Stress response silencing by an E3 ligase mutated in neurodegeneration.

Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis, yet their prolonged activation induces apoptosis and disrupts organismal health1-3. How stress responses are turned off at the right time and place remains poorly understood. Here we report a ubiquitin-dependent mechanism that silences the cellular response to mitochondrial protein import stress. Crucial to this process is the silencing factor of the integrated stress response (SIFI), a large E3 ligase complex mutated in ataxia and in early-onset dementia that degrades both unimported mitochondrial precursors and stress response components. By recognizing bifunctional substrate motifs that equally encode protein localization and stability, the SIFI complex turns off a general stress response after a specific stress event has been resolved. Pharmacological stress response silencing sustains cell survival even if stress resolution failed, which underscores the importance of signal termination and provides a roadmap for treating neurodegenerative diseases caused by mitochondrial import defects.

Protein engineering of a nanoCLAMP antibody mimetic scaffold as a platform for producing bioprocess-compatible affinity capture ligands.

Protein A affinity chromatography is widely used for the large-scale purification of antibodies because of its high yield, selectivity, and compatibility with NaOH sanitation. A general platform to produce robust affinity capture ligands for proteins beyond antibodies would improve bioprocessing efficiency. We previously developed nanoCLAMPs (nano Clostridial Antibody Mimetic Proteins), a class of antibody mimetic proteins useful as lab-scale affinity capture reagents. This work describes a protein engineering campaign to develop a more robust nanoCLAMP scaffold compatible with harsh bioprocessing conditions. The campaign generated an improved scaffold with dramatically improved resistance to heat, proteases, and NaOH. To isolate additional nanoCLAMPs based on this scaffold, we constructed a randomized library of 1 × 1010 clones and isolated binders to several targets. We then performed an in-depth characterization of nanoCLAMPs recognizing yeast SUMO, a fusion partner used for the purification of recombinant proteins. These second-generation nanoCLAMPs typically had a Kd of <80 nM, a Tm of >70 °C, and a t1/2 in 0.1 mg/ml trypsin of >20 h. Affinity chromatography resins bearing these next-generation nanoCLAMPs enabled single-step purifications of SUMO fusions. Bound target proteins could be eluted at neutral or acidic pH. These affinity resins maintained binding capacity and selectivity over 20 purification cycles, each including 10 min of cleaning-in-place with 0.1 M NaOH, and remained functional after exposure to 100% DMF and autoclaving. The improved nanoCLAMP scaffold will enable the development of robust, high-performance affinity chromatography resins against a wide range of protein targets.

A peptide interfering with the dimerization of oncogenic KITENIN protein and its stability suppresses colorectal tumour progression.

The stability of a protein, as well as its function and versatility, can be enhanced through oligomerization. KITENIN (KAI1 C-terminal interacting tetraspanin) is known to promote the malignant progression of colorectal cancer (CRC). How KITENIN maintains its structural integrity and stability are largely unknown, however. Here we investigated the mechanisms regulating the stability of KITENIN with the aim of developing therapeutics blocking its oncogenic functions. We found that KITENIN formed a homo-oligomeric complex and that the intracellular C-terminal domain (KITENIN-CTD) was needed for this oligomerization. Expression of the KITENIN-CTD alone interfered with the formation of the KITENIN homodimer, and the amino acid sequence from 463 to 471 within the KITENIN-CTD was the most effective. This sequence coupled with a cell-penetrating peptide was named a KITENIN dimerization-interfering peptide (KDIP). We next studied the mechanisms by which KDIP affected the stability of KITENIN. The KITENIN-interacting protein myosin-X (Myo10), which has oncogenic activity in several cancers, functioned as an effector to stabilize the KITENIN homodimer in the cis formation. Treatment with KDIP resulted in the disintegration of the homodimer via downregulation of Myo10, which led to increased binding of RACK1 to the exposed RACK1-interacting motif (463-471 aa), and subsequent autophagy-dependent degradation of KITENIN and reduced CRC cell invasion. Intravenous injection of KDIP significantly reduced the tumour burden in a syngeneic mouse tumour model and colorectal liver metastasis in an intrasplenic hepatic metastasis model. Collectively, our present results provide a new cancer therapeutic peptide for blocking colorectal liver metastasis, which acts by inducing the downregulation of Myo10 and specifically targeting the stability of the oncogenic KITENIN protein.

