E malaria vaccine contains the hepatitis B virus surface antigen and may thus serve as a potential hepatitis B vaccine. To evaluate the impact of RTS,S/AS01E when implemented in the Expanded Program of Immunization, infants 8–12 weeks old were randomized to receive either RTS,S/AS01E or a licensed hepatitis B control vaccine (HepB), both co-administered with various combinations of the following childhood vaccines: diphtheria-tetanus-acellular pertussis-Haemophilus influenzae type b, trivalent oral poliovirus, pneumococcal non-typeable Haemophilus influenzae protein D conjugate and human rotavirus vaccine. Long-term persistence of antibodies against the circumsporozoite (CS) protein and hepatitis B surface antigen (HBsAg) were assessed, together with the immune memory response to the HB antigen following a booster dose of HepB vaccine. Subgroups receiving RTS,S or the HepB control vaccine were pooled into RTS,S groups and HepB groups, respectively. One month post-HepB booster vaccination, 100% of participants in the RTS,S groups and 98.3% in the control groups had anti-HBs antibody concentrations ≥10 mIU/mL with the geometric mean concentrations (GMCs) at 46634.7 mIU/mL  and 9258.2 mIU/mL , respectively. Forty-eight months post-primary vaccination anti-CS antibody GMCs ranged from 2.3 EU/mL to 2.7 EU/mL in the RTS,S groups compared to 1.1 EU/mL in the control groups. Hepatitis B priming with the RTS,S/AS01E vaccine was effective and resulted in a memory response to HBsAg as shown by the robust booster response following an additional dose of HepB vaccine. RTS,S/AS01E when co-administered with PHiD-CV, HRV and other childhood vaccines, had an acceptable safety profile.RTS,S/AS01 E is a Plasmodium falciparum malaria vaccine intended for routine immunization of infants and children in malaria-endemic regions in Sub-Saharan Africa as part of the Expanded Program on Immunization (EPI). The vaccine is designed to complement currently available measures to fight P. falciparum malaria and may therefore substantially contribute to existing malaria control programs. The RTS,S/AS01E vaccine consists of the RTS,S ‘hybrid’ antigen; in which the central repeat region of the Plasmodium falciparum circumsporozoite (CS) protein, referred to as ‘R’, and the T-cell epitopes of the CS protein (‘T’) are fused to the hepatitis B virus surface antigen (HBsAg) referred to as ‘S’. The vaccine is formulated with the AS01E Adjuvant System. RTS,S/AS01E induces antibody responses to the CS protein and to HBsAg.1RTS,S/AS012 Chronic hepatitis B is a risk factor for liver cirrhosis and liver cancer, and transmission of the virus occurs by exposure to the blood or body fluids of infected individuals.2 Given the health burden of chronic hepatitis B, vaccination in infants of Sub-Saharan African countries has become key to protect against the disease. RTS,S/AS01E contains the HBsAg and may thus serve as an additional hepatitis B vaccine dose. In line with the recommendation to vaccinate all infants against hepatitis B virus,3 RTS,S/AS01E was co-administered in recent studies with diphtheria-tetanus-whole cell pertussis (DTPw)-based pentavalent vaccines, which contains the hepatitis B surface antigen.5 The data from these studies show that co-administration of RTS,S/AS01E with licensed vaccines containing the hepatitis B surface antigen has an acceptable safety profile and no deleterious effect on anti-hepatitis B virus immune response.The hepatitis B virus causes life-threating infections worldwide and poses a public health problem in Sub-Saharan Africa.E and its potential for substantial impact against both clinical and severe malaria, the European Medicines Agency (EMA) provided a positive scientific opinion for the RTS,S vaccine in children aged 6 weeks to 17 months (at the time of the first dose) in 2015.6 In 2016, the World Health Organization (WHO) recommended pilot implementation of the vaccine in children as of 5 months of age in 3 to 5 moderate-to-high malaria transmission settings in Sub-Saharan Africa.7Based on the positive benefit-risk balance of RTS,S/AS01et al.8 support the indication of the RTS,S/AS01E vaccine for immunization of infants against hepatitis B in settings where prevention of P. falciparum malaria is also being sought. These results demonstrated that RTS,S/AS01E was non-inferior to a licensed HepB vaccine in terms of anti-HBs seroprotection rates at one month post-primary vaccination. Immune responses to PHiD-CV co-administered with RTS,S/AS01E were non-inferior to PHiD-CV co-administered with HepB for 9 out of 10 vaccine serotypes . Immune responses to HRV co-administered with RTS,S/AS01E were non-inferior to HRV co-administered with HepB. RTS,S/AS01E when co-administered with PHiD-CV, HRV and other vaccines included in the EPI had an acceptable safety profile during the 26 month follow-up period.8Primary study results, published in Valéa E vaccine (up to 48 months post-primary vaccination series), the presence of anti-HBs immune memory and the anti-HBs immune response to HepB booster vaccination given 4 years after primary vaccination.As part of the long-term follow-up of this phase III study, we assessed long-term antibody persistence and safety of the RTS,S/AS01E to that induced by primary vaccination with a licensed hepatitis B virus vaccine . The study was conducted at the Clinical Research Unit of Nanoro in Burkina Faso, and the Malaria Research Center Agogo Presbyterian Hospital/Kwame Nkrumah University of Science and Technology in Ghana. Infants 8–12 weeks old were randomized to receive either RTS,S/AS01E or a licensed HepB control, both co-administered (concomitantly or staggered) with the following vaccines; diphtheria-tetanus-acellular pertussis-Haemophilus influenzae type b , trivalent oral poliovirus vaccine , pneumococcal non-typeable Haemophilus influenzae protein D conjugate vaccine  and human rotavirus vaccine .8 The follow-up study was conducted at both study sites in Burkina Faso and Ghana between November 2011 and February 2017. All children received a HepB booster vaccination 48 months post-primary vaccination. The HepB vaccine contained 10 µg of purified hepatitis B surface antigen adsorbed onto aluminum hydroxide containing 0.25 mg of aluminum. RTS,S consists of the central R repeat region and the c-term T cell epitopes of the CS-protein (named “RT”) and hepatitis B surface antigen (named “S”). Per molecule of RT, there are 5 molecules of S (of which 1 S in fusion with RT and 4 “free” S). Molecular weights are quite similar for RT and S, which means that in a 25 µg RTS,S dose there is approximately 4.2 µg RT and 20.8 µg S. AS01 is a liposome-based adjuvant comprising 3-O-desacyl-4′-monophosphoryl lipid A (MPL – produced by GSK), a Toll-like receptor 4 ligand and QS-21, a saponin extracted from the bark of the Quillaja saponaria Molina tree .This phase III, open, randomized study (NCT01345240) was designed to compare the anti-HBs immune response induced by primary vaccination with RTS,S/AS01E  or HepB (control groups) primary vaccination regimens (both co-administered in various combinations with different study vaccines),8 were pooled in RTS,S groups and control groups for the analysis and comparison of HepB antibody persistence and booster response.Immunogenicity and safety data were collected up to one month after HepB booster vaccination. Details about the co-administered vaccines can be found in the et al.8During the follow-up period, we collected blood samples and assessed the persistence of the immune response against HBsAg at 12, 24, 36 and 48 months post-dose 3. To evaluate the memory response to HBs antigen, HBs antibody titers were measured one-month after administration of the HepB booster dose. Anti-CS antibodies persistence was also measured at 12, 36 and 48 months post-dose 3. More information about the study design and the study vaccines can be found in Valéa www.clinicaltrials.gov (NCT01345240) and a protocol summary is available at http://www.gsk-clinicalstudyregister.com (GSK study number 113681).We performed the study in accordance with the principles of Good Clinical Practice, the Declaration of Helsinki and all applicable regulatory requirements. The concerned ethics committees and regulatory authorities reviewed and approved the study protocol, including amendments, and the informed consent form. Parents or legal guardians of each child gave written informed consent. The study is registered at E in terms of anti-HBs antibodies compared to a primary vaccination regimen with HepB, and of anti-CS antibodies. Other immunogenicity objectives were to study the antibody responses to HBsAg induced by the booster dose of the HepB vaccine. The safety objective included collection of serious adverse events (SAEs) and potential immune-mediated disorders (pIMDs) up to study end.The immunogenicity objectives during this follow-up study included the assessment of long-term antibody persistence following previous vaccination with RTS,S/AS019 An additional analysis was done using a threshold of ≥ 100 mIU/mL for consistency with other clinical studies and given the potential for low anti-HBs levels to mask significant infection.10 Considering that antibody responses to the RF1 epitope of the HepB surface antigen are indicative of the virus-neutralizing capacity of the humoral immune response,12 we quantified RF1-like antibody levels using an in-house developed enzyme-linked immunosorbent assay (ELISA) based competition assay with plate adsorbed HBsAg at the CEVAC laboratory . The assay cutoff was 33 EU/mL. We determined antibodies against CS by ELISA at the CEVAC laboratory . Due to revalidation with new assay agents, the cutoff for the CS assay was changed from 0.5 EU/mL used for month 1 and month 12 post dose 3 to 1.9 EU/mL applied for month 36 and month 48 post-dose 3.We measured antibodies against HBs using a chemiluminescence enzyme immunoassay  with a cutoff of 6.2 mIU/mL at GSK Biologicals . An anti-HBs concentration ≥10 mIU/mL is considered seroprotective.All solicited adverse events (AEs) were reported during the 7-day period following each vaccine dose and all unsolicited AEs during the 30-day period following each vaccine dose. All SAEs with causal relationship to vaccination, fatal SAEs and AE of special interest (AESI) including potential pIMDs from study week 0 until one-month post-booster HepB vaccination were described.We based the immunogenicity analysis on the according-to-protocol (ATP) cohort for immunogenicity analysis of follow-up 1  and follow-up 2 . The ATP cohort for immunogenicity analysis FU1 and FU2 contained all evaluable participants from the ATP cohort for analysis of immunogenicity excluding participants who did not follow inclusion criteria of