CX3CL1, also known as fractalkine, is best known for its signaling activity through interactions with its cognate receptor CX3CR1. However, its intrinsic function that is independent of interaction with CX3CR1 remains to be fully understood. We demonstrate that the intracellular domain of CX3CL1 (CX3CL1-ICD), generated upon sequential cleavages by α-/β-secretase and γ-secretase, initiates a back signaling activity, which mediates direct signal transmission to gene expression in the nucleus. To study this, we fused a synthetic peptide derived from CX3CL1-ICD, named Tet34, with a 13-amino acid tetanus sequence at the N terminus to facilitate translocation into neuronal cells. We show that treatment of mouse neuroblastoma Neuro-2A cells with Tet34, but not its scrambled control (Tet34s), induced cell proliferation, as manifested by changes in protein levels of transcription factors and progrowth molecules cyclin D1, PCNA, Sox5, and Cdk2. Further biochemical assays reveal elevation of phosphorylated insulin receptor β subunit, insulin-like growth factor-1 receptor β subunit, and insulin receptor substrates as well as activation of proliferation-linked kinase AKT. In addition, transgenic mice overexpressing membrane-anchored C-terminal CX3CL1 also exhibited activation of insulin/insulin-like growth factor-1 receptor signaling. Remarkably, we found that this Tet34 peptide, but not Tet34s, protected against endoplasmic reticulum stress and cellular apoptosis when Neuro-2A cells were challenged with toxic oligomers of β-amyloid peptide or hydrogen peroxide. Taken together, our results suggest that CX3CL1-ICD may have translational potential for neuroprotection in Alzheimer’s disease and for disorders resulting from insulin resistance. The type-1 transmembrane chemokine CX3CL1, also known as fractalkine, is known to exert its signaling function by binding to its cognate receptor CX3CR1 . In the To address these unmet questions, we first designed a synthetic peptide, named Tet34, which was tagged with a 13-amino acid tetanus sequence at the N terminus to facilitate its specific binding to GT1b ganglioside receptors, which are expressed on the surface of neuronal cells . We founIn addition, we examined downstream signaling molecules in the insulin/insulin-like growth factor 1 (IGF-1) pathways and found significantly elevated phosphorylation of Foxo-3. Foxo proteins, a subgroup of the Forkhead family of transcription factors characterized by a conserved forkhead helix loop DNA-binding domain (FOX), have diverse cellular functions and are known to have roles in stress, aging, apoptosis, and cell-cycle regulation , 13, 14.A, illustration of peptide sequences). This peptide, named as Tet34, and the control peptide, Tet34s, which has the scramble order of sequence within the CX3CL1-ICD region, were tested for their ability to penetrate into neuronal cells. Since CX3CL1 C-terminal antibody would only recognize Tet34 but not Tet34s, a batch of these peptides were also tagged with Alexa 488 (Alexa488-Tet34 or Alexa488-Tet34s) for monitoring neuronal cell uptake. We showed that Alexa488-Tet34 was more effective in penetration into mouse neuroblastomas N2A cells than Alexa488-Tet34s at concentrations of 125 nM to 200 nM . During the monition between 12 and 72 h, clear uptake of Alexa488-Tet34 was seen at 24 h and peaked around 48 h.A synthetic peptide was designed from the sequence of CX3CL1-ICD and fused with a 13-amino acid Tet sequence derived from tetanus toxin at the N terminus to improve its specific uptake by neurons and neural stem cells  to primary neuron cultures. After treatment for 12 and 24 h, cells were fixed and stained with microtubule-associated protein 2 (MAP2). Alexa488-Tet34 signal was robustly detected in neuronal cell bodies after 12 h at a concentration of 1000 nM (C). At the same concentration, Alexa488-Tet34s was not easily detected, suggesting that the sequence of CX3CL1-ICD facilities the uptake of Tet34, although slightly different uptake kinetics between primary hippocampal neurons and N2A cells.We also examined uptake of Alexa488-Tet34 in primary neurons cultured from E16.5 mouse hippocampus at varying concentrations and time points. Alexa488-Tet34 and Alexa488-Tet34s were added at concentrations of 50, 500, and 1000 nM at 7 days  1000 nM C. At theTo determine if Alexa488-Tet34 uptake was specific to neurons, we waited until 12 DIV without treatment of AraC to allow proliferation of glial cells. After treatment for 12 h, we began to observe uptake of Alexa488-Tet34 by MAP2-marked neuron, but much less by astrocytes, labeled by GPAP antibody or Iba1-labeled microglia or Olig1-labeled oligodendrocytes . Again, D), indicating that Tet34 likely has a role in upregulation of cellular proliferation.During the cellular assay, we noted that N2A cells, when treated with Alexa488-Tet34, grew more densely. Therefore, N2A cells were treated with nonfluorescent Tet34 peptides at the concentrations of 1 μM for 12, 24, and 36 h to monitor cell proliferation by EdU incorporation assay. Cells treated with Tet34 had significantly increased EdU incorporation at 24 and 36 h compared with untreated controls or Tet34s-treated cells D, indicaA and B), indicating that Tet34 has a desired signaling activity. Elevated TGFβ2 and TGFβ3 expression induced a significant increase in phosphorylated Smad1 and Smad2 levels at 48 h but not total Smad expression levels (A and B). Thus, we demonstrated that fusion of Tet sequence to CX3CL1-ICD retains the activation of the TGFβ/Smad signaling pathway.To determine how Tet34 upregulates cell proliferation, we first examined whether this fusion Tet34 peptide retains the activity of inducing TGFβ signaling, similar to the expression of CX3CL1-ct in mice . We founn levels , A and BA). A corresponding increase in the level of phosphorylated InsRβ was more evident. This increase of phosphorylated InsRβ likely induced activation of its downstream molecules insulin receptor substrate-1 and -2 ; the increase was visibly more at the 48 h treatment. Phosphorylated IRS1 was significantly increased and confirmed by quantification . We also noted an increase in the levels of insulin growth factor 1 receptor (IGF-1R), detected by the antibody specific to the β-subunit (IGF-1Rβ). Levels of pIGF-1Rβ were notably increased as well (A and B).Our further biochemical exploration revealed that Tet34 treatment of N2A cells significantly upregulated expression of InsRβ subunit, which is phosphorylated when activated by insulin , 16, 17,Fig. 2A and B). Akt phosphorylation is known to be one of the key regulators of Forkhead transcription factors (Foxos) (A and B). Phosphorylation of Foxo1 was moderate but significantly increased as well, indicating downregulation of Foxo1 and Foxo3 activity.We then further examined downstream molecules and detected activation of PDK1 and Akt, as demonstrated by an increase in their phosphorylated levels but not total PDK1 or Akt , A and B (Foxos) . The act (Foxos) , 19. Tet (Foxos) , A and BWe also repeated the same set of experiment by including pharmacological inhibition of Akt activation with compound MK2206 , indicatA). Increased phosphorylation of insulin receptor was the most prominent, whereas the increase of pIGF-1Rβ was also visible . These increases were further confirmed by quantification .Tg-CX3CL1-ct were generated by utilizing the tetracycline-inducible promoter as described previously . This prFig. 3A, total IRS1 and IRS2 levels were visibly elevated, as was phosphorylated IRS1 levels. While total PDK1 levels were slightly elevated, phosphorylated PDK1 increased significantly (A), indicating a strong activation of PDK1 . While total Akt levels were not changed, elevation in Akt phosphorylation was significant. Activity of the transcription factors Foxo1 and Foxo3 was both repressed, as the levels of both phosphorylated Foxo1 (pFoxo1) and phosphorylated Foxo3 (pFoxo3) were significantly increased with no changes in total Foxo1 and Foxo3 levels (A and B).Their downstream signaling molecules were also examined by the Western blot analysis. As shown in Figure 3ficantly A, indicaTogether, these results support that CX3CL1-ICD has a role in the activation of both IGF-1Rβ and InsRβ signaling. Insulin/IGF signaling in brains, specifically in hippocampus, has been shown to regulate spatial learning and memory , and thiA and B). This corroborates our previous findings of Tg-CX3CL1-ct/tTA mice exhibiting enhanced neuronal proliferation and maturation in the SGZ of the dentate gyrus . Sox5 is a protein known to control cell cycle progression (C and D); Sox9 is shown to promote both basal progenitor proliferation and gliogenesis in developing neocortex . Unlike results in Tg-CX3CL1-ct/tTA mice, elevation of PCNA levels was the most obvious, whereas Cdk2, cyclin D1 levels were significantly increased. Together, these results show that Tet34 treatment has a potent signaling activity, which promotes cellular proliferation, in addition to neural differentiation.In our cellular assays, we showed that the levels of Sox2 and phosphorylated Sox2, Sox5, Sox8, and Sox9 were elevated even after 24 h treatment in N2A cells , E and FA and B). We showed that while the levels of cyclin-dependent kinase inhibitors p21 and p27 were significantly reduced (A and B), consistent with the report that p21 and p27 are transcriptionally regulated by Foxos  are active neurogenic niches in the murine brain. Majority of the newborn cells in the SGZ and SVZ undergoes apoptosis in different stages of neuronal differentiation and maturation into neurons . In line1–42 (briefed as Aβ thereafter) for 12 h. We showed that Akt protein levels, and its kinase activity, were decreased by Aβ treatment. Correspondingly, less phosphorylation of its substrate, Foxo3, was detected (A). N2A cells treated with Tet34 along with Aβ showed clear resilience to Aβ-mediated reduction of pAkt and pFoxo3 levels, which were confirmed by quantification (B).A previous study showed that Aβ treatment of N2A cells induces cellular stress and apoptosis in a dose-dependent manner , 31. To detected A. N2A cefication B.Figure C and D). Changes in Bax were less obvious. Tet34-treated N2A cells appeared to protect cellular apoptosis as levels of p53, Bad, and Bim were significantly lowered when comparing with Tet34s or mock-treated conditions with or without Aβ challenges. Relatively, Tet34 had less of an attenuating effect on Aβ-mediated Bax activation (p = 0.0437) (C and D). Reduction of cytochrome c release by Tet34 treatment was substantial when comparing Tet34 treated with two control groups (C and D); Aβ induced increase in cytochrome c release, and this toxic effect