to the Sec7 domain of BIG1/BIG2 increases their catalytic activity. Our results support a model in which a flippase component of the deformation machinery impacts the activity of the GEF component of the coating machinery.

A substrate-interacting region of Parkin directs ubiquitination of the mitochondrial GTPase Miro1.

Mutations in the gene encoding for the E3 ubiquitin ligase Parkin have been linked to early-onset Parkinson's disease. Besides many other cellular roles, Parkin is involved in clearance of damaged mitochondria via mitophagy - a process of particular importance in dopaminergic neurons. Upon mitochondrial damage, Parkin accumulates at the outer mitochondrial membrane and is activated, leading to ubiquitination of many mitochondrial substrates and recruitment of mitophagy effectors. While the activation mechanisms of autoinhibited Parkin have been extensively studied, it remains unknown how Parkin recognises its substrates for ubiquitination, and no substrate interaction site in Parkin has been reported. Here, we identify a conserved region in the flexible linker between the Ubl and RING0 domains of Parkin, which is indispensable for Parkin interaction with the mitochondrial GTPase Miro1. Our results explain the preferential targeting and ubiquitination of Miro1 by Parkin and provide a biochemical explanation for the presence of Parkin at the mitochondrial membrane prior to activation induced by mitochondrial damage. Our findings are important for understanding mitochondrial homeostasis and may inspire new therapeutic avenues for Parkinson's disease.

Enantiomeric Agonists of the Type 2 Cannabinoid Receptor Reduce Retinal Damage during Proliferative Vitreoretinopathy and Inhibit Hyperactive Microglia In Vitro.

Microglia are resident immune cells of the central nervous system (CNS) and propagate inflammation following damage to the CNS, including the retina. Proliferative vitreoretinopathy (PVR) is a condition that can emerge following retinal detachment and is characterized by severe inflammation and microglial proliferation. The type 2 cannabinoid receptor (CB2) is an emerging pharmacological target to suppress microglial-mediated inflammation when the eyes or brain are damaged. CB2-knockout mice have exacerbated inflammation and retinal pathology during experimental PVR. We aimed to assess the anti-inflammatory effects of CB2 stimulation in the context of retinal damage and also explore the mechanistic roles of CB2 in microglia function. To target CB2, we used a highly selective agonist, HU-308, as well as its enantiomer, HU-433, which is a putative selective agonist. First, β-arrestin2 and Gαi recruitment was measured to compare activation of human CB2 in an in vitro heterologous expression system. Both agonists were then utilized in a mouse model of PVR, and the effects on retinal damage, inflammation, and cell death were assessed. Finally, we used an in vitro model of microglia to determine the effects of HU-308 and HU-433 on phagocytosis, cytokine release, migration, and intracellular signaling. We observed that HU-308 more strongly recruited both β-arrestin2 and Gαi compared to HU-433. Stimulation of CB2 with either drug effectively blunted LPS- and IFNγ-mediated signaling as well as NO and TNF release from microglia. Furthermore, both drugs reduced IL-6 accumulation, total caspase-3 cleavage, and retinal pathology following the induction of PVR. Ultimately, this work supports that CB2 is a valuable target for drugs to suppress inflammation and cell death associated with infection or sterile retinopathy, although the magnitude of effector recruitment may not be predictive of anti-inflammatory capacity.

Monitoring ROS Responsive Fe3O4-based Nanoparticle Mediated Ferroptosis and Immunotherapy via 129Xe MRI.

The immune checkpoint blockade strategy has improved the survival rate of late-stage lung cancer patients. However, the low immune response rate limits the immunotherapy efficiency. Here, we report a ROS-responsive Fe3O4-based nanoparticle that undergoes charge reversal and disassembly in the tumor microenvironment, enhancing the uptake of Fe3O4 by tumor cells and triggering a more severe ferroptosis. In the tumor microenvironment, the nanoparticle rapidly disassembles and releases the loaded GOx and the immune-activating peptide Tuftsin under overexpressed H2O2. GOx can consume the glucose of tumor cells and generate more H2O2, promoting the disassembly of the nanoparticle and drug release, thereby enhancing the therapeutic effect of ferroptosis. Combined with Tuftsin, it can more effectively reverse the immune-suppressive microenvironment and promote the recruitment of effector T cells in tumor tissues. Ultimately, in combination with α-PD-L1, there is significant inhibition of the growth of lung metastases. Additionally, the hyperpolarized 129Xe method has been used to evaluate the Fe3O4 nanoparticle-mediated immunotherapy, where the ventilation defects in lung metastases have been significantly improved with complete lung structure and function recovered. The ferroptosis-enhanced immunotherapy combined with non-radiation evaluation methodology paves a new way for designing novel theranostic agents for cancer therapy.

