Parkinson’s disease (PD) is associated with motor and non-motor symptoms and characterized by aggregates of alpha-synuclein (αSyn). Naturally occurring antibodies (nAbs) are part of the innate immune system, produced without prior contact to their specific antigen, and polyreactive. The abundance of nAbs against αSyn is altered in patients with PD. In this work, we biophysically characterized nAbs against αSyn (nAbs-αSyn) and determined their biological effects. nAbs-αSyn were isolated from commercial intravenous immunoglobulins using column affinity purification. Biophysical properties were characterized using a battery of established in vitro assays. Biological effects were characterized in HEK293T cells transiently transfected with fluorescently tagged αSyn. Specific binding of nAbs-αSyn to monomeric αSyn was demonstrated by Dot blot, ELISA, and Surface Plasmon Resonance. nAbs-αSyn did not affect viability of HEK293T cells as reported by Cell Titer Blue and LDH Assays. nAbs-αSyn inhibited fibrillation of αSyn reported by the Thioflavin T aggregation assay. Altered fibril formation was confirmed with atomic force microscopy. In cells transfected with EGFP-tagged αSyn we observed reduced formation of aggresomes, perinuclear accumulations of αSyn aggregates. The results demonstrate that serum of healthy individuals contains nAbs that specifically bind αSyn and inhibit aggregation of αSyn in vitro. The addition of nAbs-αSyn to cultured cells affects intracellular αSyn aggregates. These findings help understanding the role of the innate immune systems for the pathogenesis of PD and suggest that systemic αSyn binding agents could potentially affect neuronal αSyn pathology.  Parkinson’s disease (PD) is the second-most common neurodegenerative disorder. The characteristic pathological changes consist of dopaminergic neuronal loss in the substantia nigra, gliosis, and intraneuronal alpha-synuclein (αSyn) pathology, comprising Lewy bodies and Lewy neurites . αSyn isSNCA and localized in presynaptic terminals [αSyn is a natively unfolded protein of 140 amino acids encoded by the gene erminals . αSyn coerminals ,18. The erminals . The C-terminals ,21, and erminals . The discovery of αSyn pathology in PD led to a plethora of immunization studies ,23. DiffNaturally occurring antibodies (nAbs) are part of the innate immune system, produced without contact to the specific antigen they recognize and polyreactive . The majIntracellular αSyn aggregates are transported towards the perinuclear aggresome and degraded by autophagy ,48. In tGiven the importance of better understanding PD pathogenesis and providing effective therapies for patients, reproducing findings is of particular importance in Translational Neurosciences . Basic rnAbs-αSyn were isolated from IVIG as previously described . BrieflyThe indicated amounts of recombinant αSyn were spotted onto nitrocellulose membranes , dried for 15 min and blocked for 1 hour at 20 °C once in RotiBlock . Each dot blot was incubated with either 0.2 µg/mL nAbs-αSyn, FT or anti-αSyn antibody  over night at 4 °C and washed 3 times with Tris buffered saline with 0.05% Tween-20 (TBS-T) for 30 min. As secondary antibody, horse reddish peroxidase (HRP) coupled to anti-human IgG antibodies  was used to detect the antibodies of human source (nAbs-αSyn and FT). Anti-mouse antibodies  were used to detect the anti-αSyn antibody. Blots were developed with Super Signal West Femto Max Sensitivity Substrate  on Bio-Rad ChemiDoc. . The experimental setup is schematically depicted in Figure 1a.Wells of high-binding 96-well ELISA plates  were coated with 50 µL of 3 µg/mL recombinant αSyn  in phosphate-buffered saline (PBS). After 24 h at 4 °C, wells were blocked with 180 µL blocking buffer  for additional 24 h at 4 °C. Antibodies were added to the wells in a series of decreasing concentrations starting from 4 µg/ml. Plates were incubated with samples for 1 h at 20 °C and shaken on an orbital platform shaker  at 100 rpm. Afterwards, 50 µL/well biotinylated goat anti-human detection antibody was added to the wells . After 1 h at 20 °C and 100 rpm, the samples were incubated with 50 µL/well of streptavidin-peroxidase  and shaken for 20 min at 20 °C and 100 rpm. The assay was developed using 50 µL/well Tetramethylbenzidine (TMB) . After 20 min, the reaction was stopped by adding 25 µL/well of 5% sulfuric acid . Absorbance at 450 nM was measured using a microplate reader . All incubation steps were performed in the dark. After each step up to and including TMB addition, the wells were washed three times with 300 µL 1× PBS + 0.05% Tween-20 using an Amersham Biotrak II Plate Washer .SPR measurements were performed using a Biacore T200 instrument  at 25 °C with PBS/0.05% Tween-20, pH 7.4 as running buffer. For preparation of the flow cells, a CM5 sensor chip  was activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) (0.2 M/0.05 M). Recombinant full-length αSyn (200 ng/mL) was diluted in 10 mM sodium acetate, pH 4.0. It was immobilized to a final level of 125 reactive units (RU), and the flow cell was deactivated with 1 M ethanolamine-HCl, pH 8.5. A reference flow cell was activated and deactivated only. Afterwards, IVIG, FT and nAbs-αSyn at concentrations ranging from 39 nM to min. 5 μM were injected once followed by 5 cycles of regeneration with 5 M urea. All samples were injected over the flow cells for 300 s, followed by a dissociation step of 900 s. The sensorgrams were double referenced. Evaluation was performed by plotting the respective response levels against the applied nAbs-αSyn concentrations in a steady-state fit. For data evaluation, the sensorgrams were calculated in the Biacore T200 Evaluation Software 3.1 .2, 10 μM ThT, and 0.05% NaN3, reflecting intracellular potassium and magnesium concentration, as well as intracellular pH and ionic strength. A glass ball of 2.85–3.45 mm size was added per well to improve mixing. Per well, a sample volume of 125 μL sample was used. For evaluation, the mean of triplicate measurements was referenced to the highest fluorescence of 25 μM WT αSyn.ThT aggregation assays were conducted as described before  in Corni2O and then dried again with a stream of N2 gas before imaging.AFM images were taken with a Nanowizard 3 atomic force microscope  in intermittent contact mode (AC mode) in air, using silicon cantilever and tip  with a typical tip radius of 9 ± 2 nM, a force constant of 26 N/m and a resonance frequency of approximately 300 kHz. The images derive from a manual observer-blind estimation, and provide qualitative characteristics. The image processing was performed using JPK data processing software (version spm-5.0.84): for each of the presented height profiles, a polynomial fit was subtracted from each scan line first independently and then using limited data range. For the sample preparation, solutions containing fibrils were diluted to a concentration of 1 µM  in water and 5 μL samples of the diluted solution were deposited on freshly cleaved muscovite mica and left to dry for at least 30 min. The samples were carefully washed with 50 µL of double distilled HHEK293T (RRID: CVCL_0063) cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and 1% penicillin-streptomycin. ‘HEK’ cells are listed in version 8.0 of the Database of Cross-Contaminated or Misidentified Cell Lines. Our HEK293T cells were validated in November 2017 by analysis of 21 genetic loci . Cells were plated on poly-L-lysine-coated glass cover slips or on plastic plates. Transfection was performed 18–20 h after plating using Metafectene . Unless noted otherwise, subsequent analysis was completed 24 h after transfection.CytoTox 96 Non-Radioactive Cytotoxicity Assay  was performed according to the manufacturer’s protocol to measure release of lactate dehydrogenase (LDH) as a marker of cellular viability, as described before . HEK293TTo assess metabolic activity a CellTiter-Blue (CTB) Cell Viability Assay was performed according to manufacturer’s protocol as described before . HEK293TA53T-αSyn was flexibly tagged with EGFP by using the interaction of a PDZ domain (post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), and zonula occludens-1 protein (zo-1)) with its six amino acid-binding motifs. PDZ-EGFP was coexpressed with αSyn to which a six amino acid PDZ binding domain was added at the C-terminus .Analysis of cells expressing EGFP-tagged αSyn was carried out as previously described ,54. For P < 0.05 was considered significant. In graphs, p-values are depicted as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.The study was not pre-registered. For assignment of experimental groups, no special randomization methods were employed. Sample sizes were determined by past experience and not by a statistical sample size calculation . For statistical analysis and data presentation, we used GraphPad Prism 5.0 and 6.0 . Graphs represent mean SEM. The tests used for comparison in each graph are noted in the figure legend . The experiments were replicated independently at least three times with three replicates, representative graphs are shown. nAbs-αSyn were isolated from commercial human IVIG preparations using gravity flow affinity purification as described previously . nAbs-αSKD) was not determined from the binding kinetics but from a steady state fit, yielding a value of KD = (4.22 ± 0.95) × 10−6 M. nAbs-αSyn showed a higher affinity than IVIG and FT  and incubated with the prey protein (the indicated probes containing antibodies) in the fluid phase  . Next, wIn this work, we characterized nAbs for αSyn that were purified from commercial immunoglobulin preparations using biophysical methods and cultured cell lines. nAbs-αSyn bound αSyn specifically and were able to affect the subcellular distribution of αSyn, suggesting that they can be taken up by cells.nAbs-αSyn can be isolated from IVIG preparations, as we and others have demonstrated ,45,46. IKD [KD of 4 µM is an order of magnitude higher than the value previously determined from the binding kinetics [nAbs-αSyn were isolated from IVIG using gravity flow affinity purification, i.e., they are defined by binding recombinant αSyn protein. In order to obtain an estimate of the affinity of nAbs-αSyn for αSyn, we measured the association and dissociation by SPR. Although the Langmuir model used previously to calculate the KD  is perfekinetics . It shouBinding of nAbs-αSyn to αSyn is further confirmed by the fact that they inhibit aggregation of αSyn in vitro . ImportaOur study focuses on the behavior of nAbs-αSyn. In addition, more than 50 non-naturally occurring antibodies against αSyn have been established and described, including engineered antibodies and antibody fragments to target αSyn for various purposes in the field of research, diagnostics, therapeutic approaches, and biomarkes . These aWith regard to the potential mechanism of aggregation inhibition, we have shown here that binding of α-synuclein monomers can lead to potent, substoichiometric inhibition of aggregation, in the case when the 1:1 complex of α-synuclein and binding protein acts as an inhibitor of nucleation processes . MoreoveIn order to test whether inhibition of αSyn aggregation observed in vitro translates to an effect in living cells, we used the HEK293T cell line. Adding nAbs-αSyn to these cells did not alter viability . We analIn summary, we confirmed the presence of nAbs that specifically bind αSyn monomers in IVIG preparations obtained from healthy donors. These nAbs alter αSyn aggregation and fibril conformation in vitro and affect intracellular αSyn processing. Collectively, these findings are consistent with an involvement of the innate immune system in the pathogenesis of PD and with potential therapeutic effects of antibody preparations in patients with PD. Yet, several important questions remain unsolved, such as the effects of nAbs on microglia and astrocytes in the brain, the role of peripheral as opposed to central αSyn deposits for PD pathogenesis, and the species or epitopes of αSyn that are targeted by nAbs.


