Paeonia lactiflora Pall. is an important ornamental plant with high economic and medicinal value, which has considerable development prospects worldwide. The lack of efficient tissue culture techniques and genetic transformation systems has become a master obstacle for P. lactiflora research. The purpose of the present study focuses on obtaining an efficient and stable genetic transformation method using callus as the receptor and exploring an efficient protocol for callus induction and proliferation associated with P. lactiflora. Callus induction and proliferation were performed using MS medium with various concentrations of 2,4-Dichlorophenoxyacetic acid , 1-Naphthaleneacetic acid (NAA), 6-Benzylaminopurine (6-BA) and thidiazuron (TDZ). The sensitivity of callus to kanamycin and cefotaxime was determined. Several parameters such as Agrobacterium cell density, infection time and co-culture duration were studied to optimize transformation efficiency. Agrobacterium strains EHA105 and pBI121 binary vector harboring the β-glucuronidase (GUS) gene were used for transformation. Expression of the GUS reporter gene was detected by GUS assay, polymerase chain reaction (PCR) and Quantitative Real-time PCR (RT-qPCR). The MS medium containing 1.0 mg·L-1 NAA, 0.5 mg·L-1 2,4-D and 0.5 mg·L-1 TDZ was optimal for callus induction and MS medium containing 0.5 mg·L-1 NAA, 1.0 mg·L-1 2,4-D and 0.5 mg·L-1 TDZ was the best for callus proliferation. The concentrations of kanamycin and cefotaxime used for screening positive callus were 125 mg·L-1 and 200 mg·L-1, respectively. Among various combinations analyzed, the best transformation result was obtained via the 25 min of infection of Agrobacterium at 0.6 OD600 and 3 d of co-culture. Overall, this study provided technical support and theoretical guidance for improving the callus induction and proliferation efficiency and the study of gene function in P. lactiflora. Paeonia lactiflora Pall.) is a perennial herbaceous plant of the genus Paeonia  for 45 d to obtain sterile seedlings. All medium and agar used in this article were purchased from Beijing Solarbio Science & Technology Co., LTD. All plant growth regulators (PGRs) were purchased from Shanghai Yien Chemical Technology Co., LTD.Herbaceous peony hybrid seeds (‘Fen Yu Nu’×’Xi Shi Fen’) were harvested in the germplasm resource nursery of Shenyang Agricultural University, Liaoning, China. Full and large seeds were chosen and washed under the running tap water for 10 min. After soaking in distilled water for 48 h, the seed coat was removed. Subsequently, they were transferred to a clean bench for further sterilization. The seeds were dipped in 75% (v/v) ethanol for 30 s, and rinsed 2 times with sterile distilled water. Followed by 15 min sterilization with sodium hypochlorite solution , and the seeds were finally rinsed three times in sterile distilled water. After sterilization, the much of endosperm in each seed was removed to leave isolated embryo with a bit of endosperm, which was cultured on Murashige and Skoog (MS) medium supplemented with 0.5 mg·L−2·s−1 photosynthetic photon flux density of cool white fluorescent tubes.The culture vessels were 100 mL Erlenmeyer flasks with 30 mL of medium. Plants were grown in a tissue culture room at 25 ± 2°C with a 16 h light/8 h dark photoperiod in 50 μmol·m-1 6-Benzyleaminopurine (6-BA), 1.0 mg·L-1 1-Naphthylacetic acid (NAA) and 1.0 mg·L-1 2,4-dichlorophenoxyacetic acid . Since cotyledons had the best callus induction effect and were easy to obtain, the effects of different PGRs on callus induction were studied by taking sterile cotyledons as explants. According to the results of the preliminary experiment, they were transferred to MS medium containing various concentrations of PGRs alone or in combination, including 6-BA , 2,4-D , NAA  and Thidiazuron (TDZ)   Table 1.-1 TDZ, 1.0 mg·L-1 NAA and 0.5 mg·L-1 2,4-D to observe their proliferation abilities. For the best proliferation effect, the effects of different PGRs on callus proliferation were studied by taking sterile cotyledons as explants. They were transferred to MS medium containing 2,4-D , NAA  and TDZ  alone or in combination  or Cef  to determine the concentration of antibiotic needed for the effective selection of transgenic callus. The callus growth state was observed after 14 d of culture. The Kan and Cef were purchased from Beijing Solarbio Science & Technology Co., LTD.The tolerance limits of cotyledone-induced callus to antibiotic kanamycin (Kan) and cefotaxime (Cef) were assessed prior to Agrobacterium tumefaciens EHA105 culture was added to a liquid Luria Bertani (LB) medium containing different concentrations of Cef  with three biological replicates. After 48 h of incubation, the OD600 value was determined by ultraviolet (UV) spectrophotometer .The 20 μL of overnight Agrobacterium strain EHA105 purchased from Shanghai Angyu Technology Co., LTD. 1000 ng pBI121-GUS vector plasmid was added to 100 μl of Agrobacterium EHA105 competent cells and gently mixed, then placed in ice, liquid nitrogen and water at 28°C for 5 min, and finally another ice bath for 5 min. 700 μl LB liquid medium was added to the above products and incubated for 3 h in an incubator at 28°C by shaking. After the shock culture, the bacteria were dipped and coated on solid LB medium containing 50 mg·L-1 Kan and incubated upside down for 2 days at 28°C. Single colonies on the above medium were selected for PCR validation to obtain positive strains. Then a positive transformant was picked and inoculated overnight in 2 mL fresh liquid LB medium containing 50 mg·L-1 rifampicin and 50 mg·L-1 Kan at 28 °C in 200×g. The overnight culture liquid was transferred to a 50 ml liquid LB medium at a ratio of 1:50 and then cultured until the Agrobacterium cell density (OD600) reached 0.2-1.0, and then the culture was centrifuged at 5000×g for 10 min. The resuspension solution containing 5% sucrose and 100 μM filter-sterilized acetosyringone was used to resuspend the Agrobacterium as the infection solution. The healthy callus was soaked in the above prepared infection solution for infection, and the callus was gently shaken to make it fully infected. After infection, the callus was transferred to sterile filter paper until almost dry, and then transferred to the co-culture medium , dark culture was carried out at 28 °C. After three times of rinse in sterile water containing Cef of 200 mg·L-1 and blotting on sterile filter paper, the calluses were cultured on MS medium containing 0.5 mg·L-1 NAA, 1.0 mg·L-1 2,4-D, 0.5 mg·L-1 TDZ and 200 mg·L-1 Cef. Again, three times of rinse in sterile water containing Cef of 200 mg L-1 and blotting on sterile filter paper, the calluses were transferred to a selection medium . After 49 d of culture, part of the resistant callus was taken for GUS staining and Molecular identification. To improve transformation efficiency, the key factors affecting transformation efficiency were optimized, including Agrobacterium cell density , infection duration  and co-culture duration .The binary vector pBI121-GUS purchased from Shanghai Maokang Biotechnology Co., LTD. was transformed into the GUS histochemical assay was conducted according to the described procedure . The resGUS gene (1182bp): forward primer(5’-ATACCGAAAGGTTGGGCAGG-3’), and reverse primer(5’-ATAACGGTTCAGGCACAGCA-3’). The PCR procedure were as followed: pre-denaturation at 94°C for 3 min, followed by 35 cycles at 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min, and final extension at 72°C for 5 min. The amplified products were analyzed by electrophoresis in 1.2% (w/v) agarose gels. Total RNAs of callus were extracted by RNAprep pure Plant Kit . The cDNAs were synthesized by PrimeScript™ RT Master Mix kit  . The RT-qPCR primers were designed with the Primer Premier 5.0 software based on the GUS gene: forward primer (5’-AAGAGCGATGAGCACGAGTA-3’), and reverse primer (5’-AGAAATCAAACAACCCACGA-3’). PlACTIN (GenBank accession no. JN105299.1) was used as an internal control (® Premix Ex Taq™ II (Tli RNaseH Plus) . The reactions were accomplished according to the following method, holding stage: 95°C for 30 s; cycling stage: 40 cycles of 95°C for 5 s, 60°C for 30 s; melt curve stage: 95°C for 15 s, 60°C for 1 min, 95°C for 15 s. Each experiment was performed with three biological and technical replicates. The results were analyzed according to the 2−ΔΔCt methods, and exhibited by GraphPad Prism 8.0.Genomic DNA was extracted from the transformed and non-transformed callus using an extraction kit . The sequence of primers used for  control . The ActThree biological replicates were performed for all experimental treatments in this article. We used SPSS22.0 software to perform one-way analysis of variance (ANOVA) and Duncan’s method for multiple comparisons of the obtained data to determine whether the differences between the data were significant. The important data involved in the paper are calculated as follows: Induction rate = the number of inducted explants/total explants × 100%, Mortality rate = number of dead explants/total explants × 100%, Proliferation coefficient = weight of fresh proliferated callus/weight of inoculated callus, Browning rate = number of browned explants/total explants × 100%, GUS expression rate = the number of GUS-positive receptors/total number stained × 100%.The cotyledons are the most likely to produce callus Figure 2-1 2,4-D. The calluses in Group 1 were in a better growth state and presented green and full of vitality  was in Group 3, which was observed from the 1.0 mg·L-1 TDZ , infection duration  and co-culture duration  were carried out to select the optimum transgenic system. Then, the GUS staining was used for the identification of transgenic callus after selection culture for 49 d . In the same way, we also found that 25 min of infection and 3 d of co-culture led to the highest GUS expression rate, with 68.33% and 61.67%, respectively. Therefore, OD600 = 0.6, 25 min for infection and co-culture for 3 d were chosen as the optimal Agrobacterium transformation conditions.Calluses induced by cotyledons with good and consistent growth were selected for ifferent Figure 6GUS gene in the P. lactiflora genome. Non-transgenic callus genomic DNA did not show any amplified fragments  and Cef (50-400 mg·L-1) in plants of different genera  can be found in the article/Supplementary Material.Agrobacterium-mediated transformation conditions screening experiment. XL and RF were involved in GUS staining experiment. This article was written by SD, XL and SG. All authors contributed to the article and approved the submitted version.XS, SD and XL designed the project. WC, QP, XL, SD and RF completed the callus induction and proliferation experiment. SD, RX and XL completed


These results indicate a unique role for TAK1 in differentiating and proliferating retinal cells.The factors necessary for the differentiation of cell types within the retina are incompletely understood. The transforming growth factor beta (TGF-β) superfamily, including TGF-β1 and 2, the bone morphogenetic proteins, and the activins have all been implicated in differentiation; however, the mechanisms by which these factors affect differentiation are only partially understood. The studies herein focus on a potential role for transforming growth factor β-activated kinase 1 (TAK1), a hub kinase that lies at the intersection of multiple signaling pathways, in the differentiation of cell types within the chick retina. Previous studies have focused predominantly on the role this kinase plays in the inflammation process and axonal growth. TAK1 is downstream of multiple signaling pathways that are critical to development of the central nervous system, including transforming growth factor β (TGFβ), bone morphogenetic proteins (BMPs), and activins. The present study indicates that activated TAK1 is found throughout the developing retina; however, it is localized at higher levels in dividing and differentiating cells. Further,  It has ex ovo retinal cultures were treated with vehicle or TAK1 inhibitor (5Z)-7-Oxozeaenol. Quantitation of retinal cell types 24 h following treatment indicated an increase in both differentiated and proliferating cells with a small increase in cell death. The results of this study have led us to hypothesize a dual role for TAK1 in retinal development; TAK1 does appear to play a role in differentiation; however, we have identified a novel role for TAK1 in the proliferation of retinal progenitor cells.The study described herein investigates the role of TAK1 in chick retinal development. Using immunofluorescence, we describe localization patterns of activated phospho-TAK1 (pTAK1) at critical stages of retinal development from the optic cup up to embryonic day 18 (E18). To investigate a potential role for TAK1 in retinal