Toll-like receptors (TLRs) are essential for the protection of the host from pathogen infections by initiating the integration of contextual cues to regulate inflammation and immunity. However, without tightly controlled immune responses, the host will be subjected to detrimental outcomes. Therefore, it is important to balance the positive and negative regulations of TLRs to eliminate pathogen infection, yet avert harmful immunological consequences. This study revealed a distinct mechanism underlying the regulation of the TLR network. The expression of sex-determining region Y-box 4 (Sox4) is induced by virus infection in viral infected patients and cultured cells, which subsequently represses the TLR signaling network to facilitate viral replication at multiple levels by a distinct mechanism. Briefly, Sox4 inhibits the production of myeloid differentiation primary response gene 88 (MyD88) and most of the TLRs by binding to their promoters to attenuate gene transcription. In addition, Sox4 blocks the activities of the TLR/MyD88/IRAK4/TAK1 and TLR/TRIF/TRAF3/TBK1 pathways by repressing their key components. Moreover, Sox4 represses the activation of the nuclear factor kappa-B (NF-κB) through interacting with IKKα/α, and attenuates NF-kB and IFN regulatory factors 3/7 (IRF3/7) abundances by promoting protein degradation. All these contributed to the down-regulation of interferons (IFNs) and IFN-stimulated gene (ISG) expression, leading to facilitate the viral replications. Therefore, we reveal a distinct mechanism by which viral pathogens evade host innate immunity and discover a key regulator in host defense. Pathogeneses caused by microbial infections are the results of complex interactions between invading pathogens and infected hosts. Upon presentation of the pathogen, the host innate immune response is initially elicited and subsequently regulated through interactions with intracellular adaptors, membrane-bound receptors, and intrinsic crosstalk among signaling pathways . Many cyvia either the MyD88-dependent pathway that stimulates the nuclear factor kappa-B (NF-κB) and induces the tumor necrosis factor-α (TNFα), interleukin-1 (IL-1), and IFNs, or MyD88-independent pathway that activates IFN regulatory factors (IRFs) and IFNs [Inductions of IFNs are mediated by the Toll-like receptor (TLR) family, and activations of TLRs allow associated adaptors to trigger downstream events ,7. Five and IFNs ,15. MyD8and IFNs . IRAK4 sand IFNs ,18. Actiand IFNs ,20. TLRsand IFNs ,22.TableGiven the critical role of TLR in innate immunity, it is logical that TLR signaling is targeted by the pathogens in order to evade host immunity. Many cellular factors have been identified to repress TLR signaling through different mechanisms, including extracellular, transmembrane, and intracellular regulations and protein degradation . TLR sigSox4 belongs to the SRY-related HMG box (Sox) family that comprises 20 members in human and mouse . It contNP gene reverse primer. Levels of the sex-determining region Y-box 4 (Sox4) mRNA and IAV NP mRNA were detected by real-time PCR.Clinical throat swab specimens from 27 influenza A virus (IAV)-infected patients and 20 healthy individuals were collected from Wuhan Children’s Hospital . The patients were not suffering from any concomitant illnesses and did not express any serological markers suggestive of autoimmune disease. All specimens were treated with TRIzol reagent according to the protocol provided by the manufacturer (Invitrogen). Total RNAs of the specimens were extracted and reverse transcribed to cDNA using random primers and IAV g for 10 min at room temperature (RT). The middle layer was transferred to another new centrifuge tube and diluted with RPMI-1640. The remaining red blood cells were removed using red blood cell lyses buffer purchased from Sigma-Aldrich . The pure PBMCs were centrifuged at 1,500 × g for 10 min at RT and cultured in RPMI-1640.Blood samples of healthy donors were collected from Wuhan General Hospital of Guangzhou Military . To isolate peripheral blood mononuclear cells (PBMCs), blood cells were separated from blood samples and diluted in RPMI-1640 purchased from Gibco . Diluted blood cells (5 ml) were added gently to a 15 ml centrifuge tube with 5 ml lymphocyte separation medium  purchased from MP Biomedicals , and centrifuged at 2,000 × The study was conducted according to the principles of the Declaration of Helsinki and approved by the Institutional Review Board of the College of Life Sciences, Wuhan University, in accordance with its guidelines for the protection of human subjects. All participants provided written informed consent to participate in the study.2.Human hepatoma cell line (HepG2), human hepatoma cell line (Huh7), normal liver cell line (L02), human lung epithelial cell line (A549), human rhabdomyosacroma cell line (RD), and human embryonic kidney cell line (HEK 293 T) were purchased from American Type Culture Collection (ATCC) . Human hepatoma cell line (Huh7.5.1) was kindly provided by Dr. Francis V Chisari of Scripps Research Institute, USA. Cells were cultured in DMEM (Dulbecco’s modified Eagle medium) purchased from Gibco  supplemented with 10% heat-inactivated FBS  (Gibco) at 37°C in a humidified atmosphere of 5% COHuman enterovirus 71 (EV71) strain (Xiangyang-Hubei-09) was isolated previously by our group (GenBank accession no. JN230523.1) and the virus stock was propagated in RD cells . Sendai Sox4 (NM_00317.2) and the two deletion mutants Sox4ΔHMG and FLAG-Sox4ΔTAD were subcloned into pCMV-tag2A to generate pFLAG-Sox4, pFLAG-Sox4ΔHMG, and pFLAG-Sox4ΔTAD, respectively. The CDS of ubiquitin was subcloned into pcDNA3.1-Myc to generate pMyc-Ub, as described previously [The CD (coding sequence) of eviously .TLR1, TLR3, TLR4, TLR5, and TLR7 were subcloned into pGL3-Basic to generate pTLR1-Luc, pTLR3-Luc, pTLR4-Luc, pTLR5-Luc, and pTLR7-Luc, respectively. All plasmids were confirmed by sequencing analysis and the resulting proteins were verified by Western blotting.pFLAG-TLR3, pFLAG-TLR4, pFLAG-TLR7, and pFLAG-TLR9 were purchased from Beijing Zhongyuan, Ltd. . pFLAG-p65, pFLAG-p50, pFLAG-IRF3, pFLAG-IRF7, pGFP-p65, pGFP-p50, pGFP-IRF3, pGFP-IRF7, pIFN-β-Luc, and pISRE-Luc were kindly provided by Dr. Ying Zhu of Wuhan University. The full-length promoters of Antibodies against Mx1 , PKR (sc-707), OAS1 , TLR5 , and TLR10  were purchased from Santa Cruz Biotechnology . Antibodies against TLR1 (#2209), TLR2 (#2229), TLR3 (#2253), TLR4 (#2246), TLR6 , TLR7 (#2633), TLR8 , TLR9 (#2254), ubiquitin (#3933), MYD88 (#4283S), IRAK4 (#4363), and IRAK1 (#4504S), and NF-kB Pathway Sampler Kit, were purchased from Cell Signaling Technology . Antibodies against FLAG (F3165) and GFP (G1546) were purchased from Sigma-Aldrich .Sox4 (siR-Sox4) were designed and synthesized by RiboBio  and tested in a previous report [siRNAs specific to s report . TNF-α . RNA (1 μg) was used as a template to synthesize cDNA using random primers  and moloney murine leukemia virus (MMLV) reverse transcriptase (1 μl)  at 42°C for 60 min, which was then denatured for 10 min at 75°C; the total reaction volume was 20 μl.Total RNA was extracted from cells or transfected cells using TRIzol reagent according to the protocol provided by the manufacturer (Invitrogen). DNA was removed from the sample using on-column DNase I treatment at 37°C for 30 min. RNA was washed with 75% ethanol and redissolved in DEPC ddHReal-time PCR (RT-PCR) was performed using SYBR Green PCR master mix in a Light Cycler 480 . After an initial incubation at 95°C for 5 min, the reaction mixtures were subjected to 40 cycles of amplification under the following conditions: 94°C for 15 s, 56°C for 15 s, and 72°C for 20 s. The fluorescence was measured at this step to assess the quality of the primers, which was followed by a final melting curve step from 50°C to 95°C. Each sample was run in triplicate, and the threshold cycles (Cts) were averaged and normalized to endogenous glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The relative amount of amplified product was calculated using the comparative Ct method. The primers used in this study are listed in g for 5 min at 4°C to remove cell debris. Then the concentration of protein in each sample was determined using a Bradford assay kit . Cell lysates (100 μg) were electrophoresed using 10% sodium dodecyl phosphate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes . Nonspecific sites were blocked using 5% nonfat dried milk in PBS containing 0.05% Tween 20 (PBST) for at least 1 h at RT, and then the membranes were incubated with specific primary antibodies for at least 3 h at RT or overnight at 4°C. The bound primary antibodies were detected by incubation with the appropriate secondary antibodies for 45 min at RT. The blots were analyzed using a luminescent image analyzer .Cells were harvested at 48 h post-transfection or at the indicated treatment times, washed once with ice-cold phosphate-buffered saline (PBS), and re-suspended in lyses buffer  supplemented with 1 × protease inhibitor cocktail . Lysates were sonicated on ice and then centrifuged at 10,000 × For dual-luciferase assays, the expression of Renilla luciferase was used as a reference under the control of an independent cytomegalovirus (CMV) promoter, and that of firefly luciferase was under the control of the inserted target gene’s promoter. The ratio of firefly luciferase activity to Renilla luciferase activity reflects the final relative luciferase activity for each sample. Cells were transfected with the indicated plasmids and luciferase reporter plasmid for 48 h, after which the cells were washed twice with ice-cold PBS. Luciferase lyses buffer (100 µl)  was added to each well of a 24-well plate. Cells were lysed for 10 min at RT, after which 50 µl of each sample was transferred to a new centrifuge tube and mixed with 15 µl of the corresponding luciferase assay substrate . Luciferase activity was typically measured for 10 s using a luminometer . All assays were performed