dataesr/scientific-paragraphs-categorization huggingface dataset:
Acknowledgements Research reported in this publication was supported by the  National Institute of General Medical Sciences  of the  National Institutes of Health  under Award Number  R35GM139587  (to K. A. A. ). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was supported by a public grant overseen by the  French National Research Agency (ANR)  as part of the  "Investissements d'Avenir" program  ( Labex NanoSaclay , reference:  ANR-10-LABX-0035 ) and by the  French   ANR-20-CE19-0020  and  ANR-14-CE08-0017. Financial support from an  UAL-FEDER  grant ( UAL2020-FQM-B2073 ) is also acknowledged.
23 For example, in AD brains, tau is found to be phosphorylated on more than 20 positions, mostly serine and threonine residues, and these PTMs impair the protein interaction and its stabilization of microtubules. 24, 25    to the disordered nature of tau, the tau/MT assembly is a fuzzy complex,  26, 27  which remains difficult to characterize with classical structural biology approaches. In particular, the disordered CTTs do not appear in the tubulin structures obtained by crystallography or cryo-EM that are currently deposited in the Protein Data Bank 28 (PDB). In this perspective, we combined homology modeling and molecular dynamics (MD) simulations to build a complete model, including the CTTs, of the complex formed by the β/α/β tubulin heterotrimer and the tau R2 domain for two tubulin isotypes (βI/αI/βI and βIII/αI/βIII).
Building the tubulin C-terminal tails and selecting starting models for the simulations We used the structure of the complex formed by two β-and one α-tubulin subunits bound with the R2 domain of tau (a 30-residues fragment ranging from Lys274 to Gly304) obtained by cryo-EM (pdb: 6CVN)  29  as a template. As this structure does not account for the tubulin CTTs, we used Modeller v10. 2  30, 31  to build two different isotypes, βI/αI/βI and βIII/αI/βIII, using sequences from sheep tubulin (which were chosen to fit experiments from experimental collaborators) for the αI (gene TUBAIA, Uniprot 32 accession number D0VWZ0) and βI (gene TUBB, Uniprot accession number W5PPT6) subunits, and from human tubulin (since the sheep sequence is not available) for the βIII subunit (gene TUBB3, Uniprot accession number Q13509), see Table  1.
Material and methods This retrospective study analyzed the phenotypic data of APP duplication carriers detected in France in the CNRMAJ center (Rouen University Hospital) from a nation-wide recruitment since 2006. Patients or legal representatives provided informed written consent for genetic analyses, in a medical and research setting (RBM 02-59, EudraCT 2009-010884-18) or GMAJ, NCT01622894, respectively approved by Paris Ile de France II and CPP Nord Ouest I ethics committees. This study was also conducted in accordance with the Declaration of Helsinki.
We included all patients exhibiting APP locus duplication including probands and their relatives. Before diagnosis, patients had neurological examination, neuropsychological assessment, and some had cerebral MRI and CSF AD biomarker analysis. Clinical data were retrieved from patients' medical charts provided by each referring clinician. The MRI patterns of these patients were compared to a control group of 40 APP-negative CAA controls (by sequencing exons 16 and 17 and by QMPSF) referred either to CNRMAJ, Rouen or to the Department of Genetics, Lariboisière Hospital, Paris, and fulfilling radiological criteria for probable CAA  [18].
The polyhistidine (6XHis) tag is one of the most ubiquitous fusion tags used for the puri cation of recombinant proteins. The 6XHis motif enables the tagged protein to bind metal ions such as Co 2+ , Ni 2+ , Zn 2+ and Cu 2+ , which can be advantageously used for protein puri cation with immobilized metal a nity chromatography (IMAC). Despite its widespread use, high-resolution protein structures encompassing a metal-bound 6XHis motif rarely occur in the worldwide Protein Data Bank (PDB). Here, we obtained a 2. 5 Å resolution crystal structure of a single chain variable fragment (scFv) antibody bearing a C-terminal tag composed of a sortase motif (LPETG), followed by a 6XHis and a TwinStrep tag (WSHPQFEK[G 3 S] 3 WSHPQFEK). The protein crystallized in the presence of cobalt (II) and its structure reveals a novel tetramerization motif (TetrHis) stabilized by 8 Co 2+ ions clustered within a small region of space.
