Hydrogen sulfide -dependent cell death, which triggered by increased H2O2 availability in Arabidopsis thaliana catalase-deficient mutants cat2 displaying around 20% wild-type catalase activity. H2S generation and its producing enzyme L-cysteine desulfhydrase (LCD/DES) were found to transient increase in response to intracellular oxidative stress. Although introducing the mutation of des1, an important LCD, into the cat2 background produced little effect, H2S fumigation not only rescued the cell death phenotype of cat2 plant, but also attenuated SA accumulation and oxidation of the glutathione pool. Unexpectedly, the activities of major components of ascorbate–glutathione pathway were less affected by the presence of H2S treatment, but decreased glycolate oxidase (GOX) in combination with accumulation of glycolate implied H2S treatment impacts the cellular redox homeostasis by repressing the GOX-catalyzed reaction likely via altering the major GOX transcript levels.Here, we assessed the effects of H2S and peroxisomal H2O2 production that has implications for the understanding of the multifaceted roles of H2S in the regulation of oxidative stress responses.Our findings reveal a link between HThe online version contains supplementary material available at 10.1186/s12870-022-03490-3. These environmental stresses influence plant yield and quality by affecting various cellular and whole-plant processes particularly through either reducing photosynthesis or the availability of water for basic cellular functions . Moreoveproteins .2S has been gradually accepted as a key messenger involved in a vast number of physiologically important processes in photosynthetic organisms, such as stress resistance, autophagy, stomatal movement [2S in different subcellular sites including cytosol, chloroplasts, mitochondria, and nuclei [2S increases through enzymatic reactions mainly including the activation of sulfur assimilation pathway or the cysteine-degrading enzymes such as D-cysteine desulfhydrase (DCD) and L-cysteine desulfhydrase (LCD/DES) [2S biogenesis [2S is capable of reacting with different ROS, thereby potentially severing as an antioxidant involving stress regulation [2S [2S could activate the transcriptional and/or translational levels of antioxidant enzymes and key biosynthetic enzymes of their substrates, to enhance ROS-processing capacity under many stress conditions. Indeed, numerous studies found that the major components including ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathione reductase (GR) in ascorbate–glutathione pathway could be key regulatory nodes by H2S [2S into several plant species under certain stress conditions was also observed to activate the expressions of L-galactose dehydrogenase, glutamate cysteine ligase and glutathione synthetase for ascorbate and glutathione synthesis, respectively [2S is proposed to be involved in generating cysteine persulfides on proteins, a process termed persulfidation which may lead to the alteration of protein functions. For example, H2S can drive persulfidation of certain antioxidant enzymes such as APX and the other peroxidase to potentiate their scavenging activities of ROS [2S-controlled redox balance could operate via up-regulation of antioxidant defense enzymes at the multiple levels, its effects on ROS biosynthesis in higher plants is much less understood except for the case studies that DES1-derived H2S participates in guard cell ABA signaling through modulating NADPH oxidase activity [Other than its toxicity, Hmovement –7. The pd nuclei –13. WhenLCD/DES) , Other fogenesis . Furthergulation . Howevertion [2S , might bs by H2S –20. Treaectively . As wells of ROS , 24. Altactivity , 26.2S and oxidative stress was only based on the analyses of effects of H2S on abiotic stress-triggered indirectly ROS accumulation and redox imbalance. In view of its importance in the context of oxidative stress, the aim of this work was to study the role of H2S in the regulation of oxidative responses by utilizing the catalase2 (cat2) knockout mutants to directly induce stress responses such as salicylic acid (SA)-dependent cell death [2O2 is produced in peroxisomes from O2 by the reaction of glycolate oxidase (GOX)-catalyzed glycolate oxidation during photorespiration.In photosynthetic organisms, oxidative stress triggered by enhanced accumulation of ROS is common to environmental stress responses. Till now, studies on investigating the interactions between Hll death , 28, whe2O2 availability due to catalase deficiency in cat2 knockout mutants led to the activation of intracellular oxidative stress responses mainly including the induction of SA-dependent cell death [2S production in response to increased H2O2 availability in cat2 knockouts. H2S generation showed a marked transient increase in cat2 mutants  transcripts, indicators of SA-dependent pathways, in cat2 des1 and cat2 mutants were indistinguishable after twenty days of growth , GLUTATHIONE S-TRANSFERASE TAU 24 (GSTU24), URIDINE DIPHOSPHATE GLYCOSYLTRANSFERASE 74E2 (UGT74E2), ALTERNATIVE OXIDASE 1D (AOX1D), ZINC-FINGER OF ARABIDOPSIS THALIANAA 10 (ZAT10) and ZINC-FINGER OF ARABIDOPSIS THALIANA 12 (ZAT12) were selected [cat2 mutants [cat2 des1 leaves were comparable to those from single cat2 mutants , dehydroascorbate reductase (DHAR) and glutathione reductase (GR) in cat2 des1 were comparable to those observed in cat2 . As shown in Fig. 2O2-elcited lesion formation and SA accumulation in the cat2 background -catalyzed glycolate oxidation during photorespiration is metabolized by CAT. Decreased GOX activity thus might limit H2O2 accumulation. To examine whether the more reduced redox environment in the presence of H2S is due to the alteration of GOX activity, we compared the activities of GOX and the contents of its substrate glycolate between Col-0 and cat2 in the absence or presence of H2S treatments. In agreement with our previous results and others [cat2 leaves, but glycolate remained at Col-0 levels. H2S-fumigated cat2 mutants displayed significantly lower GOX than did the control treatment of cat2 mutants, accompanying by elevated levels of glycolate  with decreased levels of persulfidation relative to wild-type plants [2S signaling. Alternatively, this could raise another possibility that other and/or unidentified factors/pathways promoting H2S generation may compensate for the loss of function of DES1 if they have redundant roles in the regulation of oxidative stress. Indeed, several candidate genes, encoding the cysteine desulfhydrase, have been implicated in enhancing resistance to many stresses such as heavy metal stress, salt, drought and cold stress, pathogen attack, and preventing ROS accumulation [Our previous work demonstrated that increased Htathione , both oftathione . Glutathone [2O2 , 31, 34.stresses . Hence, immunity , 41, 42.ignaling , failed e plants , 43, indmulation –48. For mulation .2S led to the alteration in oxidative stress. Unlike other studies that reported that H2S modulated oxidative stress through elevating antioxidant capacities especially for the components in the ascorbate–glutathione pathway [2S in regulating stress responses. The photorespiratory GOX-generating H2O2 accounts for about 70% of the total pool and is therefore the most crucial H2O2 source in photosynthesizing C3 leaves [2S-treated cat2, jointly with decreased GOX activities and increased reduction status of glutathione  and homozygous knockout lines including cat2 (SALK076998) [atg5 (SAIL_129B07) [des1 (SALK_103855) [cat2 des1 atg5 and a double mutant cat2 des1 were generated for this study. Above mutant lines used in this study were verified by PCR. All specific PCR primers used for genotyping are listed in Supplemental Table S−2 s−1 at leaf level, temperatures of 22 ºC day/20 ºC night, 65% humidity. Our study complied with relevant institutional, national, and international guidelines and legislation, and no specific permits were required to collect the plant samples. Samples were rapidly frozen in liquid nitrogen and stored at -80ºC until analysis. Unless otherwise stated, data are means SE of at least three independent samples from different plants.K076998) , atg5 S_129B07)  and des1_103855) used in t29B07  an2S fumigation was carried out as described in Wei et al. [2O) were used as a donor of H2S. In a sealed glass desiccator (volume 3 L), 16-day-old seedling lines were treated with gaseous H2S for 4 h per day, which was released from the 0.5 mM NaHS aqueous solution (200 mL). The aqueous solution of NaHS at 0 mM was set as the control. The NaHS solution and its control treatment were renewed each day and treated leaves were collected at designated time intervals for analyses.The procedure of Hi et al. . Solutio2S quantification was performed as described by Singh et al. [2. The homogenate was centrifuged at 15,000 g for 15 min at 4ºC and 0.1 mL of the supernatant was used for the H2S quantification, in an assay mixture containing 1.88 mL extraction buffer and 0.02 mL of 20 mM 5,5’-dithiobis(2-nitrobenzoic acid). The reaction mixture was incubated at room temperature for 2 min and the absorbance was read at 412 nm. H2S was quantified based on a standard curve prepared with NaHS.Hh et al. . 0.5 g p3 dissolved in 1.2 N HCl and 0.1 mL 20 mM N,N-dimethyl-p-phenylenediaminedihy drochloride dissolved in 7.2 N HCl. The formation of methylene blue was determined at 670 nm. LCD enzymatic activity was calculated using a standard curve of known concentrations of NaHS.The activity of LCD was measured as described previously . Freshlycat2 in the absence or presence of H2S, cat2 des1 or cat2 des1 atg5, was quantified using IQmaterials software. Total SA was extracted as described in Langlois-Meurinne et al. [Percentage lesion area in e et al. . SA was e et al. . IdentifTotal RNA was extracted with TRIzol reagent (Takara) following the manufacturer’s instructions. To avoid potential genomic DNA contamination, RNA was treated with RNase-free DNase I (Takara). RNA quality and concentration were determined by gel electrophoresis and estimated using a nanodrop spectrophotometer at 260 nm respectively. Reverse transcription and first-strand cDNA synthesis were performed using the All-in-One cDNA Synthesis SuperMix (Bimake). qPCR was performed according to Zhang et al. . In all 2PO4 (pH 5.6), 40 μL extract and 55 μL H2O. The absorbance at 265 nm was read before and 5 min after adding 0.2 U of AO. Total ascorbate was measured after incubation of 100 μL neutralized extract with 140 μL 0.12 M NaH2PO4 (pH 7.5), 10 μL 25 mM DTT for 30 min at room temperature. Both of above assays were done in triplicate for each extract. According to a standard extinction coefficient of 14 mM−1 cm−1, absorbance changes in A265 were converted to quantities of ascorbate.Oxidized and reduced forms of ascorbate and glutathione were quantified using the plate-reader method described by Queval and Noctor . Leaf sa2PO4, 10 mM EDTA (pH 7.5), 10 μL 10 mM NADPH, 10 μL 12 mM DTNB, 10 μL of neutralized extract and 60 μL water. After shaking, the reaction was started by addition of 10 μL GR. GSSG measurement was based on the same principle except that extract aliquots were incubated with VPD for 30 min at room temperature and were centrifuged twice for 15 min at 4ºC before assay. The increase in absorbance at 412 nm was monitored for 5 min and all assays were done in triplicate for each extract. Rates were converted to quantities of glutathione using standard curves generated by assay of known concentration in the same plate.The method for glutathione measurement relies on GR-dependent reduction of DNTB by GSH monitored at 412 nm and is used to measure either total glutathione (GSH + GSSG) or GSSG. Specific assay of GSSG was done by pre-treatment of extract aliquots with 2 μL of 2-vinylpyridine (VPD), which efficiently complexes GSH. To measure total glutathione, the mixture contained 100 μL of 0.2 M NaHThe glycolate oxidase activity was performed by monitoring the formation of colored O-dianisidine radical cation in the presence of sodium glycolate at 440 nm according to protocol detailed by Waszczak et al. . Glycola2O2 monitored at 240 nm, DHAR as GSH-dependent formation of ascorbate from DHA at 265 nm, APX as H2O2-dependent ascorbate oxidation at 290 nm, and GR as GSSG-dependent NADPH oxidation at 340 nm. Bradford’s method was adopted for measuring the protein concentration, and bovine serum albumin was used as standard [Spectrophotometric assay to measure enzyme activities in the glutathione-ascorbate pathway was performed as described previously . Brieflystandard .t-test. Calculations were performed on a minimum of three independent datasets, assuming two samples equal variance and a two-tailed distribution. For comparing multiple treatments, different letters indicate significant differences using multiple pairwise t-test comparisons at P < 0.05.The significance of differences was determined by Student’s Additional file 1: Figure S1. Effects of des1 mutation on major antioxidative enzyme in cat2 mutants. (a) CAT. (b), APX. (c), DHAR. (d), GR. Samples of each line were taken after 20 days of growth. Values are means ± SE of three biological replicates. Different letters indicate significant differences by multiple pairwise t-test comparisons at P < 0.05.Additional file 2: Table S1. List of primers and restriction enzymes used in this study.