in triplicate, and the data are expressed as means ± SD (standard deviation).g for 5 min at 4°C to remove cell debris. One-fourth of the supernatant was used as input. Then the remaining supernatants were collected, pre-cleared using protein G Sepharose beads , and incubated with the indicated antibodies overnight at 4°C. The supernatants were then mixed with protein G sepharose beads  for 2 h at 4°C. The immunoprecipitates were centrifuged at 2,000 × g for 2 min at 4°C, washed five times with RIPA lyses buffer, eluted with 1% SDS buffer, boiled in loading buffer for 5 min, and then analyzed using SDS-PAGE and Western blotting.Human embryonic kidney (HEK293T) cells were seeded in dishes (10 cm diameter) and co-transfected with the indicated plasmids for 2 days. Cells were lysed using radioimmunoprecipitation assay (RIPA) buffer  were centrifuged at 10,000 × g for 2 min at 4°C for collection, washed with dialysis buffer, and eluted with elution buffer (1% SDS and 100 mM NaHCO3). Then the resulting supernatant was incubated at 67°C for 5 h to reverse formaldehyde cross-linking with a final concentration of 0.1 M NaCl, and proteins were removed by adding proteinase K for 1 h at 45°C. DNA was precipitated with ethanol and extracted three times with phenol/chloroform. Pellets were re-suspended in TE buffer and subjected to PCR amplification using the corresponding primers.Cells were seeded in dishes (10 cm diameter) and transfected with the indicated plasmids. Chromatin immunoprecipitation (ChIP) assays were performed according to the X-ChIP protocol (Abcam). Formaldehyde was added to the culture medium to a final concentration of 1% for 5 min at RT and then a final concentration of 125 nM glycine was added to stop the cross-linking reaction for 5 min at RT. The cells were washed twice with ice-cold PBS, scraped, centrifuged, and lysed in ChIP lyses buffer . Lysates were sonicated on ice and the debris was removed by centrifugation. One-fourth of the supernatant was used as DNA input. The remaining supernatant was diluted 10-fold with dilution buffer, pre-cleared using protein G Sepharose beads , and incubated with the indicated antibodies overnight at 4°C. The supernatant was then mixed with protein G Sepharose beads  for 2 h at 4°C. Immunoprecipitated complexes were centrifuged at 2,000 × 2, 10 mM KCl, 0.5 mM dithiothreitol, and 200 mM sucrose) for 15 min at 4°C with tube flipping. A final concentration of 0.5% NP-40 was then added to the lysates with tube flipping for 5 s and the lysates were centrifuged at 16,000 × g for 5 min at 4°C. The supernatant, as cytoplasm extract, was transferred to a fresh tube. The sediment was rinsed with buffer A, resuspended in one volume of buffer B  with tube flipping for 15 s, and incubated on a shaking platform for 30 min at 4°C. The nuclei were centrifuged at 16,000 × g for 5 min at 4°C and the supernatants were collected. Cocktail protease inhibitor was added to each buffer. The cytoplasm and nuclear extracts were stored at −80°C until use.Cells were seeded in six-well plates and transfected with the indicated plasmids. At 48 h post-transfection, cells were washed twice with ice-cold PBS, collected, and lysed in two volumes of buffer A  cells were co-transfected with the indicated plasmids for 36 h and treated with the protein synthesis inhibitor, cycloheximide (CHX), at a final concentration of 50 μg/ml for the indicated times before harvest. Cells were lysed in Western blot lyses buffer with proteinase inhibitor cocktail. Lysates were sonicated on ice and the debris was removed by centrifugation. Then whole cell extracts were prepared as described above and were used for Western blotting.For ubiquitination assays, HEK293T cells were seeded in dishes (10 cm diameter), co-transfected with the indicated plasmids for 36 h, and treated with the proteasome inhibitor MG-132 at a final concentration of 20 μM for 9 h. Cells were lysed in RIPA buffer with proteinase inhibitor cocktail and sonicated gently three times on the ice. Cell lysates were centrifuged to remove the debris and the supernatants were divided into two aliquots: one aliquot (5%) was used as a whole cell extract for Western blotting, while the other (95%) was incubated with the indicated antibodies overnight at 4°C and then incubated with protein G Sepharose beads for 2 h at RT. The precipitates were washed five times with RIPA buffer and then the bound proteins were eluted with 1% SDS buffer, boiled in loading buffer for 5 min, and analyzed using SDS-PAGE and Western blotting.t-tests to distinguish the equality of means. Means were illustrated using histograms with error bars representing the SD; a P value of <0.05 was considered statistically significant.All experiments were reproducible and repeated at least three times with similar results. Parallel samples were analyzed for normal distribution using Kolmogorov-Smirnov tests. Abnormal values were eliminated using a follow-up Grubbs test. Levene’s test for equality of variances was performed, which provided information for Student’s Sox4 mRNA was induced in human embryonic kidney cells (HEK 293 T) infected with vesicular stomatitis virus (VSV), human hepatoma cells (Huh7.5.1) infected with hepatitis C virus (HCV), human lung adenocarcinoma cells (A549) infected with influenza A virus (IAV), and human rhabdomyosarcoma cells (RD) infected with enterovirus 71 (EV71)   –d. Moreoividuals , and the 0.7581) . TherefoVP1 mRNA in intracellular extracts of infected cells and viral load in extracellular extracts of infected cells were stimulated by Sox4 and inhibited by siR-Sox4 . Similarly, viral protein expression was upregulated by Sox4 and downregulated by siR-Sox4 in GFP-VSV-infected cells . In addisiR-Sox4  and K. TIFN-β (a type I IFN), IFN-λ1 (a type III IFN), and IFN-induced GTP-binding protein (MxA) (an ISG) is then evaluated during SeV infection. The results indicated that all the cell lines used had activated IFN-pathway and were suitable for this study (IFN-β promoter and IFN-stimulated response element (ISRE) in L02 and HepG2 cells , 2ʹ,5ʹ-oligoadenylate synthetase 1 (OAS1), and Mx1, were repressed by Sox4 in L02, HepG2, and PBMCs during SeV stimulation . The activation of is study –d. Then,G2 cells  and f. Ind PBMCs  and h. Mmulation –k, but emulation . Therefothe IFN-β promoter stimulated by SeV could be repressed by a specific inhibitor of NF-kB (BAY11 7082)  , suggest11 7082) , indicat11 7082) , indicat11 7082) , suggest11 7082) , demonstSox4 contains an HMG domain that binds to targeted genes and a TAD domain that activates targeted genes . We specp65, p50, IRF3, and IRF7 mRNAs were not affected by Sox4  and TLR/TRIF/TRAF3/TBK1 (MyD88-independent) pathways ,48. HereMyD88 mRNA (Stimulation of TLRs triggers MyD88, which subsequently recruits IRAK4, IRAK1, and TRAF6 to form a complex to initiate signal transduction. We further determined the role of Sox4 in the formation of the MyD88 complex. The interactions of MyD88 with IRAK4 and IRAK1 were enhanced by SeV and repressed by Sox4 , top, anD88 mRNA –e and MyD88 mRNA –h were aD88 mRNA –h. In adD88 mRNA . These rD88 mRNA , suggestD88 mRNA , indicatTLR1, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10 mRNAs, but not TLR2, were repressed by Sox4 in L02 cells and PBMCs (TLRs mRNAs except TLR2 and TLR8 were inhibited by Sox4 in HepG2 cells (Inductions of IFNs are mediated by TLRs and activation of TLRs triggers downstream events of host innate immunity ,7. Internd PBMCs  and b, aG2 cells . SimilarG2 cells  and e. TG2 cells . All TLRG2 cells . In addiG2 cells . FurtherG2 cells , indicatTLR1, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10 (TLR promoters.Sox4 is a transcription factor and binds preferentially to the AACAAAG/CTTTTGTT motif leading to activate transcription of targeted genes . Interesnd TLR10 , suggestTLR promoters, we constructed mutant promoters of TLR1, TLR3, TLR4, TLR5, and TLR7 by site-directed mutagenesis of Sox4-binding sequences (TLR1 activity, but not MT-TLR1 (TLR3 function, but not MT-TLR3 (TLR4 activity, but not MT1-TLR4, and up-regulated MT2-TLR4 and MT3-TLR4 (TLR5 activity, but not MT1-TLR5, MT2-TLR5, or MT3-TLR5 (TLR7 activity, but not MT1-TLR7 or MT2-TLR7, and enhanced MT3-TLR7 (TLR promoters are required for the regulation of TLRs and demonstrated that Sox4 represses TLRs at transcription level through binding and inhibiting TLR promoters (To confirm the specific binding of Sox4 to equences –f. The w MT-TLR1 ; repress MT-TLR3 ; down-reMT3-TLR4 ; attenuaMT3-TLR5 ; and redMT3-TLR7 . Taken tromoters .Figure 8IFNs and ISGs expression with stimulation, which suggests that Sox4 plays a role in attenuating the IFN-related pathway. Then, we confirmed Sox4 performs this repression through four aspects on the upstream pathway: 1. Sox4 inhibits NF-kB activity by interacting with IKKα/α complex and repressing IκBα phosphorylation, ubiquitination, and degradation. 2. Sox4 facilitates proteasome-mediated ubiquitination-related degradation of NF-kB and IRF3/7 proteins. 3. Sox4 attenuates the phosphorylation of IRAK4, TAK1, and TBK1 to repress the activation of TLR/MyD88/IRAK4/TAK1 and TLR/TRIF/TRAF3/TBK1 pathways. 4. Sox4 downregulates the transcription of all TLRs excepting TLR2 and MyD88 by directly binding to their promoters.TLRs are mediators critical for the regulation of host immunity to combat pathogen infections ,50. Howevia different mechanisms [TLRs initiate IFN/JAK/STAT signaling through regulating TLR/MyD88/IRAK4/TAK1 and TLR/TRIF/TRAF3/TBK1 pathways . Sox4 machanisms ,26,27, tchanisms . Most TLchanisms ,55. TLR3Taken together, we identified a master regulator that hijacks host innate immunity by repressing TLRs signaling networks at multiple stages. The discovery of a master negative regulator and its direct implications in host defenses provides insights into our understanding of pathogen infection and host immunity and provides future possibilities for targeting signaling molecules for new therapeutics in human infections and associated diseases.