immunogenicity during the first and second follow-up time, respectively . Immunogenicity analysis was performed by pooled groups for the anti-HBs immune response. The total vaccinated cohort (TVC) included all participants with at least one vaccine administration documented. We performed the safety analysis on the TVC per treatment actually administered. The geometric mean concentration (GMCs) calculations were performed by taking the anti-log of the mean of the log concentration transformations. Antibody titers/concentrations below the cutoff of the assay were given an arbitrary value of half the cutoff for the purpose of GMC calculation.Valea et al.8 705 participants  were enrolled in the primary study and randomized. Among them, 675 completed the visit 24 months post-primary vaccination. Six hundred and forty-three participants completed the study at one month post-booster HepB .The seropositivity rates and GMCs for anti-CS antibodies one month following primary vaccination were already described in Valea The percentage of participants with anti-HBs antibody concentrations ≥10 mIU/mL remained high between month 1 and month 48 post-dose 3 in the RTS,S groups , and was lower in the control groups  . The perBetween month 1 and month 48 post-dose 3, anti-HBs antibody GMCs declined sharply in both the RTS,S groups (from 6412.7 mIU/mL to 669.8 mIU/mL) and the control groups (from 377.4 mIU/mL to 30.8 mIU/mL) . Forty-eOne month post HepB booster vaccination, 100% of participants in the RTS,S groups, and 98.3% in the control groups had antibody titers ≥10 mIU/mL; 99.4% in the RTS,S group, and 95.6% in the control groups had antibody titers ≥100 mIU/mL . Anti-HBOne month post-primary vaccination, 97.5% of participants in the RTS,S groups had anti-HBs RF1 antibody titers ≥33 EU/mL compared to 35.3% in the control groups. One month post-HepB booster vaccination, 95% of participants had antibody concentrations above this cutoff in the RTS,S groups compared to 72.8% in the control groups . Anti-HBNo SAEs considered causally related to study vaccination and no pIMDs were reported during the entire study period. In the first 24 months of follow-up, 5/425 (1.2%) fatal SAEs occurred in the RTS,S, versus 3/280 (1.1%) in the control groups (age of onset is between 2 and 26 months). Over approximately 4 years between study start and one month post- HepB booster vaccination, 12/425 (2.8%) fatal SAEs occurred in the RTS,S groups (5 in girls and 7 in boys) and 3/280 (1.1%) in the control groups  (Supplementary Table 2). No pattern/explanation for this difference could be identified and most of the deaths were due to various infectious diseases (Supplementary Table 2).The present follow-up study indicated a general steady decline in anti-CS antibody levels over the 49-month follow-up period. This observation indicates that no anti-CS boosting was occurring following natural exposure to malaria.E was non-inferior to 3 dose priming with a licensed HepB vaccine.8 This study showed that anti-HBs antibody GMCs declined from one month post-primary vaccination to 48 months post-primary vaccination in all groups. Antibody GMCs remained however 22-fold higher 48 months post-primary vaccination in participants who received the RTS,S/AS01E vaccine compared to participants who received the licensed HepB control vaccine, as expected from the higher HepB dose antigens and the adjuvant (AS01E) provided with the RTS,S/AS01E vaccine.We previously demonstrated that the immune response against HBsAg induced by 3-dose priming with RTS,S/AS01E, or a licensed HepB vaccine. Consistent with previous experience in children who received HepB vaccine, HBs antibody titers may wane to low or undetectable concentrations 5–15 years following primary vaccination,13 and persistence is dependent on peak anti-HBs levels.14 Nevertheless, immune responses to booster vaccination remained robust, even if levels drop below 10 mIU/mL, as previously demonstrated.14 In the present study, the booster response was robust irrespective of the primary vaccination schedule, suggesting that all participants mounted immune memory to HBsAg.A HepB booster dose 48 months post-primary vaccination induced a robust immune response in participants who received 3 priming doses of either RTS,S/AS01Detectable anti-HBs antibody titers pre-primary vaccination in about 20% of participants may represent maternal transfer of antibodies. We believe that interpretation of the results was not affected by these findings as to our knowledge, maternal antibodies may have very little impact on a booster response to an additional HepB dose 48 months after primary vaccination. Additionally, prevalence of children with detectable anti-HBs antibody titers seemed balanced between RTS,S groups.12 antibody responses to the RF1 epitope of the HBsAg were assessed and were higher in the RTS,S groups compared to the control groups, as shown by the seropositivity rates and GMCs one month after both primary and booster vaccination. This is consistent with previous results in infants 6–12 weeks of age at first vaccination, at one month post-primary vaccination with 3 doses of RTS,S/AS01E, according to a 0,1,2-month schedule.4 In the current study, higher anti-HBs RF1-like antibody GMCs were observed in both groups following booster vaccination compared to one month post-primary vaccination, indicative of a booster response. A correlated of protection exists for HepB immunization. Whether the higher anti-HBs RF1-like antibody GMCs following booster vaccination in the RTS,S groups compared to the control groups will result in a clinically meaningful benefit in terms of longer-term protection remains unknown and the clinical benefit may be limited.To assess the virus-neutralizing capacity of the humoral immune response,E was well tolerated. While the numbers of deaths were similar between groups up to 24 months post-primary vaccination (1.2% (5) versus 1.1% (3) in RTS,S and control groups, respectively), differences emerged later on during the study period (1.9% (7) versus 0 in the RTS,S and control groups, respectively). Among these 7 fatal SAEs reported after M24, 2 bacterial meningitis cases and 2 severe malaria cases were reported (Supplementary Table 2). There were no SAEs, including SAEs with a fatal outcome, that were considered causally-related to RTS,S/AS01E and no pIMDs reported during the entire study follow-up period. More data on meningitis cases, severe malaria and mortality overall and by gender will be coming from the ongoing large-scale pilot implementations of the vaccine in Malawi, Ghana and Kenya, as recommended by the WHO. No gender-specific mortality imbalance was observed within the RTS,S groups.Overall, RTS,S/AS01Follow-up of a large pool of children for 4–5 years is difficult to achieve and presents a clear strength of this study. A limitation of the study is the open label design. Overall, there were no non-responders and seroprotection against HepB was achieved in participants irrespective of the treatment group they were allocated to.Anti-HBs priming with the RTS,S/AS01E malaria vaccine is effective and results in a memory response to HBsAg as shown by the booster response leading to anti-HBs antibody GMCs after the additional dose of HepB vaccine 4 years post-priming at least as high as the licensed HepB vaccine.Click here for additional data file.


Here, we report that peptides corresponding to a predicted amphipathic α-helix in the prodomain N terminus adopt helical structure in a membrane-mimetic environment. This effect was greatly enhanced by an R46L substitution representing an atheroprotective PCSK9 loss-of-function mutation. A helix-disrupting proline substitution within the putative α-helical motif in full-length PCSK9 lowered LDL binding affinity >5-fold. Modeling studies suggested that the transient α-helix aligns multiple polar residues to interact with positively charged residues in the C-terminal domain. Gain-of-function PCSK9 mutations associated with familial hypercholesterolemia (FH) and clustered at the predicted interdomain interface  inhibited LDL binding, which was completely abolished in the case of the R496W variant. These findings shed light on allosteric conformational changes in PCSK9 required for high-affinity binding to LDL particles. Moreover, the initial identification of FH-associated mutations that diminish PCSK9's ability to bind LDL reported here supports the notion that PCSK9-LDL association in the circulation inhibits PCSK9 activity.Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a ligand of low-density lipoprotein (LDL) receptor (LDLR) that promotes LDLR degradation in late endosomes/lysosomes. In human plasma, 30–40% of PCSK9 is bound to LDL particles; however, the physiological significance of this interaction remains unknown. LDL binding  PCSK9 result in familial hypercholesterolemia (FH), whereas loss-of-function (LOF) mutations are associated with life-long reductions in plasma LDL-C and significant protection from cardiovascular heart disease (–Circulating liver-derived proprotein convertase subtilisin/kexin type-9 (PCSK9)1– disease –6. Theradisease –, 8.A). Autocatalytic processing in the endoplasmic reticulum releases the prodomain, which remains noncovalently bound and blocks substrate access to the active site, rendering mature PCSK9 catalytically inert  in the N terminus of the PCSK9 prodomain  or abolished (R496W) the ability of PCSK9 to bind LDL in vitro. These data provide molecular insight into disease-causing mutations in the pathogenesis of FH and suggest that LDL association exerts an overall inhibitory effect on circulating PCSK9 activity.We have previously mapped critical LDL-binding determinants rodomain . This rerodomain , 26, hasrodomain , 28. A rrodomain . Herein,B shows the crystal structure of PCSK9 in complex with the EGF-A domain of LDLR , both of which are evolutionarily well-conserved in mammals as well as other vertebrates . To test potential roles in LDL binding, plasmids encoding human PCSK9 with a deletion of residues 33–40  or replacement of the hydrophobic stretch and a neighboring Arg-46 residue with a glycine-serine linker were constructed and transfected into HEK293 cells. Secreted WT and mutant PCSK9 proteins present in the conditioned medium were then incubated with isolated human LDL particles followed by Optiprep density gradient ultracentrifugation to separate LDL-bound and unbound PCSK9. Deletion of the acidic tract (Δ33–40) nearly abolished PCSK9-LDL binding, whereas replacement of the hydrophobic residues with the Gly/Ser 41–46 linker reduced binding by >60% (A and B), suggestive of a functional overlap between these sequences. As recently reported (C). Helical wheel modeling revealed characteristics