development, embryonic day 6 (E6) Fertilized white leghorn chicken eggs were procured from University of Michigan and stored at 16°C in a BOD  incubator (Jeio tech: IL-11A). Eggs were incubated at 37°C for the specified periods of time . Eyes used for immunohistochemistry were enucleated, extraocular tissues removed in 1X phosphate-buffered saline (PBS) and fixed overnight in 4% paraformaldehyde diluted in 0.1M phosphate buffer, pH7.4. Samples were rinsed twice in 1X PBS at room temperature incubated overnight with increasing concentrations of sucrose solutions in order: 5%, 10%, 15%, and 20% sucrose made in 0.1M phosphate buffer, pH 7.4. Samples were then placed in 20% sucrose and optimal cutting temperature (OCT) medium at a ratio of 3:1 . The samples were oriented in the 3:1 solution oriented so that sections would be taken through the naso-temporal axis. They were then placed in dry ice to freeze and stored for future sectioning at −80°C. Samples were sectioned using a Leica CM3050 S cryostat at a thickness of 12 µm and placed on vectabond-coated  superfrost plus slides  and then stored at −80°C.Slides with cryosections previously stored at −80°C were thawed to room temperature (RT) and the perimeter of the sections was lined using a peroxidase anti-peroxidase  pen (EleFor co-labels with antibodies against phospho TAK1 (pTAK1) and other antibodies raised in rabbit, citrate antigen retrieval with tyramide amplification was done following permeabilization with methanol. Briefly, sections were incubated with 10 mM sodium citrate with 0.05% Tween pH 6.0for 1 h at 63°C, then allowed to cool for 20 min followed by two rinses of with 1X PBS. Sections were subsequently treated for 2 min with 1% sodium borohydride  in 1X PBS and then blocked with 10% donkey or goat serum in 1X PBS containing 0.25% Triton X-100 for 30 min. Primary antibodies were then diluted 5 to 10 times normal concentration in a solution of 2% donkey or goat serum and 0.025% triton X-100 diluted in 1X PBS. After incubation overnight at 4°C, sections were rinsed with 1X PBS and incubated with 3% solution of hydrogen peroxide in methanol for 15 min. Sections were washed with 1X PBS twice for 2 min before being incubated with biotinylated secondary antibody  in 1X PBS in the dark for 1 hour at room temperature. Sections were then washed using HRP streptavidin  in 1X PBS 1:1000 (and 1:500 for pSMAD1) for an hour in the dark. Afterwards, slides were then washed twice in 0.1M Tris-HCl, 0.15M NaCl, 0.05% Tween-20 (TNT) buffer and incubated with fluorescein plus amplification reagent diluted 1:300 in 1X tyramide signal amplification (TSA) amplification diluent  for 5 min in the dark. Slides were subsequently washed twice in TNT buffer followed by two 2 min rinses in 1X PBS, counterstained with Hoechst solution and mounted with aqua polymount and cover slipped. All images were acquired using an Olympus Fluoview FV 1000 confocal microscope.Chick retinas were dissected out and placed in lysis buffer  (Cas # 329-98-6) for 20 min on ice. Tissue lysates were then centrifuged at 14,000 rpm for 15 min at 4°C and the supernatant was collected for protein estimation using bicinchoninic acid (BCA) assay  , 23225. 2 at 50 rpm. Following this, retinal explants were either treated with vehicle (DMSO) as a control, or with 2 µM TAK1-specific inhibitor, (5Z)-7-Oxozeaenol (Tocris) (Cat #3604). In some experiments, ex ovo cultures were treated with a second TAK1 inhibitor known as Takinib at a concentration of 100p.m. . Following treatment with vehicle or inhibitor, retinal explants were incubated at 37°C on a shaker with 5% CO2 at a speed of 50 rpm for 24 h. Subsequently, the 24 well plate containing the explants was removed from the incubator, retinal transplants were transferred into a new 24-well plate containing 4% paraformaldehyde in 0.1M phosphate buffer, pH 7.4 and incubated on ice for 15 min. Afterwards, retinal transplants were rinsed with 1X PBS for 10 min in the 24 well plate on ice. Whole retinal explants were transferred to wells with increasing concentrations of sucrose: 5%, 10%, 15%, and 20% sucrose in 1X PBS, incubating on ice for 1–2 h. Retinal explants were then placed into 3:1 OCT: sucrose solution and into an embedding mold. Embedded retinal explants were flash frozen on dry ice until frozen solid. Samples were stored at −80°C until ready to be sectioned.The method for retinal explants was adapted from . BrieflyN = 3 retina for each vehicle and TAKi-treated retina) was taken using either an Olympus Fluoview FV 1000 or a Keyence semi-automated microscope. Each of the markers used in this study was quantitated by counting the number of cells immunolabeled in every 10th cryosection through the treated and control retinas. The number of immunolabeled cells was expressed as percentage of total number of cells labeled with Hoechst. Measurements of retinal width was determined in every 10th cryosections of E6 vehicle or TAKi-treated retinal cultures (N = 3 for each control and TAKi-treatments). Images of retinas were imported into ImageJ/FIJI using the measurement tool from the outer edge of the developing retina up to and including the nerve fiber layer. Students t-test was used to test for statistically significant differences between vehicle and TAKi-treated retinas. One-way ANOVAs were performed in some instances using GraphPad Prism version 9.0 for Windows with a Tukey post-hoc analysis .Cryosections were double-labeled with antibodies specific for a given protein and Hoechst 33342, a UV fluorescent stain that labels all fixed nuclei stain, which labels all cell nuclei. Digital images of the central region of each retina , E5, E8, E15, and E18. E3 is representative of the stages when the retina is populated primarily with proliferative progenitors. E5 is representative of stages when early retinal cell types, such as cone photoreceptors and retinal ganglion cells are present, in addition to proliferative progenitors. E8 is a mid-developmental stage when most cell types have been generated in the retinal fundus and distinct outer and inner plexiform layers are beginning to form. By E15, there is a fully laminated retina; however, cells are still differentiating and refining connections. By E18, a mature retina is present, although there is evidence that some cell types continue to develop .Retinal cryosections were labeled using an antibody that recognizes activated TAK1, phosphorylated at threonine 184/187 (pTAK1) along with a Hoechst label to visualize nuclei in the tissue. Overall, pTAK1 appeared to be localized throughout the retina, but was more heavily localized in actively dividing and differentiating cells. At E3, pTAK1 heavily labeled dividing cells at the scleral edge of the retina and weakly labeled retinal pigmented epithelium (RPE), retinal neuroblasts, and lens . The RPESections from each stage treated with isotype specific IgG in place of the primary antibody showed weak background label at the scleral edge of the retina at E5, E12-18 and weak label in the outer plexiform label at E12. Otherwise, the retina was negative for any immunofluorescence . Retinal+ cells all appeared to be co-labeled with pTAK1. To determine if pTAK1 was also present in retinal progenitors not actively dividing, sections from E3, E5, E8, and E18 embryos were double-labeled for SRY (sex determining region Y)—box 2 (SOX2) and pTAK1. At early stages (E3 and E5), SOX2 labels all retinal progenitors, while in later stages (E8 and E18) SOX2 labels Mϋller glial cells, astrocytes, and cholinergic amacrine cells. E3 and E5 retinal sections co-labeled with SOX2 showed a high degree of overlap with pTAK1 , LIM1/2 , CHX10 (bipolar cells), AP2α (amacrine), and BRN3A (ganglion cells). At E8, TAK1 was co-localized with cells positive for SOX2, LIM1/2, CHX10, AP2α, and BRN3A . A subpol bodies .N = 3) indicated 31.3% ( ± 3.52% SD) of the cells were pJNK+, whereas the percentage of pJNK+ cells in TAKi-treated retinas (N = 3) was 17.45% ( ± 2.03% SD)  .+ cells in sections from TAKi-treated retinas  indicated that vehicle-treated retinas had 2.70% ( ± 0.25% SD) cells were CC3+, while TAKi-treated retinas contained 4.67% ( ± 0.36% SD) (Previous investigations in some tissues have shown TAK1 to be involved in cell death . To inve retinas  in compa retinas . In both.36% SD) .+ cells at the scleral edge, and it was apparent that some aberrantly labeled cells localized within to the GCL were also present  pH3+ cells , a modification that is highly correlated with chromosomal condensation occurring in mitosis and meiosis . Section present . Quantif3+ cells .Localization of retinal progenitor cells was carried out localizing and quantitating cells positive for progenitor marker SOX2 in vehicle and TAKi-treated ex-ovo retinas . In vehit-test indicates a statistically significant difference between the two means (p < 0.0001). Moreover, when the number of sections that measured in 50 µm increments from 0 to 300 µm was counted, it became clear that the majority of sections measured between 150 and 300 µm whereas a large majority of vehicle-treated sections measured between50-150 µm , while the mean thickness of the TAKi-treated retinas was 192.6 ( ± 52.2) . A t-tes0-150 µm . Last, e0-150 µm .N = 4 retinas), whereas TAKi-treated cells per section averaged 12.68 . Likewise, immunolabel for NAPA73 in vehicle-treated retinas showed an average of 10.91 cells per section  and TAKi-treated samples showed an average of 21.80 cells per section . Brn3a + cells showed a similar increase, with vehicle-treated retinas showing 3.15 cells per section  and TAKi-treated an average of 11.30 per section . Islet1, a marker that has been shown to label cells undergoing differentiation, labels primarily ganglion cells at early stages, but later is found in most cells of the retina (N = 4 retinas) labeled cells per section. In comparison, TAKi-treated retinas contained an average of 15.92 . There were clearly more cells expressing markers found in early differentiating retinal cell types within the TAKi-treated retinas in comparison to vehicle-treated retinas.To determine if there was a change in the number of cells expressing differentiation-specific markers, we treated ex-ovo retinal cultures with vehicle or TAKi, localized and quantitated cells expressing visinin, islet1, NAPA73, and BRN3A . The ex-e retina . For thiTo increase confidence that TAK1 inhibitor 5Z-7-oxozeaenol was specifically affecting TAK1 activity, experiments quantitating cleaved caspase 3, pJNK, SOX2, Islet1, and Visinin were repeated using the highly selective and potent TAK1 inhibitor Takinib . ResultsEx vivo E6 retinal cultures treated with TAK1 inhibitors, 5Z-7-oxozeaenol or Takinib, for 24 h showed an increase in 1) progenitor cells (both actively undergoing mitosis and those that were not actively dividing), 2) apoptotic cells, and 3) cones and ganglion cells.The present study focused on the role of TAK1 in the differentiation of cells in the developing chick retina. Immunolocalization of pTAK1 with markers present in progenitor and differentiating retinal cells indicated that pTAK1 was widely expressed in progenitor (dividing and non-dividing) and differentiating cells of the retina, although expression in dividing progenitors and differentiating cell populations was heavier than expression noted in progenitors that were not actively dividing. ex-ovo retinal cultures indicated there were increases in cell type-specific differentiation, proliferating progenitors, and  in apoptosis. Together, these results suggest that TAK1 may not be limited to a role in differentiation but may play a broader role in the cell cycle. TAK1 has some revealing links to the cell cycle. TAK1 has been shown to directly affect levels of both cyclins D1 and A, although none have been shown in the central nervous system  that TAK1 plays in the differentiating retina. We found widespread localization of pTAK1 in both proliferating and differentiation cells in the chick retina and treatment of the E6 retina with a TAK1 inhibitors indicated that TAK1 plays a role in proliferation and differentiation of early born retinal cells. The novel observation that inhibition of TAK1 increases both differentiation of early born retinal cell types as well as proliferating cells together with the knowledge that TAK1 is a hub kinase that is downstream of multiple pathways has led us to hypothesize that perhaps TAK1 is playing a role that common amongst all the upstream factors. In regards to the TGF-β superfamily, it may be important to activate both the canonical and non-canonical signaling pathways. To obtain cell type-specific differentiation in addition to removal of cells from the cell cycle. Thus future studies will focus on teasing apart the role(s) of canonical and non-canonical pathways in the retina .