An acetylene inhibition assay did not show the denitrification of N2 to N2O by this bacterium. When N2O was injected as the only nitrogen source, this bacterium did not assimilate N2O. A polymerase chain reaction demonstrated that this bacterium did not have the typical nosZ gene. This bacterium belonged to Chitinophagaceae, but did not belong to known families that include bacteria with the atypical nosZ. This is the first study to show that a non-denitrifier actively reduces N2O, even under high pH conditions.A nitrous oxide (N N2O absorbs infrared radiation, and its potential to cause global warming is 298-fold that of carbon dioxide and, thus, is regarded as the most important ozone-depleting substance in this century , other GHG also influence climate change  and incomplete denitrifiers (NO3–/NO2– → NO → N2O) contribute to the emission of N2O. Complete denitrifiers possess the nosZ gene, which encodes N2O reductase  in soil is limited, even in relatively fertile soils because these nitrogen sources are competitively assimilated by plants and other microorganisms  (3 (0.52 g L–1) was used as the medium in the culture-based N2O reduction assay (2O (2SO4 and 1 M KOH that was gelled with 0.5% (w/v) gellan gum and then autoclaved. The same medium was used in subsequent experiments. N2O levels were measured as described in a previous study  sucrose and 5 mM KNO 4.5–7.5 . However2O-consuming microorganisms, colonies were isolated as described in a previous study (2O gas (GL Sciences) was injected using a gas-tight syringe into the headspace of gas chromatography vials to a final concentration of 2,000 ppmv. After incubations for 0, 1, and 4 days at pH 8.5, N2O concentrations in the headspace gas were measured. The results obtained showed that strain A (Sac-f1) exhibited the greatest consumption of N2O  acetylene gas and N2O  were injected into the headspace of the assay vials immediately after the inoculation of the isolated bacterium, and media were then incubated at 25°C for 0, 1, 2, 3, and 6 weeks. The concentration of N2O was measured after these incubation periods. N2O concentrations did not decrease with the acetylene gas treatment, which confirmed that the bacterium reduced N2O  using nosZ gene-specific primers (nosZ-1111F and nosZ-1773R) . PCR amplicons using the specific primers were purified by agarose gel electrophoresis. Pseudomonas denitrificans NBRC 12442 was used as the positive control. This bacterium did not have a nosZ gene (The DNA of this bacterium was extracted using an Isoplant II DNA Extraction kit (Nippon Gene), and the Z-1773R) . The 16SosZ gene , and theChitinophagaceae. The closest species to the isolated bacterium was Chitinophaga eiseniae (96.35% similarity). A phylogenetic analysis was performed based on the neighbor-joining method using MEGA X , followed by incubations for 0, 1, 2, and 3 weeks. N2O was injected as described earlier. This isolated bacterium reduced N2O at pH in the range of 4.5 to 9.0, with the optimum pH being 8.5 . Since the application of nitrogen fertilizers, such as urea, to farmlands results in the largest increase in pH  An Unknown Non-denitrifier Bacterium Isolated from Soil Actively Reduces Nitrous Oxide under High pH Conditions. https://doi.org/10.1264/jsme2.ME20100


A recombinant form of PCSK9_55 was efficiently produced but displayed impaired secretion that resulted in its intracellular trapping. However, the nonsecreted PCSK9_55 was able to induce degradation of LDLR, though with 50% lower efficiency than PCSK9_62. Collectively, our data show that 1) PCSK9_55 is formed extracellularly; 2) PCSK9_55 has a shorter half-life; 3) there is a small intracellular pool of PCSK9_55 that is not secreted; and 4) PCSK9_55 retained within the cell maintains a reduced efficiency to cause LDLR degradation.Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates cholesterol metabolism by inducing the degradation of hepatic low density lipoprotein receptors (LDLRs). Plasma PCSK9 has 2 main molecular forms: a 62 kDa mature form (PCSK9_62) and a 55 kDa, furin-cleaved form (PCSK9_55). PCSK9_55 is considered less active than PCSK9_62 in degrading LDLRs. We aimed to identify the site of PCSK9_55 formation (intracellular vs. extracellular) and to further characterize the LDLR-degradative function of PCSK9_55 relative to PCSK9_62. Coexpressing PCSK9_62 with furin in cell culture induced formation of PCSK9_55, most of which was found in the extracellular space. Under the same conditions, we found that  Elevated low density lipoprotein (LDL) cholesterol (LDL-C) levels are a major risk factor for cardiovascular disease (CVD) , 2, 3. TPCSK9 is mainly produced by the liver and undergoes several posttranslational modifications that may influence its kinetics and function , 19, 20.et al.  , 31, 32.et al.  identifiet al. . Later,   impair its proteolysis and act as gain-of-function (GOF) mutations, increasing LDLR degradation . These clinical observations first suggested that the furin-cleaved form of PCSK9 would have reduced functionality  PCSK9_55 is formed extracellularly; 2) PCSK9_55 has a shorter half-life; 3) there is a small intracellular pool of PCSK9_55 that is not secreted; and 4) PCSK9_55 retained within the cell maintains a reduced efficiency to cause LDLR degradation.In this study, we aimed to identify the site of furin-dependent PCSK9_55 formation and to further characterize its LDLR-degradative properties relative to PCSK9_62. Our results demonstrate that the coexpression of PCSK9_62 and furin leads to production of PCSK9_55 that is localized predominately in the extracellular space. Under the same conditions, the addition of a furin inhibitor with access to both intracellular and extracellular compartments selectively reduced the efficiency of cleavage only extracellularly. Our expression studies with engineered PCSK9_55 demonstrate that the intracellular pool of PCSK9_55 is not secreted to the extracellular space, suggesting that, under physiologic conditions, the intracellular PCSK9_55 originates from endocytosis of the plasma form, rather than from cleavage by intracellular furin. This was supported by a pulse-chase experiment that demonstrates PCSK9_55 first appears at the extracellular compartment. In the same experiment, we also confirmed that PCSK9_55 has a shorter half-life than PCSK9_62. We also determined that intracellular PCSK9_55 has the ability to induce the degradation of LDLRs, albeit with only 50% of the capacity compared with PCSK9_62. Taken together, our results suggest that All products were purchased from Sigma-Aldrich unless otherwise noted.2 humidified atmosphere. Composition of cell media is detailed in Human embryonic kidney HEK293T cells (ATCC® CRL-1573™) and hepatocellular carcinoma HepG2 cells (ATCC® HB-8065™) were routinely grown as previously described  at 37°C Furin inhibition was achieved using cell-permeable Decanoyl-RVKR-CMK added to cell media at 25 and 50 μM  .i) R46L (pPCSK9 62∗R46L or pIR-hPCSK9 FL + v5 (R46L)), a PCSK9 LOF mutation correlated with 15% reduction in LDL-C and 47% reduction in coronary heart disease risk (ii) R218S (pPCSK9 62∗R218S or pIR-hPCSK9 FL + v5 (R218S)), a PCSK9 GOF mutation resistant to furin cleavage and correlated to higher risk for CVD (pcDNA3 expression plasmids for human PCSK9_62 (pPCSK9 62) and PCSK9_62 lacking the prodomain  were previously developed in our laboratory . pcDNA3 ase risk , 41, and for CVD , 34, wer5 cells per well) in 6-well plates; on day 2, media was changed to serum-free media (900 μL); and cells were transfected with the DNA-agent complex  per 1.5 μg of plasmid DNA in DMEM media). Serum was withdrawn until the end of the experiment (72 h) to avoid interference. Cells and conditioned media were collected on day 5 for protein evaluations with Western blot, ELISA, or flow cytometry. Cell extracts were obtained from cells washed twice with ice-cold phosphate-buffered saline and scraped in 100 μL of lysis buffer  supplemented with 1X proteinase inhibitor ). Lysates were incubated on ice for 1 h and vortexed every 15 min and then spun at 14,000 g at 4°C for 20 min. Collected supernatants were quantified by a Lowry assay  and then stored at −20°C until used.Transient transfection was performed following the manufacturer's instructions with a nonliposomal agent. Briefly, cells were plated  were resolved in SDS-PAGE  and later transferred to nitrocellulose membranes  and blotted for human PCSK9, LDLR, furin, or actin . AntibodImage Studio Lite Ver 5.2 software (LI-COR Biosciences) was used to quantify band intensity. PCSK9 band intensity from total extracts (40 μg/lane) was normalized to actin band intensity, whereas PCSK9 band intensity from cell-conditioned media  was not normalized. Quantification of PCSK9 intensities was done separately in cell extracts and in cell media compartments due to the irrationality of normalizing by total protein content. Total PCSK9 band intensity (PCSK9_62 + PCSK9_55 or ProPCSK9 + PCSK9_62 + PCSK9_55 + PCSK9 PD) found in pPCSK9 62 (±pcDNA3) transfected cells condition was considered as the reference, and other conditions were quantified relative to it. Note that there is an ∼8X difference between the relative volume loaded from the intracellular and the extracellular compartments in the gels. Cell extracts are loaded as ∼1/6.25 of the total volume of extracts , while cell media is loaded as ∼1/50 of the total volume of media in the well .Conditioned media (9 μL) and cell extracts (20 μg) of HEK293T cell were subjected to N-glycosylation removal with the amidase PNGase F that cleaves between the innermost GlcNAc and the asparagine residue of high mannoses, following vendor's instructions , and later subjected to Western blot as described above. HepG2 cells were treated with tunicamycin   to inhibit N-glycosylation, thus generating the nonglycosylated PCSK9 form.Cell extracts (20 μg) of HEK293T cell were subjected to Endoglycosidase H (Endo H) digestion following vendor's instructions  and later subjected to Western blot as described above.Human PCSK9 was quantified in the cell extracts  and cell-conditioned media (1:10–12.5 dilution) of HEK293T cells transfected with pPCSK9 62, using a human PCSK9 ELISA kit  and following manufacturer's instructions. Total PCSK9 content in each compartment (intracellular and extracellular) was calculated based on the final volume of the cell extracts (100 μL) and cell media .2, 150 mM NaCl, 20 mM KCl and 50 mM Tris-HCl pH 7.4 in 10 μL of final volume at 37°C for 24 h) to achieve the highest yield of PCSK9 proteolysis. Reactions were stopped by chelating calcium with 4 mM EDTA. Products were analyzed by Western blot or mass spectrometry.The reaction conditions were adapted from published work , 26. PCShttps://panoramaweb.org/kqwUPs.url), a server hosted by the Department of Genome Sciences at the University of Washington (The products obtained from in vitro PCSK9 proteolysis with