Cryo-EM structures of alphavirus conformational intermediates in low pH-triggered prefusion states.

Alphaviruses can cause severe human arthritis and encephalitis. During virus infection, structural changes of viral glycoproteins in the acidified endosome trigger virus-host membrane fusion for delivery of the capsid core and RNA genome into the cytosol to initiate virus translation and replication. However, mechanisms by which E1 and E2 glycoproteins rearrange in this process remain unknown. Here, we investigate prefusion cryoelectron microscopy (cryo-EM) structures of eastern equine encephalitis virus (EEEV) under acidic conditions. With models fitted into the low-pH cryo-EM maps, we suggest that E2 dissociates from E1, accompanied by a rotation (∼60°) of the E2-B domain (E2-B) to expose E1 fusion loops. Cryo-EM reconstructions of EEEV bound to a protective antibody at acidic and neutral pH suggest that stabilization of E2-B prevents dissociation of E2 from E1. These findings reveal conformational changes of the glycoprotein spikes in the acidified host endosome. Stabilization of E2-B may provide a strategy for antiviral agent development.

Suppression of toxicity of the mutant huntingtin protein by its interacting compound, desonide.

Identifying inhibitors of pathogenic proteins is the major strategy of targeted drug discoveries. This strategy meets challenges in targeting neurodegenerative disorders such as Huntington's disease (HD), which is mainly caused by the mutant huntingtin protein (mHTT), an "undruggable" pathogenic protein with unknown functions. We hypothesized that some of the chemical binders of mHTT may change its conformation and/or stability to suppress its downstream toxicity, functioning similarly to an "inhibitor" under a broader definition. We identified 21 potential mHTT selective binders through a small-molecule microarray­based screening. We further tested these compounds using secondary phenotypic screens for their effects on mHTT-induced toxicity and revealed four potential mHTT-binding compounds that may rescue HD-relevant phenotypes. Among them, a Food and Drug Administration­approved drug, desonide, was capable of suppressing mHTT toxicity in HD cellular and animal models by destabilizing mHTT through enhancing its polyubiquitination at the K6 site. Our study reveals the therapeutic potential of desonide for HD treatment and provides the proof of principle for a drug discovery pipeline: target-binder screens followed by phenotypic validation and mechanistic studies.

Hydrogen Sulfide-Linked Persulfidation Maintains Protein Stability of ABSCISIC ACID-INSENSITIVE 4 and Delays Seed Germination.

Hydrogen sulfide (H2S) is an endogenous gaseous molecule that plays an important role in the plant life cycle. The multiple transcription factor ABSCISIC ACID INSENSITIVE 4 (ABI4) was precisely regulated to participate in the abscisic acid (ABA) mediated signaling cascade. However, the molecular mechanisms of how H2S regulates ABI4 protein level to control seed germination and seedling growth have remained elusive. In this study, we demonstrated that ABI4 controls the expression of L-CYSTEINE DESULFHYDRASE1 (DES1), a critical endogenous H2S-producing enzyme, and both ABI4 and DES1-produced H2S have inhibitory effects on seed germination. Furthermore, the ABI4 level decreased during seed germination while H2S triggered the enhancement of the persulfidation level of ABI4 and alleviated its degradation rate, which in turn inhibited seed germination and seedling establishment. Conversely, the mutation of ABI4 at Cys250 decreased ABI4 protein stability and facilitated seed germination. Moreover, ABI4 degradation is also regulated via the 26S proteasome pathway. Taken together, these findings suggest a molecular link between DES1 and ABI4 through the post-translational modifications of persulfidation during early seedling development.

Compound screening identified gossypetin and isoquercitrin as novel inhibitors for amyloid fibril formations of Vλ6 proteins associated with AL amyloidosis.