was clearly mitigated. These results are in line with the antiapoptotic effects exerted by Tet34. We further confirmed the antiapoptotic effects of Tet34 peptides on N2A cells treated with hydrogen peroxide. Tet34 attenuated the upregulation of various apoptotic markers in both 2% serum conditions as well as in hydrogen peroxide–treated groups  , C and Dl groups , C and Dd groups  and S6.E and F). The expression of these markers was lessened by Tet34 treatment in N2A cells. The 2% serum growth condition by itself also induces low level of stress, and such a low level of stress could also be alleviated by Tet34 treatment. Thus, these results demonstrate that Tet34 exerts protective effects against Aβ-mediated cellular stress and apoptosis.In addition, Aβ is known to induce endoplasmic reticulum stress in neurons . TherefoWith the increase in median life expectancy, the onset of age-dependent neurodegenerative diseases has become more prevalent, which has resulted in one of the major social and economic burdens in modern society . Thus, fB). When applied in co-cultures of primary neurons and glia, accumulation of Tet34 in neurons was evident, whereas much less was present in glial cells . We previously showed enhanced neurogenesis and significantly more mature neurons in both dentate gurus and cortical regions of Tg-CX3CL1-ct/tTA mice likely because of the activated TGFβ/Smad signaling pathway  and IGF-1Rβ (Y1134/Y1136)   and 3. H4/Y1136) . It is lvia the insulin/IGF-1/Foxo signaling.Our further investigation into the downstream targets of Foxos uncovered significantly reduced expression of apoptotic marker proteins, such as p53, Bax, and Bim. Cyclin-dependent kinase inhibitors such as p27 and p21 are transcriptional targets of Foxos , 36, andActivation of insulin signaling pathway is particularly exciting in the adult because homeostasis of insulin signaling is critical for the modulation of different cellular processes, such as cell survival, autophagy, and cell proliferation . In neurNeuronal apoptosis is associated with neurogenesis, physiologic aging, and neurodegenerative conditions , 49. Evein vivo application is imperative and may provide further insights of its dual ability to replenish neuronal loss and confer neuronal protection in various neurodegenerative diseases.In AD brains, dysregulated insulin/IGF-1 signaling has been found to contribute to neuroinflammation and oxidative stress , and ins2 for different time points before analysis.Peptide named as Tet34 has the sequence of RKMAGEMAEGLRYIPRSCGSNSYVLVPV derived from the CX3CL1 C terminus, whereas the Tet sequence for binding to neuron-specific GT1b receptor HLNILSTLWKYRC was fused to its N terminus. Tet34s has the same GT1b receptor binding sequence, but the remaining sequence has a scrambled order (HLNILSTLWKYRC-GASNVYPIMKRRRYEASLVLGPGMECSV). A batch of peptides were also custom tagged with Alexa Fluor 488 to track the kinetics of cellular and nuclear uptake (Thermo Fisher Scientific). Peptides were custom synthesized (GenScript) and solubilized in 1× PBS to attain a working solution concentration of 2 μg/μl. Multiple aliquots were prepared to avoid repeated freeze–thaw cycles and were stored at −80 °C before use. Monolayers of N2A cells were grown to 70% confluence in either 2-well chamber slides (Thermo Fisher Scientific) or 100 × 20 mm tissue culture–treated dishes (Corning) before treatment with peptides. Cells were treated with 50 nM and 2 μM of peptides in media containing 2% fetal bovine serum for immunostaining and protein analysis, respectively. Postpeptide treatment, cells were incubated at 37 °C, 5% COd-lysine (0.1 mg/ml) for 24 h at room temperature followed by washing with double-distilled water before plating. Media were exchanged after 24 h with 2% B27-supplement  and 1% glutamine in Neurobasal media. Because of omission of cytarabine (AraC) from culture media, glial cells were allowed to proliferate resulting in a mixed culture. After 12 DIV, cells were treated with 2.5 nM 488-conjugated Tet34 CX3CL1-ICD peptide or Tet34-scrambled for 12 h. Cells were fixed in cold 100% methanol, permeabilized with 0.2% Triton, and blocked in 6% normal goat serum. Slides were incubated in rabbit anti-MAP2 , rat anti-GFAP , rabbit anti-Iba1 , and rabbit anti-Olig2  primary antibodies overnight and incubated in Alexa secondary antibodies for 2 h at room temperature. Images were taken on a Zeiss confocal microscope using 63× and 20× objectives. The scale bar represents 20 μm (63×) and 50 μm (20×). Coimmunofluorescence quantification was conducted by measuring 488-pixel intensity within Map2-, Gfap-, and Iba1-positive cell bodies using ImageJ (NIH).Wildtype C57BL/6J E16.5 mouse embryos were euthanized, and brains were placed in cold Dulbecco’s PBS. The hippocampi were removed, washed with Hank's balanced salt solution, and digested in sterile-filtered papain (1 mg/ml) diluted in Neurobasal media  for 20 min. Tissues were triturated, and digestion was halted with 10% fetal bovine serum and 1% glutamine in Neurobasal media and filtered through a 70 μM cell strainer. Cells were counted in Trypan blue on a hemocytometer, and 100,000 hippocampal cells were plated onto each chamber slide. Two-well chamber slides were previously coated with poly-N2A cells were grown in 96-well tissue plates and treated with different concentrations of peptides for 24 h. Cell proliferation assays were performed using Click-iT EdU proliferation assay kits  according to the manufacturer’s instructions. Briefly, cells were seeded overnight, followed by addition of different concentrations of peptides. The EdU was added to the media after 6 h of peptide treatment. The assay was terminated at 36 h, when Edu incorporation was measured with a microplate reader at 568 nm excitation and 585 nm emission wavelength.ad libitum. The doxycycline (Sigma–Aldrich) treatment was at 0.5 mg/ml in drinking water, supplemented with 2% sucrose, and was administered to all animals used in experiment from E0 till postnatal day 45. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the UConn Health Center in compliance with the guidelines established by the Public Health Service Guide for the Care and Use of Laboratory Animals.The generation of Tg-CX3CL1-ct mice C-terminal fragment–derived CX3CL1 has been previously described . The Tg-in vitro studies, proteins were isolated from cells seeded in 6-well tissue culture–treated plates. Equal amounts of protein (50 μg) were resolved on a NuPAGE Bis–Tris gel (Invitrogen) and transferred onto a nitrocellulose membrane (Invitrogen). After protein transfer, the blot was incubated with the indicated antibodies. The sources of antibodies are p-InsRß , InsRß , p-IGF-1Rb , IGF-1Rβ , p-IRS1 , IRS-1 , IRS-2 , p-Smad2 , Smad2/3 , p-Smad1 , Smad1 , TGFβ1 , TGFβ2 , TGFβ3 , pPI3K , PI3-kinase p85 , pGSK3β , Gsk3β , PARP , cleaved caspase 9 , pFoxo1 , Foxo1 , pFoxo3 , Foxo3 , pPDK1  , PDK1 , pAKT , AKT , NeuroD1 , ASCL1 , p53 , p21 , p27 , Bax , Bim , pSox2 , Sox2 , Sox5 , Sox8 , Sox9 , HA , and CX3CL1 . Horseradish peroxidase–conjugated secondary antibodies were used and visualized using enhanced chemiluminescence (Thermo Fisher Scientific).Mouse brains were freshly dissected to isolate the hippocampus and cortex, and total proteins were extracted using modified radioimmunoprecipitation assay buffer. Routinely, at least two or three mice from each group were used for Western blot analysis. For Mouse brains were freshly dissected to isolate the hippocampus and cortex and midbrain. Total RNA was extracted using Trizol  according to the manufacturer’s protocol. About 4 μg of total RNA was used to perform complementary DNA (cDNA) transcription with High capacity cDNA Reverse Transcription kit . About 2 μl of cDNA was used for real-time PCR quantitation of IGF-1Rβ mRNA, IGF1 mRNA, and 18S rRNA with SYBR Green Master Mix . The quantitative PCR primers used were IGF-1Rβ (forward: 5′-GTG GGG GCT CGT GTT TCT C-3′ and reverse: 5′-GAT CAC CGT GCA GTT TTC CA-3′), IGF1 (forward: 5′-CCG AGG GGC TTT TAC TTC AAC AA-3′ and reverse 5′-CGG AAG CAA CAC TCA TCC ACA A-3′) and 18S rRNA (forward: 5′-TGTGCCGCTAGAGGTGAAATT-3′ and reverse 5′–TGGCAAATGCTTTCGCTTT-3′).1–42 (Biosource) was added to the cells at a 1 μM concentration and incubated for 12 h. The Aß1–42 was reconstituted according to the manufacturer's protocol. Briefly, the lyophilized peptide was dissolved in HPLC water followed by dilution in PBS. The solution was incubated at 37 °C and shaker for 24 h to obtain the neurotoxic form of Aß1–42. In our experience, we get ∼70% oligomeric Aß1–42 with this protocol. Total cell lysate was collected by radioimmunoprecipitation assay lysis buffer for Western blot evaluation of apoptotic pathways.N2A cell monolayers at 70% confluency were treated with 5 μM Tet-peptides for 24 h. Reconstituted human AßN2A cell monolayers grown to 70% confluency were treated with 2 μM Tet-peptides for 24 h in 2% serum for signaling induction. The cells were then incubated with 50 μM hydrogen peroxide  for different time points to induce apoptosis. A batch of cells were harvested at 1 h to evaluate apoptosis by Western blots. Flow cytometry using Alexa Fluor 488 Annexin V kit  was used to determine changes in the population of cells undergoing apoptosis at different time points of treatment.N2A cell monolayers at 70% confluency were treated with 2 μM Tet-peptides for 24 h in 2% serum for signaling induction. The cells were then incubated with 2 μM MK2206, Akt inhibitor  for 3 h prior to total cell lysate collection. Western blot was performed to evaluate the specificity of Tet-peptides in the induction of IGF1Rb/Akt/Foxo signaling pathway.t test. Significant p values are denoted by asterisks in the text and figures . Error bars in each case represent SEM.Quantitative data are presented as mean ± SEM. All experiments were independently repeated at least three times. Statistical analyses were conducted using Prism 6 software . Statistical comparisons between groups were analyzed for significance by one-way ANOVA with Tukey's post hoc test and Student’s All original data presented in the article will be made available for reviews when needed. Research materials will be also made available when it is required.