Herein we report the first comprehensive series of crystallographically characterised transition metal formyl complexes. In these complexes, the formyl ligand is trapped as part of a chelating structure between a transition metal  and a magnesium (Mg) cation. Calculations suggest that this bonding mode results in significant oxycarbene-character of the formyl ligand. Further reaction of a heterometallic Cr–Mg formyl complex results in a rare example of C–C coupling and formation of an ethenediolate complex. DFT calculations support a key role for the formyl-intermediate in ethenediolate formation. These results show that well-defined transition metal formyl complexes are potential intermediates in the homologation of carbon monoxide. Herein we report a comprehensive series of crystallographically characterised transition metal formyl complexes. Early studies established that these species undergo facile α-elimination to form the corresponding hydrido carbonyl complexes.13–25 The reaction is potentially reversible, but in most cases the hydrido carbonyl is thermodynamically favoured  and carbon dioxide  database reveals that despite nearly 50 years of research into transition metal formyl complexes, only a modest number have been structurally characterised  of a complete series of transition metal formyl complexes . This includes unprecedented examples of crystallographically characterised Cr, Co and Ir formyl complexes. Through reduction of a series of transition metal carbonyl complexes with a molecular magnesium hydride compound, the formyl ligand can be trapped as part of a chelating structure. Further reaction of a chromium formyl complex results in C–C bond formation and an ethenediolate species bridging the chromium and magnesium centres. DFT calculations are consistent with C–C bond formation occurring by stepwise process involving: (i) reduction of the metal formyl to generate an oxymethylene intermediate, (ii) insertion of CO, and (iii) a 1,2-hydride shift. This is an exceedingly rare example in which a well-defined transition metal formyl complex has been shown to be involved in a ethenediolate formation.45 (1) with a series of transition metal carbonyl complexes (2) in toluene solution furnished the corresponding heterometallic formyl complexes (3) in 21–68% isolated yields  NMR experiments. Further coupling was apparent in cases of spin active transition metals; the formyl ligand of 3b shows 2JW–H = 9.0 Hz, while 3g shows 2JRh–H = 2.1 Hz and 1JRh–C = 56.0 Hz. Bridging magnesium hydride resonances ranged δH = 2.86–3.17 ppm in the 1H NMR spectra, upfield of the bridging hydride in 1 (δH = 4.02 ppm). No trend between transition metal fragment and hydride resonance was observed.Reaction of a magnesium hydride dimerd yields . These r−1. 3f–h showed no stretches in this region. The isocarbonyl ligands bridging the magnesium to transition metal centre appear as strong intensity bands at 1674–1791 cm−1. Typical formyl stretches appear at a medium intensity in the range 1530–1630 cm−1, red-shifted from the carbonyl ligands.46,47The infrared data for complexes 3a–h show characteristic stretching frequencies for both the isocarbonyl and carbonyl ligands  stretch for the M–CHO fragment potentially masked by the C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>N stretch of the β-diketiminate ligand. Formyl ligand C–H bonds are observed as a weak intensity stretching frequency at 2546–2635 cm−1 for 3a, 3b, 3d, and 3f–h; formyl C–H stretches in 3c and 3e were not observed.Complexes 3a–h show stretching frequencies at 1513–1520 cmAll eight members of the series (3a–h) have been characterised in the solid state by single crystal X-ray diffraction analysis . In all P1̄ space group, with the five carbon ligands arranged in a trigonal bipyramidal geometry around the iron centre. The formyl ligand occupies the axial position with a Mg–H–Mg bridge to a cis equatorial carbonyl ligand. The iron–carbon formyl bond of 1.935(9) Å is markedly longer than the average metal–carbon bonds to the carbonyl ligands 1.81(1) Å.Complex 3c, a representative example, crystallises in the 6 transition metal fragments (3a and 3b), while some of the shortest M–C distances are recorded for the most electron rich d8 transition metal fragments (3f–h). The disorder inherent in these structures results in large estimated standard deviations on the data, but they are also reproduced by Density Functional Theory (DFT) calculations. In most cases, the hydride ligand could be located within the Fourier difference map, while the location of this atom should be treated with caution, the Mg–H–Mg bond angles varied 146(1)–160(3)°. This angle is obtuse, presumably to achieve the geometry for the bimetallic magnesium fragment to bridge two mutually cis sites of the transition metal.Within the series 3a–h, M–C bond lengths to the formyl ligand range 1.80(3)–2.19(2) Å. The longest M–C bond lengths are recorded for the most electron deficient d48–53The electronic structure and bonding interaction between the formyl ligand and transition metal centre were investigated using DFT. Calculations were performed in Gaussian 09 and Orca 4.2.1. Natural Bonding Orbital (NBO) and Extended Transition State-Natural Orbital Chemical Valence (ETS-NOCV) methods were employed to interrogate the bonding interactions in 3a–h. Structures were optimised using the wB97X-D functional, a hybrid basis set was used 6-31G** , 6-311+G*  while the SDDAll pseudopotential and associated basis sets were used to describe Mg and M centres.5(CHO)]−  shows little change in the NPA charges, supporting the idea that there is a large ionic contribution to the bonding between the transition metal and main group fragments. QTAIM calculations point to the same conclusion, with hydride (−0.8), oxygen (−1.4) and magnesium (+1.8) charges complimenting the NBO data  charges suggests that the binding interaction between the transition metal fragment and dimagnesium hydride fragment is largely ionic. The hydride (−0.8) and oxygen atoms (−0.9) directly attached to magnesium bear significant negative charge while these magnesium sites themselves are highly electropositive (+1.8). Comparison of data for 3a and 3b to the theoretical models − and monomeric 1. Such a simplification is necessary for computational cost, however this system still provides insight into the most likely steps involved in the mechanism for C–C bond formation. The overall formation of the ethenediolate from the chromium formyl complex is exergonic with . The most plausible pathway calculated for ethenediolate formation follows that originally postulated by Bercaw and co-workers -workers .37 Impor5(CHO)]− to give Int-1. Int-1 undergoes hydride-transfer from Mg to Cr–CHO viaTS-1 , forming Int-2. This step is formally a hydromagnesiation of the transition metal formyl and generates a new oxymethylene ligand. From Int-2, C–C coupling proceeds through a migratory insertion reaction that is characterised by a low energy transition state TS-2 . TS-2 proceeds to a five-coordinate chromium complex Int-3. Int-3 can then undergo a 1,2-H shift viaTS-3  to form the ethenediolate ligand which at this point is coordinated to chromium in an η1-fashion. Subsequent dissociation of CO gives the product 4a′ and occurs with translation of the ethenediolate to an η4-mode.The stepwise mechanism is initiated by the coordination of 1 to the oxygen atom of the formyl ligand of − with 1 to form a bis(formyl) intermediate was found to proceed by activation barrier of over 50 kcal mol−1, effectively ruling out this pathway.NBO analysis was performed on the key transition states on the pathway. Analysis of the hydromagnesiation step shows an increased positive charge on the H atom as it moves from Mg in Int-1 (−0.72) toward C in TS-1 (−0.51). At the same time there is an expected decrease in the WBI for the Mg–H bond . The 1,2-H atom shift occurs with re-organisation of the electron density in the {C Fig. S43. Alterna Fig. S43. For exaIn summary, we report the first comprehensive series of transition-metal formyl complexes . These include unprecedented examples of crystallographically characterised Cr, Co and Ir formyl complexes. In all cases, the metal bound formyl ligand is stabilised by a heterometallic effect. We investigated this oxycarbene electronic structure through a series of density functional theory calculations , and concluded that more electron rich metal centres likely result in more oxycarbene character, and thus stabilisation of the metal-formyl ligand. Remarkably, onwards reaction of a chromium formyl complex yields C–C coupling and an isolable ethenediolate complex. This serves as the first direct example of carbon–carbon bond formation at a well-defined and isolable metal-formyl complex.Experimental procedures, details of the calculations, and additional data can be found in the ESIJMP conducted all experimental and computational work. AJPW and JMP solved and refined single crystal XRD data. All authors contributed to writing the manuscript.There are no conflicts to declare.SC-013-D2SC02063G-s001SC-013-D2SC02063G-s002


This low affinity/selectivity interaction results in not only suboptimal anticancer activity but also induction of adverse effects. CD16a on NK cells binds to the antibody-coated cells, leading to antibody-dependent cell-mediated cytotoxicity (ADCC). Recent clinical data have shown that the increased binding affinity between mAb Fc region and CD16a receptor is responsible for significantly improved therapeutic outcomes. Therefore, the + cancer cells was measured. As effector cells, both high-affinity haNK92  and low-affinity laNK92  cells were used.To engineer BiKE, a llama was immunized, then high binding anti-CD16a and anti-HER2 VHH clones were isolated using phage display. ELISA, flow cytometry, and biolayer interferometry (BLI) data showed that the isolated anti-CD16a VHH has high affinity (sub-nanomolar) toward CD16a antigen without cross-reactivity with CD16b-NA1 on neutrophils or CD32b on B cells. Similarly, the data showed that the isolated anti-HER2 VHH has high affinity/specificity toward HER2 antigen. Using a semi-flexible linker, anti-HER2 VHH was recombinantly fused with anti-CD16a VHH to create BiKE:HER2/CD16a. Then, the ability of BiKE:HER2/CD16a to activate NK cells to release cytokines and kill HER2+ cancer than existing anti-HER2 mAbs.The data showed that the engineered BiKE:HER2/CD16a activates haNK92 and laNK92 cells to release cytokines much greater than best-in-class mAbs in the clinic. The cytotoxicity data also showed that the developed BiKE induces higher ADCC to both ovarian and breast cancer cells in comparison to Trazimera™ (trastuzumab). According to the BLI data, BiKE:HER2/CD16 recognizes a different epitope on CD16a antigen than IgG-based mAbs; thus, it provides the opportunity for not only monotherapy but also combination therapy with other antibody drugs such as checkpoint inhibitors and antibody-drug conjugates. Taken together, the data demonstrate the creation of a novel BiKE with high affinity and specificity toward CD16a on NK cells with the potential to elicit a superior therapeutic response in patients with HER2 To achieve our objective, we took advantage of a unique class of single-domain antibodies, known as VHH