Combating the worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of new variants demands understanding of the structural basis of the interaction of antibodies with the SARS-CoV-2 receptor-binding domain (RBD). Here, we report five X-ray crystal structures of sybodies (synthetic nanobodies) including those of binary and ternary complexes of Sb16–RBD, Sb45–RBD, Sb14–RBD–Sb68, and Sb45–RBD–Sb68, as well as unliganded Sb16. These structures reveal that Sb14, Sb16, and Sb45 bind the RBD at the angiotensin-converting enzyme 2 interface and that the Sb16 interaction is accompanied by a large conformational adjustment of complementarity-determining region 2. In contrast, Sb68 interacts at the periphery of the SARS-CoV-2 RBD–angiotensin-converting enzyme 2 interface. We also determined cryo-EM structures of Sb45 bound to the SARS-CoV-2 spike protein. Superposition of the X-ray structures of sybodies onto the trimeric spike protein cryo-EM map indicates that some sybodies may bind in both “up” and “down” configurations, but others may not. Differences in sybody recognition of several recently identified RBD variants are explained by these structures. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a β-coronavirus, is remarkable for its high infectivity, rapid worldwide dissemination, and evolution of highly infectious new variants , 2, 3, 4Escherichia coli and purified via metal-affinity chromatography to high purity. These sybodies behaved as monomers by size-exclusion chromatography (SEC)   EscherichTo gain insight into the precise topology of the interaction of four of these sybodies with the RBD, we determined crystal structures of their complexes: Sb16–RBD and Sb45–RBD, the ternary Sb45–RBD–Sb68 and Sb14–RBD–Sb68, and Sb16 alone. These crystals diffracted X-rays to resolutions from 1.7 to 2.6 Å . After mA and B) revealed little difference between insect-expressed (A and 3A) illustrates that complementarity-determining region 2 (CDR2) (residues 50–60) and complementarity-determining region 3 (CDR3) (residues 98–106) bestride the saddle-like region of the ACE2-binding surface of the RBD . Sb16 angulates over the RBD by 83°. However, Sb45 (B and 3B) straddles the RBD saddle in the opposite orientation, at an angle of −36°, and frames the interface with CDR2 (residues 50–59) and CDR3 (residues 97–111). Complementarity-determining region 1 (CDR1) of both sybodies (residues 27–35) lies between the CDR2 and CDR3 loops. Superposition of the two structures, based on the RBD, emphasizes the diametrically opposite orientation of the two , revealing that CDR2 of Sb16 and CDR3 of Sb45 recognize the same epitopic regions.The structure of the RBD of these complexes , A and Bxpressed  and our xpressed , 24 consxpressed A and 3A er, Sb45 B and 3B D) and Sb14–RBD–Sb68 at 1.7 Å resolution (E). The refined models revealed that while Sb14 and Sb45 interact with the ACE2 interface of the RBD, Sb68 binds the RBD at a distinct site (D and E). In the ternary complex, Sb45 binds in an identical orientation to that observed in the binary Sb45–RBD structure , but Sb68 addresses a completely different face of the RBD, similar to that bound by Fab of CR3022 on the RBD of SARS-CoV-2 , still permits Sb68 to bind to its epitope as seen in the Sb45–RBD–Sb68 structure (D).By exploring the conditions using mixtures of two or three sybodies and the RBD, we obtained crystals and solved the structures of ternary complexes consisting of Sb45–RBD–Sb68 at 2.6 Å resolution D and Sb1solution E. The renct site , D and ERS-CoV-2  and by VRS-CoV-1 . Of part  is quite different, predominantly exploiting nine CDR3, four CDR2, and one CDR1 residues at the interface (see Scrutiny of the different interfaces provides insights into the distinct ways each sybody exploits its unique CDR residues for interaction with epitopic residues of the RBD . Compil. Both Sbface see .A). Sb16 and Sb45 directly impinge on the ACE2 binding site, offering a structural rationale for their viral neutralization capacity (A).To evaluate the structural basis for the ability of these four sybodies to block the interaction of the RBD with ACE2, we superposed each of the three sybody–RBD structures onto the ACE2–RBD structure and examined the steric clashes A. Sb16 acapacity . Sb68, wcapacity A.Figure 2, 1040 Å2, 1003 Å2, 976 Å2, and 640 Å2, respectively (A–E). Sb16 and Sb45 capture more surface area than ACE2 or other published nanobody or sybody–RBD complexes (see 2) (D). The total BSA captured by Sb45 and Sb68 in the ternary complex is 1650 (1010 plus 640) Å2  interfaces between the RBD and ACE2 or the sybodies. The BSA at the ACE2–RBD, Sb14–RBD, Sb16–RBD, Sb45–RBD, and Sb68–RBD interfaces is 844 Åectively , A–E. Sbexes see . The int (see 2) D. The to 640) Å2  and is c  with ACE2 N546 carbohydrate as well as residues 313 and 316 to 218 (A). ACE2 used in the crystallographic visualization of ACE2–RBD , loops 61 to 64 with ACE2 N322 carbohydrate, and loops 87 to 89  (A), with CC values of 0.51, 0.49, and 0.58, respectively (A–C). Only two Sb45s bind to the 2-up, 1-down form of S-6P (7N0H/EMD-24106), with one on the up position of the RBD and the other on the down position of the RBD (B), with CC values of 0.51 and 0.71, respectively (D and E). It seems that Sb45 can bind all the down positions of the RBD. In particular, Sb45-Z binds well to RBD-C with higher CC values (C and E), with additional contacts to the neighboring (up position) RBD-A (A). These variations in saturation of the available conformations by Sb45 reflect the mobility of the RBD. Notably, the interfaces between Sb45 and the RBD of S-6P are the same as those in the crystal structure (7KGJ) , a stable S variant containing six beneficial proline substitutions  and acquruction) . We haveruction) A, with Cectively , A–C. On the RBD B, with Cectively , D and EC values , C and En) RBD-A A. These e (7KGJ) . MoreoveA and B). Sb14, however, reveals clashes when the Sb14–RBD complex is superposed on trimeric S in any down position (E). Sb68 could not be superposed without clashes to any RBD of the 3-down or to the 1-up, 2-down positions. The only permissible superpositions were to two in the 2-up, 1-down and to all three in the 3-up positions (F). For paired sybodies, Sb16 and Sb68 (B), or Sb45 and Sb68 (D), superposition was possible without clashes, with two or more RBDs in the up conformation. We also observed direct interactions of Sb14 and Sb68 in the Sb14–RBD–Sb68 X-ray crystal structure. Walter et al. (C).To gain additional insight into the structural consequences of the interactions of each of these sybodies with a trimeric S protein, we superposed each of the individual sybody–RBD complexes on S-6P of our cryo-EM structures (7N0G and 7N0H) . Sb16 anposition E. Sb68 cositions F. For paand Sb68 B, or Sb4and Sb68 D, superpr et al.  suggester et al. C.A). In general, the five sybodies that interact with the parental (designated WT) RBD with KD values of 6.8 × 10−9 (for Sb15) to 6.3 × 10−8 M (for Sb68) (B–E). For Sb16, Sb45, and Sb14, interaction with the N501Y mutant resulted in displacement of loops 496 to 506 by 2.0 Å, 1.0 Å, and 1.5 Å, respectively. Nevertheless, R60 of Sb16 and H103 of Sb45 maintained contact with N501Y. This suggests that N501Y mutation would not escape recognition by these sybodies. Other cryo-EM studies indicate modest effects of the N501Y substitution on binding to different antibodies (via K32 and Sb45 via R33 (D and E).The major circulating variants, specifically B.1.1.7 (United Kingdom), B.1.351 (South Africa), and P.1 (Brazil), contain mutations in the RBD that lead to increased binding affinity to ACE2 and have the potential to reduce vaccine efficacy , 41, 42.Fig. 6or Sb68)  showed dtibodies . In contE. coli systems, as noted by others . By superposition (A), Sb14 clearly belongs to class 1 because it completely covers the light chain of B38 Fab (7BZ5). Sb16 partially clashes with B38, but it primarily overlaps with the heavy chain of COVA2-39 (7JMP). It can bind both to up and down positions of the RBD in S (A). Sb45 clashes effectively with the heavy chain of COVA2-39, and our cryo-EM structures (7N0G and 7N0H) indicate that Sb45 can bind to both up and down forms of S-6P (A). By contrast, Sb68 competes mostly with the CR3022 heavy chain (6W41), VHH72 (6WAQ) (HH-U (7KN5)   and VHH-U (7KN5) , suggest  that illustrates the binding to two distinct epitopic sites. The ternary structure may also be considered as illustrative of the potential behavior of a bispecific construct linking two nanobodies. The bivalent or multivalent binding by the antibody or nanobody would be expected to increase neutralization potential . Sb45 exploits its two lengthy CDR2 and CDR3 loops that ride along both sides of the RBD surface, and Sb14 uses both CDR2 and CDR3 on the same side close to Sb68, while VHH-E uses a long CDR3 loop engaging one side of the RBD surface. Furthermore, Sb14 and Sb68 in Sb14–RBD–Sb68 (7MFU) show contacts  between two specific sybodies on the RBD surface (C), which emphasizes the potential benefit of using complementary, bivalent, or multivalent antibodies/nanobodies against the virus.The significance of the ternary structures of Sb45–RBD–Sb68 (7KLW) and Sb14–RBD–Sb68 (7MFU) is shown in a recent article  (Novagen). Expression and isolation of inclusion bodies, denaturation, and reduction were done in 6 M guanidine hydrochloride and 0.1 mM DTT as described elsewhere (The sequences encoding the RBD of the SARS-CoV-2 S protein (amino acids 333–529) were subcloned into pET21b(+) (Novagen) #145145, ) as the lsewhere . Brieflyet al.  supplemented with 25 μg/ml chloramphenicol, at 37 °C with shaking at 160 rpm for 2 h. The temperature was then decreased to 22 °C until A600 reached 0.5. Protein expression was induced by the addition of L-(+)-arabinose (Sigma) to a final concentration of 0.02% (w/v) and expression continued overnight at 22 °C and 160 rpm. The next day, cells were collected by centrifugation at 2000g for 15 min. The cell pellet was then washed twice in PBS and resuspended in the periplasmic extraction buffer ) at 4 °C for 30 min followed by the addition of TBS (pH 8.0) and 1 mM MgCl2. The cells were then centrifuged at 10,000 rpm (Fiberlite F21-8 x 507 Fixed-Angle Rotor) for 30 min. After transfer of the supernatant to a fresh tube, imidazole was added to a final concentration of 10 mM. Ni-NTA resin (QIAGEN) equilibrated with TBS was added to the supernatant and incubated for 1 h at room temperature with mild agitation. The resin was collected and washed three times with the buffer supplemented with 30 mM imidazole, and sybody proteins were eluted with 300 mM imidazole in TBS.Plasmids pSbinit encoding sybodies Sb14, Sb15, Sb16, Sb45, and Sb68  were originally reported by Walter et al.  and genelsewhere . Briefly6 viable cells/ml and grown overnight at 37 °C in Expi293 Expression Medium (Gibco). The following day, cell viability was determined, and cell density was adjusted to 3 × 106 viable cells/ml with fresh, prewarmed Expi293 Expression Medium. Transfection was then carried out as per manufacturer’s instructions using 1 μg/ml plasmid DNA. Cultures were grown for 6 days after transfection, and the supernatant was collected, filtered through a 0.22-μm filter, and passed over Ni-NTA resin for affinity purification. Further purification was accomplished by SEC using a Superose 6 10/300GL column (Cytiva) in a buffer consisting of 2 mM Tris, pH 8.0, and 200 mM NaCl. The purification of sybodies, RBDs, and S-6P is shown in Plasmid encoding S HexaPro (designated “S” throughout) was procured from Addgene (#154754)  and tranSybodies purified by Ni-NTA affinity chromatography were concentrated