of an amphipathic α-helix (AH) for these residues, with a relatively polar face and an opposing face consisting of aromatic (Tyr-38) and nonpolar amino acids , giving the helix a directional hydrophobic moment (D) ..B shows versus membrane-mimetic conditions. Synthesized peptides corresponding to residues 37–47 (DYEELVLALRS) were dissolved in phosphate buffer or in buffer containing n-dodecylphosphocholine (DPC) micelles, a widely used membrane mimetic that can induce α-helical conformation in peptides with inherent amphipathic properties (A). Proline is a potential helix breaker due to a combination of steric constraints and the inability of the backbone imino group to participate in hydrogen bond formation. Accordingly, an A44P substitution in a prodomain-derivative peptide prevented a shift to helical conformation in the presence of DPC micelles (B).Because the putative N-terminal helix is predicted to have amphipathic characteristics, a random coil-to-helix conformation change may be triggered in an amphipathic environment of a membrane surface, such as that present on lipoproteins. Therefore, we performed assessments of the secondary structure that this motif forms in aqueous operties . CD specoperties A. Prolinmicelles B.A). In each case, proline substitution inhibited the ability of secreted PCSK9 to bind to LDL by >90% (B), supporting an important structural role of helical conformation in the prodomain N terminus. We have previously characterized that uptake of PCSK9 into cells is largely dependent on its cell surface interaction with LDLR (C). Both PCSK9 proline variants also displayed LDLR-dependent cell uptake (C), indicating that these proteins were not misfolded or otherwise grossly defective. In subsequent experiments, we focused on functional effects of the A44P mutation in PCSK9 because the methyl side chain of alanine is nonreactive and less likely to be directly involved in protein function.To test the influence of peptide backbone conformation on LDL binding, we introduced proline residues at two separate positions in the putative helix (L41P and A44P) in full-length recombinant PCSK9 A. In eac by >90% B, supporith LDLR . Accordiith LDLR C. Both Pl uptake C, indicaA and B), whereas Δ31–52-PCSK9 affinity for LDL was more than 2 orders of magnitude lower than that of WT PCSK9 (A). We also assessed the affinity of PCSK9-A44P for the purified LDLR extracellular domain (LDLR-ECD), which was decreased ∼20% compared with WT PCSK9 (C). Nevertheless, when added to the medium of cultured HepG2 cells, PCSK9-A44P was able to mediate dose-dependent degradation of cell surface LDLRs at levels comparable with WT PCSK9 (D), in agreement with a recent study , which is defective in LDL binding  and hypeWT PCSK9 A. We alsWT PCSK9 C. NevertWT PCSK9 D, in agrnt study . CollectA). Helical net projections show the R46L mutation is also predicted to add additional side-chain interactions to stabilize the helical conformation . In agreement with these models, structural analysis using CD spectroscopy showed that an R46L-containing peptide representing residues 36–47 in PCSK9 presented a much stronger helical shift in a DPC micelle environment, with changes to both the intensity and position of the minimum relative to the corresponding WT peptide . The percent helicity of the peptides obtained at different DPC micelle concentrations allowed micelle binding curves to be obtained, revealing that the R46L substitution conferred an ∼8-fold higher affinity for micelles compared with the WT peptide (D and E). The R46L mutation in full-length PCSK9 did not affect binding affinity to isolated LDL particles, as unlabeled versions of WT PCSK9 and PCSK9-R46L showed nearly equal abilities to compete with dye-labeled PCSK9 for binding to LDL .The natural PCSK9 mutation R46L is a LOF variant associated with lowered plasma PCSK9 levels, an anti-atherogenic lipid profile and decreased incidence of CHD , 35, 36.Fig. 6O-sulfation . Following incubation with LDL, we were able to readily detect LDL-bound PCSK9 containing sulfated Tyr-38, whereas PCSK9-A44P containing sulfated Tyr-38 was defective in LDL binding (B). This result further suggests that helical structure in the prodomain N terminus is essential for LDL binding and also indicates that an uninterrupted hydrophobic face in the helix is not an absolute requirement. Nevertheless, hydrophobicity at this site could assist in formation of an AH motif in PCSK9 that facilitates LDL binding. In support of this hypothesis, hydrophobic substitutions (Y38F or Y38L) for the Tyr-38 residue were found to largely preserve LDL binding, whereas lower hydrophobicity (Y38A) caused a significant decrease of >50% (C). LDL binding was also decreased >60% by substitution of Tyr-38 with positively charged amino acids (Y38K or Y38R) but was less affected (decrease of ∼25%) by substitution with acidic glutamic acid (Y38E) (C).Numerous plasma proteins, such as exchangeable apolipoproteins, employ AH motifs for reversible binding to circulating lipoproteins . This fuulfation . We tookreported , a PCSK9nditions A. Follow binding B. This r of >50% C. LDL bid (Y38E) C.B) suggests that formation of the transient AH motif in the N-terminal region of PCSK9's prodomain promotes LDL association, although not necessarily through the membrane insertion of its hydrophobic face. Alternatively, the helical conformation could facilitate a protein-protein interaction, either directly with LDL-apoB100 or to an intramolecular site. To assess the latter possibility, computational molecular modeling of PCSK9 to include the missing IDR in known structures was performed using the ab initio structure prediction method implemented in ROSETTA (2QTW)  and the CHR domain. The model also predicts adoption of helical conformation in a second sequence (aa 50–58) adjacent to a Ser-47 phosphorylation site (0) described as unique in PCSK9 compared with all known structures of subtilisin-like enzymes . When introduced into recombinant human PCSK9, all three mutations greatly inhibited LDL binding in vitro (B and C). In particular, recombinant PCSK9-R496W appeared to be completely unable to bind LDL, whereas R469W and F515L substitutions lowered binding by ∼60–70% . None of these mutations greatly affected PCSK9 secretion into cell culture medium (data not shown), in agreement with a recent study . By contrast, a D374Y mutation known to increase LDLR binding affinity . To further examine the effects of the R496W mutation, we purified secreted PCSK9-R496W protein  and found that it bound LDLR-ECD in vitro with similar affinity as WT PCSK9  and also induced degradation of cell surface LDLRs with similar potency when added to the culture medium of HepG2 cells .The inability of Tyr-38 sulfation to prevent LDL binding B suggest ROSETTA , 40. ConA 2QTW)  were appion site  and with enzymes . The Argnt study , and allQTW  werein vitro depends on an IDR in the prodomain N terminus (A), which was greatly enhanced by an R46L substitution representing an athero-protective PCSK9 LOF mutation (A and B); 3) a computational three-dimensional model of PCSK9 predicted that α-helical structure in prodomain N terminus aligns multiple polar residues to interact with the CHR domain (A); and 4) several PCSK9 GOF mutations located at or near the predicted interface in the CHR domain greatly diminished LDL binding (B). Considered together, we propose a two-step process by which allosteric conformational changes in PCSK9 facilitate LDL binding: 1) the initial encounter with LDL in circulation provides lipid and/or protein contacts to trigger α-helix formation in the N-terminal IDR; 2) this structural transition aligns an interdomain interaction involving the prodomain and CHR domain, which in turn allows stable LDL association.IDRs in proteins frequently have crucial roles in regulatory and signaling processes due to increased flexibility and accessibility for post-translational modification and protein-protein interactions and in some cases undergo disorder-to-order structural transition as part of this function –45. We p–terminus . In the terminus A, which mutation ; 2) a hemutation , A and BR domain A; and 4) binding B. ConsidPCSK9 are rare in human populations and can result in substantially increased plasma LDL-C levels in FH patients  or abolish (R496W) LDL binding in vitro. The affected amino acids are clustered on a surface region in CHR domain module 1 (CM1) (aa 453–531), one of three structurally similar subdomains arranged in a pseudo-3-fold axis . The Arg-469 and Phe-515 residues do not interact with the prodomain in the model and possibly are more directly involved in binding to the LDL particle.GOF mutations in patients . As suchpatients . As an eeraction . In Fig.old axis . This saA domain . AlthougPCSK9 mutation was first identified in an individual also heterozygous for an LDLR LOF mutation (E228K), the combined effect being severe hypercholesterolemia and early-onset cardiovascular heart disease (PCSK9 mutations (∼9%) was recently reported in a Turkish FH cohort, associated with highly elevated plasma LDL-C levels and early onset CHD in seven of eight cases . Like R496W, the R469W and F515L PCSK9 mutations have been identified in FH patients (D). Given the lack of other functional effects, it is highly probable that defective LDL binding underlies the GOF phenotype associated with these PCSK9 mutations in the CHR domain. Because PCSK9 activity raises plasma LDL levels, inhibition via direct binding to LDL particles could provide a form of negative feedback control. In this scenario, PCSK9 variants that bind poorly to LDL would be less subject to this inhibition, resulting in FH.The R496W  disease . A high C levels . Using pC levels , F and Gpatients , 50, butpatients , 51, 52.patients D. Given Lipid-ordered AHs are archetypal membrane-binding motifs found in numerous plasma proteins that, like PCSK9, reversibly associate with circulating lipoproteins and regulate lipoprotein metabolism , 37. How55–in vitro with >7-fold higher affinity, identifying a negative allosteric function (A) contains a cluster of arginine residues at positions 469, 496, 499, and 510 that could interact with acidic amino acids in a disordered prodomain N terminus. This may be sufficient to maintain PCSK9 in an autoinhibited state under basal conditions, explaining why a helix-breaking A44P mutation in PCSK9 did not increase LDLR binding/degradative functions (C and D). On the other hand, adoption of rigid α-helical structure could favor certain contacts, possibly involving the Arg-496 residue identified as key to LDL binding (B). If so, this would explain why PCSK9-A44P had lowered LDL binding affinity but was not as defective in this function as Δ31–52-PCSK9, in