Persistent Endoplasmic Reticulum Stress Stimulated by Peptide Assemblies for Sensitizing Cancer Chemotherapy.

Pharmacological targeting of endoplasmic reticulum (ER) stress represents one of important methods for disease therapy, which, however, is significantly suppressed by the ER homeostatic processe. Herein, a proof-of-concept strategy is reported for persistent stimulation of ER stress via preventing ER stress adaptation by utilizing multifunctional peptide assemblies. The strategy is established via creation of peptide assemblies with ER-targeting and chaperone glucose-regulated protein 78 (GRP78)-inhibiting functions. The peptides assemblies form well-defined nanofibers that are retrieved by ER organelles in human cervical cancer cell. The underlying mechanism studies unravel that the ER-accumulated peptide assemblies simultaneously stimulate ER stress and inhibit GRP78 refolding activity and thereby promoting endogenous protein aggregation. Combining the internalized peptide assemblies with the induced protein aggregates leads to the persistent stimulation of ER stress. The persistent ER stress induced by the peptide assemblies bestows their application in sensitizing cancer chemotherapy. Both in vitro and in vivo results confirm the enhanced cytotoxicity of drug toyocamycin against HeLa cells by peptide assemblies, thus efficiently inhibiting in vivo tumor growth. The strategy reported here discloses the fundamental keys for efficient promotion of ER stress, thus providing the guidance for development of ER-targeting-assisted cancer chemotherapy in the future.

The Paf1 complex is required for RNA polymerase II removal in response to DNA damage.

Rpb1, the largest subunit of RNA polymerase II (RNAPII), is rapidly polyubiquitinated and degraded in response to DNA damage; this process is considered to be a "mechanism of last resort'' employed by cells. The underlying mechanism of this process remains elusive. Here, we uncovered a previously uncharacterized multistep pathway in which the polymerase-associated factor 1 (Paf1) complex (PAF1C, composed of the subunits Ctr9, Paf1, Leo1, Cdc73, and Rtf1) is involved in regulating the RNAPII pool by stimulating Elongin-Cullin E3 ligase complex-mediated Rpb1 polyubiquitination and subsequent degradation by the proteasome following DNA damage. Mechanistically, Spt5 is dephosphorylated following DNA damage, thereby weakening the interaction between the Rtf1 subunit and Spt5, which might be a key step in initiating Rpb1 degradation. Next, Rad26 is loaded onto stalled RNAPII to replace the Spt4/Spt5 complex in an RNAPII-dependent manner and, in turn, recruits more PAF1C to DNA lesions via the binding of Rad26 to the Leo1 subunit. Importantly, the PAF1C, assembled in a Ctr9-mediated manner, coordinates with Rad26 to localize the Elongin-Cullin complex on stalled RNAPII, thereby inducing RNAPII removal, in which the heterodimer Paf1/Leo1 and the subunit Cdc73 play important roles. Together, our results clearly revealed a new role of the intact PAF1C in regulating the RNAPII pool in response to DNA damage.

Phase transition and remodeling complex assembly are important for SS18-SSX oncogenic activity in synovial sarcomas.

Oncoprotein SS18-SSX is a hallmark of synovial sarcomas. However, as a part of the SS18-SSX fusion protein, SS18's function remains unclear. Here, we depict the structures of both human SS18/BRG1 and yeast SNF11/SNF2 subcomplexes. Both subcomplexes assemble into heterodimers that share a similar conformation, suggesting that SNF11 might be a homologue of SS18 in chromatin remodeling complexes. Importantly, our study shows that the self-association of the intrinsically disordered region, QPGY domain, leads to liquid-liquid phase separation (LLPS) of SS18 or SS18-SSX and the subsequent recruitment of BRG1 into phase-separated condensates. Moreover, our results show that the tyrosine residues in the QPGY domain play a decisive role in the LLPS of SS18 or SS18-SSX. Perturbations of either SS18-SSX LLPS or SS18-SSX's binding to BRG1 impair NIH3T3 cell transformation by SS18-SSX. Our data demonstrate that both LLPS and assembling into chromatin remodelers contribute to the oncogenic activity of SS18-SSX in synovial sarcomas.

