To develop adenoviral cell- or tissue-specific gene delivery, understanding of the infection mechanisms of adenoviruses is crucial. Several adenoviral attachment proteins such as CD46, CAR and sialic acid have been identified and studied. However, most receptor studies were performed on non-human cells. Combining our reporter gene-tagged adenovirus library with an in vitro human gene knockout model, we performed a systematic analysis of receptor usage comparing different adenoviruses side-by-side. The CRISPR/Cas9 system was used to knockout CD46 and CAR in the human lung epithelial carcinoma cell line A549. Knockout cells were infected with 22 luciferase-expressing adenoviruses derived from adenovirus species B, C, D and E. HAdV-B16, -B21 and -B50 from species B1 as well as HAdV-B34 and -B35 were found to be CD46-dependent. HAdV-C5 and HAdV-E4 from species E were found to be CAR-dependent. Regarding cell entry of HAdV-B3 and -B14 and all species D viruses, both CAR and CD46 play a role, and here, other receptors or attachment structures may also be important since transductions were reduced but not completely inhibited. The established human knockout cell model enables the identification of the most applicable adenovirus types for gene therapy and to further understand adenovirus infection biology. More than 100 types of human adenoviruses (HAdV) have been described and classified into species A–G . Other aMost receptor studies are based on non-human CHO cells with limited transferability to the human organism. Establishing a stable human receptor knockout cell line would overcome such limitations and afford the possibility to broadly analyze receptor usage on human cells. Applying the CRISPR/CAS technology , we impl®GeM Classic . All cell lines tested negative for mycoplasma using the mycoplasma detection Kit Venor®GeM Classic .Prior to the experiments, A549 cells were tested for genetic characteristics using PCR single-locus technology for authentication. Twenty-one independent PCR systems: Amelogenin, D3S1358, D1S1656, D6S1043, D13S317, Penta E, D16S539, D18S51, D2S1338, CSF1PO, Penta D, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433 and FGA, were investigated . In parallel, positive and negative controls were tested, yielding correct results. Cells were certified as the A549 human lung carcinoma cell line detected in the Leibniz Institute DSMZ, German Collection of Microorganisms and Cell Cultures online database . The A549 cell line is parental to all three established knockout cell lines: CAR-KO-, CD46-KO- and CC-KO-A549. All four cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) , supplemented with 10% fetal bovine serum  and 100 µg/mL penicillin/streptomycin . Mycoplasma contamination testing was carried out by PCR using the mycoplasma detection Kit VenorWildtype viruses HAdV-D70, HAdV-D73 and HAdV-D74 were kindly supplied by Albert Heim  ,43. HAdVFor transfection of A549 cells, we customized the plasmid pShV-CBh-Cas9 as described in . For the® LTX and PLUS™ Reagent (Invitrogen ™). Cells of 80% confluency were transfected with 2.5 µg of plasmid-DNA, diluted in 500 µL of Opti-MEM I (Gibco™), 2.5 µL of PLUS™ Reagent and 10 µL of Lipofectamin in a 6-well format. After 24 h of incubation at 37 °C and 5% CO2, the transfection mix was exchanged with growth medium. Cells were then either cultured for further use or DNA was isolated 72 h after transfection.A549 cells were plasmid-transfected using Lipofectamin6 cells diluted in 1 mL of PBS were incubated with 2 µg of the primary antibody for 10 min at 4 °C. For sorting CD46 knockout cells, we used monoclonal mouse anti-human CD46 antibody, clone MEM-258 . For sorting CAR knockout cells, the monoclonal CAR antibody CAR (E1-1) sc-56892  was used. Cells were washed twice with PBS supplemented with 0.1% BSA and 2 mM of EDTA and resuspended in 1 mL of PBS. One hundred µL of Dynabeads® Pan Mouse IgG  was added and incubated for 30 min at 4 °C by continuous tilting and rotating. The tubes were then placed on a magnet for 2 min. Cells in the supernatant were transferred to a fresh tube for further culturing, while the cell pellet collected on the magnet was discharged. Thereby, negative cells not expressing the respective surface structure (CD46 or CAR) were separated from positive cells. Magnetic cell sorting was repeated successively several times. For CAR knockout cells, magnetic cell sorting was inefficient, and thus sorting by flow cytometry (FACS) was performed. Here, 2 × 106 cells per sample were washed twice with PBS and diluted in PBS, followed by incubation with the primary antibody CAR (E1-1) sc-56892 (Santa Cruz Biotechnology Inc. USA) for 30 min at 4 °C. After washing twice with PBS, cells were incubated with the fluorophore-tagged secondary antibody goat anti-mouse IgG, F(ab′)2-APC: sc-3818 (Santa Cruz Biotechnology Inc. USA). Cells were washed again and sorted using the FACSCalibur . Sorted cells were cultured for four passages, followed by flow cytometry analysis for CAR or CD46 expression. Populations exhibiting 70% of receptor-negative cells were singularized. Therefore, a serial dilution of cells was seeded in 96-well plates and single-cell clones were picked.On day 10 post-transfection, cells were sorted by magnetic cell sorting (MACS). Therefore, 2 × 105 cells were washed twice with 1% FBS in PBS, followed by incubation with the respective antibodies. After incubation with the APC-linked secondary antibody 2-APC: sc-3818, Santa Cruz Biotechnology Inc., USA), samples were washed two times. Flow cytometric analysis was performed using the Gallios  and Software Version: Gallios 1.2 (Beckmann Coulter®). The median of fluorescence intensity (MFI) was evaluated for triplicates. Viral transduction was measured as the MFI.Flow cytometry analysis was used to assess the receptor expression of knockout cells and normal A549 cells, to measure the sorting efficiency in the process of knockout cell production. To assess receptor expression and to identify knockout clones, cells were stained with the following monoclonal antibodies directed to the human surface molecules: CD46 antibody, clone MEM-258 (GeneTex Inc. USA), and from Santa Cruz Biotechnology Inc., USA: CAR (E1-1) sc-56892, CD80 B7-1 (F-7): sc-376012, CD86 B7-2 (D-6): sc-28347 and dsg2 (AH12.2): sc-80663. Then, 1 × 10Genomic DNA from A549, A549-KO-CD46, A549-KO-CAR and A549-KO-CD46/CAR cells was isolated using the QIAamp DNA Blood Mini Kit  according to the manufacturer’s instructions.® Mutation Detection Kit  according to the manufacturer’s instructions. The resulting digests were analyzed using the CRISPR Discovery Gel Kit  in combination with a 5200 Fragment Analyzer System (Agilent) according to the manufacturer’s instructions.To analyze the genomic region of CD46, a 609 bp region spanning the CRISPR/Cas target site was amplified from isolated genomic DNA using the primer pair T7EI CD46 fw (AAT GAC AGC CAG CCA AGT TTT C) and T7EI CD46 rev (TTC GTG TCA TTC ATC TGG CA). The same approach was used for the CAR region. Here, an 829 bp region spanning the CRISPR/Cas target site was amplified using the primer pair T7EI CAR fw (CTT AAA GGA GGG CGC CAA CG) and T7EI CAR rev (AGC AAA GAA AGG CAA GCC CG). All oligonucleotides were synthesized by Integrated DNA Technologies . The resulting PCR products were then subjected to T7EI mutation detection analysis using the EnGen3 cells/well were seeded in 96-well plates. After 4 h of incubation, the growth medium was discharged from adherent cells and replaced with luciferase-tagged virus diluted in fresh medium. We used 2, 8 and 20 physical viral particles per cell. Twenty-six hours post-infection, reconstituted Nano-Glo® Luciferase Assay Reagent  was added to the cells. After 5 min of incubation at room temperature, the luminescence of the cell lysate was measured in a TECAN® Platereader. For the measurement of viral transduction by flow cytometry, 2.5 × 104 cells were seeded in 24-well plates. After 4 h of incubation, the medium was replaced with eGFP-tagged virus diluted in growth medium. Since the expression of eGFP is lower than luciferase expression efficiency in our viruses, here, we used 1 × 103 physical viral particles per cell. Forty-eight hours post-infection, cells were washed with PBS and detached with trypsin-EDTA. We resuspended cells in Dulbecco’s minimal essential medium (DMEM) containing 10% FBS, centrifuged, washed with PBS and fixated cells with 4% formaldehyde. Fluorescence was measured by analyzing 5000 single cells using a Gallios (Beckmann Coulter ®) flow cytometer and Beckmann software. Samples were excited with 488 nm and signals were detected using the FITC channel (525/40 nm). The background signal was obtained by analyzing uninfected cells (negative controls). The percentage of GFP-expressing cells was determined by selecting the region of fluorescence above the background level of auto-fluorescence from uninfected cells.For the luciferase assay, 30 × 10A549 cells are described as adenocarcinomic human alveolar basal epithelial cells, which were established from cancerous lung tissue . They arWe applied the CRSIPR/Cas9 system to generate stable human knockout cell lines for CD46 and CAR. Both CD46 and CAR exhibit varying degrees of alternative mRNA splicing, creating multiple protein isoforms [We co-transfected A549 cells with gRNA-equipped pShV-CBh-Cas9 and a plasmid encoding for eGFP expression to monitor transfection efficiency. To separate successful knockouts from unmodified cells, we performed magnetic cell sorting (MACS) using a primary antibody specific for human CD46. After the expansion of sorted cells and consecutive magnetic sorting, a subsequent FACS analysis revealed a substantial reduction in CD46-positive cells A. SubseqFor the generation of a CAR knockout cell line, we used the same approach as described for CD46. In addition, after repeated MACS, we sorted cells by FACS, yielding a population with 70% CAR-negative cells C. After To investigate adenoviral receptors, we strived to generate a human cell line lacking both important surface proteins CD46 and CAR. Therefore, the successfully cloned CD46 knockout cell line was transfected with the respective CRISPR/Cas9 plasmid-expressing gRNA for the CAR target site. Further steps were similar to the generation of the single CAR knockout cell line described above. Sixteen clones were analyzed for CAR expression . Clone 1To confirm genome editing at the CD46 and CAR target loci introduced by the CRISPR/Cas9 machinery, a T7 endonuclease I (T7EI) assay was performed. We observed a slight shift of the PCR products compared to the PCR products of the parental cell line in capillary gel electrophoresis. The results of the T7EI assay for the CD46 gene region of the single CD46 and