which an interaction with CM1 would be completely abolished (A and B). Indeed, LDL association may stabilize an autoinhibitory prodomain-CM1 interaction in PCSK9 that otherwise can be released by activating factors. Two such factors are known to improve the LDLR-binding function of PCSK9, namely acidic pH  bound LDLR function . Furtherfunction , 30, 58.function A containunctions , C and D binding B. If so,bolished , A and Bcidic pH , 16, 22 cidic pH . Heparincidic pH .PCSK9 mutations associated with plasma LDL-C levels in humans . However, it should be noted that the PCSK9 R46L allele in humans is associated with only modest lowering of plasma LDL-C (∼15%) with athero-protection likely due to life-long exposure , it did not affect LDL binding in vitro (B). On the other hand, mutation of Tyr-38 to alanine lowered PCSK9-LDL binding by >50%, whereas substitutions of higher hydrophobicity (Phe or Leu) preserved LDL binding (C and D). A possible explanation is that decreased hydrophobicity upon Tyr-38 sulfation is compensated by direct involvement of the sulfotyrosine residue in the prodomain-CM1 interaction that controls LDL binding. In a recent study, a mAb was shown to specifically bind and stabilize helical conformation in peptides derived from the N-terminal IDR in the PCSK9 prodomain  iodixanol) from Axis-Shield. EDTA-free CompleteTM protease inhibitor tablets were from Roche Applied Science, Nonidet P-40 detergent was from Biovision, and PolyJet DNA transfection reagent was from FroggaBio . All other reagents were from Sigma-Aldrich unless otherwise specified. Sodium mevalonate was prepared from mevalonic acid as described (d > 1.215 g/ml) was prepared by ultracentrifugation  supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin sulfate; Medium B contained Medium A supplemented with 10% fetal bovine serum (v/v); Medium C contained Medium A supplemented with 5% (v/v) NCLPDS; Medium D contained Dulbecco's modified Eagle's medium  supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin sulfate; sterol-depleting Medium E contained Medium D supplemented with 5% (v/v) NCLPDS, 10 μm pravastatin, and 50 μm sodium mevalonate.HEK293 and HepG2 cells (American Type Culture Collection) were maintained in monolayer culture at 37 °C and 5% CO6 tag . LDLR-ECD was enriched by affinity chromatography using TALON superflow affinity resin (Clontech) followed by gel filtration chromatography on a Superdex 200 10/300 GL column .FLAG epitope-tagged recombinant human WT PCSK9 along with A44P, R46L, R496W, and Δ31–52 variants were produced in stably transfected HEK293S cells and purified from conditioned culture medium as described previously . FluoresTM protein purification system (Bio-Rad) as per the manufacturer's instructions. Rabbit antiserum 3143 against the C-terminal 14 amino acids of LDLR was a kind gift of J. Herz . Anti-actin mouse monoclonal ascites fluid (clone AC-40) was from Sigma. The mouse anti-human transferrin receptor antibody was from Life Technologies, Inc. Secondary IRDye-labeled goat anti-mouse and anti-rabbit IgG antibodies were from LI-COR Biosciences. Rabbit antiserum 1697 raised against full-length human PCSK9 was used for immunoprecipitation and was custom-produced by Biomatik .The following antibodies were used for Western blotting. A mouse hybridoma clone expressing mAb 15A6 recognizing an epitope in the C-terminal CHR domain of PCSK9 was a generous gift from J. Horton . The IgG fraction was purified from hybridoma culture medium by Protein A chromatography on a ProfiniaTM precast gels (Thermo-Invitrogen) for electrophoresis. The size-separated proteins were then transferred to nitrocellulose membranes (Bio-Rad). Primary antibodies (described above) and IRdye800-conjugated secondary antibodies were used to detect target proteins. Signal was detected using the LI-COR Odyssey IR imaging system (LI-COR Biosciences). Quantification of the intensity of the bands was obtained using Odyssey 2.0 software (LI-COR Biosciences).Samples were loaded onto 8% SDS-polyacrylamide gels or 4–12% Tris/HEPES-SDS BoltTable S1. All desired mutations and the absence of extraneous mutations were confirmed by sequencing the entire coding region.The pcDNA3-PCSK9-FLAG vector  codes fom HEPES-KOH, pH 7.4, 150 mm NaCl, 2 mm CaCl2) and concentrated ∼10-fold on an Amicon Ultra-4 centrifugal filter unit with a 10-kDa membrane cutoff (Millipore). PCSK9 in conditioned medium preparations was quantified by Western blot analysis using purified PCSK9 as a standard and stored in single-use aliquots at −80 °C.HEK293 cells cultured in Medium B to ∼70% confluence in a 100-mm Petri dish were transfected with a total of 3 μg of PCSK9 cDNA expression vectors using PolyJet DNA transfection reagent as per the manufacturer's instructions. At 18 h post-transfection, the cells were washed and incubated with serum-free Medium A containing 1× ITS (insulin-transferrin-selenium) cell culture supplement. Conditioned media were recovered 48 h post-transfection and buffer-exchanged with HBS-C  and antioxidant (20 μm butylated hydroxytoluene) were added to the cleared plasma. LDL (d = 1.019–1.065 g/ml) was isolated using sequential potassium bromide flotation ultracentrifugation  sucrose as described  were incubated at 37 °C for 1 h. LDL-bound and free PCSK9 were then separated by Optiprep gradient ultracentrifugation according to a modified version of a protocol described previously . Tubes were centrifuged in a TLN100 rotor (Beckman Coulter) at 100,000 rpm for 2 h at 4 °C. LDL-containing fractions (600 μl) were collected by tube puncture into a 1-ml syringe. The entire LDL fraction was immunoprecipitated for 18 h at 4 °C using anti-PCSK9 rabbit polyclonal 1697 (see above) and anti-rabbit Trueblot agarose beads (Rockland). The collected beads were washed, and immunoprecipitated proteins were resolved by SDS-PAGE on 8% acrylamide gels. PCSK9 content was then quantified by Western blotting, and values were normalized to reactions containing WT PCSK9.Binding reactions (1.0-ml volume), each containing 500 μg of LDL and 1 μg of PCSK9 (in concentrated conditioned media) and 0.5% BSA (w/v) in HBS-C buffer (25 meviously . Brieflym DyLight800-labeled WT PCSK9 in HBS-C buffer containing 1% (w/v) BSA for 90 min at 37 °C with increasing amounts of unlabeled WT or mutant PCSK9. 4× loading dye  was added to the reactions, and the mixtures were resolved on 0.7% agarose gels (SeaKem LE) for 2 h at 40 V with electrode buffer of 90 mm Tris, pH 8.0, 80 mm borate, 2 mm calcium lactate. Gels were scanned directly using the LI-COR Odyssey IR imaging system (LI-COR Biosciences), and bands representing DyLight800-PCSK9 bound to LDL were quantified using Odyssey 2.0 software (LI-COR Biosciences). For PCSK9-LDLR binding analysis, partially purified LDLR-ECD (see above) was diluted in TBS-C  and blotted directly onto 0.45-μm pore size nitrocellulose membranes (Bio-Rad) using a BioDot SP slot-blot apparatus (Bio-Rad) according to the manufacturer's instructions, and blots were blocked for 30 min in TBS-C containing 5% (w/v) nonfat milk. All incubations (90 min) and subsequent washes (3 × 15 min) were carried out at room temperature with gentle oscillation in TBS-C buffer containing 2.5% (w/v) nonfat milk. Blots were incubated with 0.1 μg/ml DyLight800-labeled WT PCSK9 with increasing amounts of unlabeled WT or mutant PCSK9. Blots were then washed and scanned using the LI-COR Odyssey IR Imaging System, and band intensity was quantified using Odyssey version 2.0 software (LI-COR Biosciences). The amount of competitor PCSK9 protein required for 50% inhibition of fluorophore-labeled PCSK9 binding (iK) was determined by fitting data to a sigmoidal dose-response curve using nonlinear regression (GraphPad Prism 5 software).For PCSK9-LDL binding analysis, 0.5 mg/ml LDL was incubated with 10 nm chloroquine. Cells were then washed in ice-cold PBS-CM (PBS with 1 mm MgCl2 and 0.1 mm CaCl2), and whole-cell extracts were prepared in Tris Lysis Buffer . Extracted proteins were resolved on 8% SDS-PAGE, and LDLR, PCSK9 and actin were detected by Western blotting.HEK293 cells were cultured in Medium B in 6-well dishes to 70% confluence and then transiently transfected with 1 μg of WT human LDLR plasmid per well using PolyJet DNA transfection reagent as per the manufacturer's instructions. The following day, cells were switched to lipoprotein-deficient Medium C and treated for 2 h at 37 °C with 2 μg/ml PCSK9 (from conditioned media) in the presence of 50 μm dishes were cultured in Medium D to 60% confluence and then switched to sterol-depleting Medium E for 18–20 h. Purified, recombinant WT or A44P- or D374Y-PCSK9 was added to the medium, and cells were incubated at 37 °C for 4 h. Cell surface proteins were biotinylated with Sulfo-NHS-SS-Biotin  and captured from whole-cell extracts using NeutrAvidin-agarose beads (Pierce) as described previously  DPC micelles using a Jasco J-815 CD spectropolarimeter at 37 °C with a 0.1-cm quartz cuvette. Spectra were acquired from 200 to 250 nm using five accumulations at 20 nm/min and a data integration time of 8 s. Secondary structure deconvolution of spectra was carried out in CDPro with the CONTIN algorithm and SP43 reference set. To measure the apparent affinity of peptide-lipid interactions, peptide (30 μm) was mixed with increasing concentrations of DPC micelles, and CD spectra were collected after each increment as described above.Custom peptide synthesis (>98% purity) was by BioMatik. Lyophilized peptides were initially dissolved as stock solutions in 0.1% ammonium hydroxide, and peptide concentrations were determined by amino acid analysis  for use in mean residue molar ellipticity calculations. CD spectra were collected on 30 μ2QTW)   was usedsion 3.8 . In prepsion 3.8 , identifsion 3.8  as well sion 3.8  are const test or Student's t test, as indicated in the figure legends, using GraphPad Prism 5 software. Data used measurements from at least two distinct sample preparations for each component.Data are presented as the mean ± S.E. Two-sided statistical analysis was determined by one-sample S. K. S., A. C. F., A. M., T. K., A. V.-J., and T. A. L. data curation; S. K. S., A. C. F., A. M., N. K. G., A. V.-J., and T. A. L. formal analysis; S. K. S., A. C. F., I. A., N. K. G., A. V.-J., and T. A. L. investigation; S. K. S., A. C. F., A. M., T. K., N. K. G., A. V.-J., and T. A. L. methodology; S. K. S., A. C. F., A. M., T. K., N. K. G., A. V.-J., and T. A. L. writing-review and editing; N. K. G. and T. A. L. supervision; A. V.-J. and T. A. L. conceptualization; A. V.-J. and T. A. L. resources; A. V.-J. and T. A. L. funding acquisition; T. A. L. validation; T. A. L. visualization; T. A. L. writing-original draft; T. A. L. project administration.