furin were subjected to liquid chromatography-mass spectrometry (LC-MS). The designed method skipped the common protein digestion step used in MS, to enhance the detection of the released 7 kDa cleaved peptide in a qualitative manner, since it will only be expected in the furin-containing reaction. Two biological replicates including the products of a negative control reaction  and the experimental reaction PCSK9_62 with furin (PCSK9 55 kDa) were evaluated separately in 2 independent LC-MS analyses to detect the expected furin cleavage product containing residues 153–218 (average mass = 7730.5). With no further digestion, aliquots of 2.2 μg of PCSK9 from a control sample (no furin) and a furin-incubated sample were dried using a DNA120 SpeedVac (Thermo Scientific) and stored at −20°C until analysis. Samples were solubilized in 22 μL of 8 M urea for 24 h prior to analysis. A total of 1 μg of protein was analyzed using an LTQ Velos Pro linear ion trap (Thermo Scientific). Samples were injected onto a micro protein trap cartridge (Optimize Technologies) at a flow rate of 20 μl/min in mobile phase containing 0.1% formic acid. After 5 min, the flow was diverted to a 1 × 250 mm C4 column . Protein was eluted by increasing the acetonitrile concentration from 2% to 7.5% over 1 min and then 7.5% to 60% over 30 min, and data collection on the mass spectrometer started 10 min into the separation. The instrument used an HESI-II probe fitted with a 34 gauge metal needle, 5.0 kV source voltage, 325°C ion-transfer tube temperature, sheath gas setting of 5, full MS scans in profile mode over a range of m/z = 400–2000, and averaging of 10 μscans. Spectra acquired during elution of the PCSK9 153–218 peaks were then averaged and deconvoluted using the Manual Respect module for isotopically unresolved data in Protein Deconvolution 4.0 software (Thermo Scientific). Single-ion chromatographic traces were produced using Qual Browser software within the Xcalibur Suite (Thermo Scientific). Peptide identification was unnecessary given the fact that we used purified PCSK9. Annotated mass spectra are available at PanoramaWeb (shington .5 cells per well) in 6-well plates and maintained as detailed above. On day 2, cells were cotransfected with pPCSK9 62 + pcDNA3 or pPCSK9 62 + pFURIN as explained above. Pulse-chase experiments started on day 4 following the methods followed in  in 6-well plates. The following day, media was changed to serum-free and cells were transfected to express GFP-tagged LDLR. Serum was then withdrawn until the end of the experiment (72 h) to avoid interference. Exposure to PCSK9 was introduced either by cotransfecting cells with a plasmid expressing PCSK9 on day 2 or by adding purified PCSK9_62 to the medium  on day 4. On day 5, cells were harvested, spun , and washed with 1 mL of flow buffer . Cells were spun again and resuspended with 0.5 mL of flow buffer containing 2.5 ng/μL propidium iodide , used as a live/dead discriminator. Compensation was performed using unstained and single-stained cells. Acquisition was performed on BD FACSCanto™ II instrument using BD FACSDiva™ software, and analysis was performed using Flow Jo v10. The gating strategy followed to quantitatively monitor PCSK9 activity is detailed in HEK293T cells were plated  and a TaqMan Master mix , following vendor's recommendations. Changes in gene expression were calculated using the quantitative ΔΔCt method, normalized against RPL32, and plotted relative to the control condition.The mRNA expression levels of spliced X-box binding protein-1 (XBP-1s) was evaluated as a predictor of endoplasmic reticulum (ER) stress, following the method followed in . RNA wast-test analysis run with a 2-tail distribution in Microsoft Excel Statistical tool or Prism 8.3 . Kinetic changes studied in the pulse-chase experiments were evaluated by 2-way ANOVA. Differences with P-values < 0.05 were considered statistically significant.Values are expressed as mean ± SEM with a minimum of 3 independent experiments. Change between 2 conditions was evaluated by unpaired Student's P < 0.05) and more robustly in the extracellular compartment  . Furin also altered the PCSK9 prodomain levels, with a 146 ± 46% increase intracellularly (P < 0.01) and a 83.7 ± 2.6% reduction in the extracellular space (P < 0.001) (We induced expression of PCSK9_62 or PCSK9_55 by transient transfection in HEK293T cells lacking native PCSK9. Cotransfection with pPCSK9_62 and empty vector (pcDNA3) resulted in the expression and accumulation of ProPCSK9 and PCSK9_62 in the cell and secretion of PCSK9_62 into the media A, as exp< 0.001) C–E. Over< 0.001) B, E. Bes< 0.001) . We repe< 0.001) , and the< 0.001) .Fig. 1GeFurin-specific cleavage was corroborated by reacting purified PCSK9 (nonglycosylated) with furin and analyzing reaction products both by Western blotting and mass spectrometry  and by ei) PCSK9_62 accumulated in the extracellular space with little intracellular retention; ii) both PCSK9_62 and PCSK9_55 expressed the nonglycosylated and N-glycosylated forms; iii) furin induced the formation of PCSK9_55 from PCSK9_62, and PCSK9_55 was mainly located extracellularly; and iv) furin cleavage of PCSK9_62 induced a decrease in total PCSK9 accumulation and changes in PCSK9 prodomain.Under these conditions, P = 0.0376) when compared with nontreated cells, while causing no significant changes in the intracellular compartment (P < 0.05) more PCSK9_62 in the media when furin inhibitor was added to the pPCSK9 62 and pFURIN cotransfection condition  (P < 0.001) (P < 0.001]). Furin also induced the appearance of the ∼7 kDa peptide cleaved from PCSK9_62 to generate PCSK9_55. The released peptide was detected after 1 h of chasing intracellularly and extracellularly. Despite detecting the cleaved ∼7 kDa peptide intracellularly, PCSK9_55 was only detected in the extracellular compartment (P < 0.07), but then decayed (Kinetic changes of PCSK9 production and secretion upon furin cleavage were studied using a pulse-chase approach . Pulse-c < 0.01) A, and la< 0.001) B. Along partment A, B. Con decayed B.Fig. 3KThese data suggest that a pericellular furin pool is likely the major driver of PCSK9_55 formation in vivo. In addition, these data supports that furin induces a decrease in total PCSK9 by reducing PCSK9_62 levels and generating a product, PCSK9_55, with a shorter half-life than PCSK9_62.P < 0.05) (We engineered an expression vector to produce a recombinant form of PCSK9_55 . HEK293T < 0.05) , indicat < 0.05) .Fig. 4Inin trans with an orthologous prodomain and will be efficiently secreted , whereas PCSK9_55 only reduced LDLR levels by 28 ± 4% (P < 0.01), significantly less than the full-length protein (P < 0.01)  . We vali < 0.01) D. To det < 0.01) , using q < 0.01) .Fig. 5DeThese data indicate that intracellular PCSK9_55 retains the ability to degrade LDLRs, but its activity is reduced by 50% compared with the mature form, PCSK9_62.in trans with the orthologous prodomain that propels PCSK9 out of the cell. Finally, we demonstrate that PCSK9_55 is capable of degrading LDLRs through an intracellular pathway, although at 50% reduced activity when compared with PCSK9_62.Our study contributes to the characterization of furin-cleaved PCSK9 by demonstrating that plasma PCSK9_55 is formed in the extracellular compartment and that the intracellular pool of PCSK9_55 cannot be secreted from the cell but is still capable of degrading LDLRs through an intracellular pathway. Our in vitro model shows that overexpression of PCSK9_62 and furin in cells generates PCSK9_55 in the extracellular space. Under these circumstances, inhibition of furin selectively impacted the extracellular PCSK9_55 pool. A pulse-chase approach confirmed that PCSK9_55 can only be detected at the extracellular space and suggests a pericellular site of action for furin to cleave PCSK9_62. Moreover, our studies show that the intracellular pool of PCSK9_55 generated by recombinant expression is retained intracellularly because it fails to assemble et al. discarded the role for extracellular furin in cleaving PCSK9_62 and proposed that furin localized in the transmembrane or in the Golgi generates PCSK9_55  generated a protein that was only detected intracellularly. We tested the effect on secretion of coexpressing PCSK9 prodomain with prodomain-free PCSK9_62 or PCSK9_55. While cotransfection of the prodomain with the prodomain-free PCSK9_62 led to effective secretion of the protein, as expected , 46, 47,Changes in plasma furin levels may affect PCSK9 levels or function. Furin is a ubiquitous protein expressed in several cellular compartments including the trans-Golgi network, endosomes, and cell surface. In the ER, profurin's 83 amino acid prodomain facilitates its autocleavage where it remains bound to furin acting as a potent autoinhibitor during its transport to the trans-Golgi network/endosomes where the acidic pH promotes the autoproteolytic cleavage of the prodomain from furin, thus activating the enzyme . A transIt should be mentioned that, in humans, the ratio between PCSK9 and furin in plasma varies over 2 orders of magnitude , 54. Theet al. . Additional raw data are available upon request.Mass spectrometry raw files are available at PanoramaWeb (The authors declare that they have no conflicts of interest with the contents of this article