To assess the safety, pharmacokinetics, pharmacodynamics, and preliminary efficacy of cetrelimab (JNJ-63723283), a monoclonal antibody programmed cell death protein-1 (PD-1) inhibitor, in patients with advanced/refractory solid tumors in the phase 1/2 LUC1001 study.In phase 1, patients with advanced solid tumors received intravenous cetrelimab 80, 240, 460, or 800 mg every 2 weeks (Q2W) or 480 mg Q4W. In phase 2, patients with melanoma, non-small-cell lung cancer (NSCLC), and microsatellite instability–high (MSI-H)/DNA mismatch repair-deficient colorectal cancer (CRC) received cetrelimab 240 mg Q2W. Response was assessed Q8W until Week 24 and Q12W thereafter.Cmax) ranged from 24.7 to 227.0 µg/mL; median time to Cmax ranged from 2.0 to 3.2 h. Pharmacodynamic effect was maintained throughout the dosing period across doses. In phase 2, 146 patients received cetrelimab 240 mg Q2W. Grade ≥ 3 adverse events (AEs) occurred in 53.9% of patients. Immune-related AEs (any grade) occurred in 35.3% of patients (grade ≥ 3 in 6.9%). Overall response rate was 18.6% across tumor types, 34.3% in NSCLC, 52.6% in programmed death ligand 1–high (≥ 50% by immunohistochemistry) NSCLC, 28.0% in melanoma, and 23.8% in centrally confirmed MSI-H CRC.In phase 1, 58 patients received cetrelimab. Two dose-limiting toxicities were reported and two recommended phase 2 doses (RP2D) were defined (240 mg Q2W or 480 mg Q4W). After a first dose, mean maximum serum concentrations (The RP2D for cetrelimab was established. Pharmacokinetic/pharmacodynamic characteristics, safety profile, and clinical activity of cetrelimab in immune-sensitive advanced cancers were consistent with known PD-1 inhibitors.NCT02908906 at ClinicalTrials.gov, September 21, 2016; EudraCT 2016–002,017-22 at clinicaltrialsregister.eu, Jan 11, 2017.The online version contains supplementary material available at 10.1007/s00280-022-04414-6. In the tumor microenvironment, PD-1 activity can suppress tumor immunosurveillance and development of adaptive immune responses  non-small-cell lung cancer (NSCLC), melanoma, or colorectal cancer (CRC), as well as renal cell carcinoma, bladder cancer, small-cell lung cancer (SCLC), and gastric/esophageal cancer. The protocol was amended to limit phase 2 recruitment to PD-L1-high (≥ 50% PD-L1+ on tumor cells) NSCLC, melanoma, and MSI-high (MSI-H)/dMMR CRC determined by local or central testing. MSI-H status was centrally confirmed retrospectively using the Promega fluorescent PCR-based MSI Analysis System v1.2 . For both PD-L1 status in NSCLC and MSI-H status in CRC, only central testing data were used in this analysis.Patients with any solid tumor except lymphoma were eligible to enroll in the phase 1 part of LUC1001. Tissue sample collection for PD-L1 testing was optional in phase 1 but mandatory in phase 2. The phase 2 part of LUC1001 was initially designed to enroll patients with histologically or cytologically confirmed stage III or IV PD-L1Phase 1 included five dose-escalation cohorts and three pharmacokinetics/pharmacodynamics cohorts; patients received intravenous (IV) 80, 240, 460, or 800 mg every 2 weeks (Q2W) or 480 mg Q4W doses of cetrelimab  observed during the first 28 days on treatment. DLTs were defined as toxicities of grade 5, grade 4 (including neutropenia lasting ≥ 7 days and thrombocytopenia of any duration), or grade ≥ 3 . ALT/AST elevations meeting Hy’s law criteria and immune-related toxicities requiring treatment in excess of corticosteroids were included in the DLT definition.The clinical activity and safety of cetrelimab at the chosen RP2D in three tumor-specific groups  were assessed in phase 2 to confirm the selected doses.The primary endpoints were the safety and tolerability, and overall response rate (ORR) of cetrelimab. Secondary objectives included assessment of pharmacokinetics, pharmacodynamics, immunogenicity, and efficacy by the investigator, including the clinical benefit rate (CBR), progression-free survival (PFS), overall survival (OS), and duration of response (DOR). Selected biomarkers were examined for potential association with pharmacodynamic modulation of cetrelimab.Safety assessments included the frequency and severity of treatment-emergent adverse events (TEAEs) and immune-related adverse events (irAEs), infusion-related reactions (IRRs), vital sign measurements, clinical laboratory values, and electrocardiograms. The severity of AEs was assessed using the National Cancer Institute Common Terminology Criteria for Adverse Events v4. irAEs and IRRs were designated by the investigators. Investigators were instructed to consider all events of an inflammatory nature immune related in the absence of a clear alternative etiology.Blood samples were collected for pharmacokinetic analyses on the day of dosing both pre-infusion (within 2 h) and at the end of infusion (EOI) for the first ten doses in cohorts receiving Q2W dosing. After Dose 1 and Dose 9, additional blood samples were collected at 2 h (EOI + 2 h) and 6 h (EOI + 6 h) post infusion as well as on Days 2, 4, and 8 in the cohorts receiving Q2W dosing. After Dose 1 and Dose 5 in cohorts receiving Q4W dosing, additional samples were collected at EOI + 2 h and EOI + 6 h post infusion as well as on Days 2, 4, 8, 15, and 22. In the SC cohort, blood samples were collected for the sentinel SC dose at the following time points: pre-injection (within 2 h), post injection (PI), PI + 2 h, and PI + 6 h, as well as on Days 2, 4, 8, 15, 22, 29, and 36. Samples were also collected at the end-of-treatment (EOT) visit (≤ 30 days after the last dose) and first survival follow-up visit (~ 12 weeks after the last dose).Serum cetrelimab concentrations were measured using a validated electrochemiluminescence immunoassay method. As previously mentioned, two drug product formulations were tested at the 480 mg Q4W dose.Individual pharmacokinetic parameters were calculated using noncompartmental analysis (NCA) and descriptive statistics were provided. A two-compartmental disposition population pharmacokinetic (popPK) model with first-order elimination and zero-order IV infusion rate, parameterized in terms of clearance (CL), volume of central compartment (V1), intercompartmental CL (Q), and volume of peripheral compartment (V2), was used to describe the pharmacokinetics of cetrelimab from both phase 1 and phase 2 parts of LUC1001. Interindividual variability (IIV) was implemented on CL, V1, and V2, with random-effects correlation estimated between CL and V1. IIV was also included on residual error to allow for patient-level variations in residual error. Additionally, popPK modeling and simulations were performed using data from all dose cohorts to explore multiple phase 2 dosing regimens targeting comparable pharmacokinetic profiles and exposures similar to nivolumab and pembrolizumab.+ T cells by flow cytometry analysis. During phase 1, Dose 1 samples were taken on Day 1 before infusion and 2 h after EOI and on Day 8; Dose 2 samples were taken on Day 1 before infusion and at EOI. Samples for Dose 3 were taken on Day 1 before infusion, and for Dose 9 on Day 1 before infusion and at EOI. Dose 10 samples were drawn on Day 1 at EOI and at EOT. During phase 2, blood samples were taken before infusion on Dose 1 and Dose 5.Fresh whole blood was collected from all patients in phase 1 and from the first 40 patients in phase 2 for evaluation of PD-1 receptor occupancy (RO) on circulating CD3Ex vivo staphylococcal enterotoxin B (SEB) stimulation of IL-2 production by peripheral blood mononuclear cells was conducted to evaluate cetrelimab pharmacodynamic modulation . BrieflySerum levels of IFN-γ-inducible protein 10 (IP10) and IL-2 receptor alpha chain (IL2Ra) were measured by Meso Scale Discovery (MSD) for all patients with one pretreatment and at least one post-treatment sample collection.Serum samples were screened for anti-drug antibodies binding to cetrelimab and the titer of confirmed positive samples was reported.Tumor response was assessed by the investigators per Response Evaluation Criteria In Solid Tumors (RECIST) v1.1  every 8 ORR was defined as the percentage of patients with complete response (CR) + partial response (PR). CBR was defined as the percentage of patients with CR + PR + stable disease lasting ≥ 24 weeks . PFS wasDose escalation and recommended phase 2 dose identification were guided using a modified continual reassessment method, which was based on the probability of dose-limiting toxicities by a two-parameter Bayesian logistic regression model and escalation with overdose control principle. The sample size estimation of 180 patients for the overall study population in the dose expansion part of this study was based on the ability to detect a 79.0% success rate if the true ORR is 15.0% and 15.7% if the true ORR is 10.0% by Bayesian power, or a 43.2% success rate if the true ORR is 15.0% and 1.3% if the true ORR is 10% by Bayesian double criteria. The all-treated population, defined as patients who received ≥ 1 dose of cetrelimab, was the basis for both safety and efficacy analyses. Subpopulations of patients with NSCLC, melanoma, and CRC were analyzed individually. The ORR is presented with 2-sided 95% exact Clopper–Pearson confidence intervals (CIs). Time-to-event endpoints and corresponding 95% CIs were estimated using Kaplan–Meier methodology.At clinical data cutoff on Jul 1, 2019, the all-treated population comprised 204 patients (58 in phase 1 and 146 in phase 2). Median age was 60.0 years  and 56.9% were male , and posterolateral thoracotomy. The symptom for myasthenia gravis was grade 4 dyspnea and antiacetylcholine receptor antibody was positive. The patient received methylprednisolone 60 mg (1 mg/kg) twice daily and ipratropium bromide, along with noninvasive ventilation; however, diaphragmatic sequelae from multiple chest surgeries limited supportive respiratory therapy options.The most frequently reported TEAEs (occurring in > 15.0% of patients) were asthenia 25.5%), fatigue (21.1%), dyspnea (21.1%), pyrexia (19.6%), diarrhea (19.1%), anemia (18.6%), nausea (17.6%), decreased appetite (17.6%), cough (17.2%), and back pain (15.7%). Approximately two-thirds of patients (67.2%) experienced TEAEs deemed possibly treatment related  died within 30 days of their last cetrelimab dose. For 17 patients, the primary cause of death was disease progression. AEs were the primary cause of death for four patients and one of those AEs (the DLT of myasthenia gravis) was considered treatment related. Dyspnea was the cause of death in two patients and acute heart failure in one patient. Fifteen patients (7.4%) experienced TEAEs leading to death.The safety profile at the RP2D (240 mg Q2W IV) was similar to that in the overall all-treated population, with serious and grade ≥ 3 TEAEs that were considered treatment-related occurring in 12.3% and 14.8%, respectively. Treatment-related TEAEs at a dose of 240 mg Q2W resulted