Staphylococcus aureus is a minor disassembly factor. Cells lacking hflX do not accumulate 100S ribosomes unless they are subjected to heat exposure, suggesting the existence of an alternative pathway during nonstressed conditions. Here, we provide biochemical and genetic evidence that two essential translation factors, ribosome-recycling factor (RRF) and GTPase elongation factor G (EF-G), synergistically split 100S ribosomes in a GTP-dependent but tRNA translocation-independent manner. We found that although HflX and the RRF/EF-G pair are functionally interchangeable, HflX is expressed at low levels and is dispensable under normal growth conditions. The bacterial RRF/EF-G pair was previously known to target only the post-termination 70S complexes; our results reveal a new role in the reversal of ribosome hibernation that is intimately linked to bacterial pathogenesis, persister formation, stress responses, and ribosome integrity.The formation of translationally inactive 70S dimers  by hibernation-promoting factor is a widespread survival strategy among bacteria. Ribosome dimerization is thought to be reversible, with the dissociation of the 100S complexes enabling ribosome recycling for participation in new rounds of translation. The precise pathway of 100S ribosome recycling has been unclear. We previously found that the heat-shock GTPase HflX in the human pathogen  Hibernation-promoting factor (HPF),ribosome modulation factor (RMF), induces the dimerization of 70S ribosomes to form the translational inactive 100S complexes in all bacterial phyla with the exception of Actinobacteria and hibernation factor YfiA  are constitutively produced throughout the life cycle, as confirmed by time course immunoblotting and MS analyses of the HPF-bound 100S complexes (–e.g. post-termination recycled ribosomes) from degradation -like stalled 70S ribosomes from mRNA during thermal stress  and EF-G (encoded by fusA) proteins in the presence and absence of GTP analogs. Native 100S ribosomes were isolated to ≥70% homogeneity from methicillin-resistant S. aureus (MRSA) USA300 through a two-step density gradient fractionation and ultracentrifugation protocol. The purified RRF, EF-G, and HflX proteins or GTP alone failed to dissociate the 100S complexes (panels i–v and ix). More than 50% of the 100S ribosomes were converted into 70S monomers in the presence of RRF, EF-G, and GTP (panel vii) or HflX and GTP (panel x). The 70S ribosomes were not further dissociated by RRF/EF-G into 50S and 30S subunits because the reactions did not contain the anti-association factor IF3 (panels iii and v). This is likely due to the residual activity of the ribosome-associated RRF or EF-G because high-salt wash that otherwise removes ribosome-bound factor(s), was omitted from our 100S ribosome preparation to prevent the release of HPF. When GTP was substituted with nonhydrolyzable guanosine 5′-triphosphate (GMPPNP), which is a higher-affinity binder of ribosome-bound EF-G (panel viii). In contrast, GMPPNP induced the HflX-mediated dissociation of both the 100S and 70S complexes (panel xi), consistent with previous observations that GMPPNP is a more effective ligand of HflX in splitting the empty 70S ribosome  conversion reduces tRNA translocation (S. aureus numbering) with an aspartate  in RRF renders a temperature-sensitivity phenotype . The R29A and R59A mutants of EF-G are impaired in 100S ribosome dissociation, whereas the H572K mutant retains its splitting activity at the wildtype (WT) level (B). The dissociation of the 100S ribosome by the EF-G(H572K) is abolished upon GMPPNP substitution. The impairments caused by R29A and R59A are due in part to a decrease in GTP hydrolysis (C) and not to reduced ribosome binding . Species-specific activities of RRF and EF-G have been observed between several bacterial homologs, and noncognate RRF and EF-G pairing is nonfunctional (S. aureus EF-G(R59A) compared with that of E. coli  level B. The didrolysis C and notnctional , 47. The E. coli . We concB). Limited proteolysis showed that RRF(V116) protein was more accessible to chymotrypsin digestion and may therefore adopt different conformations than the WT RRF . In addition, RRF(V116D) bound to the ribosome and recruited EF-G as efficiently as WT RRF . Consistent with these findings, the V116D mutant allele fully complemented the growth defect of an RRF-depleted strain (D).Contrary to our prediction, RRF(V116D) showed an increase in 100S ribosome dissociation, whereas RRF(A28E) was fully functional, as expected B. Limited strain D.RRF binds to the A-site of PoTc and recruits EF-G to initiate 70S ribosome splitting. RRF null is deleterious. EF-G is also an essential factor for the translocation step during elongation . To avoifrr (locus SAUSA300_1152) and placing a hexahistidine-tagged frr under the control of an anhydrotetracycline (aTc)-inducible promoter. The His6-frr allele was then integrated into a neutral site of the S. aureus chromosome (A). The same chromosomal integration system (S. aureus knockouts. The His-tagging strategy does not impair splitting activity of S. aureus RRF (2B) nor compromises the efficiency of a reconstituted cell-free translation system containing E. coli His6-RRF (6-RRF without aTc and an upshift in the production of His6-RRF in the presence of an inducer (B). In contrast to the hflX knockout mutant, which lacked a phenotype at 37 °C but exhibited better heat tolerance at 47 °C (C), confirming the direct role of RRF deficiency. The relatively low amount of His6-RRF (compared with the WT strain) was sufficient for growth (B), implying that the WT concentration of RRF is greater than that required for normal growth.To discern between endogenous RRF and exogenously introduced RRF, we constructed an RRF-depleted strain by deleting the romosome A. The san system  has beenreus RRF  and 2B nHis6-RRF . We foun inducer B. In conat 47 °C , the depat 47 °C C, confirr growth B, implyiD). The severity of the growth defects observed on agar plates was only weakly associated with the type of gelling agents . In the absence of aTc, RRF deficiency was fully complemented by expressing WT RRF or RRF(V116D) in a plasmid but not with an empty vector (D). Using the same complementation approach, we found that an hflX expressed in trans completely restored the growth defects of the RRF-depleted strain (A) and the expression of RRF in different genetic backgrounds was not affected by the introduction of plasmid (B). These results confirm that hflX functionally overlaps with RRF/EF-G in ribosome recycling. Furthermore, HflX is not expressed during growth at 37 °C and is exclusively up-regulated upon heat shock  , in which only the supplementation of aTc supported growth D. The sey vector D. Using d strain A and the plasmid B. These Fig. S4) , explainA600 of ∼1.4) cells grown without the aTc inducer. The cultures and sample preparation were free of any of the elongation inhibitors (e.g. chloramphenicol) that are normally used to preserve polysome species. Under these conditions, the WT cells accumulated 100S ribosomes but retained a very small fraction of polysomes . In the RRF-depleted cells, PoTc could not be released from the mRNA templates, resulting in the queuing of the trailing ribosomes; these stalled polyribosomes were co-sedimented with the elongating polysomes . The lack of PoTc recycling reduced the level of ribosomal precursors available to form the 100S complexes, explaining the lower 100S peak in the RRF-depleted strain relative to the WT . The depletion of RRF was verified by immunoblotting . A very moderate effect on the 100S ribosome was observed in the hflX knockout , consistent with previous findings , providing an additional clue to potential ribosome stacking at the termination codon.We analyzed the effect of RRF depletion on ribosome pools by sucrose density gradient ultracentrifugation. Crude ribosomes were prepared from late log phase . When a termination codon was introduced at the Lys-54 position of gfp, RRF depletion promoted a low level of translational read-through, resulting in an increase in fluorescence intensity relative to the WT. The read-through was reversed upon the addition of aTc . We conclude that PoTc recycling and the formation of the 100S ribosome are interdependent and that RRF depletion reduces the translational capacity.To investigate the impact of RRF depletion on stop codon read-through, we compared the synthesis of green fluorescent protein (GFP) between the WT and the RRF-depleted strain carrying a pDM4 reporter plasmid . RRF depFig. 5The recycling of PoTc, reactivation of the silent 100S ribosomes, and prevention of ribosome degradation are cost-saving activities because ribosome biogenesis is the most energy-consuming cellular process. A recent study offered the first clue that 100S dimers are disassembled and converted into active ribosomes in a reconstituted cell-free translation system consisting solely of purified components, including RRF and EF-G, but not the HflX .S. aureus. RRF and EF-G split the 100S complexes via EF-G-mediated GTP hydrolysis, similar to the GTPase activity of HflX (m) during active growth . It. ItS. aufMet is available. Alternatively, ribosome queuing could trigger one of the ribosome rescue systems  mutant  has been extensively studied (E. coli RRF(V117D) cells are nonviable because the protein is misfolded and rapidly degraded at high temperature. Surprisingly, we found that the recombinant S. aureus RRF(V116D) protein is fully active, and perhaps more potent in dissociating 100S ribosomes in vitro (B). The overexpression of RRF(V116D) in a multicopy plasmid overcomes the growth defects of the RRF-depleted strain  and that an excess amount of RRF(V116D) can compensate for the instability of the mutant protein in vivo. Consistent with this notion, overexpression of E. coli RRF(V117D) eliminates the temperature-sensitive phenotype . These striking differences could be due to the local depletion of nutrients and moisture or the accumulation of a toxic compound on the agar plates. For instance, specific solidifying agents may sequester essential micronutrients or contain toxic ingredients. Growth defects caused by the chelation of zinc on the agar plates have been observed in a zinc transporter mutant , ruling out the contribution of a solidifying agent. It is possible that shaken liquid culture may provide additional aeration and spatial accessibility to nutrients, resulting in better growth.The phenotype of the RRF-depleted strain is more severe on solid media than in the liquid media , C and Dr mutant . To testRibosome availability is directly linked to bacterial growth and revival, and ribosome homeostasis is essential for cellular