CPEB1 Controls NRF2 Proteostasis and Ferroptosis Susceptibility in Pancreatic Cancer.

Pancreatic cancer is the deadliest malignancy with a poor response to chemotherapy but is potentially indicated for ferroptosis therapy. Here we identified that cytoplasmic polyadenylation element binding protein 1 (CPEB1) regulates NRF2 proteostasis and susceptibility to ferroptosis in pancreatic ductal adenocarcinoma (PDAC). We found that CPEB1 deficiency in cancer cells promotes the translation of p62/SQSTM1 by facilitating mRNA polyadenylation. Consequently, upregulated p62 enhances NRF2 stability by sequestering KEAP1, an E3 ligase for proteasomal degradation of NRF2, leading to the transcriptional activation of anti-ferroptosis genes. In support of the critical role of this signaling cascade in cancer therapy, CPEB1-deficient pancreatic cancer cells display higher resistance to ferroptosis-inducing agents than their CPEB1-normal counterparts in vitro and in vivo. Furthermore, based on the pathological evaluation of tissue specimens from 90 PDAC patients, we established that CPEB1 is an independent prognosticator whose expression level is closely associated with clinical therapeutic outcomes in PDAC. These findings identify the role of CPEB1 as a key ferroptosis regulator and a potential prognosticator in pancreatic cancer.

Tryptophan extends the life of cytochrome P450.

Powerfully oxidizing enzymes need protective mechanisms to prevent self-destruction. The flavocytochrome P450 BM3 from Priestia megaterium (P450BM3) is a self-sufficient monooxygenase that hydroxylates fatty acid substrates using O2 and NADPH as co-substrates. Hydroxylation of long-chain fatty acids (≥C14) is well coupled to O2 and NADPH consumption, but shorter chains (≤C12) are more poorly coupled. Hydroxylation of p-nitrophenoxydodecanoic acid by P450BM3 produces a spectrophotometrically detectable product wherein the coupling of NADPH consumption to product formation is just 10%. Moreover, the rate of NADPH consumption is 1.8 times that of O2 consumption, indicating that an oxidase uncoupling pathway is operative. Measurements of the total number of enzyme turnovers before inactivation (TTN) indicate that higher NADPH concentrations increase TTN. At lower NADPH levels, added ascorbate increases TTN, while a W96H mutation leads to a decrease. The W96 residue is about 7 Å from the P450BM3 heme and serves as a gateway residue in a tryptophan/tyrosine (W/Y) hole transport chain from the heme to a surface tyrosine residue. The data indicate that two oxidase pathways protect the enzyme from damage by intercepting the powerfully oxidizing enzyme intermediate (Compound I) and returning it to its resting state. At high NADPH concentrations, reducing equivalents from the flavoprotein are delivered to Compound I by the usual reductase pathway. When NADPH is not abundant, however, oxidizing equivalents from Compound I can traverse a W/Y chain, arriving at the enzyme surface where they are scavenged by reductants. Ubiquitous tryptophan/tyrosine chains in highly oxidizing enzymes likely perform similar protective functions.

Tryptophan-96 in cytochrome P450 BM3 plays a key role in enzyme survival.

Flavocytochrome P450 from Bacillus megaterium (P450BM3 ) is a natural fusion protein containing reductase and heme domains. In the presence of NADPH and dioxygen the enzyme catalyses the hydroxylation of long-chain fatty acids. Analysis of the P450BM3 structure reveals chains of closely spaced tryptophan and tyrosine residues that might serve as pathways for high-potential oxidizing equivalents to escape from the heme active site when substrate oxidation is not possible. Our investigations of the total number of enzyme turnovers before deactivation have revealed that replacement of selected tryptophan and tyrosine residues with redox inactive groups leads to a twofold reduction in enzyme survival time. Tryptophan-96 is critical for prolonging enzyme activity, suggesting a key protective role for this residue.

Surface cysteines could protect the SARS-CoV-2 main protease from oxidative damage.