The sequence of the motif corresponds to a 6XHis tag with a preceding ETG sequence from a sortase motif (LPETG) and a trailing sequence WSHPQ from a TwinStrep tag that were fused at the C-terminus of the expressed scFv construct (Fig. 1A ). ScFvs are composed of the variable regions of the antibody heavy and light chains (V H and V L domains) connected by a exible peptide linker, and they represent the smallest engineered antibody fragment containing the parental speci city. ScFvs can display signi cant inter-domain exibility between V H and V L domains  [13]  and have a tendency to form dimers and higher order oligomers depending on the length of the linker peptide  [14] [15] [16]. The scFv used in this work, 2A2, was designed by grafting the complementarity determining regions (CDRs) of the 2A2 antibody described to bind the lipid ceramide  [17]  onto a murine scFv scaffold with a [GGGGS] 4 linker (taken from PDB entry 5LX9 chain B [18]), with the initial aim of studying its binding to ceramide.
We used four consecutive histidines from the 6XHis of entry 2JSN  [22]  as a search motif and an arbitrarily high RMSD cut-off in order to avoid excluding structures in which histidines adopt a different conformation. Out of the 81 entries that contained at least four consecutive visible histidines and any bound zinc, copper, nickel, cobalt, cadmium or iron, we identi ed 63 structures in which at least one metal ion interacts directly with the 6XHis motif. We curated this initial dataset by removing redundancy, grouping highly similar structures together and keeping only a single representative, which yielded 27 unique X-ray crystallographic structures (Table  S1 ). We further omitted structures in which less than two histidines from the 6XHis were involved in metal coordination. Basic statistics regarding the nature, number of bound metal and their interactions with the protein are summarized in Fig.
The 36 Å Rg value obtained at low protein concentration was also roughly consistent with the Rg calculated from the crystallographic tetramer (up to 36 Å after addition of the scFv linker and missing residues at the C-terminus). Comparison of the Kratky plots from SAXS data measured at the lowest protein concentrations in the presence of EDTA versus Co 2+ ions suggests that the metal ions induce a transition from a mostly globular to a multidomain protein (Fig. 4B ). Because the strong concentration dependence of the measured Rg values precluded more detailed analysis, we next measured SEC-SAXS data with or without addition of Co 2+ ions in the SEC buffer. We hypothesized that the size-based separation of the SEC would result in higher data quality by decreasing interparticle interactions and removing potential signal contributions from small amounts of higher order oligomers or aggregates.
In most cases, a 6X His sequence was involved in crystal packing, stabilizing a dimeric state through metal coordination in conjunction with other residues located far away in the protein sequence. We were unable to identify any motif structurally homologous to TetrHis in the PDB, nor did we nd any examples of polyhistidine motifs stabilizing tetrameric states. Our analysis of SEC-SAXS data unambiguously showed that the existence of the TetrHis motif is not limited to the crystal, and that the tetrameric complex can also form in solution. In regular buffer conditions, we found that scFv 2A2 mostly adopted the classical scFv fold, but was in equilibrium with a minor population of open states resulting from the dissociation of V H and V L domains. This appears to be a frequent property of scFvs that have not been engineered for stability  [26, 27] , although to the best of our knowledge, such equilibrium has not previously been described in atomistic details by SAXS-based ensemble analysis.
X-ray Crystallization was carried out by vapor diffusion using a Cartesian Technologies pipetting system  [33]. scFv 2A2 was concentrated to 8. 5 mg/ml in 50 mM HEPES pH 7. 5 and 150 mM NaCl. The protein crystallized at 20°C in mother liquor containing 0. 01M cobalt chloride, 0. 1M MES, pH6. 5, 1. 8 M ammonium sulfate after about 5-10 days. Crystals were frozen in liquid nitrogen after being cryoprotected with mineral oil. Diffraction data were collected at X06SA beamline of the Swiss Light Source (SLS), Villigen, Switzerland, with a wavelength of 0. 999 Å. A single dataset at 2. 5 Å resolution was obtained. All data were automatically processed by xia2  [34]. Structural determination was initiated by molecular replacement using an homology model of scFv 2A2 obtained via SWISS-MODEL  [35]  as a search model in PHASER  [36].
The solution was subjected to repetitive rounds of restrained re nement in PHENIX  [37]  and Autobuster  [38]  and manual building in COOT  [39]. TLS parameters were included in the nal round of re nement. Data collection and re nement statistics are provided in Table  1 , and the nal re ned coordinates and structure factors have been deposited in the PDB with accession code 8CGE.