Monitoring ROS Responsive Fe3O4‐based Nanoparticle Mediated Ferroptosis and Immunotherapy via 129Xe MRI

AbstractThe immune checkpoint blockade strategy has improved the survival rate of late‐stage lung cancer patients. However, the low immune response rate limits the immunotherapy efficiency. Here, we report a ROS‐responsive Fe3O4‐based nanoparticle that undergoes charge reversal and disassembly in the tumor microenvironment, enhancing the uptake of Fe3O4 by tumor cells and triggering a more severe ferroptosis. In the tumor microenvironment, the nanoparticle rapidly disassembles and releases the loaded GOx and the immune‐activating peptide Tuftsin under overexpressed H2O2. GOx can consume the glucose of tumor cells and generate more H2O2, promoting the disassembly of the nanoparticle and drug release, thereby enhancing the therapeutic effect of ferroptosis. Combined with Tuftsin, it can more effectively reverse the immune‐suppressive microenvironment and promote the recruitment of effector T cells in tumor tissues. Ultimately, in combination with α‐PD‐L1, there is significant inhibition of the growth of lung metastases. Additionally, the hyperpolarized 129Xe method has been used to evaluate the Fe3O4 nanoparticle‐mediated immunotherapy, where the ventilation defects in lung metastases have been significantly improved with complete lung structure and function recovered. The ferroptosis‐enhanced immunotherapy combined with non‐radiation evaluation methodology paves a new way for designing novel theranostic agents for cancer therapy.

Regulation by the RNA-binding protein Unkempt at its effector interface

How RNA-binding proteins (RBPs) convey regulatory instructions to the core effectors of RNA processing is unclear. Here, we document the existence and functions of a multivalent RBP–effector interface. We show that the effector interface of a conserved RBP with an essential role in metazoan development, Unkempt, is mediated by a novel type of ‘dual-purpose’ peptide motifs that can contact two different surfaces of interacting proteins. Unexpectedly, we find that the multivalent contacts do not merely serve effector recruitment but are required for the accuracy of RNA recognition by Unkempt. Systems analyses reveal that multivalent RBP–effector contacts can repurpose the principal activity of an effector for a different function, as we demonstrate for the reuse of the central eukaryotic mRNA decay factor CCR4-NOT in translational control. Our study establishes the molecular assembly and functional principles of an RBP–effector interface.

Advances in IL-7 Research on Tumour Therapy.

Interleukin-7 (IL-7) is a versatile cytokine that plays a crucial role in regulating the immune system's homeostasis. It is involved in the development, proliferation, and differentiation of B and T cells, as well as being essential for the differentiation and survival of naïve T cells and the production and maintenance of memory T cells. Given its potent biological functions, IL-7 is considered to have the potential to be widely used in the field of anti-tumour immunotherapy. Notably, IL-7 can improve the tumour microenvironment by promoting the development of Th17 cells, which can in turn promote the recruitment of effector T cells and NK cells. In addition, IL-7 can also down-regulate the expression of tumour growth factor-β and inhibit immunosuppression to promote anti-tumour efficacy, suggesting potential clinical applications for anti-tumour immunotherapy. This review aims to discuss the origin of IL-7 and its receptor IL-7R, its anti-tumour mechanism, and the recent advances in the application of IL-7 in tumour therapy.

Identification of plants’ functional counterpart of the metazoan mediator of DNA Damage checkpoint 1

Induction of DNA damage triggers rapid phosphorylation of the histone H2A.X (γH2A.X). In animals, mediator of DNA damage checkpoint 1 (MDC1) binds γH2A.X through a tandem BRCA1 carboxyl-terminal (tBRCT) domain and mediates recruitment of downstream effectors of DNA damage response (DDR). However, readers of this modification in plants have remained elusive. We show that from the Arabidopsis BRCT domain proteome, BCP1-4 proteins with tBRCT domains are involved in DDR. Through its tBRCT domain BCP4 binds γH2A.X in vitro and localizes to DNA damage-induced foci in an H2A.X-dependent manner. BCP4 also contains a domain that interacts directly with NBS1 and thus acts as a functional counterpart of MDC1. We also show that BCP1, that contains two tBRCT domains, co-localizes with γH2A.X but it does not bind γH2A.X suggesting functional similarity with human PAXIP1. A phylogenetic analysis supports that PAXIP1 and MDC1 in metazoa and their plant counterparts evolved independently from common ancestors with tBRCT domains. Collectively, our study reveals missing components and provides mechanistic and evolutionary insights into plant DDR.