The SARS-CoV-2 main protease (Mpro) is responsible for cleaving twelve nonstructural proteins from the viral polyprotein. Mpro, a cysteine protease, is characterized by a large number of noncatalytic cysteine (Cys) residues, none involved in disulfide bonds. In the absence of a tertiary-structure stabilizing role for these residues, a possible alternative is that they are involved in redox processes. We report experimental work in support of a proposal that surface cysteines on Mpro can protect the active-site Cys145 from oxidation by reactive oxygen species (ROS). In investigations of enzyme kinetics, we found that mutating three surface cysteines to serines did not greatly affect activity, which in turn indicates that these cysteines could protect Cys145 from oxidative damage.

Prevalence and spectrum of germline cancer susceptibility gene variants and somatic second hits in colorectal cancer.

Colorectal cancer (CRC) is one of the most heritable cancers, and genetic factors play an important role in the increased CRC risk. However, the well-established CRC-risk genes were limited for explaining the increased risk of CRC individuals. Germline mutations in DNA damage repair (DDR) genes have also been reported to be implicated in CRC heritability. Here, we aimed to determine the prevalence and significance of germline DDR and well-established CRC-risk gene variants in CRCs with paired somatic analyses. Next-generation sequencing (NGS) was performed on tumor tissues and paired white blood cells collected from 2160 Chinese patients with CRC using well-designed 381- or 733-cancer gene panel. Germline/somatic variations were identified and assessed for pathogenicity and likely pathogenicity. Of 2160 CRCs, 136 pathogenic germline mutations in 133 patients (133/2160, 6.1%) were identified in 21 genes, including 19 out of 32 examined DDR genes. Compared with non-carriers, individuals with germline variants were prone to a higher level of microsatellite instability (MSI) and tumor mutational burden (TMB), and an earlier age of onset. Somatic sequencing identified second hits in 24/133 (18%) patients with germline variants. Among the mismatch repair (MMR) genes with germline mutations, the second hit significantly increased MSI and TMB, particularly apparent in MSH6. All MMR germline variation carriers further with a second hit were all MSI-H and had an extraordinarily high level of TMB. Collectively, approximately 6.1% of CRC patients carried pathogenic germline variants, and additional somatic second hit increases the genomic instability in CRC, whereas the more clinical significance warrants further study.

The Significance of MET Expression and Strategies of Targeting MET Treatment in Advanced Gastric Cancer.

BACKGROUND: Accurate assessment of predictive biomarker expression is critical in patient selection in clinical trials or clinical practice. However, changes in biomarker expression may occur after treatment. The aim of the present study was to evaluate the effects of chemotherapy on MET expression in gastric cancer (GC). METHODS: MET expression was examined immunohistochemically before and after treatment in 122 patients with unresectable or recurrent GC, and was evaluated according to H-score or the scoring criteria used in the MetMAb trial. MET gene amplification was assessed by chromogenic in situ hybridization (CISH). The antitumor effect of MET targeted therapy was investigated in human gastric cancer cells in vitro and in vivo, and the underlying molecular mechanisms were analyzed by western blot. RESULTS: MET expression was associated with Lauren classification as well as tumor differentiation by either scoring system. MET amplification was not associated with clinical characteristics. Of the 71 patients who had paired pre- and post-treatment tumor tissues, 28 patients (39%) were initially positive for MET expression, and 43 (61%) were negative. Twenty-five patients (35%) showed significant changes in MET expression after treatment (P=0.007). Additionally, there was a concomitant overexpression of MET and HER2 in a subset of GC patients. MET inhibitor volitinib could significantly inhibit cell proliferation and xenograft growth in vitro and in vivo in MKN45 cells with MET and phosphorylated MET (pMET) high expressions via suppressing downstream PI3K/Akt and MAPK signaling pathways. Furthermore, combination therapy targeting both MET and HER2 demonstrated a synergistic antitumor activity. CONCLUSIONS: MET expression is altered post chemotherapy and MET status should be evaluated in real-time. Both MET and pMET expressions might need to be considered for patients suitable for volitinib treatment.

PD-1 restrains IL-17A production from γδ T cells to modulate acute radiation-induced lung injury.