in dose interruptions in 29.0% of patients and discontinuation in 6.8%.Immune-related AEs were reported by the investigator for 35.3% of patients (72/204). The most common irAEs were hypothyroidism (6.9%), asthenia (4.4%), diarrhea , rash (2.9%), hyperthyroidism (2.9%), dyspnea (2.9%), pruritus (2.5%), and pneumonitis (2.5%). Grade ≥ 3 irAEs were reported for 6.9% of patients (14/204). Pneumonitis was the only grade ≥ 3 treatment-emergent irAE that occurred in two patients (1.0%); all other grade ≥ 3 irAEs occurred in a single patient (0.5%). One patient receiving 240 mg Q2W experienced serious autoimmune colitis and discontinued study treatment.IRRs were reported for 14.2% of patients (29/204). Median time to onset of first IRR was 15  days and median time to resolution of first IRR was 1  day. The majority of IRRs occurred after the first two doses. Two patients (1.0%) experienced grade ≥ 3 IRRs and one patient discontinued study treatment due to a grade 3 IRR of hypertension.The pharmacokinetics of cetrelimab were linear and dose proportional across IV doses of 80, 240, 460, and 800 mg Q2W, with moderate variability after the first dose , AUC over a dosing interval (τ) (AUCτ)] increased after each repeated IV dose until steady state appeared to be reached after four doses with the Q4W schedule and seven doses with the Q2W schedule  was higher than the mean Ctrough of 480 mg Q4W (48.2 µg/mL). The t½ calculated from NCA of steady-state pharmacokinetic data from the 480 mg Q4W groups (22.4 days) was consistent with the predicted t½ via popPK modeling (25.0 days). At Dose 9 of 240 mg Q2W during phase 2, the mean accumulation ratios of the Ctrough and Cmax were 2.86 and 1.84, respectively (n = 68). Both mean accumulation ratios were similar to those observed for 240 mg Q2W in phase 1 .The mean Body weight and albumin were identified as two of the significant covariates for CL and body weight was identified as a significant covariate for V1 (data on file).The overall prevalence of anti-cetrelimab antibodies was 1.7% (3/182 patients with available data); 2.2% of patients in the phase 2 part of the study who were treated with IV cetrelimab 240 mg Q2W developed anti-cetrelimab antibodies (3/134 patients with available data). Anti-cetrelimab antibodies did not appear to affect cetrelimab pharmacokinetics (Online Resource 5).P = 0.012) over baseline on Day 43 (Online Resource 6). Significant elevation of IL2Ra chain was observed on Day 43 in patients with stable disease (P = 0.0006) and progressive disease (P < 0.0001), but not in patients with CR or PR following treatment with 240 mg Q2W cetrelimab.In the all-treated population, IP10 concentration was significantly increased  across all doses  tested Fig. b, indicaIn the all-treated population from both phases of LUC1001, the ORR was 18.6% (38/204) and the CBR was 31.3% (64/204). Six patients (2.9%) had CR, 32 (15.7%) had PR, and 36 (17.7%) had stable disease. Median PFS was 2.8 (95% CI 1.9–3.7) months and median OS was 17.8 (95% CI 11.9–22.6) months (Online Resource 7 and Online Resource 8).KRAS mutation was identified in 3 patients (8.6%), an EGFR mutation in 3 (8.6%), and other mutations in 9 (25.7%). Nineteen patients (54.3%) expressed high levels of PD-L1 and would have been eligible to receive PD-1 inhibitor therapy in clinical practice.Of 35 patients with NSCLC in phase 1 and phase 2 included in the clinical analysis, 71.4% were diagnosed with adenocarcinoma, 22.9% with squamous cell carcinoma, 1 with large-cell carcinoma, and 1 with “other.” At screening, 94.3% of patients had stage IV NSCLC, and 97.1% had previously received chemotherapy. Mutation status was known for 15 patients (42.9%): a +) tumors, the ORR was 52.6% and the CBR was 73.7%. The overall median duration of treatment was 7.0  months for all treated patients and longer [10.7  months] among patients with PD-L1 high tumors. Median DOR could not be estimated, because there was an insufficient number of progression events at data cutoff. For all treated patients, median PFS was 7.4 [95% CI 2.43–not estimable (NE)] months and the median OS was 22.4 (95% CI 9.95–NE) months (Online Resource 7 and Online Resource 8). Response and duration of treatment for individual patients are shown in Online Resource 9 and Fig. The median follow-up was 15.7  months. In all 35 patients, the ORR was 34.3% and the CBR was 51.4%. In the subgroup of 19 patients with PD-L1-high (≥ 50% PD-L1BRAF/MEK-targeted therapy (26.0%), IFN (24.0%), and chemotherapy (36.0%). All patients had stage IV melanoma at screening except one patient (2.0%) with stage III. BRAF mutations were found in 19 patients (45.2%) and PD-L1 status was positive (> 1%) for 8 (16.0%) patients. The median duration of follow-up for all patients was 16.6  months. The overall median duration of treatment was 5.5  months for all treated patients.Fifty patients with melanoma enrolled in phase 1 and phase 2 combined, including six with uveal melanoma. Of these, 37 (74.0%) had previously received systemic treatment. Prior treatments included ipilimumab (38.0%), + (PD-L1 IHC ≥ 1%), the ORR was 50.0% and the CBR was 75.0%. Median DOR and OS could not be estimated (Online Resource 7). The median PFS for all treated patients was 5.4 (95% CI 2.73–9.95) months (Online Resource 8). Response and duration of treatment for individual patients are shown in Online Resource 10 and Fig. In all 50 patients, the ORR was 28.0% and the CBR was 46.0%. The six patients with uveal melanoma had progression as best response. Among the eight patients who were PD-L1BRAF/MEK-targeted therapy.Of 48 patients with CRC enrolled in phase 1 and 2, 21 were centrally determined to be MSI-H by IHC. Twelve patients were normal (microsatellite stable) by central laboratory and nine could not be evaluated by the central laboratory because of lack of control sample or insufficient tumor sample. Twenty-seven patients (56.3%) were dMMR by local laboratory. Overall, MSI-H testing was highly variable, with ~ 50.0% concordance between local and central laboratories. At screening, all patients with CRC had stage IV disease, and all had received prior systemic therapy; 46 patients (95.8%) received chemotherapy and one (2.1%) received Patients with MSI-H CRC had a median treatment duration of 9.9  months, while the all-treated CRC population had a median treatment duration of 2.4  months. The median follow-up was 12.1  months for the all-treated population and 12.3  months for those with MSI-H CRC.In all 48 patients with CRC, the ORR was 16.7% and the CBR was 37.5%. In patients with MSI-H CRC, the ORR was 23.8%, with PR being the best overall response for all five. The CBR was 61.9% in the MSI-H group. Median DOR and OS could not be estimated (Online Resource 8). Response and duration of treatment for individual patients with CRC are shown in Online Resource 11 and Fig. In this first-in-human, phase 1/2 study, the safety, pharmacokinetics, pharmacodynamics, and biomarkers of cetrelimab were thoroughly characterized in PD-1/PD-L1 inhibitor–naïve patients with advanced or refractory solid tumors. Detailed characterization of cetrelimab monotherapy is essential for the selection of dosing regimens to accommodate potential schedules of combination partners. In phase 1 of LUC1001, cetrelimab was well tolerated across the IV doses of 80, 240, 460, or 800 mg Q2W or 480 mg Q4W. The safety profile of cetrelimab was consistent with that of other anti-PD-1 antibodies. As expected with immune checkpoint inhibitors, immune-related AEs were observed and they were managed with corticosteroids and treatment interruption. Only two DLTs occurred during the dose-escalation phase. Across phases 1 and 2, although 137 patients (67.2%) experienced TEAEs deemed possibly treatment related, most were managed by dose interruption, and only 12 patients (5.9%) experienced treatment-related AEs resulting in treatment discontinuation. No unexpected safety signals were observed. The prevalence of IRRs was higher for cetrelimab (14.2%), as reported by the investigators, compared with that reported for pembrolizumab (0.2%) and nivolumab (6.4%) . NotablyCtrough for IV doses of cetrelimab 240 mg Q2W (66.9 µg/mL) and 480 mg Q4W (48.2 µg/mL) derived from the simulations, based on the popPK model parameters and associated interpatient variability, were similar and in range with other PD-1 inhibitors such as nivolumab [t½ of cetrelimab was aligned between NCA (22.4 days) and the popPK model (25.0 days) and conforms to the expected behavior of enhanced neonatal FC receptor–mediated recycling for IgG antibodies at around 3 weeks [t½ was 25 days for nivolumab [Serum cetrelimab steady-state ivolumab –30 (56.5ivolumab –33 (23.3 3 weeks . The repivolumab  and in tivolumab . Based oA relatively flat dose–response relationship has been demonstrated for two approved PD-1 inhibitors (nivolumab and pembrolizumab), suggesting a relatively broad therapeutic window (around 1–10 mg/kg) for the anti-PD-1 mechanism of action , 36, 37.Anti-cetrelimab antibodies were detected in a small proportion of patients in phase 1 (1.7%) and phase 2 (2.2%) and did not appear to affect the pharmacokinetics of cetrelimab. Therefore, available data suggest that anti-cetrelimab antibodies do not impact clinical activity.Preliminary efficacy data reported suggest that the efficacy profile of cetrelimab may be consistent with known profiles of PD-1 inhibitors in melanoma –45, PD-LFGFR alterations (NCT03473743), as chemotherapy or targeted therapy can release tumor neoantigens and prime the tumor microenvironment for immune response by a PD-1 inhibitor. These studies, however, are required to determine the safety and efficacy of such combinations based on the unpredictability in treating cancer and delivering meaningful new treatment options to patients.In summary, the phase 1/2 LUC1001 study characterized the safety, pharmacokinetics, pharmacodynamics, and efficacy data of cetrelimab in patients for the first time to support cetrelimab dose schedules of IV 240 mg Q2W and 480 mg Q4W. It is important to acknowledge that multiple PD-1 agents have already been approved for the treatment of PD-1/PD-L1 inhibitor–naïve patients with advanced or refractory solid tumors; cetrelimab is currently being developed to support combination studies that are ongoing. Furthermore, data from the phase 1/2 LUC1001 study cannot be directly compared with those from other trials at this time due to the small sample size and study design that is not controlled with an active comparator. The data reported here are critical for understanding cetrelimab in a clinical setting, and these findings are consistent with results with other approved PD-1 inhibitors. Based on the preliminary antitumor activity of cetrelimab in patients with advanced solid tumors in LUC1001 , studiesSupplementary file1 (DOCX 654 KB)Below is the link to the electronic supplementary material.