CD46/CAR double knockout cell lines are depicted in To investigate the receptor usage of adenoviruses from different species, we screened viruses from our virus library engineered and equipped with a luciferase reporter gene . TransduBased on the human adenocarcinoma alveolar epithelial cell line A549, three stable knockout cell lines were established carrying knockouts for CAR, CD46 or a double knockout for CAR and CD46. Transduction experiments with the well-studied adenoviruses HAd-C5 and HAdV-B35 confirmed existing data indicating that these viruses use CAR and CD46, respectively, for cell entry ,25,30. WThe A549 cell line serves as a common model in adenoviral research, representing a standard reference cell line. Originating from the human alveolar epithelium, it resembles a natural host with reliable infection rates by adenoviruses. However, as an immortalized cancer cell line, genetic alterations may result in modified protein expression compared to healthy alveolar epithelial cells. As a lung cancer-derived cell line, it is likely to express CAR on a higher level compared to healthy alveolar cells since lung cancers are shown to have elevated CAR expression levels . This huA recent study described a DSG-2 and CD46 single and double knockout cell line based on A549 cells, which was used to investigate the receptor usage of human adenovirus type 55 . This stIn this study, we screened through our engineered virus library for CAR and CD46 receptor usage by adenoviruses from species B to E. Thereby, we identified viruses that depend on one of the two receptors for cell entry. CD46-dependent viruses were HAdV-B16, -B21 and -B50 from species B1, and HAdV-B34 and -B35 from species B2. CAR-dependent viruses were HAdV-5 from species C and HAdV-4 derived from species E. The other screened viruses, HAdV-B3 and -B14 from species B1 and B2 and all species D viruses, appear to use both CAR and CD46 for cell entry, but other receptors or attachment structures may also play a role since transductions were only reduced but not completely inhibited. For adenoviruses from species D, sialic acid (SA) has been described as a receptor. A previous review described receptor usage roughly by pooling viruses from one species . This scWhether CAR plays the same role for infection of these viruses in vivo is questionable since the accessibility of CAR as a tight junction protein  in tissuFor the recently described adenoviruses HAdV-D70, HAdV-D73 and HAdV-D74, genetic analysis of fiber genes has shown a relation to other species D adenoviruses in BLAST analysis . HoweverWith the generated set of human knockout cell lines, we established a reliable tool to study adenoviral receptors in vitro. The knowledge of receptor usage is important for the development of new adenoviral vectors for gene therapy and is essential for the development of anti-infective drugs. Adenoviruses are a recognized threat to immunocompromised patients ,9,10. Ho


Cancer vaccination is a potential anti-cancer therapy which may improve clinical outcomes, particularly in combination with current immunotherapies, but to date has demonstrated only limited success. Here we set out to improve the effectiveness of peptide-based cancer vaccine design by implementing a unique approach to target peptide antigens to the cross-presentation pathway within dendritic cells. We demonstrate that the addition of a short cell penetrating peptide sequence to the vaccine construct enhances the efficiency of our vaccine and improves outcomes in both a viral and a cancer model. Our findings provide a simple strategy that may enhance the effectiveness of peptide-based cancer vaccines and the priming of anti-tumor immunity.+ cross-presenting DC, and a cell penetrating peptide (CPP) with endosomal escape properties, to enhance antigen delivery into the cross-presentation pathway. Using a prime-boost regimen, we demonstrated robust expansion of antigen-specific T cells following vaccination with our CPP-linked peptide vaccine and protective immunity against HSV-1 skin infection, where vaccine epitopes were natively expressed by the virus. Additionally, our novel vaccination strategy slowed tumor outgrowth in a B16 murine melanoma model, compared to adjuvant only controls, suggesting antigen-specific anti-tumor immunity was generated following vaccination. These findings suggest that novel strategies to target the antigen cross-presentation pathway in DC may be beneficial for the generation of anti-tumor immunity.Cross-presenting dendritic cells (DC) offer an attractive target for vaccination due to their unique ability to process exogenous antigens for presentation on MHC class I molecules. Recent reports have established that these DC express unique surface receptors and play a critical role in the initiation of anti-tumor immunity, opening the way for the development of vaccination strategies specifically targeting these cells. This study investigated whether targeting cross-presenting DC by two complementary mechanisms could improve vaccine effectiveness, in both a viral setting and in a murine melanoma model. Our novel vaccine construct contained the XCL1 ligand, to target uptake to XCR1 The success of checkpoint blockade immunotherapy (CPB) ,2,3, in The simplest form of cancer vaccines are multivalent peptide constructs containing a pool of known disease-specific antigens, to activate the immune system. These constructs can be packaged in nanoparticles to enhance their longevity and uptake by antigen-presenting cells  and deli+ T cells against viral- or tumor-specific antigens [DC are professional antigen-presenting cells and the key sentinels of the immune system responsible for the priming of cytotoxic CD8antigens . This imantigens ,28. The antigens ,30,31, santigens  has alloantigens ,34,35. Tantigens  may drivCross-presentation occurs when exogenous or extracellular antigens, taken up by specific DC and retained within cytosolic endosomes, are shuttled back into the cytoplasm via transporters such as Sec61 , for deg+ T cells, with increased efficiency compared to controls. In a Herpes Simplex Virus 1 (HSV) infection model this CD8+ response inhibited viral replication at the site of infection. Furthermore, when targeting B16 melanoma, engineered to express the vaccine antigens, this immune response delayed tumor outgrowth. Importantly, our data show that the simple addition of an endosomal escaping CPP to a peptide-based vaccine construct can enhance vaccine efficiency leading to the induction of robust anti-viral and anti-tumor immunity. This information may be of particular interest in the current development of preclinical peptide-based vaccines and the design of future cancer trials in combination with CPB immunotherapy.In this study we apply two non-overlapping techniques to target a short peptide-based vaccine construct, containing tumor-associated antigens, specifically to the cross-presentation pathway in specific DC subsets. First, we engineered a XCR1-targeting moiety using a sequence from the murine X-C Motif Chemokine Ligand 1 (XCL1) protein. Secondly, we employed our expertise in CPP development to design a CPP containing peptide vaccine, to drive endocytosed peptide antigens out of endosomes and into the cytoplasm to access the cross-presentation pathway more efficiently. Our data demonstrate that our novel vaccination strategy induces strong proliferation of antigen specific CD8290–302, a GTG spacer sequence, and the HSV glycoprotein B (gB) epitope gB498–505.The peptide vaccine was designed as a multicomponent construct comprising a recombinantly expressed protein unit, and a synthetic peptide unit. The protein unit was made up of the targeting moiety, murine XCL1 , and the protein ligation moiety, SpyCatcher  . These uThe linking sequence of the peptide subunit required redesign after results described in Peptides were synthesized by Fmoc chemistry, at Pepscan  as two separate components before being linked together via disulfide coupling. All peptides were purified by HPLC, and purity confirmed >95% by LC-MS.The first peptide component, required for linking to the targeting and ligation unit, was synthesized as a single fragment, and comprised the first two subunits, namely the SpyTag peptide ligation tag and the linker sequence. The sequences comprised SpyTag (GAHIVMVDAYKPTK) and either a short spacer with a disulfide bond, or an early endosomal Furin cleavage site RKKR, and cathepsin-B endosomal cleavage site Valine-Citrulline . All first component peptides contained a C-terminal Cysteine for coupling, with N-termini acetylated and C-termini amidated for protection. The initial first component peptides used to generate data for The second component, containing the cell-penetrating peptide and peptide antigen subunits, was synthesized as a single fragment. The cell penetrating peptide subunit sequences comprised of either 1746 (PLKPKKPKTQEKKKKQPPK-PKKPKTQEKKKKQPPKPKR), TAT (GRKKRRQRRRPQ) or no CPP sequence. The antigen subunit sequence comprised a short linker (GAS), the HSV gD epitope (IPPNWHIPSIQDA), a short linker (GTG), and the HSV gB epitope (SSIEFARL). Second component peptides were synthesized either with an N-terminal mercaptopropionic acid (mcpa) to allow for ligation to first component peptides by sulfur chemistry, or acetylated for protection for non-targeted applications. All C-termini were amidated for protection. First and second peptides were ligated together by sulfur chemistry to form a disulfide bond between the C-terminal Cysteine of component 1, and the sulfhydryl of the C-terminal mercaptopropionic acid of component 2. Conjugated peptide sequences used in manuscript are presented below: Synthetic peptide unit sequences for data in SpyT-1746-gDgB:Ac-GKPIPNPLLGLDSTGASAHIVMVDAYKPTKG-C(nh2)-mcpa- PLKPKKPKTQEKKKKQPPKPKKPKTQEKKKKQPPKPKRGASIPPNWHIPSIQDAGTGSSIEFARL-nh2; SpyT-TAT-gDgB:Ac-GKPIPNPLLGLDSTGASAHIVMVDAYKPTKG-C(nh2)-mcpa-GRKKRRQRRRPQGASIPPNWHIPSIQDAGTGSSIEFARL-nh2; SpyT-gDgB:Ac-GKPIPNPLLGLDSTGASAHIVMVDAYKPTKG-C(nh2)-mcpa-GASIPPNWHIPSIQDAGTGSSIEFARL-nh2Synthetic peptide unit sequences for data in SpyT-FCB-1746-gDgB:Ac-GAHIVMVDAYKPTKGSGTGGSTGRKKRSV(Citrulline)GC(nh2)-mcpa-PLKPKKPKTQEKKKKQPPKPKKPKTQEKKKKQPPKPKRGASIPPNWHIPSIQDAGTGSSIEFARL-nh2SpyT-FCB-TAT-gDgB:Ac-GAHIVMVDAYKPTKGSGTGGSTGRKKRSV(Citrulline)GC(nh2)-mcpa-GRKRRQRRRPQGASIPPNWHIPSIQDAGTGSSIEFARL-nh2SpyT-FCB-gDgB:Ac-GAHIVMVDAYKPTKGSGTGGSTGRKKRSV(Citrulline)GC(nh2)-mcpa-IPPNWHIPSIQDAGTGSSIEFARL-nh2Synthetic peptide unit sequences for data in 1746-gDgB:Ac-PLKPKKPKTQEKKKKQPPKPKKPKTQEKKKKQPPKPKRGASIPPNWHIPSIQDAGTGSSIEFARL-nh2.TAT-gDgB:Ac-GRKRRQRRRPQGASIPPNWHIPSIQDAGTGSSIEFARL-nh2.gDgB:Ac-IPPNWHIPSIQDAGTGSSIEFARL-nh2E. coli expression strain (BL21(DE3)) and expressed in 2YT media for 16 h at 30 ·C. Expression cultures were harvested by centrifugation at 6000 g for 30 min at 4 °C, and cell pellets stored at −80 °C until purification. The protein was isolated by resuspending the cell pellets in lysis buffer followed by sonication and purification by immobilized metal affinity chromatography, buffer exchange, ion exchange chromatography and size exclusion chromatography. Final protein yield from 10 L preparation was 11.9 mg at 99% purity .The recombinant protein targeting component of the peptide vaccine (His-muXCL1-SpyC) was expressed as a fusion protein comprising an N-terminal hexa-His sequence (HHHHHH), a HRV-3C cleavage site (LEVLFQGP), murine XCL1 , a 10-amino acid linker sequence (GGSGTGATSG), followed by the SpyCatcher ligation scaffold  and a C-His-muXCL1-SpyC Protein sequence :MGHHHHHHGATLEVLFQGPGGSVGSEVSDKRTCVSLTTQRLPVSRIKTYTITEGSLRAVIFITKRGLKVCADPQATWVRDVVRSMDRKSNTRNNMIQTKPTGTQQSTNTAVTLTGGSGTGATSGDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGGAGSWSHPQFEKGThe peptide fragments containing SpyTag sequences were solubilized in sterile PBS (pH 7.4) under sterile conditions in a biohazard hood. The solubilized peptides were then incubated with the SpyCatcher-XCL1 protein in sterile PBS (pH 7.4) overnight at 4 °C. Conjugation efficiencies were determined by SDS-PAGE gel, and were 70–90%. Ligation mixtures were used directly for animal studies.C57BL/6 female mice that express the CD45.2 allele were purchased from the Animal Resources Centre . gB-specific T-cell receptor (TCR) transgenic (gBT.I) mice and gD-specific TCR transgenic mice  that exp290–302 and the HSV glycoprotein B epitope gB498–505 (B16.gD.gB). Briefly, retroviruses were generated by transfecting the 293T cell line with pMIG-gD.gB, pMD.old.gag.pol, and pCAG-VSVG. B16 cells were transduced with 1 mL filtered retroviral supernatant in the presence of 8 μg/mL polybrene . B16.gD.gB cells were passaged routinely at 70–80% confluency and cultured in RPMI media  supplemented with 10% FCS (Sigma-Aldrich), 2 mM L-glutamine, 50 µM 2-mercaptoethanol, 100 µg/mL streptomycin and 100 U/mL penicillin  at 37 °C, 5% CO2.B16.F10 (B16) murine melanoma cells were purchased from the ATCC and engineered to express the HSV glycoprotein D epitope gD5 B16.gD.gB tumor cells, washed and injected in 100 uL sterile PBS. Tumor volume (mm3) was calculated using electronic calipers.Mice were vaccinated subcutaneously on the right hind flank with either 2.0 nmol, 0.20 nmol or 0.02 nmol of vaccine constructs, in sterile PBS: 1746-gDgB, TAT-gDgB, or gDgB alone, plus 50 ug adjuvant poly I:C . Control mice received poly I:C alone or PBS. Fourteen days later mice received a boost vaccination consisting of the same. Seven days later mice received subcutaneous inoculation of 5 × 105 Vα2+ CD8+ gBT.I T cells and 1 × 104 Vα3.2+ CD4+ gDT.II T cells were washed and resuspended in 200 µL RPMI for i.v. injection into recipient mice at least one day prior to whole-cell vaccination. Expansion of T cells was quantified in the spleen of vaccinated mice 7 days after last challenge as the % of Vα3.2+ cells of total CD45.1+CD3+CD4+ or % Vα2+ cells of total CD45.1+CD3+CD8+ cells from the live cell gate.Single cell suspensions of transgenic T cells were prepared from pooled lymph nodes from naïve female gBT.I or gDT.II mice. After negative selection with monoclonal antibodies and magnetic beads, purity of T cells were determined by flow cytometry. 1 × 10Spleen and/or lymph nodes were harvested and passed through a 70 µm metal mesh prior to red blood cell lysis. Resulting single cell suspensions were stained with monoclonal antibodies specific for mouse CD8α (53-6.7), CD45.1 (A20), Vα2 (B20.1), Vα3.2 (RR3-16) and CD4 (GK1.5) . Multi-parameter analysis was performed on a LSRFortessa (BD) and data were analyzed with FlowJo (Tree Star). Prior to acquisition cells were stained with propidium iodide  to exclude dead cells.6 PFU, was placed onto the abraded skin and covered with OpSite Flexigrid (Smith & Nephew). The flank of the mouse was wrapped with Micropore tape and then Transpore tape  to prevent removal of OpSite Flexigrid and subsequent disruption of the viral infection and wrapping was removed 48 h later.Mice were anesthetized by i.p. injection of a 1:1 Ketamine (Parnell Laboratories) and Ilium Xylazil-20 (Troy Laboratories) solution in saline and HSV infections were carried out as described previously . Briefly2 piece of skin encompassing the infection site was removed from euthanized mice and snap frozen. The presence of infectious virus in tissue samples was determined using standard PFU assays on confluent Vero cell monolayers. Thawed skin samples were diced in DMEM media (Life Technologies) and homogenized with a TissueRuptor (Qiagen) before collecting the viral lysate. A 10-fold serial dilution series of samples were tested for plaque formation to determine viral titer in the original tissue sample.A 1-cmp < 0.05, ** p < 0.01, *** p < 0.001.All statistical analyses were performed using GraphPad . Data are reported as a mean +/− standard error of mean for experiments comparing responses in individual animals. Differences in T cell proliferation were compared between groups using a Student’s t-test. Differences in survival were compared using the Log-Rank (Mantel–Cox) test. Statistical significance was indicated as * + DC and enhancing endosomal escape of the antigen payload we designed a novel vaccine construct consisting of the murine XCL1 protein linked by SpyCatcher/SpyTag conjugation and disulphide chemistry to a CPP sequence known as 1746 [498–505 (gB) and gD290–302 (gD) [+ (gBT.I) and CD4+ (gDT.II) [To test the approach of targeting both the vaccine construct to XCR1 as 1746  and the 302 (gD)    T cells + T cell expansion by greater than 2-fold over TAT-gDgB, which showed no improvement over the no-CPP vaccine. Importantly, these data suggest that addition of the FCB cleavage motif allows for proper decoupling of vaccine within endosomes, whereafter the antigenic component is carried into the cytoplasm through the endosomal escape properties of the 1746 CPP, linked to the peptide antigens, facilitating enhanced cross-presentation.We hypothesized that limited reduction of the disulphide bond within the vaccine construct was potentially occurring, leading to improper release of the antigenic subunit from the SpyTag and thus limiting endosomal escape and entry into the cross-presentation pathway. Disulphide bond reduction is feasible within the endosomal uptake pathway ,50, desp+ cross-presenting DC alone was likely to be insufficient to drive robust cross-presentation, if the antigens were restricted within endosomes. Therefore, we next tested with non-targeted CPP-vaccine constructs, as this reduced the need to uncouple vaccine components within intracellular compartments. Again we used our prime-boost vaccination strategy with 1746-gBgD, TAT-gDgB or gDgB peptide vaccines and measured the expansion of gDT.II and gBT.I T cells at 7 days post boost vaccination. Here we saw that gDT.II expansion was similar between 1746-gBgD and TAT-gDgB, with both showing improvement over the no-CPP vaccine  one week prior to performing a skin scarification and HSV infection. At a timepoint where viral recrudescence is visible as a result of viral replication in sensory neurons (D6 post infection), we harvested skin to perform viral plaque forming assays to titrate viral load at the site of infection (Having determined the effectiveness of our CPP coupled vaccine to drive vaccine-specific T cell expansion, we now wanted to see whether the CD8nfection a. We founfection b, and thnfection c. All peGiven the promising results for our endosomal escaping CPP-vaccine construct in our T cell expansion assays and HSV infection model we wanted to determine whether this strategy could generate potent anti-tumor immunity. We used a B16 melanoma model, engineered to express the vaccine epitopes gD and gB and applied the same prime boost regimen used previously in our expansion assays. The simple gDgB peptide vaccine increased overall survival by approximately 2 weeks, a non-significant increase compared to the adjuvant alone. Vaccinating with 1746-gDgB extended survival by a further two weeks a, achiev+ cDC1 and enhancing antigen entry into the cross-presentation pathway through the use of an endosomal escape CPP. We find that the latter approach is key for enhanced antigen specific CD8+ T cell expansion following vaccination and that correct endosomal cleavage and subsequent escape is critical for cross-priming to occur.Here we have reported a novel strategy to increase the efficiency of peptide-based cancer vaccines, using a dual approach of targeting the vaccine construct to XCR1+ T cell expansion, suggesting that targeting, in this setting, is not an absolute requirement. This may be explained by the fact that the skin is a relatively DC-rich organ that undergoes constant immune surveillance [DC-targeting as a route to enhance the potency of cancer vaccines has been explored previously, with targeting of antigen to DC-specific markers such as XCR1 ,34, Cleceillance  and, as + T cell expansion (Rather, the key to success in our targeted vaccine design appears to be engineering of the construct to allow decoupling of the targeting and CPP-antigen subunits. Our initial hypothesis that a disulfide linked peptide vaccine construct would be reduced in the endosome to facilitate vaccine decoupling was shown to be flawed . While ixpansion . TherefoThe use of CPP as a strategy to enhance vaccine-delivered antigen uptake and cross-presentation in DC is an area of growing research. Originating with sequences identified from nature such as the HIV-derived TAT peptide  and PeneClinical trials using novel CPP containing vaccines such as SO-11 KISIMA-01, a Phase Ib trial in stage IV colorectal cancer  utilisin+ T cell expansion. A key requirement of this approach was the correct decoupling of targeting moieties from the CPP-antigen subunit within the endosome, allowing for endosomal escape and entry into the cytosolic cross-presentation pathway. Our CPP-vaccine construct primed immunity that reduced viral load after HSV skin infection and displayed an anti-tumor effect in a preclinical melanoma model, engineered to express the vaccine antigens. These results provide preclinical proof of concept for the use of endosomal escaping CPP for the development of effective cancer vaccines.Here we demonstrated that the addition of an endosomal escape CPP to a simple peptide vaccine construct increases the efficiency of vaccination, driving strong antigen specific CD8Data in this manuscript have been published as part of provisional patent application WO/2019/134018: Vaccine conjugates and uses thereof. Jason Waithman, Shane Stone, Paul Watt.