AL amyloidosis is a life-threatening disease characterized by the deposition of amyloidogenic immunoglobulin light chain secreted from clonal plasma cells. Here we established an in-vitro screening system of amyloid inhibition of a variable domain in λ6 light chain mutant (Vλ6), Wil, and screened a food-additive compound library to identify compounds inhibiting the fibril formation. We found gossypetin and isoquercitrin as novel inhibitors. NMR analysis showed that both compounds directly interacted with natively-folded Wil, and proteolysis experiments demonstrated that these compounds conferred proteolytic resistance, suggesting that the compounds enhance the kinetic stability of Wil. Since gossypetin and isoquercitrin specifically interacted with the protein at micromolar concentrations, these compounds could be used as lead to further develop inhibitors against AL amyloidosis.

LncRNAs LCETRL3 and LCETRL4 at chromosome 4q12 diminish EGFR-TKIs efficiency in NSCLC through stabilizing TDP43 and EIF2S1.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are effective targeted therapy drugs for advanced non-small cell lung cancer (NSCLC) patients carrying sensitized EGFR mutations. The rapid development of EGFR-TKIs resistance represents a major clinical challenge for managing NSCLC. The chromosome 4q12 is the first genome-wide association study (GWAS)-reported locus associated with progression-free survival (PFS) of NSCLC patients treated with EGFR-TKIs. However, the biological significance of the noncoding transcripts at 4q12 in NSCLC remains elusive. In the present study, we identified two 4q12 long noncoding RNAs (lncRNAs) LCETRL3 and LCETRL4 which could significantly dimmish EGFR-TKIs efficiency. In line with their oncogenic role, evidently higher LCETRL3 and LCETRL4 levels were observed in NSCLC tissues as compared with normal specimens. Importantly, lncRNA LCETRL3 can interact with oncoprotein TDP43 and inhibit ubiquitination and degradation of TDP43. Similarly, lncRNA LCETRL4 can bind and stabilize oncoprotein EIF2S1 through reducing ubiquitin-proteasome degradation of EIF2S1. In particular, elevated levels of LCETRL3 or LCETRL4 in NSCLC cells resulted in stabilization of TDP43 or EIF2S1, increased levels of NOTCH1 or phosphorylated PDK1, activated AKT signaling and, thus, EGFR-TKIs resistance. Taken together, our data revealed a novel model that integrates two lncRNAs transcribed from the 4q12 locus into the regulation of EGFR-TKIs resistance in NSCLC. These findings shed new light on the importance of functionally annotating lncRNAs in the GWAS loci and provided insights to declare novel druggable targets, i.e., lncRNAs, which may unlock the therapeutic potential of EGFR-TKIs resistant NSCLC in the clinic.

5-C-Branched Deoxynojirimycin: Strategy for Designing a 1-Deoxynojirimycin-Based Pharmacological Chaperone with a Nanomolar Affinity for Pompe Disease.

In recent years, the function of pharmacological chaperones as a "thermodynamic stabilizer" has been attracting attention in combination therapy. The coadministration of a pharmacological chaperone and recombinant human acid α-glucosidase (rhGAA) leads to improved stability and maturation by binding to the folded state of the rhGAA and thereby promotes enzyme delivery. This study provides the first example of a strategy to design a high-affinity ligand toward lysosomal acid α-glucosidase (GAA) focusing on alkyl branches on 1-deoxynojirimycin (DNJ); 5-C-heptyl-DNJ produced a nanomolar affinity for GAA with a Ki value of 0.0047 µM, which is 13-fold more potent than DNJ. The protein thermal shift assay revealed that 10 µM 5-C-heptyl-DNJ increased the midpoint of the protein denaturation temperature (Tm) to 73.6 °C from 58.6 °C in the absence of the ligand, significantly improving the thermal stability of rhGAA. Furthermore, 5-C-heptyl-DNJ dose dependency increased intracellular GAA activities in Pompe patient's fibroblasts with the M519V mutation. The introduction of C5 alkyl branches on DNJ provides a new molecular strategy for pharmacological chaperone therapy for Pompe disease, which may lead to the development of higher-affinity and practically useful chaperones.

Selective stabilization of the intermediate inactivated Na+ channel by the new-generation anticonvulsant rufinamide.