Cordyceps fumosorosea strain RCEF 6672 including mannitol (1), uridine (2), adenine (3). N6-(2-hydroxyethyl)-adenosine (4). N6-(2-hydroxyethylacetate)-adenosine (5), fumosoroseanoside A (6) and B (7), ovalicin-4α-alcohol (8), 1-linoleoyl-sn-glycero-3-phosphocholine (9) and its isomer (10), fumosoroseain A (11) and its isomer (12), 5 non-ribosomal peptides (13 to 17) and 3 fatty acids (18 to 20). The compounds 5, 6, 7, 9 and 11 were prepared with preparative and semi-preparative HPLC and identified with 1D and 2D NMR. Compounds 4 and 5 were the first time identified from C. fumosorosea. Compounds 6, 7 and 11 are novel compounds. Compounds 6 and 7 showed antibacterial and antifungal activities, and 11 showed antiaging activity. All the secondary metabolites (4 to 8 and 11 to 17) have strong bioactivities indicating that the metabolites have pharmaceutical development potentiality.High-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) analysis revealed that there are 20 main components in spores and mycelia extract of The online version contains supplementary material available at 10.1186/s13568-022-01379-w. C. fumosorosea for the first time.Clarified small molecular metabolites of Identified three novel compounds with antimicrobial or antiaging activities.The fungus has development potentiality for rich in bioactive metabolites.The online version contains supplementary material available at 10.1186/s13568-022-01379-w. Cordyceps are highly valued traditional medicines or healthy foods in Asian countries for a long history , 3 poly-ketone, 3 terpene, 1 siderophore, 5 NRPs or poly-ketone gene clusters. Our primary HPLC-HRMS analysis showed that C. fumosorosea had complicated metabolites including several possible unknown compounds. To make clear the structure and bioactivity of the unknown metabolites will be able to not only reveal the utilization value of the fungus, but also help us to evaluate chemical ecological safety of the fungal pesticide. Therefore, we launched a systematic analysis and identification of the metabolites of C. fumosorosea. In this study we will focus on the unknown metabolites. Through HPLC-HRMS guided isolation we can prepare the unknown compounds directly without the disturbance of the known metabolites. Because that Cordyceps do not rot easily in the soil and are usually used as tonic medicine or food, they are presumed to contain antibiotics and antiaging compounds . HPLC grade methanol and formic acid were from Tedia Company of China . Deuterated NMR solvents were purchased from Cambridge Isotope Laboratory .3)2SO signal  or CD3OD signal  as internal standards for 1D NMR and 2D NMR.Preparative HPLC was performed using Agilent modules consisting of an autosampler PS410, two pumps M400, a UV detector 1260VWD and an automatic fraction collector 440FC. Semi-preparative HPLC was performed using Shimadzu modules consisting of an autosampler SIL-20A, two pumps LC-20AD and a PDA detector SPD-M20A. ESI-HR-MS data were obtained using an Agilent 1100 HPLC tandem 6510QTOF MS spectrometer. NMR experiments were recorded using a Bruker Advance 600 MHz spectrometer. Chemical shifts were referenced to the residual (CDC. fumosorosea infected lepidopterous larva collected in Anhui Province (China) and grown at 25 °C on PDA plates. The isolated strain was identified by morphology and sequence analysis of the ITS region of the rDNA. It has been catalogued RCEF 6672 and deposited in the Research Center for Entomogenous Fungi , Anhui Agricultural University.The fungal strain was isolated from 6). 300 μL of the suspension was inoculated on a 150 mm PDA plate, and 2000 of the plates were inoculated for mass cultivation. After 10 days cultivation the spores and surface mycelia were scraped off the plates and freeze dried. The dried spores and mycelia were extracted with methanol and ethyl acetate mixture (1:1) under 40 kHz ultrasonication for three times in 30 min, and the total solid–liquid ratio is 1: 4 (w/v). The extract was evaporated under reduced pressure, and 10.5 g pasty crude extract was obtained. With the assistance of 40 kHz ultrasonication, 8 g of crude extract was redissolved in 10 ml of methanol. The turbid liquid was centrifuged at 10,000 g for 10 min, and the supernatant was separated by preparative HPLC with an Agilent Prep-C18  column for primary isolation, and semi-preparative HPLC with an Agilent SB-C18  column for final purification. The conditions for preparative HPLC are as follows: injection volume 150 μL; flow rate 15 mL/min; elution gradient: 0–15 min, 30% to 100% methanol, 15–20 min, 100% methanol, 20—22 min, 100% to 30%. The effluents were collected every one minute for one tube and tested by HPLC-HRMS. The tube contained unknown compounds was condensed under reduced pressure and submitted to semi-preparative HPLC. The conditions for semi-preparative HPLC are as follows: injection volume 30 μL; flow rate 3 mL/min; elution gradient: 0–1 min, 40% methanol, 1–15 min, 40% to 100% methanol, 15–18 min, 100% methanol, 18–22 min, 100% to 40% methanol, 22–25 min, 40% methanol. The effluents of the unknown compounds were collected according to the m/z of the unknown compounds detected by HRMS through a 10:1 splitter.After 7 days cultivation on PDA plate, the spores of the fungus were harvested with a 0.05% Tween 80 solution to make a spore suspension  column, and the LC parameters were set as follows: injection volume, 5 μL; column temperature, 25 °C; and flow rate, 0.4 mL/min. The mobile phase was composed of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile, and a gradient elution was carried out: 0–40 min, 5–100% B, 40–45 min, 100% B, 45–55 min, 100–5% B. The eluates were monitored with a PDA performing a full wavelength scan from 200 to 600 nm, and a TOF MS with following parameter settings: gas temperature, 350 °C; drying gas, 10 L/min; nebulizer pressure, 45 psi; capillary voltage, 4000 V in positive mode and 3500 V in negative mode; fragmentor voltage, 215 V in positive mode and 170 V in negative mode; skimmer voltage, 60 V. Data acquisition was performed in the m/z range of 50–1700 Da. The effluents of the preparative HPLC were detected by HPLC-HRMS without chromatographic column.Dictionary of Natural Products (http://dnp.chemnetbase.com/). The known compounds were confirmed by UV/visible spectra whenever possible, and verified by their elution order (polarity) and structure characteristics. Some common metabolites of entomopathogenic fungi such as mannitol, adenosine and beauvericin A were confirmed with standards. Molecular formulas without corresponding compounds in the database were labeled as unknown compounds and submitted to identification with 1D and 2D NMR.The molecular formulas of the metabolites were calculated by Mass Hunter based on accurate mass and isotopic pattern recognition. Compounds were putatively identified by searching the molecular formulas against the in-house entomopathogenic fungi database and the E. coli (ATCC 25922). Amphotericin B was used as positive control for the antifungal bioassays against C. albicans strain CMCC98001 . All samples were prepared to a final concentration of 1, 2 and 3 mg/mL in 1.5% DMSO, and 10 μL were loaded on the paper discs (6.0 diameters). The width of the transparent antimicrobial zone was measured with a vernier caliper. Each experiment was repeated three times.The antimicrobial activities of the metabolites were evaluated by the disc diffusion assay according to the method reported by Wu et al.  was fed with E. coli strain OP50 according to the method reported by Onken and Driscoll  at 20 °C with different content of compounds  or 0.1% DMSO as control. The nematodes were transferred to new plates every two days to ensure adequate food, and the number of dead worms were recoded simultaneously.Lifespan was measured according to the method of Liu et al. . The synReproduction assay was carried out according to the method reported by Chen et al. . Each plC. elegans were cultivated and treated with compounds as described in lifespan assays. On the 5th, 8th, 12th and 16th day of adulthood the nematodes were counted and classified into three classes: motion A nematodes moved constantly, motion B nematodes only moved when prodded and motion C nematodes sway only head or tail if prodded.Locomotion assay was performed according to the method of Wilson et al. . 50 C. eThermotolerance assay was performed according to the Wilson’s method  are at 209 and 265 nm  and 4.27  and δc 39.1 and 62.7, which were very similar to those of N6-(2-hydroxyethyl) adenosine , δC 19.4 and 171.5 in the HMBC spectrum, which suggested 5 as an acetylated form of N6 -(2-hydroxyethyl) adenosine. The position of an acetyl moiety was determined from the HMBC correlation between δC 171.5 and δH 4.27 (H-2ʹ). Taken together, the structure of 5 was determined as N6-(2-hydroxyethylacetate) adenosine  adenosine is the known compound cordyrrole B which can significantly inhibit adipocyte differentiation and pancreatic lipase  are at 260, 310, and 360 nm  was depicted in Fig. Compound NMR Fig. , 2009. TNMR Fig. . The che7: The molecular formula was assigned as C24H28O12 based on its ESI-HRMS  were disappeared, while one more methoxy group  appeared. Meanwhile, the keto carbon at δC 205.5 shifted to a higher field δC 170.5. Obviously, the methyl group in compound 6 which combined to the carbonyl carbon of the ketone group was oxidized and formed a methyl ester group. Based on above analysis the structure of the new compound (7) was shown in Fig. Compound 9: The molecular formula was calculated as C23H44NO7P based on its ESI-HRMS , 4 unsaturated carbons , 12 methylene carbon (δC 23 to 34) and a methyl carbon (δC 14.4). The chemical shifts of the other 5 carbons including 4 methylenes  and 1 methine carbons (δC 69.9) suggested the existence of a glycerol and an ethanolamine group. Carbons δC 69.9, 67.8, 63.2 and 41.8 were split with J = 7.5, 5.7, 6.6 and 5.2 Hz respectively suggesting that they had two or three bonds coupling with the phosphorus atom of phosphate group. The coupling constants between the phosphorus atom and the second carbon (J = 7.5) and the third carbon (J = 5.7) of the glycerol moiety suggested that the esterification position is the third carbon of the glycerol group. Therefore, compound 9 is 1-linoleoyl-sn-glycero-3-phosphocholine  are at 231 nm , 4 unsaturated carbons , 12 methylene carbon (δC 25 to 34) and a methyl carbon (δC 13.48). The three methyl groups with the same chemical shift values  are consistent with the characteristics of trimethylamine. Long-range coupling between the hydrogen (δH 3.75) of the trimethylamine connected methine and the carbonyl carbon (δC 170.75) and an ethanol group . Its structure and hydrocarbon attributes are shown in Fig. Compound 6-(2-hydroxyethylacetate) adenosine and linoleoyl-glycero-phosphocholine have been reported already  antibacterial and antifungal activities compared with the negative control (the solvent) at the experimental doses from 10 to 30 μg per paper disk , and the average lifespan was extended by 11.3% at 200 μM . But the 400 μM treatment group had no significant effect on promoting reproduction (P > 0.05). Results of the locomotion tests . In the heat shock experiment .Lifespan was the main antiaging indicator of 11 can significantly prolong lifespan, increase fertility and enhance stress tolerance. This indicated that compound 11 had significant antiaging activity at the tested concentrations. Compound 11 (fumosoroseain A) is a kind of lecithin derivative which usually possess many bioactivities including anti-aging bioactivity at a suitable concentration , 1 nucleoside (3), 3 fatty acids (18 to 20) and 2 phospholipids (9 and 10). Among them adenosine (3) is one of the main healthy constituents of Cordyceps (Hu and Li 9 and 10) are nutrients with many bioactivities  is a common secondary metabolite of Cordyceps with heathy functions, and related to fungal sporulation and environmental adaption (Hu and Li 13 to 17), 2 glycosides (6 and 7), 2 alkaloids (11 and 12), 2 nucleosides (4 and 5) and 1 terpene derivate (8).Above analysis showed that there are 20 main small molecular metabolites in mycelia and spores extract of 13 to 17) are existed in most cordyceps. Among them the beauveriolides (13 and 14) have anti-aging, beta-amyloid lowering and anti-atherogenic activities, while beauvericins (15 and 17) have insecticidal, induction of cell apoptosis and ionophoric property, but they have also cytotoxicity -adenosine (4) was the first time detected from C. fumosorosea. It exists in several other cordyceps and has Ca2+ antagonists, anti-cancer, inotropic and renal protection activities, but it also induces oxidative stress  (5) adenosine is an analogue of N6-(2-hydroxyethyl) adenosine and was identified from C. fumosorosea for the first time. Ovalicin-4α-alcohol (8) which was originally reported from Metarhizium anisopliae is a arterialisation inhibitor, a vascularisation inhibitor and an antipsoriatic agent , B (7) and fumosoroseain A (11). Bioactivity tests showed that fumosoroseanoside A and B had antibacterial and antifungal activities which may contribute to the niche competition of C. fumosorosea against other bacteria and fungi which would live on the killed pests. Fumosoroseain A (11) shows anti-aging activity, suggesting that this compound has potential to be developed as antiaging agent -adenosine, N6-(2-hydroxyethylacetate)-adenosine, ovalicin-4α-alcohol, beauveriolide I, and E have also healthy or medicinal bioactivities 2SO. Figure S3.1H and 13C NMR spectrometry of Compound 7 in (CD3)2SO. Figure S4. 1D NMR and 2D NMR spectrometry of Compound 9 in CD3OD. Figure S5. 1D NMR and 2D NMR spectrometry of Compound 11 in CD3OD.