In Situ Self-Sorting Peptide Assemblies in Living Cells for Simultaneous Organelle Targeting.

Self-sorting is a common phenomenon in eukaryotic cells and represents one of the versatile strategies for the formation of advanced functional materials; however, developing artificial self-sorting assemblies within living cells remains challenging. Here, we report on the GSH-responsive in situ self-sorting peptide assemblies within cancer cells for simultaneous organelle targeting to promote combinatorial organelle dysfunction and thereby cell death. The self-sorting system was created via the design of two peptides E3C16E6 and EVMSeO derived from lipid-inspired peptide interdigitating amphiphiles and peptide bola-amphiphiles, respectively. The distinct organization patterns of the two peptides facilitate their GSH-induced self-sorting into isolated nanofibrils as a result of cleavage of disulfide-connected hydrophilic domains or reduction of selenoxide groups. The GSH-responsive in situ self-sorting in the peptide assemblies within HeLa cells was directly characterized by super-resolution structured illumination microscopy. Incorporation of the thiol and ER-targeting groups into the self-sorted assemblies endows their simultaneous targeting of endoplasmic reticulum and Golgi apparatus, thus leading to combinatorial organelle dysfunction and cell death. Our results demonstrate the establishment of the in situ self-sorting peptide assemblies within living cells, thus providing a unique platform for drug targeting delivery and an alternative strategy for modulating biological processes in the future.

Self-Amplifying Assembly of Peptides in Macrophages for Enhanced Inflammatory Treatment.

Enzyme-regulated in situ self-assembly of peptides represents one versatile strategy in the creation of theranostic agents, which, however, is limited by the strong dependence on enzyme overexpression. Herein, we reported the self-amplifying assembly of peptides precisely in macrophages associated with enzyme expression for improving the anti-inflammatory efficacy of conventional drugs. The self-amplifying assembling system was created via coassembling an enzyme-responsive peptide with its derivative functionalized with a protein ligand. Reduction of the peptides by the enzyme NAD(P)H quinone dehydrogenase 1 (NQO1) led to the formation of nanofibers with high affinity to the protein, thereby facilitating NQO1 expression. The improved NQO1 level conversely promoted the assembly of the peptides into nanofibers, thus establishing an amplifying relationship between the peptide assembly and the NQO1 expression in macrophages. Utilization of the amplifying assembling system as vehicles for drug dexamethasone allowed for its passive targeting delivery to acute injured lungs. Both in vitro and in vivo studies confirmed the capability of the self-amplifying assembling system to enhance the anti-inflammatory efficacy of dexamethasone via simultaneous alleviation of the reactive oxygen species side effect and downregulation of proinflammatory cytokines. Our findings demonstrate the manipulation of the assembly of peptides in living cells with a regular enzyme level via a self-amplification process, thus providing a unique strategy for the creation of supramolecular theranostic agents in living cells.

Crystal Structure of the Core Module of the Yeast Paf1 Complex.

The highly conserved multifunctional polymerase-associated factor 1 (Paf1) complex (PAF1C), which consists of five core subunits: Ctr9, Paf1, Leo1, Cdc73, and Rtf1, acts as a diverse hub that regulates all stages of RNA polymerase II-mediated transcription and various other cellular functions. However, the underlying mechanisms remain unclear. Here, we report the crystal structure of the core module derived from a quaternary Ctr9/Paf1/Cdc73/Rtf1 complex of S. cerevisiae PAF1C, which reveals interfaces between the tetratricopeptide repeat module in Ctr9 and Cdc73 or Rtf1, and find that the Ctr9/Paf1 subcomplex is the key scaffold for PAF1C assembly. Our study demonstrates that Cdc73 binds Ctr9/Paf1 subcomplex with a very similar conformation within thermophilic fungi or human PAF1C, and that the binding of Cdc73 to PAF1C is important for yeast growth. Importantly, our structure reveals for the first time that the extreme C-terminus of Rtf1 adopts an "L"-shaped structure, which interacts with Ctr9 specifically. In addition, disruption of the binding of either Cdc73 or Rtf1 to PAF1C greatly affects the normal level of histone H2B K123 monoubiquitination in vivo. Collectively, our results provide a structural insight into the architecture of the quaternary Ctr9/Paf1/Cdc73/Rtf1 complex and PAF1C functional regulation.