function. The identification of the dual action of RRF/EF-G in maintaining a normal translational cycle and reactivating dormant ribosomes implies that RRF is an ideal antimicrobial target for inhibiting both the proliferation and regrowth of pathogens.Table S1. The methicillin-resistant S. aureus (MRSA) USA300 strain JE2 (GenBank CP000255) was used throughout the study. The deletion mutant of hflX (SAUSA300_1198) has been described previously  at a 5:1 tube- or flask-to-medium ratio with a 1:100 dilution of an overnight seed culture. TSB agar plates were prepared using DifcoTM agar , Eiken agar , Gelrite , and agarose . When necessary, erythromycin, chloramphenicol, cadmium chloride, kanamycin, and anhydrotetracycline  were used at 5 μg/ml, 10 μg/ml, 0.15 mm, 75 μg/ml, and 400 ng/ml, respectively. The restriction-deficient S. aureus RN4220 strain was used as a plasmid passage surrogate before the plasmid was transformed into the destination JE2 derivatives. E. coli cells harboring expression vectors were grown at 16 or 37 °C in LB (Difco). Antibiotics were used at 50 (kanamycin) or 100 μg/ml . All GTP analogs were from Sigma-Aldrich. Primers were purchased from IDT DNA and are listed in Table S2.The strains and plasmids used in this study are listed in eviously . Unless 6-tagged frr (SAUSA300_1152) was PCR amplified from S. aureus JE2 genomic DNA using the primers P1209 and P1210. The fragment was cloned into the KpnI and SacI sites of pRMC2 , a site-directed QuikChange mutagenesis kit (Agilent Genomics) was employed to introduce a V116D substitution using the primers P1219/P1220.pLI50  was usedpromoter  was atta6-tagged RRF, the frr coding region was PCR amplified with P1120/P1121 primers using JE2 genomic DNA as the template. The product was cloned into the NdeI and EcoRI sites of pET28a. The A28E and V116D alleles were introduced by QuikChange mutagenesis using primer pairs P1217/P118 and P1219/P1220, respectively. To construct C terminally His8-tagged EF-G, fusA (locus SAUSA300_0532) was PCR amplified with P1167/P1168, and the fragment was cloned into the NcoI and XhoI sites of pET28a. The R29A, R59A, and H572A mutations were introduced into EF-G by QuikChange mutagenesis using primer pairs P1121/P1122, P1213/P1214, and P1215/P1216, respectively. The K54stop mutation was introduced to the gfp reporter in pDM4 by QuikChange mutagenesis using primers P1255/P1256.The pET28a vector was used to overexpress HflX, RRF, and EF-G recombinant proteins. pET28-HflX has been described previously . To consA260) of ribosomes were layered on a 5–25% sucrose gradient that was prepared on a BioComp Gradient Master. The samples were centrifuged at 210,000 × g at 4 °C in a SW41 rotor in a Beckman Coulter Optima XPN-100 ultracentrifuge for 3 h. Fractionation was performed using a Brandel fractionation system equipped with a UA-6 UV detector. The 100S ribosome peaks were collected and centrifuged in a SW55 Ti rotor at 246,000 × g at 4 °C for 18 h. The ribosome pellet was resuspended in low-salt buffer 2, 100 mm KOAc, 0.5 mm phenylmethylsulfonyl fluoride, 1 mm DTT) and quantified according to the A260 value (1 A260 = 23 pmol/ml of 70S).Crude ribosomes were isolated from late log-phase JE2 strains by cryo-milling methods , 25. FifIn vitro 100S dissociation samples were assembled with a 0.2 μm 100S ribosomes, 2 μm proteins , and 2 mm GTP analogs in a 100-μl reaction. The reactions were incubated at 37 °C for 30 min before being spun through a 5–20% sucrose gradient. Ribosome profiles were recorded in real time traces probed at an absorbance of 254 nm. For quantification of 100S to 70S ratios, the baseline was first defined as the global minimum excluding the free nucleic acids and protein peak. The boundary of 100S–70S were manually selected from the trough between the peaks. The area under a peak was calculated by ImageJ and divided to obtain the ratio.A600 ∼ 1.4 and harvested by rapid filtration and freezing in liquid nitrogen without the addition of chloramphenicol typically used to arrest translation in the ribosome profiling experiments  of RNase A (Ambion AM2271) at 25 °C for 1 h prior to ultracentrifugation.For ribosome queuing analysis, cells were grown in TSB at 37 °C until eriments . Frozen eriments . Four A2A600 of ∼0.4, after which isopropyl 1-thio-β-d-galactopyranoside was added at a final concentration of 0.5 mm, and induction was performed at 16 °C for 16 h before harvest. Polyclonal antibodies were raised in rabbits by Josman, LLC.The overexpression and purification of the His-tagged recombinant proteins have been described in detail previously , 31 and S. aureus total lysates were prepared in a bead beater as described previously (hflX knockout), and anti-EF-G .Total eviously . Immunobeviously , 66. AntiColorLock Gold kit (Innova Biosciences) was used to determine the GTP hydrolysis of EF-G variants by measuring the amount of free Pi. Detailed protocols were reported previously (The Peviously .S. aureus cells harboring the pDM4 or pDM4(K54stop) plasmid were grown at 37 °C in TSB in the presence or absence of 400 ng/ml of aTc. Cells were harvested at an A600 of ∼0.3 by centrifugation. The pellets were resuspended and washed three times with sterile 1× PBS buffer to remove background fluorescence from TSB medium. The cell density was adjusted to an A600 of ∼0.2. Two-hundred microliters of the cell suspension was transferred to a black 96-well–flat-bottom plate (Corning Costar®) in triplicate, and GFP fluorescence was measured on a TECAN SPARK plate reader at excitation and emission wavelengths of 485 and 535 nm, respectively. The final fluorescence units were calculated by subtracting the fluorescence from the JE2 background and dividing the results by the final A600 to correct for cell density. The experiments were repeated five times. Statistical significance was evaluated with unpaired t test.All data are contained within the manuscriptA. B. data curation; A. B. and M.-N. Y. formal analysis; A. B. validation; A. B., K. E. S., and M.-N. Y. investigation; A. B. visualization; A. B., K. E. S., and M.-N. Y. methodology; A. B. and M.-N. Y. writing-original draft; M.-N. Y. conceptualization; M.-N. Y. resources; M.-N. Y. supervision; M.-N. Y. funding acquisition; M.-N. Y. project administration; M.-N. Y. writing-review and editing.


We herein isolated each disruptant of these four genes to determine their functions in more detail. Disruptants of ver-1, ordB, and hypA genes commonly accumulated VA in their mycelia. In contrast, the verA gene disruptant accumulated a novel yellow fluorescent substance (which we named HAMA) in the mycelia as well as culture medium. Feeding HAMA to the other disruptants commonly caused the production of aflatoxins B1 (AFB1) and G1 (AFG1). These results indicate that HAMA pigment is a novel aflatoxin precursor which is involved at a certain step after those of ver-1, ordB, and hypA genes between VA and DMST. HAMA was found to be an unstable substance to easily convert to DMST and sterigmatin. A liquid chromatography-mass spectrometry (LC-MS) analysis showed that the molecular mass of HAMA was 374, and HAMA gave two close major peaks in the LC chromatogram in some LC conditions. We suggest that these peaks correspond to the two conformers of HAMA; one of them would be selectively bound on the substrate binding site of VerA enzyme and then converted to DMST. VerA enzyme may work as a key enzyme in the creation of the xanthone structure of DMST from HAMA.In the biosynthesis of aflatoxin,  Aspergillus flavus and Aspergillus parasiticus ,18O-meth pathway .A. nidulans, A. versicolor and various fungal species are known to produce sterigmatocystin (ST) as a final product fusion structure of the xanthone and dihydrobisfuran moieties  the verA gene is involved in the pathway from HAMA to DMST through a regiospecific xanthone ring formation. We also investigated accumulating substances in the deletion mutant of A. nidulans stcP gene − (100%) and 747.3 [2M−H]− (32%), indicating that the molecular mass of HAMA is 374  (− (100%), and 355 [M−H2O−H]− (55%) (+ (100%), 357 [M − H2O + H]+ (11%) (− (100%), 355 [M−H2O−H]− (58%) in APPI-MS spectra  on LC chromatograms were coincident to those of authentic samples of DMST and STM, respectively, and their molecular masses were commonly 310 in APPI-MS spectra B-c,d. Thd sample B, LC excverA disruptant culture medium by LC-MS using condition [C]. Two main peaks were observed at 27.2 min and 32.0 min on LC chromatograms (+ (100%)  C-e,f. ThstcP disruptant of A. nidulans FGSC26 −, which has the deletion of the 18-kb region from the pks gene to fas-1 in the AF gene cluster of strain NR-1  and verA-R2922 [5′-TGCGGCCCTGAGCTTCT-3′]. The 2.0-kb ptrA probe was amplified from pPTRI plasmid  using the primers as described  and ordB-L-KpnI-R [5′-CGGGGTACCGCCCACCTCTTCGTACCTAG-3′]. Pair 2: ordB-R-HindIII-R [5′-CCCCAAGCTTGCAATTGTGTAGTCTTCTCTGG-3′] and ordB-R-SalI-F [5′-AACGCGTCGACGTTGACGGAGGATCTGTTAGC-3′].To delete the KpnI for the 3′-flanking region and HindIII/SalI for the 5′-flanking region, and then inserted into the pSP72-ptrAKpnI/EcoRV and HindIII/XhoI sites to give a pORDB-L/R, which was then linearized by BglII and Eam1105I. The final linear replacement construct, which contained the 2-kb selectable marker ptrA flanked by a 1.4-kb fragment of the 3′-flanking region and a 1.35-kb fragment of the 5′-flanking region was used for transforming the SYS4 strain. Fungal protoplast preparation and transformation with DNA were performed as described  and hypA-L-HindIII-F [5′-CCCCAAGCTTCATGATACAGATTGAGTGCGAC-3′]. Pair 2: hypA-R-KpnI-F [5′-CGGGGTACCCGTATCTCAGTTATGCAATGTCTC-3′] and hypA-R-R [5′-AGTCCAATGCCGTCAAC-3′].To delete the SalI/HindIII for the 3′-flanking region and KpnI for the 5′-flanking region, and then inserted into the pSP72-ptrA XhoI/HindIII and KpnI/EcoRV sites to give a pHYPA-L/R, which was then linearized by BglII. The final linear replacement construct, which contained the 2-kb selectable marker ptrA flanked by a fragment of the 3′-flanking region and a fragment of the 5′-flanking region was used for transforming the SYS4 strain. The transformation, screening, and characterization of the PT-resistant transformants were performed as described above.The resulting PCR products were successively cut with ver-1 gene in the genome of A. parasiticus SYS4, we made the ver-1 gene disruption construct in a two-step procedure. A 1.1-kb fragment of the 5′-flanking region of ver-1 gene including the 5′ non-coding upstream sequence of the gene and the coding sequence of norA gene and a 1.1-kb fragment of the 3′-flanking region of the ver-1 gene including the 3′-non-coding downstream region of ver-1 and the coding sequence of verA gene were amplified by PCR