Small angle x-ray scattering Small angle X-ray scattering measurements of scFv 2A2 at 2. 1, 4. 2 and 8. 5 mg/ml were performed at the SWING beamline of the French national synchrotron facility (SOLEIL). Data was collected at 15°C, a wavelength of 1. 0332 Å and a sample-to-detector distance of 1. 99 m. For batch measurements, the scattering from the buffer alone was measured before and after each sample measurement and was used for background subtraction with PRIMUS from the ATSAS package  [40]. For SEC-SAXS measurements, scFv 2A2 samples at 8. 5 mg/ml were loaded onto a Superdex200 5/150 column (GE Healthcare) previously equilibrated in 50 mM HEPES pH 7. 5 and 150 mM NaCl with or without addition of 5mM CoCl 2. The measured SAXS images were normalized to the transmitted intensity and azimuthally averaged by using the in-house software Foxtrot (https://www.
8 µs for the monomer and ≈ 2. 0 µs for the tetramer. Snapshots were extracted every 1 ns from each trajectory, leading to the generation of 2760 models of the monomer and 2042 models of the tetramer. For each model from both ensembles, theoretical SAXS patterns were calculated with CRYSOL  [45]      unique PDB structures containing at least 4 consecutive histidines and a bound metal. The structures are classi ed according to the nature of the bound metal (A), the number of bound metal ions in the assembly (B), the number of protein chains that contribute coordinating residue sidechains (C), and whether the assembly involves a short linear sequence motif or a 3-dimensional motif (with residues located far away within the protein sequence) (D). E and F. Nickel-dependent dimeric assemblies of SlyD from Thermus thermophiles, observed in PDB entries 4odp and 3cgm.
3 His residues located at the Cterminus of the construct are involved in metal coordination, along with 2 His from a 6XHis tag located 3 residues downstream, and 1 His from the 6XHis tag of a symmetric-related molecule. G, H and I. Nickeldependent trimeric assemblies observed in PDB entries 2fg9, 6wxa and 7c3a. In each case, 3 x 2 His residues from 6XHis tags assemble to form a octahedral coordination sphere around a single nickel ion.
We processed the ISTD-normalized raw data using the MetaboR script (https://github. com/ DirtyHarry80/MetaboR). Figure  5 , below, shows the plots generated using the script and this dataset. Then, we performed the absolute quantification of 4-aminobutyric acid, glutamine and tryptophan in the HT29 Ctrl and Met samples. The calculation details are outlined in Supplemental section, Table  S6. Figure  6  shows tryptophan quantification.
QUANTIFICATION AND STATISTICAL ANALYSIS Basic data analysis For relative quantification, the peak areas obtained for each compound are subtracted from the blank measurement and then divided by the ISTD area measured in that sample. Instead of subtracting its signal, the blank can be used to select the metabolites for further analysis (i. e. , all metabolites significantly above the blank). The significance should be decided based on statistic testing (e. g. , t-test) and a fold-change threshold (we suggest at least factor 10). Agilent users will require the Mass Hunter Workstation Quantitative Analysis software to perform peak integration, blank subtraction, and ISTD normalization. Similar software is provided by other vendors (e. g. , Chromeleon for Thermo MS or LabSolutions for Shimadzu MS).
These analyses are efficiently done using dedicated R language packages, for example Metabo AnalystR. 10  To provide an example on how to use MetaboAnalystR, we deposited an R script and a test dataset in the public repository GitHub, accessible at the following address: https://github. com/ DirtyHarry80/MetaboR. git. Note: To be used in MetaboAnalystR, data need to be saved as tab-delimited text files (decimals separated by a point) and must follow several criteria. For example, the R code lines, as outlined in MetaboR, will only work if the data table format respects a specific layout. For more details, see the MetaboR information on Github. Depending on the data pre-normalization strategy, the function ''Normalization'' needs different parameter adjustments (in the MetaboR script, the parameters of ''Normalization'' are set to "Quan-tileNorm", "NULL", "AutoNorm" because we already normalized the data using ISTD and we wish to apply ''QuantileNorm'' and scale the data using "AutoNorm'').
Data and code availability The dataset supporting the current study has been included in Tables S3-S6 in the Supplemental section. The code used to create Figure  5  and the test dataset were deposited in the public repository GitHub, accessible at the following address: https://github. com/DirtyHarry80/MetaboR. git (https://doi. org/10. 5281/zenodo. 7962986). The instrument specific raw data are available from the corresponding author on request.
