Periodontitis is one of major oral diseases, which has no consensus on early screening tool. This study aimed to compare the association and screening ability of S100A8 and S100A9 in saliva, blood and gingival crevicular fluid (GCF) for periodontitis status.We recruited 149 community Korean adults, 50 no or initial periodontitis (NIPERIO) and 99 established periodontitis (PERIO). Using clinical attachment loss and a panoramic radiograph, stage II–IV of new classification of periodontitis proposed at 2018 was considered cases as PERIO. Enzyme linked immunosorbent assay kit was used to quantify S100A8 and S100A9. T-test, analysis of covariance, Mann–Whitney test and correlation analysis were applied to compare the relationship of S100A8 and S100A9 in saliva, blood, and GCF for periodontitis. Receiver operating characteristic curve was applied for screening ability.p < 0.05). However, S100A8 and S100A9 in GCF were higher in NIPERIO (p < 0.05). The screening ability of salivary S100A8 was 75% for PERIO, while that of GCF S100A8 was 74% for NIPERIO. Salivary S100A8 was positively correlated to blood S100A8 (p < 0.05).Among S100A8 and S100A9 in saliva, blood and GCF, S100A8 in saliva was significantly higher in PERIO than in NIPERIO (Salivary S100A8 could be a potential diagnostic marker for established periodontitis and be useful for screening established periodontitis.The online version contains supplementary material available at 10.1186/s12903-021-01749-z. Periodontitis, the major oral disease, is a polymicrobial infectious disease that is related with systemic inflammation, destroys supporting tissue around the tooth and ultimately leads to tooth loss , 2. SystEarly detection of periodontitis is necessary for public health in a preventive dimension, because it leads to tooth mobility, tooth loss, mastication deficiencies and digestive problems . PeriodoBiofluids like blood, saliva, urine, tears have been used as source of biomarkers for certain disease . ScientiS100A8 and S100A9 are a subgroup of molecules within the broader family of S100 calcium-binding protein and it has ability to bind with zinc. These proteins are mostly expressed on neutrophils and monocytes or macrophages . PreviouHence, the present study aimed to compare the association and screening ability of S100A8 and S100A9 in saliva, blood and GCF according to the periodontitis status.This cross-sectional study randomly selected participants from public advertisement. All of the participants voluntarily provided a written informed consent. The Institutional Review Board for Human Subjects of Seoul National University Dental Hospital reviewed and granted the ethical consideration for this study CRI17009). This study was organized according to the checklist of items included in STROBE   showed that S100A8 in saliva  was 8.0 ± 10.2 for established periodontitis (PERIO) patients versus 6.1 ± 8.3 for no or initial periodontitis (NIPERIO) participants. Under the condition of type I error of 0.05, type II error of 0.8 and ratio of 2 between PERIO and NIPERIO participants, the sample size of this study was estimated as total of 147 (98 PERIO and 49 NIPERIO). Sample size was estimated 18 (12 PERIO and 6 NIPERIO) for S100A8 in GCF (0.4 ± 0.4 for NIPERIO vs. 0.15 ± 0.3 for PERIO) and 27 (18 PERIO and 9 NIPERIO) for S100A8 in blood (600 ± 240 for PERIO vs. 490 ± 210 for NIPERIO). The estimated sample size for S100A9 was 141 (94 PERIO and 47 NIPERIO) in saliva (1.2 ± 1.9 for NIPERIO vs. 1.6 ± 2.1 for PERIO), 21 (14 PERIO and 7 NIPERIO) in GCF (0.2 ± 0.2 for NIPERIO vs. 0.08 ± 0.2 for PERIO) and 39 (26 PERIO and 13 NIPERIO) in Blood (159 ± 28 for NIPERIO vs. 170 ± 24 for PERIO). Finally, we decided the total samples size of this study was 147 (98 PERIO and 49 NIPERIO).The total of 149 community Korean adults, 50 no or initial periodontitis and 99 established periodontitis were recruited. The inclusion criteria for participants of this study were five-fold. (1) Those who agreed to take periodontal examination including clinical attachment loss (CAL) and panoramic radiograph according to the new international periodontal classification guideline by American Academy of Periodontitis and European Federation of Periodontology , (2) ThoTrained dentists examined CAL and radiographic bone loss by using panoramic radiograph . CAL was calculated by adding up pocket depth and gingival recession using a UNC-15 probe in all of the natural teeth except 3rd molar. Periodontal status was categorised according to the guidelines of 2017 American Association of Periodontology—European Federation of Periodontology workshop in Periodontology . Among o ≥ 4 mm. BOP was evaluated using the guideline in a previous study [Plaque index (PI), pocket depth (PD) and bleeding on probing (BOP) were considered as periodontal clinical parameters. PI was evaluated by Turesky modification of the Quigley–Hein Index . PD was us study . Finallyg for 15 min at 4 °C, aliquoted supernatants into 1 ml in sterilised 1.5 ml Eppendorf tube and stored the aliquoted saliva samples in – 80 °C deep-freezers for further analysis.Information about the standard sampling protocol for saliva collection was provided to each participant: not to brush tooth, not to drink or eat one hour before sampling. In order to maintain consistency of samples, we collected unstimulated whole saliva using passive drooling method for 10 min in a 50 ml conical tube. We centrifuged the saliva by 2600×g for 15 min at 4 °C) and supernatants were aliquoted into 1 ml in sterilised 1.5 ml Eppendorf tube. The tubes with GCF samples were stored at stored at − 80 °C for further analysis.Radiographic evaluation and periodontal probing using UNC-15 probe were applied to decide the deepest pocket among all teeth pockets. GCF samples were obtained from the deepest pocket. During GCF sampling, we tried to avoid blood and saliva contamination. After isolating the tooth with cotton rolls, three absorbent paper points  were gently inserted in the same deepest pocket for 30 s. Paper points were immediately placed in a cryovial containing 1 ml of phosphate buffer saline in pH 7.4, which were centrifuged (2600×g for 15 min at 4 °C) and supernatants (plasma) were aliquoted into 1 ml in sterilised 1.5 ml Eppendorf tube. The tubes were then stored at − 80 °C for further analysis.Blood of 4 ml was drawn by venepuncture by a trained medical technologist. The blood samples were centrifuged  kit  with validation for cell culture supernatant and blood sample through manufacturer’s instruction. Standard curve was drawn using standard S100A8 and S100A9 supplied by the manufacturer. GCF and saliva samples were diluted on concentration dependent using reagent diluent provided by manufacture  and diluted sample concertation for S100A8 and S100A9 were calculated from standard curve of S100A8 and S100A9. Similarly, blood samples were diluted on concentration dependent manner with reagent diluent  and concentrations of S100A8 and S100A9 were estimated using standard curve. We decided the standard dilution rate that falls on the range of 500–1000 pg/ml on pilot study.We considered sociodemographic factors such as sex and education, behavioural factors such as smoking and drinking, and systemic health information including obesity, diabetes, hypercholesterolemia and hypertension as confounders. Face to face interview, laboratory blood analysis and physical examination were applied to collect the data for confounders. We dichotomised following variables: education (until middle school vs. above high school), smoking (smoker encompassing past and current smoker versus non-smoker who has never smoked during the lifetime), drinking . We classified four systemic health components as follows: (1) obesity: body mass index (BMI) calculated as kg of body weight divided by square meter of height ≥ 25, (2) diabetes: high plasma glucose level (> 126 g/dl) or having anti-diabetic medication, (3) hypercholesterolemia: high plasma cholesterol level (> 240 mg/dl) or having anti-hypercholesterolemia medication, and (4) hypertension: systolic > 130 mmHg or diastolic > 85 mmHg or having anti-hypertensive medication. The blood pressure was measured by physicians in the sitting position using mercury manometer. We assayed biochemical variables using 12-h fasting blood samples drawn at recruitment.p value < 0.05. Data were analysed using Statistical Package for Social Sciences version 25 .The distribution of characteristic variables by periodontitis status (NIPERIO vs. PERIO) were addressed using mean values with standard deviations (SD) for continuous variables, and frequencies and proportion for categorical variables. Chi-square test was applied for categorical variables. Kolmogorov–Smirnov (K–S) test was applied to evaluate the normal distribution for continuous variables. When variables were in normal distribution, parametric tests were applied, otherwise non-parametric tests were applied. T-test were performed to evaluate difference in continuous variables with normal distribution. Mann–Whitney (M–W) test were applied for continuous variables without normal distribution. The relationships between values in the blood, GCF and saliva were analysed with Spearman’s correlation test. Since number of participants were big (n = 149), Analysis of covariance (ANCOVA) was applied to estimate adjusted mean with standard error (SE) of S100A8 and S100A9 levels after controlling for age, sex, education, smoking, drinking, diabetes, hypercholesterolemia, hypertension and obesity. The receiver operating characteristic (ROC) curve was applied for estimating c-statistics (area under the curve: AUC) as screening ability of S100A8 and S100A9 for periodontitis status. Statistical significance was set at p < 0.05) (Table p > 0.05).The participants of this study aged from 21 to 77 years. The PERIO participants were older, more males, more hypertensive and more obese than the NIPERIO participants (p < 0.05)  Table .p < 0.05). The representative level of S100A8 in saliva was higher in PERIO participants than in NIPERIO participants  . However, S100A9 in GCF was lower in PERIO participants than in NIPERIO participants . S100A9 in saliva and blood showed no difference.S100A8 and S100A9 in saliva, blood and GCF were not in normal distribution  (Table p > 0.05). In blood, the adjusted value of S100A8 and S100A9 were not significantly different according to periodontitis status . However, the adjusted values of S100A8 and S100A9 in GCF were higher by around 2.5 times in NIPERIO participants than in stage II and stage III–IV periodontitis participants .The adjusted value of S100A8 in saliva, after controlling for confounders, was also higher by 1.6 and 1.8 times in stage II and stage III–IV periodontitis participants than in NIPERIO participants  Table . That ofp < 0.05) . However, S100A9 in saliva was negatively correlated to that in GCF among PERIO patients .Scatter plot showed that S100A8 in saliva was positively correlated to that of blood , while that of salivary S100A9 was 0.50 (p > 0.05). S100A8 and S100A9 in GCF showed high screening ability of 0.74 and 0.62 for NIPERIO , respectively. S100A8 and S100A9 in blood showed non-significant c-statistics of 0.5 (p > 0.05).ROC curve showed that salivary S100A8 had highest screening ability for PERIO among S100A8 and S100A9 in saliva, blood, and GCF Fig. . The scrOur data showed that salivary S100A8 had the most appropriate screening ability for periodontitis among S100A8 and S100A9 in saliva, blood and GCF. To the best of our knowledge, this is the first evidence that salivary S100A8 could be the best maker for screening established periodontitis after comparing among S100A8 and S100A9 in saliva, blood and GCF. This result was supported by the previous evidence that salivary S100A8/9, calprotectin, was a significant maker for periodontitis .Comparing with the previous study, our study had some advantage. Firstly, this study compared S100A8 and S100 A9 levels among saliva, blood and GCF. Secondly, sufficient 149 number of participants were randomly recruited from the general population and there was no selection bias. Thus, our results could be generalized. Thirdly, age, sex, smoking, drinking, education, diabetic, hypercholesterolemia, hypertension and obesity were considered as confounders for the adjustment. Fourthly, physical and dental examination were performed by physicians and trained dentists using UNC-15 probes and a panoramic radiograph. Fifthly, periodontitis was classified according to the recent New international classification of periodontitis . FinallyOur data showed that elevated levels of S100A8 in saliva were significantly associated with PERIO in Korean adults. A recent Korea study reported that salivary S100A8 levels were higher by 70% in periodontal disease than that of healthy participants . S100A8 S100A9 involved in the regulation of inflammatory processes and immune response . CalprotOur data showed that S100A8 in saliva and blood was positively correlated each other. This link was higher in healthy adults compare to in periodontitis patients. These results showed the evidence that saliva represented local and systemic inflammation via GCF and blood, while blood represented only systemic inflammation. Hence, salivary S100A8 showed highest screening ability among S100A8 and S100A9 in saliva, GCF and blood. Contrary to previous studies , 31, salOur data showed that the screening ability of S100A8 for established periodontitis was 0.73 of c-statistics, which was higher than the previous Korean study  with 0.6There are some limitations of this study. Firstly, samples were analysed using ELISA were stored more than one month. Long term storage of saliva might influence on the detection of salivary protein . SecondlOverall, elevated level of salivary S100A8 protein concentration could be a valid marker for established periodontitis screening. Thus, S100A8 salivary kit will be useful for screening established periodontitis. Further prospective studies including periodontal treatment will be indicated for elucidating the prognostic effect of salivary S100A8 for the promotion of periodontal health.Additional file 1. STROBE Statement—Checklist of items that should be included in reports of observational studies.