using two sets of primer pairs with endonuclease restriction sites underlined to facilitate sub-cloning. Pair 1: norA-XhoI-F [5′-ATGCATTTGCTCGAGCCAACGGACTTACCC-3′] and ver-1-up-PstI-R [5′-AAAACTGCAGCTCTGCCTCTATCCAAAGCC-3′]. Pair 2: ver-1-down-KpnI-F [5′-GGGGTACCCGCTATATACTCGTGGGTGA-3′] and verA-BglII-R [5′-GCCGGACGAGAATTGCTGGGAGATCTTCAG-3′]. A 2-kb selectable marker ptrA gene was amplified by PCR using the primer pairs pPTRI-KpnI-F [5′-GGGGTACCGGGCAATTGATTACGGGATCCCA-3′] and pPTRI-PstI-R [5′-AAAACTGCAGTGACGATGAGCCGCTCTTGC-3′] and pSP72-ptrA. The resulting three PCR products were combined by ligase, and the final linear replacement construct was used for transforming SYS4 strain. The transformation, screening, and characterization of the PT-resistant transformants were performed as described above.To delete the v/v), and the extract was analyzed by TLC.After the tip cultures of SYS4 or each of the disruptants and then centrifugation, metabolites in the culture medium were extracted with an equal volume of chloroform, and the resulting extract was analyzed by TLC or HPLC. For detection of metabolites in mycelia, the mycelia in the tip was extracted with 800 µL acetone, and the resulting acetone extract was air-dried by keeping it at room temperature in dark condition. The resulting debris was solubilized by adding 100 µL of benzene:acetonitrile . Fluorescence of the metabolites on the TLC plate was observed under 365-nm UV light and photographed with an Olympus C-745 digital camera. In some cases, the TLC plate was observed under 300-nm UV light and photographed with Fluor-S™ MultiImager  for the detection of AFs. For the characterization of substances derived from HAMA or metabolites, strain NIAH-26, ordB-, hypA-, and verA-disruptants were also analyzed by TLC using solution B containing benzene:ethyl acetate  with 10% acetic acid.The extracts of culture medium and mycelial extract were spotted onto a silica gel TLC plate  and then developed with solution A containing chloroform:ethyl acetate: 90% formic acid , and the flow rate was 1 mL min−1 − strain was cultured with the treated HAMA, and the resulting culture medium was analyzed by HPLC. The acid and alkaline stabilities of the pigments were also examined by changing the pH of the culture medium of verA disruptant to pH 1.5 or pH 11.0 by adding 1 M HCl or 1 M NaOH, followed by incubation at room temperature for 3 h. After the pH of the solution was readjusted by adding the same volume of 1 M NaOH or 1 M HCl, respectively, the resulting solution was used for the feeding experiment with strain NIAH 26 using the tip culture method.To test the stability of HAMA, we examined the heat stability of the pigment after the culture medium of the 2 gas in a microtube; the debris was then solubilized in methanol. These procedures of drying and solubilization were repeated several times. The resulting solutions were analyzed by HPLC using the ODS column and solution D.For the determination of the effect of the repetition of drying followed by solubilization, we dried the extract of the TLC-purified HAMA after the elution of the TLC spot with ethyl acetate with NverA disruptant was analyzed by liquid chromatography-mass spectrometry (LC-MS) using three conditions as follows:The pigment HAMA that accumulated in 3CN; gradient: 0 min 10% B, 2 min 10% B, 10 min 100% B, 13 min 100% B, 15 min 10% B; Detection: DAD 220–340 nm MaxPlot; MS + ESI (m/z 100–1000), −ESI (m/z 100–1000).Condition [A]: HPLC condition column: YMC-UltraH Pro C18 , solvent A: 0.1% formic acid in water, solvent B: 0.1% formic acid in CH−1.Condition [B]: The LC-MS 2010A system  consisted of an LC-VP separation module equipped with an SPD-M10AVp photodiode array (PDA) detector and a single-quadrupole mass spectrometer with an atmospheric pressure photoionization (APPI) source probe. Solvent A, 5 mM ammonium acetate in water; solvent B, methanol; gradient: 0 min 10%, 2 min 10%, 17 min 95%, 22 min 95%, 23 min 10%, and 30 min 10%. MS + APPI (m/z 100–500), −APPI (m/z 100–500) An Inertsil column  was used, and the flow rate was 0.2 mL minv/v).Condition [C]: the LC-MS conditions were the same as those in condition [B] except that the carrier solvent was set as an isocratic mixture of methanol and 5 mM ammonium acetate in water  and FGSC26 (the wild strain) as described [5) of either strain were cultured in oat flake medium (3 g oat flakes and 0.5 mL water) at 28 °C for 5 days. Metabolites of fungus ware extracted twice with the same volume of acetone-chloroform solution. The resulting pooled extract was concentrated and then analyzed by silica-gel TLC using benzene:acetic acid . Compounds on the TLC plate were visualized by spraying 10% (wt./vol) aluminum chloride solution in ethanol and heating. For the quantitative measurement of the metabolites, the part corresponding to either spot of STM or DMST was scraped and extracted with ethyl acetate. The extract was then analyzed by HPLC using an ODS column and solution D.We analyzed the metabolites produced by escribed  with minverA gene in the aflatoxin gene cluster is involved in the step to make the xanthone structure of demethylsterigmatocystin as well as dihydrodemethylsterigmatocystin in aflatoxin biosynthesis.We clarified that


Cytotoxic T lymphocytes kill cancer or virally infected cells by exocytosis of lytic granules. This leads to perforin-mediated granzyme entry into the target cell, consequently killing the target cell. Granzymes and perforin are activated by cysteine cathepsins whose activity is regulated by the protein inhibitor cystatin F. Since cystatin F can be secreted by a range of cancer and immune cells in tumour microenvironments, we here investigated whether extracellular cystatin F can be taken up by and affect the function of cytotoxic T lymphocytes. We demonstrated cystatin F uptake into cytotoxic T lymphocytes, down-regulation of target peptidases, and reduced target cell killing. Overall, our results indicate that cystatin F is an important mediator that can impair the killing efficiency of cytotoxic T lymphocytes and thus suggest that it is a possible target for cancer immunotherapy.Cystatin F is a protein inhibitor of cysteine cathepsins, peptidases involved in the activation of the effector molecules of the perforin/granzyme pathway. Cystatin F was previously shown to regulate natural killer cell cytotoxicity. Here, we show that extracellular cystatin F has a role in regulating the killing efficiency of cytotoxic T lymphocytes (CTLs). Extracellular cystatin F was internalised into TALL-104 cells, a cytotoxic T cell line, and decreased their cathepsin C and H activity. Correspondingly, granzyme A and B activity was also decreased and, most importantly, the killing efficiency of TALL-104 cells as well as primary human CTLs was reduced. The N-terminally truncated form of cystatin F, which can directly inhibit cathepsin C (unlike the full-length form), was more effective than the full-length inhibitor. Furthermore, cystatin F decreased cathepsin L activity, which, however, did not affect perforin processing. Cystatin F derived from K-562 target cells could also decrease the cytotoxicity of TALL-104 cells. These results clearly show that, by inhibiting cysteine cathepsin proteolytic activity, extracellular cystatin F can decrease the cytotoxicity of CTLs and thus compromise their function. Cytotoxic T lymphocytes CD8+ (CTLs) and natural killer (NK) cells are crucial players in the immune response against virally infected cells and tumour cells . The maiIn cytotoxic lymphocytes, cathepsin C is the major progranzyme convertase, activating progranzymes by removing a dipeptide at their N-terminal end . In the In immune cells, cystatin F, an endogenous protein inhibitor, was described to play an important role in regulating cysteine cathepsin activity . CystatiIn cytotoxic cells, cystatin F is localised in cytotoxic granules in which it co-localises with its target cathepsins C, H, and L ,15,16 inIn this study, we demonstrate that extracellular cystatin F decreases CTL cytotoxicity, likely by affecting the activation of granzymes, effector molecules of the perforin/granzyme pathway. We first demonstrate internalisation of cystatin F into TALL-104 cells and interaction of internalised cystatin F with cathepsin C. We show that extracellular cystatin F attenuates the activities of cathepsins C, H, and L and granzymes A and B, while it does not affect perforin processing. Finally, we demonstrate that cystatin F secreted from target cells can also affect the cytotoxicity of CTLs.We first assessed recombinant cystatin F uptake into TALL-104 cells, a model cell line of cytotoxic T lymphocytes . WesternIn cytotoxic lymphocytes, cystatin F regulates the activities of cathepsins C, H, and L, which are implicated in granzyme and perforin activation ,13. The Next, we aimed to determine whether lower cathepsin C and H activity translates to decreased granzyme activity. Granzyme A and B activity was determined in whole-cell lysates of TALL-104 cells treated with full-length or N-terminally truncated cystatin F. Extracellularly added full-length cystatin F significantly inhibited the activity of granzymes A and B d,e in TANext, we tested whether treatment of TALL-104 cells with full-length or N-terminally truncated cystatin F affects their cytotoxic potential by calcein-AM release assay. The ability of TALL-104 cells to kill target K-562 cells was significantly reduced after treatment with either form of cystatin F f,g.Lastly, to further extend our findings in a more physiological setting we tested the effect of full-length cystatin F on granzyme activity and cytotoxicity of primary human SEA/SEB-stimulated CTLs. Indeed, activities of granzymes A and B were significantly decreased a,b. ImpoSince cystatin F is a potent inhibitor of cathepsin L, which is involved in the maturation of cathepsin C , we nextGiven the effect of cystatin F on TALL-104 cytotoxicity, we assessed whether the presence of cystatin F in the target cells themselves inhibits the cytotoxic effect of lymphocytes, thus conferring protection to target cells. Target K-562 cells that do not express endogenous cystatin F were incubated with recombinant cystatin F. Western blot analysis revealed that full-length as well as N-terminally truncated cystatin F were both internalised into target K-562 cells within 4 h . When cyCystatin F is a protein inhibitor of cysteine cathepsins that is found intracellularly in the lysosomal/endosomal pathway as well as extracellularly ,13. The Extracellular cystatin F was demonstrated to be internalised by several cell types, including human HeLa and HEK-293 cell lines ,14 and mTo confirm the effect of internalised cystatin F on target