Biochemical insights into Paf1 complex-induced stimulation of Rad6/Bre1-mediated H2B monoubiquitination.

The highly conserved multifunctional polymerase-associated factor 1 (Paf1) complex (PAF1C), composed of five core subunits Paf1, Leo1, Ctr9, Cdc73, and Rtf1, participates in all stages of transcription and is required for the Rad6/Bre1-mediated monoubiquitination of histone H2B (H2Bub). However, the molecular mechanisms underlying the contributions of the PAF1C subunits to H2Bub are not fully understood. Here, we report that Ctr9, acting as a hub, interacts with the carboxyl-terminal acidic tail of Rad6, which is required for PAF1C-induced stimulation of H2Bub. Importantly, we found that the Ras-like domain of Cdc73 has the potential to accelerate ubiquitin discharge from Rad6 and thus facilitates H2Bub, a process that might be conserved from yeast to humans. Moreover, we found that Rtf1 HMD stimulates H2Bub, probably through accelerating ubiquitin discharge from Rad6 alone or in cooperation with Cdc73 and Bre1, and that the Paf1/Leo1 heterodimer in PAF1C specifically recognizes the histone H3 tail of nucleosomal substrates, stimulating H2Bub. Collectively, our biochemical results indicate that intact PAF1C is required to efficiently stimulate Rad6/Bre1-mediated H2Bub.

Supramolecular Antagonists Promote Mitochondrial Dysfunction.

Mitochondrion-targeting therapy exhibits great potential in cancer therapy but significantly suffers from limited therapeutic efficiency. Here we report on mitochondrion-targeting supramolecular antagonist-inducing tumor cell death via simultaneously promoting cellular apoptosis and preventing survival. The supramolecular antagonist was created via coassembly of a mitochondrion-targeting pentapeptide with its two derivatives functionalized with a BH3 domain or the drug camptothecin (CPT). While drug CPT released from the antagonist induced cellular apoptosis via decreasing the mitochondrial membrane potential, the BH3 domain prevented cellular survival through facilitating the association between the supramolecular antagonists and antiapoptotic proteins, thereby initiating mitochondrial permeabilization. Both in vitro and in vivo studies confirmed the combinatorial therapeutic effect arising from the BH3 domain and CPT drug within the supramolecular antagonist on cell death and thereby inhibiting tumor growth. Our findings demonstrate an efficient combinatorial mechanism for mitochondrial dysfunction, thus potentially serving as novel organelle-targeting medicines.

Qki activates Srebp2-mediated cholesterol biosynthesis for maintenance of eye lens transparency.

Defective cholesterol biosynthesis in eye lens cells is often associated with cataracts; however, how genes involved in cholesterol biosynthesis are regulated in lens cells remains unclear. Here, we show that Quaking (Qki) is required for the transcriptional activation of genes involved in cholesterol biosynthesis in the eye lens. At the transcriptome level, lens-specific Qki-deficient mice present downregulation of genes associated with the cholesterol biosynthesis pathway, resulting in a significant reduction of total cholesterol level in the eye lens. Mice with Qki depletion in lens epithelium display progressive accumulation of protein aggregates, eventually leading to cataracts. Notably, these defects are attenuated by topical sterol administration. Mechanistically, we demonstrate that Qki enhances cholesterol biosynthesis by recruiting Srebp2 and Pol II in the promoter regions of cholesterol biosynthesis genes. Supporting its function as a transcription co-activator, we show that Qki directly interacts with single-stranded DNA. In conclusion, we propose that Qki-Srebp2-mediated cholesterol biosynthesis is essential for maintaining the cholesterol level that protects lens from cataract development.