or nanobody, which was first found in the blood of camelidae family  and laNK92  cells as effector cells and compared with Trazimera™ (trastuzumab). Trastuzumab is an FDA-approved mAb against HER2 where its Fc region interacts with CD16a receptor with low affinity and selectivity. Here, we used NK92 cell line because it is available as both high-affinity  and low-affinity  variants, is FDA-approved for human use, and has a broad range of anticancer activity. The ability of BiKE:HER2/CD16a to activate NK92 cells was determined by inspecting the CD107a degranulation and measuring the release of cytotoxic proteins and cytokines  using flow cytometry and ELISA, respectively.The crystallizable fragment (Fc) of IgG-based monoclonal antibodies binds to Fcγ receptors (FcγRs) that are expressed on the surface of leucocytes and required for the efficacy of most antibody drugs. The FcγRs are divided into two main categories of activating and inhibitory, which bind to Fc regions of antibodies with different affinities. Activating receptors include FcγRI/CD64, FcγRIIa/CD32a, FcγRIIc/CD32c, FcγRIIIa/CD16a, and FcγRIIIb/CD16b and the inhibitory ones include FcγRIIb/CD32b . Furtherimmunity . Consequimmunity , 10. Thee family , 12. We The list of cells, antibodies and reagents used in this study are provided in + haNK92 (V176) and CD16a+ laNK92 (F176) cells were purchased from ATCC and cultured in α-MEM supplemented with 12.5% Fetal Bovine Serum (FBS), 12.5% Horse Serum, 0.2 mM myo-inositol, 0.02 mM Folic Acid, 100 U/ml penicillin-streptomycin, 0.1 mM 2-Mercaptoethanol, and 100 U/ml IL-2. BT474 (HER2+ breast cancer) was purchased from ATCC and maintained in Hybri-Care Media supplemented with 10% FBS and 100 U/ml penicillin-streptomycin. JIMT-1, HER2+ breast cancer cell line resistant to anti-HER2 antibodies (+ ovarian cancer) cell line was obtained from ATCC and maintained in McCoy’s 5A (Modified) Medium supplemented with 10% FBS. Neutrophils were purchased from HemaCare and cultured in DMEM-10% FBS for an hour, then immediately used for downstream application. B cells were purchased from Charles River and cultured in RPMI-1640 supplemented with 10% fetal calf serum (FCS) and 2 mM L-Glutamine.CD16atibodies , was kin6 cells/ml in 100 ml of FreeStyle™ 293 expression media using 250 ml Reusable Spinner Flask for 30 min. The PiggyBack expression vectors were then complexed with linear polyethylenimine (PEI) 25K. For every 106 cells, 1 µg plasmid was mixed with 4 µg of PEI (1:4 w/w ratio) in Opti-MEM and incubated at room temperature (RT) for 20 minutes. Next, the plasmid:PEI complexes were added to the seeded cells dropwise under constant stirring. The flask was incubated at 37°C with 5% CO2 for 24h. The next day, two-fold fresh FreeStyle media was added to the transfected cells. Protein expression continued for 8 to 10 days, or until the viability dropped below 85%, whichever came first. To extract and purify proteins, cells were centrifuged at 10,000 g, 10 min, 4°C, followed by supernatant collection and filtration through a 0.45 µm filter to remove the cell debris. Then, 300 µL of Ni-NTA resin was equilibrated with buffer , mixed with the supernatant, and incubated at 4°C overnight. The next day, the supernatant/Ni-NTA mixture was poured onto a chromatography column and washed using 20 ml of washing buffer . Finally, rCD16a and rHER2 proteins were eluted using 500 µl of elution buffer . The purity and molecular weight of the purified proteins were estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE).The genes encoding rCD16a (UniProt ID P08637) and rHER2 ectodomain (Uniprot ID P04626) were synthesized by VectorBuilder  and then cloned into two separate PiggyBac mammalian expression vectors under EF-1α promoter. The mammalian expression system was selected because the rCD16a ectodomain is a glycosylated protein. A secretory signal sequence was designed at the proteins’ N-terminal and a His-Tag at their C-terminal to facilitate purification from the culture media by Ni-NTA column chromatography  . A female llama was immunized jection) .via SuperScript™ IV First-Strand Synthesis system. After that, the generated cDNA was used as a template in the Nested PCR reaction using a set of primers  of llama library was inoculated into 100 ml of 2xYT supplemented with 100 µg/ml carbenicillin and 2% Glucose. Once the OD600nm reached 0.6, 500 µl of the helper phage, VCSM13, was added to the culture and incubated at 37˚C without shaking for 60 min. The infected bacteria were harvested by centrifugation  and supernatant was discarded. The pellet was resuspended in 100 ml of 2xYT (without Glucose), supplemented with 100 µg/ml carbenicillin and 50 µg/ml kanamycin, and incubated at 37˚C overnight with shaking. The next day, bacteria were spun down at speed of 10,000 g for 20 min at 4˚C. Then, 10 ml of PEG6000 (20% W/V)/2.5 M NaCl solution was added to the 40 ml supernatant and kept on ice for 60-120 minutes. Finally, the recombinant phages were recovered via centrifugation  followed by washing three times with DPBS.Four rounds of phage display were performed to reach specific anti-CD16a and anti-HER2 libraries. First, 2 ODThe purified phages were used in polyclonal phage ELISA. In this experiment, all the incubations were at room temperature (RT) and a shaking speed of 700 rpm unless mentioned otherwise. The bait protein (either CD16a or HER2) was coated into a 96-well plate overnight at 4˚C. The next day, after blocking with 2% skimmed milk, ~100 µl of recombinant phages were added and incubated at RT for 30 min. To remove the non-specific VHHs, the plate was washed ten times with 0.1% DPBS-T followed by ten times washing with DPBS. Finally, the binders were recovered using 50 µl of 0.25% Trypsin (enzymatic elution), which was neutralized by 4-(2-Aminoethyl) benzene-1-sulfonyl fluoride (AEBSF). The purified phages were used in equal quantities to infect 100 µl of TG1 bacteria for one hour at 37˚C, which was then transferred into 100 ml of LB media, supplemented with 100 µg/ml carbenicillin, followed by overnight bacterial culture.To select the top binders, 190 colonies were randomly selected to be cultured in 1 ml of LB media, supplemented with 100 µg/ml carbenicillin and 2% Glucose, in a Deep-Well 96-well plate. To keep the culture oxygenated, a ventilated adhesive plate was used to cover the plate. The following day, 1 ml of fresh LB media, supplemented with 100 µg/ml carbenicillin, was inoculated with 10 µl of overnight culture. After four hours, the expression was induced using 1 mM Isopropyl β- d-1-thiogalactopyranoside (IPTG). The expression continued overnight at 28°C. The next day, the bacteria were harvested using centrifugation . The supernatant was discarded and the pellet was stored at -20°C. Next, the periplasmic extract was prepared using three cycles of freeze-thaw . Next, 500 µl of DPBS was added to the pellet and placed on a shaker for 30 min following centrifugation . Finally, the supernatant (400 µl) was transferred to a new Deep-Well 96-well plate. The periplasmic extract was used in ELISA to find the top binding candidates.® M200 PRO plate reader.For anti-CD16a ELISA, the plate was coated using 100 µl of 5 µg/mL streptavidin overnight at 4°C. The next day, after one round of washing, 50 µl of biotinylated CD16a and CD16b were added and incubated for 30 minutes. For anti-HER2 ELISA, 100 µl of 1µg/ml HER2 protein and three other members of the Heregulin superfamily, including HER1, HER3, and HER4 were used to coat Nunc MaxiSorp™ high protein-binding capacity 96-well ELISA plate and incubated overnight at 4°C. After three times washing, 100 µl of the periplasmic extract was added. To detect the bound VHH, the secondary antibody, HRP anti-HAtag antibody (1:10000 dilution) was added. To develop color, 50 µl of 1-Step™ Turbo TMB-ELISA substrate solution was added and incubated in darkness for 15 min. After stopping the reaction using 50 μL of 1N HCl, the plate was read at 450 nm wavelength using Tecan Infinite2 and 100 μg/ml carbenicillin, was inoculated by 7.5 ml of overnight culture grown at 37°C. After the OD600nm reached 0.6-0.8, 0.5 mM IPTG was added to induce expression. The culture was then incubated at 28˚C overnight. The next day, the media was spun down using a centrifuge , the pellets were resuspended in 10 ml of TES buffer , and incubated at 4˚C for one hour while shaking. Next, 15 ml of TES/4 buffer (10 ml TES buffer was added to 30 ml distilled water) was added and incubated at 4˚C for 45 min while shaking. The bacteria were pelleted via centrifugation  and the supernatant was loaded onto the Ni-NTA column. The column was washed with 30 ml of wash buffer . The purified VHH was eluted using elution buffer . The purity of the eluted VHH was evaluated by SDS-PAGE.Anti-HER2 and anti-CD16a VHHs were sequenced by GeneWiz  and then cloned into pHEN6  expression vectors. The pHEN6 was chemically transformed into WK6 E. coli expression system, followed by the selection of the highest expressing transformant using western blot analysis. Next, 750 ml of Terrific Broth (TB), supplemented with 0.1% Glucose, 1 mM MgClvia HMA  as a linker. WELQut protease site, c-myc, and His-Tag sequences were designed at the BiKE’s C-terminal to facilitate its purification and detection. The BiKE:HER2/CD16a (E5C1) construct was then synthesized and cloned into a pHEN6 expression vector by GenScript , followed by transformation into WK6®E. coli expression host. Next, 750 ml of Terrific Broth (TB) containing 100 μg/ml carbenicillin was inoculated with 7.5 ml of overnight culture. After 16 – 18h, when the OD600nm reached ~5, 1 mM IPTG was added to induce protein expression. The expression continued at 37°C for three hours and the bacterial culture was centrifuged at 10,000 g for 10 min at 4°C). The pellet was resuspended in lysis buffer  (3 ml per gram of pellet) and incubated at 4°C for 30 min while shaking. The bacterial suspension was then sonicated  for 30 min on ice. Next, the cell debris was pelleted via centrifugation , the supernatant was loaded onto a Ni-NTA column and washed with 30 ml of wash buffer . To elute BiKE, the column was equilibrated with 20 ml of DPBS and then eluted using 10 U/mL of WELQut protease. The purity of the eluted protein was measured using SDS-PAGE. The molecular weight of the purified BiKE and its monomeric status was evaluated at Rutgers Center for Advanced Biotechnology and Medicine core facility using liquid chromatography/mass spectroscopy (LC-MS).To express BiKE:HER2/CD16a, the gene encoding the construct was designed as a single chain peptide by fusing anti-CD16a VHH (C1 clone) with anti-HER2 VHH (E5 clone) The purified VHHs were subjected to functional analysis using ELISA. For anti-CD16a VHH ELISA, 50 µl of biotinylated CD16a, CD16b-NA1, CD16b-NA2, and CD32b proteins were added to the streptavidin-coated plate and incubated for 45 min at RT. Similarly, for anti-HER2 VHH ELISA, HER2, HER1, HER3, and HER4 proteins were used to coat ELISA plates. Following washing, 100 µl of 100 nM purified VHHs were added and incubated at RT for one hour. Next, 100 µl anti-HA tag HRP conjugated secondary antibody was added. Finally, the substrate was added for color development, and the plate was read at OD450nm.The binding of purified BiKE:HER2/CD16a to HER2 and CD16a antigens was also evaluated using ELISA. For this purpose, 50 µl of biotinylated CD16a or HER2 was added to the streptavidin-coated plate and incubated for 45 min at RT. Followed by washing and blocking, 100 µl of purified VHH or BiKE ranging from 1000 to 0 nM were added and incubated at RT for one hour. Next, 100 µl anti-cMyc tag HRP conjugated secondary antibody  was added and the plate was read at OD450 nm.