BACKGROUND: Combining radiotherapy (RT) with programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) inhibitors has been shown to enhance anti-tumor effects in the treatment of non-small cell lung carcinoma (NSCLC). Pulmonary toxicity is a major adverse effect of thoracic RT in NSCLC patients, whether it is administered alone or in combination with PD-1/PD-L1 inhibitors. This study aimed to evaluate the potential pulmonary toxicity of RT combined with concurrent PD-1 inhibitor and to clarify the underlying mechanisms. METHODS: Radiation-induced lung injury (RILI) was induced in C57BL/6 mice by given 24 Gy in three fractions on consecutive days, with or without concurrent injection of anti-PD-1 antibody. On days 3, 7, 14, and 28 after the first exposure to irradiation, lung tissue and peripheral blood samples were collected from the mice. Histological injury was analyzed, and inflammatory cell infiltration and interleukin (IL)-17A production in the lung tissues were quantified. RESULTS: Mice that received irradiation with concurrent administration of anti-PD-1 antibody had the highest histological score for RILI. In the murine lung tissues, the levels of PD-1 and IL-17A expression were increased in γδ T cells but not in the other CD3+ T cells after irradiation. Concurrent blockade of PD-1 enhanced IL-17A production from γδ T cells in the lung tissues after irradiation. In the mice with acute RILI, concurrent administration of anti-PD-1 antibody exaggerated pulmonary inflammation, with significantly increased levels of neutrophilic infiltration and IL-17A detected in both the lung and blood. CONCLUSIONS: PD-1 could restrain IL-17A production from γδ T cells to modulate acute RILI. The concurrent administration of anti-PD-1 antibody aggravates the severity of acute RILI. More attention should be paid to pulmonary toxicity in patients undergoing thoracic RT with concurrent anti-PD-1 immunotherapy.

Two Tryptophans Are Better Than One in Accelerating Electron Flow through a Protein.

We have constructed and structurally characterized a Pseudomonas aeruginosa azurin mutant Re126WWCuI , where two adjacent tryptophan residues (W124 and W122, indole separation 3.6-4.1 Å) are inserted between the CuI center and a Re photosensitizer coordinated to the imidazole of H126 (ReI(H126)(CO)3(4,7-dimethyl-1,10-phenanthroline)+). CuI oxidation by the photoexcited Re label (*Re) 22.9 Å away proceeds with a ∼70 ns time constant, similar to that of a single-tryptophan mutant (∼40 ns) with a 19.4 Å Re-Cu distance. Time-resolved spectroscopy (luminescence, visible and IR absorption) revealed two rapid reversible electron transfer steps, W124 → *Re (400-475 ps, K 1 ≅ 3.5-4) and W122 → W124•+ (7-9 ns, K 2 ≅ 0.55-0.75), followed by a rate-determining (70-90 ns) CuI oxidation by W122•+ ca. 11 Å away. The photocycle is completed by 120 µs recombination. No photochemical CuI oxidation was observed in Re126FWCuI , whereas in Re126WFCuI , the photocycle is restricted to the ReH126W124 unit and CuI remains isolated. QM/MM/MD simulations of Re126WWCuI indicate that indole solvation changes through the hopping process and W124 → *Re electron transfer is accompanied by water fluctuations that tighten W124 solvation. Our finding that multistep tunneling (hopping) confers a ∼9000-fold advantage over single-step tunneling in the double-tryptophan protein supports the proposal that hole-hopping through tryptophan/tyrosine chains protects enzymes from oxidative damage.

A novel PADRE-Kv1.3 vaccine effectively induces therapeutic antibodies and ameliorates experimental autoimmune encephalomyelitis in rats.