The crystal structure of the FERM and C-terminal domain complex of Drosophila Merlin.

NF2/Merlin is an upstream regulator of hippo pathway, and it has two states: an auto-inhibited "closed" state and an active "open" form. Previous studies showed that Drosophila Merlin adopts a more closed conformation. However, the molecular mechanism of conformational regulation remains poorly understood. Here, we first confirmed the strong interaction between FERM and the C-terminal domain (CTD) of Merlin, and then determined the crystal structure of the FERM/CTD complex, which reveals the structural basis of Merlin adopting a more closed conformation compared to its human cognate NF2. Interestingly, we found that the conserved lipid-binding site of Merlin might be masked by a linker. Confocal analyses confirmed that all putative lipid-binding site are very important for the membranal location of Merlin. More, we found that the phosphomimic Thr616Asp mutation weakens the interaction between FERM and CTD of Merlin. Collectively, the crystal structure of the FERM/CTD complex not only provides a mechanistic explanation of functionally dormant conformation of Merlin may also serve as a foundation for revealing the mechanism of conformational regulation of Merlin.

Snf5 and Swi3 subcomplex formation is required for SWI/SNF complex function in yeast.

The SWI/SNF chromatin remodeling complex, which alters nucleosome positions by either evicting histones or sliding nucleosomes on DNA, is highly conserved from yeast to humans, and 20% of all human cancers have mutations in various subunits of the SWI/SNF complex. Here, we reported the crystal structure of the yeast Snf5-Swi3 subcomplex at a resolution of 2.65 Å. Our results showed that the Snf5-Swi3 subcomplex assembles into a heterotrimer with one Snf5 molecule bound to two distinct Swi3 molecules. In addition, we demonstrated that Snf5-Swi3 subcomplex formation is required for SWI/SNF function in yeast. These findings shed light on the important role of the Snf5-Swi3 subcomplex in the assembly and functional integrity of the SWI/SNF complex.

Treatment with Humanized Selective CD19CAR-T Cells Shows Efficacy in Highly Treated B-ALL Patients Who Have Relapsed after Receiving Murine-Based CD19CAR-T Therapies.

PURPOSE: CD19 chimeric antigen receptor (CAR)-T therapy has shown impactful results in treatment of B-cell malignancies. However, immune recognition of the murine scFv may render subsequent infusion(s) ineffective. Also, nonselective expansion of both CAR-transduced and nontransduced T cells during the production stage affects the yield and purity of final products. Here, we aim to develop a humanized selective (hs) CD19 CAR to solve the above problems.Experimental Design: A CD19 hsCAR was designed, which incorporated a short selective domain between the humanized heavy chain and light chain. The CAR was examined for its property, and then trialed in 5 highly treated B-ALL patients. RESULTS: hsCAR possessed around 6-fold higher affinity to CD19 versus murine CAR (mCAR). Incubation with selective domain-specific mAbs (SmAb) selectively expanded CAR-transduced T cells, and led to a higher proportion of central memory T cells in the final products. SmAb-stimulated CD19 hsCAR-T cells exhibited superior antitumor cytotoxic functions in vitro and in vivo. Autologous (n = 2) and allogeneic donor (n = 3, with hematopoietic stem cell transplantation) hsCAR-T cells were infused into 5 patients who had relapsed after receiving mCAR-T treatments. Two patients received mCAR-T treatments twice previously but the second treatments were ineffective. In contrast, subsequent hsCAR-T treatments proved effective in all 5 patients and achieved complete molecular remission in four, including one with extramedullary disease with central nervous system involvement. CONCLUSIONS: hsCD19 CAR-T treatment shows efficacy in highly treated B-ALL patients who have relapsed after receiving CD19 mCAR-T therapies.