using Amicon 10K MWCO concentrators and purified on a Sepax SRT-10C SEC100 column at a flow rate of 1 ml/min. Monomeric sybodies elute at a retention volume of 11 to 12.5 ml from the Sepax SRT-10C SEC100 column. Monomeric peak fractions were collected and analyzed by SDS-PAGE. Analytical SEC of the RBD–sybody complexes was performed on a Shodex KW-802.5 column at a flow rate of 0.75 ml/min in TBS buffer (pH 8.0). .SPR experiments were performed on a Biacore T200 system (Cytiva) at 25 °C in 10 mM Hepes, pH 7.2, 150 mM NaCl, 3 mM EDTA, and 0.05% Tween-20. The RBD was immobilized on a series S CM5 sensor chip (Cytiva) by amine (N-hydroxysuccinimide/1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) coupling to flow cells. For background subtraction, a reference cell was mock coupled. Binding and kinetic studies were performed multiple times for each sybody. Graded and increasing concentrations of SB16, SB45, and SB68 were injected over the RBD-immobilized surface at a flow rate of 30 μl/min, with an association time of 120 s and dissociation time of 2000 s. Binding data were analyzed by surface site affinity distribution analysis with EVILFIT  and werem values for the RBD, S, Sb14, Sb15, Sb16, Sb45, and Sb68 . Each well contained 2- to 4-mg protein in the buffer  and 5× SYPRO Orange  in a total volume of 20 ml. After an initial 2-min hold at 25 °C, the plate was heated to 99 °C at a rate of 0.05 °C/s. The data were analyzed with Protein Thermal Shift Software, v1.3 (Invitrogen), to obtain Tand Sb68 .sptlabtech.com). The crystals of SB16–RBD and SB45–RBD complexes and Sb16 alone were observed within 1 week using Protein Complex (QIAGEN) and Wizard Classic 4 (Rigaku). The conditions for Sb16–RBD were either 0.1 M Hepes, pH 7.0, 15% PEG 20000, or 0.1 M Hepes, pH 7.0, and 18% PEG 12000, and for Sb45–RBD, were 18% PEG 12000 and 12% PEG 8000, 0.1 M Hepes, pH 7.5, and 0.2 M NaCl. The crystallization condition for Sb14–RBD–Sb68 was 12% PEG 8000, 0.1 M Mops, pH 7.5, and 0.1 M magnesium acetate. Sb16 alone crystallized in 20% PEG 4000, 0.1 M MES, pH 6.0, and 0.2 M LiSO4. We also screened mixtures of two or three sybodies with the RBD. The crystals of Sb45–RBD–Sb68 were obtained after 1 month after mixing the three proteins in an equimolar ratio in 10% PEG 8000 and 0.1 M sodium cacodylate, pH 6.0.Purified sybodies  and RBDs were mixed in an approximate 1:1 M ratio to a final concentration of 8 mg/ml. The protein mixtures were incubated on ice for 1 h before screening. Initial screening for crystals was carried out using the hanging-drop vapor-diffusion method using the mosquito robotic system (manaslu.fiserlab.org/MMM (H domain of 7BZ5. Molecular replacement solutions were found using Phaser (https://niaidsis.niaid.nih.gov), provided courtesy of Peter Sun, National Institute of Allergy and Infectious Diseases. Hinge calculates an ellipsoid  for the indicated domains and reports the angle between the long axes of the adjacent domains as the hinge angle. BSA calculations were performed with PISA (https://www.ebi.ac.uk/pdbe/pisa/). The final structures for the RBD–SB16 and RBD–SB45 complexes showed Rwork/Rfree (%) of 25.4/27.7 and 18.6/21.6, respectively, and for SB16 alone, Rwork/Rfree of 23.7/25.1. Data collection and structure refinement statistics are provided in Crystals of protein complexes were optimized with slight adjustments of the concentration of PEG components. The crystals were cryoprotected in the mother liquor containing 5% ethylene glycol and 5% glycerol and flash-frozen in liquid nitrogen for data collection. Diffraction data were collected at the Southeast Regional Collaborative Access Team beamline 22ID-D at the Advanced Photon Source, Argonne National Laboratory, and the data were processed with XDS . Multipl.org/MMM ), using g Phaser , 54. Subg Phaser . CDR loog Phaser . In part2. Two raw datasets were collected on two frozen grids: one with 1780 micrographs and one with 7945 micrographs.Freshly purified S-6P was incubated with Sb45 in a 1:3 M ratio and repurified by SEC. Negative stain screening was accomplished with a Tecnai 12 120-keV microscope (Thermo Fisher). We screened several sybody–S complexes for good negative staining images, and complexes of Sb45–S gave the best data. The protein complexes were concentrated to 0.7 to 1 mg/ml, and 3 μl of the sample was applied onto holey-carbon cryo-EM grids , which had been glow-discharged for 60 s, blotted for 3 s, and plunge-frozen into liquid ethane with a Vitrobot (Thermo Fisher Scientific) at 4 °C and 95% humidity. Cryo-EM data in selected grid regions were collected on a Titan Krios 300-keV microscope (Thermo Fisher). Images were acquired automatically with SerialEM  on a BioAb initio 3D reconstruction” classification and divided into two or four subgroups. After removing the particles of unrecognized or “defective” shape, a total of 417,460 particles with shape resembling S remained. These particles were subjected to “homogeneity refinement,” followed by “CTF global and local refinement” and “nonuniform refinement.” No symmetry was imposed aside from C1 during the map refinements. The map after refinement could reach 2.84 Å resolution by the gold-standard FSC estimation with a 0.143 cut-off criterion. We then further identified the two conformations of S-6P as previously described (C), and one subclass of 61,062 particles showed the conformation of “2-up, 1-down” (C). The maps of “1-up, 2-down” and “2-up, 1-down” were refined at 3.02 Å and 3.34 Å resolution, respectively. The local resolution plots for each map are shown in D and E. The maps are deposited in the Electron Microscopy Data Bank as EMD-24105 and EMD-24106.All image processing, 2D class, 3D reconstruction, and map refinements were performed with cryoSPARC, v3.1 and v3.2 , 36, 37.escribed . One subescribed C, and on 1-down” C. The maAn initial model for S-6P was generated using PDB ID: 6XKL and was fitted as a rigid body into the map using Chimera  followedwww.pdb.org) under accession numbers 7MFU, 7KGK, 7KGJ, 7KLW, and 7MFV for Sb14–RBD–Sb68, Sb16–RBD, Sb45–RBD, Sb45–RBD–Sb68, and Sb16, respectively. The cryo-EM maps of SB45+S-6P  and Sb45+S-6P  have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-24105 and EMD-24106, respectively, and their respective models under 7N0G and 7N0H.All data are included in the article or the supplemental material. The X-ray structure factors and coordinates are deposited at the Protein Data Bank (This article contains The authors declare that they have no conflicts of interest with the contents of this article.


The characteristics of Prussian blue analogues PBAs in different aqueoussystems are analyzed.The relationship between structure and performance of PBAs is summarized.The measures to improve electrochemical property of PBAs are proposed. In the applications of large-scale energy storage, aqueous batteries are considered as rivals for organic batteries due to their environmentally friendly and low-cost nature. However, carrier ions always exhibit huge hydrated radius in aqueous electrolyte, which brings difficulty to find suitable host materials that can achieve highly reversible insertion and extraction of cations. Owing to open three-dimensional rigid framework and facile synthesis, Prussian blue analogues (PBAs) receive the most extensive attention among various host candidates in aqueous system. Herein, a comprehensive review on recent progresses of PBAs in aqueous batteries is presented. Based on the application in different aqueous systems, the relationship between electrochemical behaviors  and structural characteristics  is analyzed and summarized thoroughly. It can be concluded that the required type of PBAs is different for various carrier ions. In particular, the desalination batteries worked with the same mechanism as aqueous batteries are also discussed in detail to introduce the application of PBAs in aqueous systems comprehensively. This report can help the readers to understand the relationship between physical/chemical characteristics and electrochemical properties for PBAs and find a way to fabricate high-performance PBAs in aqueous batteries and desalination batteries. As is known to all, energy is the source of economic development and the material basis for human survival. Therefore, energy has always been a common concern of the world. Traditional energy sources have brought many changes to the development of human society and the world. However, the excessive use of traditional energy produces carbon dioxide and other pollutants, causing serious ecological problems. To solve these environmental problems, scientists are beginning to explore renewable energy sources to reduce environmental pollution and meet the needs of social development –5. Howev. first explored the reversible inserted/extracted electrochemical property of K+ in K2FeIIFeII(CN)6 and opened the door of PBAs as electrode material ·wH2O in which A is the alkali metals and M and R represent the transition metal. It is worth noting that most of the R-site in PBAs for the aqueous battery is replaced by Fe atom. Here, the classic PBAs structure is illustrated in Fig. xM[R(CN)6]·wH2O is taken for example as a cathode. During the discharging process, host ions in the electrolyte are inserted into PBAs, which causes the R or M and R atoms to simultaneously undergo a reduction reaction to release energy. In the charging process, host ions are extracted from PBAs with R or M and R atoms simultaneous undergoing oxidation reaction to store energy. The charging and discharging process can be reflected by the following chemical reaction equation:With a metal–organic open framework, PBAs possess a general composition of AFurthermore, Fig. 3+/M2+ and Fe3+/Fe2+ couples, which means that PBAs can provide more capacity. The theoretical capacities of different PBAs in characteristic aqueous systems are summarized in Fig. −1. But the theoretical capacity utilization of NiFe-PBAs, CuFe-PBAs and ZnFe-PBAs is only 50% because Ni, Cu and Zn in PBAs are electrochemically inert. In addition to these structural advantages, PBAs also have some structural defects that need to be addressed. During the preparation process of PBAs through co-precipitation, it will inevitably bring some interstitial water and vacancies in PBAs crystals, and Fig. 6 vacancies, its random distribution can cause the distortion of PBAs lattice, thus reducing the structural stability. Especially for reduced-form PBAs, due to the oxidation reaction of transition metal elements, the structural instability is more serious. Furthermore, as shown in Fig. For PBAs, it possesses the 3D cubic structure and tunable chemical compositions that give PBAs some advantages when inserting ions with large hydrated ionic radius. Firstly, there are many available “A” sites with large interstitial (~ 4.6 Å diameter) in the body center of PBAs lattice. Meanwhile, the PBAs possess a special ions channel with approximately 3.2 Å diameter in the (100) direction , 88. The+, Li+, K+, H+ and NH4+ batteries. For aqueous Na+ and K+ batteries, PBAs usually show excellent cyclic stability due to the better structural compatibility and moderate hydrated radius for Na+ and K+. Usually, these PBAs in aqueous Na+ and K+ batteries can maintain more than 80% of the initial capacity within 300 cycles. Although PBAs have poor cyclic stability in aqueous Li+ batteries due to the large hydration ion radius of Li+ , mixed-ion batteries composed of Li+ and other metal ions have great application prospects in the improvement in energy density. Besides, the excellent rate performance and cycling stability of PBAs in aqueous H+ batteries and aqueous NH4+ batteries have also attracted more and more attention in recent years due to the special transport mechanism of H+ in aqueous electrolyte and the good structural compatibility of NH4+ and PBAs. This section will comprehensively introduce the application of PBAs in these aqueous monovalent-ion batteries.The application of PBAs in aqueous monovalent-ion batteries can be divided into different fields: aqueous Na+ batteries (ASIB), they can be divided into single-metal-atom redox PBAs  and double-metal-atom redox PBAs . The single-metal-atom redox PBAs mean that only Fe atom can undergo the redox reaction and provide capacity during the insertion and extraction of Na+. Therefore, as shown in Fig. +. Herein, for better comparison, PBAs in ASIB are cataloged into two groups: PBAs with single-atom redox and PBAs with double-atom redox.For the PBAs in aqueous NaxM[Fe(CN)6]y·nH2O, these types of PBAs belong to the single-metal-atom redox PBAs. The typical single-metal-atom redox nickel hexacyanoferrate (K0.6Ni1.2Fe(CN)6·3.6H2O, NiFe-PBA) was firstly synthesized by Cui and co-workers through spontaneous precipitation in aqueous solution as electrode materials for ASIB 0.87·3.02H2O and construct a practical ASIB with NaTi2(PO4)3 6]0.87·3.02H2O has advantages in reversible capacity and cycle stability compared with cubic Na1.21Ni[Fe(CN)6]0.86·3.21H2O [1.45Ni[Fe(CN)6]0.87·3.02H2O, the addition of chelating agents and surfactants reduces the crystallization rate and makes the crystal shape of monoclinic Na1.45Ni[Fe(CN)6]0.87·3.02H2O more uniform, thus increasing the contact area between the electrode and the electrolyte and shortening the migration path of ions. Thirdly, a slower reaction rate leads to the formation of higher crystallinity product with less interstitial water and vacancy, thus accelerating Na+ insertion/extraction rate. Moreover, an ex situ X-ray photoelectron spectroscope (XPS) at different charge/discharge states in Fig. + extraction/insertion. The result shows that the valance change only occurs in Fe atom, which fully explained the Ni atom in NiFe-PBAs with no electrochemical activity. Although NiFe-PBAs show acceptable performance in terms of electrochemical stability, its low redox potential is not suitable for aqueous energy storage applications.Later, Zhu and co-workers also synthesized Na-rich Na·3.21H2O . The reaFor the redox potential (E) of host ions in PBAs, there is a qualitative relationship :2\documeEredox is related to the potential of the redox couple itself. Eion-host is related to the interaction between host ions and PBAs framework, which usually increases as the radius of the ion enlarges. Eion-ion is related to the repulsion between host ions in PBAs. Since the redox couple of single-metal-atom redox PBAs is only [Fe(CN)6]3−/[Fe(CN)6]4−, the redox potential will not differ much between them when the same ion is inserted in PBAs. However, the increase of electrons in the antibonding orbital (eg) of M atom in PBAs can cause [Fe(CN)6]3−/[Fe(CN)6]4− redox potential to move toward more positive values ) as cathode for ASIB [2Cu1-xNix[Fe(CN)6] cathode, it can exhibit a discharge capacity of 56 mAh g−1 with a capacity retention of 96% after 1000 cycles. Impressively, its redox potential can be adjusted from 0.6 to 1.0 V with the increased content of Cu.The value of e values . Thus, ti2(PO4)3 . Impressfor ASIB . For thi3[Fe(CN)6]2 as cathode, NaTi2(PO4)3 as anode, and NaClO4–H2O–polyethylene glycol as electrolyte [+ intercalation mechanism in Zn3[Fe(CN)6]2 and its basic morphology. For this battery, its energy density can reach up to 59 Wh kg−1 due to the high output voltage of 1.6 V. Besides, the energy density of ZnFe-PBAs can be further improved by improving the synthesis method. Yao and co-workers adopt a two-step method of electrodeposition and water bath treatment grew K2Zn3[Fe(CN)6]2·9H2O on carbon cloth (CC@ZnFe-PBAs) [2Zn3[Fe(CN)6]2·9H2O cubes are uniformly deposited on carbon cloth 3 to form a full cell. In general, the single-metal-atom redox PBAs inherit the advantage of high electrochemical stability that makes it possible to be used as ASIB electrode. However, considering the specific capacity, double-mental-atom redox PBAs are more competitive.For ZnFe-PBAs, it has great potential in improving energy density due to its high redox potential. Recently, Liu and co-workers reported a full cell that uses Znctrolyte . Figure Fe-PBAs) . The sch3+/Fe2+) releases its redox capacity. In contrast, double-metal-atom redox PBAs have the advantage in capacity because both Fe3+/Fe2+ and M3+/M2+ can provide capacity. As typical double-metal-atom redox PBAs, NaFeFe(CN)6 [3[Fe(CN)6]2 2  are initity Fig. . Through1.33Fe[Fe(CN)6]0.82 with low defect and water content by a multi-step crystallization method [1.33Fe[Fe(CN)6]0.82 exhibits a higher specific capacity of 125 mAh g−1 at 2 C. Even at 20 C, it still can present a highly efficient Na storage reversibility with a desirable capacity of 102 mAh g−1 6 (Na1.85Co[Fe(CN)6]0.99·2.5H2O) 0.81 and explored the influence of electrolyte concentration on electrochemical performance [0.13Mn[Fe(CN)6]0.81/NaTi2(PO4)3 full battery in 1 M NaClO4 and 17 M NaClO4, they find the electrochemical window in 17 M NaClO4 is 2.8 V which is higher than the 1.9 V in 1 M NaClO4 and the initial charge/discharge capacities can raise to 124/116 mAh g−1 6]0.81, the structural collapse can be described in the following chemical reaction:Besides the defects and interstitial water of PBAs, electrolyte concentration is also one of the factors affecting the electrochemical performance of PBAs. Take MnFe-PBAs for example, Okada and co-workers synthesized Naformance . For Na02g and e2g orbitals. Compared with t2g orbitals, the variation of electrons in the e2g orbital has more influence on the crystal structure due to the orientation of the e2g orbitals along the axis of the bond 0.86∙γ0.14∙2.16H2O and Na1.33Mn[Fe(CN)6]0.79∙γ0.21∙1.88H2O 3 3 . Impress+ 6·3.6H2O by co-precipitation method to insert K+ 6·2H2O nanocubes 3 . It exhictrolyte . Althoug+ batteries possess huge application potential and thus received more attention in further optimizing its electrochemical performance. The most mainstream methods are controlling the size of particle and achieving higher crystallinity. The influence of size of particle on performance can be summarized as follows: Firstly, small crystals provide shorter diffusion paths and thus diffusion rates of alkali ions are improved. Secondly, the strain produced by volume changes during ion charge and discharge processes is minimized, which in turn improves rate capability and stability. Thirdly, the increase of the surface area of PBAs can effectively improve electron transport and structural resilience 0.63∙□0.37∙3.4H2O as cathode and explore its electrochemical performance in 2.0 M H2SO4 electrolyte [6]0.63∙□0.37∙3.4H2O exhibits a capacity of 48 mAh g−1 at 5000 C that is the half capacity in 1 C. In addition, it shows an unprecedented long cycle performance that can retain 60% of its initial capacity at 500 C after 730,000 cycles. Later, they find that the Ni[Fe(CN)6]0.60∙□0.40∙4.5H2O also exhibits excellent cycle and rate performance when it used as electrode for aqueous H+ batteries [6]0.60∙□0.40∙4.5H2O still possesses well rate performance that can deliver ∼50% of theoretical capacity at 50 C. Therefore, the application of PBAs in aqueous H+ batteries at low temperature also has a broad development prospect.For PBAs, it will produce a lot of crystal water in the crystal lattice when using the co-precipitation method. In addition, due to some defects in PBAs, these defects will expose M atoms in PBAs outside the unit cell and combine with water molecules. Then, these water molecules bonded with M atoms can communicate crystal water in each lattice and form a hydrogen bond network in PBAs. Therefore, Hctrolyte . Impressatteries . Further+, NH4+ as a non-metallic charge carrier also has received more and more attention in recent years. In 2011, Cui and co-workers explored the insertion performance of NH4+, Na+ and K+ in K0.9Cu1.3Fe(CN)6 and K0.6Ni1.2Fe(CN)6 0.88 as cathode and 3,4,9,10-perylenetetracarboxylic diimide (PTCDI) as anode. Due to the good structure stability of (NH4)1.47Ni[Fe(CN)6]0.88, it exhibits an initial capacity of 51.3 mAh g−1 and retains 74% at 5 C after 2000 cycles. However, due to the limited stability of the anode, the cycling stability of this full cell is worse that can retain 67% at 3 C after 1000 cycles. Later, Huang et.al. proposed a novel aqueous Zn2+/NH4+ hybrid battery that uses the K0.72Cu[Fe(CN)6]0.78·3.7H2O as cathode, zinc as anode in 1 M (NH4)2SO4/0.1 M ZnSO4 electrolyte 0.78·3.7H2O shows an excellent cyclic stability that can retain 78% at the current density of 1800 mA g−1 after 1000 cycles. Impressively, due to the stability of the zinc anode in the electrolyte, the full cell shows a better cycle stability that can retain 76.5% at the current density of 1800 mA g−1 after 1000 cycles. Thus, the zinc anode is worthy of consideration in future studies of PBAs in aqueous NH4+ batteries. Aside from the CuFe-PBAs and NiFe-PBAs, the FeFe-PB (Fe[Fe(CN)6]0.88·□0.12·2.8H2O) in aqueous NH4+ battery also has well electrochemical performance [6]0.88·□0.12·2.8H2O exhibits a nearly zero strain characteristic when it hosts NH4+. Thus, its cyclic retention rate can reach up to 78% after 50,000 cycles. By increasing the agitation rate during the preparation of PBAs, Huang and co-workers synthesized NaFeIIIFeII(CN)6 with ball-cutting morphology 0.7·3.6H2O [3+/Fe2+ and CuII/CuI experience solid-state redox when Mg2+ inserts into K0.1Cu[Fe(CN)6]0.7·3.6H2O. About the PBAs in aqueous full Mg2+ batteries, Xia and co-workers recently used Na1.4Ni1.3Fe(CN)6·5H2O cathode and polyimide anode to construct a full battery 2 is more suitable to be used in aqueous Zn2+ batteries than aqueous Na+ and K+ batteries because K2Zn3[Fe(CN)6]2 has better structural stability in aqueous Zn2+ electrolyte [2Zn3[Fe(CN)6]2-based full battery was successfully assembled the Zn anode, achieving a high output voltage of 1.7 V and an energy density of 100 Wh kg−1. Later, they explored the effect of Zn3[Fe(CN)6]2 with different morphologies for Zn2+ insertion [3[Fe(CN)6]2 with different morphologies are shown in Fig. 2+ diffusion and Zn3[Fe(CN)6]2 structure stability.About the PBAs in aqueous Znctrolyte . Besidesnsertion . Figure 2+ battery, Wang and co-workers demonstrated for the first time that Zn2+ can reversibly insert/extract into/from KCuFe(CN)6 nanocube 6]0.72·3.7H2O) can provide 90% of theoretical capacity with a capacity retention of 96.3% after 100 cycles at 1 C in a dilute electrolyte of 20 mM ZnSO4 solution 0.66⋅3.7H2O as cathode material for Ca2+ storage [0.02Cu[Fe(CN)6]0.66⋅3.7H2O exhibits an excellent long cycle performance that can retain 88% of its initial capacity at 5 C after 2000 cycles. In addition, the K0.02Cu[Fe(CN)6]0.66⋅3.7H2O also exhibits excellent rate performance in aqueous Ca2+ batteries. Its capacity can be retained to 65% when the current density increases from 0.2 C to 20 C, while that of in aqueous Mg2+ battery can only be maintained to 33%. This difference in rate performance can be summarized in two points: First, Ca2+ with a smaller charge density will encounter less resistance when it is inserted/extracted into/from the PBAs. Second, hydrated Ca2+ binds fewer water molecules on the surface, speeding up the transport of Ca2+in PBAs. Similarly, the K0.02Cu[Fe(CN)6]0.64·9H2O also demonstrated same excellent Ca2+ storage performance in aqueous Ca2+ battery, which can retain 94% of its initial capacity at 5 C after 1000 cycles 0.65·2.6H2O