The nuclear envelope (NE) safeguards the genome and is pivotal for regulating genome activity as the structural scaffold of higher-order chromatin organization. NE had been thought as the stable during the interphase of cell cycle. However, recent studies have revealed that the NE can be damaged by various stresses such as mechanical stress and cellular senescence. These types of stresses are called NE stress. It has been proposed that NE stress is closely related to cellular dysfunctions such as genome instability and cell death. Here, we found that an endoplasmic reticulum (ER)-resident transmembrane transcription factor, OASIS, accumulates at damaged NE. Notably, the major components of nuclear lamina, Lamin proteins were depleted at the NE where OASIS accumulates. We previously demonstrated that OASIS is cleaved at the membrane domain in response to ER stress. In contrast, OASIS accumulates as the full-length form to damaged NE in response to NE stress. The accumulation to damaged NE is specific for OASIS among OASIS family members. Intriguingly, OASIS colocalizes with the components of linker of nucleoskeleton and cytoskeleton complexes, SUN2 and Nesprin-2 at the damaged NE. OASIS partially colocalizes with BAF, LEM domain proteins, and a component of ESCRT III, which are involved in the repair of ruptured NE. Furthermore, OASIS suppresses DNA damage induced by NE stress and restores nuclear deformation under NE stress conditions. Our findings reveal a novel NE stress response pathway mediated by OASIS. It had been thought that the nuclear envelope (NE) is stable during interphase . HoweverRecent studies have established that NE rupture is repaired by a series of factors that mediate the reassembly of NE components , 12. A cOASIS (CREB3L1) is an ER-resident transmembrane transcription factor belonging to the CREB/ATF family , 18. We In this study, we showed that OASIS specifically accumulates at damaged NE and can act in response to NE stress. The discovery of this novel pathway that responds to NE stress is important not only for understanding cellular response to damaged NE, but also for finding clues to develop therapeutic strategies for nuclear envelopathies.OASIS is mainly localized to the ER under normal conditions. Interestingly, OASIS is also enriched in close proximity to the nucleus . To elucPrevious studies demonstrated that reduced Lamin B1 level results in disorganization of the nuclear lamina and it can be visualized as a discontinuous localization of Lamin A/C or the formation of nuclear blebs accompanied by chromatin herniation , 8. We gWe also analyzed the localization of OASIS on the nuclear blebs using the expression vector for OASIS tagged with Flag at its N-terminus (Flag-OASIS). Flag-OASIS also accumulated at the periphery of the nuclear blebs where Lamin A/C were absent, just like mVenus-OASIS Fig. . We obseNext, we investigated whether the OASIS accumulated at damaged NE is the full-length or cleaved form because OASIS is cleaved at the transmembrane domain in response to ER stress Fig.  19–21].–21.19–21All the OASIS family members share similar domain structures and activation processes . Thus, wOur previous work demonstrated that OASIS is degraded by ubiquitin-proteasome pathway under normal condition and is stabilized upon ER stress . Thus, wTo test whether OASIS also accumulates at damaged NE induced by different types of NE stress, we applied additional models of NE stress. Transwell migration assay can recapitulate the cell migration under constricted environments such as packed tissues or capillaries. As cells migrate through the porous membrane filter device with a small pore size such as Transwell, nuclear blebs are formed at the leading edge of the nucleus on the bottom side Fig.  9, 10]., 10.9, 1A recent study established that compressive force immediately induces NE rupture, thus can be used to monitor acute NE stress response Fig.  13]. We. We13]. It has been shown that the clinically used HIV protease inhibitor Nelfinavir impairs the maturation of Lamin A and induces the loss of NE integrity . To testTo identify the factors that colocalize with OASIS at damaged NE, we investigated the localization of a wide variety of proteins in shLMNB1 cells expressing Flag-OASIS. First, we investigated the localization of the nuclear pore complexes (NPCs) at the nuclear blebs where Flag-OASIS accumulates. NPCs were almost undetectable at the periphery of the nuclear blebs where Flag-OASIS accumulated. Fig. . The linA subgroup of INM proteins named the LEM domain proteins share a conserved motif that is exposed to the nucleoplasm . LEM domOasis knockout (KO) mice [Oasis KO mice revealed lower circularity and higher aspect ratio compared with those of WT at the bottom side of Transwell, respectively  mice . The nucely Fig. . The rolte Fig. S. It has te Fig. S; 3 μm. Tte Fig. S; 8 μm. TIn this study, we showed that an ER-resident transmembrane transcription factor, OASIS, accumulates on damaged regions of NE using several models of NE stress. Notably, Lamin proteins are depleted from the regions where OASIS accumulates, indicating their mutually exclusive localization pattern at the damaged NE Figs. –4. DisruOasis knockout primary cultured astrocytes revealed increased DNA damage only when they migrate through small size of pore  OASIS specifically accumulates on the damaged NE where Lamin proteins are depleted in response to various types of NE stress; (2) OASIS colocalizes with the components of the LINC complex and the factors involved in NE repair; and (3) OASIS suppresses DNA damage induced by NE rupture and maintains the nuclear morphology under NE stress. These findings suggest that OASIS is a key molecule that responds to NE stress and suppresses nuclear deformation as well as DNA damage caused by cellular migration under constricted environments.Oasis−/− (KO) mice (C57BL/6) were previously established in our laboratory [Oasis KO mice, littermates derived from the mating of Oasis+/− mice were used. The experimental procedures and housing conditions for animals were approved by the Committee of Animal Experimentation, Hiroshima University.The boratory . For com2, 95% humidified air atmosphere. U251MG cells were maintained under the same conditions except that Eagle’s Minimum Essential Medium (Gibco) was used as the basal medium. The shRNA expression vector for LMNB1  and empty vector pLKO.1 were used for the production of lentiviruses. HeLa cells were infected with the produced lentiviruses and stable cell lines were established by puromycin selection at 3 μg/ml. The expression vector of Flag-tagged full-length (U251-OASIS-FL) and N-terminus (U251-OASIS-N) of OASIS and empty vector (U251-Con) were linearized, transfected into U251MG cells, and stable cell lines were established by puromycin selection at 2 μg/ml. Primary astrocytes were derived from WT and Oasis KO mice and maintained as described previously [HeLa cells and U2OS cells were maintained in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% (v/v) heat-inactivated fetal bovine serum and penicillin/streptomycin at 37 °C in a 5% COeviously . HeLa cehttp://n2t.net/addgene:54640) [NheI-BsrGI sites (pmVenus-C1). The expression vector of mVenus fused to human OASIS at its N-terminus (mVenus-OASIS) was constructed by PCR amplification of the cDNA of OASIS using the following primer sets and was cloned into pmVenus-C1 at XhoI-KpnI sites. 5′-CTGAACTCGAGGTATGGACGCCGTCTTGGAAC-3′ (OASIS-forward), 5′-TCGTAGGTACCCTAGGAGAGTTTGATGGTGG-3′ (OASIS-reverse). The expression vectors of GFP-MAN1 and GFP-CHMP7 were constructed by PCR using the following primer sets and were cloned into pEGFP-C1 at BglII-SalI sites. 5′-GAAGATCTGCGGCGGCAGCAGCTTCGGC-3′ (MAN1-forward), 5′-AACGCGTCGACTCAGGAACTTCCTTGAGAATTGGTTAGG-3′ (MAN1-reverse), and 5′-GAAGATCTTGGTCCCCGGAGCGGGAGGCCGAG-3′ (CHMP7-forward), 5′-AACGCGTCGACCTACAATGGCTTTAGAGTCGGTTCC-3′ (CHMP7-reverse). The mScarlet-I (mSCI) expression vector (pmSCI-C1) was constructed by AgeI-BsrGI digestion using the plasmid containing cDNA of mSCI, a gift from Dorus Gadella  [XhoI-EcoRI site [BsrGI site. 5′-GAGCTGTACAAGTCTAGTCCTGAGCTTCCCACCGAAGATG-3′ (mini-Nesp2-N-forward), 5′-GCCTGTACAAATGAAATTCTAGAAGTAGAATAAATTTTTTCTCCAAAATG-3′ (mini-Neps2-N-reverse). Expression vectors for other epitope-tagged OASIS, Luman, and AIbZIP had been constructed as previously described [http://n2t.net/addgene:101772) and LAP2β-AcGFP  were gifts from Daniel Gerlich and Eric Schirmer, respectively [A rapidly maturating mutant of mVenus, mVenusQ69M , was gene:54640) , and thee:98831) . The expoRI site . 5′-ATCCescribed , 39. Expescribed . The expectively . Flag-OLg for 5 min, the protein concentrations of the supernatants were determined using a bicinchoninic acid assay (BCA) kit . Equal amounts of proteins (10 μg) were used for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). For immunoblotting, the following antibodies were used: anti‐β‐actin , anti‐Lamin B1 , and anti-OASIS monoclonal antibody produced with a hybridoma as described previously (1:1000) [Proteins were extracted from the indicated cells in a cell lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% SDS, and Protease inhibitor cocktail Set V . The lysates were briefly sonicated. After centrifugation at 16,000x(1:1000) .The cells were grown on human fibronectin-coated coverslips or glass-bottom dishes  and fixed in 4% paraformaldehyde for 10 min. After the fixation, the cells were permeabilized in 0.5% Triton‐X 100 for 3 min, followed by blocking with 5% normal goat serum for 60 min. The following antibodies were used as the primary antibodies: anti-KDEL , anti-Lamin A/C , anti‐HRD1 , anti‐Flag M2 , anti-DYKDDDDK , anti‐HA , anti-Myc , anti‐Lamin B1 , mAB414 , anti‐GFP , anti-Nesprin-2 , anti-LAP2β antibodies , and anti-γH2AX antibodies . Incubation with primary antibodies was performed at 4° overnight. For secondary antibodies, goat anti-mouse or -rabbit IgG F(ab′)2 fragments conjugated with Alexa 488 or 568 were used (Thermo Scientific). Confocal fluorescent images were acquired by FV1000D  or LSM780 . Image processing and image analysis were performed by ImageJ . For quantification of signal intensity of Flag-OASIS, nuclear masks were generated with the signal of Lamin A/C staining. The ROIs for damaged NE of nuclear blebs and intact NE of main nuclei, of which width was set at 0.39 μm, were generated by using the functions of Dilate, Erode, and Image Calculator. The ROIs for 0.39 μm outside of these regions were generated using the functions of Dilate and Image Calculator. For analysis of γH2AX positive cells, the cells that have at least one prominent γH2AX focus were judged as positive. The cells in which γH2AX signal is spread to entire nucleus were omitted from quantification.Constricted migration assay was performed using Transwell with a 3 μm pore size , as described previously . HeLa ceϕ27 mm glass-bottomed dish (Matsunami Glass) and cultured overnight again. Then the medium was supplemented with 3 μm polystyrene beads and an 18 × 18 mm cover glass attached with a silicon mold was placed on the glass bottom dish. The silicon mold was pressed by a 10 cm tissue culture dish and subjected to immunofluorescence staining as described above.Cellular compression assay was performed as described previously with minor modifications . HeLa ceU251-Con cells, U251-OASIS-FL cells, and primary astrocytes were plated in 2-well silicone chamber  and cultured until they become confluent. Then silicon chambers were removed and the relative positions of leading edges was measured to calculate migration rates.t‐test was used. To compare the rates of the indicated events between two samples, Fisher’s exact test was used. To compare numbers of discontinuous region of Lamin A/C, nuclear circularity, and nuclear aspect ratio, Mann–Whitney’s U-test was used. Microsoft Excel and R (version 4.0.2) were used via RStudio (version 1.3.1073) for statistical analysis [To compare the expression level of Lamin B1 between control cells and shLMNB1, the expression level of Flag-OASIS between DMSO and MG-132 treated cells in shLMNB1 cells, the migration rates between WT and Oasis KO astrocytes, and the migration rates between U251-Con cells and U251-OASIS-FL cells, two tailed Student’s analysis , 43.Figure S1 Localization mVenus-OASIS in U2OS cells.Figure S2 Localization of OL-Chimera in shLMNB1 cells.Figure S3 OASIS does not affect cellular migration.