PARP1-DNA co-condensation drives DNA repair site assembly to prevent disjunction of broken DNA ends

DNA double-strand breaks (DSBs) are repaired at DSB sites. How DSB sites assemble and how broken DNA is prevented from separating is not understood. Here we uncover that the synapsis of broken DNA is mediated by the DSB sensor protein poly(ADP-ribose) (PAR) polymerase 1 (PARP1). Using bottom-up biochemistry, we reconstitute functional DSB sites and show that DSB sites form through co-condensation of PARP1 multimers with DNA. The co-condensates exert mechanical forces to keep DNA ends together and become enzymatically active for PAR synthesis. PARylation promotes release of PARP1 from DNA ends and the recruitment of effectors, such as Fused in Sarcoma, which stabilizes broken DNA ends against separation, revealing a finely orchestrated order of events that primes broken DNA for repair. We provide a comprehensive model for the hierarchical assembly of DSB condensates to explain DNA end synapsis and the recruitment of effector proteins for DNA damage repair.

ADP-Hep-Induced Liquid Phase Condensation of TIFA-TRAF6 Activates ALPK1/TIFA-Dependent Innate Immune Responses.

The ALPK1 (alpha-kinase 1)-TIFA (TRAF-interacting protein with fork head-associated domain)-TRAF6 signaling pathway plays a pivotal role in regulating inflammatory processes, with TIFA and TRAF6 serving as key molecules in this cascade. Despite its significance, the functional mechanism of TIFA-TRAF6 remains incompletely understood. In this study, we unveil that TIFA undergoes liquid-liquid phase separation (LLPS) induced by ALPK1 in response to adenosine diphosphate (ADP)-β-D-manno-heptose (ADP-Hep) recognition. The phase separation of TIFA is primarily driven by ALPK1, the pT9-FHA domain, and the intrinsically disordered region segment. Simultaneously, TRAF6 exhibits phase separation during ADP-Hep-induced inflammation, a phenomenon observed consistently across various inflammatory signal pathways. Moreover, TRAF6 is recruited within the TIFA condensates, facilitating lysine (K) 63-linked polyubiquitin chain synthesis. The subsequent recruitment, enrichment, and activation of downstream effectors within these condensates contribute to robust inflammatory signal transduction. Utilizing a novel chemical probe (compound 22), our analysis demonstrates that the activation of the ALPK1-TIFA-TRAF6 signaling pathway in response to small molecules necessitates the phase separation of TIFA. In summary, our findings reveal TIFA as a sensor for upstream signals, initiating the LLPS of itself and downstream proteins. This process results in the formation of membraneless condensates within the ALPK1-TIFA-TRAF6 pathway, suggesting potential applications in therapeutic biotechnology development.

Cytosolic sorting platform complexes shuttle type III secretion system effectors to the injectisome in Yersinia enterocolitica

Bacteria use type III secretion injectisomes to inject effector proteins into eukaryotic target cells. Recruitment of effectors to the machinery and the resulting export hierarchy involve the sorting platform. These conserved proteins form pod structures at the cytosolic interface of the injectisome but are also mobile in the cytosol. Photoactivated localization microscopy in Yersinia enterocolitica revealed a direct interaction of the sorting platform proteins SctQ and SctL with effectors in the cytosol of live bacteria. These proteins form larger cytosolic protein complexes involving the ATPase SctN and the membrane connector SctK. The mobility and composition of these mobile pod structures are modulated in the presence of effectors and their chaperones, and upon initiation of secretion, which also increases the number of injectisomes from ~5 to ~18 per bacterium. Our quantitative data support an effector shuttling mechanism, in which sorting platform proteins bind to effectors in the cytosol and deliver the cargo to the export gate at the membrane-bound injectisome.

Structural basis for competitive binding of productive and degradative co-transcriptional effectors to the nuclear cap-binding complex

The nuclear cap-binding complex (CBC) coordinates co-transcriptional maturation, transport, or degradation of nascent RNA polymerase II (Pol II) transcripts. CBC with its partner ARS2 forms mutually exclusive complexes with diverse "effectors" that promote either productive or destructive outcomes. Combining AlphaFold predictions with structural and biochemical validation, we show how effectors NCBP3, NELF-E, ARS2, PHAX, and ZC3H18 form competing binary complexes with CBC and how PHAX, NCBP3, ZC3H18, and other effectors compete for binding to ARS2. In ternary CBC-ARS2 complexes with PHAX, NCBP3, or ZC3H18, ARS2 is responsible for the initial effector recruitment but inhibits their direct binding to the CBC. We show that invivo ZC3H18 binding to both CBC and ARS2 is required for nuclear RNA degradation. We propose that recruitment of PHAX to CBC-ARS2 can lead, with appropriate cues, to competitive displacement of ARS2 and ZC3H18 from the CBC, thus promoting a productive rather than a degradative RNA fate.