Na + /K + -ATPases are also known to genetically interact with UBE3A in an AS mouse model, and a reduction of their levels rescues electrophysiological and morphological abnormalities in the hippocampus of AS model mice  [29]. Other putative UBE3A substrates have been reviewed elsewhere  [3, 39, 40]. Of these, signi cant interest was at some point directed towards ARC and Ephexin-5, two proteins claimed to share a highly variable "UBE3A-target sequence" with Sacsin, a protein whose ubiquitination fraction was claimed to be increased by UBE3A on a proteomic dataset referred to by Greenberg and colleagues  [41, 42]. This yet unpublished proteomic dataset was obtained using a HA-Ub mouse  [43] , which to our knowledge has never before or after been used to generate any ubiquitin proteomics data.
One whole brain was collected for each of the three experimental replicates from both control and UBE3A overexpressing mice. After trypsin digestion, TMT labelled peptides of each sample type were analysed by LC-MS/MS. Highly reproducibly TMT data (Supplementary Fig. 3A ) allowed the identi cation and quanti cation of 5244 protein groups (Supplementary Table  1A ). Gene Ontology (GO) analysis of the total identi ed proteins determined by g:Pro ler showed a highly signi cant enrichment of proteins within the cerebral cortex, cerebellum and hippocampus (respective p-values of 10 - 248 , 10 - 215 and 10 - 197 ), as expected. Furthermore, quanti cation with MaxQuant and Perseus software con rmed a signi cant increase of UBE3A peptides in the UBE3A overexpressing mouse brain (Supplementary Table  1B ).
Similar levels of ubiquitinated material were enriched in all replicas of both experimental conditions as observed by western blotting and silver staining analysis of the pull-down samples (Supplementary Fig. 4A  and 4B ). In order to detect by MS the proteins isolated by Neutravidin pull-down, samples were rst fractionated by SDS-PAGE, Coomassie-stained and all gel lanes cut into four slices as illustrated in Supplementary Fig. 4C. Next, proteins were in-gel digested with trypsin, and the resulting peptides analysed by shotgun proteomics in a Q Exactive HF-X LC-MS/MS system. Finally, MS-derived raw data was processed using MaxQuant and the obtained label free quantitative (LFQ) intensities output analysed with Perseus software. A total of 1344 protein groups were identi ed with at least 1 unique peptide and quanti ed by this LFQ proteomics approach (Supplementary Table  1A ).
Venn diagrams showed a very high overlap (in the range of 87-92%) between proteins identi ed across replicas of the same experimental condition (Supplementary Figs. 4D). Multi-scatter plots correlating measured LFQ values for all 6 samples (Supplementary Fig. 4E ) indicate that the similarity between distinct replicas of the same experimental condition is higher (Pearson correlation coe cient (r) 0. 986-0. 992) than between distinct datasets (r ranging from 0. 887-0. 893). Imputation of missing values was performed as earlier described  [46] ; these imputations allow for a correct statistical analysis of the data  [50]. Multiscatter plots across whole brain samples quanti ed by TMT (Supplementary Fig. 3A ) or the brain ubiquitome quanti ed by LFQ (Supplementary Fig. 4E ) indicate that UBE3A overexpression did not cause major changes in the overall correlation of quanti ed intensities.
Comparison of the whole brain proteome and its ubiquitome When comparing across both proteomic datasets, more than 85% of the proteins detected in the ubiquitome analysis had also been detected in the whole mice brain extracts (Fig. 2C ). When analysing for enrichment (relative to the TMT dataset) by g:Pro ler, several molecular functions were enriched in the brain total ubiquitome dataset, the most predominant term being cell adhesion molecule binding with a p value of 10 - 17. Furthermore, we noted the cellular compartment terms vesicle, membrane and synapse to be predominantly enriched (p values of 10 - 34 , 10 - 16 and 10 - 8 , respectively) in the brain ubiquitome relative to the brain proteome. These ndings support previous work indicating that ubiquitination in the brain might particularly regulate synaptic function by acting on endocytic and lysosomal pathways  [51].