However, the importance of the equivalent Rpt prolines for eukaryotic proteasome assembly was unknown. Here we showed that the equivalent proline is highly conserved in Rpt2, Rpt3, and Rpt5, and loosely conserved in Rpt1, in deeply divergent eukaryotes. Although in no case was a single Pro-to-Ala substitution in budding yeast strongly deleterious to growth, the rpt5–P76A mutation decreased levels of the protein and induced a mild proteasome assembly defect. Moreover, the rpt2–P103A, rpt3–P93A, and rpt5–P76A mutations all caused synthetic defects when combined with deletions of specific proteasome base assembly chaperones. The rpt2–P103A rpt5–P76A double mutant had uniquely strong growth defects attributable to defects in proteasome base formation. Several Rpt subunits in this mutant formed aggregates that were cleared, at least in part, by Hsp42 chaperone-mediated protein quality control. We propose that the conserved Rpt linker prolines promote efficient 26S proteasome base assembly by facilitating specific ATPase heterodimerization.The proteasome is a large protease complex that degrades many different cellular proteins. In eukaryotes, the 26S proteasome contains six different subunits of the ATPases associated with diverse cellular activities family, Rpt1–Rpt6, which form a hexameric ring as part of the base subcomplex that drives unfolding and translocation of substrates into the proteasome core. Archaeal proteasomes contain only a single Rpt-like ATPases associated with diverse cellular activities ATPase, the proteasome-activating nucleotidase, which forms a trimer of dimers. A key proteasome-activating nucleotidase proline residue (P91) forms  Saccharomyces cerevisiae) that form a heterohexameric Rpt ring in the order Rpt1-2-6-3-4-5 , Nas6 (p28/gankyrin/PSMD10), Rpn14 (PAAF1), and Hsm3 (S5b/PSMD5) .A)  , 13. TheFig. 1A) , 15. SimA) , 17.FiguMethanocaldococcus jannaschii, the PAN ring arranges in a trimer-of-dimers configuration (cis-conformation in one subunit of the dimer and trans-conformation in the other (B)  are in a cis-conformation . A. AMethanormation . The higno acids . Despitenfolding , 21. Celnfolding .M. jannaschii PAN–P91A and PAN–P91G mutant proteins in vitro was unsuccessful because the complexes were unstable, further highlighting the importance of this residue in PAN ring assembly  in ARC-N (consisting of CC and OB domains) was found to significantly reduce the ability of the complex to inhibit aggregation of denatured citrate synthase and luciferase, suggesting that the conserved proline is important for activity (An attempt to characterize recombinant assembly . In anotactivity .S. cerevisiae (C) , Rpt2 (P103), Rpt3 (P93), and Rpt5 (P76) in revisiae C 24). D. DS. cerS. cerevisiae. Furthermore, we provide evidence for distinct contributions of the Hsp42 chaperone and the Not4 ubiquitin ligase in promoting base assembly in yeast expressing proline-to-alanine mutations in both Rpt2 and Rpt5 by suppression of the aggregation of these subunits.Here, we show that, collectively, the conserved linker proline residues in Rpt2, Rpt3, and Rpt5 are important for proper proteasome base assembly in A; cis-peptide conformation. This should kink the CC–OB linker and facilitate helix interaction and CC formation with the appropriate trans-Rpt partner . To investigate if these mutations affect proteasome assembly, we subjected whole-cell lysates from these strains to native gel immunoblot analyses. Despite their lack of obvious growth impairment, the rpt3–P93A and rpt5–P76A mutants exhibited defects in proteasome assembly based on excess accumulation of free lid subcomplex, CP and Blm10-CP  (C and D). The rpt5–P76A mutant suffered a more pronounced assembly impairment based on the decreases in doubly capped 26S proteasomes and the Rpt4–Rpt5 intermediate (D). We also observed a characteristic smear or doublet near the position where the Rpt4–Rpt5 complex normally migrates (D). These species might represent either a unique Rpt5-containing assembly intermediate or a dead-end complex.To determine the importance of these prolines nditions B. To invgulator) , C and Drmediate D. We alsmigrates D. These hsm3Δ; loss of the Hsm3 RP assembly chaperone (RAC) has the strongest effect on growth of any single RAC mutant . The rpt–PA single mutations might not be sufficiently detrimental to require Adc17 for enhanced base assembly. In support of this, we found that adc17Δ displayed a slight synthetic defect with the more deleterious rpt2–P103A rpt5–P76A double mutant (B). In addition, there is likely redundancy among the three chaperones affecting the Rpt3–Rpt6 module. Nas6 and Rpn14 could still promote proper assembly of Rpt3–Rpt6 in the absence of Adc17. No additional growth defects were observed when these single Rpt mutations were crossed into yeast strains with the CP assembly chaperone deletions pba1Δ or pba4Δ. Notably, rpt1–P96A, which affects the ATPase subunit with the least conserved linker proline, did not exhibit synthetic defects with any tested base assembly chaperone deletion . This was not observed when rpn4Δ was combined with rpt1–P96A, rpt2–P103A, or rpt3–P93A (A). The growth defects of rpn4Δ rpt5–P76A were paralleled by strongly reduced 26S proteasome formation in vivo (B). Quantitative RT-PCR analysis also revealed slight but consistent increases in proteasome subunit transcript levels in rpt5–P76A relative to RPT5 cells, as expected if Rpn4-induced transcription was partially compensating for reduced RP base assembly in the mutant (B). When we made Ala substitutions in the two residues flanking Rpt5–P76 (Rpt5–L75A and Rpt5–Y77A), little or no synthetic defect was seen with RPN4 deletion, demonstrating the specificity of the rpn4Δ rpt5–P76A interaction (C).Of the four individual Pro-to-Ala mutants analyzed,  reduced , 26. Indpt5–P76A A. This wpt3–P93A A. The gr in vivo B. Quantie mutant B. When weraction C.Figure Escherichia coli and determined the solubility of these proteins via a pelleting assay. For this, we lysed bacterial cells expressing each protein under nondenaturing conditions and subjected the lysates to centrifugation. The supernatant (S) fraction contained soluble proteins, and the pellet (P) fraction included aggregated proteins. Rpt5–P76A had a much higher propensity to aggregate than WT Rpt5, as most of the mutant protein was in the P (D). This observation was paralleled by the finding that steady-state levels of soluble Rpt5 were lower in rpn4Δ rpt5–P76A than in rpn4Δ RPT5 yeast (E). These data suggest that Rpt5–P76A is prone to misfolding and aggregation. Rpt5–P76A aggregation is associated with proteasome assembly defects in mutant yeast cells and consequently, upregulation of proteasome subunit genes via Rpn4 to compensate for the depletion of the compromised mutant subunit. To investigate if there is a defect in Rpt5–P76A binding to Rpt4 in yeast, we conducted anti-FLAG immunoprecipitations using Nas2–3xFLAG expressed in RPT5 and rpt5–P76A cells. As noted above, Nas2 forms a ternary assembly complex with Rpt4 and Rpt5 , resulted in a growth defect, which was severely exacerbated at an elevated temperature  + 4 μM canavanine plates, although the defect was not nearly as severe as that of rpt2,5PA .In the archaeal PAN ATPase, the single PAN–P91A mutation disrupts all subunits of the hexamer but in particular the three “assembly . We nextFig. 4A; . To deterpt2,5PA mutant had a pronounced proteasome assembly defect characterized by accumulation of free CP, Blm10-CP, and lid subcomplex and decreased levels of singly (RPCP) and especially doubly capped (RP2CP) 26S proteasomes (C). In addition to these species, the double mutant also accumulated a number of novel Rpt2-containing subcomplexes, which might be dead-end complexes (C). Interestingly, these subcomplexes were also found in hsm3Δ RPT2 cells and at higher levels in hsm3Δ rpt2–P103A . Based on anti-Rpt5 immunoblotting, the Rpt4–Rpt5 complex is also present at 30°C but not at 37 °C. This assembly intermediate may be less stable or unable to form at elevated temperatures (C). Consistent with the decrease in level of full proteasomes in rpt2,5PA, the mutant cells exhibited lower total proteasomal peptidase activity (D) and an increased accumulation of cellular ubiquitin conjugates (E). Defects in proteasome assembly and activity in the double mutant were worse at elevated temperatures (C–E).The teasomes C. In addomplexes C. Interet2–P103A . Because mutants ; Fig. S1unstable C. Based eratures C. Consisactivity D and an njugates E. Defecteratures , C–E.rpt2,5PA did not decrease at either a permissive or nonpermissive temperature (A). In fact, overall levels of subunits increased despite the strong reduction in fully formed proteasomes in the mutant strain, hinting at a reduced elimination of defective proteasome subunits/subcomplexes. Indeed, we observed an accumulation of high-molecular-weight Rpt5-containing species that could be ubiquitinated forms of Rpt5-PA (A). To confirm this, we conducted ubiquitin pull-down assays and found higher levels of ubiquitinated Rpt5 in the rpt2,5PA mutant than in the WT strain (B).Analyses of steady-state levels of proteasome subunits revealed that the overall levels of subunits in perature A. In fac Rpt5-PA A. To conT strain B.Figure rpt2,5PA cells because of the presence of these mutant Rpt5 species. Indeed, when NOT4 was replaced with the catalytic mutant not4–L35A, the growth defect of rpt2,5PA cells was partially suppressed (C). The enhanced growth correlated with a reduction in ubiquitinated Rpt5 in rpt2,5PA not4–L35A cells based on ubiquitin pull-down assays (D). Not4 regulation of proteasome assembly is likely specific to RP base mutants as not4–L35A did not suppress the temperature sensitivity of either pre9Δ (CP subunit) or sem1Δ (lid subunit) mutant (It was previously reported that Rpt ring assembly is regulated through selective ubiquitination of Rpt5 by the E3 ligase Not4 . When ubppressed C. The enn assays D. Not4 r) mutant .rpt2,5PA did not decrease even at elevated temperatures (A), and levels of full proteasomes and soluble Rpt intermediates/subcomplexes were further reduced at elevated temperatures (C), we speculated that Rpt subunits in this mutant have the propensity to be sequestered either for storage for future use or as insoluble aggregates for degradation or elimination via mother cell retention. We conducted aggregation assays using yeast whole-cell lysates to determine if the subunits form insoluble aggregates at elevated temperatures (A). We found that the double mutant had a higher P-to-S ratio for Rpt2 