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Renal fibrosis is a common feature of all types of chronic kidney disease (CKD) and is tightly regulated by the TGF-β/Smad3 pathway. Let-7i-5p belongs to the let-7 microRNA family with diverse biological functions. It has been reported that let-7i-5p suppresses fibrotic disease in the heart, lungs, and blood vessels, while the role of let-7i-5p in renal fibrosis remains limited. In this study, we aimed to investigate the role of let-7i-5p in renal fibrosis in a mouse model of unilateral ureteral obstruction (UUO) and TGF-β1–stimulated renal tubular cell line TCMK1. The RNA-targeting CRISPR/Cas13d system was used to knock down let-7i-5p. Renal injury and fibrosis were determined by histological analysis, RT-PCR, Western blot, and immunostaining. Our results have shown that in the kidneys after UUO, the expression of let-7i-5p was significantly increased along with notable tubular injury and interstitial fibrosis. Electroporation of let-7i–targeting Cas13d plasmid efficiently knocked down let-7i-5p in kidneys after UUO with reduced tubular injury, fibrotic area, and expression of fibrotic marker genes α-SMA, fibronectin, and Col1a1. In TGF-β1–stimulated TCMK1 cells, knockdown of let-7i-5p by Cas13d plasmid transfection also blunted the expression of fibrotic marker genes. Most importantly, the genomic locus of let-7i showed enriched binding of Smad3 as revealed by chromatin immunoprecipitation. In TCMK1 cells, the overexpression of Smad3 can directly induce the expression of let-7i-5p. However, the deletion of Smad3 abolished TGF-β1–stimulated let-7i-5p expression. Collectively, these findings suggest that let-7i-5p is a Smad3-dependent microRNA that plays a pathogenic role in renal fibrosis. Let-7i-5p could be a promising target for the treatment of CKD-associated renal fibrosis. The global incidence of chronic kidney disease (CKD) is about 13% which casts a non-negligible burden on the healthcare system . Renal fvia phosphorylation. Phosphorylated Smad2/3 translocate to the nucleus to modulate target gene transcription , microRNAs function to degrade or inhibit the translation of the target mRNAs . Let-7 iervation . For exaervation . The miRervation . Among td Col5a2 . Let-7i d Col5a2 . In the d Col5a2 . In contd Col5a2 . Despitein vitro and in vivo (Ruminococcus flavefaciens XPD3002 (designated as RfxCas13d) can knock down target RNA with superior efficiency and specificity when compared with the conventional RNA interference–based technologies . Furthermore, we also discovered that let-7i-5p is a target gene of the pro-fibrogenic TGF-β/Smad3 pathway.The CRISPR-Cas13d vector targeting pri-let-7i was constructed by cloning the guide RNA sequence  under U6 promoter of the backbone vector pLVX-U6-RfxCas13d . The recombined vector was designated as pCas13d-let-7i for short. The Smad3 overexpression plasmid pcDNA3.1-Smad3 was previously described in Male C57BL/6J mice, 8–10 weeks old, were purchased from GemPharmatech Co., Ltd. . The mice were adaptively fed for 1 week. Then, unilateral ureteral obstruction (UUO) or sham operation was performed on the right kidney as previously described . Seven dThe electroporation-mediated gene delivery was performed 1 day before the UUO operation. The animal was anesthetized with pentobarbital sodium. After shaving the dorsal hair, an incision (of about 0.5 cm) was made to expose the right kidney. Then, the renal artery and vein were clamped with an arterial clamp. Then, 100 μg of let-7i-5p knockdown plasmid  was slowly delivered into the kidney by retrograde renal vein injection through a 31-G needle. Electroporation was performed with the CUY21EDIT II Electroporator . Briefly, the kidney was clamped with a pair of tweezer-like electrodes. An electronic pulse of 50 V was applied four times. After electroporation, the artery clamp was removed and the kidney was replaced into the abdominal cavity. The incision was sealed, and the animal was placed in a warm place, waiting to wake up. For the sham operation, only 100 μl of saline was injected, followed by electroporation. The electroporation efficiency in the kidney was monitored by electroporating a plasmid coding the enhanced green fluorescent protein (EGFP). Electroporation following the aforementioned parameters can realize ubiquitous EGFP expression in the cortex tubules .The paraffin-embedded kidney tissue was cut into 4-μm-thick sections. Hematoxylin and eosin (HE) staining and Masson staining were performed as previously described . The tub2 and 100% humidity. All experimental treatments were performed in the 6-well plate. For the treatment with TGF-β1, TCMK1 cells of 80%–90% confluence were starved in the medium containing 0.5% FBS for 12 h. Then, 5 ng/ml of TGF-β1 was added to stimulate for the indicated time. For plasmid transfection, TCMK1 cells of about 50% confluence were transfected with the indicated plasmid (4 μg in each well) by polyethylenimine reagent. The medium was replaced 24 h after transfection.The mouse renal tubular cell line TCMK1 was purchased from ATCC  and cultured in DMEM  + 10% fetal bovine serum  + 1% penicillin–streptomycin  at 37°C with 5% CO−∆∆Ct method. The expression levels of mRNA-coding genes were normalized with GAPDH while the expression level of let-7i-5p was normalized with U6. Primers used in PCR amplification are listed in Supplementary Table S1.Total RNA of the kidney tissue or TCMK1 cells was isolated with the TRIzol reagent . For the detection of protein-coding gene and U6, 1 μg of RNA was used for cDNA synthesis with random hexamers by the HiScript III 1st Strand cDNA Synthesis Kit  followed by PCR amplification with Taq Pro Universal SYBR qPCR Master Mix . For the detection of let-7i-5p, cDNA was synthesized from 1 μg of RNA with the let-7i-5p–specific stem-loop primer (5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACAGC-3′) by the miRNA 1st Strand cDNA Synthesis Kit  followed by PCR amplification with miRNA Universal SYBR qPCR Master Mix . The relative expression of an indicated gene is calculated by the 2Protein was isolated with the RIPA lysis buffer and quantitated by the BCA method. Then, 20 μg of protein was separated in 10% SDS-PAGE gel and transferred to the PVDF membrane. After blocking with 5% fat-free milk powder, the indicated primary antibody was applied to incubate at 4°C overnight. For the detection of phosphorylated protein, 2.5% bovine serum albumin (BSA) was used for blocking and primary antibody incubation. The next day, the membrane was washed with TBST and incubated with HRP-conjugated secondary antibody at room temperature (RT) for 1 h. After washing with TBST, signals were developed with ECL Western Blotting Substrate  and visualized by ChemiScope 600 EXp system . ImageJ 1.47v software  was used to quantitate the relative expression of the indicated protein by normalizing it to GAPDH. Antibodies used in Western blot are listed in Supplementary Table S2.2O2 for 15 min at RT. After further blocking with 2.5% BSA, the primary antibody was applied to incubate at 4°C overnight. The next day, after washing with PBS, the downstream manipulations were performed with the Universal Mouse/Rabbit HRP-conjugated Polymer Detection Kit  following the manufacturer’s instruction. DAB was used for chromogenesis. Images were taken with a light microscope .The paraffin tissue section was dewaxed in xylene and rehydrated in gradient ethanol solution. Antigen retrieval was performed in 10 mM citrate solution (pH 6.0) by boiling in a microwave oven for 10 min. Endogenous peroxidase was blocked by treatment with 3% HFor immunofluorescence, the OCT-embedded fresh kidney tissue was cut into 4-μm-thick sections and fixed with 4% paraformaldehyde for 10 min. After permeabilization with 2.5% Triton X-100 for 15 min, the section was blocked with 2.5% BSA for 1 h. The primary antibody was incubated at 4°C overnight. The next day, the section was washed with PBS and incubated with fluorescent secondary antibody for 1 h. The nuclei were counterstained with DAPI. Images were captured with a fluorescent microscope . Antibodies used in immunohistochemistry are listed in Supplementary Table S2.TCMK1 cells were seeded into the 10-cm culture dish. When the confluence reached 90%, the cells were starved in a medium with 0.5% FBS for 6 h followed by stimulation with 5 ng/ml TGF-β1 for 30 min. The cells were then harvested for chromatin immunoprecipitation (ChIP) assay with a rabbit anti-Smad3 antibody  with the Magnetic Bead ChIP Kit . The chromatin precipitated by the Smad3 antibody was subjected to DNA purification which was used for PCR analysis (ChIP-PCR) or sequencing (ChIP-seq). ChIP-seq and downstream bioinformatic analysis was performed as previously described . The disThe Smad3 knockout (Smad3KO) TCMK1 cells were generated with CRISPR/Cas9-mediated gene knockout. Briefly, TCMK1 cells were transfected with the vector pX330-Smad3 which co-expressed Cas9 protein and mouse Smad3-targeting gRNA (with gRNA target sequence of 5′-GTCCCCAGCACACAATAACT-3′). Besides, this vector also co-expressed EGFP. After transfection, the EGFP-tagged cells were sorted with flow cytometry  and subjected to single cell–derived clonal expansion. DNA fragment flanking the edition site of the Smad3 locus was amplified by PCR in those successfully expanded cell clones and analyzed by Sanger sequencing . Cell clones with presumable deletion of Smad3 were further validated by Western blot analysis.t-test was used for comparison between two groups while one-way ANOVA with LSD test was applied for multigroup comparison. p < 0.05 was regarded as statistically significant. GraphPad Prism 5 software was used for statistical analysis and graph drawing.All quantitative data were presented as mean ± standard derivation (SD). A student’s As shown in in vitro and in vivo . Nevertheless, little is known about the mechanism regulating let-7i-5p expression. TGF-β signaling promotes renal fibrosis in CKD primarily through the downstream transcriptional factor Smad3 . It has Tsc1) to activate the mammalian target of rapamycin (mTOR) signaling in UUO mice  to promote renal fibrosis in UUO mice and folic acid–induced CKD mice. In contrast to the pro-fibrotic effect on renal disease, let-7i-5p seems to play an anti-fibrotic role in the fibrotic diseases of the heart, vasculature, and lungs (Col1a2, Col3a1, Col4a1, and Col5a2 (Tgfbr1 (It is reported that let-7i-5p promotes renal fibrosis by targeting tuberous sclerosis complex subunit 1 (UUO mice . Furthernd lungs . In hyped Col5a2 . In vasc (Tgfbr1 . These i (Tgfbr1 . Therefoin situ organ electroporation technology to realize the efficient knockdown of endogenous microRNA in vivo. Together with the knockdown study in TCMK1 cells, our results suggest that the CRISPR/Cas13d is a promising technology for the rapid functional study of microRNA in vitro and in vivo.CRISPR/Cas13d is thought to be a powerful tool to knock down RNA . As the Conclusively, findings in this study demonstrate that let-7i-5p is a pro-fibrotic microRNA and is regulated by the TGF-β/Smad3 pathway in renal fibrosis.