as host material to reversibly store Al3+ [0.03Cu[Fe(CN)6]0.65·2.6H2O. Furthermore, they confirmed the shielding effect of crystal water and hydration shell of Al3+ on electrostatic repulsion between Al3+ and K0.03Cu[Fe(CN)6]0.65·2.6H2O, which lays a foundation for the insertion of Al3+ in PBAs. Later, Gao et al. used Al2(SO4)3 solution as aqueous electrolyte to explore the Al3+ reversible insertion characteristic in nanosized KCu[Fe(CN)6]·8H2O 6]·8H2O can release a discharge capacity of 46.9 mAh g−1 at 400 mA g−1 with retention of 54.9% after 1000 cycles. In addition to reducing electrostatic repulsion, PBAs with small particle size can also improve the migration rate of Al3+ by reducing the diffusion distance. This is proved by Wills and Pang et al. via the investigation on CuFe(CN)6 6 6]·2.6H2O is related to a single-phase reaction without producing polycrystalline structure. In addition, they also found that the capacity fading is not mainly caused by the collapse of PBAs structure, but the transfer of Ni in K0.02Ni1.45[Fe(CN)6]·2.6H2O to Al foil anode, resulting in interface instability between anode and electrolyte. Obviously, this study provides a new idea for us to solve capacity fading problem of PBAs upon multivalent ions uptake.For the insertion of Alore Al3+ . Figure H2O Fig. b 10]. A. A3+ in N)6 Fig. e and K2CN)6 Fig. d, f, resatteries . They cl2+, Sr2+, Fe2+, Co2+, Ni2+, Cu2+, Pb2+, Y3+, La3+, Ce3+, Nd3+ and Sm3+ in PBAs also has been explored. For Ba2+ and Sr2+, Cui and co-workers explored their insertion performance in NiFe(CN)6 0.65·2.6H2O [0.03Cu[Fe(CN)6]0.65·2.6H2O has a serious decay process when inserting Co2+, Ni2+ and Cu2+ resulting in poor cycling performance. The main reason for this phenomenon is that partial replacement of these ions with the high-spin transition metal atom in K0.03Cu[Fe(CN)6]0.65·2.6H2O results in irreversible phase transition. Therefore, the main problem for the insertion of these ions into PBAs is to resolve this irreversible phase transition. It should be noted that the insertion and extraction of Fe2+ in FeFe-PB do not produce this irreversible structural phase change. In 2019, Ji et al. used Fe[Fe(CN)6]0.73∙□0.27∙3.6H2O as cathode, Fe as anode and FeSO4 solution as electrolyte to build a full cell 0.65·2.6H2O, they all show similar voltammogram, which means that they have the same insertion sites in K0.03Cu[Fe(CN)6]0.65·2.6H2O. Impressively, they both can deliver a capacity of approximately 60 mAh g−1 at 0.2 C. Although these lanthanide metal ions can be reversibly inserted/extracted in/from PBAs, they are rarely studied due to their low natural reserves and high cost. It is worth noting that the insertion of Pb2+ and Y3+ possesses the fastest ion migration kinetics and most reversible electrochemistry among these uncommon divalent and trivalent metal ions, respectively [2+ in K0.03Cu[Fe(CN)6]0.65·2.6H2O, it exhibits an initial capacity of 55 mAh g−1 and retains 73% at 5 C after 2000 cycles. When the K0.03Cu[Fe(CN)6]0.65·2.6H2O inserting Y3+, it exhibits an initial capacity of 40 mAh g−1 and retains 87% at 5 C after 2000 cycles. Obviously, the insertion of these two ions in PBAs possesses great exploration value and is waiting to be studied.In addition to these common metal ions, Cui et alectively . For the.) and these electrochemical performances of various PBAs in aqueous battery are summarized in Table From the above introduction, it can be clearly found that for PBAs, their electrochemical performance  is closely related to its structure  possesses a small number of accessible ions in the interface of electrode and electrolyte. Therefore, the mass transfer rate of ions in this kind of desalination batteries is slow, and the desalination process can only be guaranteed under the low current density, which is usually about 10 A m−2 , 142. In0.5MnO2 battery, Na+ and Cl− are captured in electrode materials through a complex four-step charge and discharge progress and the removal rate of Na+ can reach 25%  (NaNiHCF) and Na2Fe[Fe(CN)6] (NaFeHCF) for better cation capture. The morphology of NaNiHCF and NaFeHCF is revealed in Fig. −1 at 0.1 A m−2 6/K3Fe(CN)6. Na+ removal depends on the reaction between K4Fe(CN)6/K3Fe(CN)6. Since the liquid-form PBAs possess larger surface, it is more favorable for hosting ions. Thus, impressive salt removal efficiency can be achieved at 85% that is at least two times higher than most desalination batteries reported till now.For traditional desalination battery like Ag/Naeach 25% . During  battery . Figure m−2 Fig. d. Meanwh cathode . Impress+ insertion in cathode and Cl− insertion in anode), it possesses the potential to avoid using semi-permeable membrane. Soon after, membrane-free Na3Ti2(PO4)3/Bi desalination battery was put forward 2·nH2O as cathode and Bi as anode to enable membrane-free desalination, and Fig. As is well known, the membranes play an important role in existing desalination technologies. For reverse osmosis, it requires a semi-permeable membrane for only transporting water molecules but blocking other ions to ensure that the desalination process takes place –148. For forward . Since Ci2(PO4)3 . Thus, a2NiFe(CN)6 via co-precipitation /NaCu[Fe(CN)6] symmetrical cell to optimize the desalination performance in brackish water  cell has the highest specific capacity with 81 and 820 mAh g−1  based on the mass of cathode and anode, respectively. Therefore, PBAs-based desalination batteries inherit the potential to be explored. However, only few PBAs can be used in desalination batteries due to poor structural stability of PBAs, such as ZnFe-PBA and MnFe-PBA. Thus, future research can be focused on improving the stability of PBAs to promote more applications of PBAs in desalination batteries. More importantly, we should recognize that this electrochemical desalination technology is a bit too simple for large-scale application. Therefore, in the future exploration of electrochemical seawater desalination, we can develop a hybrid technology that can combine multiple desalination systems to achieve better results.Table + with small hydrated ionic radius and large energy density makes it more suitable for inserting in PBAs lattice. Although Li+ is a superstar in non-aqueous batteries, it is unsuitable to be hosted in PBAs in aqueous electrolyte due to its large hydration radius, causing structural damage upon inserting/extracting into/from PBAs. However, co-insertion of Li+ with other metal ions possesses great research significance due to synergistic effect. For K+, PBAs show excellent rate performance because of the small hydrated ionic radius of K+ among monovalent ions. In addition, the application of PBAs in aqueous non-metallic ion battery also exhibits excellent performance. In aqueous H+ batteries and NH4+ batteries, PBAs exhibit excellent rate performance and cycling stability, respectively, due to the special transport mechanism of H+ in aqueous electrolyte and the good structural compatibility among NH4+ and PBAs. As for the application of PBAs in aqueous multivalent ions battery, the vacancies of PBAs can be used as insertion site to contain large volume of hydrated multivalent ions, and crystal water in PBAs also has a certain shielding effect on the electrostatic repulsion of multivalent ions in PBAs structure. Therefore, the application of PBAs in aqueous multivalent-ion battery also has application potential. Although PBAs have a good performance in the application of aqueous monovalent battery, it still has great room for improvement in capacity delivery, cycle stability, rate performance, etc. Future research can be considered in the following aspects. (1) The control of vacancies and interstitial water: Although co-precipitation, the most widely used preparation method of PBAs, is simple and efficient in operation, the faster crystallization rate will bring more vacancies and interstitial water. The presence of defects and interstitial water will lead to the reduction of redox sites in PBAs and the stability of PBAs structure, thus resulting in the reduction of capacity and the degradation of cycling performance. Therefore, during the preparation of PBAs, the reaction rate can be reduced by adding chelating agents or adopting multi-step crystallization to improve the crystallinity of PBAs. (2) The suppression of structural change: Generally, PBAs display a transition between one or two crystal types during the insertion and extraction of host ion. Due to the large insertion sites and ion channels of PBAs, such a degree of phase transition will not have a great impact on the structural stability for PBAs. However, for some PBAs such as MnFe-PBAs, the asymmetric distribution of electrons in the d orbitals of Mn3+ will lead to the generation of John–Teller distortion, resulting in the phase transformation between three crystal types. This type of structural change has a great impact on the stability of PBAs structure, thus reducing its cyclic stability. In general, this distortion can be well controlled by Mn being partially replaced by Fe to reduce Mn density in PBAs. Therefore, the influence of such multiple-phase reactions on the structural stability of PBAs can be well solved by metal substitution. (3) The enhancement of electronic conductivity: In fact, PBAs as electrode material are poorly conductive. Therefore, it can be combined with various carbon materials or conductive polymers to improve its electronic conductivity. However, these conductive additive agents and binders do not provide capacity in the electrochemical reaction, thus reducing the energy density of PBAs. Actually, improving the conductivity of PBAs on the premise of ensuring the energy density can be solved by directly depositing PBAs onto carbon materials, which are also acted as current collector. This method not only avoids the use of binder and conductive agent, but also achieves the mixing of PBAs and conductive current collector, which has great development potential in improving the conductivity and energy density of the electrode. (iv) The optimization of electrolyte: As shown in Fig. ., which will produce toxic components in the electrolyte. Therefore, acidic electrolyte should be selected in general to improve the stability of PBAs and avoid the generation of toxic components.In this review, the recent progress of PBAs as electrode for aqueous monovalent-ion and multivalent-ion batteries is discussed. Meanwhile, the application of PBAs in aqueous desalination batteries is also introduced. PBAs have received a lot of attention for electrode materials in aqueous batteries due to their 3D rigid framework and facile synthesis. Among various host ions, NaFor the application of PBAs in aqueous multivalent-ion battery, besides the four points discussed above, there is another aspect that needs special attention. Although the crystal water in PBAs has a certain shielding effect on the electrostatic interaction between multivalent ions and PBAs, the presence of crystal water has negative impact on the structural stability and capacity delivery of PBAs. Therefore, it becomes important to explore the balance between the content of crystal water and the cycling life of PBAs. In addition, most of the current researches on the reaction mechanism of aqueous battery are based on ex situ characterization methods. Therefore, it is also important to use a variety of in situ characterization methods to explore the insertion/extraction mechanism of host ions.3[Fe(CN)6] shows the best performance, which widens the way for the future research of PBAs in desalination battery. Moreover, PBAs-based multi-channel desalination battery also shows excellent salt removal efficiency in brackish water through special structural design, which provides a clear direction for desalination battery in brackish water in future. In general, PBAs as a desalination battery electrode have the following advantages: (i) For PBAs, they have large ion channels and well electrochemical reversibility, which enable PBAs-based desalination battery to achieve better desalination performance with simplified desalination steps. (ii) PBAs have good chemical stability under acidic conditions, which enables it to form a practical desalination battery with traditional Cl− storage electrode and realize the membrane-free. (iii) Except for large ion channel, PBAs also have small particle size, which makes it suitable to purify brackish water in desalination battery. For the future application of PBAs in desalination batteries, the simplification of desalination steps and the development of membrane-free desalination batteries are the promising directions. Furthermore, in order to get better desalination performance, it is suggested to take the following methods: (i) preparing high-crystallinity, low-defect and small-sized PBAs, (ii) exploring PBAs with new structure, such as MnMn-PBA, CoCo-PBA and CrCr-PBA and their derivatives, and (iii) combining PBAs-based desalination battery with other systems to form hybrid desalination system.As desalination battery is a special kind of aqueous battery, the development of PBAs in conventional aqueous battery also promotes its application in desalination battery. Among various PBAs, Zn/K