Na+ channels undergo multiple inactivated states with different kinetics, which set the refractory period of neuronal discharges, but isolating the intermediate inactivated state has been challenging. Most classical Na+channel-inhibiting anticonvulsants bind to the fast inactivated state to reduce Na+currents and cellular excitability. These anticonvulsants have the slow binding kinetics and thus necessitate long depolarization for drug action, a "use-dependent" effect sparing most normal activities. Rufinamide is a new anticonvulsant targeting Na+channels, and has a therapeutic effect on Lennox-Gastaut syndrome (LGS) which is refractory to classicalNa+channel inhibitors. The efficacy on LGS, whose epileptiform discharges largely involve short depolarization or bursts, is primarily due to the very fast binding kinetics of rufinamide. Could the very fast kinetics of rufinamide lead to indiscriminate inhibition of neuronal activities ? Onhippocampal neurons from male and female mice, wefound that rufinamide most effectively shifts the Na+channel inactivation curve if the inactivating pulse is 1 s, rather than 0.1 or 18 s, in duration. Rufinamide also shows a maximal slowing effect on the recovery kinetics from the inactivation driven by modest depolarization (e.g. -60 mV) of intermediate length (e.g. 50-300 ms). Consistently, rufinamide selectively inhibits the burst discharges at 50-300 ms on a plateau of ∼-60 mV. This is mechanistically ascribable to selective binding of rufinamide to an intermediate inactivated state withan apparent dissociation constantof ∼40 µM. Being the first molecule embodying the evasive transitional gating state, rufinamide could have a unique anti-seizure profile with a novel form of use-dependent action.

Phosphorylation-Induced Ubiquitination and Degradation of PXR through CDK2-TRIM21 Axis.

Pregnane X receptor (PXR) is a member of the nuclear receptor superfamily that is activated by a variety of endogenous metabolites or xenobiotics. Its downstream target genes are involved in metabolism, inflammation and processes closely related to cancer. However, the stability regulation of PXR protein resulting from post-translational modification is still largely undefined. In the present study, primary mouse hepatocytes, hepatoma HepG2 cells and HEK 293T cells were used to investigate gene expression and protein interactions. The role of kinases was evaluated by RNA interference and overexpression constructs with or without PXR phosphorylation site mutations. The activity of CYP3A4 and P-gp was determined by enzymatic and substrate accumulation assays. It was found that E3 ubiquitin ligase TRIM21 mediates the ubiquitination and degradation of PXR and plays an important role in regulating the activity of PXR. On this basis, PXR phosphorylation-associated kinases were evaluated regarding regulation of the stability of PXR. We found cyclin dependent kinase 2 (CDK2) exclusively phosphorylates PXR at Ser350, promotes its disassociation with Hsp90/DNAJC7, and leads to subsequent TRIM21-mediated PXR ubiquitination and degradation. As well-known CDK inhibitors, dinaciclib and kenpaullone stabilize PXR and result in elevated expression and activity of PXR-targeted DMETs, including carboxylesterases, CYP3A4 and P-gp. The suppressed degradation of PXR by CDK2 inhibitors denotes dinaciclib-induced promotion of PXR-targeted genes. The findings of CDK2-mediated PXR degradation indicate a wide range of potential drug-drug interactions during clinical cancer therapy using CDK inhibitors and imply an alternative direction for the development of novel PXR antagonists.

A review on prevention of glycation of proteins: Potential therapeutic substances to mitigate the severity of diabetes complications.

Non-enzymatic reaction involving carbonyl of reducing sugars and amino groups in proteins produces advanced glycation end products (AGEs). AGE accumulation in vivo is a crucial factor in the progression of metabolic and pathophysiological mechanisms like obesity, diabetes, coronary artery disease, neurological disorders, and chronic renal failure. The body's own defense mechanism, synthetic inhibitors, and natural inhibitors can all help to prevent the glycation of proteins. Synthetic inhibitors have the potential to suppress the glycation of proteins through a variety of pathways. They could avoid Amadori product development by tampering with the addition of sugars to the proteins. Besides which, the free radical scavenging and blocking crosslink formation could be another mechanism behind their anti-glycation properties. In comparison with synthetic substances, naturally occurring plant products have been found to be comparatively non-toxic, cheap, and usable in an ingestible form. This review gives a brief introduction of the Maillard reaction; formation, characterization and pathology related to AGEs, potential therapeutic approaches against glycation, natural and synthetic inhibitors of glycation and their probable mechanism of action. The scientific community could get benefit from the combined knowledge about important molecules, which will further guide to the design and development of new pharmaceutical compounds.