Whole brain proteome changes induced by UBE3A Given the fold-change compression of TMT-based MS acquisition, instead of prede ning a fold change threshold in the volcano plot to categorize proteins as enriched, reduced or non-changed, using Perseus software  [50]  we applied a Student´s t-test with permutation based false discovery rate (FDR) correction (FDR cutoff = 0. 01, s0 fuzz factor = 0. 1). A total of 52 proteins appeared signi cant enriched in the UBE3A-OE mouse brain, while another 52 proteins appeared depleted (Fig. 3A , Supplementary Table  1B ). Relative to the total proteome, g:Pro ler analysis identi ed a very signi cant enrichment of synaptic proteins in the brain extracts of UBE3A-OE mice, as well as of proteins regulating calmodulin binding and of proteins involved in chorea and status epilepticus, with a concomitant depletion of nucleic acid binding proteins being identi ed (Fig.
Interestingly, of the 122 putative UBE3A substrates, 82 had LFQ values in both control and overexpression samples with the other 40 being detected as ubiquitinated only upon UBE3A overexpression (Supplementary Table  1G ). A graphic representation of their LFQ intensity increase (Fig. 4D ) suggests that these UBE3A substrates went missing on the control sample due to their values being too close to the detection limit threshold in the control brains. And indeed, PRM allowed the detection in the control sample (see Supplementary Table  1G  and Fig. 5A ) of a number of these proteins not detected in the LFQ experiments. In order to further challenge the list of 122 candidate UBE3A substrates, a permutation-based FDR correction was applied using Perseus to this dataset, with standard parameters of FDR cutoff and an s0 of 0.
Two E2 enzymes depleted of their active carrier form in the UBE3A overexpressing brains are also shown. Actual fold change, -log 10 (p-value) and number of unique peptides are shown for both whole brain lysates (TMT experiment) and biotin-based pull-downs (LFQ experiment). Proteins with empty values are those not identi ed in the TMT experiment. Previously described involvement in LTP (as per number of papers), seizures (as being identi ed per GO terms in g:Pro ler, or via Pubmed, Pm) or NDDs (as per number of unique mutations recorded in the ClinVar database) are indicated in the Functional Evidence column. When looking more closely to the size of the identi ed proteins in the ubiquitome we identi ed a signi cant shift on the molecular weight of the proteins enriched upon UBE3A overexpression (Fig.
Indeed, absolutely no correlation is observed between ubiquitination and abundance changes for the proteins identi ed in this work with changing UBE3A levels (Figs. 2D and Supplementary Fig. 5B ), discarding changes in protein abundance  [52]  as a plausible approach for globally identifying UBE3A substrates. Having considered the possibility that this lack of correlation would be caused by using the bioUb strategy for the identi cation of ubiquitinated substrates, we analysed various diGly ubiquitome datasets from the literature on which full proteome data were also available (data not shown). The lack of correlation observed for the UBE3A dataset here appears to be a rather universal feature (manuscript in preparation).
4G ), in addition to its canonical E2 enzyme UBE2L3. After the early observation that substrates of the ubiquitin E3 ligase APC/C shared a common recognition motif  [70] , it has often seem that nding such a recognition motif for every E3 ligase would help us resolve the signalling pathways they regulate. Despite earlier work claiming to have identi ed a UBE3A substrate recognition motif, the answer to how does UBE3A recognise its substrates remains unresolved. However, analysing the dataset of candidate UBE3A substrates identi ed here in the brain mouse, however, we noted a signi cant enrichment of large Mw proteins (Fig. 4E ). The Mw of proteins whose ubiquitination is increased by UBE3A is signi cantly bigger, either upon comparison to all proteins identi ed in the brain, to all identi ed ubiquitinated proteins, or to the proteins that appear less ubiquitinated upon UBE3A overexpression.
Generation of the transgenic mice Generation of BirA and bioUb mice were already described in  Lectez et al. , (2014). Mouse Ube3a cDNA (GenBank accession number NM_011668), corresponding to the longest mouse Ube3a isoform (Uniprot ID: O08759-1 or NCBI reference: NP_035798. 2), was ampli ed from a pCAG-IRES-EGFP plasmid kindly provided by Dr. Zhi-Qi Xiong  [72]. First, the HECT domain of Ube3a was ampli ed and inserted between the EcoRI and NotI sites of pCAG-bioUb, after removal of the bioUb insert found between the same restriction sites  [44]. This rst step was carried out in order to generate an AgeI site on the pCAG plasmid. Then, the full Ube3a sequence was ampli ed with high-delity PCR and cloned between the AgeI and NotI sites of the pCAG vector. Correct DNA sequence was con rmed by sequencing.