Very low-protein (VLP) diets negatively impact calcium (Ca) metabolism and absorption. The objective of this study was to investigate the effect of supplemental branched-chain amino acids (BCAA) and limiting amino acids (LAA) on Ca digestibility, absorption and reabsorption in pigs fed with VLP diets. Forty-eight piglets were assigned to six treatments: positive control (PC), negative control (NC), and NC containing LAA 25%, LAA 50%, LAA + BCAA 25% (LB25) and LAA + BCAA 50% (LB50) more than recommendations.3. LB50 tended to increase vitamin D receptor transcript and protein in the gut, but decreased mRNA or protein abundance of parathyroid hormone 1 receptor (PTH1R), calbindin 1 (CALB1), cytochrome P450 family 27 subfamily B member 1 and occludin in small intestine. LB50 increased the transcript of cytochrome P450 family 24 subfamily A member 1 and PTH1R but decreased the transcript of transient receptor potential cation channel subfamily V member 5, CALB1 and solute carrier family 17 member 4 in kidney.Relative to PC or NC, LB25 and LB50 had higher digestibility of Ca and plasma Ca and phosphorus (P), but lower plasma vitamin DOverall, BCAA increased Ca digestibility through regulating the transcellular and paracellular Ca absorption in the gut and reabsorption in kidney during protein restriction. The nitrogen (N) excretion from modern swine production adversely influences the environment and human health . Very loWe recently showed that the branched-chain amino acid (BCAA) metabolism is significantly affected in pigs fed with VLP diets . BCAA in+/H+ exchanger 3 (NHE3) inhibitor could suppress Leu-enhanced Ca transport and sodium efflux from basolateral membrane suggesting that the BCAA-enhanced Ca transport is dependent to NHE3 pathway [Nutrients restriction  and prot pathway . The datP = 0.99), but at the end of study the LB25 had higher body weight than L25 . Further, LB50 had higher final body weight (15.62 ± 0.98 kg) than NC (13.22 ± 0.98 kg) and L50 (11.40 ± 0.91 kg) (P < 0.01). Chromium oxide  was supplemented to all diets as a marker for measuring the nutrients digestibility [All procedures performed in the current study were in accordance with guidelines of Animal Care and Use Committee at Oklahoma State University and were approved by this committee (ACUP #AG-17-27). The animals, housing, diets and experimental design were previously described by authors . Brieflyg for 15 min at 4 °C. The collected plasma and serum samples were aliquoted and stored at − 80 °C until future analysis. Following blood collection, all pigs were euthanized by CO2 asphyxiation method, duodenum, jejunum, and kidney samples were collected, rinsed in distilled water, snap-frozen in liquid nitrogen and stored at − 80 °C until analysis.After mixing the diets, feed samples from all bags of each treatment (~ 1 kg) were collected and stored at − 20 °C until analysis. Fecal grab samples were collected by rotational transfer of pigs from their individual pens to metabolic crates for 24 h during N2 and N3 phases. All pigs had free access to feed and water in metabolic crates. The collected samples were stored at − 20 °C until further analysis. Fecal consistency score was determined weekly with 0  =  formed, normal; 1  =  soft, wet cement consistency; 2  =  runny or watery; 3  =  mucoid diarrhea; 4  =  bloody diarrhea, as previously described . As we dCrd: chromium amount in the diet, Crf: chromium amount in the feces, Nf: the nutrient amount in the feces, Nd: nutrient amount in the diet.As we described previously , 21, cheConcentration of Ca , P , alkaline phosphatase , and blood urea nitrogen  in plasma (> 300 μL) were detected using a chemistry analyzer system  following calibration  . The abs3  and serum FGF-23  concentrations were measured according to manufacturers’ protocols. Using Epoch microplate spectrophotometer , the VD3 and FGF-23 absorbance were detected at 450 nm. The intra-assay coefficient of variation for VD3 and FGF-23 were 3.97% and 1.52%, respectively.Plasma vitamin DCYP24A1) [PTH1R) [THRA) [SLC34A1) [SLC34A2) [SLC34A3) [SLC17A4) [TRPV5) [TRPV6) [CALB1 [VDR [2+ ATPase 1b (PMCA1b) [OCLN [ ZO1 [-∆∆CT method, the relative abundances of target genes transcript were obtained.The relative mRNA abundances of key molecules involved in Ca and P absorption and excretion in duodenum and kidney were determined using quantitative PCR (qPCR) . BrieflyCYP24A1) , parathy [PTH1R) , thyroid) [THRA) , solute SLC34A1) , solute SLC34A2) , solute SLC34A3) , solute SLC17A4) , transie [TRPV5) , transie [TRPV6) , CALB1 [) [CALB1 , VDR [40(PMCA1b) , OCLN [4b) [OCLN , ZO1 [42LN [ ZO1  (Table 1g for 15 min), protein concentration of samples was determined using Bradford assay. Then, protein homogenates were mixed with 2 × Laemmli buffer  and incubated at 95 °C for 3 min. The protein extract  derived from each sample was fractioned using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis . The constant voltage of 130 V was applied for gel electrophoresis for 1:15 h. After separation, the proteins were transferred into the nitrocellulose membranes applying the voltage of 100 V for 1:30 h. The membranes were blocked with 8% (m/v) skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 2 h at room temperature with mild agitation. Then, the membranes were incubated with added primary antibodies  in jejunum and kidney , 45. Brit-test followed by a Benjamini-Hochberg correction with 0.1 false discovery rate for six preplanned comparisons including PC vs. LB25, PC vs. LB50, NC vs. LB25, NC vs. LB50, L25 vs. LB25, and L50 vs. LB50. All values were presented as mean ± standard error of the mean with a P ≤ 0.05 considered statistically significant and 0.05 < P ≤ 0.1 considered as tendency.As we previously described , 14, forP = 0.07), whereas dietary treatments significantly affected AFD of Ca in N3 phase  and that tended to be significant in N3 (P = 0.09). While AFD of P was ~ 9% and 14% lower in LB25 and LB50 than PC during N2 ; and it tended to be higher in LB50 relative to PC  in LB25 Fig. . JejunalThe effect of diet on relative protein abundance of renal NHE3 was significant; however, no significant differences were found among diets Fig. . The pro3, but increased Ca and P concentration, 3) LB25 and LB50 decreased the transcript or protein abundance of CALB1 and CYP27B1 in gut, 4) LB50 tended to increase the protein abundance of VDR in jejunum, 5) LB50 decreased the transcript of OCLN and PTH1R in duodenum, 6) LB50 increased the transcript of CYP24A1 and PTH1R, but decreased the transcript of TRPV5 and LB25 increased the gene expression of CYP24A1 and SLC34A2 in kidney, 7) LB25 and LB50 reduced transcript of CALB1 and SLC17A4 in kidney. Overall, the supplementation of BCAA greater than recommended levels into the VLP diet improved Ca digestibility and increased plasma Ca and P concentration through regulating the transcellular and paracellular pathways of Ca absorption in the gut and Ca and P reabsorption in kidney.Feeding pigs with VLP diets is an effective dietary strategy to reduce N excretion from modern swine production , 3; howeIt is well documented that not only protein intake but also dietary individual AA have modulatory impacts on Ca metabolism through the mechanisms related to Ca absorption, excretion, and hormonal changes , 47. WheSupplementation of BCAA together with LAA higher than recommended levels  increased plasma Ca and P concentrations. In support of our data, addition of BCAA keto acids into the VLP regimen of patients with chronic renal failure, significantly increased serum P levels . The inc3 2D3) during transcriptional events in peripheral tissues, including intestine [3 that eventually lead to inhibiting the Ca absorption [3 in the current study. VD3 along with parathyroid hormone plays a crucial role in maintaining the Ca homeostasis through increasing the intestinal Ca absorption [3 in LB25 and LB50 pigs is likely the result of a negative feedback loop that inhibits the release of this hormone along with parathyroid hormone and Ca absorption when blood Ca is elevated [The mechanisms that underlie the increased Ca digestibility and intestinal absorption in response to BCAA supplementation during protein restriction is unknown. Amino acids are involved in Ca absorption through different mechanisms including alteration in the metabolism of enterocytes and activation of extracellular receptors or acceptors , activatntestine  and is intestine . The incntestine . A negatsorption . LB25 ansorption , 65. Theelevated . The mecCYP24A1 and in kidney. The active form of vitamin D is converted to 24-hydroxylated inactive product under the action of 1,25-hydroxyvitamin-D3–24-hydroxylase encoded by CYP24A1 gene [3 concentration as well. Reduced concentration and active form of VD3 might contribute to decreasing Ca absorption in gut and Ca reabsorption in kidney as a compensatory response to increased blood Ca concentration. PTH1R is involved in urinary excretion of phosphate in kidney [PTH1R in LB50 pigs is indicative of a greater excretion of phosphate in kidney in an effort to maintain the P homeostasis through reducing the increased plasma P levels. Further, LB50 decreased the transcript of renal TRPV5, CALB1, and SLC17A4, which are all involved in reabsorption of Ca and P [Calcium homeostasis is under coordinated regulation of intestinal, renal and bone physiology . Little 4A1 gene . The incn kidney . The higCa and P , 71. DecTo our knowledge, this is the first study assessing the effect of protein deficient diets supplemented with BCAA higher than recommended levels on Ca digestibility and pathways involved in its (re)absorption. Supplemental BCAA improved the digestibility of Ca and the concentration of Ca and P during protein restriction. Our data provide evidence that BCAA are acting via both transcellular and paracellular pathways in gastrointestinal tract and control renal Ca and P reabsorption to regulate the circulating Ca and P concentration. It is remained to be determined whether BCAA influence the Ca homeostasis through bone or other potential pathways in gastrointestinal tract.