Sorafenib attenuates liver fibrosis by triggering hepatic stellate cell ferroptosis via HIF-1α/SLC7A11 pathway.

OBJECTIVES: Evidences demonstrate that sorafenib alleviates liver fibrosis via inhibiting HSC activation and ECM accumulation. The underlying mechanism remains unclear. Ferroptosis, a novel programmed cell death, regulates diverse physiological/pathological processes. In this study, we aim to investigate the functional role of HSC ferroptosis in the anti-fibrotic effect of sorafenib. MATERIALS AND METHODS: The effects of sorafenib on HSC ferroptosis and ECM expression were assessed in mouse model of liver fibrosis induced by CCl4 . In vitro, Fer-1 and DFO were used to block ferroptosis and then explored the anti-fibrotic effect of sorafenib by detecting α-SMA, COL1α1 and fibronectin proteins. Finally, HIF-1α siRNA, plasmid and stabilizers were applied to assess related signalling pathway. RESULTS: Sorafenib attenuated liver injury and ECM accumulation in CCl4 -induced fibrotic livers, accompanied by reduction of SLC7A11 and GPX4 proteins. In sorafenib-treated HSC-T6 cells, ferroptotic events (depletion of SLC7A11, GPX4 and GSH; accumulation iron, ROS and MDA) were discovered. Intriguingly, these ferroptotic events were not appeared in hepatocytes or macrophages. Sorafenib-elicited HSC ferroptosis and ECM reduction were abrogated by Fer-1 and DFO. Additionally, both HIF-1α and SLC7A11 proteins were reduced in sorafenib-treated HSC-T6 cells. SLC7A11 was positively regulated by HIF-1α, inactivation of HIF-1α/SLC7A11 pathway was required for sorafenib-induced HSC ferroptosis, and elevation of HIF-1α could inhibit ferroptosis, ultimately limited the anti-fibrotic effect. CONCLUSIONS: Sorafenib triggers HSC ferroptosis via HIF-1α/SLC7A11 signalling, which in turn attenuates liver injury and fibrosis.

Thermal Unfolding of the Human Serotonin Transporter: Differential Effect by Stabilizing and Destabilizing Mutations and Cholesterol on Thermodynamic and Kinetic Stability.

Folding-deficient mutants of solute carrier 6 (SLC6) family members have been linked to human diseases. The serotonin transporter [(SERT)/SLC6A4] is an important drug target in the treatment of depression, anxiety, and obsessive-compulsive disorders and-with structural information in several conformational states-one of the best understood transporters. Here, we surmised that thermal unfolding offered a glimpse on the folding energy landscape of SLC6 transporters. We carried out molecular dynamic (MD) simulations to understand the mechanistic basis for enhanced and reduced stability, respectively, of the thermostabilized variant SERT-Y110A/I291A/T439S, which had previously been used for crystallization of human SERT in the outward-facing state, and of the folding-deficient SERT-P601A/G602A. We also examined the hydrophobic mismatch caused by the absence of cholesterol to explore the contribution of cholesterol to protein stability. When compared with wild type SERT, the thermodynamic and kinetic stability of SERT-Y110A/I291A/T439S was enhanced. In the other instances, changes in these two components were not correlated: the mutations in SERT-P601A/G602A led to a drop in thermodynamic but an increase in kinetic stability. The divergence was even more pronounced after cholesterol depletion, which reduced thermodynamic stability but increased the kinetic stability of wild type SERT to a level comparable to that of SERT-Y110A/I291A/T439S. We conclude that the low cholesterol content of the endoplasmic reticulum facilitates progression of the folding trajectory by reducing the energy difference between folding intermediates and the native state. SIGNIFICANCE STATEMENT: Point mutations in solute carrier 6 (SLC6) family members cause folding diseases. The serotonin transporter [(SERT)/SLC6A4] is a target for antidepressants and the best understood SLC6. This study produced molecular dynamics simulations and examined thermal unfolding of wild type and mutant SERT variants to understand their folding energy landscape. In the folding-deficient SERT-P012A/G602A, changes in kinetic and thermodynamic stability were not correlated. Similarly, cholesterol depletion lowered thermodynamic but enhanced kinetic stability. These observations allow for rationalizing the action of pharmacochaperones.