Amplifying cancer chemoimmunotherapy through F4/80+CD11c+ DC-like macrophages induced by micellar docetaxel together with a TLR7/8 nanoagonist

Chemotherapy-based combinational immunomodulation has been developed to inflame the “cold” tumor immune microenvironment (TiME) for enhanced triple-negative breast cancer (TNBC) chemoimmunotherapy. However, the potential of docetaxel-based chemotherapy combined with immunomodulation by TLR7/8 agonists remains undiscovered. Herein, we first constructed micellar docetaxel (DTX-m) and a TLR7/8 nanoagonist suitable for systematic administration, R848 nanoparticles (R848 NPs). By adjusting the administration sequence, the combination of DTX-m and R848 NPs can efficiently inhibit tumor growth in vivo in a rodent triple-negative breast cancer model. The results of a mechanistic study reveal that alternate sequential administration promotes the polarization of macrophages to F4/80+CD11c+ DC-like macrophages and cumulatively promotes the secretion of CXCL10 and proinflammatory cytokines, which boost the recruitment of NK cells and APCs to reinflame the tumor immune microenvironment, further facilitating the recruitment and proliferation of effector T cells and amplifying tumor chemoimmunotherapy. Administration sequence regulates the therapeutic outcome by steering the polarization of macrophage phenotypes.

FABP4 Suppresses the Progress of Multiple Myeloma through Enhancing Alarming Function of Bone Marrow Resident Memory T Lymphocytes

Multiple myeloma (MM) is a hematological malignancy which characterized by malignant plasma cells resident in the bone marrow (BM). The BM microenvironment plays critical roles in the invasion, proliferation and migration of myeloma cells. Despite the therapies of MM improved in recent years, this disease is still incurable and requires precise treatment. Resident memory (TRM) cells are a distinct tissue-localized T cell lineage that is crucial for protective immunity in peripheral tissues. Recent studies reveal TRM cells as a vital component of the host immune response to cancer. TRM-like tumor-infiltrating lymphocytes (TILs) can be found in a wide range of human cancers, where they portend improved prognosis. However, the precise role and mechanism of bone marrow TRM cells in MM still remain elusive. In present study we first found that the accumulation of T RM cells (CD8 +CD44 +CD69 +CD62L -) in BM newly diagnosed MM (NDMM) patients was significantly higher than that of relapse recurrent MM (RRMM) ( Figure A), which was further confirmed by the immunohistochemistry (IHC) staining analysis ( Figure B). By using Seahorse XF e analyzer we found a decrease of oxygen consumption rate (OCR) of T RM cells in RRMM comparing to that of NDMM ( Figure C). Since fatty acid metabolism is important in β oxidation, we further investigated the expression of fatty acid binding proteins (FABP4/FABP5) in T RM cells and showed that FABP4 was dominant in T RM cells of NDMM patients ( Figure D). FABP4 +/+ mice and their relative controls FABP4 -/- mice were adopted to further explore the role and mechanism of T RM cells in MM progression. The abundance of genes related fatty acids β oxidation was significantly decreased in FABP4 -/- T RM cells ( Figure E). The tumor cell viability was decreased in myeloma cells co-cultured with FABP4 -/- T RM cells in the presence of bortezomib ( Figure F). Moreover, the expression of cytotoxic cytokines including IFN γ, TNFα and CXCL10 was significantly higher in FABP4 -/- T RM cells ( Figure G). The alarming function of T RM cells was further enhanced via recruiting more effector T (CD8 +CXCR3 +) cells ( Figure H), therefore the apoptosis of tumor cells was elevated in BM of MM patients. In summary, T RM cells are necessary and sufficient for long-lived protection against tumors. T RM cells derived FABP4 exerts anti-tumor immunity through prolonging the survival of T RM cells and enhancing the recruitment of more effector T cells to suppress the tumor progression ( Figure I). This study suggests that T RM cells might serve as a novel therapeutic target in MM and treatment specific targeting FABP4 might shed light on the development of potential therapeutic strategies for treating MM especially relapse patients. Acknowledgement This project is supported by the Sun Yat-sen University Hundred Talents Program (PT19200101), Natural Science Foundation of Guangdong Province (2022A1515010290).