NKX3.1, a prostate-specific tumor suppressor, is either genomically lost or its protein levels are severely downregulated, which are invariably associated with poor prognosis in prostate cancer (PCa). Nevertheless, a clear disconnect exists between its mRNA and protein levels, indicating that its post-translational regulation may be critical in maintaining its protein levels. Similarly, AURKA is vastly overexpressed in all stages of prostate cancer (PCa), including castration-resistant PCa (CRPC) and neuroendocrine PCa (NEPC), although its transcripts are only increased in ~ 15% of cases, hinting at additional mechanisms of deregulation. Thus, identifying the upstream regulators that control AURKA and NKX3.1’s levels and/or their downstream effectors offer an alternative route to inhibit AURKA and upregulate NKX3.1 in highly fatal CRPC and NEPC. AURKA and NKX3.1 have not linked to each other in any study to date.A chemical genetic screen revealed NKX3.1 as a direct target of AURKA. AURKA-NKX3.1 cross-talk was analyzed using several biochemical techniques in CRPC and NEPC cells.We uncovered a reciprocal loop between AURKA and NKX3.1 in CRPC and NEPC cells. We observed that AURKA-mediated NKX3.1 downregulation is a major mechanism that drives CRPC pathogenesis and NEPC differentiation. AURKA phosphorylates NKX3.1 at three sites, which degrades it, but AURKA does not regulate NKX3.1 mRNA levels. NKX3.1 degradation drives highly aggressive oncogenic phenotypes in cells. NKX3.1 also degrades AURKA in a feedback loop. NKX3.1-AURKA loop thus upregulates AKT, ARv7 and Androgen Receptor (AR)-signaling in tandem promoting highly malignant phenotypes. Just as importantly, we observed that NKX3.1 overexpression fully abolished synaptophysin and enolase expression in NEPC cells, uncovering a strong negative relationship between NKX3.1 and neuroendocrine phenotypes, which was further confirmed be measuring neurite outgrowth. While WT-NKX3.1 inhibited neuronal differentiation, 3A-NKX3.1 expression obliterated it.NKX3.1 loss could be a major mechanism causing AURKA upregulation in CRPC and NEPC and vice versa. NKX3.1 genomic loss requires gene therapy, nonetheless, targeting AURKA provides a powerful tool to maintain NKX3.1 levels. Conversely, when NKX3.1 upregulation strategy using small molecules comes to fruition, AURKA inhibition should work synergistically due to the reciprocal loop in these highly aggressive incurable diseases.The online version contains supplementary material available at 10.1186/s12929-021-00765-z. Aurora kinase A (AURKA), a serine/threonine kinase, is essential for mitosis in normal cells. AURKA is overexpressed in numerous solid and hematological cancers. AURKA protein is increased in 100% of prostate intraepithelial neoplasia (PIN) lesions, and in vast majority of prostate tumors > 94%) , indicat% , indicThe present study focuses on one such feedback loop, which was discovered between AURKA and a tumor-suppressor NKX3.1 in CRPC and NEPC cells. We have identified NKX3.1 as a direct substrate of AURKA by employing a pioneering global screen , 15–18. While the tumor-suppressive functions and loss of NKX3.1 are well established, a contradiction exists between its mRNA and protein levels in PCa. Most of the studies reported that mRNA levels of NKX3.1 are either increased or unchanged in PCa tissues compared to normal tissues . In contC4-2, 22Rv1, HEK-293T and Phoenix cells were purchased from American Type Culture Collection  and maintained according to the manufacturer’s instructions. 49F cells were obtained from Dr. Amina Zoubeidi and maintained in RPMI medium, 10% FBS along with 10 μM enzalutamide . The details of the antibodies used in this study are included in Additional file Cloning of human AURKA and NKX3.1 shRNAs have been reported before , 28. LenCloning of AURKA and NKX3.1 were reported before , 28. KinEscherichia coli. To remove the background signal, the AURKA-TPX2 complex was treated with kinase buffer  containing ATP (100 μM) for 1 h at 30 °C. Subsequently, the beads were washed three times with kinase buffer, followed by the addition of ~ 2 μg of 6x-His-tagged WT type or mutant NKX3.1 and 2 μCi of [γ-32P] ATP for 25 min. The reaction mixture was boiled in SDS-PAGE dye for 5 min, proteins were separated using SDS-PAGE and exposed to X-Ray film.6x-His-tagged AURKA was purified using Ni-NTA beads. 6x-His-TPX2 was expressed and isolated from Immunofluorescence was performed as before . PCa celReal-time qPCR experiments were conducted as reported before . The priThe cells were seeded in 6-well plates for 12 h prior to infection. Subsequently, corresponding retro- or lentiviruses were added for an additional 32 h. Cycloheximide (20 μg/ml) was then added for the times indicated in the figures prior to lysis. The cell lysates were subjected to Western blot analysis.Ubiquitylation assay was performed as described before . Briefly2, and 1 mM PMSF) and placed on ice for 10 min, followed by centrifugation at 3000 rpm at 4 °C (10 min). To separate the nuclear fraction, the pellet was resuspended in buffer B  and 1 mM PMSF). The suspension was homogenized using a 27½ gauge needle (ten times). The lysates were placed on ice for 30 min and the nuclear fraction was separated by centrifugation at 24,000×g at 4 °C for 20 min. The cytosolic and nuclear extracts were further analyzed by Western blotting [C4-2 and 22Rv1 cells were washed twice with chilled PBS, resuspended in buffer A . Statistical significance of difference was determined by the one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test. P < 0.05 was considered statistically significant.As NKX3.1 was identified as an AURKA target in a global screen, we inspected whether AURKA directly phosphorylates NKX3.1 in vitro. AURKA in complex with its activator TPX2 was incubated with recombinant 6x-His-NKX3.1, which resulted in the phosphorylation of the latter, indicating that NKX3.1 is a substrate of AURKA  and performed an in vitro kinase assay using AURKA-TPX2. While WT NKX3.1 was robustly phosphorylated, 3A-NKX3.1 showed minimal phosphorylation, confirming that AURKA only phosphorylates these three sites on NKX3.1 Fig. B.We subsequently tested whether AURKA phosphorylates NKX3.1 in cells. HA-tagged WT and 3A-NKX3.1 were ectopically expressed in C4-2 cells, followed by alisertib treatment for 12 h. HA-tagged proteins were isolated and their phospho-Ser levels were analyzed using a phospho-Ser antibody. While WT NKX3.1 showed robust phosphorylation, it was completely abolished upon alisertib treatment, indicating that NKX3.1 is phosphorylated by AURKA in C4-2 cells  at the Nkx3.1 locus results in hyperplasia and eventually PIN formation. At the molecular level, loss of a single Nkx3.1 allele prolongs the proliferative stage of dividing luminal cells, causing hyperplasia [Nkx3.1 controls discrete subsets of genes, thus, loss of one Nkx3.1 allele results in complete loss of some target genes, while other genes require loss of both copies [NKX3.1 is predominantly expressed in prostate luminal epithelial cells, and promotes cellular differentiation and lineage plasticity . The loserplasia . Most imh copies .While the loss of NKX3.1 protein is a hallmark of PCa in clinical specimens and mouse models, the accompanying NKX3.1 mRNA levels show little correlation with its protein levels . AdditioLike NKX3.1, AURKA mRNA levels also show little correlation with protein levels in PCa, underscoring that both their regulation at the protein level is critical. NKX3.1 binds AURKA and triggers its ubiquitylation. 3A-NKX3.1 is resistant to AURKA-mediated degradation, hence, it effectively degrades AURKA and reverses oncogenic phenotypes. Thus, genomic loss of NKX3.1 could be a dominant factor contributing to AURKA protein upregulation in a significant percentage of PCa. While the exact mechanism by which NKX3.1 promotes the ubiquitylation of AURKA remains unclear, it could be at least partly mediated by AKT/GSK3b/FBXW7 pathway . FBXW7 iRecent studies have uncovered a few mechanisms by which AURKA upregulates AR and ARv7 signaling in CRPC. AURKA stabilizes YBX1 causing upregulation of AR protein and ARv7 mRNA levels . We showSeveral studies have shown that AURKA inhibition downregulates AKT activation, however, the molecular players mediating this response largely remain unknown. Previously, we identified that AURKA directly phosphorylates and degrades PHLDA1 in breast cancer cells . PHLDA1 AURKA overexpression or amplification is a hallmark of NEPC. In contrast, little is known about the role of NKX3.1 in NEPC. Nkx3.1 was genomically lost in p53- and Rb-deficient mouse prostate tumors exhibiting neuroendocrine phenotypes . SimilarOur studies indicate that NKX3.1 loss could be a major mechanism causing AURKA upregulation in CRPC and NEPC and vice versa. As upregulating NKX3.1 due to genomic loss requires gene therapy, targeting AURKA using specific inhibitors such as alisertib, provides a powerful therapeutic opportunity to maintain NKX3.1 protein levels in CRPC and NEPC. Conversely, when NKX3.1 upregulation strategy using small molecules comes to fruition, AURKA inhibition is expected to work synergistically with NKX3.1 upregulation due to the feedback loop in these highly aggressive diseases.Additional file 1. Additional Table S1. List of antibodies used in this study. Additional Table S2. Primer sequences of real-time qPCR primers.


Salmonella Typhimurium host, instigated by the ORFan gene pid (for phage P22 encoded instigator of dgo expression) and resulting in derepression of the host dgoRKAT operon. The pid gene is highly expressed in phage carrier cells that harbor a polarly located P22 episome that segregates asymmetrically among daughter cells. Here, we discovered that the pid locus is fitted with a weak promoter, has an exceptionally long 5′ untranslated region that is instructive for a secondary pid mRNA species, and has a 3′ Rho-independent termination loop that is responsible for stability of the pid transcript.Many phage genes lack sequence similarity to any other open reading frame (ORF) in current databases. These enigmatic ORFan genes can have a tremendous impact on phage propagation and host interactions but often remain experimentally unexplored. We previously revealed a novel interaction between phage P22 and its  Next, 50 µL of the mixture was added to 2 mL of LB broth and grown for 6 h at 37 °C under shaking conditions. Phages were extracted and propagated on LT2 as plaques in LB soft agar or as lysates in LB. This procedure was repeated eight times to obtain eight pools of mutagenized phage lysate. The mutagenized phage lysate was propagated on stationary phase culture of LT2K7 on green indicator plates . After oExpression of β-galactosidase (LacZ) was visualized by adding 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside  to agar plates (40 µg/mL). β-Galactosidase hydrolyses X-Gal to an insoluble blue precipitate. For clear visualization of spotted phage populations, we pressed agar plates onto Whatman filter papers  before photographing them. 4)2SO4, 6 g/L Na2HPO4, 3 g/L KH2PO4, 3 g/L NaCl, 0.1 mM CaCl2, 1.0 mM MgCl2, and 0.003 mM FeCl3 [600 of the sample .Population-level fluorescence measurements were obtained in a Fluoroskan Ascent FL , equipped with a filter pair with an excitation wavelength of 485 nm and an emission wavelength of 520 nm. A multi-well microtiter plate was filled with 200 µL of stationary phase cultures grown in AB-glucose medium (2 g/L (NHmM FeCl3  and suppp = 0.05) were carried out using the JMP software , and differences were considered significant when p ≤ 0.05.All fluorescence measurements were performed in triplicate. Statistical analyses, histograms, and Tukey HSD tests  for 90 min. Infection was stopped by adding 20% volume of stop mixture , followed by snap-freezing in liquid nitrogen. Following this, total RNA was extracted according to the hot phenol method, and the remaining DNA was digested via DNase I treatment . Subsequently, 7 µg of total RNA per sample was resolved on a 6% polyacrylamide gel containing 7 M urea and transferred to an Amersham Hybond-XL  membrane. After UV-crosslinking, transcripts of interest were detected by radiolabeled oligonucleotides Sal_5S_rRNA (5S rRNA probe for loading control), Pid_5UTR, and/or Pid_ORF  by T4 polynucleotide kinase  for 1 h at 37 °C. After removing unincorporated 32P-γ-ATP with a Microspin G-25 column , 7 µL of the labelled oligonucleotide was added to 17 mL EKONO hybridization buffer  and hybridized to the membrane, overnight at 42 °C in a hybridization oven. Radioactive signals were imaged with a Typhoon 9400 , and the membrane was stripped for re-probing by immersing it in 500 mL hot H2O/5 mL 10% SDS for 7 min.Exponential phase cultures of  Pid_ORF . The lab