Bacteroides fragilis were considered as potential candidates in addition to the known BF9343_3149, but only this one showed glycolipid synthase activity. The enzyme was expressed as a SUMO fusion protein to produce soluble protein. It is a non-processive glycosyltransferase that prefers UDP-Gal over UDP-Glc as a donor substrate, and maximum activity was found at pH 7.3 and around 30–35 °C. It does not require metal cations for activity as other GT4 enzymes, but Zn2+ inactivates the enzyme. The reaction occurs when the ceramide lipid acceptor is solubilized with BSA (100% conversion) but not when it is presented in mixed micelles, and anionic lipids do not increase activity, as in other membrane-associated glycolipid synthases. Further protein engineering to increase stability and activity can make feasible the enzymatic synthesis of α-GalCer for biomedical applications.Glycolipids are complex molecules involved in important cellular processes. Among them, the glycosphingolipid α-galactosylceramide has proven to be of interest in biomedicine for its immunostimulatory capabilities. Given its structural requirements, the use of ceramide glycosyltransferase enzymes capable of synthesizing this molecule under in vivo or in vitro conditions is a potential production strategy. Several GT4 enzymes from  Sphingomonas paucimobilis hexanoyl]-D-erythro-sphin-gosine) was provided by the synthesis facility at the Institut de Química Avançada de Catalunya, Barcelona, Spain. UDP-Galactose sodium salt was obtained from Merck. DH5α (a)E. coli. Synthetic sequences were obtained from GeneArt  and cloned using restriction enzymes NcoI and XhoI into pET28a vector to obtain the expression vectors pET28a-HisTag-BF9343_XXXX. Positive transformants were selected on LB plates with 50 µg/mL of kanamycin. Successful plasmid constructions were verified by Sanger sequencing.pET28a vector-Protein sequences were obtained from the UniProt database and then translated to DNA code optimized for (b)pET22b-Strep-SUMO vector-Sequence BF9343_3149 was later cloned into a pET22b vector containing a N-terminal fused Strep-tag and SUMO protein. Cloning was carried out by Circular Polymerase Extension cloning (CPEC), amplifying the linear vector and the insert containing BF9343_3149 with primers sharing homologous regions, and CPEC reaction to obtain pET22b-SUMO-BF9343_3149-StrepTag. Positive transformants were selected on LB plates with 100 µg/mL of ampicillin. Successful plasmid construction was verified by Sanger sequencing.B. fragilis GT sequences were cultured in 300 mL of LB containing kanamycin at a final concentration of 50 µg/mL, inoculated at an initial OD of 0.05 and incubated at 37 °C until OD 1. At this point, protein expression was induced by adding IPTG to a final concentration of 1 mM, and cultures were incubated at 25 °C for 4 h. The cells were then harvested at 5000× g for 25 min and washed with 15 mL of NaCl 0.9%. The cell pellets were kept at −20 °C until use. pET28a-HisTag-BF9343_XXXX.-BL21(DE3)Star cells harboring pET28a containing pET22b-SUMO-BF9343_3149-StrepTag.-BL21(DE3)Star cells harboring pET22b-SUMO-BF9343_3149-StrepTag were grown in 600 mL of LB containing ampicillin at a final concentration of 100 µg/mL, inoculated at an initial OD of 0.05 and incubated at 37 °C until OD = 1. At this point, protein expression was induced by adding IPTG to a final concentration of 0.1 mM, and cultures were incubated at 30 °C for 4 h. The cell pellets were obtained as previously described.2HPO4, 150 mM of NaCl, pH 7.5) supplemented with 1mM of PMSF (phenylmethylsulfonyl fluoride). The cells were disrupted by sonication in a Soniprep 150 sonifier at 4 °C . The lysate was centrifuged at 25,000× g for 60 min. The supernatant was recovered and filtered (0.45 μm) before purification with a StrepTrap HP 1 mL column . The protein was eluted with lysis buffer with 2.5 mM of d-desthiobiotin. Eluted fractions were combined and dialyzed using Amicon Ultra-15 10 kDa (Millipore) with lysis buffer. The final retained fraction was recovered, and volume was adjusted to 1 mL with lysis buffer. Protein was quantified using the Bradford assay. Frozen cell pellets from the 600 mL cultures were thawed to room temperature and resuspended in 15 mL of lysis buffer , as described by Orive et al. 2020 [Glycolipid synthase activity from fresh cell-free extracts or purified enzyme was measured with Cer-NBD as an acceptor and UDP-Gal (or UDP-Glc) as a donor by HPLC with fluorescence detector 2HPO4, 150 mM of NaCl, pH 7.5) and the appropriate amount of enzyme in a 200 µL final reaction volume. It was preincubated at 37 °C for five minutes and the reaction was started by adding UDP-Gal. Sampling time was adjusted depending on enzyme concentration. Aliquots were withdrawn and mixed with methanol (MeOH:sample 8:2) and centrifuged to eliminate debris. The samples were then analyzed by HPLC. Chromatographic peaks were assigned by co-injection with independent standards. Initial rates were obtained from the linear progress curve of product formation. Initial rates were determined from triplicate assays.Ceramide-NBD with BSA: Reactions were performed with 25 µM of Cer-NBD, 25 µM of BSA, 1.25 mM of UDP-Gal, phosphate buffer (50 mM of Na(b)2 stream and the lipid mixture dried under vacuum for 2 h. When the reactions were performed, the lipid film was solubilized to homogeneity in 80 µL of phosphate buffer solution  by extensive vortexing and ultrasound bath for 5 min, and kept on ice for 1 min. This cycle was repeated 8 times. Vesicle formation was analyzed using ZetaSizer . For the enzymatic reaction, 25 µL of enzyme was added to 60 µL of freshly prepared vesicles solution. The mixture was kept for 30 min on ice and preincubated for 5 min at 37 °C. The reaction was started by adding 12.5 µL of UDP-Gal and incubated at 37 °C. The reaction conditions were: 1.25 mM of UDP-Gal, 25 µM of Cer-NBD, DOPC/DOPG vesicles ; 3:1 mM of DOPG, phosphate buffer , and enzyme (0.92–2.75 µM) in a 200 uL reaction volume. Aliquots were withdrawn every 30 min for 90 min, stopped as before and analyzed by HPLC.Ceramide-NBD in vesicles: Ceramide-NBD, DOPG and/or DOPG (matrix lipid) were dissolved in chloroform in a glass vial using the desired amounts for each experiment, as indicated. Chloroform was evaporated under a NUDP-Gal (0.1–2.5 mM) and ceramide-NBD (5–300 µM) with an equimolar amount of BSA were individually varied, maintaining constant the other components. Initial rates vs. donor or acceptor concentrations were fitted to a Michaelis–Menten equation with substrate inhibition by non-linear regression, using Prism 8 software , from which the kinetic parameters were derived. Tris buffer was used when analyzing metal binding to avoid precipitation of divalent cations. The cell pellet was resuspended with 20 mM of Tris buffer with 200 mM of NaCl, 1 mM of EDTA and 1 mM of DTT at pH 7.5 supplemented with 1 mM of PMSF. Enzyme purification was carried out following the same protocol as before, but with lysis buffer, 20 mM of Tris buffer and 200 mM of NaCl at pH 7.5. The reactions were performed with 25 µM of Cer-NBD, 25 µM of BSA, 1.25 mM of UDP-Gal, the appropriate amount of enzyme, Tris buffer  with or without EDTA (200 mM), and the addition of metal ions (0.25–1 mM) in a 200 µL final reaction volume. It was preincubated at 37 °C for five minutes, and the reaction was started by adding UDP-Gal.B. fragilis αGalCer_GT was obtained from Uniprot A0A380YRQ3. UDP-Gal structure was taken from PDB 5M7D. The complex αGalCer_GT·UDP-Gal was generated by docking with AUTODOCK VINA [The AlphaFold model of OCK VINA . Both thOCK VINA : polar hOCK VINA .


Saccharomyces cerevisiae Drg1. These structures reveal that Arx1 and the eukaryote-specific rRNA expansion segment ES27 form a joint docking platform that positions Drg1 for efficient extraction of Rlp24 from the pre-ribosome. The tips of the Drg1 N domains thereby guide the Rlp24 C terminus into the central pore of the Drg1 hexamer, enabling extraction by a hand-over-hand translocation mechanism. Our results uncover substrate recognition and processing by Drg1 step by step and provide a comprehensive mechanistic picture of the conserved modus operandi of AAA-ATPases.The AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis that initiates cytoplasmic maturation of the large ribosomal subunit. Drg1 releases the shuttling maturation factor Rlp24 from pre-60S particles shortly after nuclear export, a strict requirement for downstream maturation. The molecular mechanism of release remained elusive. Here, we report a series of cryo-EM structures that captured the extraction of Rlp24 from pre-60S particles by  The AAA-ATPase Drg1 assembles at the pre-60S ribosomal particle to release the ribosomal maturation factor Rlp24. Here, single-particle cryo-EM and 3D variability analysis dynamically visualize Rlp24 release by hand-over-hand substrate translocation. The pre-60S particles are subjected to massive structural rearrangements during maturation, including removal of pre-rRNA spacers, formation of the polypeptide exit tunnel (PET) and incorporation of the 5S pre-rRNA10. Thereafter, loading of Nmd3 provides the nuclear export signal (NES) for the exportin Crm1, also known as Xpo1, which mediates transport through the nuclear pore complex11.The assembly of ribosomes is a major activity in all cells. It is evolutionary conserved and tightly coordinated with cell cycle progression and proliferation. Eukaryotic ribosome biogenesis starts in the nucleolus by transcription of a precursor RNA (pre-rRNA) that contains the information for the 18S, 5.8S and 25S ribosomal RNAs (rRNAs) in yeast. Co-transcriptional pre-rRNA cleavage in the nucleolus separates the maturation paths of the small 40S and large 60S ribosomal subunits  per monomer and is closely related to human p97 and yeast Cdc48 (refs. 20). Mutations in SPATA5, the human ortholog of Drg1, cause developmental and neurological defects, underlining the protein’s importance for higher eukaryotes24.The initial event of cytosolic maturation is the release of the shuttling protein ribosomal-like protein 24 (Rlp24) by the essential hexameric type-II AAA-ATPase diazaborine resistance gene 1 (Drg1)12. However, only ATP hydrolysis in D2 is essential for viability and release of Rlp24 (ref. 16). The D2-domain-specific Drg1 inhibitor diazaborine therefore blocks Rlp24 extraction and prevents cell growth and proliferation17. The mechanism of Rlp24 release by Drg1 was unknown.The interaction with the unstructured Rlp24 C-terminal domain stimulates ATP hydrolysis in both AAA domains of Drg1, which drives the release reactionHere, we visualize by cryo-electron microscopy (cryo-EM) how Drg1 is recruited to the pre-ribosome to extract Rlp24. Rlp24 is captured by the Drg1 N domains and AAA domains and extracted by hand-over-hand translocation. Our data uncover orchestrated conformational changes of the AAA-ATPase during substrate translocation and provide a structural basis for the substrate-processing mechanism.S. cerevisiae strain, with separately purified Drg1 in vitro and collected single-particle cryo-EM data -tagged Bud20 as bait from a leptomycin B (LmB)-sensitive ata Fig. . LmB covort Fig. . These port Fig. . The incort Fig. . ResiduaTo capture Drg1 in the substrate-bound state, we used the slowly hydrolyzable ATP-analog ATPγS to prevent full extraction of Rlp24. This enabled us to identify Drg1-bound pre-60S particles and the AAA-ATPase alone Fig. . The str28. The particles also contain the shuttling proteins Mrt4, Nog1, Tif6, Rlp24 and Bud20  for Nsa2 that forms two helices at the base of the P-stalk. , with mainly negatively charged surfaces  as well as between a second Arx1 loop (203–219 aa) and the linker connecting the Drg1 NN and NC subdomains (118–127 aa).In Drg1, the N-domain cleft is bound by the Arx1 loop region (561–568 aa) Fig. . 