Several point mutations of GCAP1 have been associated with impaired calcium sensitivity that eventually triggers progressive retinal degeneration. In this work, we demonstrate that the recombinant human protein presents a highly dynamic monomer-dimer equilibrium, whose dissociation constant is influenced by salt concentration and, more importantly, by protein binding to Ca2+ or Mg2+. Based on small-angle X-ray scattering data, protein-protein docking, and molecular dynamics simulations we propose two novel three-dimensional models of Ca2+-bound GCAP1 dimer. The different propensity of human GCAP1 to dimerize suggests structural differences induced by cation binding potentially involved in the regulation of retGC activity.The guanylyl cyclase-activating protein 1, GCAP1, activates or inhibits retinal guanylyl cyclase (retGC) depending on cellular Ca In photoreceptor cells, light photons are transformed into electric signals by the coordinate interplay of a complex network of proteins, regulated by different chemical messengers .2+ concentration (<100 nM) GCAP1 EF2 can bind at least one Mg2+ ion [2+ through the cyclic nucleotide-gated (CNG) channels determining GCAP1 Ca2+-bound state (EF2-4 bind 3 Ca2+ ions) that inhibits retGC1 activity. During evolution, GCAP1 N-terminal EF-hand (EF1) lost the capability to bind calcium and became involved in the binding of retGC1 and modulation of its activity [Guanylyl cyclase-activating protein 1 (GCAP1) belongs to the calmodulin superfamily and hosts 4 EF-hand motifs (EF1–EF4). At low CaMg2+ ion ,4. Activactivity ,6,7.2+ determining the deregulation of retGC1 at physiological calcium levels, with progressive degeneration of the cone photoreceptors [2+ in the respective GCAP1 variants [So far, 22 pathological point mutations of GCAP1 have been identified, which are associated with an autosomal dominant cone or cone-rod dystrophy . Such mueceptors ,10. Nevevariants ,12. Recevariants .It is known that purified GCAP1 displays both monomeric and dimeric species . Recentl2+ or Mg2+ binding and by ionic strength.In this work, we performed several experiments on recombinant human (h) GCAP1, to thoroughly analyze the dimeric assembly. Microscale thermophoresis, size exclusion chromatography (SEC) and small-angle X-ray scattering (SAXS) data confirmed the presence of a monomer-dimer equilibrium in solution influenced by Ca2+-bound form presented in this work, recapitulate all current experimental lines of evidence from us and other authors and might have implications on the regulatory activity of GCAP1.Using the ZDOCK program  for rigiE. coli strain BL21-CodonPlus RP  together with a pBB131 plasmid carrying the coding sequence for yeast N-myristoyltransferase (NMT). The hGCAP1 construct has the E6S mutation needed for the post-translational myristoylation.To produce the N-terminal myristoylated hGCAP1, the recombinant plasmid pET11a carrying the cDNA sequence was co-transformed in the Cell cultures were grown at 37 °C in 2 L of LB medium containing 100 µg/mL ampicillin (Amp), 30 µg/mL kanamycin (Kan), and 34 µg/mL chloramphenicol (Cm). The myristic acid (220 µM) was added when the culture reached the OD600 = 0.4. Fifteen min later, the expression of hGCAP1 and NMT was induced by the addition of 1 mM IPTG. Cells were harvested after 3 h at 37 °C and frozen at –20 °C.E. coli cells were resuspended in lysis buffer  and lysed by high-pressure cell disruptor  at 25 KPSI. The crude extract was centrifuged at 38,700× RCF for 30 min and filtered through a 0.45 µM filter. The clarified soluble extract was loaded in a HiPrepTMQ XL 16/10  in loading buffer  and eluted with a linear gradient of NaCl (from 0.2 to 1 M).Typically, 3 g of 2, and one cOmplete protease inhibitor tablet. All the purification steps were performed using an ÄKTA pure 25L  at 4 °C and analyzed with SDS-PAGE.Fractions corresponding to hGCAP1 were pooled, concentrated, and passed through a HiLoadTM 16/600 SuperdexTM75 pg  equilibrated with 20 mM Tris/HCl pH 8.0, 100 mM NaCl, 5 mM DTT, 1 mM CaClTo obtain the apo form of hGCAP1, the protein was further purified using a hydrophobic interaction chromatography, decreasing the NaCl concentration from 300 mM to 0 mM. EGTA was added at 4 mM concentration both in the loading and the elution buffer.2+ were recorded in a Jasco J-810 spectropolarimeter  equipped with a Jasco temperature controller module PFD-425S. All measurements were performed in a 0.1 cm path length quartz cuvette at 0.2 mg/mL protein concentration in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM DTT, added with 2 mM Ca2+ or 2 mM EGTA. Far-UV spectra were recorded from 190 to 260 nm, at 20 and 95 °C (temperature was increased in ramp mode at 1 °C/min).Far-UV Circular Dichroism (CD) spectra of hGCAP1 with and without Ca2+-bound hGCAP1 the protein buffer was exchanged into 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM DTT, 5 mM CaCl2 using a PD-10 Desalting Column . Calcium was substituted with 5 mM MgCl2 and 2.5 mM EGTA to study the Mg2+-bound protein.To study the monomer-dimer equilibrium of the CaDifferent protein dilutions were prepared  and injected (200 μL) in a SuperdexTM75 Increase 10/300 GL column  with 0.8 mL/min flow rate.Assuming a linear dependency of Ve from the logarithm of the Mw (Ve = A × log(Mw) + B) and assuming that in the presence of a monomer-dimer equilibrium the apparent Mw is related to the percentage of each species through the equation Mw = (%dim × 22.9 + 22.9) we get the relation Ve = A × log((%dim × 22.9 + 22.9)) + B. Describing the monomer-dimer equilibrium through the dissociation constant Kd we arrive at the 3 parameters  equation to fit the Ve vs. concentration (c) curve:2 supplied with 0.05% Tween20. Labeled hGCAP1 (10 nM) was added in each of the sixteen dilutions and the reactions were loaded in sixteen capillaries. After confirming that the fluorescence of each capillary was constant, the MST traces were recorded at 24 °C with 40% laser excitation power and 40% MST power. The human GCAP1 was labeled with the red fluorescent dye NT-647-NHS with the Monolith NTTM Protein Labeling kit according to the manufacturer protocol . The NT-647-NHS carries a reactive NHS-ester group that reacts with primary amines (lysine residues) to form a covalent bond, achieving about 1:1 ratio of labeled protein to dye. The dye solution was prepared at 30 µM in the supplied labeling buffer. GCAP1 (10 µM) was diluted in a 1:2 ratio with the labeling solution and incubated at room temperature for 30 min. Sixteen serial dilutions of unlabeled protein were prepared from 46.6 µM to 11.4 nM in 20 mM Tris-HCl, 100 mM NaCl, pH 7.5, 1 mM DTT, 5 mM CaCl2, 2.5 mM EGTA, pH 7.5, 1 mM DTT buffer supplied with 0.05% Tween20 to study the magnesium-bound hGCAP1.The same experiments were repeated using a 20 mM Tris-HCl, 150 mM KCl, 5 mM MgClAll the MST experiments were carried out with a Monolith NT.115 (Nano Temper Technologies). Kd values were calculated as the average of the triplicate experiments analyzed with MO.Affinity Analysis 3 software.hGCAP1 were concentrated to 175 µM in 20 mM Tris-HCl pH 8, 5 mM 2-mercaptoethanol, and filtered through a 0.22 µm filter . Eighty microliters of protein solution were used to measure hydrodynamic radius by DLS. Measurements were performed using a DynaPro instrument  in a thermostatic cuvette at 10 °C.Size-Exclusion Chromatography (SEC) combined with Multi-Angle Light Scattering (MALS) detection was performed in an HPLC system composed by a Waters 515 HPLC Pump, a Waters 2487 Dual λ Absorbance detector , a Wyatt Dawn Heleos MALS and a Wyatt Optila T-rEX differential refractive index detector .2, as mobile phase, at a flow rate of 0.8 mL/min. Molar masses were calculated by means of the Astra V software vs. 5.3.4.20 (Wyatt), using a dn/dc value of 0.185.200 μL of 2 mg/mL GCAP1 samples were run on a Superdex 75 Increase 10/300 GL column , by using 20 mM Tris-HCl, pH 8, 5 mM 2-mercaptoethanol, with/without 1 mM CaCl2), magnesium buffer , EGTA buffer . All the measurements were performed in a Varian Cary Eclipse fluorescence spectrophotometer using a 280 nm excitation wavelength, 300–400 nm emission wavelength, setting the slit at 10 nm, and the temperature at 25 °C.The Trp fluorescence of the hGCAP1 was measured diluting the protein concentration at 3 µM in the followed three different buffers: calcium buffer  at ESRF beamline BM29 with Pilatus 1M detector with 1 s exposure time.The 2700 frames of hGCAP1 in EGTA  were collected at 20 °C: the selected peak comprised 47 frames (1372–1419) that were scaled in groups of three and subtracted from the buffer (frames 15–194) considering each slightly different concentration (from UV absorbance) before subtraction.2+  and Ca2+/Mg2+  were collected at 4 °C. The selected peak of hGCAP1 in Ca2+/Mg2+ at the higher protein concentration comprised 41 frames . The data analysis was performed using programs PRIMUS 3.0 [http://www.bioisis.net)).The SEC-SAXS data of hGCAP1 in MgIMUS 3.0  and ScÅt2+-loaded hGCAP1 reported in [Different possible dimeric conformations were generated using the equilibrated structure of Caorted in  as the morted in  with a sorted in . The sta3) comprising 30673–33644 water molecules, 93–96 K+ ions, 13–14 Mg2+ ions, and 95–101 Cl− ions for a total of 98566–107500 atoms, depending on the specific dimer. The protocols for energy minimization, equilibration, and production were the same as in Marino et al. [The three dimers  were then subjected to 200 ns all-atom MD simulations using GROMACS v. 2016.1  with CHA2 with respect to SAXS data over the last 100 ns of the trajectory was calculated using the program CRYSOL 2.8 [saxns_fit2eom script; http://xray.utmb.edu/SAXNS) that builds a mixed model selecting different structures along the simulations to enhance the agreement with SAXS data.For each trajectory, 2000 frames were extracted at 100 ps intervals and the average χYSOL 2.8  both conYSOL 2.8   was selected as the representative model for each dimer, namely d1′, d2′, and d3′, respectively.From each trajectory, the frame displaying the lowest absolute χ0 = 3.86–0.39 × ZD-s).In such dimers, the two chains were separated and subjected again to rigid-body docking using ZDOCK 2.3 . For eac0) was calculated. The persistence of electrostatic interactions over the 200 ns trajectories was calculated using PyInteraph 1.0 software [Finally, in silico mutagenesis of the variants known to interfere with the dimerization process, namely H19R, Y22D, F73E, and V77E , on bothsoftware , which cThe recombinant hGCAP1 was expressed in E. coli BL21-CodonPlus RP cells with N–terminal myristoylation (Myr). In such conditions, we expected an efficient cut of the N-ter Met (>97%)  followed2+ was detected using circular dichroism spectroscopy. The ellipticity at 220 nm of the Ca2+/EGTA protein varied from –26/–27 mdeg (at 20 °C) to –19/−22 mdeg (at 95 °C)  . In both2+ bound hGCAP1 (Ve = 10.89 mL) with that of 5 reference proteins  it was possible to estimate its molecular weight at 49 kDa, compatible with a dimeric assembly. In these experimental conditions, the hGCAP1 concentration at the elution peak, calculated from UV absorbance at 280 nm (Mw/ε = 1.07 mg/mL), was 17.7 μM. Reducing the protein concentration, the SEC peak shifted toward higher Ve , suggesting the presence of a monomer-dimer equilibrium.We used analytical size exclusion chromatography (SEC) to study the monomer-dimer equilibrium of hGCAP1 . Comparing the elution volume (Ve) of the Ca2+  at low concentration (20 nM) was monomeric. Therefore, the addition of increasing amounts of unlabeled protein-induced changes in labeled protein fluorescence depending on the formation of the dimeric assembly . In thisAdditional SEC experiments were performed varying the concentration of apo hGCAP1 , in a buffer with low ionic strength . These experiments showed constant values of the elution volumes, indicating the loss of the monomer-dimer equilibrium and the presence of a single quaternary assembly. Multi-angle light scattering coupled with SEC (SEC-MALS) analysis showed an Mw corresponding to the hGCAP1 monomer  to the salt-free buffer was sufficient to induce again the monomer-dimer equilibrium, with a protein molecular weight calculated from MALS of about 29 kDa  demonstrating that the binding of calcium is a determinant factor to promote GCAP1 dimerization.2+ or EGTA showed similar values of maximum emission wavelength . Such values are correlated with significant solvent exposure of tryptophan, as