Oxidative DNA lesions, such as those induced by hydrogen peroxide (H2O2), are often thought to require similar processing events, yet far less is known about how cells process oxidative damage during replication. Here we show that replication is not disrupted by H2O2-induced DNA damage in vivo. Following an initial inhibition, replication resumes in the absence of either lesion removal or RecF-processing. Restoring DNA synthesis depends on the presence of manganese in the medium, which we show is required for replication, but not repair to occur. The results demonstrate that replication is enzymatically inactivated, rather than physically disrupted by H2O2-induced DNA damage; indicate that inactivation is likely caused by oxidation of an iron-dependent replication or replication-associated protein that requires manganese to restore activity and synthesis; and address a long standing paradox as to why oxidative glycosylase mutants are defective in repair, yet not hypersensitive to H2O2. The oxygen-sensitive pausing may represent an adaptation that prevents replication from occurring under potentially lethal or mutagenic conditions.UV irradiation induces pyrimidine dimers that block polymerases and disrupt the replisome. Restoring replication depends on the  All cells must accurately duplicate their genomes to reproduce. However, DNA damage can block the replication machinery and prevent it from completing this task, resulting in mutations if the wrong base is incorporated, rearrangements if replication resumes from the wrong site, or cell lethality if the block to replication cannot be overcome (reviewed in ). In ordEscherichia coli, the disruption of replication at UV-induced lesions involves uncoupling of the leading and lagging strand, exposure of the nascent DNA to exonucleolytic degradation, and the dissociation of several components of the replisome, including Pol III, the beta clamp, and the tau complex, whereas both helicase and primase remain associated with the branched replication fork DNA . These e glycol ).2O2 , HL921 (SR108 Δ(srlR-recA)306::Tn10), HL922 (SR108 recB21recC22 argA81::Tn10), HL952 (SR108 uvrA::Tn10), CL008 (SR108 recG258::Tn5), CL544 (SR108 recR6212::cat), CL577 (SR108 ruvC53eda-51::Tn10), CL579 (SR108 recF6206::tetR), and CL915 (SR108 recN::cat) have been previously described (nth::kanRnei::cat) was constructed by P1 transduction of the nei::cat allele from CL1005 (SR108 nei::cat (nth::kanR (nth::kanRnei::cat fpg::tetR) was constructed by P1 transduction of the fpg::tetR allele from CL1778 (SR108 fpg::tetR (xthA::cat) was constructed by gene replacement using the recombineering strain DY329 . All strains used in this work are summarized in Supplementary Table S1.SR108 .600 of 0.3, and then treated with 10 mM H2O2. At the times indicated, 0.1-ml aliquots of each culture were removed and serially diluted in 10-fold increments into DGCthy medium. Triplicate 10-µl aliquots of each dilution were then spotted on Luria-Bertani (LB) plates supplemented with 10 µg/ml thymine (LBthy). Viable colonies were counted following overnight incubation at 37°C.Fresh overnight cultures were diluted 1:100 in Davis medium supplemented with 0.4% glucose, 0.2% casamino acids and 10 µg/ml thymine (DGCthy), grown at 37°C to an OD2/s. Fresh overnight cultures were diluted 1:100 in DGCthy medium and grown at 37°C to an OD600 of 0.3. At this time, 0.1-ml aliquots of each culture were removed and serially diluted in 10-fold increments into DGCthy medium. Triplicate 10-µl aliquots of each dilution were then spotted on LBthy plates and irradiated with increasing doses of UV as indicated. Viable colonies were counted following overnight incubation at 37°C.UV irradiation used a 15-W germicidal lamp (254 nm) at an incident dose of 0.9 J/m600 of 0.3. Where indicated 200 µM manganese (II) chloride (MnCl2·4H2O) was added to the medium as well. At this time, cultures were irradiated with an incident dose of 50 J/m2 and then returned to 37°C to allow recovery. For H2O2 challenge, fresh overnight cultures were diluted 1:100 in DGCthy medium supplemented with MnCl2 as indicated, grown at 37°C to an OD600 of 0.3, and then treated with 10 mM H2O2 for 5 min at 37°C. Cells were filtered on 0.45-µm membranes (Fisherbrand) to remove excess H2O2 from the medium, resuspended in fresh DGCthy medium supplemented with manganese as appropriate to initial growth conditions and returned to 37°C for the duration of the time course. For both treatments, a 0.75-ml aliquot was transferred at the times indicated to an equal volume of 2× 200 mM NaCl, 20 mM Tris, pH 8.0, 40 mM EDTA, pH 8.0 (NET). Cells were pelleted and resuspended in 0.14-ml of lysis buffer  and incubated for 30 min at 37°C. Then, 0.01-ml 10 mg/ml ProteinaseK and 0.01-ml 20% Sarkosyl were added to the samples and incubation was continued for 30 min at 37°C. The samples were then extracted once with four volumes of phenol:chloroform, followed by two volumes of chloroform, and dialyzed against 200-ml 1 mM Tris, pH 8.0, 1 mM EDTA, pH 8.0 for 30 min using 47-mm Millipore 0.025-µm pore disks.For UV irradiation, fresh overnight cultures were diluted 1:100 and grown at 37°C in DGCthy medium to an OD2O2-treated samples, 15 µl of each DNA sample was treated with reaction buffer  supplemented with either no enzyme or 0.53 µM Fpg glycosylase and 2.7 µM Endo III for 1 h at 37°C. Enzyme preparations were titrated using purified undamaged genomic DNA as a template. The highest enzyme concentration that did not exhibit nonspecific activity on undamaged DNA was used. For the preparations in our lab, this corresponded to 2 U T4 Endo V, 0.53 µM Fpg glycosylase and 2.7 µM Endo III. Treated samples were then electrophoresed on a 0.5% alkaline agarose gel in 30 mM NaOH, 1 mM EDTA at 30 V for 16 h, stained and visualized with ethidium bromide. The intensity of each high-molecular-weight band was determined using ImageQuant software (GE Biosciences). The fraction of lesion-free DNA fragments was quantified as a ratio of high-molecular-weight DNA in the T4 Endo V- or Fpg/Endo III-treated band (EnzT) to the band without enzyme treatment (NoEnzT) at each time point, T. To normalize for any nicks or AP sites present in the DNA before UV or H2O2 treatment, the ratio obtained at each time point was divided by the ratio calculated at the pretreatment time point as follows: (EnzT/NoEnzT)/R0, where R0 is (EnzT/NoEnzT) at the time immediately before UV or H2O2 addition.For UV-irradiated samples, 15 µl of each DNA sample was treated with reaction buffer  supplemented with either no enzyme or 2 U T4 endonuclease V  for 1 h at 37°C. For H600 of 0.25–0.35. Where indicated manganese was added to the medium as described above. At this time, half of the cells were mock irradiated, while the other half of the culture was irradiated with 50 J/m2.For experiments using UV irradiation, overnight cultures were diluted 1:100 and grown at 37°C in DGCthy to an OD2O2, overnight cultures were diluted 1:100 and grown at 37°C to an OD600 of 0.25–0.35 in DGCthy supplemented with 200 µM MnCl2 where indicated. At this time, half of the cells were mock treated, while the remaining culture was exposed to 10 mM H2O2 for 5 min at 37°C. Following either mock or H2O2 treatment, cells were filtered on 0.45-µm membranes to remove excess H2O2 from the medium and resuspended in fresh DGCthy medium either supplemented with Mn or not based on initial growth conditions.For experiments using H2O2 experiments, cultures were returned immediately to 37°C after treatment to allow recovery and continued growth. At the times indicated, duplicate 0.5-ml aliquots of culture were pulse-labeled with 0.5 µCi/ml [3H]thymidine for 2 min at 37°C. Cells were then lysed, and the DNA was precipitated in cold 5% trichloroacetic acid and filtered onto Millipore glass fiber filters. The amounts of 3H on each filter were determined by scintillation counting.For both UV irradiation and H2 where indicated and grown without amp selection to OD600 of 0.5. At this time, cultures were either UV-irradiated with an incident dose of 50 J/m2 and then returned to 37°C to allow recovery, or treated with 10 mM H2O2 for 5 min at 37°C, filtered on 0.45-µm membranes to remove excess H2O2 from the medium, then resuspended in fresh DGCthy medium and returned to 37°C for the duration of the time course. At the times indicated, a 0.75-ml aliquot was transferred to an equal volume of 2× NET. Total DNA (genomic and plasmid) was then purified from these cultures as described above.Cells containing the plasmid pBR322 were grown overnight in DGCthy medium supplemented with 100 µg/ml ampicillin (amp). A 0.2-ml aliquot of this culture was pelleted and resuspended in 20-ml DGCthy with 200 µM MnCl32P-labeled pBR322 plasmid prepared by random prime labeling (Agilent) using dCTP  and visualized using a Storm 840 PhosphorImager (GE Biosciences) and its associated ImageQuant software.DNA samples were dialyzed against 200-ml 1 mM Tris, pH 8.0, 1 mM EDTA, pH 8.0 for 30 min using 47-mm Millipore 0.025-µm pore disks and digested with PvuII restriction endonuclease (Thermo Fisher Scientific) overnight at 37°C. Samples were then extracted once with one volume of chloroform, loaded directly on a 0.4% agarose gel in 1× TBE (Tris-borate-EDTA) and DNA was separated initially at 1 V/cm for 15 h. For the second dimension, lanes were excised, rotated 90°, recast in 1% agarose in 1× TBE and electrophoresed at 6.5 V/cm for 7 h. DNA in the gel was transferred to Hybond N+ nylon membrane by standard Southern blotting techniques, and the plasmid DNA was detected using https://doi.org/10.25387/g3.13426079.Strains used in these experiments are available upon request and can be found in Supplementary Table S1. Supplemental material is available at figshare: 2O2-induced DNA damage in vivo, we compared the replication occurring in H2O2-treated wild-type cells to that occurring in fpg nei nth mutants, which lack the three predominant oxidative DNA glycosylases. Consistent with previous reports  was determined for each time point and expressed relative to the rate immediately prior to treatment. In both wild-type and fpg nei nth cells, a rapid inhibition of DNA replication was observed immediately following H2O2 treatment. Surprisingly, replication rapidly resumed even in the absence of lesion removal and the fpg nei nth mutant restored DNA synthesis with kinetics that were modestly altered or reduced relative to wild-type cells , to monitor repair . Immedir repair  and measobserved .in vivo, and imply that these lesions do not disrupt replication. However, an inhibition of replication is clearly observed immediately following exposure to H2O2. This latter observation would appear to suggest that replication is disrupted by these lesions. We considered two possibilities to explain these apparently contradictory observations. It is possible that other forms of H2O2-induced DNA damage, not recognized by these glycosylases, disrupt replication and are responsible for the inhibition that is observed. Alternatively, the inhibition could result from an oxidative sensor or another aspect of the cellular oxidative stress response that is not directly associated with DNA damage.Thus, unlike UV-induced damage, the results demonstrate that lesions removed by Fpg, Endo III, or Endo VIII do not prevent replication from progressing recF mutants. In contrast to other DNA damage hypersensitive mutants, the defect in recF pathway mutants is specific to their ability to restore replication following disruption . Other hypersensitive mutants, including recBCD, recG, and ruvABC process and restore replication normally following disruption, arguing that cell lethality in these mutants arises from other causes  mutants , DNA sy2O2 stress, we compared the structural intermediates that arise on replicating plasmids after treatment with H2O2 to that occurring after UV irradiation using 2D agarose gel electrophoresis. To this end, wild-type cultures of E. coli containing the plasmid pBR322 were either exposed to H2O2 or irradiated with UV, as before. At various times following treatment, total genomic DNA was isolated, digested with the restriction enzyme, PvuII, to linearize the plasmid just downstream of its origin of replication, then separated and analyzed by 2D agarose gel electrophoresis. Non-replicating plasmid molecules migrate as a linear 4.4-kb fragment and form the prominent spot observed on the gels. Replicating molecules form Y-shaped structures that migrate more slowly due to their larger size and nonlinear shape, and appear as an arc radiating out from the prominent linear spot  and that manganese was needed to re-metallate these or alternative enzymes before genomic DNA replication can resume. These observations would be consistent with the results presented here and suggest that an oxidized ‘sensor’ protein is responsible for the observed inhibition rather than H2O2-induced lesions. The sensor could be a replication protein, or a protein that associates with the replisome to prevent DNA synthesis from progressing during oxygen stress. Alternatively, the sensor could be a repair protein(s) that is inactivated to prevent repair, thereby preventing replication from resuming. To examine this latter possibility, we compared the ability of cells to repair H2O2-induced lesions under varying manganese growth conditions. To this end, cultures were grown in defined medium supplemented with 200 µM MnCl2 or without additional metals, treated with H2O2, and monitored for the recovery of replication and repair of the H2O2-induced lesions as before. Although the absence of manganese reduced the ability of the cells to resume replication, it did not alter the rate or time that the H2O2-induced lesions were removed . The moncreases , 1987. ‘2O2 hypersensitivity of recBC mutants, which are defective in the repair of double-strand breaks (Alternatively, the modest SOS induction may suggest that oxidative damage induces double-strand breaks that are removed from the replication fork and arise elsewhere in the genome. This latter explanation would explain the lack of requirement for RecF which is not required for the repair of double-strand breaks and would also explain the Hd breaks . Many ird breaks .recF mutants to H2O2 treatment, which are impaired in resuming replication after disruption (It is possible that the oxygen-sensitive pausing of replication represents an adaptive response to prevent genomic replication from occurring during periods of oxygen stress when potentially lethal or mutagenic damage is present. Pausing replication to allow more time for repair to occur has been demonstrated to be beneficial both for reducing mutations and increasing survival . This masruption . In the 2O2 treatment, several of which can be reactivated following re-metallation with manganese, which is insensitive to oxidation, to restore function (There are other examples of oxygen sensors with the most notable example being OxyR, which following oxidation, transcriptionally upregulates genes associated with the oxidative stress response . Many mofunction . The senThis study was supported by National Science Foundation grant MCB1916625. Conflicts of interest: None declared.