Dual functions of STAT3 in LPS-induced angiogenesis of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a long-term consequence of chronic inflammatory liver injury. Hepatic injury is associated with a defective intestinal barrier and increased hepatic exposure to bacterial products including lipopolysaccharide (LPS), which promotes hepatocarcinogenesis. Despite its clinical significance, the molecular mediator linking chronic inflammation with HCC development remains to be clarified. In this study, we explored the significant dual functions of active signal transducer and activator of transcription 3 (STAT3) in LPS-induced angiogenesis of HCC. The in vitro effects of active STAT3 in tumor cells and endothelial cells were assessed using angiogenesis assay, ELISA, confocal assay, flow cytometry and western blot. The in vivo role of active STAT3 was assessed in xenografts model in nude mice. Here we report a novel mechanism by which LPS/STAT3 signaling promotes the angiogenesis of HCC both in vitro and in vivo. STAT3 activated by LPS increases the production of vascular endothelial growth factor (VEGF) by tumor cells, which not only promotes the proliferation of HCC cells but also stimulates the migration and tubulogenesis of endothelial cells through STAT3 activation and hence promotes angiogenesis in HCC. Our findings not only provide a potential mechanism by which bacterial infection enhances HCC oncogenesis through promoting the angiogenesis in liver, but also suggest that targeting STAT3 might be an effective therapeutic strategy in HCC treatment considering the dual roles of STAT3 in angiogenesis.

Supramolecular Nanofibers of Drug-Peptide Amphiphile and Affibody Suppress HER2+ Tumor Growth.

Antibody-based medicines and nanomedicines are very promising for cancer therapy due to the high specificity and efficacy of antibodies. However, antibody-drug conjugates and antibody-modified nanomaterials frequently suffer from low drug loading and loss of functions due to the covalent modification of the antibody. A novel and versatile strategy to prepare supramolecular nanomaterials by the coassembly of an affibody (antiHER2) and drug-peptide amphiphiles is reported here. During the enzyme-instructed self-assembly process, the drug-peptide amphiphile can coassemble with the affibody, resulting in supramolecular nanofibers in hydrogels. The drug loading in the supramolecular nanofibers is high (>30 wt%), and the stability of antiHER2 is significantly improved in the nanofibers at 37 °C (>15 d in vitro). The supramolecular nanofibers exhibit high affinity for HER2+ cancer cells and can be efficiently taken up by these cells. In a mouse tumor model, the supramolecular nanofibers abolish HER2+ NCI-N87 tumor growth due to the good accumulation and retention of nanofibers in tumor. This study provides a novel strategy to prepare nanomedicines with high drug loading and high specificity.

The STAT3 inhibitor S3I-201 suppresses fibrogenesis and angiogenesis in liver fibrosis.

Liver fibrosis is a common pathological response to chronic hepatic injury. STAT3 is actively involved in the fibrogenesis and angiogenesis seen in liver fibrosis. S3I-201 (NSC 74859) is a chemical inhibitor of STAT3 activity, which blocks the dimerization of STAT3, STAT3-DNA binding and transcription activity. This study evaluated the effects of S3I-201 against liver fibrosis. S3I-201 inhibited the proliferation, migration, and actin filament formation in primary human hepatic stellate cells (HSCs), as well as the expression of α-SMA, collagen I and TIMP1 in both primary HSC and in a CCl4-induced fibrosis mouse model. S3I-201 induced both apoptosis and cell cycle arrest in the HSC cell line (LX-2). S3I-201 inhibited the expression of fibrogenesis factors TGFß1 and TGFßRII, as well as the downstream phosphorylation of Smad2, Smad3, Akt and ERK induced by TGFß1. In addition to fibrogenesis, both in vitro and in vivo assays showed that S3I-201 inhibited angiogenesis through expression suppression of VEGF and VEGFR2. Moreover, S3I-201 also had a synergistic effect with sorafenib, an FDA approved liver cancer drug, in the proliferation, apoptosis, angiogenesis and fibrogenesis of HSC. S3I-201 suppressed liver fibrosis through multiple mechanisms, and combined with sorafenib, S3I-201 could be a potentially effective antifibrotic agent.

Paf1 and Ctr9 subcomplex formation is essential for Paf1 complex assembly and functional regulation.