The development of persistent endoplasmic reticulum (ER) stress is one of the cornerstones of prostate carcinogenesis; however, the mechanism is missing. Also, alcohol is a physiological ER stress inducer, and the link between alcoholism and progression of prostate cancer (PCa) is well documented but not well characterized. According to the canonical model, the mediator of ER stress, ATF6, is cleaved sequentially in the Golgi by S1P and S2P proteases; thereafter, the genes responsible for unfolded protein response (UPR) undergo transactivation.Cell lines used were non-malignant prostate epithelial RWPE-1 cells, androgen-responsive LNCaP, and 22RV1 cells, as well as androgen-refractory PC-3 cells. We also utilized PCa tissue sections from patients with different Gleason scores and alcohol consumption backgrounds. Several sophisticated approaches were employed, including Structured illumination superresolution microscopy, Proximity ligation assay, Atomic force microscopy, and Nuclear magnetic resonance spectroscopy.trans-Golgi matrix dimeric protein GCC185 as a Golgi retention partner for both S1P and S2P, and in cells lacking GCC185, these enzymes lose intra-Golgi situation. Progression of prostate cancer (PCa) is associated with overproduction of S1P and S2P but monomerization of GCC185 and its downregulation. Utilizing different ER stress models, including ethanol administration, we found that PCa cells employ an elegant mechanism that auto-activates ER stress by fragmentation of Golgi, translocation of S1P and S2P from Golgi to ER, followed by intra-ER cleavage of ATF6, accelerated UPR, and cell proliferation. The segregation of S1P and S2P from Golgi and activation of ATF6 are positively correlated with androgen receptor signaling, different disease stages, and alcohol consumption. Finally, depletion of ATF6 significantly retarded the growth of xenograft prostate tumors and blocks production of pro-metastatic metabolites.Herein, we identified the We found that progression of PCa associates with translocation of S1P and S2P proteases to the ER and subsequent ATF6 cleavage. This obviates the need for ATF6 transport to the Golgi and enhances UPR and cell proliferation. Thus, we provide the novel mechanistic model of ATF6 activation and ER stress implication in the progression of PCa, suggesting ATF6 is a novel promising target for prostate cancer therapy.The online version contains supplementary material available at 10.1186/s13046-021-02066-7. The link of intracellular stresses to tumorigenesis and tumor growth promotion has been the subject of a decades-long debate. The environment of the endoplasmic reticulum (ER) undergoes significant modifications in response to neoplastic transformations, including oxidative stress, DNA damage, aerobic glycolysis, and calcium deprivation –3. TheseActivating Transcription Factor 6 (ATF6) governs one important UPR branch. ATF6 is a type II transmembrane protein of the ER. In response to ER stress, 90 kDa ATF6 (p90) dissociates from GRP78/BiP and translocates to the Golgi to be cleaved in its luminal domain by site-1 protease (S1P). Afterward, the N-terminus of ATF6 is cleaved by site-2 protease (S2P), releasing the 50 kDa cytosolic domain (p50). The p50 ATF6 moves to the nucleus, where it initiates transcription of the genes involved in the resistance of ER stress and UPR . SeveralRecent investigations indicate that Golgi disorganization is a hallmark of cancer progression –18. AddiUsing different models of ER stress in various PCa cell lines and analyzing hundreds of prostate tumor samples, we discovered that Golgi proteases S1P and S2P can stay and function in the ER, thereby facilitating the cleavage of ATF6 and amplifying UPR. Additionally, we demonstrated that this mechanism is particularly important in the context of how alcohol affects the progression of PCa.6 cells in 50 μl of plain RPMI medium plus 50 μl Matrigel (Corning). In another series of xenograft models, mice were injected with LNCaP cells transfected with control or ATF6 shRNA. Tumor diameter was measured weekly throughout the study. This protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at UNMC.Male athymic nude mice  were individually housed in filter-top cages at the University of Nebraska Medical Center (UNMC) animal facility and consumed food and fresh tap water ad libitum. Food consumption and body weights were recorded weekly. The animals received alcohol orally in drinking water. The amount of alcohol was increased gradually from 4 to 14% within one week. Control mice received water with the appropriate isocaloric amount of sucrose. On the first day of 14% EtOH administration, both groups were inoculated with LNCaP (c-28) cells by subcutaneous injection in the flank 5 × 10Tissue sections from the normal prostate were obtained from US Biomax and Novus Biological. Also, sections were provided through the Department of Pathology and Microbiology (IRB protocol # 304–16-EP) at the University of Nebraska Medical Center, the Johns Hopkins University School of Medicine (Prostate Cancer Biorepository Network), and the Vernadsky Crimean Federal University (Russia). Patient questionnaires asked for a) preferred alcoholic beverage(s); b) an average number of drinks consumed in a week within the last five years; c) frequency of heavy episodic drinking and d) duration of heavy drinking occasions. We monitored the PLA signal in the pairs of proteins, S1P/GCC185 and S2P/GCC185, in normal prostate and PCa cells from patients with the same grade and Gleason score who are non-drinking patients (who do not drink or drink less than once per month) versus patients who regularly consume alcohol at a moderate level  or at a heavy level . Lysates isolated from normal prostate cells and tumors of PCa patients were obtained from Protein Biotechnologies (USA).These include: Antibodies and reagents; Cell culture and EtOH treatment; Soft agar assay for colony formation; Cell migration assay; Immunohistochemistry; Confocal immunofluorescence microscopy; Three-dimensional structured illumination (3D-SIM) microscopy and image analysis; AFM imaging and image analysis; In situ Proximity Ligation Assay (PLA); NMR data collection and analysis; Isolation of Golgi membrane fractions by sucrose gradient centrifugation; Isolation of microsomal fraction; Immunoprecipitation (IP), plasmid constructions, and transfection; Quantitative gene expression analysis by qRT-PCR; Quantification and statistical analysis.P < 0.001, P < 0.01, pairwise Wilcoxon with Bonferonni-Hochberg multiple test). Overall, the data presented in Fig. To characterize the level of ATF6-mediated ER stress, we measured the expression of cleaved ATF6 in non-malignant prostate epithelial RWPE-1 cells, androgen-responsive LNCaP cells c-24 (hereafter LNCaP cells), and androgen-refractory PC-3 cells. As shown in Fig. trans-Golgi fraction; in contrast, only marginal amounts of these proteases were detected in the cis-medial-Golgi membranes  and then S2P and GCC185 (~ 0.72)   and GCC185 (trans-Golgi). In LNCaP cells, the Pearson coefficient in pairs S1P/GCC185 and S2P/GCC185 was significantly higher than that in S1P/giantin and S2P/giantin, respectively   . In the  t test) . To vali t test) . To evalNext, using LNCaP cells, we validated the complexes S1P↔GCC185 and S2P↔GCC185 by detecting both S1P and S2P in the GCC185 IP sample  of GCC185 in LNCaP cells    (We further verified the segregation of S1P and S2P from Golgi by structured illumination superresolution microscopy (SIM), which allows to create 3D reconstructed images with a lateral resolution of ~ 110 nm, approximately twice that of diffraction-limited instruments. First, in LNCaP cells, we measured colocalization of S1P and S2P with the ER marker calreticulin before and after GCC185 KD. Contrary to the control, cells with depleted GCC185 demonstrated a high level of colocalization between both proteases and calreticulin ( t test) . Finally t test) . In sum,3 and 547 ± 15 nm3, corresponding to the monomeric and dimeric GCC185, respectively , which was correlated with Gleason scores, according to a significant difference found between Gleason score groups 2–5 , 6–7, and 8–10   Fig. A-D. Howest) Fig. E. Then, st) Fig. .Fig. 4CP < 0.001, Kruskal-Wallis omnibus test) . Consistent with the data presented in Fig. A quantitative in situ proximity ligation assay (PLA) was carried out in PCa samples to corroborate these data by an alternative sensitive technique. PLA can visualize protein-protein interaction by the red fluorescence emanating from the proximity (below 40 nm) of these two proteins. Here, we measured the PLA-specific red fluorescent spots to detect the proximity of S1P and S2P to GCC185. In addition, we sought to assess whether the level of segregation of these proteases from the Golgi correlates with the aggressive potential of PCa. The technical details of the experiments and our modification for the image processing are described in us test) . It was us test)  , the Spearman correlation coefficient rs was calculated to determine the ATF6-AR relationship. In all groups, we detected a strong positive correlation between these two parameters; namely, rs was 0.71 for normal tissue, 0.48 for NAT, 0.64 for BPH, 0.59 for BPH LG PIN, 0.63 for BPH HG PIN, and 0.59 for PCa samples (P < 0.001 for every group)  and 22Rv1 cells .Androgens and androgen receptor (AR) signaling pathways are commonly considered the main oncogenic drivers of prostate carcinogenesis . Moreovey group) . SimilarThese data imply that a lack of ATF6 could have a direct impact on AR signaling and subsequent tumor proliferation. We performed siRNA-mediated KD of ATF6 in LNCaP cells, and as shown in Fig. P < 0.01, t test). Next, in LNCaP (C-25-27) cells, we performed the stable KD of ATF6α using shRNA; the tumorigenicity of these cells was assessed following subcutaneous injection into the flank of nude mice .To assess the proliferation rate and the ability of cells to metastasize, we monitored the anchorage-independent growth by soft agar analysis, as previously described . In ATF6LNCaP C-2-27 cellsLNCaP C-2-27 cellsLNCaP C-2-27 cellsP < 0.001, t test). In EtOH-treated cells, we detected a decline in the expression of GCC185 and the development of ER stress, which was indicated by the elevated expression of GRP78  to evaluate the level of intranuclear ATF6. We found that in the nuclei of xenograft tumor cells from alcohol-fed mice, the intensity of