discussed by Vivian and Callis [2+ concentration (5 mM) we registered a 2 nm redshift of the emission peak (345.2 nm), indicating that Ca2+ binding slightly increases the solvent exposure of Trp ) without Ca2+  showed a Mw, estimated from scattering intensity relative to water at zero angle (I0 = 18.29 ± 0.06), of 21.1 kDa [2 = 2.62) was greatly improved by the addition of the missing 17 C-terminal residues   to 43.7 (at 51.1 μM).In particular, CaThe SEC-SAXS data of calcium bound GCAP1 collected at the higher protein concentration ~76 uM; , correspThe three-dimensional structural model for hGCAP1  was obtained by homology modeling using the X-ray structure of cGCAP1  as a teIn view of these lines of evidence, the homology modeled and equilibrated structure of hGCAP1 described by Marino and Dell’Orco  was used2 calculated on the final 50 ns of the MD simulations , whereas d1 exhibits a poorer agreement (<χ2> = 13.45). The ranking changes when considering in the models also the conformations of C-ter, with d1 and d2 being in comparable agreement with SAXS  and d3 in disagreement (<χ2> = 13.79)  and d3 as the worst (χ2 = 2.37)  of every monomer. The average χulations  shows th= 13.79) . To take = 2.37) B.To summarize, the agreement with SAXS data of d1 and d2 improves by adding the C-ter amino acids and it gets even better when protein dynamics are explicitly considered. On the contrary d3—the best dimer in the absence of C-ter—worsens considerably when taking into account the contribution of the C-ter end.2 with respect to the SAXS data , under the assumption that mutations do not significantly perturb the structure of each interacting molecule. The scores of SAXS and ZDOCK analysis are reported in For each MD simulation, the structural conformation showing the lowest χ0 values are high for all the dimers but are especially apparent for d1′, for which a 4.5 to 5.8 kcal/mol effect was predicted, which would strongly shift the equilibrium toward the monomeric form.All the dimers predicted by the rigid-body docking algorithm accounting for shape and electrostatic complementarity and desolvation of residues buried at the dimer interface (ZDOCK 2.3) resulted in high-score poses, and 16–24 of the 12,000 poses reconstituted within 1 Å  with respect to d1′ (219.4 Å2) with a similar shape complementarity score (2487 vs. 2541) were measured. Interestingly, F73 was found to be involved in a π-stacking interaction in both interfaces, specifically with Y22 in dimer d1′ and with F65 in dimer d2′. Finally, the static analysis confirmed the electrostatic interactions involving D72 specifically with R93 in d1′  and with K97 in d2′ (charge-reinforced H-bond), which could contribute to the highest docking score (ZD-s) of the first dimeric assembly , a slightly increased buried solvent accessible surface area for d2′ .SAXS data on hGCAP1 at a low concentration (10.9 µM) without Ca2+ ions or, to a greater extent, by three Ca2+ ions. Indeed, we observed that at low salt concentration the addition of a small amount (1 mM) of calcium is sufficient to push the monomeric state toward the monomer-dimer equilibrium. In conditions closer to the physiological state, i.e., at medium salt concentration and in the Ca2+-bound state, hGCAP1 displays a monomer-dimer equilibrium with Kd~9 μM. When the 3 calcium ions are exchanged with one/two Mg2+ the higher electrostatic repulsion screen decreases the dimerization Kd (~60 μM).Purified hGCAP1 analyzed with different experimental techniques shows the equilibrium between monomer and dimer. Since the protein has a theoretical isoelectric point of 4.4 and is negatively charged at physiological pH, we expect a prevailing effect of electrostatic repulsions at low salt concentration shifting the equilibrium towards the monomeric state, as observed in SEC-MALS experiments. The electrostatic repulsion is partially shielded by binding one/two MgInterestingly, also the chicken protein, despite being monomeric in the crystal structure, displays a similar dynamic monomer-dimer equilibrium (not shown), showing that this property is common to different GCAP1 orthologs.2+ can also explain the protein resistance to trypsin with respect to the unbound state observed in previous works [The shift toward dimeric assembly favored in the presence of Caus works ,42.The differences in monomer-dimer equilibrium suggest conformational changes induced by GCAP1 ligation state as observed with Trp fluorescence experiments, that are likely related to GCAP1 regulatory activity on retGC1, as suggested by thorough MD simulations .0 values, compatible with a shift of hGCAP1 to its monomeric form. It is interesting to highlight that the higher destabilization is associated with the substitution involving H19 for both d1′ and d2′  and 19-fold higher than that in Mg2+ (~60 μΜ), therefore hGCAP1 would rather bind to its target regulator than to itself under both conditions. Dimerization of hGCAP1 could then rather serve as a regulatory mechanism to prevent an excessive diffusion to the outer segment in the absence of RetGC, and not necessarily a condition to achieve a 2:2 complex. An updated estimate of the intracellular concentration of both GCAP1 and retGC in human photoreceptors is therefore necessary to clear up the actual molecular scenario.Monomer–dimer equilibrium in proteins is determined by a subtle balance between stabilizing and destabilizing forces . Based o μM, see  is 3-fol2+ or Mg2+ are reflected in the observed changes of the dimer dissociation constant. Such conformational changes are likely related to the mechanisms of GCAP1 to control the activity of the retGC1 partner, and therefore their investigation here described represents a step forward to dissect the structural bases of the regulatory mechanism of GCAP1.GCAP1 structural changes related to the presence of Ca


ATP13A2, a late endo-/lysosomal polyamine transporter, is implicated in a variety of neurodegenerative diseases, including Parkinson’s disease and Kufor–Rakeb syndrome, an early-onset atypical form of parkinsonism. Loss-of-function mutations in ATP13A2 result in lysosomal deficiency as a consequence of impaired lysosomal export of the polyamines spermine/spermidine. Furthermore, accumulating evidence suggests the involvement of ATP13A2 in regulating the fate of α-synuclein, such as cytoplasmic accumulation and external release. However, no consensus has yet been reached on the mechanisms underlying these effects. Here, we aimed to gain more insight into how ATP13A2 is linked to α-synuclein biology in cell models with modified ATP13A2 activity. We found that loss of ATP13A2 impairs lysosomal membrane integrity and induces α-synuclein multimerization at the membrane, which is enhanced in conditions of oxidative stress or exposure to spermine. In contrast, overexpression of ATP13A2 wildtype (WT) had a protective effect on α-synuclein multimerization, which corresponded with reduced αsyn membrane association and stimulation of the ubiquitin-proteasome system. We also found that ATP13A2 promoted the secretion of α-synuclein through nanovesicles. Interestingly, the catalytically inactive ATP13A2 D508N mutant also affected polyubiquitination and externalization of α-synuclein multimers, suggesting a regulatory function independent of the ATPase and transport activity. In conclusion, our study demonstrates the impact of ATP13A2 on α-synuclein multimerization via polyamine transport dependent and independent functions. SNCA gene (encoding αsyn) can cause autosomal dominant PD [2O2) and ferrous chloride (FeCl2) induces αsyn aggregation [Parkinson’s disease (PD) is the most common neurodegenerative movement disorder, with prominent loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the presence of proteinaceous inclusions termed Lewy bodies . Dopamininant PD . In addiinant PD . αsyn isinant PD ,8. Due tinant PD . On the inant PD . Membraninant PD . Additioregation . Moreoveregation .Caenorhabditis elegans was antagonized by ATP13A2 [ATP13A2 is a P5B-type ATPase that is highly expressed in the human brain with highest expression in the SNpc , where i ATP13A2 . Moreove ATP13A2 . On the  ATP13A2  and AAV- ATP13A2 . Taken tWe recently reported that ATP13A2 is a lysosomal polyamine exporter with the highest affinity for spermine (SPM) . PolyamiDeficiency of ATP13A2 and familial ATP13A2 mutations have been reported to induce αsyn aggregation ,26,28,392 and H2O2 induces αsyn aggregation [Next, we examined the effect of ATP13A2 on αsyn under cellular stress. We previously showed that oxidative Fenton stress caused by FeClregation . Here, wWe further investigated whether ATP13A2 affects the level of membrane-associated αsyn since polyamines electrostatically interact with biological membranes to bridge and shield the membrane surface charges that possibly contribute to the aggregation of membrane particles resistant to the repulsive forces . MembranWe then wondered whether the observed effect of ATP13A2 WT on αsyn multimers was mediated by increased degradation. Therefore, we treated our cells with 10 µM of the lysosomal inhibitor chloroquine (CQ) for 24 h and found that autophagy inhibition did not affect the level of αsyn multimers in ATP13A2 WT cells compared to control conditions . We alsoWe recently showed that ATP13A2 is a lysosomal polyamine exporter with the highest affinity for SPM. At high concentrations, polyamines induce cell toxicity, which is exacerbated by ATP13A2 loss due to lysosomal dysfunction and rupture . It was Interestingly, while treatment with SPM did not affect αsyn levels at the membrane c,d, it rWe previously reported that high concentrations of SPM induce the loss of lysosomal membrane integrity in ATP13A2 knockout (KO) cells . Here, wOf note, high CQ concentrations also induced a pronounced increase of αsyn multimers independent of ATP13A2 , while lSpreading of aggregated αsyn is considered as a key event in PD progression. Since ATP13A2 was reported to regulate the biogenesis of exosomes and promote αsyn secretion ,27, we aIn this study, we investigated in detail the relationship between ATP13A2 and αsyn multimerization, membrane localization, and secretion in basal conditions compared to conditions of oxidative stress and lysosomal rupture. Despite several previous studies having already considered the potential link between ATP13A2 and αsyn, such as those showing that deficiency of ATP13A2 leads to αsyn accumulation, as well as the protective effect of ATP13A2 against αsyn-induced neurotoxicity ,22,28, tOur recent identification of SPM as a substrate of ATP13A2 sheds new light on the link between ATP13A2 and αsyn . PolyamiWe also found that deficiency in ATP13A2 increases αsyn multimerization in basal conditions. The levels of αsyn multimers induced by oxidative stress or SPM were increased in conditions of ATP13A2 KD and could be completely prevented upon overexpression of ATP13A2 WT, but