Previous studies have confirmed that selective blockade of Kv1.3 channels could modulate the activities of pathogenic T cells and microglia/macrophages, which play key roles in experimental autoimmune encephalomyelitis (EAE). In this study, we designed an anti-Kv1.3 vaccine (PADRE-Kv1.3) to explore its protective role in EAE rat models. When the vaccine was applied in EAE rats, clinical scores and several staining techniques were used to evaluate the severity of the disease. T cell subtypes and related cytokines, as well as microglia/macrophage activation were assayed through flow cytometry, qRT-PCR or immunofluorescence staining, respectively. We herein showed that rats and mice developed high titers of anti-Kv1.3 antibodies and appeared no abnormal manifestations after the PADRE-Kv1.3 vaccine treatment. In EAE models, the vaccine treatment effectively alleviated the clinical severity and lessened pathological damages in the central nervous system (CNS). In addition, we found the vaccine significantly decreased the number of pathogenic T cells (Th17 and IFN-γ-producing T cells) and the production of related pro-inflammatory cytokines (IL-17A, IFN-γ and IL-1ß), but increased the number of protective T subsets (CD4+IL-10+ T cells and Treg cells) in the spleen or CNS. Moreover, the infiltration of microglia/macrophages significantly reduced and these cells shifted toward anti-inflammatory M2 subtype in the CNS after the vaccine treatment. Thus, we demonstrated that the PADRE-Kv1.3 vaccine could induce therapeutic anti-Kv1.3 antibodies and ameliorate EAE in rats effectively and safely, which provides a new field of vision for the protection and therapy of multiple sclerosis.

Topiramate modulates post-infarction inflammation primarily by targeting monocytes or macrophages.

AIMS: Monocytes/macrophages response plays a key role in post-infarction inflammation that contributes greatly to post-infarction ventricular remodelling and cardiac rupture. Therapeutic targeting of the GABAA receptor, which is enriched in monocytes/macrophages but not expressed in the myocardium, may be possible after myocardial infarction (MI). METHODS AND RESULTS: After MI was induced by ligation of the coronary artery, C57BL/6 mice were intraperitoneally administered with one specific agonist or antagonist of the GABAA receptor (topiramate or bicuculline), in the setting of presence or depletion of monocytes/macrophages. Our data showed that within the first 2 weeks after MI, when monocytes/macrophages dominated, in contrast with bicuculline, topiramate treatment significantly reduced Ly-6Chigh monocyte numbers by regulating splenic monocytopoiesis and promoted foetal derived macrophages preservation and conversion of M1 to M2 or Ly-6Chigh to Ly-6Clow macrophage phenotype in the infarcted heart, though GABAAergic drugs failed to affect M1/M2 or Ly-6Chigh/Ly-6Clow macrophage polarization directly. Accordingly, pro-inflammatory activities mediated by M1 or Ly-6Chigh macrophages were decreased and reparative processes mediated by M2 or Ly-6Clow macrophages were augmented. As a result, post-infarction ventricular remodelling was attenuated, as reflected by reduced infarct size and increased collagen density within infarcts. Echocardiographic indices, mortality and rupture rates were reduced. After depletion of monocytes/macrophages by clodronate liposomes, GABAAergic drugs exhibited no effect on cardiac dysfunction and surrogate clinical outcomes. CONCLUSION: Control of the GABAA receptor activity in monocytes/macrophages can potently modulate post-infarction inflammation. Topiramate emerges as a promising drug, which may be feasible to translate for MI therapy in the future.

Electron Tunneling through Pseudomonas aeruginosa Azurins on SAM Gold Electrodes.

Robust voltammetric responses were obtained for wild-type and Y72F/H83Q/Q107H/Y108F azurins adsorbed on CH(3)(CH(2))(n)SH:HO(CH(2))(m)SH (n=m=4,6,8,11; n=13,15 m=11) self-assembled monolayer (SAM) gold electrodes in acidic solution (pH 4.6) at high ionic strengths. Electron-transfer (ET) rates do not vary substantially with ionic strength, suggesting that the SAM methyl headgroup binds to azurin by hydrophobic interactions. The voltammetric responses for both proteins at higher pH values (>4.6 to 11) also were strong. A binding model in which the SAM hydroxyl headgroup interacts with the Asn47 carboxamide accounts for the relatively strong coupling to the copper center that can be inferred from the ET rates. Of particular interest is the finding that rate constants for electron tunneling through n = 8, 13 SAMs are higher at pH 11 than those at pH 4.6, possibly owing to enhanced coupling of the SAM to Asn 47 caused by deprotonation of nearby surface residues.