3DVA re17, binds Arx1 similarly as the wild type does. As Arx1 does not affect in vitro ATPase activity of Drg1 strain  contacts the substrate chain above the pore loops . This suggests that diazaborine fixes Drg1 in the symmetric form and prevents conversion to the active conformation.Our dataset contains both a symmetric and an asymmetric conformation of free Drg1 Fig. . While tThe free asymmetric Drg1 hexamers in a staircase-like arrangement are highly similar to the particle-bound state, but with much higher local resolution (up to 3.2 Å). Intriguingly, the pore harbors a polypeptide chain engaged by pore loops of several protomers, confirming that this is the active substrate-processing state Fig. . The fre33. Four protomers  motif into the adjacent nucleotide-binding site  still has the nucleotide bound in D2, albeit there is no contact to the substrate. This presumably reflects a transition state prior to repositioning of this protomer. D2 of the clockwise adjacent protomer (P+1) is, at this stage, also not contacting the substrate.Upon releasing the nucleotide from the seam D2, the whole D2 domain is lifted. The rise of the helical subdomain elevates the α/β subdomain of the clockwise P+1) D2 domain, and the D2 pore loop of this domain moves toward the substrate, providing the next grip in top position of all D2 pore loops. At this stage, the seam is still detached, and thus has the highest degree of freedom to move. The counterclockwise P–1 D2 domain is now in the lowest position and thus the next in line to take the current seam’s place. In the starting frame, nucleotide hydrolysis in the P–1 D2 is still blocked by the inserted ISS, presumably to prevent premature hydrolysis. Nucleotide dissociation in the seam D2 fully retracts the arginine finger from the nucleotide in the P–1 site and partially retracts the ISS  D1 domain, because the helical subdomain of the seam tilts the D1 α/β subdomain of protomer F Fig. . This haThe structural changes in the D2 domain of the seam protomer cause leftward movement of the D1 helical domain, which is why the D1 and D2 rings are rotated by ~30° against each other. This pulls down the D1 α/β domain and the associated N domain of the seam protomer and causes rotation of the helical domain of protomer C, which affects positioning of the N domain of D. Thus, the ATP-loading state of the seam protomer also modulates N-domain substrate engagement in protomer D. As Drg1 is bound through the N domain of protomer D, positioning of the AAA-ATPase relative to the pre-ribosome will change during the cycle of ATP binding and hydrolysis, explaining its blurry appearance in our particle-bound structure.In summary, nucleotide dissociation in the seam D2 domain initiates a cascade of conformational changes that remotely triggers substrate engagement in the adjacent protomers. Consecutive grabbing and releasing of the substrate by the pore loops creates directionality and pulls the substrate chain through the pore in a stepwise manner.KD) of 2.7 µM  of 46 nM and a Hill coefficient of 3.5 ± 0.9, indicating positive cooperativity.To assess the mechanism of recognition, we quantified binding of Drg1 to the pre-60S particle using surface plasmon resonance  . This enabled us to measure release of the pre-ribosome from chip-bound Bud20-TAP and thereby quantify substrate processing by Drg1. We injected Drg1 in the presence of 1 mM ATP and measured the release of the particles from the sensor chip. With 37.5 nM Drg1 and ATP, we observed a linear decrease (~1 RU/second) of chip-bound particles , fully blocked the release in the presence of ATP. However, lower RU levels were reached as compared with ATPγS, suggesting that the drug interfered with pre-ribosome binding 46 or VAT (PDB: 5VCA)48 structures five protomers are associated in both domains. Thus, we captured an intermediate state in which the AAA domains of one protomer (protomer F in our structure) are asynchronous  from the α/β subdomain of the adjacent protomer  per yeast cell50.With our new SPR setup, we could measure, in real-time, both binding of Drg1 to the pre-ribosome in the presence of ATPγS and ATP-dependent extraction of Rlp24 through the concomitant release of Bud20-TAP. The measured release of 1 RU/second corresponds to ~1.5 pre-ribosomal particles per Drg1 hexamer per second or ~90 particles per minute . Thus, oTaken together, our data visualize the assembly of a AAA-ATPase on a large and complex target structure and dissect the chain of events during mechanical extraction of its specific substrate. This provides an important step forward to understand the dynamics and conserved modus operandi of these sophisticated biomechanical devices.Plasmids and strains generated in this study are available from the corresponding authors upon request. Materials, chemicals and reagents are listed in Supplementary Table Yeast and bacterial strains are listed in Supplementary Table Saccharomyces cerevisiae leptomycin sensitive (crm1T539C) mutant strain chromosomally expressing Bud20-GFP and the mCherry-tagged nuclear membrane protein Nic96 was grown to OD600 0.6 in SD medium supplemented with all amino acids. Thereafter, LmB (135 ng/µl) and/or diazaborine (10 µg/ml) were added, and cells were inspected by fluorescence microscopy after 30 minutes of treatment. As negative control, untreated cells were inspected. A Leica DM6 equipped with a ×100 HC PL APO (1.4) objective was used for fluorescence imaging.The S. cerevisiae mutant strain (crm1T539C) using Bud20-TAP as bait. Cells were grown to late log-phase (OD600 1–1.2) and then treated with 135 ng/µl µM LmB for 30 minutes. Cells were collected by centrifugation  and stored at −20 °C. Affinity purification via the Tandem affinity purification (TAP) tag was performed as described52: crude extracts were prepared by breaking the cells in a Merkenschlager bead mill in the presence of 0.6 mm glass beads (4 minutes with CO2 cooling every 30 seconds) in buffer A ). The homogenate was centrifuged at 40,000g, 4 °C, 30 minutes. For cryo-EM analysis, export-competent particles were purified using homemade magnetic beads containing covalently linked rabbit IgG and subsequent TEV protease cleavage38. For SPR analysis, particles were purified on calmodulin resin using the calmodulin binding peptide of the TAP tag and subsequently eluted with EGTA. The particle concentration of the eluate was estimated by OD260 measurement of the RNA content in the sample.Pre-60S particles were isolated from a LmB-sensitive 16: the expression strain was inoculated to a starting OD600 of 0.01, incubated at 30 °C at 110 r.p.m. in baffled flasks. Protein expression was induced by immediate addition of 0.025 µM CuSO4, and cells were collected after 24 hours. Affinity purification was performed as described18: frozen cells were thawed in lysis buffer  and 0.5 mM PMSF) and disrupted by vigorous shaking with 0.6 mm glass beads in a beadmill (Merckenschlager). Homogenates were centrifuged twice at 40,000g at 4 °C for 15 and 30 minutes, respectively. Crude extracts were incubated for 90 minutes at 4 °C with GSH-agarose beads (Sigma Aldrich) for affinity purification of GST-tagged Drg1. After consecutive washing steps , the protein was eluted. For elution, the GST tag was cleaved off using Prescission protease  overnight at 4 °C on a rotator in elution buffer suitable for the respective experiment. Protein concentration was measured using the Bradford assay (Biorad) with BSA calibration curve.GST-tagged wild-type Drg1 and the EQ1 (E346Q) variant were overexpressed in yeast as describedFor reconstruction of the Drg1–pre-60S complex, purified Drg1 was eluted in cryo-elution buffer , adjusted to 1.75 mg/ml (12 µM) and mixed in a 1:1 ratio with 200 nM export-competent pre-60S particles in the presence of 2 mM ATPγS and incubated for 10 minutes prior to plunge freezing. Quantifoil (R1.2/1.3) copper grids were glow-discharged for 60 seconds using an ELMO glow discharge system. Grids were loaded with 4 µl of the Drg1–pre-60S mixture and plunge frozen in liquid ethane using an FEI Vitrobot Mark IV at 4 °C and an environment with 100% humidity. The blotting force was set to 6, along with a blotting time of 7 seconds.−1) and dose fractionation. One movie contains 54 images resulting in a total dose of 60 e–/Å2 with a total exposure time of 4.84 seconds. The microscope was operated at 300 kV in nanoProbe energy-filtered transmission electron microscopy (EFTEM) mode at a nominal magnification of ×81,000. The dataset was acquired using SerialEM53 with an active beam tilt and astigmatism compensation. The acquisition scheme was designed to collect nine holes once per stage movement. Since the resulting maps showed preferred orientation, an additional, 34°-tilted dataset was collected with the same settings. The acquisition scheme was adapted to three holes per stage movement along the tilting axes. The required tilt angle was calculated with Relion v3.0.Cryo-EM data for the Drg1–pre-60S complex were collected on a FEI Titan Krios G3i in conjunction with a Gatan K3 BioQuantum direct electron detector using a slit width of 20 eV. The camera was operated in counting mode using hardware binning (pixel size of 1.07 Å pixel54). Micrograph preprocessing included motion correction (patch motion correction) and CTF determination (patch CTF estimation). Micrographs and power spectra were individually inspected using the manually curate exposures function to exclude low quality micrographs due to ice contamination, devitrification or strong drift.Image processing was mostly performed in Cryosparc v3.0 (ref. 55).For the Drg1–pre-60S complex, 6,214 high-quality micrographs from the untilted dataset as well as 4,241 micrographs from the tilted dataset were used for particle picking. Initial 2D class averages generated from manually picked particles of the Drg1–pre-60S complex were used for template picking in both datasets. Selected 2D classes from both datasets were combined, resulting in an initial set of 3,645,306 particles. After multiple rounds of 2D classification, 1,782,014 particles were used to generate an initial ab initio 3D model. Subsequently, multiple rounds of heterogeneous refinement, as well as 3DVA followed by clustering, were performed to separate pre-ribosomal particle populations. On the basis of the conformation of the L1 stalk, export-competent Bud20-TAP particles (closed L1) were differentiated from earlier Bud20-TAP populations or mature 60S subunits (both open L1). Heterogeneity analysis was performed with CryoDRGN v.3.0.2  were picked. Initial 2D class averages generated from manually picked particles were used for automated template picking generating an initial set of 3,148,330 particles. Multiple rounds of 2D classification were used to clean the dataset and remove pre-ribosomes. 