The evolutionarily conserved multifunctional polymerase-associated factor 1 (Paf1) complex (Paf1C), which is composed of at least five subunits (Paf1, Leo1, Ctr9, Cdc73, and Rtf1), plays vital roles in gene regulation and has connections to development and human diseases. Here, we report two structures of each of the human and yeast Ctr9/Paf1 subcomplexes, which assemble into heterodimers with very similar conformations, revealing an interface between the tetratricopeptide repeat module in Ctr9 and Paf1. The structure of the Ctr9/Paf1 subcomplex may provide mechanistic explanations for disease-associated mutations in human PAF1 and CTR9. Our study reveals that the formation of the Ctr9/Paf1 heterodimer is required for the assembly of yeast Paf1C, and is essential for yeast viability. In addition, disruption of the interaction between Paf1 and Ctr9 greatly affects the level of histone H3 methylation in vivo. Collectively, our results shed light on Paf1C assembly and functional regulation.

Basal condensation of Numb and Pon complex via phase transition during Drosophila neuroblast asymmetric division.

Uneven distribution and local concentration of protein complexes on distinct membrane cortices is a fundamental property in numerous biological processes, including Drosophila neuroblast (NB) asymmetric cell divisions and cell polarity in general. In NBs, the cell fate determinant Numb forms a basal crescent together with Pon and is segregated into the basal daughter cell to initiate its differentiation. Here we discover that Numb PTB domain, using two distinct binding surfaces, recognizes repeating motifs within Pon in a previously unrecognized mode. The multivalent Numb-Pon interaction leads to high binding specificity and liquid-liquid phase separation of the complex. Perturbations of the Numb/Pon complex phase transition impair the basal localization of Numb and its subsequent suppression of Notch signaling during NB asymmetric divisions. Such phase-transition-mediated protein condensations on distinct membrane cortices may be a general mechanism for various cell polarity regulatory complexes.

Self-Assembly Molecular Chaperone to Concurrently Inhibit the Production and Aggregation of Amyloid ß Peptide Associated with Alzheimer's Disease.

Amyloid ß peptide (Aß) plays a crucial role in the pathogenesis of Alzheimer's disease (AD). Currently, decreasing Aß production and preventing Aß aggregation are thought to be important strategies in anti-AD therapy. However, inhibiting Aß production or aggregation in isolation is not sufficient to reverse the neurodegenerative process of AD patients in clinical testing. Here, a self-assembly molecular chaperone (SAMC) consisting of γ-secretase inhibitor DAPT and mixed-shell polymeric micelles is devised, serving as a bifunctional suppressor of AD. This two-in-one combinational system can simultaneously inhibit Aß production and aggregation, which would contribute to enhancing the therapeutic effect by decreasing Aß levels. Decorating a neuron-specific RVG29 peptide onto the surface, the DAPT-incorporated SAMC can specifically target neuronal cells and, thus, will relieve the strong side effect of DAPT on normal cells. Therefore, this combination strategy holds great potential to open up an avenue for AD treatment.

The conformation change and tumor suppressor role of Merlin are both independent of Serine 518 phosphorylation.

Merlin functions as a tumor suppressor and suppresses malignant activity of cancer cells through multiple mechanisms. However, whether Serine 518 phosphorylation regulates the conformation of Merlin as well as the open-closed conformational changes affect Merlin's tumor inhibitory activity remain controversial. In this study, we used different mutants to mimic related conformational states of Merlin and investigated its physiological functions. Our results showed that the phosphorylation at Serine 518 has no influence on Merlin's conformation, subcellular localization, or cell proliferation inhibitory activity. As a fully closed conformational state, the A585W mutant loses the ability to recruit Lats2 to the cell membrane, but it does not affect its subcellular distribution or cell proliferation inhibitory activity. As a fully open conformational state, mimicking the conformation of Merlin isoform II, the ΔEL mutant has the same physiological function as the wild type Merlin isoform I. Collectively, we provide for the first time in vivo evidence that the function of Merlin, as a tumor suppressor is independent of its conformational change.