+ SKOV-3 and HER2¯ MDA-MB-231 cancer cells were cultured followed by incubation with the purified anti-HER2 VHH or BiKE. Approximately 106 cells were counted and washed using DPBS-1% FBS. Then, equivalent to 100 nM of trastuzumab, pertuzumab, anti-HER2 VHH, or BiKE was added to the cells and incubated on ice for 60 min. After the washing step, FITC-conjugated anti-Human IgG or anti-Histag antibody was added and incubated on ice for 60 min. Cells were washed three times and then analyzed using Beckman Coulter Gallios flow cytometer at Rutgers Flow cytometry core facility.HER2+), neutrophils (CD16b+), and B cells (CD32b+) were cultured according to the vendors’ protocols. Cells were harvested and washed once with DPBS supplemented with 2% FBS (2% DPBS-FBS). Next, the non-specific interaction of antibody Fc region with Fc receptors was blocked using Human TruStain FcX™ to eliminate background staining.NK92 cells (CD16a6 NK92 cells (CD16a+) and neutrophils (CD16b+) were stained with 3G8 mAb and C1 VHH on ice for 60 min. Cells were washed three times and incubated on ice with PE-conjugated Rabbit anti-mouse antibody and anti-HisTag antibody for 45 min to label bound 3G8 and C1 VHH, respectively. Samples were then analyzed by Beckman Coulter CytoFLEX Flow Cytometer. The same method was used to evaluate the ability of BiKE to recognize CD16a on NK92 cells.To evaluate the selectivity of anti-CD16a VHH (clone C1) toward CD16a antigen, 0.5 x 10To evaluate the interaction of anti-CD16a VHH (clone C1) with CD32b on B cells, B Cells were stained with AT10 (Anti-CD32a/b mAb) and C1 VHH, as mentioned above. Next, cells were stained with FITC-conjugated Rabbit anti-mouse antibody and anti-HisTag antibody to label bound AT10 and C1 VHH, respectively. Samples were then analyzed by Beckman Coulter CytoFLEX Flow Cytometer.D) and constant rates of association (Kon) and dissociation (Koff) of VHHs and BiKE, an Octet RED96e (Sartorius) Biolayer Interferometer (BLI) located at Biophysics Core Facility at Princeton University was used. An Octet® Streptavidin (SA) Biosensor was soaked for at least 10 min in DPBS supplemented with 0.1% Casein, biotinylated CD16a, CD16b-NA1, CD16b-NA2, and HER2 antigens were loaded onto the streptavidin (SA) biosensor until 1 nm shift was reached. Next, the sensor was dipped into the washing buffer (DPBS + 0.05% Tween 20 + 0.1% Casein) for 2 min to reach the baseline. Then, the sensor was submerged into wells containing 60, 30, 15, 7.5, 3.75, 1.875, and 0 nM of purified anti-CD16a VHHs or BiKE for 5 min (association step). Then, in the dissociation step, sensors were dipped into the washing buffer for 5 min to acquire data  were under either adherent or non-adherent conditions. To do ADCC under the adherent condition, 103 target cancer cells were mixed with 100 nM of mAb or BiKE and then added to a non-treated 96-well plate. Then, different numbers of haNK92 cells were added to each well to obtain the E:T ratios of 16, 8, 4, 2, 1, 0.5, and 0. The plate was incubated at 37°C for four hours. Next, alamarBlue ™ HS Cell Viability Reagent was added and incubated at 37°C for two to three hours. Finally, the plate was read by a Tecan Infinite M Plex plate reader and the cell viability was measured using the following formula:To do ADCC under the non-adherent condition (in suspension), 5×10[(Fluorescent Intensity of Test Group - Fluorescent Intensity of NK only)/(Fluorescent Intensity of Target Cells only)] × 100SKOV-3 cells were seeded in a tissue culture treated 96-well plate at the density of 10,000 cells per well. The next day, 10 nM BiKE or trastuzumab was added to the plate and incubated at 37˚C for 30 min. Next, effector cells (laNK92 or haNK92) were added at E:T ratio of 4. After two hours (Perforin and Granzyme B) and 24h (TNF-α and IFN-γ), plates were centrifuged down at 2000 g for 10 min to pellet the cells. Then, the supernatant was transferred into a non-treated 96-well plate. The amount of cytokine release was quantified using Quantikine ELISA kit and Perforin Human ELISA Kit following manufacturer’s protocol. The data are presented as mean ± SD (n=3).4 SKOV-3 cells were seeded in a 96-well plate and incubated overnight. The next day, a serial dilution of BiKE and trastuzumab ranging from 0 to 100 nM were added and incubated for 30 min at 37°C. Then, laNK92 (GFP+) or haNK92 was added at E:T ratio of 4 and the plate was incubated at 37°C for two hours. Next, Fc Blocker was added to the plate and incubated at 37˚C for additional one hour. Afterwards, APC-conjugated anti-CD107a antibody was added and incubated for one hour in an incubator. Finally, the plate was centrifuged down, and the supernatant was discarded. The cells were washed twice to remove excess antibodies and data was acquired by the Beckman Coulter CytoFLEX Cytometer. For data analysis, the GFP+ (laNK92) was gated to distinguish between effector and target cells. Next, the surfaced CD107a was quantified on GFP+ cell population.First, 10rCD16a and rHER2 were genetically engineered and expressed in 293-F mammalian expression system, followed by purification. The SDS-PAGE analysis of purified proteins revealed the purity of both proteins to be >95% , 16. ThePost immunization, llama blood was drawn and PBMCs were isolated, followed by cDNA and phagemid library generation, which were used in phage display. After four rounds of phage display, 190 colonies were selected, and lysed to obtain periplasmic extracts , were us+), neutrophils (CD16b+/CD16a¯), and B cells (CD32b+/CD16a¯) were used as cell controls, whereas 3G8 mAb (anti-CD16a/b mAb) and AT10 (anti-CD32a/b mAb) were used as antibody controls. The results of this experiment showed that C1 VHH effectively bound to CD16a receptor on NK92 cells without cross-reactivity with neutrophils (NA1) and B cells  and MDA-MB-231 (HER2¯) cancer cells. The flow cytometry data showed that the selected E5 anti-HER2 VHH bound to HER2 on the surface of SKOV-3 cells but not MDA-MB-231 cells   and 2.25 × 10-9 (M) for E5 VHH and BiKE:E5C1, respectively , high-affinity antibodies have KD values in the sub-nanomolar range (10-9 M). Therefore, both BiKE:E5C1 and E5 VHH are considered high-affinity antibodies.The affinity of the E5 and C1 VHHs, as well as BiKE:E5C1 toward their corresponding antigens, were quantified using BLI. First, the affinity of E5 VHH toward HER2 antigen was measured, then compared with affinity of BiKE:E5C1 toward HER2. The BLI sensograms showed the K-9 (M) and 2.7 × 10-9 (M) for C1 VHH and BiKE:E5C1 toward CD16a antigen, respectively (-4 to 10-6 M) toward CD16b-NA1, whereas they had high affinities toward CD16b-NA2 antigen , and laNK92 plus BiKE:E5C1  followed by measurement of cell viability. The results of this experiment showed that trastuzumab significantly increased cytotoxicity of laNK92 cells at most E:T ratios; however, in all three tested cell lines, the ADCC of BiKE:E5C1 was superior to trastuzumab , a cell toxicity assay was performed. As effector cells, both laNK92 (F176) and haNK92 (V176) cells were used. It has been shown that the CD16a (V176) has a relatively higher affinity toward the Fc region of mAbs . HER2+ cstuzumab . As a re+ cancer cells were seeded in adherent condition as mentioned above and treated with haNK92 cells alone, haNK92 plus trastuzumab , or haNK92 plus BiKE:E5C1 , followed by cell viability measurement. Cancer cells were also treated under the same conditions at a fixed E:T ratio using different concentrations of antibodies. The results of these experiments showed that ADCC with BiKE:E5C1 was superior to trastuzumab not only at different E:T ratios (To determine whether BiKE:E5C1 provides an advantage in terms of ADCC over trastuzumab in patients with high affinity NK (V176) cells, a cell toxicity assay using haNK92 (V176) was performed. HER2+ cancer cells in suspension, representing circulating cancer cells, by haNK92 cells, an ADCC assay under non-adherent conditions was performed. In this experiment, both cancer cells and haNK92 cells were seeded in non-adherent plates, followed by the addition of either BiKE:E5C1 or trastuzuamb. The results of this study showed BiKE:E5C1 assisted in the killing of HER2+ cancer cells by haNK92 cells more effectively than trastuzumab  (D of ~8 × 10-7) with cross-reactivity with CD16b and CD32b receptors (IgG-based antibodies mediate the clearance of cancer cells s (ADCP) , 23. Fors (ADCP) . Howevereceptors . As a reeceptors , 26. Addeceptors , 28.+) cells with negligible binding to neutrophils and B cells  to generate BiKE:E5C1. Several studies have demonstrated that less flexible linkers yield higher agonistic activity , respectively (-7 and 8×10-7 (M), respectively. Since clinical data have shown that patients with CD16a (V176) allele responded better to antibody therapies than those with CD16a (F176) allele (-9 (M), which is a ten-fold improvement over IgG1 (-9 (M) and 36-fold higher than margetuximab, we expect to observe better therapeutic response with BiKE:E5C1 than margetuximab in future clinical trials.We then examined the ADCC of Trastuzumab and compared it with BiKE:E5C1 using laNK92 and haNK92 cells. Approximately 10% of humans are homozygous for the higher affinity CD16a (V176/V176) allele, whereas the rest of the human population are either low affinity homozygous (F176/F176) or heterozygous (F176/V176) . The binectively . To be c) allele , severalver IgG1 . Howeverver IgG1 . Conside+ cancer cells with concentrations at least 100 times less than trastuzumab , which is in Phase I clinical studies for the treatment of lymphomas (NCT04101331)  . In gene+ NK92 cells, the engineered BiKE:E5C1 induced the release of higher amounts of Perforin, Granzyme B, TNF-α, and IFN-γ, in comparison to trastuzumab leading to higher ADCC with BiKE:E5C1. Since BiKE:E5C1 recognizes a different epitope on CD16a antigen than trastuzumab, the opportunity for combination therapy with other antibody drugs such as checkpoint inhibitors and antibody-drug conjugates arises. Overall, the data demonstrate the creation of a new BiKE with the potential to elicit a superior therapeutic response in patients with HER2+ cancer than existing anti-HER2 mAbs.Our data show that the engineered BiKE:E5C1 has high affinity and specificity toward CD16a with negligible cross-reactivity with CD16b-NA1 and CD32b. Upon activation of CD16The original contributions presented in the study are included in the article/AH: Conceptualization, Methodology, Validation, Formal analysis, Resources, Writing - Review & Editing, Visualization, Supervision, Project administration, Funding acquisition. SN: Methodology, Validation, Formal analysis, Investigation, Writing - Original Draft, Visualization. GL: Methodology. SE: Investigation. GY: Methodology, Investigation. VV: Methodology, Investigation. All authors contributed to the article and approved the submitted version.