not by the transport-deficient mutant DN. This is an interesting finding given the high oxidative stress levels in PD brain and highlights the importance of ATP13A2 in the prevention of αsyn multimerization in conditions of oxidative stress.Under physiological conditions, αsyn populates an ensemble of confirmations by interacting with proteins or binding to membranes, mediating a variety of cellular events , whereasKD or KO of ATP13A2 may result in impaired autophagy, which has been linked to αsyn accumulation ,46. HoweSpreading of pathological αsyn contributes to the neurodegenerative phenotype in a number of cell and animal models. Previous studies suggested the involvement of ATP13A2 in the biogenesis of exosomes and αsyn externalization ,27, but We used the artificial, transport-deficient mutant ATP13A2 DN as a control in several of our experiments, which enabled us to dissect polyamine transport-dependent and -independent functions of ATP13A2 . ATP13A22. After lentiviral transduction, cells were selected with blasticidin  or puromycin (3 µg/mL). To inhibit proteasomal or lysosomal activity, cells were treated for 24 h with 1 μM MG132  or 10 μM chloroquine , respectively. For cell lysis, cells were washed with phosphate-buffered saline (PBS) and lysed in lysis buffer  1 mM, Triton 1%, glycerol 10%, and protease inhibitor (PI) cocktail ). Cell lysates were cleared by centrifugation at 12,000× g for 15 min and further analyzed via immunoblotting.Human neuroblastoma SH-SY5Y cells with stable expression of human αsyn WT were generated using pCHMWS-αsyn-IRES-puro lentiviral vectors (LVs), and cells were selected with puromycin . Next, these selected cells were transduced with human ATP13A2-WT-IRES-puromycin, human ATP13A2-D508N-IRES-puromycin, fLuc-IRES-puromycin (control), sh-ATP13A2-blasticidin (KD), and sh-fLuc-blasticidin (KD control) LVs to obtain ATP13A2 overexpression or KD cells. All LVs were generated by the Leuven Viral Vector Core (LVVC) as described previously ,47. Cell2  and 100 µM H2O2  in DMEM-complete ). After 72 h, cells were washed with PBS and scraped for immunoblotting or intact cell cross-linking.To induce αsyn aggregation, oxidative stress was applied to the cells as described by Macchi et al. with a few modifications . One daySPM  was prepared to a final stock concentration of 200 mM in 0.1 M 3-(N-morpholino)propane sulfonic acid (MOPS)-KOH (pH 7.0) . For theg for 15 min, the cells were resuspended in 200 µL of PBS + PI buffer. Next, we incubated the cells with 0.5 μM DSG  or 1 μM DSP  for 30 min at 37 °C. The reaction was quenched with 10 µL of Tris-HCl buffer  for 15 min at room temperature. Then cells were sonicated twice at 30 Hz for 15 on–off intervals of 10 s each (1 s pulse on/off) .Cells were washed twice with PBS and harvested with PBS with PI cocktail (PBS + PI buffer). After centrifuging at 1000× g for 15 min, the cells were resuspended in 500 µL of PBS + PI buffer and sonicated twice at 30 Hz for 15 on–off intervals of 10 s each (1 s pulse on/off). After sonication, cells were centrifuged at 340,000× g at 4 °C for 15 min, and the supernatant was collected as the cytosolic fraction. The pellets were washed three times with PBS + PI buffer and centrifuged at 340,000× g for 15 min; then, the pellets were dissolved in PBS + PI + 1% Triton buffer. After centrifugation at 340,000× g at 4°C for 15 min, the supernatant was collected as membranous fraction. For intact cell cross-linking combined with membrane fractionation experiments, cells were treated with cross-linker first, followed by membrane fractionation.Cells were washed twice with PBS, scraped, and harvested with PBS + PI buffer. After centrifuging at 1000× g for 15 min to remove cells, 15,000× g for 30 min to remove cell debris, filtration of apoptotic bodies through a 0.22 µm filter, and a final spin at 140,000× g for 3 h at 4 °C. Supernatant was decanted, and nanovesicles were collected in 50 µL of cell lysis buffer for immunoblotting. For intact nanovesicle cross-linking, 9 × 15 cm dishes cells were plated. The nanovesicles were dissolved in 200 µL of PBS + PI buffer, followed by cross-linking with DSP or DSG.Cells were plated in 3 × 15 cm dishes and allowed to attach overnight. The next day, the cells were washed twice with PBS, and DMEM with 1% exosome-depleted fetal bovine serum (FBS) was added to each plate. The medium was collected 24 h later, and nanovesicles were purified using differential centrifugation: 300× To assess lysosomal membrane integrity, cells were seeded in 12-well plates; the next day, cells were incubated with 5 µg/mL acridine orange  for 15 min at 37 °C. Thereafter, medium was discarded, cells were washed with PBS, and fresh medium was added. Then, cells were treated with the indicated concentrations of SPM or the positive control chloroquine (500 µM) for 2 h at 37 °C. Lastly, cells were collected and resuspended in PBS containing 1% bovine serum albumin (BSA). The mean fluorescence of 10,000 events was captured using an Attune Nxt  flow cytometer.After determination of protein content using a bicinchoninic acid (BCA) assay , cell extracts containing 15 µg of total protein were separated on 4–15% precast midi protein gels  and electroblotted onto polyvinylidene difluoride membranes . Membranes were first incubated with 0.4% paraformaldehyde (PFA) for 15 min for better αsyn binding, then blocked with PBST with 5% milk for 30 min and incubated with the following primary antibodies: anti–α-Syn , anti-α-tubulin , anti-ubiquitin , anti-GAPDH , anti-β-catenin , anti-Hsc70 , anti-Hsp90 , anti-LC3 , and anti-ATP13A2 . After overnight incubation with primary antibodies at 4 °C, blots were washed three times with PBS/1%Triton and incubated with horseradish peroxidase-conjugated secondary antibodies  for 1 h. Detection was performed using chemiluminescence  and an LAS-4000 imaging system .t-test) to compare values to the hypothetical value of 1. All error bars represent the standard error of the mean (SEM). Statistical significance was set at *** p < 0.001, ** p < 0.01, and * p < 0.05.Figures shown are representative of at least three independent experiments. Images were analyzed by ImageJ 1.49v, and data were analyzed using Graph Pad Prism 6 . Statistical analysis was performed using a one-way ANOVA test with Bonferroni to compare selected pairs of columns or column statistics (one-sample Our findings indicate that ATP13A2 regulates αsyn multimerization induced by oxidative stress or SPM via a mechanism involving maintenance of lysosomal membrane integrity, regulation of αsyn membrane association, and UPS-mediated αsyn degradation. Our findings shed new light on the link between αsyn and ATP13A2 and provide an explanation for the protective role of ATP13A2.


FESEM and TEM images clearly showed that nanospheres are composed of ultra-thin MoS2/C nanosheets coated with carbon layer and possess an expanded interlayer spacing of 0.98 nm. As anodes for LIBs, MoS2/carbon nanospheres deliver an initial discharge capacity of 1307.77 mAh g−1 at a current density of 0.1 A g−1. Moreover, a reversible capacity of 612 mAh g−1 was obtained even at 2 A g−1 and a capacity retention of 439 mAh g−1 after 500 cycles at 1 A g−1. The improved electrochemical performance is ascribed to the hierarchical porous structure as well as the intercalation of carbon into lattice spacing of MoS2, which offers fast channels for ion/electron transport, relieves the influence of volume change and increases electrical conductivity of electrode. Meanwhile, the expanded interlayer spacing of MoS2 in MoS2/C can decrease the ion diffusion resistance and alleviate the volumetric expansion during discharge/charge cycles.To overcome the deficiency of the volume expansion of MoS In the past decades, lithium-ion batteries (LIBs) highlight the advantages of high energy density, long service life and environmental compatibility, which has great applications in renewable energy storage, electronic devices and energy vehicles , 2. Neve−1 –5, which3S4 [2 [2 [2 [2 [2 [2), a semiconductor with a typical two-dimensional (2D) layered structure, has been the most studied material in this family. More importantly, the larger interlayer distance (d = 0.62 nm) of MoS2 than graphite (d = 0.34 nm) can accelerate the process of Li+ intercalation/extraction, putting up a high storage capacity of 670 mAh g−1 [2 as LIBs is hindered from the poor cycling stability. This is because that the relatively low electronic/ionic conductivities and the electrochemical degradation of the active MoS2 materials due to a polysulfide shuttling effect result in the capacity loss and poor rate capability [2 and introducing the conductive carbonaceous materials [2 and maintain the electrode structure stability, for example, MoS2/graphene [2/CNTs [2/carbon nanofibers [2/RGO [Recently, the transition metal sulfides  have be[2 [2 [2 , 13. Amo mAh g−1 –16. Unfopability , 18. To aterials , 19–21. graphene , 23, MoS [2/CNTs , MoS2/canofibers , MoS2/RGs [2/RGO , etc.2/C-based nanocomposites, giving full play to their respective advantages to improve the cycling stability. Li et al. reported a novel 2D MoS2/C hybrid nanosheet superstructure consisting of the alternative layer-by-layer interoverlapped single-layer MoS2 and mesoporous carbon [2/m-C interface contact result in the maximization of synergistic interaction. Their group also projected 3D ordered macroporous MoS2/carbon flexible electrodes by assembling MoS2/C nanostructure on carbon cloth with few-layered MoS2 nanosheets homogenously embedded into the interconnected carbon wall through the use of polystyrene (PS) nanospheres as the macropore template [2 nanosheets onto the polypyrrole-derived carbon nanotubes (PCN) substrate and the coating of outer carbon layer onto the nanosheets to fabricate PCN@MoS2@carbon sandwiched architecture [2 nanosheets are sandwiched between hollow PCN and thin carbon layer. Sun et al. successfully prepared 1T-MoS2/C hybrids that consist of smaller and fewer-layer MoS2 nanosheets via a facile hydrothermal method with a proper glucose additive [2/C anodes deliver superior cycling stability (maintain 870 mAh g−1 after 300 cycles at 1 A g−1) and high rate performance (a reversible capacity of 600 mAh g−1 at 10 A g−1). The superior electrochemical performance can be attributed to the higher intrinsic conductivity of 1T-MoS2 and thin carbon layers covered on the surface with an enlarged interlayer spacing of 0.94 nm. Given the above, the modified MoS2/C-based nanocomposites can indeed optimizing the electrochemical properties.Based on the above considerations, recent researches mostly focus on constructing novel MoSs carbon . The unitemplate . The fleitecture . In the additive . The 1T-2/carbon nanospheres. The nanospheres are self-assembled from ultra-thin MoS2/C nanosheets coated with carbon layer, resulting in forming internal interconnected channels and exposing more