Gold nanoparticles (AuNPs) decorated with biologically relevant molecules have variety of applications in optical sensing of bioanalytes. Coating AuNPs with small nucleotides produces particles with high stability in water, but functionality-compatible strategies are needed to uncover the full potential of this type of conjugates. Here, we demonstrate that lipoic acid-modified dinucleotides can be used to modify AuNPs surfaces in a controllable manner to produce conjugates that are stable in aqueous buffers and biological mixtures and capable of interacting with nucleotide-binding proteins. Using this strategy we obtained AuNPs decorated with 7-methylguanosine mRNA 5’ cap analogs and showed that they bind cap-specific protein, eIF4E. AuNPs decorated with non-functional dinucleotides also interacted with eIF4E, albeit with lower affinity, suggesting that eIF4E binding to cap-decorated AuNPs is partially mediated by unspecific ionic interactions. This issue was overcome by applying lipoic-acid-Tris conjugate as a charge-neutral diluting molecule. Tris-Lipo-diluted cap-AuNPs conjugates interacted with eIF4E in fully specific manner, enabling design of functional tools. To demonstrate the potential of these conjugates in protein sensing, we designed a two-component eIF4E sensing system consisting of cap-AuNP and 4E-BP1-AuNP conjugates, wherein 4E-BP1 is a short peptide derived from 4E-BP protein that specifically binds eIF4E at a site different to that of the 5’ cap. This system facilitated controlled aggregation, in which eIF4E plays the role of the agent that crosslinks two types of AuNP, thereby inducing a naked-eye visible absorbance redshift. The reported AuNPs-nucleotide conjugation method based on lipoic acid affinity for gold, can be harnessed to obtain other types of nucleotide-functionalized AuNPs, thereby paving the way to studying other nucleotide-binding proteins. Moreover, they strongly absorb in the visible region of the light spectrum3, and most importantly, their surfaces are easily functionalized with various moieties, including biomolecules4. One of the key features of surface-functionalized gold-nanoparticles is their ability to undergo controlled aggregation induced by intermolecular interactions, which results in a change in the color of the AuNP solution from deep-red to purple or blue5. This phenomenon has been extensively used to develop AuNP-based sensors and molecular probes. AuNP-based colorimetric probes have previously been used for heavy-metal ion detection6, imaging protein conformational changes7, and studies of biomolecular interactions8. Modifying AuNP surfaces with small nucleotides has been shown to provide certain advantages for stability10. For example, Zhao et al. prepared AuNPs in the presence of ATP as a stabilizing molecule and found that such NPs are more stable in aqueous solution compared to citrate-stabilized AuNPs10. We envisaged that nucleotide-decorated AuNPs can also be harnessed to optically sense nucleotide binding proteins, provided that the ligands can be introduced in a controllable manner that is also compatible with biological interactions. In this work, we aimed to test this idea using mRNA 5’ cap analogs as model nucleotides.Gold nanoparticles (AuNPs) have many distinctive properties that make them particularly useful for sensor development. At proper doses they are biocompatible and not cytotoxic7G cap is a nucleotide structure present at the 5'-end of eukaryotic mRNA, and consists of N-7-methylated guanosine connected to an mRNA chain via a 5'–5' triphosphate bridge11. Cap takes part in several important cellular processes through multiple interactions with cap-binding proteins12, which participate in mRNA maturation, transport, translation and degradation14. Eukaryotic translation initiation factor 4E (eIF4E) plays a key role in ribosome recruitment during protein biosynthesis15, serving as the cap-binding part of the eIF4F pre-initiation protein complex16. eIF4E is often overexpressed and up-regulated in cancer cells, therefore it has been identified as a cancer marker and therapeutic target18. Nathan et al. used Western blot analyses and the immunohistochemical staining of surgical margins of head and neck squamous cell cancers (HNSCCs) to show eIF4E overexpression in margins otherwise defined as lacking histological alterations due to tumor growth19. Therefore, the authors postulated that significant molecular changes occur in advance of any histological signs of tumor recurrence, thereby identifying eIF4E as a highly sensitive marker for HNSCC growth. Zhou et al. showed that a relationship exists between eIF4E overexpression, angiogenesis, and vascular invasion of cancer cells20. Elevated eIF4E expression was found to correlate with worse overall and disease-free patient survival, which confirmed eIF4E level to be an independent prognostic factor for breast cancer. To date, eIF4E expression levels have been detected either by Western-blot or immunohistochemical methods. Both of these techniques are rather time-consuming and, in the case of immunohistochemistry, requires an invasive procedure to obtain samples. Consequently, the development of novel and fast methods for determining eIF4E concentration levels in biological samples is a crucial objective.m25, and other types of (nano)materials26, and here we found it shows sufficient chemical stability to be compatible with nucleotide chemistry. We characterized the cap-modified AuNPs for physicochemical and biochemical properties and optimized the cap-ligand density on the AuNP surfaces by the addition of a neutral diluting molecule, which achieved both sufficient AuNP stability and proper recognition by eIF4E. We then investigated the binding of eIF4E by the conjugates using UV–Vis spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM). In parallel, we modified AuNPs with a small 4E-BP128 peptide to create conjugates interacting with eIF4E at a different site to the cap-AuNPs30. Finally, we used both types of conjugate to create a proof-of-concept colorimetric sensor based on nucleotide-decorated AuNPs, where binding of the protein causes a change visible with a naked eye.Herein, we describe the synthesis of AuNPs functionalized with eIF4E-binding nucleotides and peptides, and their evaluation as colorimetric molecular probes for the detection and quantification of eIF4E. First, cap-AuNPs were prepared from dinucleotide cap analogs modified with lipoic acid and citrate-stabilized AuNPs in a two-step shell-exchange process. Lipoic acid has been previously used for modification of gold surfaces31  and two structurally similar dinucleotides carrying guanine or adenine instead of N7-methylguanine  ester of lipoic acid, purified, and characterized using HPLC, mass spectrometry (MS), and NMR spectroscopy adenosine 5’-monophosphate. The dinucleotides were then conjugated with the 33. The particles were close to monodispersed (± 10% size deviation), with diameters of 17.6 ± 1.5 nm determined by TEM or 17.0 ± 1.9 nm as calculated using UV–Vis spectral data and previously reported equations34. Our initial attempts to prepare AuNP-cap conjugates by the direct functionalization of citrate AuNPs with cap analogs were unsuccessful due to particle aggregation under the experimental conditions. Hence, we used a two step-protocol, which included modifying the AuNP surfaces with Tween80 (a detergent)35, followed by the addition of the cap analog  that reflects the apparent binding affinity of eIF4E for modified AuNPs  and ApppA (1c) . Unexpectedly, G-cap-AuNPs and A-cap-AuNPs also showed increases in RH in the presence of eIF4E, albeit slightly smaller than that observed for the cap-AuNPs , therefore impairing differentiation between m7G, G, and A caps. As such, we next optimized the AuNPs surface composition in favor of specific binding.The interaction of AuNPs with eIF4E was independently investigated by DLS. First, the size of differently decorated AuNPs was determined. Incubating AuNPs with cap analogs resulted in an increase in the hydrodynamic radius RH Fig. F, suggesNPs Fig. G. These 7G-cap-TL-AuNP conjugate  as functions of eIF4E concentration . As negative controls, the 1:1 m7G-cap-TL-AuNP conjugate was titrated against an unspecific protein .A series of titration experiments was then conducted in order to compare the relative affinities of variously diluted conjugates for eIF4E; ECion Fig. A. The lo7G-cap-TL-AuNP conjugate to bind to eIF4E41. TEM images in the presence of eIF4E revealed the formation of a distinctive ~ 3 to 4-nm-thick halo around the conjugate, whereas no such formation was observed in the absence of protein  and synthetized peptide-modified AuNPs that target the 4E-BP1/eIF4G binding site in eIF4E30. After preliminarily screening several eIF4G- and 4E-BP1-derived peptides (data not shown), we selected a peptide derived from the 4E binding protein (4E-BP1) modified with cysteine at its N-terminus. 4E-BP1 is a translation initiation repressor30 that binds to eIF4E and inhibits its ability to form the eIF4F translation initiation complex43. The selected 4E-BP1 peptide forms a helical structure upon binding to eIF4E without interfering with its cap-binding activity29  and incubation for another 24 h. Titration experiments involving the 4E-BP1-AuNP conjugate and eIF4E revealed significant AuNP aggregation, which proceeded over time and did not produce a stable signal at any timepoint  peptide sequence as a diluting molecule and AuNP-stabilizing agentIn control experiments using the 4E-BP1-AuNP conjugates and BSA protein such aggregation was not observed Fig. D, sugges7G-cap-TL-AuNP conjugate (0.6 nM) was incubated for 1 h at room temperature with various concentrations of eIF4E, after which an equal volume of the 4E-BP1-AuNP conjugate was added to the mixture to a final concentration of 0.6 nM and incubated for another hour. As a result, the AuNP absorption band shifted toward longer wavelengths, with the most significant shift observed for the m7G-cap-conjugate in the presence of the highest concentration of eIF4E   showing great increase in RH  Figure A, S10B. jugate 0. nM was i RH Fig. . Changesjugate 0. nM was i7G-cap-TL-AuNP conjugate resulted in smaller absorption band shifts, as expected  to develop a two component sensing system that enabled UV–Vis and naked-eye eIF4E detection.In this study, we demonstrated an approach that enables AuNP surfaces to be modified with nucleotides in a controllable, adjustable, and functionality-compatible manner, and relies on the use of lipoic-acid-modified nucleotides. The utility of this approach was demonstrated by modifying AuNPs with 7-methylguanine RNA cap analogs, which are specific binders of the eIF4E oncogenic translation factor. The abilities of these conjugates to interact with eIF4E was confirmed by UV–Vis, DLS, and TEM studies. The properties of the obtained mOverall, our study highlighted the potential utility of AuNPs in research on cap-binding proteins, taking advantage of the exceptional sensitivities of their UV–Vis absorption properties to changes in monolayer size and composition, as well as intermolecular aggregation. These properties, combined with the potential use of cap-functionalized AuNPs for imaging proteins and protein complex assemblies by TEM, can be harnessed to create novel tools for eIF4E detection and eIF4E inhibitor discovery. The developed approach can be also adjusted to other cap-binding proteins or even de-capping enzymes, provided that properly designed hydrolysis-resistant compounds are used to functionalize AuNPs. The reported AuNP-modification method based on the lipoic acid moiety serving as a molecular anchor with affinity for gold, can be used to obtain other types of nucleotide-functionalized AuNPs, thereby paving the way to studying other nucleotide-binding proteins using similar approaches. Finally, the concept of protein-mediated crosslinking between two types of conjugate can be also adopted to other proteins that bind multiple ligands or allosterically regulated proteins.Lipoic acid and Tris were purchased from Sigma-Aldrich, and all other reagents and solvents were procured from commercial sources. Reactions involving microwave radiation were conducted