This revealed substantial, spontaneous conformational changes on a slow time scale, with parts of the POTRA2 domain approaching the lipid bilayer and the protein’s surface loops. Specifically, our data indicate that an amphipathic POTRA2 β hairpin can insert into the β barrel. We propose that these motions enlarge the channel and initiate substrate secretion. Our data propose a solution to the conundrum how TpsB transporters mediate protein secretion without the need for cofactors, by utilizing intrinsic protein dynamics.The Two-Partner secretion pathway mediates protein transport across the outer membrane of Gram-negative bacteria. TpsB transporters belong to the Omp85 superfamily, whose members catalyze protein insertion into, or translocation across membranes without external energy sources. They are composed of a transmembrane β barrel preceded by two periplasmic POTRA domains that bind the incoming protein substrate. Here we used an integrative approach combining  Bordetella pertussis is a model TPS system, in which the FhaC transporter is necessary and sufficient to mediate the translocation of the adhesin FhaB across the outer membrane (The Two-Partner Secretion (TPS) pathway is dedicated to the export of large proteins notably serving as virulence factors . The Tpsmembrane .Omp85 proteins are composed of N-terminal POTRA (polypeptide transport associated) domains - two in the case of TpsB transporters - followed by a 16-stranded transmembrane β barrel, which for FhaC is the FhaB translocation pore . The POTAb and CdiBEc have shown very similar folds to that of FhaC, albeit with slightly different positions of H1 in the barrel  or JCB571 (dsbA::kan) (E. coli BL21 (DE3-omp5) (ΔlamB ompR)  on pFc3 . pSphB1αβ is a derivative of pT7SB1αβ-S (bA::kan)  were usemB ompR)  for over on pFc3 . Overexp on pFc3 . ptacFhaM2lk-His . pFJD63 promoter . Its der7SB1αβ-S  with a 67SB1αβ-S  after thin vivo, the pFc3 variants were introduced in E. coli JCB571. The recombinant bacteria were grown at 37°C in minimum M9 medium containing 0.1% casaminoacids under agitation. The cells were collected by centrifugation when the optical densities at 600 nm (OD600) of the cultures reached 0.8. The cell pellets were resuspended in 50 mM sodium phosphate (pH 6.8) containing 10 mM N-ethylmaleimide and lysed using a Hybaid ribolyzer apparatus (50 s at speed 6). The membranes were collected by ultracentrifugation of the clarified lysates at 90,000 g for 1 h. The pellets were resuspended in loading buffer without reducing agent and separated into two aliquots, with dithioerythritol (DTE) added at 25 mM to one of them before heating at 70 °C for 10 min. FhaC was detected using anti-FhaC antibodies (To monitor disulfide (S-S) bond formation tibodies  with alkE. coli JCB570 or JCB571 harboring a pFJD63 derivative and ptacFha44-His were diluted to OD600 of 0.3 in LB and grown under agitation with 0.1% arabinose for 20 min to produce FhaC. The bacteria were collected by centrifugation, resuspended in prewarmed LB without arabinose and grown to OD600 of 0.8 before adding IPTG at 1 mM to induce the expression of Fha44. Culture aliquots were collected 5 and 20 min thereafter and placed on ice. After centrifugation to pellet the bacteria, Fha44 was affinity-purified from the supernatants with Ni-NTA beads . The membrane extracts were prepared and FhaC was detected as above. Fha44 was detected by immunoblotting using anti-6His antibodies, the ECL kit of Amersham  and the Amersham Imager 600 (GE) with 1 s exposure. The amounts of Fha44 in supernatants were quantified with ImageJ.For the secretion assays, overnight cultures of N66I were performed as described , 1 g/L15N-NH4Cl, 1 g/L15N,2H-isogro (Sigma) and 13C-α-ketobutyric acid (Sigma) to achieve u-, Ile-δ1(13CH3) isotope labeling (E. coli BL21 (DE3-omp5) and purified from β-octylglucoside (bOG) extracts using Ni2+ affinity chromatography. For BfrG 300 mM NaCl was added to improve solubility.The production and purification of the FhaC derivatives and of Fha30escribed . Expresslabeling . For spiE. coli polar lipids were prepared as described  of escribed . Where iE. coli BL21 (DE3), with an induction of 3 h at 28 °C. For NMR experiments, scaffold proteins were expressed in M9 minimal medium in D2O using 2H-glucose as carbon source to suppress their signals in the -based NMR spectra. The bacteria were broken using a French press in 50 mM Tris-HCl (pH 8), 300 mM NaCl (TN buffer), 1% Triton X100 (TNX buffer), and the clarified lysates were subjected to Ni2+ affinity chromatography. After successive washes in TNX, TN buffer with 50 mM cholate, 20 and 50 mM imidazole, the proteins were eluted in TN buffer with 400 mM imidazole, concentrated by ultrafiltration and dialyzed against 20 mM Tris-HCl (pH 8), 200 mM NaCl and 0.1 mM EDTA. DMPC and DMPG at a 2:1 ratio were solubilized in chloroform, lyophilized overnight and resuspended to 25 mM in 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.5 mM EDTA, 50 mM cholate. For NMR experiments, deuterated (d54-) DMPC and DMPG  were used. FhaC, the scaffold protein and the lipids were mixed at a ratio of 1:3:180, cholate was added to 15 mM, and incubation was performed for 1 h at room temperature. Biobeads were added progressively, and the incubation was continued at 4 °C overnight. The nanodiscs were collected by ultracentrifugation and concentrated by ultrafiltration. For NMR experiments, the buffer was exchanged to 100 mM NaPi in D2O pH* 7.2 using a 2-ml ZebaSpin column (7 kDa MWCO).Nanodiscs were prepared with the MSP1D1 and MSP1E3D1 scaffold proteins  produced1H Larmor frequency (18.8 and 22.3 T magnetic field) (Bruker Biospin) at a MAS frequency of 50 kHz. Sample temperature was kept at about 17°C as judged by the chemical shift of the bulk water resonance. Spectra were indirectly referenced to 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) via the lipid methylene proton resonance, which appears at 1.225 ppm under our experimental conditions. Typical pulse lengths for 1H and 13C 90° pulses were 2.1 and 3.8 µs, respectively. For cross-polarization (CP), radiofrequency (RF) field strengths were 21 and 30 kHz for 1H and 13C, respectively, with a 50-to-100% ramp on 1H and a duration of 1.5 ms. 1H-detected 2D 13C-1H dipolar hCH correlation spectra . For the 2D hChH correlation spectrum, a 1H–1H mixing time of 6.4 ms using radio frequency driven recoupling (RFDR) (1H RF 120 kHz). 13C R1ρ spectra (13C spinlock field strengths from 1.2 to 10 kHz (applied on-resonance with isoleucine δ113C methyls) and 5 spinlock durations from 2.5 to 80 ms, with a 1H 180° pulse in the middle of the spinlock to suppress chemical shift anisotropy/dipolar coupling cross-correlated relaxation , reconstituted into E. coli polar lipid liposomes. These spectra were referenced to spectra recorded on FhaC187R1 after reduction of MTSL by a 3.5-fold molar excess of ascorbic acid (FhaC187R1red) and FhaCC220 carrying a diamagnetic MTSL analogue, respectively  solid-state NMR rotors  using an ultracentrifugation device  (Giotto  spectra  were typI scheme  was usedg (RFDR)  was appl spectra  were reclaxation . For solectively .13C-1H heteronuclear multiple-quantum coherence (HMQC) or SOFAST-HMQC  at 32°C sample temperature. Standard AST-HMQC  experimebic acid .NMR spectra were processed with TopSpin 4.0.3 (Bruker Biospin) or NMRPipe  and anal1 methyl signals, Ile-to-Val mutations were used in all cases except for Ile114 in domain POTRA1, which was identified by attenuation of its signal in the spectrum of paramagnetic FhaC195R1 (average distance of Ile114 Cδ1 to the paramagnetic center modeled on residue 195: 16.6 Å), and Ile420, which was assigned by exclusion.For assignment of isoleucine (Ile) δR2,eff were extracted from experimental R1ρ values using separately recorded R1 experiments .To generate relaxation dispersion curves, effective transverse relaxation rates eriments . R2,effexchange  fitted r220R1/FhaC220R1dia and FhaC187R1/FhaC187R1red for solid-state experiments, FhaC195R1/FhaC195R1red in case of the solution-state experiments, respectively) were calculated. These para-versus diamagnetic signal intensity ratios do not normalize to one in our case. In the solid-state experiments, this can be explained by different amounts of protein integrated into liposomes during reconstitution (FhaC220R1 vs FhaC220R1dia), as well as different amounts of sample transferred to the NMR rotor (FhaC220R1 vs FhaC220R1dia as well as FhaC187R1 before and after reduction). Both in solid and solution state, spectroscopic factors also play a role . This is equivalent to normalizing signals within each spectrum to a reference signal whose intensity is unaffected by PRE effects. We then only analyzed relative signal attenuation levels, instead of attempting to extract quantitative distance measures. Error bars of PRE intensity ratios were calculated based on the root-mean-standard deviation of the spectral noise using standard error propagation. To test for significant differences between these intensity ratios, p values for comparisons were calculated from z scores, assuming errors of intensity ratios to be normally distributed. To estimate expected distances between FhaC Ile residues and the MTSL label, and consequently relative PRE attenuation levels, ensembles of 200 MTSL conformations compatible with labeling on FhaC residues 187, 195, and 220 were calculated using the mtsslSuite web server or PyMOL plugin (http://www.mtsslsuite.isb.ukbonn.de/) and the FhaC crystal structure (PDB 4QKY) with the corresponding residue changed to Cys in PyMOL . Distances from the average position of the paramagnetic center as given by mtsslSuite to Ile Cδ1 nuclei were calculated using PyMOL.For PRE NMR experiments, ratios of peak intensities in spectra of para- and diamagnetic species (FhaCn delays ). For thL plugin  (http://t ― (π)pump ― τ2 –t ― (π)probe ― τ2 ― (echo)] was employed with a 200-ns τ1 delay and τ2 delays ranging from 3,200 ns to 7,000 ns depending on the sample  EPR experiments were performed as described . Pulsed-e sample . Probe pe sample , and thetructure .