A and MgB) significantly enhances its catalytic efficiency, calcium binding suppresses its function. However, how the coordination of different metal ions affects its catalysis has not been defined. Here, we report a new crystal structure of ARH3 complexed with its product ADP-ribose and calcium. This structure shows that calcium coordination significantly distorts the binuclear metal center of ARH3, which results in decreased binding affinity to ADP-ribose, and suboptimal substrate alignment, leading to impaired hydrolysis of PAR and mono(ADP-ribosyl)ated serines. Furthermore, combined structural and mutational analysis of the metal-coordinating acidic residues revealed that MgA is crucial for optimal substrate positioning for catalysis, whereas MgB plays a key role in substrate binding. Our collective data provide novel insights into the different roles of these metal ions and the basis of metal selectivity of ARH3 and contribute to understanding the dynamic regulation of cellular ADP-ribosylations during the DNA damage response.ADP-ribosylation is a reversible and site-specific post-translational modification that regulates a wide array of cellular signaling pathways. Regulation of ADP-ribosylation is vital for maintaining genomic integrity, and uncontrolled accumulation of poly(ADP-ribosyl)ation triggers a poly(ADP-ribose) (PAR)–dependent release of apoptosis-inducing factor from mitochondria, leading to cell death. ADP-ribosyl-acceptor hydrolase 3 (ARH3) cleaves PAR and mono(ADP-ribosyl)ation at serine following DNA damage. ARH3 is also a metalloenzyme with strong metal selectivity. While coordination of two magnesium ions (Mg We further found that MgA and MgB play key but different roles for proper substrate alignment and substrate binding, respectively. Together, our results provide new insights into the metal-dependent mechanism and function of ARH3.Here, to address these previously unanswered questions on ARH3, we combined biochemical, biophysical, mutational, and structural tools. We found that Mg2+-dependent catalytic mechanism and metal preference of ARH3 has been proposed by several groups   , Table 1nd Ca2+) , indicatomplexes , 22, 23.2+ to ARH3 increases its binding affinity to ADPR nearly 60-fold, in comparison to that of the metal-free enzyme state (in the presence of EDTA), KDMg of 1.42 μM versus KDEDTA of 80.65 μM , which is consistent with the similar level of PAR hydrolysis activity in the presence of Mg2+ or Mn2+ , about a sixfold reduction in ADPR-binding affinity compared with that with Mg2+  and an increase in entropy (TΔS) (2+ used for ITC (5 mM) is saturating, given that the Ca2+-dependent inhibitory was not further enhanced at higher Ca2+ concentrations . Taken together, these results are consistent with the metal selectivity of ARH3 that enables its specific substrate recognition.The addition of MgA and B, . The ADPFig. 1C,  22). In. In2+ toFig. 1D, . Unlike py (TΔS) . The contrations . OverallO-linkage in substrates  and ADPR are positioned in a way similar to those found in the Mg2+-bound form (B and D). However, the overall coordination geometry of the dimetallic catalytic center is remarkably different from that found in the Mg2+-bound form.We previously reported the ligand-driven conformational switch in ARH3 that enables specific recognition and cleavage of the 1″-bstrates . Further of ARH3 , 22 and  of ARH3 , we reasFig. 2A, . Two met2+-bound form where both Mg2+ ions have a similar octahedral coordination geometry with six ligands, the coordination architecture of CaA and CaB shows a notable difference  and is likely because of the longer cationic diameter of calcium (radius of 0.99 Å) compared with magnesium (radius of 0.65 Å)  is substantially longer than that of MgA in the Mg2+-bound form (2.23 Å).Unlike the MgC and D, . CaB is Fig. 2C, . This is 0.65 Å) . In cont2+ complex, the orientation of 1″-OH group of the terminal ribose, corresponding to the 1″-O-linkage in uncleaved substrates, is tilted toward CaB (B). With respect to the Mg2+-bound form, the 1″-OH group is rotated ∼10° in the Ca2+-bound form. This rotation induces a concomitant ∼1 Å displacement of 1″-OH toward CaB. As a result, the 1″-OH group is directly coordinated to CaB, whereas 2″-OH bridges the dimetallic center by interacting with both Ca2+ ions (C).In the ARH3–ADPR–Caward CaB B. With ra2+ ions C.2+-bound form (C and D). In the Mg2+-bound form, this μ-aqua ligand simultaneously engages both Mg2+ ions and the 2″-OH. This μ-aqua ligand is missing in three of four ARH3–ADPR–Ca2+ complexes in the asymmetric unit, and only a very weak electron density was found in the last ARH3 molecule. Instead, two Ca2+ ions are bridged by 2″-OH of the terminal ribose (C), which causes the tilted confirmation of the terminal ribose. This absence of the bridging water ligand may explain the shorter metal–metal distance in the Ca2+-bound form than in the Mg2+-bound form (3.1 Å [Ca2+–Ca2+] versus 3.3 Å [Mg2+–Mg2+])  is missing in the Caund form , C and Dl ribose C, which +–Mg2+]) .2+ metal center and the tilted conformation of 1″-OH, a position for the scissile O-linkage in substrates, are likely to interfere with a precise alignment of substrates for nucleophilic attack, resulting in suppression of enzymatic activities of ARH3. These results also support the hypothesis that a subtle change in the metal-coordination arrangement can result in a detrimental effect on ARH3 functions.Taken together, our findings suggest that the distorted di-Ca2+, whereas Asp78 and Asp316 coordinate only MgA and MgB, respectively but do not directly interact with ADPR. We purified these mutants (D) and monitored their PAR and serine MARylation hydrolytic activities in the presence of Mg2+, EDTA, and Ca2+  with alanine. Asp77 and Asp314 interact with both ADPR and Mg mutants D and monand Ca2+ .Figure 32+ effectively suppresses PAR hydrolysis by ARH3, which is in good agreement with published results (A) .The addition of a metal-chelating EDTA or Ca results A . Collectively, these results suggest that metal-coordinating residues in the active site of ARH3 are critical for both PAR and MAR hydrolysis, and de-MARylation function of ARH3 appears to require a more strict structural integrity to specifically recognize and/or cleave MARylated substrates.While the WT enzyme efficiently reversed the serine-linked MARylated PARP1 substrate, all tested ARH3 mutants lacked enzymatic activity , B and CA. Thus, Asp77 appears to be important for the maintenance of integrity of MgA and the optimal orientation of the terminal ribose for catalysis. Similarly, on the other side of the active site, Asp314 interacts with 3″-OH of the terminal ribose and MgB. Asp314 seems to contribute to the integrity of MgB, given that the substitution of Asp314 with glutamate eliminates MgB from the active site . This lack of MgA causes a large-scale rearrangement in the active site. First, the terminal ribose is rotated ∼37° using MgB as a pivot (B). Because of this rotation, which is an even greater degree of rotation than that found in the Ca2+-bound form of ARH3 . This significantly rotated conformation of ADPR in ARH3D77A, with respect to ARH3WT, is likely to interfere with a correct substrate positioning for catalysis . Together, the coordination of MgA appears to contribute to the optimal alignment of substrate, while it sacrifices the overall substrate-binding affinity of ARH3.Structural analysis of ARH3 missing , A and B a pivot B. Becaus of ARH3 , the 2″-Fig. 5A, . As a reD314A, similar to ARH3D314E (B is missing (D). Unlike ARH3D77A, the orientation of ADPR in ARH3D314A is nearly identical to that in ARH3WT, and critical residues for the formation of the binuclear metal center occupy nearly identical positions (E). This finding is in line with the proposed important role of MgA for precise substrate alignment. The geometry of MgA is also maintained in an octahedral shape as seen in ARH3WT (F). Overall, in contrast to ARH3D77A, ARH3D314A appears to show a limited effect on the alignment of the terminal ribose . Consistent with these findings, ARH3D314A shows a 44-fold decrease in ADPR-binding affinity compared with ARH3WT , our careful structural analysis showed that Ca2+ coordination in ARH3 results in significant rearrangements in geometry, coordination, and metal–ligand distances of the binuclear metal center, explaining the impaired ARH3 activities in the presence of Ca2+  and 2″-OH, and CaA shows a very relaxed octahedral geometry . The significantly distorted active-site structure and reduced binding affinity in the Ca2+-bound form of ARH3 are consistent with the proposed roles of MgA and MgB in substrate alignment and binding. For example, the highly relaxed CaA geometry, compared with the Mg2+-bound form, likely interferes with an optimal substrate alignment in ARH3 and following nucleophilic attack on the distal ribose. In addition, Ca2+ ions can reduce the chemical reactivity of metal-bound water molecules. Previous studies reported that a Ca2+-bound water molecule has a higher pKa (12.9) in comparison to that bound to Mg2+ (11.4) or Mn2+ (10.9) (2+ (radius of 0.99 Å) (2+ (radius of 0.65 Å) and Mn2+ (radius of 0.80 Å), along with its preference for a higher coordination number (up to eight ligands), might cause steric interference with the coordinated oxygen atoms, possibly perturbing the stability of the transition state.In contrast to MgFig. 2D, , Ca2+ io ligands , 36. In + (10.9) . Consequ+ (10.9) . Finally 0.99 Å) , compare2+ ions in ARH3. Although several mutations were previously reported . Given the missing MgA, a complete loss of enzymatic activity, and an increased ADPR-binding affinity in ARH3D77A (A), MgA appears to play an important role in catalysis by sacrificing the substrate-binding affinity. Also, these findings raise the possibility that Asp77 may function as a catalytic residue. In contrast, ARH3D314A, in which MgB is missing, shows nearly identical active-site structure, but an impaired ADPR-binding affinity, which is comparable to the metal-free ARH3 , serine MAR (B), O-acetyl-ADPR , the DNA-binding domain , and the PARP1C catalytic domain  of human PARP1 were purified as described previously  by Gene Universal Inc. All plasmids were sequenced, and mutations were confirmed. The biotinylated PARP1C and ARH3 mutant proteins were purified using the same protocol as ARH3WT. A gene for human HPF1 was synthesized and cloned into the pET21b-His6-pps by Gene Universal Inc. HPF1 was purified as described previously  or EDTA (Sigma–Aldrich). The WT or mutants of ARH3 were placed in a cell at 50 μM concentration, and ADPR was placed in the injection syringe at 660 μM concentration. Overall, 26 injections (10 μl per injection except for the first sample [5 μl]) were administered with an interval of 5 min between peaks to allow the baseline to be stabilized. Control experiments were conducted under the same condition to determine the heat of dilution by injecting ADPR to buffers, which shows no significant heat generation  in a buffer containing 100 mM NaCl, 50 mM Tris (pH 7.5), 10 mM MgCl2, and 2 mM DTT. ARH3 proteins  with final concentrations of 2 μM were mixed with the PARylated PARP1C substrate, followed by incubation for 1 h in 37 °C. Reactions then were stopped by adding 4× SDS-loading dye (Bio-Rad) and visualized by Coomassie blue staining of SDS-polyacrylamide gels.A gel-based PAR-turnover assay was performed to evaluate the enzymatic activity of ARH3eviously . Briefly+ (Sigma–Aldrich) in a buffer containing 100 mM NaCl, 50 mM Tris (pH 7.5), 10 mM MgCl2, and 2 mM DTT. The reaction then was passed through a PD-10 column to remove excess β-NAD+. PARP inhibitor, Olaparib (SelleckChem), at 8 μM was then added before performing dePARylation of the substrate by incubating with 10 nM PARG for 30 min in 37 °C. The generated serine-linked MARylated PARP1C was passed through a PD-10 column to remove protein-free PAR and ADPR units, followed by aliquoting and storing in −80 °C. ARH3 proteins at a final concentration of 60 nM were treated into the serine-MARylated substrate in a buffer containing 50 mM Tris–HCl (pH 7.5), 50 mM NaCl, and 5 mM MgCl2, followed by incubation for 1 h in 37 °C. Reactions then were stopped by adding 4× SDS-loading dye and resolved by 10% SDS-polyacrylamide gels.To generate serine-linked MARylated substrate, 4 μM of PARP1C was first PARylated in the presence of 4 μM PARP1 DBD, 8 μM HPF1, 2 μM double-stranded DNA, and 400 μM β-NADTo visualize and quantify the level of serine-MARylation, we performed Western blotting on samples. Reactions were transferred to polyvinylidene fluoride membranes. Polyvinylidene fluoride membranes first were blocked by incubation in PBS with Tween-20 (PBST) buffer containing 5% nonfat skim milk for 1 h at room temperature. After one washing step with PBST, membranes were incubated with anti–pan-ADPR antibody , anti-ARH3 N-terminal antibody (Aviva System Biology) (1/1000 dilution), and antibiotin antibody  overnight at 4 °C. After three steps of wash with PBST, blots were incubated with horseradish peroxidase–conjugated polyclonal secondary anti-rabbit antibody  for 1 h at room temperature. Afterward, membranes were washed three times with PBST and finally visualized by enhanced chemiluminescence detection kit using the iBRIGHT-FL1000 imager (Thermo Fisher). Bands were quantified using ImageJ software .WT, ARH3D77A, and ARH3D314A (10 mg/ml) were cocrystallized with 5 mM ADPR (Sigma–Aldrich) and 5 mM Ca2+ (for ARH3WT) or Mg2+ (for ARH3D77A and ARH3D314A) by hanging-drop vapor diffusion  before flash cooling in liquid nitrogen for data collection.The ARH3iffusion . BrieflyWT–ADPR–Ca2+ complex crystals  diffracted to a resolution of 1.75 Å, ARH3D77A–ADPR–Mg2+ complex crystals (P1) diffracted to a resolution of 1.85 Å, and ARH3D314A–ADPR–Mg2+ complex crystals (P1) diffracted to a resolution of 1.80 Å. X-ray datasets were collected with an Eiger 16M detector (Dectris) and processed using HKL2000  as a search model. Models were manually built using Coot (Rfactor of 14.9% and Rfree of 18.8% for ARH3WT–ADPR–Ca2+, Rfactor of 18.1% and Rfree of 22.7% for ARH3D77A–ADPR–Mg2+, and Rfactor of 18.8% and Rfree of 22.7% for ARH3D314A–ADPR–Mg2+. Crystallographic data are shown in Structures were determined by molecular replacement using MOLREP , 44 in ting Coot  and refiing Coot ; RfactorWT–ADPR–Ca2+, ARH3D77A–ADPR–Mg2+, and ARH3D314A–ADPR–Mg2+ complexes have been deposited in the Protein Data Bank under accession numbers 7L9F, 7L9H, and 7L9I, respectively.Atomic coordinates and structure factors of the ARH3This article contains The authors declare that they have no conflicts of interest with the contents of this article.