in 10-mL heavy-walled glass pressurized vials and snap-on caps. Microwave heating was performed in a CEM Discover single-mode microwave cavity, in dynamic power mode with a maximum power of 10 W and at 50 ± 0.5 °C. Reaction mixtures were stirred with a magnetic stir bar during irradiation. Temperature, pressure, and power were monitored during the course of each reaction using the provided software (standard infrared temperature sensor).3-form) column. The column was loaded with the reaction mixture and washed through with excess water to remove metal(II) salt/EDTA complexes. Nucleotides were then eluted using a linear gradient of triethylammonium bicarbonate (TEAB) in deionized water. The compounds were isolated as triethylammonium (TEA) salts after evaporation under reduced pressure with repeated additions of ethanol to decompose TEAB. Yields were calculated on the basis of either sample weight or (preferably) optical milliunits (mOD) of the product. Optical units were measured in 0.1 M phosphate buffer (pH 7 or pH 6 for m7G nucleotides) at 260 nm. Analytical HPLC was performed on an Agilent Technologies Series 1200 instrument using a (RP) Supelcosil LC-18-T HPLC column  with a linear 0–50% methanol gradient in 0.05 M ammonium acetate buffer (pH 5.9) in 7.5 min, or 0–25% methanol in 0.05 M ammonium acetate buffer (pH 5.9) in 15 min, with UV-detection at 260 nm and fluorescence detection (excitation at 280 nm and detection at 337 nm or 450 nm). Semi-preparative HPLC was performed on the same instrument equipped with a Discovery RP Amide C-16 HPLC column  with linear 0–100% acetonitrile gradients in 0.05 M ammonium acetate buffer (pH 5.9) in 120 min, with UV-detection at 260 nm and fluorescence detection (excitation at 280 nm and detection at 450 nm).Synthesized nucleotides were purified by ion-exchange chromatography on a DEAE-Sephadex A-25  and the triethylammonium salt of AMPPEG13  were mixed with ZnCl2  in DMSO (7.9 mL). The mixture was microwave heated  in 20 min intervals until complete conversion to the product was observed by analytical HPLC. The reaction was terminated by the addition of a solution of EDTA  and NaHCO3  in water (72 mL). The reaction product was purified using ion-exchange chromatography on DEAE-Sephadex. The collected eluate was evaporated to afford 3 750 mOD of m7GpppAPEG13 as a solid contaminated with ZnCl2. The product was repurified using semi-preparative RP HPLC. The collected eluate was lyophilized to afford 236 mg (0.23 mmol) of m7GpppAPEG13 as the sodium salt. Yield: 50%.The sodium salt of mPEG13  were mixed with ZnCl2  in DMSO (4 mL). The mixture was microwave heated for 2.5 h and terminated by the addition of a solution of EDTA  and NaHCO3  in water (20 mL). The reaction product was purified by ion-exchange chromatography on DEAE-Sephadex. The collected eluate was evaporated to afford 2 536 mOD (94.3 μmol) of GpppAPEG13 as a solid. Yield: 43%.The sodium salt of GDP-Im  and the triethylammonium salt of AMPPEG13  were mixed with ZnCl2  in DMSO (4 mL). The mixture was microwave heated for 2 h and terminated by the addition of a solution of EDTA  and NaHCO3  in water (36 mL). The reaction product was purified by ion-exchange chromatography on DEAE-Sephadex. The collected eluate was evaporated to afford 3 250 mOD  of ApppAPEG13 as a solid. Yield: 62%.The sodium salt of ADP-Im  and the triethylammonium salt of AMPN,N,N′,N′-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU 1 equiv.), with stirring for 30 min at room temperature. The nucleotide was concurrently dissolved in DMSO to a concentration of 0.1 M, after which the activated lipoic acid was added in 2 equiv. portions to the nucleotide solution in 20 min intervals, until complete conversion to the product was observed by analytical HPLC. The reaction was terminated by tenfold dilution with water and neutralization with acetic acid. The white excess lipoic acid sediment was removed by centrifugation, the reaction product was purified by semi-preparative RP-HPLC, and the collected eluate was lyophilized.The N-hydroxysuccinimide (NHS) ester of lipoic acid was prepared by dissolving lipoic acid in DMSO to a concentration of 0.6 M and adding triethylamine  and 7GpppAPEG13  was separately dissolved in DMSO (300 μL). Coupling was performed according to the general procedure, with activated lipoic acid (14 equiv.) added to the reaction mixture. Reaction was terminated after 2 h by the addition of water (6 mL) and the reaction product was purified by semi-preparative RP-HPLC. The collected eluate was lyophilized to afford 6.9 mg (5.61 μmol) of m7GpppAPEG13Lipo as a solid. Yield: 20%.Lipoic acid , TEA , and TSTU  were dissolved in DMSO (1.01 mL), and the ammonium salt of mm/z calculated for C39H61N11O21P3S2- [M–H]− 1176.27032, measured 1176.27079, Δm/z = 0.40 ppm; 1H NMR  d ppm 1.30  1.44—1.59  1,63  1.75  1.86  1.99  2.18  2.39  3.09  3.14  3.22  3.54  3.58—3.67  3.69  3.99  4.24–4.31  4.34–4.41  4.49  4.51  4.68  5.88  6.01  8.20  8.41  8.92 . 31P NMR ; d ppm − 25.38  − 13.89  − 13.77 .HRMS ESI(−) PEG13  was concurrently dissolved in DMSO (500 μL). Coupling was performed according to the general procedure, with activated lipoic acid (10 equiv.) added to the reaction mixture. The reaction was terminated after 3 d by the addition of water (11 mL) and the reaction product was purified by semi-preparative RP-HPLC. The collected eluate was lyophilized to afford 16.6 mg (13.7 μmol) of GpppAPEG13Lipo as a solid. Yield: 21%. HRMS ESI(−) m/z calculated for C38H59N11O21P3S2− [M–H]− 1162.25467, measured 1162.25328, Δm/z = 1.20 ppm; 1H NMR  d ppm 1.29  1.42–1.60  1,62  1.75  1.85  1,99  2.17  2,38  3.04—3.18  3.22  3,53  3.58–3.75  4.18–4.33  4.33–4.39  4.48  4,52  4,64  4.69  5.81  6,06  7.98  8,21  8.41 . 31P NMR  d ppm − 22.34  −10.84 .Lipoic acid , TEA , and TSTU  were dissolved in DMSO (1.69 mL), and the triethylammonium salt of GpppAPEG13  was concurrently dissolved in DMSO (1.5 mL). Coupling was performed according to the general procedure, with activated lipoic acid (10 equiv.) added to the reaction mixture. The reaction was terminated after 2 d by the addition of water (24 mL). The reaction product was purified by semi-preparative RP-HPLC. The collected eluate was lyophilized to afford 6.6 mg (5.5 μmol) of ApppAPEG13Lipo as a solid. Yield: 3.4%. HRMS ESI(−) m/z calculated for C38H59N11O20P3S2− [M–H]− 1146.25976, measured 1146.25823, Δm/z = 1.33 ppm; 1H NMR  d ppm 1.26  1.38–1.56  1.58  1,74  1.83  1.95  2.15  2.36  3.03–3.16  3,20  3.52  3.57—3.71  4.23—4.38  4,49  4,51  4,60  4.65  6.01  6.03  8.12  8.12  8.25  8,31 . 31P NMR  d ppm − 22.32  − 10.97  −10.87 .Lipoic acid , TEA , and TSTU  were dissolved in DMSO (3.37 mL), and the triethylammonium salt of ApppAN-Hydroxysuccinimide (NHS) ester of lipoic acid was prepared by dissolving lipoic acid  in of DMSO (1.7 mL) to a concentration of 0.6 M, after which TEA  and TSTU  were added and stirred for 30 min at room temperature. Tris(hydroxymethyl)aminomethane  was concurrently dissolved in DMSO (3.2 mL), after which the activated lipoic acid was added to the Tris solution in 2 equiv. portions in 20 min intervals. After 24 h, undissolved Tris sediment was removed by centrifugation and DMSO (14 mL) was added to the reaction mixture to a concentration of 50 mM.The 4 · 3H2O  was added to deionized water (25 mL) to a concentration of 0.25 mM. The reaction mixture was heated to reflux with continuous stirring and a 5% aqueous solution of trisodium citrate  was added and the mixture was further heated under reflux for 20 min. The reaction was terminated by cooling to room temperature.HAuClThe 4E-BP1 peptide sequence was: CCALNN-GG-TRIIYDRKFLMECRNA.1 mM 4E-BP1 peptide stock solution was prepared by dissolving 4E-BP1 in a 1:1 (v/v) mixture of 2,2,2-trifluoroethanol and PBS buffer (pH 7.2). The stock solution was stored at − 18 °C until required.7GpppAPEG13Lipo, GpppAPEG13Lipo, or ApppAPEG13Lipo in water . To obtain Tris-Lipo dilution, appropriate amount of 50 mM solution of Tris-Lipo in DMSO was mixed with m7GpppAPEG13Lipo water solution before mixing with AuNPs to final molar ratio of the ligands of 1:1, 1:2, 1:5, 1:20 or 1:100. The mixture was left for 24 h at room temperature, without stirring. The AuNP solution was shielded from light in every step. Excess ligands and Tween80 were then removed by centrifugation  and the supernatant was collected. The AuNP-cap analog conjugate was then dissolved in HEPES buffer  and filtered through a 0.42-µm filter.A gold nanoparticle solution was gently mixed with a Tween80 solution (3.6 mg/mL) in phosphate buffer  in a 1:1 ratio. The mixture was then left at room temperature for at least 1 h to ensure physical adherence of Tween80 to the AuNP surfaces. The AuNP/Tween80 solution (1.9 mL) was then mixed with mA gold nanoparticle solution was gently mixed with a CALNN peptide solution (0.35 mM) in PBS buffer (pH 7.2) at a 10:1 ratio. The mixture was then left at room temperature overnight for the CALNN peptide to physically adhere to the AuNP surfaces. The AuNP/CALNN solution (2 mL) was mixed with a 4E-BP1 stock solution  and the mixture was left for 24 h at room temperature, without stirring. The AuNP solution was shielded from light. Excess 4E-BP1 and CALNN was removed by centrifugation  and the supernatant was collected. The AuNP-4E-BP1 conjugate was then dissolved in HEPES buffer  and filtered through a 0.42-µm filter.A 0.5 mL aliquot of each conjugate solution (0.6 nM in HEPES buffer) was added into a standard UV–Vis cuvette at room temperature. The conjugate solution was titrated with increasing concentrations of eIF4E protein (0.5 nM to 2.56 µM). After each addition of protein, the mixture was incubated for 3 min at room temperature without stirring, after which the absorption was measured. Absorption spectra were recorded in the 400–800 nm range on the above-mentioned UV-1800 Shimadzu instrument with 0.5 nm resolution.The AuNP-cap analog conjugate (0.6 nM in HEPES buffer) was incubated for 1 h at room temperature with various concentrations of eIF4E protein (5 nM to 2.56 µM). An equal volume of the AuNP-4E-BP1 peptide conjugate solution (0.6 nM in HEPES buffer) was then added, and the mixture was incubated at room temperature for another hour, after which absorption was measured.For the AuNP conjugates, 20 µL of freshly prepared AuNP solution was cast onto a carbon-coated Formvar 300-mesh copper grid and left to dry in air. AuNP TEM images were acquired on a Zeiss Libra 120 EFTEM transmission electron microscope.A 600 pM solution of the m7G-cap-TL-AuNP conjugate  was applied to a glow-discharged carbon-coated 300-mesh cooper TEM grid and stained with 1% uranyl acetate. TEM images were acquired with Tecnai T12 BioTWIN 120 kV Electron Microscope (FEI) with acceleration voltage of 120 kV.The particle sizes of the prepared AuNP conjugates were determined by adding 50 µL (0.15 nM of AuNPs in HEPES buffer) of each sample to a standard low-volume DLS cuvette for measurement at 25 °C. The size distribution of each sample was recorded in triplicate using a 658-nm-wavelength laser. The hydrodynamic radii of the AuNP conjugates were measured using a Wyatt DynaPro NanoStar DLS analyzer.Zeta potentials of AuNP conjugates were determined by adding 1 ml of each sample (0.15 nM of AuNPs in HEPES buffer) to a Zeta deep cell for measurement at 24˚C. Zeta potential of each sample was recorded in triplicate using Zetasizer nano ZS, Malvern.7GpppAPEG13Lipo, CALNN and 4E-BP1 peptides) were measured as dry powders. AuNP conjugate samples were prepared by centrifugation , removal of the supernatant containing excess ligand moieties and/or buffer, dissolving the sample in 1 ml of pure water, second centrifugation followed by supernatant removal and lastly by freeze-drying the sample until completely dry. Dry AuNP conjugate powder was transferred directly to the ATR accessory crystal prior to measurement. FTIR spectra were recorded using Shimadzu Fourier Transform Infrared Spectrophotometer IRPrestige-21. Presented spectra are an average of 60 scans.FTIR spectra of AuNP conjugates were recorded using ATR sampling technique at room temperature. Samples of ligands used for AuNP surface modifications (Tris-Lipo, mSupplementary Figures.