the ATF6’s IF was elevated compared to the control group .These data indicate the contribution of ATF6-mediated ER stress in EtOH-induced disorganization and activation of the GSK3β → HDAC6-P → HSP90 → AR axis. Then, we estimated the proliferation rate by anchorage-independent analysis. As shown in Fig. P < 0.01, pairwise Wilcoxon with Bonferonni-Hochberg multiple test). The difference in the PLA signal between these two groups of alcohol-consuming patients was also significant . Also, it is known that PCa patients with cribriform growth at Gleason grade 4 have a worse prognosis than those with ‘poorly formed glands’ [P < 0.05, Mann-Whitney test). Next, we tested whether a difference between these cribriform patterns can also be detected in the ATF6 signaling. We measured the level of intranuclear ATF6 IF signal in cribriform patterns and found a significant difference between the groups of non-alcoholic and heavy drinkers; however, non-alcoholic patients did not differ from the patients who moderately consumed alcohol .Previously, we found that tumor tissue from PCa patients with the same stage of disease displayed different levels of Golgi fragmentation, which was positively correlated with the rate of alcohol consumption  , 36. Thi glands’ . Here, w1H-13C HSQC), we detected a total of 106 spectral features across all cell media samples (five repeats for control and EtOH-treated LNCaP cells). Iterative class PCA models were calculated to identify potential outliers, with all control and EtOH-treated samples projected within the respective 95% confidence interval (CI). Supervised OPLS-DA analysis could differentiate control samples from EtOH-treated through one predictive component and one orthogonal component with R2Y = 0.846, Q2Y = 0.562  and P = 0.026 (1000 permutations)  . The varSearches across available databases/repositories – Human Metabolome Database (HMDB), Chenomx, Platform for RIKEN Metabolomics (PRIMe) – identified discriminatory NMR features associated with 20 unique metabolites. The intake of phenylalanine, N-Acetyl-beta-D-glucosamine, 2-oxoglutarate/α-ketoglutarate, glutamine, glutamate, acetate, N-acetylglutamine, lysine, and valine were observably increased in EtOH-treated samples. Conversely, the consumption of serine, glyceraldehyde/glycerose, cystine, tyrosine, ornithine, asparagine, 2-oxobutyrate/α-ketobutyrate, citrate, alanine, ethyl-oxaloacetate, and isoleucine were observably increased in control samples .In ATF6 KD cells, the consumption of glycine was reduced Table S2). This would be expected given the lower proliferation rate of these cells Fig. D-G, and . This woA list of predicted intermediates – modules, enzymes, and reactions – from FELLA PT analysis were mapped to known genes (64 proteins / 102 genes) and used to perform Gene Ontology (GO) analysis  of EtOH-Our study uncovered a previously unexplored mechanism of how prostate cancer cells sustain ER stress injury in the face of Golgi disorganization. We found that S1P and S2P proteases relocate to the ER, thus obviating the need for ATF6 transport to the Golgi. This facilitates the cleavage of ATF6 and subsequent UPR, thereby accelerating cell proliferation Fig. . Indeed,trans-Golgi dimeric protein GCC185 as the Golgi retention partner for S1P and S2P. While we cannot exclude the contribution of other players, for instance, Rab and ARF proteins, to the situation of these proteases in the Golgi, our data unequivocally pointed to the direct interaction of both S1P and S2P with GCC185. Contrary to S1P and S2P, the expression of this golgin was reduced in PC-3 cells and the PCa tissue with a high Gleason score, implying the potential use of S1P, S2P, and GCC185 as the prognostic markers for this disease. Moreover, AFM analysis showed that in PC-3 cells, GCC185 persists in the Golgi in its monomeric form. On the one hand, this is an indication of events associated with ER stress, when stalled ER-to-Golgi trafficking may block posttranslational modification of Golgi matrix proteins, including their dimerization [Our findings corroborate other researchers’ publications, suggesting that S1P and S2P may act in the ER after ER and Golgi stresses induced by Brefeldin A , 23, albrization , 25, 45.rization . Howeverrization . It thisThis study provides compelling evidence that alcohol consumption can contribute to the development of sublethal ER stress in PCa cells. The effect of EtOH-induced Golgi disorganization echoes the results observed in ER stressed cells: Golgi disorganization, downregulation of GCC185, increase of GRP78 and cleaved ATF6, and translocation of S1P and S2P to the ER. The size of the xenograft tumor in EtOH-fed mice is larger than in the control group, as well as the expression of intranuclear ATF6. Our metabolomics data indicate that EtOH specifically activates several pro-oncogenic pathways, which are blocked upon the depletion of ATF6. Thus, the present study sheds new light on the crosstalk between alcohol-induced ER stress and cancer cells’ ability to survive and grow.Finally, for the first time, we observed that synthesis of AR and its transactivation significantly relies on the ATF6-mediated UPR: in PCa tissue samples, which exhibit fragmented Golgi phenotype, we found a strong positive correlation between the expression of ATF6 and intranuclear AR. By shifting S1P and S2P to ATF6 and simplifying the ER stress pathway, cancer cells can rapidly provide an appropriate level of UPR to compensate for the need for AR protein itself and subsequent AR-triggered proliferation. In light of our recent publication indicating a link between Golgi fragmentation and the transactivation of AR , this seWe found that advanced PCa cells utilize an alternative mechanism of ATF6-mediated UPR, thus simplifying stress response and promoting the maturation of proteins responsible for the growth of the prostate tumor, including those involved in AR signaling. Activation of ATF6 correlates with the severity of this pathology and level of alcohol consumption. Depletion of ATF6 significantly blocks the growth of a prostate tumor, suggesting its potential implication in PCa therapy.Additional file 1: Fig. S1. . IF staining of RWPE-1 cells to detect colocalization of (A) S1P (green) and (B) S2P (green) with different trans-Golgi markers: GCC185 (red), GCC88 (red), TMF (red), and Golgin-245 (red). All images were acquired with the same imaging parameters, nucleus – blue, DAPI; bars, 10 μm. White boxes indicate the cell enlarged and shown at the right. (C) Quantification of the Pearson coefficient of colocalization for the cells presented in A and B . (D) GCC185, GCC88, TMF, and Golgin-245 W-B of the protein complexes from S1P and S2P IP samples prepared from RWPE-1 cells. Fig. S2. (A) Morphological staining of the Golgi by giantin in control and Tg-treated RWPE-1 and LNCaP cells, and non-treated PC-3 cells; bars, 10 μM. (B) Quantification of percent of cells with disorganized Golgi from A . (C) GRP78 W-B of RWPE-1, LNCaP (non-treated and Tg-treated), and PC-3 cell lysates; β-actin as a loading control.  S1P and S2P W-B of the ER (D) and Golgi (E) fractions isolated from LNCaP cells: control and Tg-treated. HSP70 and GM130 were used as a loading control for the ER and Golgi, respectively. Fig. S3. (A) S1P and S2P antibody was validated in the lung cancer tissue samples according to manifucture’s (Abcam) recommendation.  Immunohistochemical staining of S1P (B) and S2P (D) on the tissue samples from BPH and PCa patients. At least five representative areas were selected from the tumor area and normal tissue adjacent to tumor (NAT). Red boxes indicate the area enlarged and shown below.  Quantification of the expression of S1P (C) and S2P (E), presented as a ratio of the total intensity to the area (mm2). The details are described in the Methods section. Data are presented as medians (min – max); **P < 0.001, *P < 0.01, Mann-Whitney test. The number of patients counted for S1P: NAT – 10, BPH – 8, and PCa – 6; for S2P: NAT – 8, BPH – 11, and PCa – 7. Fig. S4. (A) Processing of PLA images using ImageJ software. Fig. S5. (A-F) Immunostaining of ATF6 (green) and AR (red) in the tissue: (A) normal prostate (B) Normal prostate tissue Adjacent Tumor (NAT), (C) BPH, (D) BPH with Low Grade Prostatic Intraepithelial Neoplasia (BPH LG PIN), (E) BPH with High Grade Prostatic Intraepithelial Neoplasia (BPH HG PIN), and (F) PCa. All images were acquired with the same imaging parameters, nucleus – blue, DAPI; bars 10 μm. Using ImageJ, each nucleus was outlined using the DAPI channel. Fig. S6.  Immunostaining of ATF6 (green) and AR (red) in LNCaP (A) and 22Rv1 (B) cells. All images were acquired with the same imaging parameters, nucleus – blue, DAPI; bars, 10 μm. Fig. S7. (A) AR W-B of the nuclear fraction from 22Rv1 cells: control, treated with ETOH, and ATF6 siRNA followed by EtOH. (B) HDAC6-P and HSP90-Ac W-B of the lysate of cells from A. Fig. S8. (A) Migration of LNCaP cells: control, treated with 50 mM EtOH for 96 h, and treated with EtOH in the presence of 100 nM ATF6 siRNAs; bars, 200 μm. Cell migration was measured via the Transwell chamber assay with an 8-μm pore size; an equal number of LNCaP cells (5 × 104) in the three groups were seeded into the upper chamber with 200 μl of serum-free medium. (B) Quantification of the migrated cells for the cells presented in A . (C) ATF6 W-B of the lysate from scramble or ATF6 siRNA-transfected LNCaP cells. Fig. S9. Supervised OPLS-DA analysis  between Ctrl and EtOH-treated samples of LNCaP cell media based on 106 metabolic features from 2D 1H-13C HSQC NMR spectra and created with MVAPACK (http://bionmr.unl.edu/mvapack.php) . (A) Scores plot (n = 10). (B) Model statistics – R2Y = 0.846 and Q2Y = 0.562 (7-fold CV). (C) VIP plot – 36 features (VIP > 1.0). (D) Permutation results (n = 1000) – P value = 0.026)). Table S1: List of identified metabolites from 36 discriminatory features (VIP > 1.0) from supervised OPLS-DA analysis between Ctrl and EtOH-treated samples of the LNCaP cells media based on 106 metabolic features from 2D 1H-13C HSQC NMR spectra. Table S2: List of discriminatory metabolites  across conditions  of LNCaP cell media based on 878 metabolic features acquired by 2D 1H-13C HSQC NMR experiments. Table S3: List of predicted intermediates (64 proteins / 102 genes) from KEGG ORA/PT pathway analysis with ‘FELLA’ and discriminatory NMR metabolites between Ctrl and EtOH-treated samples of the LNCaP cells media.