3D classification by heterogeneous refinement resulted in a final set of 114,728 particles that was used for refinement and 3DVA3DVA  allows sorting of structural variants from complex particle mixtures. It was performed on the refined particles in Cryosparc v3.0 with a filter resolution of 7 Å, followed by a 3D Var display job with eight frames. Visualization was performed as a volume series in UCFS ChimeraX v1.3. Conformational heterogeneity in the final particle populations for both the free Drg1 hexamers as well as the Drg1–pre-60S complex was detected and visualized using 3DVA.57) for further processing. Finally, maps were post-processed using DeepEMhancer58.After homogeneous refinement in Cryosparc v3.0, particles were transferred to RELION v3.0 using pyEM v0.5 (ref. 7NKU)17. Model building was performed in Coot v0.9.6 (ref. 59), followed by refinement using Phenix v1.18.2-3874 (ref. 60) and ISOLDE v1.2.2 (ref. 61).As an initial model the symmetric Drg1 hexamer was used  were taken from PDB 6N8K27. An initial model for L12 was taken from the mature 80S ribosome (PDB 4V6I). As an initial model for the ES27 rRNA segment, PDB 3IZD was used. Adjustment and real-space refinement of these models was performed in Coot v0.9.2 and phenix refine v1.18.2-3874, respectively. For molecular visualization, UCSF chimera v1.14 (refs. 63) and ChimeraX v1.3 were used65.For the Bud20-TAP particle, initial models of the following components were taken from published early cytoplasmic pre-60S particles: Arx1 was taken from PDB 6RZZdrg1-18 mutant after 1 hour of incubation at 37 °C as described previously12. After TEV cleavage, the eluate from 2 L of culture was incubated with purified Drg1 (150 µg) in the presence of 1 mM ATPγS for 30 minutes on a rotator at room temperature. Afterwards, one-third of the sample was supplemented with 1.5 mM isotopically labeled crosslinking reagent A  and one-third with 1.5 mM isotopically labeled Crosslinker reagent B (bis(sulfosuccinimidyl)suberate, BS3-d0/BS3-d12, Sigma). One-third was further purified via Calmodulin sepharose beads without crosslinking. Crosslinking reactions were incubated at 30 °C in a thermomixer at 300 r.p.m. After 30 minutes, the reactions were quenched with 5 µl of a 1 M NH4HCO3 stock solution for further 10 minutes at 30 °C. Subsequently, samples were concentrated in a speedvac and stored at −20 °C.Pre-60S particles were purified via Arx1-TAP from a thermosensitive 28. In short, samples were dried , resuspended in 8 M urea, reduced, alkylated and digested with trypsin (Promega). Digested peptides were separated from the solution and retained by a solid-phase extraction system . Crosslinked peptides were enriched by size exclusion chromatography (SEC) using an ÄKTAmicro chromatography system  equipped with a Superdex Peptide 3.2/30 column . For each crosslinked sample four fractions were measured in technical duplicates. Therefore, the elution fractions 0.9–1.0 and 1.0–1.1 ml were pooled and the three elution fractions 1.1–1.2, 1.2–1.3 and 1.3–1.4 ml were analyzed separately by liquid chromatography with tandem MS (LC–MS/MS). Absorption levels at 215 nm of each fraction were used to normalize peptide amounts prior to LC–MS/MS analysis.Crosslinked samples were processed essentially as describedm/z, and a maximum injection time of 50 ms. The most intense precursor ions (intensity ≥ 5.0 × 103) with charge states 3–8 and monoisotopic peak determination set to ‘peptide’ were selected for MS/MS fragmentation by CID at 35% collision energy in a data-dependent mode. The duration for dynamic exclusion was set to 60 seconds. MS/MS spectra were analyzed in the ion trap at a rapid scan rate.LC–MS/MS analysis was carried out on an Orbitrap Fusion Tribrid mass spectrometer (Thermo Electron). Peptides were separated on an EASY-nLC 1200 system (Thermo Scientific) at a flow rate of 300 nl/minute over an 80-minute gradient . Full-scan mass spectra were acquired in the Orbitrap at a resolution of 120,000, a scan range of 400–1,500 66 in ion-tag mode, with a precursor mass tolerance of 10 ppm against a database containing ribosomal proteins and known assembly factors . For each experiment, only unique crosslinks were considered and only high-confidence crosslinked peptides that were identified with a delta score (deltaS) below 0.95 and an ld-Score above 32, translating to an FDR ≤ 0.5, were selected for this study. Crosslink networks were visualized with xiNet67.MS raw files were converted to centroid files and searched using xQuest/xProphetS. cerevisiae Arx1 was expressed as an N-terminal GST fusion from pGEX-6P-1 in Escherichia coli Rosetta (DE3) pLysS cells. Five hundred milliliters of LB medium (+100 µg/ml ampicillin and 40 µg/ml chloramphenicol) were inoculated to a starting OD600 0.04 and grown at 37 °C (170 r.p.m.). At an OD600 of 0.4, the culture was shifted to a shaking water bath at 16 °C, and after 30 minutes, heterologous protein expression was induced with 0.4 mM IPTG for 18 hours. Collected cells were washed once with aqua bidest and stored at −80 °C. For cell lysis, cells were resuspended in lysis buffer ) and incubated with 1 mg/ml lysozyme for 45 minutes prior to sonification. After removal of cell debris by centrifugation , the supernatant was incubated with GSH-agarose beads (Sigma Aldrich) at 4 °C for 60 minutes. Afterwards, beads were washed twice with lysis buffer, once with washing buffer  and twice with storage buffer . The beads with the bound GST-Arx1 were resuspended in storage buffer, portioned, shock frozen in liquid nitrogen and stored at −80 °C.12 as bait protein were included as additional reference. After five binding-buffer washing steps, followed by centrifugation , Arx1 and co-purifying Drg1 were separated from the beads by Prescission protease treatment  overnight, which cleaves off the GST tag. Samples using GST or GST-Rlp24C as baits were eluted by addition of 20 mM free GSH (Sigma Aldrich). The eluates were analyzed on a NuPAGE 4–12% Bis-Tris gel (Invitrogen).GST-Arx1 or GST-Rlp24 beads were thawed and washed once with binding buffer . Eighty micrograms Drg1 were incubated with the GST-tagged bait proteins on the beads in binding buffer (120 µl) in the presence of either 1 mM ATP or ATPγS or no nucleotide for 60 minutes at room temperature under constant rotation. Empty GST was included as control for unspecific binding. For the GST-Arx1 pulldown, samples using GST-Rlp24C68 as reported previously16. Essentially, purified Drg1 was eluted in 20 mM HEPES-KOH, 150 mM potassium acetate, 5 mM magnesium acetate, 0.1% Tween-20, 1 mM DTT, pH 6.8). HIS6-tagged Rlp24C or the indicated variants thereof were heterologously expressed in E. coli and purified as described in ref. 16. The activity of 5 µg/100 µl Drg1 was measured either alone  or in the presence of 2 µg (0.8 µM) HIS6-Rlp24C and/or 2.7 µg (0.4 µM) Arx1. All samples contained 1 mM ATP (Sigma Aldrich). The released phosphate was quantified using the Malachite Green Phosphate Assay kit (Bioassay Systems). The absorbance of the samples at 600 nm was measured at a GeniusPro TECAN plate reader using a Microsoft Excel data collection plugin (XFluor4 v4.51). The specific activity (µmol ATP/hour/mg Drg1) of all samples was normalized to the Drg1 basal activity to display relative activities. Three biological replicates were measured with three technical replicates to determine mean and s.d.Drg1 ATPase activity was measured using the Malachite green phosphate assaydrg1Δ, drg1Δ/arx1Δ or rlp24Δ) carrying the wild-type genes with their endogenous promoter and terminator on URA3 (pRS316) plasmids were transformed with plasmids expressing the indicated variants of Drg1, Arx1 or Rlp24. For plasmid shuffling, the transformed strains were grown in SD-leu (pRS315 plasmids) or in SD-his-leu (pRS315 and pRS313 plasmids) and spotted in a serial dilution on selective media and 5-FOA agar plates. For overexpression of Drg1 variants from the CUP1 promoter, the cells were spotted on selective medium containing different concentrations of CuSO4.Shuffle strains , which was also used as running buffer additionally supplemented with 1 mM ATPγS (or 1 mM ATP for the release assay). Then, 75–10,000 nM Drg1 supplemented with 1 mM ATPγS was injected with each cycle, composed of 180 seconds association, 120 seconds dissociation and 60 seconds regeneration of the chip surface with 1 M NaCl. Two biological replicates were measured on two separate CM5 chips with two technical replications (n = 4). For binding-affinity determination, measured RUs were normalized to the maximal response of each series (= 100%) and plotted over the concentration in Graphpad prism v3.03. The KD was determined by non-linear regression (one site binding hyperbola).SPR measurements were performed on a Biacore X100 . To analyze binding of Drg1 to the Rlp24C domain, purified GST-Rlp24C was immobilized as ligand on a CM5 sensor chip using the amine coupling kit (both Cytiva). Analogously, GST alone was immobilized in the reference flow cell. Drg1 was purified as described and eluted in elution buffer . For binding-affinity determination, measured RUs were normalized to the maximal response of each series (=100%) and plotted over the concentration in GraphPad prism v3.03. EC50 and Hill slope were determined by non-linear regression .To measure binding of Drg1 to the export-competent particles, IgG antibodies were immobilized in both flow cells of CM3 chips (Cytiva) by amine coupling. Subsequently, purified export-competent particles still carrying the Protein A moiety of the TAP tag were captured  on the IgGs prior to injection of increasing concentrations of Drg1. All samples contained 1 mM ATPγS. Each injection cycle was composed of a fresh particle capturing step, followed by injection of Drg1 , and finally two 30-second pulses of 10 mM glycine-HCl pH 2.2 to regenerate the binding surface. Three biological replicates were measured on three separate chips . Given that the specific responses produced by biomolecules are largely independent of size69 and that the CM3 sensor chip used for the experiment provides only half the surface volume with the thickness of the dextran matrix being 50 nm, we calculated that a response of ~1,500 RU obtained by the injection of the 2.1 MDa pre-ribosomal particle corresponds to a surface concentration of 0.14 µM. This would give a concentration of ~9.5 nM for 1 RU of the 2.1-MDa pre-ribosomal particle. Finally, we related ΔRU/s to the Drg1 concentration (37.5 nM monomeric Drg1 corresponding to 6.25 nM hexamer) to estimate the rate of particle release per Drg1 hexamer. Four 37.5-nM injections from four biological replicates were used for the quantification (n = 4).For the release assay, particles were analogously captured via IgGs, followed by injection of different Drg1 concentrations either containing 1 mM ATPγS (binding control), 1 mM ATP or 1 mM ATP + 100 µg/ml diazaborine. Linear segments (20 seconds) of the 37.5 nM Drg1 injections in the presence of ATP were used to quantify ΔRU/s. To determine the change in the number of bound particles, we used the manufacturer’s estimate  that an SPR signal of 1 RU approximately corresponds to a surface concentration of 1 pg/mmThe experiments described in Figures Further information on research design is available in the Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41594-022-00832-5.Supplementary InformationSupplementary Table 2Reporting SummaryPeer Review FileSupplementary Movie 1Substrate translocation by Drg1Supplementary Movie 2Drg1 on pre-60S particleSupplementary Table 1Crosslinking MS data


Likewise, gold nanoparticles (AuNPs) were added to form MoS2-PEG/Au to investigate its potential application as a theranostic nanomaterial. These nanomaterials were fully characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), photoelectron X-ray spectroscopy (XPS), Fourier-transformed infrared spectroscopy (FTIR), cyclic voltammetry and impedance spectroscopy. The produced hierarchical MoS2-PEG/Au microstructures showed an average diameter of 400 nm containing distributed gold nanoparticles, with great cellular viability on tumoral and non-tumoral cells. This aspect makes them with multifunctional characteristics with potential application for cancer diagnosis and therapy. Through the complete morphological and physicochemical characterization, it was possible to observe that both MoS2-PEG and MoS2-PEG/Au showed good chemical stability and demonstrated noninterference in the pattern of the cell nucleus, as well. Thus, our results suggest the possible application of these hybrid nanomaterials can be immensely explored for theranostic proposals in biomedicine.In this work, flower-like molybdenum disulfide (MoS The Nyquist plot is a plot of the real part of impedance (Z′) vs. imaginary part of impedance (−Z′′). The electrochemical impedance spectroscopy (EIS) technique is an important tool for evaluating the behavior of electrode material at the interface electrolyte-electrode . To exam2-PEG electrode shows an ERS value of 256.4 Ω while MoS2-PEG/Au presents a higher value 339.2 Ω, which can justify the better capacitive performance of the MoS2-PEG electrode. The charge transfer resistance, which is measured from the semi-circle along the real axis from high to medium frequency is also lower in the MoS2-PEG electrode than in the MoS2-PEG/Au one. At low frequency, the Nyquist plots showed a linear