–2 mbar and 1.21 × 10–2 mbar for the source, trap and transfer regions unless indicated otherwise. Collision-induced unfolding (CIU) ion mobility experiments were performed with 50 V sampling cone; 50–200 V trap collision energy; 42 V trap DC bias; and 15 V transfer collision energy. Pressures throughout the instrument were 9 and 1.46 × 10–2 mbar for the source and trap/transfer collision cells. All spectra were processed with Masslynx v4.1 (Waters). Collision cross section (CCS) calibration was performed using GDH, ADH, ConA and PK as proteins standard as described . Samples were directly infused with nano-electrospray ionization with in-house-prepared gold-coated borosilicate glass capillaries with a spray voltage of +1.4 kV. Spectra were recorded on a quadrupole TOF instrument  optimized for transmission of native, high-m/z protein assemblies. Critical voltages and pressures throughout the instrument were 50, 10, 150 and 15 V for the sampling cone, extraction cone, trap and transfer collision cell, respectively, with pressures of 9 mbar, 1.47 × 10escribed . It shouSynthesized peptides were dissolved in DMSO to a final concentration of 100 mM and added to the protein sample at final concentrations of 10 μΜ FhaC and 100 μM peptide. To correct for non-specific and detergent-specific binding, SphB1-αβ was run at identical concentrations and conditions. For both proteins the fraction of peptide-bound protein was calculated based on peak intensities, after which the binding to the decoy protein was subtracted to correct for non-specific binding.dsbA- background, such that S-S bonds could spontaneously form after FhaC biogenesis as a result of its conformational changes in the membrane.We previously obtained hints that motions of the H1 helix and the L6 loop affect the conformation of the POTRA2 domain . To obta195 + Cys224, Cys176 + Cys224, Cys48 + Cys224, Cys48 + Cys545 and weakly for Cys167 + Cys224, indicating S-S bond formation within specific pairs of engineered Cys residues (48 + Cys224), in the absence of reducing agent, we reproducibly obtained two species migrating above and below the reduced form, probably corresponding to different conformations of the denatured cross-linked protein. In contrast, no loop other than L1 or L8 was found to cross-link with these periplasmic regions, and none cross-linked with the Cys residue in the POTRA1 domain. Thus, in vivo, the last portion of the H1-POTRA1 linker can be found close to the extracellular loops L1 and L8 that immediately follow and precede the first and last β−barrel strands, B1 and B16, respectively, and the b5-b6 β hairpin and the H4 helix of the POTRA2 domain can be found close to the extracellular loop L1, indicating that these periplasmic elements approach the β−barrel seam in specific conformers.We combined Cys residues at the extracellular surface at positions 224 in L1, 290 in L3, 342 in L4, 391 in L5, 503 in L7 and 545 in L8 with periplasmic Cys residues in the POTRA2 domain at positions 167, 176, 187, and 195, in the linker at position 48, and in the POTRA1 domain at position 86 . None ofresidues . For Fha1H, 13C-isotope labeling of isoleucine (Ile) δ1 methyl groups  relaxation dispersion experiments probing exchange between states with different chemical shifts on the µs time scale  or in a sample with a diamagnetic MTSL analog attached to the same residue (FhaC220R1dia), respectively .To specifically probe for alternative FhaC conformations, we performed PRE NMR experiments in which a paramagnetic MTSL spin label is attached to an engineered Cys in the protein, and attenuation of NMR signals of nuclei within a radius of about 25–30 Å around the MTSL probe can be detected even if they only transiently approach the probe . To probectively . While a220R1dia, in agreement with expectations, residues more than 35 Å away from the position of the paramagnetic MTSL tag modelled onto the FhaC crystal structure  exhibited the highest Ipara/Idia ratios, while residues expected to be within 16–25 Å of the paramagnetic center  showed attenuation of their NMR signals  detergent micelles and in proteoliposomes. The spectra of FhaC187R1+503R1 and FhaC195R1+503R1 in proteoliposomes exhibited signal broadening compared with the proteins in bOG , the spectral changes for the other two proteins most likely reflect proximity of the spin probe at positions 187 and 195 to the lipid bilayer.We introduced a Cys residue at position 503 in the extracellular L7 loop, using it as a fixed reference position in the protein, as in previous PELDOR experiments , and com195R1+503R1 and FhaC187R1+503R1, the main populated states correspond to distance distributions between the two spin probes that are consistent with distances calculated using MTSL rotamer libraries attached to the corresponding residues of the FhaC crystal structure   to chara domains . Thus, tC4+C391 in which H1 is locked inside the barrel by an S-S bond and thus cannot move out  and L + H4  of the POTRA2 domain, b2-b3 of the POTRA1 domain, Lk, a non-structured peptide from the linker region between H1 and the POTRA1 domain, and the N-terminal β hairpin of the FhaB transport substrate, Fha-NT , all three peptides increased the CCS of the compact state of FhaC by rather small increments of 91–92 Å2  and in a barrel-distal region of the POTRA2 domain (Asp165-Lys171) with Cys, and we determined the effects of these mutations on secretion activity (157-Cys245 and Cys184-Cys245 S-S bonds were compatible with FhaC secretion activity (165 + Cys171 substitutions abolished it in a dsbA+ background, but not in a dsbA- background. Therefore, FhaC activity requires flexibility of the H4 helix N terminus, most likely because this region moves in the course of secretion. In contrast, transport activity does not require disruption of the interface between the first periplasmic turn of the barrel and barrel-proximal elements of the POTRA2 domain.The alternative FhaC conformers suggested by our data imply a re-orientation of the POTRA2 domain relative to the barrel. To probe this activity . S-S bonactivity , whereasAs Omp85 proteins are thought to perform their functions in the absence of an energy source in the periplasm, their postulated conformational cycling must involve low energy barriers between conformers, as reported for BamA . Here, wAll structural elements of TpsB transporters are connected with one another, structurally and functionally, and their motions appear to be coupled . In the In this work we obtained evidence for further conformational changes involving the POTRA2 domain. Thus, EPR, NMR, and S-S cross-linking data revealed that parts of the POTRA2 domain approach the barrel seam and the extracellular side of FhaC in specific conformers, and structural MS experiments indicated the binding of a synthetic peptide corresponding to the last portion of POTRA2 to the β barrel. We propose that this peptide binds to the B1 strand of the partially unzipped barrel seam based on the following reasoning. The number of unfolding transitions we observed in CIU experiments in FhaC and control β barrel proteins corresponds to the number of their soluble domains, which are generally more likely to unfold in these experiments than lipid- or detergent-embedded β barrels . At highOur EPR, NMR, and S-S crosslinking results indicate that the last portion of the POTRA2 domain approaches the extracellular side of the protein spontaneously. Since the b5-b6 peptide immediately precedes the B1 strand of the barrel, interaction of these regions in a partially opened β−barrel seam is likely facilitated in the native protein compared to the situation in MS experiments. The b5-b6 hairpin is amphipathic and exhibits suitable charge partitioning for β augmentation of the B1 strand. Such an interaction would be in line with the molecular mechanisms of BamA and Sam50, in which the unzipped B1 strand templates folding of client proteins by β augmentation .We thus propose that the spontaneous conformational changes of the POTRA2 domain initiate secretion as follows. Exit of H1 facilitates partial unzipping between B1 and B16, coupled with partial unfolding of the POTRA2 domain and a swing motion of its b5-b6 hairpin - and possibly also H4 - towards the barrel seam. Partial disruption of the barrel seam enables binding of part of the b6 strand of the flipped POTRA2 hairpin to barrel strand B1. Since the groove between H4 and b5 is the substrate binding site , this coAlthough the next secretion steps are speculative, we propose the following model based on earlier work. The substrate downstream of the segment bound to b5 forms a hairpin inside the channel, and the entropic cost of confining a portion of the unfolded polypeptide facilitates its diffusion toward the surface . We havein vitro translocation assay, it has been shown that FhaC mediates protein secretion without the need for cofactors or energy (Using an r energy , and thir energy , FhaC car energy .According to our model, the POTRA2 domain partially breaks up during secretion and must thus reassemble after secretion is completed. This may be mediated by interactions between the periplasmic turn T1 of the barrel and barrel-proximal residues of the POTRA2 domain. The observation that replacement of these interactions by S-S bonds is compatible with FhaC activity indicates that they represent fixed points in the protein. By anchoring the POTRA2 domain to the barrel, these interactions may ensure that FhaC can regain its resting conformation after secretion, which is necessary to limit outer membrane permeability. Consistent with this hypothesis, disrupting the conformation of the periplasmic turn T1 yielded transient, very large channels as detected in electrophysiology experiments . Stabiliin vitro and in vivo approaches and establishes mechanistic links between TpsB transporters and Omp85 insertases.In summary, we propose a novel mechanism for initiation of protein transport in TPS systems based on large-scale, spontaneous conformational dynamics of the TpsB partner. Our model integrates and explains currently available data from several complementary


S. cerevisiae. Instead, we found that the CP subunits α1, α2, and α4 each form independent small complexes. Purification of such complexes containing α4 revealed the presence of chaperones of the Hsp70/Ssa and Hsp110/Sse families. Consistently, certain small complexes containing α1, α2, and α4 were not formed in strains lacking these chaperones. Deletion of the SSE1 gene in combination with deletions of PRE9 (α3), PBA3, or UMP1 genes resulted in severe synthetic growth defects, high levels of ubiquitin-conjugates, and an accumulation of distinct small complexes with α subunits. Our study shows that Hsp70 and Hsp110 chaperones cooperate to promote the folding of individual α subunits and/or their assembly with other CP subunits, Ump1, and Pba1-Pba4 in subsequent steps.Whereas assembly of the 20S proteasome core particle (CP) in prokaryotes apparently occurs spontaneously, the efficiency of this process in eukaryotes relies on the dedicated assembly chaperones Ump1, Pba1-Pba2, and Pba3-Pba4. For mammals, it was reported that CP assembly initiates with formation of a complete α-ring that functions as a template for β subunit incorporation. By contrast, we were not able to detect a ring composed only of a complete set of α subunits in  Thermoplasma, the proteasome of which is formed only by one kind of α and β subunits, led to the proposal of a biogenesis model according to which the formation of a homo-heptameric ring of α subunits would create a platform for β ring incorporation [Rhodococcus, with a more complex proteasome composed of two distinct α and two different β subunits, this model apparently does not apply. Instead, α-β dimers were initial intermediates leading to 15S PC formation [Methanococcus maripaludis are able to form single ring structures, functional proteasomes were even assembled from mutant α subunits incapable of forming such α rings indicating that assembly occurred in a α ring-independent manner [S. cerevisiae, the proteasome of which is also constituted of 14 distinct subunits, there is no report identifying α-rings as assembly intermediates. Instead, precursors containing Ump1 [The ubiquitin/proteasome system (UPS) of eukaryotic cells provides an essential proteolytic control system, malfunctions of which are linked to various human diseases ,2. A cenporation . Howeverormation . Anothert manner . In mammt manner ,27. In ting Ump1  and β2, ing Ump1 . More reing Ump1 .In the present work, we dissected early assembly steps that lead to the 15S PC in yeast. We found that α1, α2, and α4 subunits assemble into small complexes, formation of which depends on Hsp70 and Hsp110 chaperones. These complexes accumulate either in the absence of the α3 subunit, or