Genome wide analysis revealed conserved domains involved in the effector discrimination of bacterial type VI secretion system

Type VI secretion systems (T6SSs) deliver effectors into target cells. Besides structural and effector proteins, many other proteins, such as adaptors, co-effectors and accessory proteins, are involved in this process. MIX domains can assist in the delivery of T6SS effectors when encoded as a stand-alone gene or fused at the N-terminal of the effector. However, whether there are other conserved domains exhibiting similar encoding forms to MIX in T6SS remains obscure. Here, we scanned publicly available bacterial genomes and established a database which include 130,825 T6SS vgrG loci from 45,041 bacterial genomes. Based on this database, we revealed six domain families encoded within vgrG loci, which are either fused at the C-terminus of VgrG/N-terminus of T6SS toxin or encoded by an independent gene. Among them, DUF2345 was further validated and shown to be indispensable for the T6SS effector delivery and LysM was confirmed to assist the interaction between VgrG and the corresponding effector. Together, our results implied that these widely distributed domain families with similar genetic configurations may be required for the T6SS effector recruitment process.

Promoting the Recruitment, Engagement, and Reinvigoration of Effector T Cells via an Injectable Hydrogel with a Supramolecular Binding Capability for Cancer Immunotherapy.

T cells play a basic and key role in immunotherapy against solid tumors, and efficiently recruiting them into neoplastic foci and sustaining long-term effector function are consistent goals that remain a critical challenge. Here, an injectable alginate-based hydrogel with abundant β-cyclodextrin (ALG-βCD) sites is developed and intratumorally injected to recruit CCR9+ CD8+ T cells (a subset of T cells with robust antitumor activity) via the trapped chemokine CCL25. In the meantime, an intravenously injected adamantane-decorated anti-PD1 antibody (Ad-aPD1) would hitchhike on recruited CCR9+ CD8+ T cells to achieve the improved intratumoral accumulation of Ad-aPD1. Moreover, the Ad-PD1 and Ad-PDL1 antibodies are immobilized in the ALG-βCD hydrogel through supramolecular host-guest interactions of Ad and βCD, which facilitate engagement between CD8+ T cells and tumor cells and reinvigorate CD8+ T cells to avoid exhaustion. Based on this treatment strategy, T cell-mediated anticancer activity is promoted at multiple levels, eventually achieving superior antitumor efficacy in both orthotopic and postsurgical B16-F10 tumor models.

Ubiquitinated H2A.X-Induced RNF168 Condensation Promotes DNA Double-Strand Break Repair and Tumor Radioresistance

Ubiquitination of histone is an essential process involved in DNA damage response (DSB) serving as scaffolds for DNA repair proteins, but how these factors are recruited so quickly and regulated in a spatiotemporal manner remains poorly understood. Liquid-liquid phase separation (LLPS) has recently emerged as a mechanism for membraneless condensation driven by multivalent interactions. In this study, we aimed to investigate the LLPS potential of RNF168, an E3 ligase essential for DSB repair, and the mechanism underlying its-mediated tumor radio-resistance. The intrinsic disordered domain (IDR) of RNF168 was determined by the PONDR website. The LLPS properties were validated by droplet formation in vivo and in vitro. RNF168-mEGFP were expressed in Escherichia coli and purified with GST tag. The synthesized K63-linked ubiquitin chains were added to mimic the interactions between RNF168 and radiation-induced ubiquitinated-histone. Effects of RNF168 LLPS on downstream proteins were verified by immunofluorescence. RNF168-mEGFP recombinant protein formed liquid-like droplets in vivo and co-localized with γ-H2A.X foci after irradiation. The droplet's fluorescence recovered quickly after photobleaching, which could be abolished by 1,6-hexanediol treatment or ATP deprivation. Purified RNF168-mEGFP protein also condensed in vitro, and the size and number of droplets were related to protein concentration, salt concentration, pH, and temperature. Condensation of RNF168 was dependent on the IDR (323-459 amino acid), and more importantly, enhanced by synthesized K63-linked ubiquitin chains. LLPS of RNF168 was required for recruitment of RNF168 to DSB and RNF168-mediated γ-H2A.X ubiquitination. LLPS deficiency of RNF168 resulted in decreased recruitment of 53BP1, BRCA1, and RAP80 proteins, resulting in impaired DSB repair and genomic instability. Notably, higher expression of RNF168 was correlated with a poorer response to neoadjuvant radiochemotherapy in rectal cancer patients. Finally, RNF168 condensate-induced tumor radioresistance was further verified in the xenograft model. RNF168 undergoes LLPS at the DSB site, which is determined by both the IDR domain and the interaction with K63-linked ubiquitin chains. Radiation-induced RNF168 condensation accelerates the accumulation of RNF168 and promotes the recruitment of downstream effectors to DSB, resulting in enhanced DSB repair and tumor radioresistance.