range with a slope of near 45°, known as Warburg resistance, where the frequency signal depends on the ion transportation in the electrolyte [2-PEG electrode, suggesting a lower ion diffusion resistance in the structure of the electrode [2-PEG/Au electrode shift along the x-axis toward more resistive values. Therefore, the better charge transmission and the ion diffusion through the electrode material structure help to explain the higher capacitance of the MoS2-PEG electrode.As a comparison, the MoSctrolyte . The exalectrode  while im2-PEG or MoS2-PEG/Au could be able to promote reduction of cellular viability in normal and tumoral cells under four different concentrations. The Green Monkey Kidney cell line VeroCCL-81 was used as non-tumoral model and the results applying MoS2-PEG showed discrete cellular viability reduction after 24 h when exposed to 10 µg/mL (2.1 ± 0.2)% and 5 µg/mL (5.2 ± 0.1)%, respectively, as shown in 2PEG/Au demonstrated decreased cellular viability in all concentrations tested, such as (12.2 ± 0.3)% for 100 µg/mL, (19.9 ± 0.9)% for 50 µg/mL, (14.4 ± 0.4)% for 10 µg/mL and (2.5 ± 0.1)% for 5 µg/mL, respectively, compared to 65.5% ± 0.1% of the death control cells. After 48 h no cellular viability reduction was observed for cells exposed to MoS2-PEG and smaller decrease of cellular viability was observed in cells exposed to MoS2-PEG/Au at 100 µg/mL (3.2 ± 0.1)%, 50 µg/mL (5.5 ± 0.2)%, 10 µg/mL (5.0 ± 0.1)% and 5 µg/mL (6.9 ± 0.1)%, respectively, as shown in The potential cytotoxicity of a given nanomaterial is a prerequisite for biomedical applications. In this study we assessed whether MoS2-PEG and MoS2-PEG/Au in the cellular viability of tumoral cells, the human squamous carcinoma A431 cell line and the human pharynx carcinoma FaDU cell line were used. For A431 cells no difference in the percentage of cellular viability was observed for MoS2-PEG in 24 h, as shown in 2-PEG/Au at 48 h showing (3.2 ± 0.1)% decreasing for 100 µg/mL and (4.2 ± 0.2)% for 50 µg/mL, respectively, as shown in panels (c) and (d). No alterations in the cellular morphology were observed as shown in To study the effects of MoS2-PEG showed a decrease of 3.8 ± 0.3% for 100 µg/mL and (11.8 ± 0.2)% for 50 µg/mL at 24 h, as shown in 2-PEG/Au the most pronounced cellular viability decreasing were observed for 100 µg/mL (6.5 ± 0.9)% and 50 µg/mL (11.61 ± 0.12)% at 48 h, as shown in panels (c) and (d). After contact with both nanomaterials FaDU cells demonstrated morphological changing when compared to control cells, as shown in 2-PEG/Au in the number and shape of their nuclei were assessed by Hoechst 33,342 nuclear staining. Interestingly, no differences were observed neither in nuclei number nor in nuclei morphology even at the highest concentration applied, when compared to control cells as shown in Another tumoral model was used and MoS2-PEG and MoS2-PEG/Au all cell lines presented very low damage what suggests the safety of these nanomaterials in vitro. Gold nanoparticles have been largely studied in biomedicine due to their well-known biocompatibility [2-PEG and MoS2-PEG/Au. Nonetheless, this change in morphology did not alter cellular viability or nuclear number as demonstrated in 2-PEG/Au. Although both nanomaterials exhibited good dispersion in water they agglomerated in culture medium, maybe due to the presence of protein and salt. However, MoS2-PEG and MoS2-PEG/Au structures were able to adsorb in the cellular membranes and it is possible that a small amount of these nanomaterials carried into the cells which explains the alterations in cellular viability percentages. It was already shown that 2D MoS2 nanoparticles do not promote deleterious effects in cellular viability or induce genetic defects in HEK293f cells [2-PEG and MoS2-PEG/Au themselves decrease cellular viability, they can be functionalized for theranostic purposes. We believe that MoS2-PEG/Au may possess potential application as a platform for theranostic, especially for cancer screening and photothermal therapy, because transition-metal dichalcogenides also enhance fluorescence through FRET and MoS2 nanosheets coated with folic acid had demonstrated efficient detection of miRNA-21 expression in a unique MCF-7 and HeLa cancer cells lines, working as a fluorescent nanoprobe. Based on the lack of cellular injuries demonstrated by our data both nanocomposites could have their surfaces modified to improve their dispersibility in aqueous solution. As an example, MoS2-PEG could be investigated as a new contrast agent for radiological imaging, and MoS2-PEG/Au could be functionalized to promote drug-delivery. The carefully physicochemical and biological characterizations of nanomaterials for biomedical applications are the first steps for a such proposal [2-PEG/Au for these purposes. Then, this initial study paves the way for other works in nanobiomedicine, for instance by incorporating dyes and/or biomolecules to MoS2-PEG/Au by taking advantages of the excellent optical properties of AuNPs and MoS2 as well.Our results demonstrated that both nanomaterials are biocompatible in normal cells, as well as in tumoral cells. Despite the high concentrations applied, for instance, 100 µg/mL and 50 µg/mL the cellular viability was kept above 80%. Moreover, at 48 h of contact with MoStibility . It seem3f cells . In thisproposal , and our2-PEG and MoS2-PEG/Au were successfully synthesized and widely characterized with different physicochemical techniques. By using TEM and SEM, it was possible to verify hierarchical flower-like structures, with a lateral size of up to 400 nm. Through the EDS and XPS studies, it was also observed that AuNPs were distributed throughout the material. MoS2-PEG/Au sample showed a lower capacitance value compared to MoS2-PEG; this fact may be associated with the low wettability of this electrode as observed in its contact angle. In this case, the presence of AuNPs could be related to increase the MoS2-PEG hydrophobicity, allowing only partial contact between the electrode and the electrolyte. However, in both cases the produced samples showed chemical stability and biocompatibility, due to the cell viability achieved above 80% for different times. Thus, its potential use as multifunctional nanomaterial for theranostic applications is an important subject that needs to be better explored.MoS


PI3Kα is a dimeric lipid kinase consisting of a catalytic subunit p110α and a regulatory subunit p85α. It controls cell proliferation and survival and is an important therapeutic target for cancer. However, the development of effective drugs against PI3Kα requires a level of structural information that is currently unavailable, because the extreme flexibility of PI3Kα interferes with structural analysis. Nanobodies were used in conjunction with chemical cross-linking to generate insights into the identity and the positions of the most flexible domains of PI3Kα and into mechanistic aspects of positional flexibility. The studies also reveal the existence of a previously unreported structural conformation of PI3Kα. Nanobodies and chemical cross-linking were used to gain information on the identity and positions of flexible domains of PI3Kα. The application of chemical cross-linking mass spectrometry (CXMS) facilitated the identification of the p85 domains BH, cSH2, and SH3 as well as their docking positions on the PI3Kα catalytic core. Binding of individual nanobodies to PI3Kα induced activation or inhibition of enzyme activity and caused conformational changes that could be correlated with enzyme function. Binding of nanobody Nb3-126 to the BH domain of p85α substantially improved resolution for parts of the PI3Kα complex, and binding of nanobody Nb3-159 induced a conformation of PI3Kα that is distinct from known PI3Kα structures. The analysis of CXMS data also provided mechanistic insights into the molecular underpinning of the flexibility of PI3Kα. Class I phosphoinositol 3-kinases (PI3Ks) are a family of lipid kinases, each composed of a regulatory and a catalytic subunit. Class IA of PI3K consists of three isoforms of the catalytic subunit, p110α, p110β, and p110δ, that are bound to the regulatory subunit p85α or one of its isoforms, p55α, p50α, or p85β . The dimPI3Ks control signaling events that are essential for cell growth and survival, and PI3K activity can be an important factor in tumor formation . Startin361116Detailed structural information could make an important contribution to these efforts. Crystallography, NMR, and hydrogen–deuterium exchange have provided valuable data on large parts but not all of the PI3Kα complex , 21–31. 2132A llama was immunized, and purified lymphocytes were used to generate a variable domain of heavy chain (VHH) library in phage. Three consecutive rounds of phage display were performed. Each was followed with an ELISA-based binding screen to obtain PI3Kα-specific nanobodies A. From tThe binding kinetics were measured using surface plasmon resonance (SPR) and binding affinities calculated from the kinetic rates . The effSI Appendix, Fig. S1). Truncations of the regulatory domain p85α were expressed in HEK293T cells and pulled down using nanobodies expressed in Escherichia coli, purified, and bound to magnetic resin. These experiments showed that Nb3-126 binds to the BH domain, while Nb3-142 and Nb3-159 bind to the nSH2 domain. This is surprising as the BH domain has not previously been identified as having a role in activation.The domains bound by the nanobodies were identified through coimmunoprecipitation (3 and DSG) were chosen to increase the cross-linking coverage of the entire complex. Purified PI3Kα associated with individual nanobody complexes was subjected to chemical cross-linking with either BS3 or DSG. The resulting products were evaluated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) to ensure almost complete cross-linking of PI3Kα with each nanobody . The reaction products were then digested by trypsin and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to map the cross-links within nanobody-bound PI3Kα complexes. Four experimental replicates were performed for each nanobody-bound complex for both BS3 and DSG. The CXMS analysis identified a total of 982, 738, and 602 cross-links within the complexes of PI3Kα with Nb3-126 . Comparing the cross-links assigned between PI3Kα and different nanobodies, we found both shared and unique residues in the nanobodies that were cross-linked with PI3Kα. For nanobody Nb3-126, the high-frequency cross-links were with Gln1, Ser52, and Lys53; for nanobody Nb3-159, residues Gln1, Ser63, and Lys65 were cross-linked at high frequency with PI3Kα . The cross-links of nanobody Nb3-142 are located outside the antigen binding domain and therefore do not provide information on the Nb3-142 binding site. The results with nanobodies Nb3-126 and Nb3-159 are in concordance with the coimmunoprecipitations, suggesting these nanobodies bind to the BH and nSH2 domains of p85, respectively.To validate the results of the coimmunoprecipitations and to explore nanobody-induced conformational and functional changes in PI3Kα, CXMS experiments were performed to obtain the cross-link maps for PI3Kα in complex with different nanobodies. Two complementary amine–amine cross-linkers . The Nb3-126 complex results in a high-resolution structure of the catalytic core of PI3Kα .We have performed a three-dimensional (3D) variability analysis (3DVA) of all three nanobody-bound structures using cryoSPARC. In addition to the average structure, cryo-EM can also provide information on the variability of the complex. Previous work has shown that there are a range of displacements of the ABD and iSH2 domains . ComplexSI Appendix, Fig. S7). At one extreme, Nb3-159 adopts a conformation similar to other PI3Kα complexes. At the opposite extreme . This raises the possibility that the ABD and iSH2 domains can adopt conformations that are very different from those seen in crystallography.It is unclear if this structure can occur without binding of a nanobody, but a