Inhibition of cytoplasmic EZH2 induces antitumor activity through stabilization of the DLC1 tumor suppressor protein.

mRNA expression of the DLC1 tumor suppressor gene is downregulated in many lung cancers and their derived cell lines, with DLC1 protein levels being low or absent. Although the role of increased EZH2 methyltransferase in cancer is usually attributed to its histone methylation, we unexpectedly observed that post-translational destabilization of DLC1 protein is common and attributable to its methylation by cytoplasmic EZH2, leading to CUL-4A ubiquitin-dependent proteasomal degradation of DLC1. Furthermore, siRNA knockdown of KRAS in several lines increases DLC1 protein, associated with a drastic reduction in cytoplasmic EZH2. Pharmacologic inhibition of EZH2, CUL-4A, or the proteasome can increase the steady-state level of DLC1 protein, whose tumor suppressor activity is further increased by AKT and/or SRC kinase inhibitors, which reverse the direct phosphorylation of DLC1 by these kinases. These rational drug combinations induce potent tumor growth inhibition, with markers of apoptosis and senescence, that is highly dependent on DLC1 protein.

MINDY1 promotes breast cancer cell proliferation by stabilizing estrogen receptor α.

Breast cancer is the most commonly diagnosed malignant tumor among females. Estrogen receptor α (ERα) is initially expressed in 70% of breast cancers and is a well-known target of endocrine therapy for ERα-positive breast cancer. In the present study, we identified MINDY1, a member belongs to the motif interacting with Ubcontaining novel DUB family (MINDY), as a potential deubiquitylase of ERα in breast cancer. There was a positive correlation between ERα and MINDY1 protein levels in human breast cancer tissues. We found that high expression of MINDY1 was associated with poor prognosis. MINDY1 interacted with ERα, thereby mediating the deubiquitination of ERα and increased its stability in a deubiquitylation activity-dependent manner. MINDY1 depletion significantly decreased the ERα protein level and ERα signaling activity in breast cancer cells. Specifically, MINDY1 associated with the N-terminal of ERα via its catalytic domain, thus inhibiting K48-specific poly-ubiquitination process on ERα protein. In addition, MINDY1 depletion led to growth inhibition and cell cycle arrest of ERα-positive breast cancer cells. Finally, overexpression of ERα could rescue the MINDY1 depletion-induced growth inhibition both in vitro and in vivo, suggesting that MINDY1 promotes breast carcinogenesis through increasing ERα stability. Overall, our study proposed a novel post-translational mechanism of ERα in supporting breast cancer progression. Targeting the MINDY1 may prove to be a promising strategy for patients with ERα-positive breast cancer.

Water-soluble pillar[5]arene sulfo-derivatives self-assemble into biocompatible nanosystems to stabilize therapeutic proteins.

Pillar[5]arenes containing sulfonate fragments have been shown to form supramolecular complexes with therapeutic proteins to facilitate targeted transport with an increased duration of action and enhanced bioavailability. Regioselective synthesis was used to obtain a water-soluble pillar[5]arene containing the fluorescent label FITC and nine sulfoethoxy fragments. The pillar[5]arene formed complexes with the therapeutic proteins binase, bleomycin, and lysozyme in a 1:2 ratio as demonstrated by UV-vis and fluorescence spectroscopy. The formation of stable spherical nanosized macrocycle/binase complexes with an average particle size of 200 nm was established by dynamic light scattering and transmission electron microscopy. Flow cytometry demonstrated the ability of macrocycle/binase complexes to penetrate into tumor cells where they exhibited significant cytotoxicity towards A549 cells at 10-5-10-6 M while maintaining the enzymatic activity of binase.