active sites for electronic/ionic transmission and lithium storage. As a consequence, when employed as anode material in half cell LIBs, the as-prepared open porous structure of MoS2/C nanospheres exhibits remarkable lithium storage properties, including high specific capacity, long cycling performance, as well as fast rate capability.Herein, we demonstrate a facile, easy-operated and high effective hydrothermal method toward hierarchical MoS2/C nanospheres was based on a previous procedure with modifications [2MoO4), 3 g of thiourea (CH4N2S) and 1 g of polyvinyl pyrrolidone (PVP) were dissolved in 30 mL deionized water with magnetic stirring to form a uniform solution. Then, we added 0.2 g of dopamine hydrochloride (DPH) into the above mixture with forming a red suspension. After continuously stirring for 30 min, the resulting suspension was put into a 50-mL Teflon-lined stainless steel autoclave kept at 200 °C for 18 h, followed by cooling down to room temperature naturally. The black precipitates were collected and washed with deionized water and absolute ethanol by centrifugation method, and dried under vacuum at 60 °C for overnight. Lastly, MoS2/C nanospheres were obtained by the calcination of black precipitates in argon atmosphere at 700 °C for 3 h. For comparison, we bought commercial pure MoS2 powder from Aladdin.The synthesis of MoSications . Typicalλ = 0.15406 nm) at the 2θ range of 5°–80° with a scanning rate of 4° min−1. N2 adsorption/desorption isotherms and Brunauer–Emmett–Teller (BET) surface area were performed by a Micromeritics ASAP 2020 analyzer. The Raman spectrums were tested on a LabRAM HR800 Raman spectrometer equipped with a laser light at 532 nm. The content of carbon in the MoS2/C nanospheres was determined by a simultaneous DSC/TGA analyzer  at a heating rate of 10 °C min−1 from 25 to 700 °C with a flow of air. The element composition and chemical state of the materials were evaluated through X-ray photoelectron spectroscopy . The detailed morphologies and microstructure of the samples were examined using field emission scanning electron microscopy , and transmission electron microscopy , respectively.The X-ray diffraction (XRD) patterns were measured using a TD-3500 X-ray diffractometer with Cu/Ka radiation (N-methyl-2-pyrrolidinone (NMP) solution, the active materials (MoS2/C or MoS2) with conductive additive (acetylene black) and polymer binder (PVDF) in a 8:1:1 mass ratio to form a homogeneous slurry. We coated the slurry on a copper foil and dried in a vacuum oven at 80 °C for 4 h. Subsequently, the electrodes were punched into circular disks and dried in a vacuum oven at 120 °C for 12 h. 1M LiPF6 dissolved in ethylene carbonate (EC) and dimethyl carbonate (DMC) with the volume ratio of 1:1 was selected as the electrolyte. The electrochemical performance was implemented using a battery testing system (Neware BTS-610) in a cut-off voltage window of 0.01–3 V at different current densities. Cyclic voltammetry (CV) curves were carried on an electrochemical workstation (CHI 760E) between 0.01 and 3 V at a scanning rate of 0.2 mV s−1.The capacities and cycling properties of the as-prepared samples were performed by CR2032 coin-type cells assembled in an argon-filled glove box with lithium sheet as the counter electrode and a Celgard 2400 microporous polypropylene film as the separator. The working electrodes were obtained by mixing in 2/C nanospheres is shown in Fig. 2MoO4 supplies molybdenum ion, CH4N2S provides sulfur source, and DPH is carbon source. Polydopamine carbon materials have better electrochemical performance and better conductivity. PVP acts as the dispersant and stabilizer. In hydrothermal process, surfactant PVP preferentially adsorbs along MoS2 nanograin boundary, causing MoS2 crystal nucleus anisotropic growth and forming MoS2 nanosheets. Due to the large specific surface area and high surface energy of MoS2 nanosheet/carbon precursor, MoS2 nanosheets are self-assembled into nanospheres. Finally, MoS2/C nanospheres are prepared by calcination in inert atmosphere.The preparation process diagram of hierarchical porous MoS2 and MoS2/C nanospheres. The patterns of commercial MoS2 are consistent with the standard card of hexagonal 2H-MoS2 (JCPDS 87-2416). The diffraction peaks at about 2θ = 32.8° and 58.5° can be indexed to (100) and (110) planes of MoS2 in both commercial MoS2 and as-prepared MoS2/C [2/C at around 2θ = 7.9° and 18.3°, which are pointed to expanded (001) and (002) crystal planes [2/C and the participation of DPH contributed to the expanded interlayer spacing. As shown in Fig. 2 adsorption/desorption isotherms analysis, the BET surface area of MoS2/C was concluded to be 16.59 m2 g−1. The pore size distribution  and 1605 cm−1 (G band) can be direct to carbon phase [sp3-hybridization on the graphitic plane, while G band is ascribed to crystalline graphitized carbon [2/C composites. The 4.2% weight loss before 100 °C corresponds to the evaporation of adsorbed free water in the samples, and 37.54% weight loss reveals the oxidation of MoS2 to MoO3 in air [2 and carbon are determined to be 4.2, 64.78 and 31.02 wt%, respectively.Figure d MoS2/C , 29. Addl planes . The exp of MoS2 , 29, 30,on phase , 21, 31.d carbon , 32. The3 in air , 15. Hen2/C in Fig. d. Two major peaks centered at 229.8 and 233.1 eV are ascribed to Mo 3d5/2 and Mo 3d3/2 of Mo4+ in MoS2/C [s. And the rest of peak at 236.3 eV can be indexed to Mo6+ indicating the formation of C–O–Mo bond between MoS2 and carbon, which is consistent with other MoS2/C composites [p3/2 and S 2p1/2 of S2− in MoS2[s is shown in Fig. XPS was investigated to assess the element composition and valence states of MoSn MoS2/C , 29. Anomposites , 30, 33.− in MoS2. The dec2/C. The representative FESEM and TEM images of MoS2/C are displayed in Fig. 2/C nanosheets with an average diameter of 130–200 nm. The FESEM district elemental mapping analysis  pattern indexes a diffraction ring to the polycrystalline nature of the MoS2  of 66.18%. Moreover, the undesirable CE and capacity loss approximately 33% probably originate from the irreversible decomposition of electrolyte and the generation of SEI film on the electrode surface [−1 and 836.13/810.92 mAh g−1, respectively. This enhanced CE reveals the electrochemical reversibility of MoS2/C anode is good.The as-obtained MoSm LixMoS2, 36. AnoS2 to Mo , 15, 25,i2S to S , 37. In  surface . The sec2/C and commercial MoS2 anodes under the current density of 0.1 A g−1 for 100 cycles. Commercial MoS2 presents initial charge/discharge specific capacities of 671.70/952.52 mAh g−1, which is far from the MoS2/C composites (865.54/1307.77 mAh g−1). This is because the existence of carbon in MoS2/C improve the conductivity and the surface/interfacial storage of Li originated from the porous nanosphere structure and the reversible formation/decomposition of polymeric gel-like film (SEI) [2/C and MoS2 anodes show discharge specific capacities of 587.18 and 350 mAh g−1 with high CE of approximate 99%. The rate capability of two samples was also evaluated at different current densities ranging from 0.1 to 2 A g−1 in Fig. 2/C retains high discharge capacities at higher current densities: 878 mAh g−1 at 0.1 A g−1, 806 mAh g−1 at 0.2 A g−1, 733 mAh g−1 at 0.5 A g−1, 673 mAh g−1 at 1 A g−1, 633 mAh g−1 at 1.5 A g−1 and 612 mAh g−1 at 2 A g−1 after 10 cycles. When revaluated at current density of 0.1 A g−1, the discharge capacity rapidly reaches 754 mAh g−1 and remains 876 mAh g−1 after 40 cycles, which is almost close to that of first 10th cycle, suggesting the outstanding rate performance and structural stability of MoS2/C. As for MoS2, the discharge capacities from 0.1 A g−1 to 2 A g−1 after 10 cycles are 320 and 55 mAh g−1 with enormous capacity loss about 83%. The results reveal that the electrical conductivity of commercial MoS2 is no significant improvement due to the addition of activated carbon, thereby not achieving the effect of rapid charge and discharge. This is because simple physical mixing cannot effectively improve the electrical conductivity of commercial MoS2, but can accomplish the ideal goal by carbon coating like as-obtained MoS2/C.Figure lm (SEI) . After 1−1. In order to activate electrodes, the cells are tested at a low current density of 0.05 A g−1 for first two cycles. The MoS2/C exhibits high discharge capacities of 515, 443 and 439 mAh g−1 at 1.0 A g−1 for 100th, 300th, 500th cycles, respectively. Compared with the previously reported MoS2 anode materials from Table 2/C nanospheres have better electrochemical performance and they will show great potential to replace graphite anode materials. It is worth noting that the capacity curve of MoS2/C as a whole is relatively stable without particularly obvious decline except from the first few cycles, illustrating preeminent long-term cycling stability. However, the MoS2 anodes suffer from tremendous capacity loss with low discharge capacities of 114, 109 and 138 mAh g−1 at the same cycles. As a result, MoS2/C still exhibits more superior electrochemical properties than commercial MoS2, although activated carbon with the same relative weight ratio is introduced into MoS2 electrode. This can be attributed to the following advantages. I. MoS2/C composites possess open porous architecture that the nanosphere are self-assembled by ultra-thin MoS2/C nanosheets coated with thin carbon layer, which facilitate the intimate contact between electrode and the electrolyte and fast electron and ion transport. Meanwhile, open porous architecture is advantageous to form internal interconnected channels and expose increased number of active sites for electronic/ionic transmission and lithium storage. II. The thin carbon layer derived from carbonization of DPH can not only act as a stable supporting matrix to impede the aggregation of MoS2 nanosheets, but also improve the overall conductivity of the material. III. The expanded interlayer spacing of MoS2 in MoS2/C can decrease the ion diffusion resistance and alleviate the volumetric expansion during discharge/charge cycles.The long-term cycling performance of two samples is shown in Fig. 2/carbon nanospheres self-assembled by ultra-thin MoS2/C nanosheets by a facile hydrothermal method followed by annealing. Benefiting from the rational structure design, the MoS2/C provides fast channels for ion/electron transport and maintains high stability and conductivity of the whole electrode in lithium storage. Furthermore, the intercalation of C into interlayer spacing of MoS2 can accommodate the volume expansion to make sure the integrality of electrode and enhance electronic conductivity. The as-fabricated MoS2/C anode achieves high specific capacity (1307.77 mAh g−1 at 0.1 A g−1), excellent long cycling performance (439 mAh g−1 at 1.0 A g−1 for 500 cycles) and fast rate capability (612 mAh g−1 at 2 A g−1).In this work, we have fabricated hierarchical porous MoS