similar density map is seen in the 3D classification of Nb3-142   . The resFig. S8) . AlthougThe CXMS results contain cross-links within the PI3Kα complex, which make it possible to extract valuable data on the interactions of PI3Kα domains, notably the unmodeled SH3, BH, and cSH2 domains of p85α. In a previous study, we described three extradensity domains (EDs), ED1, ED2, and ED3, that probably correspond to SH3, BH, and cSH2, respectively. However, these EDs could not be assigned to individual domains of p85α with certainty . EDs havPrevious structural studies have analyzed changes of the catalytic subunit p110α induced by the interaction with the regulatory subunit p85α under various experimental conditions. These investigations emphasize a dominant role of the nSH2 domain of p85α in regulating enzyme activity of PI3Kα –47. AlthThe persistent technical challenge for the cryo-EM analysis of PI3Kα is the positional flexibility of the BH, SH3, and cSH2 domains of the regulatory subunit p85. The mobility of these domains also creates major obstacles to defining therapeutic targeting sites for allosteric inhibitors. We have used nanobody binding and chemical cross-linking experiments with the goal of stabilizing the domains which are flexible relative to the catalytic core of PI3Kα. While neither of these methods have resulted in a completely stable complex, the acquired CXMS data allowed us to identify interactions between the stable catalytic core and the flexible domains previously named ED1, ED2, and ED3 . The anaConsidering the interactions of the individual nanobodies with PI3Kα, we find stimulating, inhibiting, and neutral effects on enzyme activity. Nanobody Nb3-126 stimulates PI3K activity through interaction with the BH domain. This effect does not involve a displacement of the nSH2 domain. The activation results from a destabilization of the inhibitory cSH2 domain interactions as seen in the electron density confidence map in Nanobody Nb3-142 inhibits activity of the complex through binding to the nSH2 domain. This interaction stabilizes the cSH2–catalytic core interactions and could also prevent binding to the cell membrane. The neutral nanobody Nb3-159 binds to the nSH2 domain and induces flexibility in the position and conformation of the ABD and iSH2 domains but causes no net change in activity. The 3DVA shows that binding of Nb3-159 results in a continuum of structures. Most notable is a structure showing extreme conformational changes, especially in the iSH2 and ABD domains but also affecting other domains of p110α. Although this conformation resulted from nanobody binding, there is a possibility that such structures could also be induced by other protein–protein interactions with PI3Kα.The linkages between domains of p85 are long and a major factor contributing to positional flexibility. The intervening sequences between the SH3, BH, nSH2, iSH2, and cSH2 domains have large, disordered segments and can access a complex 3D space. The domains of p85α are not hinged regions that move in a single plane but rather independent functional domains connected by spaghetti-like peptides. This is also reflected in the CXMS and HADDOCK predictions which show that it is not possible to satisfy all cross-links within a single docked model. Rather, the flexibility generates numerous cross-links, but not all are possible simultaneously. This flexibility may be essential to adopt conformations necessary for binding the diverse upstream receptor tyrosine kinases (RTKs) that initiate the activation of PI3Kα.We see our work on advancing the structural knowledge of PI3Ks as part of a broader scientific and technical development that is applying cryo-EM to this family of lipid kinases. This includes the analysis of the p110γ–p101 complex of class IB of PI3K , the recA llama was subcutaneously injected on days 0, 7, 14, 21, 28, and 35 with recombinant human phosphoinositide 3-kinase α (150 µg per injection) and Adjuvant P (GERBU). On day 40, about 100 mL of anticoagulated blood were collected from the llama for lymphocyte preparation.8 independent transformants. Eighty-three percent of the transformants contain the vector with the correct insert size of the VHH-encoding sequences as determined by DNA sequencing.A VHH library was constructed from the llama lymphocytes to screen for the presence of antigen-specific nanobodies. To this end, total RNA from peripheral blood lymphocytes was used as a template for first-strand cDNA synthesis with an oligo(dT) primer (ThermoFisher Scientific). The VHH encoding sequences were amplified by PCR using this cDNA, digested with PstI and NotI, and cloned into the PstI and NotI sites of the phagemid vector pMECS (ThermoFisher Scientific). The resulting VHH library is called Core 120 and consists of about 8.5 × 103, pH 8.2). The enrichment of antigen-specific phages was assessed by ELISA screening after each round of panning by comparing the number of phagemid particles eluted from antigen-coated wells with that eluted from negative (uncoated and blocked) control wells (The Core 120 library was panned for three rounds using solid-phase plates  coated wol wells . This re6 tag and amber stop codon was used for nanobody expression and purification. A 10 mL Luria-Bertani (LB) medium (Beyotime) overnight culture was diluted 1:100 into 1,000 mL fresh TB medium  in the presence of 100 µg/mL ampicillin, 2 mM MgCl2, and 0.1% glucose at 37 °C. When the culture reached an optical density at 600 nm between 0.6 and 0.9, nanobody expression was induced by 1 mM isopropyl ß-D-1-thiogalactopyranoside (Beyotime), and the temperature was decreased to 28 °C for 16 h. Bacteria were harvested by centrifugation at 5,000 rpm for 25 min, and the pellet was resuspended in 12 mL TES buffer  by pipetting, followed by shaking on ice for 1.5 h. Eighteen mL TES/4 buffer (TES diluted four times with ddH2O) were then added to the suspension and incubated on ice for another 1.5 h with shaking. After centrifuging for 30 min at 35,000 rpm (4 °C), the supernatant was bound to Ni-NTA resin (Cytiva) for 1 h at 4 °C. The resin was subsequently loaded onto a gravity flow column (Sangon Biotech), washed with PBS, and eluted with buffer A . The eluate was concentrated to 1 mL using an Amicon 10 kDa centrifugal filter (Millipore) and injected into a Superdex 16/60 200 pg gel filtration column (Cytiva) preequilibrated with buffer B . Fractions of gel filtration were collected, concentrated, aliquoted, and kept at −80 °C until use.The phagemid vector pMECS with a HisThe method of SPR was used as previously described  using a 70 value (4.8 mM). Nanobody  was mixed with PI3Kα and PIP2 substrate (14.5 μL) and incubated for 10 min before addition of ATP working buffer (5 μL) contained in the kit. The reaction was allowed to proceed for 30 min in the dark at RT before introducing the stop solution and detection mixture. The plates were then incubated for 3 h in the dark and read using an EnVision multilabel plate reader (PerkinElmer).The activity of PI3Kα in the presence of a given nanobody was determined by the PI 3-Kinase HTRF Assay (Millipore) as reported previously . BrieflyThe methods of PI3Kα expression and purification were described previously . PI3Kα–nThe purified PI3Kα–nanobody complex (2.5 μL) at a concentration of 1 mg/mL was applied to glow-discharged holey carbon grids  and subsequently vitrified by plunging into liquid ethane using a Vitrobot Mark IV (ThermoFisher Scientific). Automatic data collection was performed on a Titan Krios equipped with a Gatan K3 Summit direct electron detector (ThermoFisher Scientific). Dose-fractionated image stacks were subjected to beam-induced motion correction and dose-weighting using MotionCor2.1 . A sum o3 or DSG (ThermoFisher Scientific) in buffer B for 1 h at 25 °C. For the Nb3-159 and Nb3-142 complexes, 0.5 mg/mL of proteins were prepared and cross-linked with 1 mM BS3 or DSG. Cross-linking was quenched by the addition of 20 mM NH4HCO3 (Sigma) at RT, followed by desalting with PD Spintrap G-25 column  and air-dried in a SpeedVac concentrator (Labconco). In each cross-linking condition, the reaction was conducted in four replicates. Cross-linked protein samples were then subjected to SDS-PAGE to evaluate the cross-linking efficiency.The Nb3-126 complex was concentrated to 1 mg/mL and cross-linked with 1 mM BS4HCO3. Subsequently, they were reduced with 5 mM tris(2-carboxyethyl)phosphine (ThermoFisher Scientific) for 20 min at RT and alkylated with 10 mM iodoacetamide (Sigma) for 20 min at RT. The denatured proteins were digested using trypsin (Promega) at an enzyme-to-sample ratio of 1:50 at 37 °C overnight. After being acidified with 1% final concentration of formic acid (Sigma), the supernatant was desalted using UltraMicro Spin Column, Silica C18 (The Nest Group). Finally, peptide samples were lyophilized with a SpeedVac concentrator (Labconco) and redissolved with 0.1% formic acid (FA) before LC-MS/MS analysis.The air-dried samples were resuspended in 8 M urea (Sigma) and 20 mM NHLC-MS/MS analysis was carried out on an EASY-nLC 1200 system coupled to a Q Exactive HF mass spectrometer (ThermoFisher Scientific). The peptide samples were loaded onto an analytical column (200 mm × 75 μm) packed with C18-AQ 1.9 μm C18 resin (Dr. Maisch GmbH) at a flow rate of 300 nL/min and separated using a 120 min gradient: 0 to 30 min from 5 to 15% B, 30 to 70 min from 15 to 25% B, 70 to 110 min from 25 to 40% B, 110 to 115 min from 40 to 100% B, and 115 to 120 min 100% B . The top 20 most intense precursor ions from each full scan  target 3e6, scan range 300 to 1,800 mass-to-charge ratio) were isolated for higher-energy collisional dissociation MS2 analysis  with a dynamic exclusion time of 30 s. Precursors of charge states 3+ to 8+ were selected for MS2 data acquisition.Software pLink 2.3.9  was usedThe cryo-EM structure of PI3Kα (Protein Data Bank [PDB] ID 7MYN)  was dockFigures were produced using Chimera  and ChimDataset S1.A model of the full-length PI3Kα complex was generated using the Nb3-142–bound structure, the Nb3-142 CXMS data, and the SH3 (PDB ID 1PNJ), BH (PDB ID 6D82), and cSH2 (PDB ID 1QAD) domains. Probable interactions were determined with DisVis 2.0 using the Nb3-142 model with each domain individually, employing the full set of cross-links . Proteing for 10 min, resuspended in 50 mM Tris⋅HCl, pH 7.0, 5% glycerol, and 50 mM NaCl, and lysed using sonication. The resulting suspension was then centrifuged at 15,000 × g for 1 h at 4 °C. The supernatant was purified using His-Trap FF column (Cytiva) and eluted with 300 mM imidazole in 50 mM Tris⋅HCl, pH 7.0, 5% glycerol, and 50 mM NaCl. Using Amicon Ultra 10 kDa centrifugal filters (Millipore), the buffer was exchanged to 50 mM Tris⋅HCl, pH 7.0, 5% glycerol, and 50 mM NaCl.Nanobody sequences were synthesized using G-block synthesis (IDT). These were cloned into the NcoI and SalI sites of pET-28a (Novogene). The resulting plasmids were transformed into BL21 (DE3). Bacteria were grown to an OD600 of 0.5 in LB medium (BD Biosciences), induced with 1 mM IPTG (Sigma-Aldrich), and incubated for 4 h at 37 °C. The bacteria were harvested by centrifugation at 6,000 × The purified protein was bound to DynaBeads His-Tag (Life Technologies) and rinsed three times with lysis buffer . The PI3Kα complex was produced in HEK293T cells by transfection of pHIV constructs using Lipofectamine 3000 (ThermoFisher Scientific). After 48 h, cells were lysed using lysis buffer. The PI3Kα complex consisting of full-length p110α with N-terminal HA-tagged p85α full-length or residue ranges 75 to 720, 307 to 720, or 1 to 608 was mixed with the prepared nanobody-bound DynaBeads by incubation for 10 min at RT with agitation. These complexes were then washed three times with wash buffer  and eluted using wash buffer with 300 mM imidazole, separated on an SDS-PAGE gel, and blotted to PVDF (Millipore). The resulting blots were probed with anti-His tag and anti-HA tag antibodies  to determine binding.Supplementary FileSupplementary File