and Rpt5 subunits than WT, suggesting that these subunits aggregate in the mutant (B). Unlike Rpt2 and Rpt5, aggregation was not as prominent for the CP subunit α4 and was absent for lid subunit Rpn12 in the mutant strain (A). Rpt3 also aggregated in the mutant but Rpt4 did not (A). However, overall levels of Rpt4 seemed to be lower in the mutant in samples collected from saturated cultures, suggesting that Rpt4 expression might be suppressed or that it is selectively degraded (A). The smears observed predominantly above Rpt2 and Rpt3 monomers in total protein (T) and P fractions in Rpt2 and Rpt3 immunoblots are likely primarily SDS-resistant aggregates that are recognized by the Rpt2 and Rpt3 antibodies. Ubiquitin pull-down assays suggested that the smear above Rpt3 monomer does contain a small fraction of ubiquitinated Rpt3 although the levels were similar in both WT and mutant strains (B). In addition to proteasome subunits, we also examined potential aggregation of the RACs Hsm3, Nas2, Nas6, and Rpn14. Only Nas2 appeared to aggregate slightly more in rpt2,5PA than WT cells (versus 7–9 in each blot).Because overall levels of Rpt subunits in eratures A, and leeratures C, we speeratures A. We foue mutant B. Unliket strain A. Rpt3 a did not A. Howevedegraded A. The sm strains B. In addrpn5ΔC) forms aggregates at elevated temperatures, which are regulated by the protein quality control (PQC) machinery, most notably heat-shock protein Hsp42  . Levels of Rpt2-containing intermediates also decreased slightly but reproducibly in hsp42Δ rpt2,5PA relative to rpt2,5PA cells, suggesting either fewer of these potential dead-end complexes form or, if functional, greater incorporation into higher-order complexes (B). We also observed a small increase in the Rpt4–Rpt5 complexes in hsp42Δ rpt2,5PA cells along with reduced free lid accumulation, consistent with weakly enhanced proteasome RP assembly (B). Aggregation assays showed slightly reduced aggregation of Rpt2, Rpt5, and possibly also the Nas2 chaperone, which would be consistent with enhanced formation of the Nas2–Rpt4–Rpt5 assembly module  and CP (pre9Δ) mutants (A previous study showed that a lid mutant (in Hsp42 . DeletioC mutant . We askef rpn5ΔC A 28). T. Trpn5ΔCy assays , B and Complexes B. We alsassembly B. Aggregy module D and S9. mutants . These frpt2,5PA mutant, we created an rpt2,5PA strain with both Not4 and Hsp42 inactivated and compared its growth with rpt2,5PA strains with mutation of either Not4 or Hsp42 alone. Inactivation of both Hsp42 and Not4 simultaneously further enhanced the growth of rpt2,5PA cells (A). This growth enhancement is consistent with a further reduction in aggregated Rpt5 in the P (B). Hence, these factors appear to modulate proteasome assembly by at least partly separate pathways.To investigate the epistatic relationship between the enhanced growth caused by inactivation of Not4 E3 ligase activity  or Hsp42PA cells A. This gin the P B. Hence,cis-peptide bonds, which would create a kink in the linker that allows the upstream helical domain of the subunit to form a CC more readily with its (trans) ATPase partner . For most of the mutants, proteasome assembly appears normal or nearly so. Despite the lack of a growth defect, the rpt5–P76A mutant did display a modest proteasome assembly deficiency (D). The lower solubility of Rpt5–P76A is reminiscent of the aggregation seen with the homologous archaeal PAN–P91A mutation . It is difficult to disentangle reduced solubility of Rpt5–P76A protein and defective heterodimerization with Rpt4 as the primary cause of the rpt5–P76A base assembly defect because they are related. Quantitative binding analysis with separately purified Rpt4 and Rpt5 will be required to determine if Rpt5–P76A has lower affinity toward Rpt4, although our finding that recombinant Rpt5 expressed in E. coli forms homo-oligomers would complicate such an analysis (unpublished observations).Nas2 coprecipitation analysis revealed similar ratios of Rpt4 and Rpt5 in mutation . There amutation . Second,mutation B. It is cis” subunits of the archaeal homohexamer. Consistent with such a quantitative effect, base assembly is far more severely disrupted in the double rpt2–P103A rpt5–P76A mutant (C). This may provide additional flexibility in CC formation between Rpt1 and Rpt2. We note the presence of a glycine residue in place of proline in the Rpt4 and Rpt6 “trans” subunits, which may also facilitate correct CC formation in their respective heterodimers. The glycine in Rpt4 (G104) is particularly well conserved.Assembly of an Rpt heterohexamer in eukaryotes, unlike a homohexamer, may limit growth and assembly defects because of single Pro-to-Ala mutations; introduction of a single PAN–P91A mutation, by contrast, effectively disrupts all three “A mutant . Other fA mutant C. This mrpt2–P103A, rpt3–P93A, and rpt5–P76A mutations have strong synthetic defects with hsm3Δ and with other select RAC gene deletions . The reduced accumulation of these unique complexes without an increase in higher-order complexes and full proteasomes at elevated temperature further suggests that these complexes are unstable and subsequently degraded and/or sequestered into aggregates.The  defects . We spec strains C. The rerpt2,5PA strain, suggesting that the misassembled ATPase subunit is marked for ubiquitin-dependent degradation. Expression of the Not4–L35A ubiquitin ligase catalytic mutant partially suppresses the growth defect and levels of ubiquitinated Rpt5–P76A in rpt2,5PA mutant, consistent with the presence of a base assembly defect and a role for Not4 in regulating base assembly in this mutant . Th. Thrpt2,rpt2,5PA double mutant (B). We attempted to tag the N termini of Rpt2 and Rpt5 with GFP to track these aggregates via microscopy, but the resulting strains were inviable. Nevertheless, we found that deletion of HSP42 partially suppressed growth defects of rpt2,5PA, although not to the extent seen with the lid mutant rpn5ΔC (A) . Indeed, our data indicate that Hsp42 is a general regulator of proteasome assembly, as revealed by the ability of hsp42Δ to also suppress other RP base (cim3-1 and rpt4–G106D) mutants and a CP (pre9Δ) mutant . This finding provides evidence for two distinct but complementary pathways that regulate the assembly of the proteasome base, at least when components of the base are defective.Rpt2 and Rpt5 subunits are found in aggregates in the e mutant B. We attt rpn5ΔC A 28). T. Trpt2,5 mutants , A and B) mutant . Interesassembly  but alsoassembly , 33, 34 assembly , 36. Notrpt2,5PA A. This fcis-peptide bonds specifically in one subunit of each eukaryotic Rpt heterodimer, which facilitates their dimerization with the correct “trans” subunits, presumably through enhanced CC formation. Yeast cells have multiple mechanisms that allow them to tolerate mutations in the highly conserved linker prolines of the three predicted “cis” subunits Rpt2, Rpt3, and Rpt5. These include the Rpn4-dependent transcriptional feedback loop and Rpt heterodimer-specific assembly chaperones. On the other hand, PQC mechanisms that result in ubiquitination and degradation of mutant or misassembled subunits of the proteasome or the sequestration of aberrant assembly intermediates into Hsp42-dependent aggregates such as insoluble protein deposits enhance growth deficiencies of these mutants. Together with the recent description of cotranslational assembly of Rpt heterodimers, our results point to the importance of multiple mechanisms, which are likely intertwined, to ensure efficient and high-fidelity assembly of the eukaryotic 26S proteasome.In summary, our data are consistent with the hypothesis that the highly conserved Rpt linker prolines promote formation of HIS3 cassette as described previously  on plasmids bearing a URA3 selectable marker. Strains were then transformed with plasmids carrying either a TRP1 or LEU2 selectable marker and expressing either WT or mutant rpt alleles. The resulting strains were then cured of the original URA3 plasmid by counterselection on 5-fluoroorotic acid. The list of yeast strains and plasmids used can be found in Yeast strains were made following standard procedures . Yeast heviously . BecauseYeast strains were grown in a yeast extract-peptone-dextrose (YPD)-rich medium or SD media to saturation overnight. The next day, strains were diluted in sterile water at 0.2 units of absorbance at 600 nm in a final volume of 1 ml. Samples were then spotted in a 6-fold dilution series on the appropriate plates and incubated at various temperatures, and growth was monitored over several days.2 were ground using a mortar and pestle until a fine powder formed. The resulting powder was collected in a pre-chilled tube and incubated in proteasome extraction buffer  for 10 min with occasional vortexing. Samples were then centrifuged at 22,000g for 10 min to remove unlysed cells and cell debris. The resulting S were collected, and the protein concentration was determined using the bicinchoninic acid (BCA) assay conducted according to manufacturer's instructions (Thermo Fisher Scientific). Fifty microgram samples were electrophoresed in 4% nondenaturing polyacrylamide gels (native PAGE). Gels were either overlayed with a fluorogenic substrate, Suc-LLVY-AMC (Sigma-Aldrich) or were used in immunoblot analyses. Details of the experimental procedures for the in-gel peptidase assay are as described (E), 10 μg of each S was run in denaturing SDS gels and subjected to immunoblotting.Yeast extracts for nondenaturing gel analyses were prepared as previously described with slight modifications . Yeast cescribed . For anaescribed E, 10 μg g for 1 min, and S were discarded. Pelleted cells were resuspended in 1× SDS-PAGE sample buffer containing 4% β-mercaptoethanol (BME) and heated at 100 °C for 5 min followed by centrifugation at 10,000g for 1 min; 10 to 15 μL of the S was resolved in discontinuous SDS gels and subjected to immunoblot analyses.Yeast extracts for denaturing gel analyses were prepared as previously described with slight modifications . Yeast cg for 5 min at 4 °C. The S was transferred into a new tube. The P was washed once with 600-μL lysis buffer and recentrifuged as above; the lysis buffer was removed, and the P was resuspended in 600-μL lysis buffer. UN and IN cell P were resuspended in 150 μL of 1× SDS sample buffer containing 1% BME. T, S, and P samples were brought to 1× concentration of SDS sample buffer containing 1% BME . All samples were heated at 100 °C for 5 min followed by centrifugation at 10,000g for 1 min. Fifteen microliter of UN and IN and 30 μL of T, P, and