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved to display particular patterns of genetic diversity in the genome across geographical regions. These variations in the virus and genetic variation in human populations can determine virus transmissibility and coronavirus disease 2019 (COVID-19) severity. Genetic variations and immune differences in human populations could be the driving forces in viral evolution. Recently emerged SARS-CoV-2 variants show several mutations at the receptor binding domain in the spike (S) glycoprotein and contribute to immune escape and enhanced binding with angiotensin 1-converting enzyme 2 (ACE2). Since ACE2 and transmembrane protease serine 2 (TMPRSS2) play important roles in SARS-CoV-2 entry into the cell, genetic variation in these host entry-related proteins may be a driving force for positive selection in the SARS-CoV-2 S glycoprotein. Dendritic or liver/lymph cell-specific intercellular adhesion molecule (ICAM)-3-grabbing non-integrin is also known to play vital roles in several pathogens. Genetic variations of these host proteins may affect the susceptibility to SARS-CoV-2. This review summarizes the latest research to describe the impacts of genetic variation in the viral S glycoprotein and critical host proteins and aims to provide better insights for understanding transmission and pathogenesis and more broadly for developing vaccine/antiviral drugs and precision medicine strategies, especially for high risk populations with genetic risk variants. According to a meta-analysis, the fatality rate of COVID-19 is around 3% [The recently identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the pandemic of coronavirus disease 2019 (COVID-19), which began at the end of 2019 and is ongoing [ABO blood group gene, and Ellinghaus and colleagues further confirmed that patients with blood group A showed higher risk than others [ACE2 and TMPRSS2 may provide the driving force for viral evolution, therefore causing positive selection for these emerging mutations in the SARS-CoV-2 S glycoprotein. Additionally, dendritic or liver/lymph cell-specific intercellular adhesion molecule (ICAM)-3-grabbing non-integrin (DC/L-SIGN) has been known to play vital roles for several pathogens, including SARS-CoV [SARS-CoV-2 is the third coronavirus to cause a pandemic, the other two being SARS-CoV  and Middn others . Anothern others . Howevern others ,15. Seven others , South An others , Brazil n others , and Norn others , respectSARS-CoV . TherefoIn this review, we aim to compile knowledge and current advances on the impacts of genetic variations in the viral S glycoprotein and critical host proteins on the susceptibility to SARS-CoV-2 infection and immune escape. This understanding is crucial for controlling the pandemic through enhanced surveillance and vaccine development.SARS-CoV-2 is one of the coronaviruses (CoV) and is an enveloped and positive-sense ssRNA (~30 kb) virus which belongs to the Betacoronavirus genus, Nidovirales order. Two replicase open reading frames (ORFs) encoded by ORF1a (~13.2 kb) and ORF1b (~8.1 kb) occupy at least two-thirds of the CoV genome A. The poC-terminal region of the CoV genome. The SARS-CoV-2 S glycoprotein contains a furin recognition cleavage site  which provides efficient proteolytic processing into S1 and S2 [Four structural protein genes are located in the 1 and S2 . Additio1 and S2 ,25,26. T1 and S2 ,28. With1 and S2 , and the1 and S2 ,31. The 1 and S2 ,33. Seve1 and S2 .The first step of CoV infection of a target cell is the binding of the viral RBD of the S glycoprotein to the cell membrane receptor, ACE2 B. The SAThe continuous transmission of SARS-CoV-2 has caused rapid accumulation of mutations in the S glycoprotein across geographical regions. One of the predominant mutations, D614G, was found to be circulating and rapidly spreading outside China in the early pandemic. The recently emerged variants in the United Kingdom (B.1.1.7), South Africa (B.1.351), North America (B.1.429), and Brazil (B.1.1.28) exhibit several mutations in the S glycoprotein, especially in the RBD. B.1.1.7 carries eight mutations in the S glycoprotein which include H69/V70 deletion (ΔH69/V70), Y144 deletion (ΔY144), N501Y, A570D, P681H, T716A, S982A, and D1118H. B.1.351, recently found in South Africa, carries three mutations located in the RBD region which are K417N, E484K, and N501Y. B.1.1.28 (P.1 and P.2 lineages) in Brazil exhibits a different pattern of mutations. The P.1 lineage has the same RBD mutations as South Africa, whereas the P.2 lineage has independently accrued the spike E484K mutation . Those vThe impact of D614G on viral transmissibility has been widely studied due to its emergence in the early pandemic and its worldwide presence. The D614G mutation is of rising concern, as it has the potential to affect SARS-CoV-2 infectivity through changes to RBD structure, S1/S2 subunit interaction, viral entry, and immune response . BecerraN-terminal domain (NTD) of the S glycoprotein. The ΔH69/V70 deletion has been found globally, however, tracking of SARS-CoV-2 sequences has shown it to be mainly circulating in Europe. A single round infectivity experiment showed that SARS-CoV-2 carrying either ΔH69/V70 or ΔH69/V70 combined with N501Y can enhance infectivity in 293T/hACE2 cells. Additionally, a virus carrying the ΔH69/V70 mutation exhibits more S glycoprotein incorporated in the virion [ΔH69/V70 are located at the e virion . A recene virion . The lose virion ,59,60. Te virion .S477N is located in the RBD and has been found to enhance binding with ACE2 . The E48The ΔY144 and the L242/L244 deletions (ΔL242/244) are located at the NTD of the S glycoprotein and show a loss of binding ability with neutralizing antibodies ,76. StarThere are several mutations which appear in more than 0.1% of sequences circulating across geographical regions, including ΔH69/V70 and Δ144 in S1 NTD; K417N/T/V, N439K, L452R, Y453F, S477N, E484K, and N501Y in RBD; D614G in S1 CTD; P681H and V1176F in S2. ΔH69/V70, Δ144, N439K, Y453F, and N501Y are found circulating mainly in Europe. K417N/T/V is carried by B.1.351 and B.1.1.28 which are mainly circulating in Africa and Europe. L452R is carried by B.1.429 which is mainly circulating in North America and Europe. S477N is mainly circulating in Europe and Oceania. E484K is carried by several current circulating variants and is found in Africa (33.8%) and Europe (30.8%), however, there are also more than 10% distributed in North and South Americas . P681H iSeveral functionally important mutations have been found to be circulating across geographical regions and co-occurring with other variants. E484K is one of the most concerning mutations which exhibits increased dynamics in several severe transmission regions. Recent evidence has shown that SARS-CoV-2 carrying E484K or K417N  in the S glycoprotein could contribute to broad immune escape from monoclonal antibodies and human convalescent sera ,76. The ACE2 gene contains 18 exons located in chromosome X. ACE2 consists of three domains: (1) N-terminal peptidase domain (residues 19-615), (2) C-terminal collectrin-like domain , and (3) end with a hydrophobic transmembrane region and an intracellular segment of 43 residues [ACE2 are significantly associated with blood pressure in different populations [The residues ,85. ACE2residues ,87, reguresidues , and medresidues . ACE2 haresidues . Severalulations ,92. Addiulations ,93.ACE2 between different populations may contribute to susceptibility to SARS-CoV-2. According to a recent report by Darbani and colleagues, 34 ACE2 variants have been defined with importance for SARS-CoV-2 entry and infection [ACE2 allele frequencies included six interaction-booster variants  and eight interaction-inhibitor variants  which have been shown to vary significantly between populations. In addition, Darbani and colleagues showed that more than half of the variants were found in males, which may explain previous clinical observations showing higher mortality rates in males [ACE2 intron variants and ten other protein intron variants  which showed association with higher ACE2 expression levels by genetic analysis of expression quantitative trait loci (eQTLs) [CLTRN and the CA5B genes. Future study is required to clarify the correlation between the ACE2 gene regulatory network and genetic variation.A previous study on a group 2 coronavirus demonstrated that the correlation between viral receptor genetic variation and viral binding activity can affect host susceptibility . Similarnfection . The ACEin males ,100. Howin males . Cao and (eQTLs) . IntriguACE2 genetic analysis by Stawiski and colleagues identified nine ACE2 variants which could increase susceptibility to SARS-CoV-2 and 17 ACE2 variants which displayed protective roles on SARS-CoV-2 infection by structural computational analysis [ACE2 identified by recent studies, including key residues of ACE2 binding with SARS-CoV-2 RBD and residues with potential to affect binding, are summarized in ACE2 missense variants in ACE2 rare missense variants in the population do not disrupt the interaction with the SARS-CoV-2 S glycoprotein. Hashizume and collegues identified seven ACE2 missense variants which exist in Asian but not in American and European populations. They further demonstrated that these ACE2 missense variants have a limited effect on SARS-CoV-2 infectivity in vitro [ACE2 genetic variants with high allele frequencies are associated with the higher expression level of ACE2 in Asian populations. ACE2 expression is found to be significantly lower in North America, Europe, and Africa, in decreasing order [Another systematic analysis . The misin vitro . In adding order . Additiong order . FurtherTMPRSS2 gene contains 14 exons located in chromosome 21. TMPRSS2 is mainly expressed on the luminal side of the prostate epithelium. The expression level is regulated by androgens, and overexpression of TMPRSS2 can be found in prostate cancer tissue [TMPRSS2 could affect its expression level, protein structure, and functions, further affecting individual susceptibility to SARS-CoV-2 infection. A systematic investigation of TMPRSS2 variants identified 13 intron variants, two exon variants (coding regions), and six 3′UTR variants that can affect TMPRSS2 structure and function. rs12329760 and rs75603675 (both missense variants) potentially affect TMPRSS2 structure and post-translational modifications, respectively. Six 3′UTR variants  potentially affect the miRNA target activity [The r tissue . In addir tissue ,108. A cr tissue . TMPRSS2r tissue . The firr tissue . The secactivity . Recentlactivity . TMPRSS2activity .ACE2 and TMPRSS2 are believed to affect individual susceptibility to SARS-CoV-2. However, a large scale clinical epigenetic study is needed to further confirm the effect of genetic variation on the susceptibility to SARS-CoV-2 infection.Taken together, based on current evidence, genetic variations of Mycobacterium tuberculosis, and Dengue infection [DC/L-SIGN genetic variation and susceptibility to SARS-CoV-2 infection.DC-SIGN is a C-type lectin receptor expressed on dendritic cells. A DC-SIGN related receptor called L-SIGN (or CD209L and DC-SIGNR) is expressed on lymph node and liver cells. The function of DC/L-SIGN is to recognize high mannose glycans on the cell and the pathogen surface ,114. Mornfection ,119,120.nfection . NotablyTMPRSS2 and susceptibility to SARS-CoV-2 in human populations. For DC/L-SIGN, future study is required to understand the correlation between genetic variants and the severity of COVID-19, especially focusing on -336G and the homozygous/heterozygous forms of L-SIGN. Continuing and enhancing surveillance, monitoring evolutionary changes of SARS-CoV-2 in different populations, and understanding the impact of mutations on viral transmissibility and immune escape ability are urgently needed to provide guidance on controlling and measuring transmission. Additionally, the acceleration of COVID-19 vaccine roll-out to the public is urgently needed to prevent SARS-CoV-2 evolution against the current vaccines.The pandemic of COVID-19 has caused more than 111 million confirmed cases and more than 2.4 million deaths globally as of 22 February 2021 since the first case was reported from Wuhan, China. The confirmed cases and deaths are rising quickly, and the fast evolution and transmission of SARS-CoV-2 has generated several particular mutations across geographic regions ,123,124.