when the dedicated chaperones Ump1 or Pba1-Pba4 are missing. The Hsp70-Hsp110 chaperone system likely promotes the correct folding of individual subunits and/or the assembly with other subunits and dedicated chaperones.PRE6 and SCL1 under the control of the PGAL promoters were grown to an OD600 of 0.6 in media containing galactose, washed with sterile distilled water, resuspended in media containing glucose, and further incubated for the indicated periods of time. Spot assays were prepared as follows: 2 mL of overnight cultures were diluted in 5 mL of the appropriate media and grown until an optical density at 600 mm (OD600) of about 0.8 was reached. The cultures were then diluted with sterile water to OD600 of 0.5 in a total volume of 200 μL. From this suspension, sequential 5 μL of 1:10 dilutions were spotted onto plates, and the cells allowed to grow for 1–3 days. An SSE1 disruption cassette was obtained by amplifying the TRP1 gene from pFA6a-TRP1 [sse1∆::TRP1 strain (PR93) was prepared by gene replacement in the strain JD47-13C. Construction of a set of strains, each with an individual endogenous chromosomal locus of one of the 14 CP subunits encoding genes modified such that a C-terminally tagged version is expressed, was performed as follows. Using primers that contained flanking EcoRI and KpnI sites, 5’∆ fragments of the respective genes were amplified by PCR. These sites were then used to insert the amplified fragments into integrative plasmids containing sequences encoding epitope tags followed by the terminator sequence of the CYC1 gene (TCYC1). These plasmids were based upon YIplac128 (LEU2 marked), YIplac211 (URA3 marked), or YIplac204 (TRP1 marked) [S. cerevisiae genome. The resulting strains contained one copy of the respective gene expressed from its natural promoter and fused in-frame to the tag-coding sequence, followed by TCYC1, in addition to a 5’∆ fragment of the same gene without promoter. The epitope tags used were 2xHA (“HA”) or FLAG-6His (“FH”) [PRE6 and SCL1 with the PGAL1 promoter (PGALS) [kanMX4 marker). Correct insertion of PGALS in front of the desired genes was verified by analytical PCR [Yeast strains used in this work are listed in A6a-TRP1 . The sse marked) . Each ofs (“FH”) . All pla (PGALS) . In thes (PGALS)  containiical PCR . 2, 2 mM ATP, and 15% glycerol) [600 0.8–1.2) were harvested at 3000× g rcf, washed with cold water, frozen in liquid nitrogen, and stored at −80 °C. The cells were lysed with glass beads  in extraction buffer by vortexing. Cell debris was removed by centrifugation at 15,800× g rcf at 4 °C. The protein content in the supernatant was determined using the Bradford protein assay from Bio-Rad . For gel filtration studies, yeast cells from 200 mL exponentially growing cultures were collected by centrifugation (OD600 0.8–1.2). Extraction buffer was added in a proportion of 2 mL/g of pelleted yeast cells. Cell paste was ground to powder in a mortar in the presence of liquid nitrogen. After centrifugation at 31,000× g rcf for 10 min at 2 °C, the supernatant was subjected to a second centrifugation at 60,000× g rcf for 30 min at 4 °C. The protein concentrations in the extracts in parallel experiments were adjusted to 5 mg/mL using extraction buffer.Total protein crude extracts analyzed by native electrophoresis or by gel filtration were prepared in 26S buffer  . For a nSamples containing 1 mg of total protein in a total volume of 200 μL were separated on a Superdex200 column equilibrated with 26S buffer and coupled to an ÅKTA FPLC . The flow rate was 0.35 mL/min, and fractions of 0.5 mL were collected. The Superdex200 column was calibrated using the following standards: ferritin (440 kDa), catalase (232 kDa), bovine serum albumin (67 kDa), and ovalbumin (43 kDa); dextran blue was used to monitor the void volume .600 of 1.2. The cells were pelleted and frozen in liquid nitrogen. The cells were reduced to powder with the help of a mortar in the presence of liquid nitrogen with 26S buffer with protease inhibitors  in the proportion 2 mL/g wet weight. Cell debris were removed by centrifugation at 30,000× g rcf for 30 min at 4 °C. The supernatant was subjected to a buffer exchange with the help of a PD-10 column  equilibrated with FLAG buffer . The eluted proteins were added to 1 mL of anti-FLAG resin equilibrated with 15 mL FLAG buffer for 2 h and 30 min in batch with rotation at 4 °C. The suspension was transferred to an empty column. The flow-through was collected, and the FLAG resin was washed 3 times with 20 mL FLAG buffer. The bound proteins were eluted sequentially with 1 mL elution buffer containing FLAG peptide at a concentration of 50 µg/mL, 100 µg/mL, and 200 µg/mL. The eluted material was concentrated with a 15 mL Centricon Biomax 5K . Two hundred microliters of the concentrated protein solution were injected in a Superdex200 column equilibrated with 26S buffer. Proteins from the relevant steps were analyzed by SDS-PAGE. After silver staining, the detected bands were excised and analyzed by LC-MS at the Centre of Molecular Medicine Cologne (CMMC) Central Bioanalytics Unit (ZBA).Cells from the strain JM12 expressing α4/Pre6-FH were grown at 30 °C in a culture volume of 4 L in SD without tryptophan until an ODm/v) bromophenol blue). Eight micrograms of total protein per lane were applied in Tris-HCl 4–15% gradient gels . The native PAGE was run in an ice box at 16 mA, until the bromophenol blue dye reached the edge of the glass plate in 25 mM Tris-HCl, 192 mM glycine, pH 8.3. The gels were incubated for 15 min in transfer buffer  SDS, and 20% (v/v) methanol) containing additionally 1% (m/v) SDS. The proteins were blotted at 0.8 mA/cm2 for 2 h. For SDS-PAGE, the protein samples were boiled for 3 min in the presence of 2% SDS and 0.1 M 2-mercaptoethanol, then subjected to 12% SDS-PAGE, and thereafter transferred onto a PVDF membrane (Millipore) in a dry blot system . The blots were incubated with either mouse anti-ubiquitin P4D1 , mouse anti-HA 16B12, rat 3F10-POD conjugated, or M2 anti-Flag monoclonal antibodies  and were processed as described [Native samples, freshly prepared, were diluted in native gel sample buffer . Smaller assembly intermediates en route to the 15S PC can be well analyzed in a gel filtration column such as Superdex200 that separates globular proteins in the range of 10–600 kDa. We prepared distinct strains each containing a C-terminally HA-tagged version of one of the 14 CP subunit. From each strain, we analyzed crude extracts by gel filtration. Complexes such as 20S CP or 26S proteasomes were eluted largely in the void volume of the column  tag, we performed an affinity purification followed by a gel filtration A,B. The Bands derived from fraction 25 were excised from the gel and analyzed by mass spectrometry C. The maS. cerevisiae [Proteasome PCs associated with the Hsp70 chaperones were first observed for mammalian proteasomes . More rerevisiae . These crevisiae . The autrevisiae . SSE1 gene causes slow growth, whereas sse2 null mutants have apparently no growth defect. The combination of both deletions is lethal [sse1∆ strain, and compared the total protein crude extracts from this strain and the respective WT by native PAGE. As expected, HA signals were detected in the 15S PC, as well as in the 20S and 26S proteasomes, in both backgrounds. In addition, in the WT, α4 was detected in two fast migrating bands, named α4 complex 0 (α40) and α4 complex +C (α4+C)    A. StrikiC (α4-C) A. Extrac and +C) B. In anomplex 0 α and α4 c of α4-C C. PRE6 gene was switched to PGAL1, and the α4 subunit tagged with HA. Cells grown in galactose overexpress PRE6 and grow like WT. A switch to glucose results in the repression of α4 subunit synthesis. We followed the repression process over time and could observe that 20S CPs, once formed, did not disappear over a longer period of time. By contrast, similar to the short-lived 15S PC, the newly identified α4-containing complexes rapidly disappeared and were hardly detectable after 2 h of repression  combined either with WT SSA1 or with a temperature-sensitive (ts) version of SSA1 . We HA-tagged α1 and α4 in these strains and the respective WT. Protein crude extracts were analyzed by native PAGE and anti-Ha Western blotting. Compared to the WT, the absence of Ssa2, Ssa3, and Ssa4 (∆∆∆) resulted in a reduction of +C complexes containing α1 or α4 . We additionally asked whether a more direct effect of the deletion SSE1 on proteasome biogenesis can be detected. To address this question, we compared proteasomal complexes containing an HA-tagged version of the β2/Pup1 subunit in WT and sse1∆ cells by gel filtration , but it also interacts through a large surface with α5 (buried area: 1309 Å), and with the β4 and β5 subunits, although with the two last ones only through much smaller surfaces  . Curiousteasomes . In compThe formation of an α ring as a template for β subunit incorporation is the currently prevailing model for eukaryotic proteasome assembly ,10,11. IOur study reveals that Hsp70 and Hsp110 chaperones contribute to efficient assembly of early complexes involving α1, α2, and α4 subunits. These chaperones likely promote proper folding of the nascent subunits and/or prevent spontaneous self-association or aggregation. The ability to self-assemble is evident from in vivo data showing that two α4 subunit can incorporate next to each other in an α ring in strains lacking the α3 subunit ,47. The Trypanosoma brucei α5 [E. coli were reported to result in the formation of two or four stacked homo-heptameric rings, respectively. These findings demonstrated that multiple surfaces also of these subunits promote homomeric interactions either within a ring or between rings. The yeast α1, α2, and α4 subunits may similarly also be able to self-associate forming unproductive homomers. The observation that complexes containing individual ones of these subunits, but lacking the Hsp70 and Hsp110 chaperones (designated “αx0”), eluted from a gel filtration column between the 67 kDa  and the 232 kDa markers  is consistent with this possibility. Our data are insufficient to estimate the oligomeric states of the subunits in these complexes. Whether they correspond to homo-heptameric rings (~200 kDa) as reported for other α subunits (see above) has to be further investigated. Another open question is whether the observed αx0 complexes are dead-end products or could serve as a resource that may release free subunits for proper CP assembly under certain conditions, possibly assisted by Hsp70 and Hsp110 chaperones [Aside from the above-mentioned observation made for α4 in yeast and human cells, expression of protozoan rucei α5  or humanrucei α5  in E. coaperones .C disappeared giving rise to complex α4-C (We have shown here that α1 and α4 form complexes with Ssa1 and Sse1 in an ATP/ADP manner. Hsp70-assisted polypeptide folding is thought to occur in ADP/ATP-dependent cycles with substrate binding occurring in the ADP-bound state, whereas substrate release occurs when the chaperone is in the ATP-bound state ,51. For lex α4-C C. Taken together, our identification of complexes of α1, α2, or α4 subunits with Hsp70 and Hsp110 suggests that these two chaperones might cooperate to prevent unproductive homomeric interactions of these α subunits and thereby increase the efficiency of CP assembly. Consistent with this notion is the observation that increased amounts of precursor complexes were detected in cell lacking Sse1 E. Dissec