S were resolved in 10% denaturing gels, and the gels were stained with GelCode Blue Stain Reagent (Thermo Fisher Scientific) and imaged.Competent Rosetta DE3 cells transformed with either pET15b–6His–Rpt5 or pET15b–6His–Rpt5–P76A plasmid were grown in LB + 100 μg/ml ampicillin media overnight at 37 °C. Cultures were diluted 1:100 in fresh LB + ampicillin medium and grown to absorbance at 600 nm = 0.6 to 0.8. One unit of culture at absorbance at 600 nm was removed as an uninduced (UN) sample. Cultures were then induced with 0.2 mM IPTG  and grown at 16 °C overnight. One unit of culture at absorbance at 600 nm was harvested as an induced (IN) sample. Another 1.5 ml aliquot from each culture was harvested for aggregation assays and resuspended in 700 μL lysis buffer  and incubated at 4 °C for 30 min. Samples were then sonicated 6 × 10 s with 10-s incubations on ice between each sonication round; 100-μL aliquot from each sample was transferred into a new tube and represented T. The remaining samples (600 μL each) were centrifuged at 21,0002. Cells were ground using a mortar and pestle until a fine powder was formed. Cell powder was resuspended in ice-cold lysis buffer ) and vortexed intermittently during a 10-min incubation on ice. Samples were centrifuged at 3000g for 30 s to remove cell debris.Yeast cultures were grown in YPD or SD media (with casamino acids) overnight. The next day, cultures were diluted to absorbance at 600 nm = 0.2 in YPD or SD media (with casamino acids) and grown to the midexponential or saturation phase. Cells were harvested and washed with sterile cold water and flash-frozen in liquid Ng for 20 min at 4 °C in a Beckman Coulter TLA-55 rotor. S were transferred to fresh tubes. P were then washed with the lysis buffer and recentrifuged as above. S were discarded, and the resulting P were resuspended in half the volume of the S to make the P fraction. T, S, and P samples were brought to 1× concentration of SDS sample buffer containing 1% BME . Samples were heated at 100 °C for 5 min followed by centrifugation at 10,000g for 1 min. Equal volumes of T, S, and P samples were loaded onto SDS gels and subjected to immunoblot analyses. The P sample loaded was therefore twice as concentrated as those of T and S samples to account for potentially low levels of proteins in P that might be difficult to detect.S were transferred to fresh tubes. BCA assays were conducted to determine T concentrations. Protein concentration was normalized across all samples tested. A small aliquot was set aside as T. The remaining normalized S were centrifuged at 100,000After samples were resolved in denaturing SDS or nondenaturing polyacrylamide gels, proteins in the gels were transferred to PVDF membranes (Millipore). Immunoblots were analyzed using primary antibodies against α4/Pre6 (D. Wolf); Rpt1 (W. Tansey); Rpt2 (Enzo Life Sciences); Rpt3 (Enzo Life Sciences); Rpt4 (W. Tansey); Rpt5 (Enzo Life Sciences); Rpn2 (M. Glickman); Rpn12 (D. Finley); the RACs Hsm3, Nas2, Nas6, and Rpn14 . Two micrograms of total RNA was reverse-transcribed using the iScript cDNA Synthesis Kit (Bio-Rad). The resulting cDNA was subjected to quantitative PCRs using iQ SYBR Green Supermix (Bio-Rad) and analyzed on a LightCycler 480 (Roche). Each quantitative PCR was conducted in three technical triplicates. All experiments were conducted according to manufacturers' instructions.Yeast cultures were grown in SD media overnight. Cultures were diluted to absorbance at 600 nm = 0.2 in SD media and grown to the midexponential phase. Cells corresponding to one unit of absorbance at 600 nm were harvested and washed with sterile ice-cold water. Total RNA was extracted from the cells using an RNeasy Mini Kit (Qiagen) and eluted in 50-μL nuclease-free water. Contaminating DNA was subsequently removed from the samples using the DNA-B, we transformed WT and rpt2,5PA strains with pUB175 (expressing untagged ubiquitin) or pUB221 (expressing His6-tagged ubiquitin). The yeast ubiquitin genes in these plasmids are expressed under the control of a copper-inducible CUP1 promoter. Overnight cultures grown at 30 °C were diluted to absorbance at 600 nm = 0.2 and grown in 175-ml SD-URA for 2.5 h at 37 °C. For the experiment shown in Figure 5D, rpt2,5PA NOT4 and rpt2,5PA not4-L35A cells were transformed with pUB175 or pUB221. Overnight cultures grown at 30 °C were diluted to absorbance at 600 nm = 0.2 and grown in 175-ml SD-URA-LYS for 2.5 h at 30 °C. Cultures were then induced with 0.5 mM CuSO4  and grown for another 4 h. Five microliters of each culture was harvested, washed with sterile water, and saved.To determine if proteasome subunits were ubiquitinated at nonpermissive temperatures, we conducted ubiquitin pull-down assays with slight modifications from a previously outlined protocol . For theFigure 52HPO4/NaH2PO4, 10 mM imidazole, pH 8.0) followed by cell disruption with glass beads for 6 × 20 s using a vortexer at top speed with 30-s breaks on ice between each round. Samples were centrifuged at 1690g for 15 min to remove cell debris. S were collected, and the T concentration of each sample was determined via Bradford assay (Bio-Rad). T was normalized to 2 mg across all samples tested and incubated with 0.25 ml of 50% Ni-NTA resin (Qiagen) for 2 h. The resin was subsequently pelleted, and the S was aspirated off. The remaining beads were washed three times with 1-ml buffer A followed by three washes with 1-ml buffer A/TI  and finally once with 1-ml buffer TI. The beads were resuspended in 0.20-ml 2× SDS sample buffer (containing 0.2-mM imidazole and 8% BME) and subsequently boiled for 5 min.The remaining culture fractions were harvested by centrifugation, washed with sterile water, and resuspended in 2-ml buffer A  and boiled for 10 min. Bradford assays were conducted to determine protein concentration, and 10 μg of each sample was resuspended in 2× sample buffer (containing 0.2 mM imidazole and 8% BME) and further boiled for another 5 minutes. Ten micrograms of the input sample and 30 μL of each pull-down sample were loaded onto 10% denaturing gels and subjected to immunoblot analyses.RPT5 and rpt5–P76A strains expressing endogenously tagged Nas2–6xGly–3xFLAG were diluted to absorbance at 600 nm = 0.2 and grown in 100-ml YPD to the midexponential phase at 30 °C, washed with ice-cold water, and subsequently frozen in liquid nitrogen and stored at −80 °C. Frozen cells in liquid N2 were ground using a mortar and pestle until a fine powder formed. Cell powders were incubated in buffer F  on ice and vortexed intermittently for 10 min. Samples were then centrifuged at 20,000g for 10 min to remove cell debris. The resulting S was transferred into a fresh tube, and protein concentrations were determined via the BCA assay.Overnight cultures of T was normalized to 4.94 mg in 1.2-ml final volume for each sample. Hundred microliters T was set aside as the input. The remaining samples were incubated with 100-μL anti-FLAG resin on a rotating mixer at 4 °C for 2 h. The anti-FLAG resin was centrifuged, and the S was removed. FLAG resin was washed thrice with 1-ml buffer F. Bound proteins were subsequently eluted with 300-μL buffer F containing 200 μg/ml 3xFLAG peptide. Input samples and FLAG eluates were brought to 1× concentration sample buffer containing 1% BME and heated at 100 °C for 5 min. Eight microliters input and 30-μL pull-down eluate were loaded onto 12% denaturing gels and subjected to immunoblot analyses.All data are contained within this article.This article contains The authors declare that they have no conflicts of interest with the contents of the article.


However, the mechanisms responsible for photoreceptor death remain unclear. Here, we tested two hypothesized biochemical events that may underlie photoreceptor death: (i) the failure to prevent aberrant activation of retinal guanylyl cyclase (RetGC) by calcium-sensor proteins (GCAPs) versus (ii) the reduction of GMP phosphorylation rate, preventing its recycling to GDP/GTP. We found that GMP converts to GDP/GTP in the photoreceptor fraction of the retina ∼24-fold faster in WT mice and ∼400-fold faster in rd3 mice than GTP conversion to cGMP by RetGC. Adding purified RD3 to the retinal extracts inhibited RetGC 4-fold but did not affect GMP phosphorylation in wildtype or rd3 retinas. RD3-deficient photoreceptors rapidly degenerated in rd3 mice that were reared in constant darkness to prevent light-activated GTP consumption via RetGC and phosphodiesterase 6. In contrast, rd3 degeneration was alleviated by deletion of GCAPs. After 2.5 months, only ∼40% of photoreceptors remained in rd3/rd3 retinas. Deletion of GCAP1 or GCAP2 alone preserved 68% and 57% of photoreceptors, respectively, whereas deletion of GCAP1 and GCAP2 together preserved 86%. Taken together, our in vitro and in vivo results support the hypothesis that RD3 prevents photoreceptor death primarily by suppressing activation of RetGC by both GCAP1 and GCAP2 but do not support the hypothesis that RD3 plays a significant role in GMP recycling.Retinal degeneration-3 protein (RD3) deficiency causes photoreceptor dysfunction and rapid degeneration in the  RD3 defferences , making ferences  and rd3 rd3/rd3 mice (genotype synonymous with Rd3-/-), where a nonsense mutation similar to LCA12 in humans truncates RD3 .  . The removal of GCAP1 or GCAP2 alone increased the average rd3/rd3 photoreceptor nuclei count to 68% and 57%, respectively , whereas deletion of both GCAPs most efficiently offset the degeneration, preserving 86% of RD3-deficient photoreceptors .Figure 1neration , 18, 19.neration , 22, 23,neration . Simultad Guca1b , respect retinas . Plana-B . We emphasize that we estimated only the depletion of GTP to GMP via RetGC1/PDE6 activity, hypothetically counteracted by RD3, not the depletion of the GTP via GTPase activities, such as that of transducin .We further compared GMP phosphorylation and cGMP production rates in the normal and pothesis   from its phosphorylated products, [3H]GDP and [3H]GTP, using TLC on polyethyleneimine (PEI) cellulose (B). Incubation with the retinal extract resulted in a complete conversion of [3H]GMP to the phosphorylated species (C), indicating a lack of interference from any reversible reactions potentially capable of affecting the analysis. The concentrations of GMP and ATP in the assay  by far exceeded the respective 14 μM km,GMP and 0.4 mM km,ATP values for the retinal guanylate kinase reported by Hall and Kuhn .To avoid potential nonspecific effects of RD3 on a complex enzymatically coupled spectrophotometric assay used in study by Wimberg et al. , we direeceptors , with a eceptors . The metubstrate A from itellulose B. Incuba species C, indicaand Kuhn . [3H]GMPn period , D and EA), GMP was phosphorylated at the rate of 32 nmol/min/retina . Therefore, to evaluate the fraction of GMP-phosphorylating activity in the photoreceptor layer, we compared the WT (Rd3+/+) retinas with the Rd3+/+ retinas from CORD6 transgenic mouse line 362 . In the latter case, GMP was phosphorylated at the rate ∼10 nmol/min/retina. After subtracting the activity associated with the inner retina, the GMP phosphorylation rate corresponding to the photoreceptor layer equaled 24.7 nmol GMP/min/retina, comprising nearly 70% of the activity in the whole retina. We also used retinas from rd3/rd3 mice aged 7 months, lacking the photoreceptor layer at that age , to determine the fraction of guanylate kinase activity in Rd3-/- inner retina (Rd3+/+ line 362 mice (p > 0.999). In young rd3/rd3 mouse retinas retaining ∼71% of the photoreceptors , the GMP to GDP/GTP conversion rate was ∼28 nmol GMP/min/retina, not significantly different from that in WT retinas . After subtracting the activity in the inner retina, the average rd3/rd3 photoreceptor-specific activity comprised ∼18 nmol GMP/min/retina , reducing it 4-fold at 5 μM (A). We did not detect any effect on the GMP phosphorylation rate in WT retinas or in Rd3+/+ retinas lacking photoreceptors using the same preparation of RD3 (B). In case the endogenous RD3 retinas masked the effect of added RD3, we also tested homozygous rd3/rd3 (Rd3-/-) retinas . Again, contrary to the hypothesis , RD3 at the same concentration that strongly inhibited RetGC activity did not affect the rate of GMP phosphorylation in the mouse retinas lacking endogenous RD3 (B).The observed GMP phosphorylation rate in the retinas, regardless of the presence or absence of the endogenous RD3, exceeded that of the GTP to cGMP conversion by guanylyl cyclase. The RetGC activity in WT retinas averaged 0.9 nmol cGMP/min/retina, whereas in rd3/rd3 mice, it was reduced to 0.06 nmol/min/retina . Adding  at 5 μM A. We didn of RD3 B. In cas retinas . To maxipothesis  , then rearing rd3 mice in constant darkness from birth would have to reduce the pace of degeneration, by preventing activation of both PDE6 and RetGC. We hence randomized rd3/rd3 mice from several litters at birth and housed them either under a regular 12-hour-dark/12-hour-light (30–50 lux) cycle or in complete darkness. Prolonged rearing in the dark did not improve electroretinography (ERG) in rd3/rd3 mice compared with their littermates housed in the normal cyclic lighting conditions. The ERG a-wave (negative voltage deflection directly produced by hyperpolarizing photoreceptors) was equally reduced in both cases (A). There was no difference in the extent of photoreceptor degeneration, either. After 2.5 months of rearing in the dark, the reduction of photoreceptor nuclei count per 100 μm in rd3/rd3 mice was exactly the same as in their littermates reared in the normal light cycle , a ∼59% loss compared with WT retinas (p < 0.0001)  .Figure 6rd3/rd3 mice, even though the latter still retain detectable RetGC activity and even demonstrate rudimentary photoresponses (2+GCAPs (rd3/rd3 photoreceptors (rd3 and LCA12 photoreceptors degenerate because they cannot suppress RetGC activity in the inner segment in the absence of RD3 (A). However, Plana-Bonamaisó et al. proposed that it was specifically Mg2+GCAP2 that triggers the apoptotic process in the inner segment of rd3 photoreceptors . These results do not support the hypothesis , the events downstream of the aberrant RetGC activation remain unclear at this point. Hypothetically, cGMP in photoreceptor inner segment could prematurely activate cGMP-gated CNG channels produced in the endoplasmic reticulum and cause aberrant release of Ca2+ from the reticulum. Alternatively, the process could involve cGMP-dependent protein kinases. These and other possible scenarios would need to be evaluated in future in-depth studies.Whereas the deregulation of RetGC Fig. 1et al.  to be of particular interest, because it possibly suggested a different explanation for the protective effect of deleting GCAPs in rd3 photoreceptors. Conceivably, deletion of GCAPs could, by reducing RetGC activity, decrease the rate of GTP consumption by the cyclase (and hence GMP production by PDE6). However, we also reasoned that to deplete GTP via RetGC activity (B), the rate of GMP phosphorylation would have to be much slower than the rate of GTP consumption by the cyclase or would at least have to become much slower in the absence of RD3. Our direct comparison of the rates of the GTP→cGMP conversion by RetGC and GMP phosphorylation shows that the two are indeed drastically different but in a manner directly opposite from that presumed by the hypothesis: GMP phosphorylation in the retina occurs at least 30-fold faster than the GTP consumption by RetGC , because GMP phosphorylation rate does not change in rd3 photoreceptors . Both GTP conversion to cGMP by RetGC and cGMP hydrolysis by PDE6 are suppressed in the dark and accelerated in the light , the photoreceptors in rd3/rd3 mice degenerated in the dark just as severely as did those of their littermates housed under normal-illumination conditions  RD3 protects photoreceptors against degeneration by counteracting GCAP-dependent stimulation of the retinal membrane guanylyl cyclase; (ii) primarily GCAP1 and to a lesser extent GCAP2 both contribute to the triggering of photoreceptor degeneration in the absence of RD3; (iii) the rate of GMP phosphorylation in photoreceptors drastically exceeds the rate of GTP-to-GMP conversion via the RetGC/PDE6 pathway; and (iv) the role of RD3 in protecting photoreceptors from degeneration does not involve stimulation of GMP phosphorylation.To summarize, the results of our study support the hypothesis depicted in Figure 1rd3/rd3 mouse strains were purchased from JAX Research/Jackson’s Laboratory; GCAP1-/- (Guca1a-/-) and GCAP2-/- (Guca1b-/-) mice harboring the respective gene disruption were developed as previously described  as previously described  and then with 2.5% glutaraldehyde/2.5% formaldehyde in PBS. The eyes were surgically removed and fixed overnight in 2.5% glutaraldehyde/2.5% formaldehyde/PBS solution (Electron Microscopy Sciences) at 4 °C. The fixed eyes were washed in PBS, soaked in PBS overnight, processed for paraffin embedding, sectioned (5 μm thickness), and stained with hematoxylin/eosin . The retinal sections were photographed using an Olympus Magnafire camera mounted on an Olympus BX21 microscope. The photoreceptor nuclei in the outer nuclear layer of the retina were counted from 425 μm fragments of the retina between the optic nerve and the periphery, averaged from 2 to 3 fragments per retina, and the density of the nuclei per 100 μm length was averaged from multiple mice of each genotype as indicated in the figures.E. coli strain (Stratagene/Agilent Technologies) induced by isopropyl–β–D-thiogalactopyranoside, extracted from the inclusion bodies, and purified by salt precipitation and dialysis as previously described in detail (The recombinant RD3 was expressed from a Novagen pET11d vector in a BL21(DE3) Codon Plus n detail , 40, 41.E. coli strain  harboring a pBB131 plasmid coding for a yeast N-myristoyl transferase and purified by calcium precipitation, butyl–Sepharose and Sephacryl S–100 chromatography as described previously in detail (Myristoylated bovine GCAP1 was expressed from pET11d vector in a BLR(DE3) n detail , 43. The2, and stored at −70 °C before their use in the cyclase activity assays conducted under infrared illumination. The guanylyl cyclase activity was assayed as previously described in detail , 100 μM zaprinast and dipyridamole, 10 mM creatine phosphate and 0.5 unit of creatine phosphokinase (Sigma Aldrich) was incubated at 30 °C for 12 min. The reaction was stopped by heat-inactivation at 95° for 2 min, and a 5-μl aliquot from each reaction was chromatographed by TLC on fluorescently backed PEI cellulose plates (Merck) in 0.2 M LiCl. The spot containing the resultant [32P]cGMP and [8-3H]cGMP control radioactivity was excized from the plate and the [32P] and [3H] radioactivity eluted with 2 M LiCl were counted using liquid scintillation. Data fitting was performed using Synergy Kaleidagraph 4 software.Mouse retinas for RetGC activity measurements were excised from the dark-adapted 3.5-week-old mice under infrared illumination (Kodak number 11 infrared filters) using a dissecting microscope fitted with an Excalibur infrared goggles as described , 19, wran detail  with modn detail . In brie3H]5’GMP substrate was prepared from 200 μCi [8-3H]cGMP (Perkin Elmer). The [8-3H]cGMP solution in 50% ethanol, 7 Ci/mmol, was dried to remove ethanol from the solution, dissolved in 120 μl of 50 mM HEPES KOH buffer (pH 7.5) containing 100 mM NaCl, 2 mM MgCl2, and 1 mM DTT and incubated for 30 min at 30 °C with 2 μg purified bovine PDE6 holoenzyme . After the incubation, PDE6 was heat-inactivated for 2 min at 95°. At least 98% of [8-3H]cGMP was converted to [8-3H]GMP when tested by TLC on PEI cellulose plates in 0.2 M LiCl  was incubated at 30 °C for 15 to 20 min. The reaction was stopped by adding of 5 ul of 5% trifluoroacetic acid, neutralized by adding 5 μl of 0.4 M Na2CO3, and 5-μl aliquots were chromatographed on PEI cellulose TLC plates developed in 0.5 M LiCl. The spots containing 3H-GDP and 3H-GTP were excised from the PEI cellulose plate, and the [3H] radioactivity eluted with 0.5 ml 2 M LiCl was counted using liquid scintillation in 10 ml of scintillation cocktail. Data fitting was performed using Synergy Kaleidagraph 4 software.[8-2 M LiCl . Mouse r5 photoisomerizations/rod) 1-ms 505-nm light pulses were delivered through infrared camera-guided corneal electrode/LED light source. The a-wave amplitude was measured 7 ms after the flash.ERG was performed in the dark as previously described in detail , 31 usinpost-hoc Tukey’s honestly significance difference (HSD) test and nonpaired/unequal variance Student’s t test were performed using Synergy Kaleidagraph 4 and StatPages on-line resource. Data normality and homogeneity of variances before applying the ANOVA/post hoc testing were verified using the respective Kolmogorov-Smirnov and Levene’s tests.ANOVA and adizhoor@salus.edu) upon reasonable request.The data referred to in this manuscript are contained within the manuscript. Unprocessed data can be available from the corresponding author (The authors declare that they have no conflicts of interest with the contents of this article.