Photinus pyralis (Ppy); one mutant named Donor is defective in the second half reaction, while the other mutant named Acceptor exhibited low activity in the first half reaction. To date, Ppy has been used in the system; however, its thermostability is low. In this study, to improve the stability of the system, we applied Fluc from thermostabilized Luciola lateralis to FlimPIA. We screened suitable mutants as probes for FlimPIA and obtained Acceptor and Donor candidates. We detected the interaction of FKBP12-FRB with FlimPIA using these candidates. Furthermore, after the incubation of the probes at 37°C for 1 h, the luminescence signal of the new system was 2.4-fold higher than that of the previous system, showing significant improvement in the stability of the assay.The protein–protein interaction assay is a key technology in various fields, being applicable in drug screening as well as in diagnosis and inspection, wherein the stability of assays is important. In a previous study, we developed a unique protein–protein interaction assay “FlimPIA” based on the functional complementation of mutant firefly luciferases (Fluc). The catalytic step of Fluc was divided into two half steps: D-luciferin was adenylated in the first step, while adenylated luciferin was oxidized in the second step. We constructed two mutants of Fluc from  Subsequently, the excited OxL emits light . The Donor maintains the activity in the first half-reaction but is defective in the second half-reaction, while the Acceptor has a low activity in the first half-reaction but mostly retains the activity in the second-half reaction. Therefore, the Donor can produce the reaction intermediate, LH2-AMP, while producing almost no OxL. However, the Acceptor cannot produce LH2-AMP but can retain the catalytic activity for the oxidation of the intermediate. When the Donor and Acceptor are each fused to an interacting protein, the interaction of the proteins induces the approximation of the two mutant enzymes. Based on this principle, previously, we succeeded to detect 1) the interaction between FK506-binding protein 12 (FKBP12) and FKBP-rapamycin-associated protein (FRB), which interact depending on an immunosuppressant rapamycin, and 2) that between p53 and its inhibitor Mdm2, which controls apoptosis, senescence, DNA repair and cell growth. Each PPI results in a higher rate of LH2-AMP catalysis into OxL by the Acceptor, leading to the higher light emission (The protein–protein interaction (PPI) assay is a key technology, not only in basic biology but also for practical purposes in drug screening , diagnosts light   Fluc (LlL) with mutations for thermostability and organic solvent tolerance  was obtained from Biosynth AG . Rapamycin was purchased from LKT Laboratories , and 3-(N-morpholino) propanesulfonic acid (MOPS) was purchased from Dojindo . Oligonucleotides were synthesized by Eurofins . A QuikChange Site-Directed Mutagenesis Kit was obtained from Agilent . An In-Fusion HD Cloning Kit was purchased from Takara-Bio . Restriction enzymes and E. coli SHuffle T7 express lysY were obtained from New England Biolabs . E. coli JM109 competent cells were from SciTrove . Overnight Express Autoinduction System and BugBuster Master Mix were obtained from MERCK . Other reagents of the highest grade available were purchased from Kanto Chemicals , Fujifilm-Wako Pure Chemical Industries , or Nacalai-Tesque , unless otherwise indicated.Firefly luciferin -fused LlL(K443A), LlL(K443A) was amplified by polymerase chain reaction (PCR) using pET26-LlL(K443A) as a template and the primers LlLNotG4S-for and LlLXho-rev. The amplified fragment was digested with eviously . To yielTo acquire the FRB-LlL(S440X/K529Q) library, QuikChange site-directed mutagenesis was performed using pET32-FRB-LlL(K529Q) as a template and LlL447_KpnI and LlLS440X-KpnI-r as primers. Because bulky amino acids such as leucine and phenylalanine at the position of 440 were suitable as the Ppy Acceptor, the primer LlLS440X-KpnI-r was designed to selectively encode these residues at this position. To yield the FRB-LlL(S440M/K529X) library, the full-length pET32-FRB-LlL (S440M/K529Q) was amplified and site-directly mutated via PCR using the primer set of LlL530X-for and LlLS530X-rev. As the primers encoded 15 nt each of homologous sequences at the ends, both ends of the amplified fragment were recombined by In-Fusion cloning. To yield the FKBP-LlL(H245X/K443A) library, the full-length pET32-FKBP-LlL(K443A) was amplified and site-directly mutated via PCR with the primer set of LlLH245X-for and LlLH245X-rev. Both ends of the amplified fragment were recombined using In-Fusion cloning. To obtain the FKBP-LlLH245E/K443A library, pET32-FKBP-LlL(H245E/K443A) was amplified and site-directly mutated via PCR using the primer set of LlLK443X-for and LlLK443X-rev. Both ends of the amplified fragment were recombined using In-Fusion cloning, according to the manufacturer’s protocol. To confirm that each resultant library contained sufficient variation of the mutants, the DNA sequences of several clones were analyzed.To obtain FRB-LlL(S440F/K529Q) and FRB-LlL(S440W/K529Q), pET32-FRB-LlL (S440M/K529M) was amplified and site-directly mutated by PCR using the primers LlLS440FW-KpnI and LlL447-KpnI-r, and both ends of the amplified fragment were fused by In-Fusion cloning. pET32-FKBP-LlL(H245E/K443A/I530R) and pET32-FKBP-LlL(H245E/K443A/I530K) were obtained similarly using pET32-FKBP-LlL(H245E/K443A) as a template and LlLI530X-for and LlLI530X-rev as primers.E. coli SHuffle T7 Express lysY and purified using the TALON metal affinity resin, as described previously , wherein the concentration of MgSO4 and pH were previously optimized for FlimPIA (2 and ATP). Rapamycin was dissolved in dimethyl sulfoxide (DMSO), and the same amount of DMSO (0.1%) was added as a vehicle to the negative sample. In the optimized buffer conditions, 1 mM of coenzyme A was added to reduce the amount of spontaneously produced dehydroluciferyl-AMP (L-AMP), which acts as a competitor of LH2-AMP and inhibits light emission. The addition of coenzyme A results in the conversion of L-AMP to dehydroluciferyl-coenzyme A, which does not act as a competitor . The colonies were selected and cultured using the Overnight Express Autoinduction System in a 96-well culture plate at 30°C overnight. The pellet of each clone was lysed in 50 µl of BugBuster Master Mix, according to the manufacturer’s protocol. The lysates were centrifuged, and the supernatants were collected. To estimate the adenylation activity, each supernatant (10 µl) was added to 90 μl of reaction buffer containing 150 µM of LH2 and 2 mM ATP. To estimate the oxidative luminescent activity, 90 µl of the reaction buffer containing 200 nM LH2-AMP was added to each supernatant (10 µl).2-AMP  To analyze the first half reaction, ATP and LH2 were added to the lysates of E. coli. 3) To assess the second half reaction, LH2-AMP was added to the lysates. 4) The ratios of the luminescence intensity of 2) and that of 3) were calculated. When the ratio was high, the clone was suitable as a Donor, whereas when the ratio was low, the clone was suitable as an Acceptor.Because the mutations of H245D and K529Q were not functional for LlL-based FlimPIA, we decided to perform screening for more suitable mutants by the following procedure. 1) The saturation mutagenesis libraries, K529X, S440X, H245X, and K443X were screened. -based Acceptor, an FRB/S440X/K529Q library was created, and 96 colonies were selected for screening. Among them, five clones, namely, FRB/S440N/K529Q (two clones), FRB/S440K/K529Q (two clones), and FRB/S440M/K529Q, were selected. The proteins of FRB/S440S/K529Q, FRB/S440N/K529Q, FRB/S440K/K529Q, and FRB/S440M/K529Q were expressed in E. coli and purified. Using each protein as the Acceptor and FKBP/Ppy Donor, FlimPIA was performed , FRB/S440M/K529M, and FRB/S440M/K529N. FlimPIA was performed using each FRB/LlL Acceptor candidate and FKBP/Ppy Donor. As a result, all candidates acted as the Acceptors, and the maximum S/B ratio of K529M was remarkably higher than that of the other candidates .E/K443A (three clones), FKBP/H245A/K443A, FKBP/H245S/K443A, and FKBP/H245P/K443A. FlimPIA was performed using each candidate and FRB/LlL(S440M/K529M). Among these candidates, only H245E/K443A showed increased luminescence intensity upon Rap addition , FKBP/H245addition . HoweverR) and FRB/LlL (H245E/K443A/I530K) were expressed in E. coli and purified. Because I530R is effective for the stabilization of ATP in Ppy  library. Among 288 clones, H245E/K443R (three clones), H245E/K443G (two clones), H245E/K443V, H245E/K443P, and H245E/K443A were selected. FlimPIA was performed using each candidate and the FRB/Ppy Acceptor (K529Q/S440F) . HoweverP in Ppy , we hypoP in Ppy .2-AMP for FlimPIA; therefore, the lower production of LH2-AMP after the incubation might not remarkably change the result of FlimPIA.Because of the low intensity and S/B ratio of the FKBP/LlL Donor (H245E/K443A) and FRB/LlL Acceptor (S440M/K529M), the stability of the heterologous pair of the Ppy Donor and LlL Acceptor was compared with that of the LlL Donor and LlL Acceptor pair . FlimPIA2, and 1 mM coenzyme A, the maximum S/B ratio reached more than 6  , which p