Luteolin attenuates cancer cell stemness in PTX-resistant oesophageal cancer cells through mediating SOX2 protein stability.

Cancer drug resistance is a formidable obstacle that enhances cancer stem-like cell properties, tumour metastasis and relapse. Luteolin (Lut) is a natural flavonoid with strong antitumor effects. However, the underlying mechanism(s) by which Lut protects against paclitaxel-resistant (PTX-resistant) cancer cell remains unknown. Herein, we found that Lut significantly attenuated the stem-like properties of PTX-resistant cancer cells by downregulating the expression of SOX2 protein. Additionally, further study showed that Lut could inhibit the PI3K/AKT pathway to decrease the phosphorylation level of AKT(S473) and UBR5 expression, which is an ubiquitin E3 ligase that promotes SOX2 degradation. In addition, Lut also inhibited PTX-resistant cancer cell migration and invasion by blocking epithelial-mesenchymal transition (EMT). Importantly, Lut inhibited the tumorigenic ability of oesophageal PTX-resistant cancer cells and showed no obvious toxicity in vivo. Thus, Lut has potential as a promising agent for drug-resistant oesophageal cancer therapy.

SPOP mutation induces DNA methylation via stabilizing GLP/G9a.

Mutations in SPOP E3 ligase gene are reportedly associated with genome-wide DNA hypermethylation in prostate cancer (PCa) although the underlying mechanisms remain elusive. Here, we demonstrate that SPOP binds and promotes polyubiquitination and degradation of histone methyltransferase and DNMT interactor GLP. SPOP mutation induces stabilization of GLP and its partner protein G9a and aberrant upregulation of global DNA hypermethylation in cultured PCa cells and primary PCa specimens. Genome-wide DNA methylome analysis shows that a subset of tumor suppressor genes (TSGs) including FOXO3, GATA5, and NDRG1, are hypermethylated and downregulated in SPOP-mutated PCa cells. DNA methylation inhibitor 5-azacytidine effectively reverses expression of the TSGs examined, inhibits SPOP-mutated PCa cell growth in vitro and in mice, and enhances docetaxel anti-cancer efficacy. Our findings reveal the GLP/G9a-DNMT module as a mediator of DNA hypermethylation in SPOP-mutated PCa. They suggest that SPOP mutation could be a biomarker for effective treatment of PCa with DNA methylation inhibitor alone or in combination with taxane chemotherapeutics.

Ubiquitination Flow Repressors: Enhancing Wound Healing of Infectious Diabetic Ulcers through Stabilization of Polyubiquitinated Hypoxia-Inducible Factor-1α by Theranostic Nitric Oxide Nanogenerators.

Current treatments for diabetic ulcers (DUs) remain unsatisfactory due to the risk of bacterial infection and impaired angiogenesis during the healing process. The increased degradation of polyubiquitinated hypoxia-inducible factor-1α (HIF-1α) compromises wound healing efficacy. Therefore, the maintenance of HIF-1α protein stability might help treat DU. Nitric oxide (NO) is an intrinsic biological messenger that functions as a ubiquitination flow repressor and antibacterial agent; however, its clinical application in DU treatment is hindered by the difficulty in controlling NO release. Here, an intelligent near-infrared (NIR)-triggered NO nanogenerator (SNP@MOF-UCNP@ssPDA-Cy7/IR786s, abbreviated as SNP@UCM) is presented. SNP@UCM represses ubiquitination-mediated proteasomal degradation of HIF-1α by inhibiting its interaction with E3 ubiquitin ligases under NIR irradiation. Increased HIF-1α expression in endothelial cells by SNP@UCM enhances angiogenesis in wound sites, promoting vascular endothelial growth factor (VEGF) secretion and cell proliferation and migration. SNP@UCM also enables early detection of wound infections and ROS-mediated killing of bacteria. The potential clinical utility of SNP@UCM is further demonstrated in infected full-thickness DU model under NIR irradiation. SNP@UCM is the first reported HIF-1α-stabilizing advanced nanomaterial, and further materials engineering might offer a facile, mechanism-based method for clinical DU management.