SOD3+/+) and SOD3 knock-out (SOD3−/−) mice. Interestingly, B cells isolated from SOD3−/− mice showed higher secretion of IgE, whereas, the use of DETCA, a known inhibitor of SOD3 activity, reversed the inhibitory effect of SOD3 on IgE production. Similarly, SOD3 was found to reduce the proliferation, IgE isotype switch, ROS level, and CCL17 and CCL22 productions in B cells. Furthermore, SOD3 was found to suppress both LPS/IL-4 and anti-CD40/IL-4-mediated activation of downstream signaling such as JAK1/JAK3, STAT6, NF-κB, p38, and JNK in B cells. Taken together, our data showed that SOD3 can be used as an alternative therapy to restrict IgE-mediated allergic diseases.Immunoglobulin E (IgE) functions as a first-line defense against parasitic infections. However, aberrant production of IgE is known to be associated with various life-threatening allergic diseases. Superoxide dismutase 3 (SOD3) has been found to suppress IgE in various allergic diseases such as allergic conjunctivitis, ovalbumin-induced allergic asthma, and dust mite-induced atopic dermatitis-like skin inflammation. However, the role of SOD3 in the regulation of IgE production in B cells remains elusive. In this study, we investigated the effect of SOD3 on LPS/IL-4 and anti-CD40/IL-4-mediated secretion of IgE in murine B cells. Our data showed that SOD3 can suppress both LPS/IL-4 and antiCD40/IL-7-induced IgE secretion in B cells isolated from both wild-type ( •SOD3 suppresses LPS/IL-4 and anti-CD40/IL-4-induced secretion of IgE in B cells•SOD3 reduces the expression of IgE isotype class switch recombination genes.•SOD3 suppresses the LPS/IL-4 and anti-CD40/IL-4-induced superoxide production.•SOD3 suppresses the LPS/IL-4 and anti-CD40/IL-4-induced chemokines secretions.•SOD3 modulate JAK-STAT, p38, JNK, and NF-κB signaling pathways in B cells. However, inappropriate regulation of IgE is found to be associated with various allergic diseases and can lead to severe life-threatening anaphylaxis . Serum ISuperoxide dismutase 3 (SOD3), also known as extracellular superoxide dismutase is responsible for the maintenance of redox homeostasis in the extracellular matrix of various tissues . Along wIn this study, we aimed to determine the effect of SOD3 on IgE production in B cells. Primary naïve B cells were differentiated for IgE production and the effects of SOD3 on IgE production and underlying effects on signaling were investigated. Our data showed that SOD3 limits the IgE production in B cells with reduced proliferation and IgE isotype class switching. Thus, our study provides supportive information regarding the use of SOD3 in hyper-IgE-mediated allergic conditions.22.1SOD3+/+) and whole-body SOD3 knock-out (SOD3−/−) mice using the MACS B cell isolation kit  following manufacturer instructions. Purified B cells were then rested at least 2 h on ice and then 2 × 105 B cells were cultured in flat-bottom 96-well plates. The cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium  supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin  at 37 °C in a humidified incubator.All procedure of animal research was performed in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Rodent experiment provided by the IACUC  in the school of medicine, The Catholic University of Korea . Following the approval from The Catholic Ethics Committee of Catholic University of Korea, primary B cells were isolated from mouse spleen  of C57/BL6 wild-type  for 1 h. The cells were then treated with a combination of either 10 μg/mL of LPS  and 10 ng/mL of rh mouse IL-4  or 5 μg/mL of anti-CD40  and 10 ng/mL of rh mouse IL-4  for 5 days.2.2The levels of IgE release were measured from cell-free supernatant by ELISA method using IgE mouse ELISA kit  according to the manufacturer's instructions. Briefly, 100 μl of each standard and samples were added into pre-coated wells and were incubated at room temperature for 30 min. After incubation, the contents of the well were aspirated and washed with the provided 1X wash buffer for 5 times. Following washing, 100 μl of 1X enzyme-antibody conjugated were added to each well and the plates were incubated in dark for 30 min. The plates were then washed and 100 μl of TMB substrate solution were added to each well and incubated in dark at room temperature for 10 min. After 10 min, 100 μl of stop solution were added into each well, and absorbance was taken at 450 nm.2.35 cells/mL in 100 μL per well of culture media. Cells were then pre-treated with SOD3 for 1 h followed by treatment with either LPS/IL-4 or anti-CD40/IL-4 for 5 days. Cell proliferation was measured with BrdU cell proliferation assay kit  following the manufacturer's instruction.Isolated naïve B cells were plated in 96-well plates at 2 × 102.42. After 5 days of incubation, the cells were removed and the wells were washed 5 times with 200 μl of PBS/well. Detection antibodies were added to each well and incubated at room temperature for 2 h. The wells were then incubated with 100 μl of streptavidin-ALP for 1 h at room temperature. Substrate solution was added and developed distinct spots were analyzed with the microscope.The number of IgE secreting cells was determined by using Mouse IgE ELISpotBASIC kit  following the manufacturer's instructions. Briefly, provided PVDF plates were activated with 50 μl of 70% ethanol per well for 2 min. The plates were washed 5 times with sterile water and incubated overnight with 100 μl of anti-IgE antibody at 4 °C. The next day, coated antibodies were aspirated and washed with sterile water. The wells were then blocked with 200 μl of culture medium and incubated at room temperature for 30 min. After blocking, cell suspension with indicated stimuli was added to each well and incubated for 5 days at 37 °C in a humidified chamber with 5% CO2.5Isolated B cells were first pre-treated with SOD3  for 1 h. The cells were then treated with either LPS/IL-4 or anti-CD40/IL-4 and 5 μM of dihydroethidium (DHE) , and incubated in dark at 37 °C for 10, 30, and 60 min. Fluorescents were measured at an excitation of 500 nm and emission of 600 nm by fluorimeter.2.6Cells were harvested after 5 days of incubation at 37 °C in humidified incubator and were lysed in ice-cold radioimmunoprecipitation (RIPA) lysis buffer  containing protease and phosphatase inhibitor cocktails . Protein concentrations were determined by BCA protein assay Kit  as described by manufacturer instructions. Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membrane (PVDF). After blocking, membranes were incubated with primary antibodies such as p-p38 , p38 , P-JNK , JNK , P-JAK1 , JAK1 , P-JAK3 , JAK3  P-STAT6 , STAT6 , p-NF-κBp65 , NF-κBp65  and GAPDH  at 1:1000 dilutions for overnight at 4 °C. Membranes were then washed and incubated with horseradish-peroxidase conjugated secondary antibodies (1:5000 dilutions) for 2 h at room temperature. The blots were then detected with western blot detection kit .2.7Total RNA was isolated from cells by using TRIzol reagent . Complementary DNA was synthesized from 1 μg of total RNA using the PrimeScript™ RT reagent Kit  and RT-PCR was performed using LightCycler 96 . Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. The amplified products were analyzed by electrophoresis in a 2% agarose gel. The used primers and their sequences can be found in supplementary sections .2.8SOD3 was purified as previously described . Briefly2.9Statistical differences were analyzed by one-way analysis of variance (ANOVA) followed by Tukey test. All results represent three independent experiments. P < 0.05 was considered statistically significant .33.1in vitro. Here, we found that SOD3 significantly suppresses the production of IgE in B cells under both LPS/IL-4 and anti-CD40/IL-4 conditions (SOD3−/−) mice and also used DETCA, a known inhibitor of SOD3 activity. Our data showed that the IgE production was relatively higher in B cells isolated from SOD3−/− mice compared to wild-type (SOD3+/+) mice and the use of SOD3 downregulates the release of IgE in both SOD3−/− and SOD3+/+ mice. In addition, DETCA rescued the inhibitory effect of SOD3 on IgE production in both SOD3+/+and SOD3−/− mice under LPS/IL-4 and anti-CD40/IL-4 conditions  process for IgE production . Thus, w+/+ mice C and D.F3.4Upon encountering with antigen and IL-4, activated B cells are known to differentiate into IgE secreting plasma cells . Thus, w3.5Naïve B cells are also known to release chemokines such as CCL22 and CCL17 upon activation . Thus, w3.6The interactions of IL-4 with its receptor are known to activate JAK-STAT signaling pathways ,24, and 4Despite the important role of IgE in confronting helminthic infection, aberrant production of IgE was found to be associated with several allergic diseases ,2. The cSOD3−/−) mice compared to SOD3+/+ and the use of SOD3 limiting the release of IgE in both SOD3−/− and SOD3+/+ mice emphasized the important role of SOD3 in regulating the production of IgE. Similarly, the use of SOD3 activity inhibitor DETCA tried to rescue the inhibitory effect of SOD3 on IgE production in B cells isolated from both SOD3−/− and SOD3+/+ mice. However, DETCA could not completely reverse the inhibitory effect of SOD3. The possible reason is that SOD3 is also known to possess some effect in a non-enzymatic manner. Along with scavenging oxidative stress during inflammatory conditions, SOD3 also found to interact with receptors such as histamine 4 receptor in keratinocytes and IL-4 receptor in Th2 cells [For the activation and differentiation of B cells into IgE secreting B cells, we used two different types of antigens. Lipopolysaccharides at high concentrations are capable of T-independent activation of B cells and the use of anti-CD40 ligand activates the B cells in T-dependent manner . The useh2 cells . In addih2 cells  and alsoh2 cells  These noSOD3−/− mice showed more proliferation than SOD3+/+ mice [SOD3 has been shown to suppress the proliferation of Th17 cells . Similar+/+ mice . In addi+/+ mice ,36. Thes+/+ mice . Upon an+/+ mice . The ant+/+ mice . Our datUpon activation by antigen, stimulated B cells undergo class switch recombination (CSR) process in which the immunoglobulin heavy μ constant region exons (Cμ) are deleted and replaced by one of several sets of CH exons  to alter the isotype of the antibodies, resulting in the production of specific antibodies ,40. To u20–22. These chemokines function to recruit Th2 cells and in turn, Th2 cells produce cytokines required for B cell differentiation and antibody production [in vivo, where cytokines produced by Th2 cells and B cells work synergistically for the secretion of IgE.Upon activation, B cell is not much known to secrete various cytokines as T cells. The main cytokines produced by B cells are the chemokines such as CCL17 and CCL22 oduction . Here, wActivated B cells are known to differentiate into antibody-secreting cells, leading to adaptive immune response through the secretion of antibodies. Following ligand interaction with either Toll-like receptor (TLR) or with B cell receptor, B cells rapidly undergo proliferation and differentiate into antibody-secreting cells ,38. Our Ligation of LPS and CD40 ligand with TLR4 and CD40 receptors respectively on B cells are known to activate downstream signaling pathways such as MAPK and NF-κB and subsequently leads to the activation and proliferation of B cells . SimilarPlethora of studies suggests that SOD3 can be used as novel treatment strategy for various diseases such as cancer, diabetes, rheumatoid arthritis, chronic inflammation, aging and neurodegenerative diseases . Previou5Along with antioxidant and anti-inflammatory functions, SOD3 also has been shown to possess immunomodulatory functions in various immune cells. SOD3 was found to suppress DCs maturation through reduced expression of receptor proteins . SimilarGA contributed to the design, conceptualization, formal analysis, investigation, methodology, visualization, and writing; SKS, MJL, CWB, YHK and HYK contributed to the analysis, and writing; TYK contributed to conceptualization, supervision, and validation. All authors reviewed the manuscript.All data generated or analyzed during this study are included in this article (and its supplementary information files).The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper


Mycobacterium tuberculosis is the cause of one of the most important infectious diseases in humans, which leads to 1.4 million deaths every year1. Specialized protein transport systems—known as type VII secretion systems (T7SSs)—are central to the virulence of this pathogen, and are also crucial for nutrient and metabolite transport across the mycobacterial cell envelope3. Here we present the structure of an intact T7SS inner-membrane complex of M. tuberculosis. We show how the 2.32-MDa ESX-5 assembly, which contains 165 transmembrane helices, is restructured and stabilized as a trimer of dimers by the MycP5 protease. A trimer of MycP5 caps a central periplasmic dome-like chamber that is formed by three EccB5 dimers, with the proteolytic sites of MycP5 facing towards the cavity. This chamber suggests a central secretion and processing conduit. Complexes without MycP5 show disruption of the EccB5 periplasmic assembly and increased flexibility, which highlights the importance of MycP5 for complex integrity. Beneath the EccB5–MycP5 chamber, dimers of the EccC5 ATPase assemble into three bundles of four transmembrane helices each, which together seal the potential central secretion channel. Individual cytoplasmic EccC5 domains adopt two distinctive conformations that probably reflect different secretion states. Our work suggests a previously undescribed mechanism of protein transport and provides a structural scaffold to aid in the development of drugs against this major human pathogen. Mycobacterium tuberculosis reveals an important role of interactions with MycP5 protease for complex integrity.A cryo-electron microscopy structure of the inner membrane complex of the ESX-5 type VII secretion system of  Mycobacterium tuberculosis encodes five homologous, but functionally distinct, T7SSs that are designated ESX-1 to ESX-5. These systems translocate a number of effector proteins across the unique and impermeable diderm cell envelope3. Because of their importance for mycobacterial physiology and virulence, T7SSs are considered to be promising targets for the development of drugs for the treatment or prevention of tuberculosis4. Although T7SSs have previously been shown to form hexameric complexes5, high-resolution structural information exists only for part of the T7SS—a dimeric ESX-3 subcomplex from the nonpathogenic species Mycobacterium smegmatis7. Here we reconstituted the ESX-5 T7SS of M. tuberculosis H37Rv in M. smegmatis to obtain a structural view of the entire T7SS membrane complex from this human pathogen.M. tuberculosis ESX-5 system showed robust expression in M. smegmatis and correct assembly of the membrane complex  resulted in copurification of the conserved MycP5 protease   analysis of the try Fig. . The memcD5 Fig. . The oveM. tuberculosis is formed by three EccB5 dimers and three MycP5 proteases. The EccB5 dimers assemble in a triangle, which forms a central cavity  can be distinguished between monomers, depending on their position. EccB5 dimerization is mediated mainly through the R1 and R4 repeat domains and is further stabilized by the EccB5 C termini, which wrap around