cathepsins, we analysed cathepsin C, H, and L activity in TALL-104 cells and found that they were significantly reduced. We also compared the effect between full-length and N-terminally truncated cystatin F on their activities. N-terminally truncated cystatin F is a monomeric form and can immediately interact with cathepsins after internalisation. Conversely, full-length cystatin F forms inactive dimers that must first be processed at the N-terminus and monomerise to become active inhibitors . Both foFinally, we investigated whether cystatin F in target cells could confer protection against CTL killing. Indeed, cystatin F-pretreated target K-562 cells were killed less efficiently by TALL-104 cells. In the timeframe of the cytotoxicity assay, cystatin F-loaded target K-562 cells released a large portion of internalised cystatin F. Therefore, we propose that target cells may protect themselves from CTL killing by secreting cystatin F, which is subsequently internalised into cytotoxic cells, in which it inhibits the activation of granzymes that are crucial for efficient target cell killing. A protective role of cystatin F in target cells might be especially important in tumour microenvironments in which cystatin F could be exploited by tumour cells to evade the anti-tumour immune response. Indeed, up-regulated cystatin F expression in tumour tissue was demonstrated in patients with colorectal cancer, and was even correlated with liver metastasis and worse prognoses . A similFor western blotting and immunofluorescence microscopy, the rabbit anti-cystatin F antibody from Sigma-Aldrich  and Davids Biotechnologie GmbH  was used, respectively. Additionally, the following antibodies were used: rabbit anti-β-actin (Sigma-Aldrich), rabbit and mouse anti-glyceraldehyde 3-phosphate dehydrogenase , rabbit anti-his-tag , rabbit anti-N-terminal part of cystatin F , mouse anti-cathepsin C, mouse anti-granzyme A, and mouse anti-perforin . The mouse anti-cathepsin H antibody 1D10  and sheeFull-length and N-terminally truncated human recombinant cystatin F were produced in the human freestyle HEK293F cell line and purified as described in .8/mL for 1 h and then re-suspended in RPMI (Lonza) with 10% foetal bovine serum (Gibco) and 100 IU/mL interleukin-2 (Bachem) at a cell density of 2 × 106/mL. After 5 days SEA/SEB-specific CTLs were isolated by positive selection according to manufacturer’s instructions . The purity of CTLs was determined by flow cytometry using fluorescence labelled antibodies against CD3, CD4, CD8, CD56, CD16, CD19 and TCRα/β  and was >95%.TALL-104 cells  were cultured in Iscove’s Modified Dulbecco’s Medium  with 20% foetal bovine serum , 100 IU/mL interleukin-2 , 2.5 µg/mL recombinant human albumin (Sigma-Aldrich), and 0.5 µg/mL D-mannitol (Sigma-Aldrich). HeLa cells  were cultured in Dulbecco’s Modified Eagle Medium  with 10% foetal bovine serum (Gibco), 100 U/mL penicillin , and 100 U/mL streptomycin (Lonza). K-562 cells  and Raji cells  were cultured in RPMI-1640 (Lonza) with 10% foetal bovine serum, 100 U/mL penicillin (Lonza), and 100 U/mL streptomycin (Lonza). Primary human CTLs were isolated from peripheral blood mononuclear cells (PBMC) obtained from healthy volunteers at the Blood Transfusion Centre of Slovenia, Republic of Slovenia, according to institutional guidelines. PBMCs were stimulated with 5 µg/mL staphylococcal enterotoxin A  and 5 µg/mL staphylococcal enterotoxin B  at a cell density of 10Cells, untreated or incubated for 4 h with 100 nM full-length cystatin F, were washed once in phosphate-buffered saline (PBS), left to adhere to slides for 30 min at 37 °C, and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature (RT). After rinsing three times with PBS for 5 min each, specimens were blocked for 1 h in 5% goat serum and 0.3% Triton X-100 in PBS. Primary antibodies were diluted in 1% bovine serum albumin and 0.3% Triton X-100 in PBS to a final concentration of 10 μg/mL for anti-cystatin F, 2 μg/mL for anti-his-tag and 4 μg/mL for anti-perforin and anti-granzyme A antibodies and incubated overnight at 4 °C. Next, slides were rinsed three times in PBS for 5 min each and incubated with goat anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Fluor 555, diluted 1:1000 in the same buffer as primary antibodies, for 2 h at RT. After the final washing step, the slides were cover-slipped using Prolong Gold Antifade Mountant . Samples without one or both primary antibodies were used as controls. Images were acquired with a Carl Zeiss LSM 710 confocal microscope  with ZEN 2011 imaging software (Carl Zeiss).g for 20 min at 4 °C. Supernatants were transferred to fresh tubes, and protein concentrations were determined using the DC-Protein Assay Kit (Bio-Rad). The lysis buffer for western blot analysis comprised 50 mM Tris-HCl pH 8, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 1 mM EDTA with added protease inhibitors (Roche). The lysis buffer for cathepsin activities comprised 0.1 M citrate buffer pH 6.2 with 1% Triton X-100. The lysis buffer for granzyme activities comprised 25 mM HEPES, 250 mM NaCl, 2.5 mM EDTA, and 0.1% Nonidet p-40, pH 7.4.Cells were either untreated or incubated with 100 nM full-length or N-terminally truncated cystatin F for 4 h and 24 h or with 20 μM E64d for 17 h. Cells were then washed in PBS, lysed in lysis buffer, incubated for 30 min on ice, and centrifuged at 16,000× Samples containing 30–50 µg of cell lysate total protein were resolved in SDS-PAGE using 12% polyacrylamide gel and transferred onto nitrocellulose membranes using the trans-blot turbo transfer system (Bio-Rad) and trans-blot turbo RTA transfer kits with nitrocellulose membrane (Bio-Rad). For some experiments, samples were resolved using 12% TGX stain-free polyacrylamide gels (Bio-Rad); these were activated before the transfer with UV light for 1 min using the ChemiDoc MP System (Bio-Rad). After the transfer, the membranes were imaged for stain-free labelling of total proteins with the ChemiDoc MP System. Next, membranes were blocked in 5% non-fat dry milk in PBS (for cystatin F), 5% non-fat dry milk in tris-buffered saline with 0.1% Tween-20 , and 1.5% non-fat dry milk/0.5% bovine serum albumin in tris-buffered saline with 0.1% Tween-20 (for perforin). The primary antibodies were diluted in blocking solution at 1:500 for cystatin F and N-terminal part of cystatin F, 1:200 for cathepsin C and perforin, and 1:600 for his-tag and cathepsins H and L and incubated overnight at 4 °C. After washing, the membranes were incubated with HRP- or fluorescently conjugated secondary antibodies diluted 1:5000 in blocking solution. Membranes with HRP-conjugated antibodies were incubated with Lumi-Light western blotting substrate (Roche). Images were acquired using a ChemiDoc MP System (Bio-Rad), and quantification analysis was performed in Image Lab, version 5.1 software (Bio-Rad).6) were either left untreated or were incubated for 4 h with 100 nM recombinant full-length cystatin F with his-tag. Afterwards, the cells were washed with PBS and lysed in lysis buffer  with protease inhibitors. After incubation on ice for 30 min, the cell lysates were centrifuged for 20 min at 16,000× g, and the supernatant was transferred to a new tube. The anti-his-tag antibody was added to the cell lysate at a final concentration of 2.7 µg/mL, and the samples were incubated at 4 °C overnight with constant shaking. Dynabeads protein A (Thermo Fisher Scientific) were washed once with lysis buffer, added to the cell lysate, and incubated at RT for 20 min with constant shaking. The beads were then washed three times in lysis buffer, resuspended in SDS loading buffer containing 40 mM dithiothreitol, and boiled for 5 min. The co-immunprecipitated proteins were analysed by western blot.TALL-104 cells (50 × 10Enzyme activities were determined using the following substrates: 70 µM H-Gly-Phe-7-amino-4-methylcoumarin (AMC) (Bachem) for cathepsin C, 20 µM H-Arg-AMC (Bachem) for cathepsin H, 50 µM Z-Phe-Arg-AMC for cathepsin L (Bachem), 50 µM acetyl-Ile-Glu-Pro-Asp-AMC for granzyme B (Bachem), and 200 µM Z-Gly-Pro-Arg-AMC for granzyme A (Bachem). The following assay buffers were used: 25 mM MES, 100 mM NaCl, 5 mM cysteine, pH 6 (for cathepsin C); 100 mM MES, 2 mM EDTA, 5 mM cysteine, pH 6.5 (for cathepsins H and L); 50 mM Tris-HCl, 100 mM NaCl, pH 7.4 (for granzyme B); and 20 mM Tris, 150 mM NaCl, pH 8.1 (for granzyme A). Whole-cell lysates were first activated in assay buffer for 15 min at RT for cathepsins or for 30 min at 37 °C for granzymes. The substrate was then added, and formation of fluorescent products was measured continuously on a microplate reader Infinite M1000 . To determine cathepsin L activity, 5 µM of irreversible inhibitor of cathepsin B, CA-074 (Bachem), was added before the addition of substrate.HeLa cells were grown in 6-well plates and transfected with the pcDNA3 plasmid containing wild-type cystatin F , using P6 cells) were determined using the inverse of the number of effector cells needed to lyse 30% of target cells × 100 [To determine the effect of cystatin F on cytotoxicity of effector cells (TALL-104 cells and primary human SEA/SEB-stimulated CTLs), effector cells were first incubated for 4 h with 100 nM full-length or N-terminally truncated cystatin F. K-562 were used as target cells for TALL-104 cells, while SEA/SEB-pulsed Raji cells were used as targets for SEA/SEB-stimulated CTLs. Raji cells were pulsed with 1 µg/mL SEA and 1 µg/mL SEB for 1 h at 37 °C. Alternatively, to test the effect of cystatin F present in target cells on cytotoxicity, target K-562 cells were incubated for 4 h with 100 nM full-length or N-terminally truncated cystatin F. Next, cystatin F was thoroughly washed away, and the calcein-AM release assay  was perfls × 100 .t-test when two groups were compared or with one-way ANOVA followed by Šidák’s multiple comparisons test when more than two groups were compared. Differences were considered significant when p ≤ 0.05.GraphPad Prism 6  was used for data analysis. Statistical significance was assessed with a In this study, we have demonstrated that extracellular cystatin F upon internalisation decreases the cytotoxicity of cytotoxic T cells by attenuating the activities of cathepsins C and H and consequently the activities of granzymes A and B. Furthermore, we showed that cystatin F inactivates cathepsin C by direct inhibition and not by affecting processing from its inactive precursor. Similarly, cystatin F does not affect perforin processing by cathepsin L. Importantly, cystatin F secreted from target cells can also decrease T cell cytotoxicity, indicating that cystatin F can provide a protective mechanism for target cells with which they can avoid cytotoxic attack.