The membrane contact sites (MCSs) between the ER and late endosomes (LEs) are essential for the regulation of endosomal protein sorting, dynamics, and motility. PDZD8 is an ER transmembrane protein containing a Synaptotagmin-like Mitochondrial lipid-binding Proteins (SMP) domain. PDZD8 tethers the ER to late endosomes and lysosomes by associating its C-terminal coiled-coil (CC) with the LE Rab7. To identify the structural determinants for the PDZD8–Rab7 interaction, we determined the crystal structure of the human PDZD8 CC domain in complex with the GTP-bound form of Rab7. The PDZD8 CC contains one short helix and the two helices forming an antiparallel coiled-coil. Two Rab7 molecules bind to the opposite sides of the PDZD8 CC in a 2:1 ratio. The switch I/II and interswitch regions of the GTP-loaded Rab7 form the binding interfaces, which correlates with the GTP-dependent interaction of PDZD8 and Rab7. Analysis of the protein interaction by isothermal titration calorimetry confirms that two Rab7 molecules bind the PDZD8 CC in a GTP-dependent manner. The structural model of the PDZD8 CC–Rab7 complex correlates with the recruitment of PDZD8 at the LE–ER interface and its role in lipid transport and regulation. Most membrane lipids are synthesized in the endoplasmic reticulum (ER) and transported to other membranes by lipid trafficking pathways via vesicles or lipid transfer proteins (LTPs). Most organelles are connected with the ER network by forming membrane contact sites (MCSs), where the two membranes are closely apposed and tethered within a typical gap of 30 nm2. Non-vesicular lipid transport by soluble protein carriers at the MCSs plays a significant role in lipid distribution due to the efficiency and specificity of the lipid transport3.The lipid compositions of membranes are unique for each subcellular organelle in eukaryotes, and the proper distribution of lipids is crucial for the organellar function4. Endosomal maturation requires the sequential recruitment and activation of Rab GTPases, molecular switches that cycle between the inactive GDP- and active GTP-bound states. The GTP-loaded Rab7A (hereafter referred to as Rab7) on the LEs recruits various effectors facilitating endosome transport along microtubule tracks and their fusion with lysosomes and autophagosomes5. Rab7 recruits mainly through the switch regions, structurally diverse effector proteins such as PDZD8, FYCO1, RILP, and Rubicon5.The endocytic machinery sorts and degrades a variety of substances internalized by vesicles and vacuoles for intracellular homeostasis. The communication at the MCSs between the ER and late endosome (LE)/lysosome is critical for endosomal protein sorting, dynamics, and motility6. PDZD8 consists of an N-terminal transmembrane helix, a SMP domain, a PDZ domain, a C1 domain, and the C-terminal coiled-coil domain. PDZD8 was known to form mitochondria-associated ER membranes (MAMs) and mediate the Ca2+ dynamics in neurons6. PDZD8 also localizes at the contact sites between the ER and late endosomes/lysosomes by interacting with the LE Rab77. PDZD8 associates with an ER protein Protrudin and coordinates endocytic flow with lipid transport at the ER–endosome/lysosome interface10. The PDZD8–Rab7-Protrudin complex regulates the plus-end-directed movement of endosomes by recruiting kinesin-1, promoting the recycling and secretion of endosomes13. In contrast, the ORP1L–Rab7–RILP complex regulates the minus end-directed motility of endosomes by binding to the dynein motor complex4. The Rab-binding domains (RBDs) of PDZD8 and RILP bind only to the active form of Rab714. The RBD of RILP adopts an α-helical conformation forming a symmetric coiled-coil dimer14. The RBD of at the C-terminal region of PDZD8 is predicted to have a coiled-coil (CC) structure of about 45 amino acids. However, due to the low sequence similarity to the proteins of known structures, the structure of the PDZD8 CC and its Rab7-binding mode were elusive.PDZD8 is an ER anchored lipid transfer protein containing a lipid-binding module of the synaptotagmin-like mitochondrial-lipid-binding (SMP) domain familyIn this study, to identify the structural determinants for the GTP-dependent interaction of PDZD8 and Rab7, we determined the structure of the human PDZD8 CC and Rab7-GTP complex. The PDZD8 CC forms a flat structure of three α-helices interacting with two Rab7 molecules on opposite sides of the coiled-coil. The PDZD8 CC associates with the active Rab7 by recognizing the switch I/II and interswitch regions. Our structural model of the PDZD8 CC–Rab7 complex is consistent with the role of PDZD8 in membrane tethering and lipid transport at the membrane contact sites.5 space group appeared in a week. The structure of the PDZD8 CC–Rab7 complex was determined at 2.1 Å resolution by molecular replacement method using a structure of the GTP-bound Rab7 , followed by a lipid-binding SMP domain and a PDZ domain. The C-terminal region of PDZD8 contains a C1 domain (residue 841–891) and a coiled-coil domain (residue 1010–1100). The middle region (residue 449–841) connecting the PDZ and C1 domains is made of a long disordered loop of about 400 amino acids  on the α2-α3 helices of PDZD8. Rab7-B contacts with the 19 residues of the PDZD8 CC, forming a buried surface area of 224 Å2 . The DNA encoding human Rab7 (residue 2–195) was sub-cloned into the BamHI/XhoI site of the modified pHIS-2 vector. The dominant active GTP-binding (Q67L) and dominant-negative GDP-binding (T22N) mutants of Rab7 were prepared by point mutagenesis. Escherichia coli strain BL21(DE3) cells transformed with the plasmid encoding the PDZD8 CC or Rab7 were grown to an OD600 of 0.8 at 37 °C in LB medium. Cells were induced by adding isopropyl β-D-1-thiogalactopyranoside to a final concentration of 0.5 mM and were incubated for 12 h overnight at 20 °C prior to harvesting.The full-length clones for human PDZD8  and Rab7  were purchased from 21C Frontier Human Gene Bank . DNA encoding the C-terminal coiled-coil (CC) domain (residues 994–1123) of human PDZD8 was amplified by polymerase chain reaction and subcloned into the NcoI/XhoI site of a modified pHIS2 vectorCells expressing the PDZD8 CC were resuspended in 2X PBS buffer containing 20 mM imidazole (lysis buffer) and lysed by sonication. The supernatant containing the His-tagged PDZD8 CC was loaded to a Ni–NTA affinity column. The Ni–NTA column was thoroughly washed with the lysis buffer. The target protein was eluted from the column using a buffer containing 100 mM Tris–HCl pH 8.0 , 300 mM imidazole. The eluate was concentrated to 10 mg/ml using Amicon Ultra-15 centrifugal filter. The His-tag was cleaved by 10 international unit (IU) of thrombin protease  per 10 mg of recombinant protein. The cleaved sample was subjected to size-exclusion chromatography on a HiLoad Superdex 200 column equilibrated with 20 mM Tris–HCl pH 8.0, 150 mM NaCl. The fractions containing the PDZD8 CC were concentrated by the centrifugal filter to 10 mg/ml for crystallization. The Rab7 Q67L or T22N mutant were purified by the same procedure as the PDZD8 CC. To inhibit GTP hydrolysis of the PDZD8–Rab7 complex required for crystallization, we used a dominant active Q67L mutant of Rab7. The purified Rab7 Q67L was supplemented with the final 1 mM of GTP.2-gas stream (~ 100 K) during diffraction experiments. Native diffraction data for the PDZD8 CC–Rab7 were collected at a fixed wavelength of 0.97949 Å using an ADSC Q270 CCD detector on the 7A beamline at Pohang Light Source (PLS), Pohang Accelerator Laboratory. All data were processed and scaled using HKL-2000. The structure of PDZD8 CC–Rab7 complex was determined by molecular replacement using the structure of human Rab7 (PDB code: 1T91) as a search model. Two molecules of Rab7 were found in the asymmetric unit using the program Phaser33, and the density-modified map showed clear electron densities of the PDZD8 CC and Rab7. The final model including two molecules of Rab7 and one molecule of PDZD8 CC was refined to Rwork/Rfree values of 21.1%/25.7% using the software Phenix34. The figures of all PDB structures were drawn using the software PyMOL (https://pymol.org).To obtain the crystals of the PDZD8 CC–Rab7 complex, we mixed the purified PDZD8 CC and Rab7 proteins with a 1:1 molar ratio at a total 10 mg/ml, and incubated for 1 h at room temperature before crystallization setup. Preliminary crystallization experiments were carried out at 22 °C in 96-well crystallization plates using customized crystallization screening solutions by dispensing 0.8 μl protein solution and 0.8 μl precipitant solution. The hexagonal crystals of the PDZD8 CC–Rab7 appeared in a week using a solution consisting of 0.1 M Tris–HCl pH 8.0, 20% PEG 1000, 0.1 M NaCl in the 96 well plates. The crystallization condition was further optimized to 0.1 M Tris–HCl pH 8.0, 25% PEG 1000, 0.1 M NaCl using the hanging-drop technique in 15-well screw-cap plates. A drop consisting of 1.5 μl protein solution was mixed with 1.5 μl precipitant solution and equilibrated against 1 ml reservoir solution. High-quality crystals with dimensions of 0.1 × 0.1 × 0.15 mm appeared in a week. Crystals of the PDZD8 CC–Rab7 were cryoprotected in reservoir solution supplemented with 10% glycerol and flash-cooled by immersion in liquid nitrogen. Crystals were preserved in a cryogenic Nd, and the stoichiometry value, n, were calculated from the measured heat changes, δHi, upon the association of the MBP-PDZD8 CC and Rab7. The titration data were analyzed using the NanoAnalyze program (TA instruments) and fitted into a two-site binding model for wild type and an independent binding model for mutants.The protein–protein interaction of the PDZD8 CC and Rab7 was analyzed by isothermal titration calorimetry (ITC) using an Affinity ITC calorimeter  at 20 °C. The PDZD8 CC constructs used for the ITC experiments were fused to the His-tagged maltose-binding protein (MBP) in their N-termini for protein stability. The purified Rab7 Q67L and Rab7 T22N were loaded with 1 mM of GTP and GDP, respectively. All proteins were prepared in the identical buffer containing 20 mM Tris–HCl pH 7.5, 150 mM NaCl. The syringe was loaded with 1 mM of the MBP-PDZD8 CC and the cell was filled with 300 μl of 0.1 mM Rab7. The titration curve was obtained by injecting 2 μl × 25 aliquots of the MBP-PDZD8 CC into the cell at a time interval of 300 s. The enthalpy of reaction, ΔH0, the binding constant, KSupplementary Information.