their interacting EccB5 partner to form intermolecular hydrophobic contacts with its R1 domain  of the inner EccB5  , and loop 3, which contacts the neighbouring protease domain from the side  Fig. . Additiom10 Fig. . MycP5–Mity Fig. , which i5 is the largest in the periplasmic assembly, and covers a surface area of around 2,000 Å2 and provides a solvation-free energy gain of ΔG = −23 kcal mol−1 per dimer  are even more modest, which provides a rationale for why interactions between MycP5 and the membrane complex have so far remained unknown.The dimer interface between the inner and outer EccBmer Fig. . By cont5 on the entire structure, we analysed MycP5-free M. tuberculosis ESX-5 complexes from the same preparation  better anchor the periplasmic assembly to the membrane, while also stabilizing the dimeric unit  together form a circular raft with an inner cavity , and is therefore thought to be the central component in this nanomachinery. Each EccC protein has—in addition to two TMHs—four FtsK/SpoIIIE-like ATPase domains ) that are known to be important for secretion14. We fully resolved the twelve EccC5 TMHs in the intact M. tuberculosis ESX-5 complex; these form three four-TMH bundles, each of which belongs to the EccC5 molecules of one dimer . This NBD is bound to the cytosolic domains of inner and outer EccD5, which—in turn—are bound to EccE5 at the periphery, together forming a ‘cytosolic bridge’ , adopts two main conformations: we refer to these as extended and contracted  region, including about 380 bp upstream of eccB5, of M. tuberculosis H37Rv was amplified while adding DraI and PacI restriction sites at the 5′ and 3′ ends, respectively (primers 1 and 2), and cloned into the modified plasmid upstream of the TwinStrep tag sequence, resulting in plasmid intermediate 1. The M. tuberculosis H37Rv region spanning rv1791–rv1798 (pe19–eccA5) was amplified while adding SpeI and NdeI unique restriction sites (primers 3 and 4) and cloned downstream of the TwinStrep tag sequence into the intermediate 1, resulting in plasmid intermediate 2. Plasmid intermediate 2 was digested with SpeI and SnaBI, removing the region rv1791–rv1794 (pe19–espG5), and the region encompassing rv1785–rv1794 (cyp143–espG5) was amplified as two individual PCR products (primers 5 and 6 and primers 7 and 8). The restricted backbone and PCR products were InFusion (Takara Bio)-ligated, resulting in the final pMV-ESX-5mtb containing the entire rv1782–rv1798 (eccB5–eccA5) locus.The plasmid expressing Mycobacterium smegmatis MC2155 expressing M. tuberculosis ESX-5 was grown at 37 °C and 90 rpm in LB medium supplemented with 0.05% Tween 80 and 30 μg ml−1 streptomycin. Cultures were grown to an optical density (OD) at 600 nm of about 1.5, spun down for 15 min at 12,000g in a JLA-8.1000 rotor and subsequently washed in PBS. After culture collecting, all subsequent steps were performed at 4 °C. Washed cell pellets were resuspended in buffer A  at a concentration of about 50 OD ml−1 and lysed by passing two times through a high-pressure homogenizer (Stansted) using a pressure of 0.83 kbar. Unbroken cells were pelleted at 5,000g for 5 min and supernatants were transferred to ultracentrifugation tubes. Cell envelopes were separated from the soluble fraction by ultracentrifugation at 150,000g for 1.5 h. Following ultracentrifugation, supernatants were discarded, pellets were washed once with buffer A, resuspended in buffer A at a concentration of 750–1,000 OD ml−1, snap-frozen in liquid nitrogen and stored at −80 °C until further use. The protein concentration of the cell envelope fraction was measured by BCA assay (Pierce).M. tuberculosis ESX-5 was purified as follows: cell envelope fractions were diluted to 3 mg ml−1 in buffer B , supplemented with 0.25% DDM, 3 mM ADP–AlF3 and 6 mM MgCl2. Following solubilization, the cell envelope mixture was spun down at 100,000g for 20 min, supernatants were collected and incubated with StrepTactin resin (IBA). Beads were subsequently washed with buffer B supplemented with 0.03% DDM, 1 mM ADP–AlF3 and 2 mM MgCl2. Bound protein was eluted from the resin with buffer B supplemented with 0.03% DDM, 3 mM ADP–AlF3, 6 mM MgCl2 and 10 mM desthiobiotin. The protein concentration of the eluate was measured by Bradford assay and amphipol A8-35 was added in an amphipol:protein ratio of 5:1. After a 1-h incubation, the amphipol-containing eluate was incubated overnight (around 12–16 h) with BioBeads in a BioBeads:detergent ratio of 20:1. Subsequently, BioBeads were removed using gravity flow chromatography columns and the sample was concentrated using Amicon Ultra 0.5-ml 100-kDa spin concentrators. The concentrated sample was further purified through size exclusion chromatography (SEC), using a Superose 6 Increase column running in buffer C  supplemented with 1 mM ADP–AlF3 and 2 mM MgCl2. Size exclusion chromatography fractions were analysed by blue-native polyacrylamide gel electrophoresis (BN-PAGE) and negative-stain electron microscopy, after which the appropriate fractions were concentrated for cryo-EM using Amicon Ultra 0.5-ml 100-kDa spin concentrators. The initial Arctica sample was purified similarly, with the addition of 5% glycerol in the SEC purification step and the omission of ADP–AlF3 and MgCl2 from the purification protocol.All steps were performed at 4 °C. The 5, used in Extended Data Fig. 16. Polyclonal rabbit antisera against the peptide was raised in rabbits by Innovagen using Stimune (Prionix) as adjuvants. The antibody was used as a 1:5,000 dilution. Blots were visualized on a ChemoStar TouchMotionCor2 using ChemoStarTS.Samples consisting of either solubilized membranes or purified membrane complexes were mixed with 5% G-250 sample additive (Invitrogen), to a final concentration of about 0.2%, and run on 3–12% NativePage Bis-Tris Protein Gels (Invitrogen) according to manufacturer specifications. Gels were either stained with Coomasie R-250 or transferred to PVDF membranes and stained with appropriate antibodies . Four microlitres of diluted sample was applied to the grids and incubated for 30 s. The sample was blotted off from the side and the grid was washed briefly with 4 μl of staining solution (2% uranyl acetate) and then stained with 4 μl of the staining solution for 30 s. The stain was blotted off from the side and grids were air-dried. Grids were imaged using a Thermo Fisher Scientific Talos L120C TEM equipped with a 4K Ceta CEMOS camera using TIA 4.1.5.For the main datasets, purified sample was applied to Quantifoil R2/2, 200 mesh, copper grids floated with an additional approximately 1.1-nm layer of amorphous carbon. Four microlitres of sample was applied onto glow-discharged grids (30 s at 25 mA) and allowed to disperse for 60 s at 4 °C and 100% humidity. Grids were blotted for 4–6 s with a blot force of −5 and plunge-frozen in a liquid propane–ethane mixture, using a Thermo Fisher Scientific Vitrobot Mark V. For the initial Arctica dataset, all steps were similar, with the exception of the additional layer of amorphous carbon.2, fractionated in 38 frames over a 40-s exposure time and with a nominal defocus range of 1–2.5 μm.The initial cryo-EM dataset was collected on a 200-kV FEI Talos Arctica electron microscope equipped with a Falcon III direct electron detector running in counting mode and using Thermo Fisher Scientific EPU 1.11. A total of 853 movies were recorded with a nominal magnification of 150,000×, corresponding to a pixel size of 0.96 at the specimen level. Movies were recorded with a total dose of 40.28 electrons per A2, fractionated in 50 frames over a 3-s exposure time and with a nominal defocus range of 1–3 μm.The two high-resolution datasets were recorded using Thermo Fisher Scientific EPU 2.4 software on a 300-kV Titan Krios TEM, equipped with a Gatan K3 direct electron detector running in counting mode and a Gatan Bioquantum energy filter (slit size 10 eV). We recorded 7,984 and 9,389 movies in counting mode in the two separate sessions with a nominal magnification of 81,000×, corresponding to a pixel size of 1.1 Å at the specimen level. Movies were recorded with a total dose of 59.5 electrons per A17, unless stated otherwise. For the initial Arctica dataset, movies were motion-corrected using MotionCor218 and dose-weighted, and the contrast-transfer function (CTF) was estimated using CTFFIND419. Automated particle-picking was performed using Cryolo20 and the pretrained Janni model. Following particle extraction and several rounds of 2D classification to remove obvious artefacts, an initial de novo model was generated. The dataset was further cleaned using 3D classification and the best class was subsequently used for reference-based particle-picking. Following 2D and 3D classification (and 3D refinement in C1), the map displayed an apparent threefold symmetry and was further refined in C3. This final map displayed an estimated 13.5 Å resolution.Single-particle analysis was performed using Relion3.121. DeepEMhancer further helped to improve the observed anisotropy, overall map connectivity.In the first Krios dataset, movies were motion-corrected using MotionCor2, dose-weighted and the CTF was estimated using CTFFIND4. Automated particle-picking was performed using Cryolo with the pretrained Janni model and a low threshold. Particles were extracted and binned 4× and several rounds of 2D classification were performed followed by 3D classification with the 30 Å-filtered Arctica model as a template. The resulting particles were re-extracted without binning, CTF-corrected and polished and refined in C1, giving a map with an estimated overall resolution of 4.5 Å. For the cytosolic region, particles were recentred on the cytosolic region, re-extracted, CTF-corrected, polished and 3D-refined. Following refinement, the density accounting for individual cytosolic dimers was subtracted, resulting in a particle stack that was three times larger. Cytosolic dimers were first refined with a mask encompassing both cytosolic bridges. Subsequently, these were focus-refined using a soft mask around one of the cytosolic bridges. This map was refined using the default Relion value ‘--tau2fudge 2’ but also ‘--tau2fudge 4’, which increased the overall connectivity of the lower cytosolic area. The final map for the cytosolic bridge showed an estimated resolution of 3.3 Å and was sharpened using either Relion postprocessing or DeepEMhancer5 were processed separately. The MycP5-unbound map displayed increased heterogeneity and—following unbinned re-extraction and refinement—the particles were 3D-classified without alignment, resulting in two obvious classes of 4.5 Å and 6.7 Å resolution. Model free density modification in Phenix.Resolve_Cryo_EM22 further improved the resolution to 4.3 Å and 5.8 Å, respectively. By contrast, a similar 3D classification for the MycP5-bound map did not further classify into structurally distinct classes. Following unbinned re-extraction and refinement, the MycP5-bound map showed an overall resolution of 4 Å, which was further improved to 3.8 Å after C3 refinement. Model free density modification in Phenix.Resolve_Cryo_EM further improved the resolution of the entire C1 map to 3.8 Å and of the C3 refined map to 3.56 Å. For the periplasmic map, the centre of mass for that region was determined using Chimera23 and the particles were recentred, extracted, 3D-refined and polished to obtain the periplasmic map at an estimated 3.8 Å resolution in C1. Following 3D classification without alignment and further refinement in C3, the estimated resolution of the periplasmic map improved to 3.5 Å. To separate the two states of EccC5, particles were recentred on the lower cytosolic region, at the level of the DUF domain, polished and 3D-refined. This was followed by a masked 3D classification in which the mask contained NBD1 and NBD2 of EccC5 in the extended conformation. The two main classes were further 3D-refined unmasked and subsequently masked, leading to a map of about 4.27 Å for the extended conformation and 7.6 Å for the contracted conformation.The second Krios dataset was processed similarly, with some exceptions. Following 3D classification of the binned data against the 4.5 Å Krios map filtered to 30 Å, the two maps with and without MycP5, EccB5, EccC5 and EccD5 with Phyre224. For MycP5, Protein Data Bank (PDB) entry 4J9412 served as a structural template, and PDB entries 4KK725, 4NH014 and 6SGW6, and 6SGZ6 served as reference models for EccB5, EccC5 and EccD5, respectively. To obtain atomic models of the periplasmic part (MycP5–EccB5) of the M. tuberculosis ESX-5 complex, homology models of MycP5 and EccB5 were rigid-body-fitted into a C1 symmetry, focus-refined periplasmic M. tuberculosis ESX-5 map (Electron Microscopy Data Bank (EMDB) code EMD-12518) using the fit-in-map tool in ChimeraX (v.1.0)26. Model building, extension and interactive refinement was performed with ISOLDE (v.1.0.1)27, a molecular-dynamics-guided structure refinement tool within ChimeraX (v.1.0). The resulting coordinate file (PDB 7NPS) was further refined with Phenix.real_space_refine (v.1.18.2-3874)28 using reference model restraints, strict rotamer matching and disabled grid search. Model validation was carried out using the MolProbity web server29 and EMRinger30 within the Phenix software package. Models for the membrane-embedded region (MycP5–EccB5–EccC5–EccD5) and cytoplasmic bridge (cytosolic domains of EccC5–EccD5) (PDB 7NPT) were built in the same way, using a reconstruction of the full M. tuberculosis ESX-5 complex (EMDB EMD-12517) and a focus-refined map of the cytoplasmic domains (EMDB EMD-12520) sharpened with DeepEMhancer, respectively. Finally, a composite model was assembled by fusing the periplasmic assembly and six copies of the cytosolic bridge to the membrane-embedded region model. This composite model was then refined against the full M. tuberculosis ESX-5 complex map with C1 symmetry (PDB 7NP7 and EMDB EMD-12514) and C3 symmetry (PDB 7NPR and EMDB EMD-12517).Model building started by generating homology models for MycP5-free maps was performed with ISOLDE using the composite ESX-5 model, in which MycP5 and the periplasmic domain of EccB5 (residues 84–507) had been deleted. Adaptive distance restraints as well as torsion restraints were applied to all atoms to restrain short-range conformational changes but allow for long-range conformational movements. ISOLDE simulations for dynamic fitting of the coordinate file into EMD-12521 and EMD-12522 were performed  after which the models showed satisfying fits to the new maps without further manual intervention. MycP5-free models were further refined against the maps using Phenix.real_space_refine (v.1.18.2-3874) as stated.Modelling into MycP31. Buried surface areas between subunits were calculated by PISA32.Visualization of atomic coordinates and map volumes was performed with ChimeraX (v.1.1) and PyMOL v.2.40Further information on research design is available in the Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-021-03517-z.Supplementary InformationThis file contains Supplementary Tables 1-3 and Supplementary Figures 1-3.Reporting Summary.Peer Review FileVideo 1Overall structure of the ESX-5mtb membrane complex.Video 2The periplasmic dome of the ESX-5mtb membrane complex.Video 3The central, EccC5 gated, pore of the ESX-5mtb membrane complex.Video 4Flexibility of the MycP5 free maps and stabilization upon MycP5 binding.


Control the nanostructures of g-C3N4 can tailor its photocatalytic performance. In this paper, one-dimensional chain-like g-C3N4 was successfully synthesized by heat-induced polymerization of melamine which was saturated in ethylene glycol. The photocatalytic hydrogen production rate (HER) of the prepared g-C3N4 chain enhanced about 3 times than that of bulk g-C3N4, increasing from 9.6 μmolh−1 to 28.7 μmolh−1. The improved photocatalytic activity of the g-C3N4 chain was attributed to the advantages of porosity and nanostructure. The extraordinary nanopores result in an enlarged specific surface area for adsorption and the production of abundantly available channels for charge transfer. The one-dimensional chain-like structure can facilitate the exposure of internal/external active sites as many as possible, and induce the directional migration of charge carriers.The development of graphite-carbon nitride (g-C Furthermore, the growth of 1D nanostructures has an immense effect on improving the mechanical energy, thermal and electrical capabilities of materials. Because nanoparticles in 1D nanostructures are interconnected in three dimensions, an extremely fast interparticle, vectorially transport of photogenerated charge carriers (electrons and holes) is likely to emerge through the grain boundaries. This represents that the redox reaction sites are far away related to the photoexcitation sites, which seems to be responsible for the high activities of photocurrent generation and hydrogen production.3N4 chain nanostructures were obtained by regulating the saturation of melamine and ethylene glycol solution for photocatalytic hydrogen evolution. The enhanced photocatalytic properties are known to be caused by an extension of the life of the photoinduced charge carrier. In addition, increasing specific surface area is also a vital factor in promising photocatalytic performance. Owing to the composite method is convenient, environmentally friendly, and low-cost, it is suitable for an expanded range of practical applications.In the present work, 1D g-C3N4 chain nanostructures were prepared by a controllable approach. Firstly, excessive amounts of melamine powders (3.0 g) were dissolved in 60 ml of ethylene glycol to form a saturated solution at room temperature. Subsequently, add 1 ml of concentrated nitric acid solution to 59 ml of water, drop by drop add the solution to 20 ml of supernatant and stir continuously until a white flocculent precipitate is obtained. The white flocculent precipitate was bleached with ethanol five times to remove nitric acid and ethylene glycol. Lastly, the collected sample was transferred into the muffle furnace for heating 2 h at 550°C with a heating rate of 20°C·min−1. Meanwhile, as the contrast sample, the bulk g-C3N4 was obtained by the heating process at 550°C for 2 h with a heating rate of 20°C·min−1 of the melamine powders.The one-dimensional g-C3N4 chain nanostructures were characterized by scan electron microscopy . And specific surface areas of the one-dimensional g-C3N4 chain nanostructures were measured by a Micromeritics ASAP 2010 instrument and analyzed by the Brunauer–Emmett–Teller (BET) method. Photoluminescence (PL) spectra of photocatalysts were performed on a Jobin Yvon HR800 micro-Raman spectrometer including a 325 nm line from a He-Cd laser. UV-Vis diffuse reflectance spectra (DRS) of the samples are obtained by using a UV-Vis-IR spectrometer . Photocurrent measurements were characterized by CHI 660 E electrochemical workstation  by applying a three-electrode cell accompanied by a visible light source. A platinum filament, Ag/AgCl electrode, and 0.2 M of Na2SO4 were acted as the counter electrode, reference electrode, and an electrolyte solution, separately. The as-fabricated sample mixed a certain amount of Nafion solution was painted on FTO glass as a working electrode (the effective area was 1 cm × 1 cm).The crystal structure was characterized by X-ray diffraction  using the Cu Ka radiation (k = 1.5418 Å). The morphologies of one-dimensional g-C2 evolution was injected into a 250 ml of quartz reactor with a visible-light source irradiation. As a typical synthesis experiment, 0.1 g of photocatalyst with a certain Pt cocatalyst (1 wt%) was dispersed in a mixed solution of aqueous solution (90 ml) and methanol (10 ml). The amount of H2 evolution was measured in a gas chromatograph . The time interval of sampling was performed at 40 min during the water splitting process.Photocatalytic H3N4, in which the sample possesses a hierarchical component with a particle size of greater than 20 μm. However, in 3N4 changed greatly compared with the bulk structure, and a new one-dimensional chain structure appeared. It can be illustrated from 3N4 one-dimensional structure could reach dozens of micrometers, and the diameters of those fibers range from 1 to 2 µm. Each one-dimensional structure was separated from each other, and further secondary structures could be found. Through the observation of the morphology characteristics of the two structures, we can infer that the chain g-C3N4 material will possess a vast specific surface area, which is more conducive to enhance the photocatalytic performance. The transmission electron micrographs of chain g-C3N4 are shown in 3N4 presents a similar sponge hole that exists on the surface of the chain g-C3N4, the morphology can effectively improve the capacity in the course of the photocatalytic reaction area. Meanwhile, the product can shorten the time and distance of the charge transfer, and promote the charge separation order to improve the photocatalytic activity of the material. As we all known, in the pyrolysis period of supramolecular precursor, the by-products were formed with varieties of gases gradually released, giving rise to the nitrogen defects obtained in the framework of g-C3N4.The morphology of the as-fabricated products was scrutinized by scanning electron microscope (SEM). 3N4 and bulky g-C3N4 are expressed in X-ray diffraction (XRD) patterns for chain g-C3N4 were compared to the bulky g-C3N4 exhibit with similar characteristics. The FT-IR the bands located at 1,240, 1,321, 1,415 cm−1, and 1,570 cm−1 are mainly from the typical stretching modes of C-N heterocycles. And the band at 810 cm−1 is attributed to out-of-plane bending modes of C-N heterocycles. The C-N stretching mode has IR band at 1,635 cm−1. And the broadband near 3,200 cm−1 corresponds to the stretching modes of terminal NH2 or NH groups at the defect sites of the aromatic ring , which is approximately three times larger than pure bulky g-C3N4 (16.15 m2g−1). The inset in 3N4 and bulky g-C3N4. The pore distribution for chain g-C3N4 is mainly located at 2.46 nm, and the pore volume is 0.233 cm3g−1. But for the bulky g-C3N4, the pore volume is just 0.097 cm3g−1. The mesoporous structure and large surface area of the chain g-C3N4 are conducive to the absorption of more active substances and reactants on the surface, enhancing the photocatalytic reaction. Therefore, we can assume that the chain g-C3N4 material can have superior photocatalytic activity.The exposed surface area and pores distribution of chain g-C3N4 and bulk g-C3N4 was calculated to be approximate 2.88 and 2.93 eV, respectively. When the particle size descends to a certain value, the electron energy level near the Fermi energy level of the metal changes from quasi-continuous to discrete energy level (3N4.The UV-vis diffuse reflectance spectroscopy illustrated by gy level , and the3N4 has a one-dimensional superstructure with a wide surface area, making it a more suitable candidate material for photocatalytic H2 production. To evaluate the photocatalytic performance of chain g-C3N4 photocatalyst with the visible light irradiation, the H2 production performance was tested and compared with that of bulky g-C3N4 photocatalytic performance was compared.According to mentioned above, the prepared chain g-C2 evolution rate of the bare bulky g-C3N4 sample was measured to be 9.6 μmolh−1, separately. However, the photocatalytic properties of chain g-C3N4 were markedly improved, and the H2 generation rate increased as high as 28.7 μmolh−1. The chain g-C3N4 sample shows rather superior photocatalytic activity for H2 evolution, which could be ascribed to its vaster surface area (2 production system using chain g-C3N4 as the photocatalyst under visible light irradiation. Therefore, we studied the chemical stability of chain g-C3N4, as described in 3N4 did not decline significantly, indicating that the material has good chemical stability.As the result shown in ace area . Figure 3N4 emerge severe charge recombination, while the PL spectrum of chain g-C3N4 is intense quenched (3N4 can be restrained. The restraining of the e-h pair recombination is powerfully verified by the increased photocurrents for chain g-C3N4, as shown in 3N4 was provided with a better separation efficiency of e-h pairs.The key factor of photocatalytic reaction is the effective generation and rapid separation of photoexcited carriers . The optquenched . The reo3N4 and chain g-C3N4 were obtained through these six cycles. It can be seen that the transient photocurrent spectrum of the chain g-C3N4 electrode was much better than the bulky g-C3N4. Test results indicate that chain g-C3N4 greatly improves the separation of interface carriers. The photocurrent research consequences coincide with the impedance and photoluminescence research consequences, demonstrating that the chain g-C3N4 material will have better photocatalytic performance.To further confirm the ability of several samples to separate and transfer charges and to respond to light , we perf3N4 and chain g-C3N4 utilizing melamine as reactant materials. The chain g-C3N4 nanostructures illustrated improvement on the photocatalytic H2 production under visible light irradiation owing to unique inimitable one-dimensional structure, high specific surface area, excellent light-harvesting properties, and low recombination rate of electron-hole pairs. This simple preparation and facile composition of one-dimensional g-C3N4 nanostructures demonstrate a promising candidate for exploring more actual applications of carbon nitride.In summary, we proposed an annealing method to achieve bulky g-C