MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it Redox signalling directly regulates TDP-43 via cysteine oxidation and disulphide cross-linking. Redox signalling directly regulates TDP-43 via cysteine oxidation and disulphide cross-linking. EMBO J. (0261-4189) 2012 imex curation Cohen TJ., Hwang AW., Unger T., Trojanowski JQ., Lee VM. Only protein-protein interactions human-qbi-293 Homo sapiens qbi-293 qbi-293 crosslink cross-linking study crosslink western blot western blot Immuno blot Immuno blot Cohen TJ., Hwang AW., Unger T., Trojanowski JQ., Lee VM. EMBO J. (0261-4189) 2012 Only protein-protein interactions imex curation Redox signalling directly regulates TDP-43 via cysteine oxidation and disulphide cross-linking. Redox signalling directly regulates TDP-43 via cysteine oxidation and disulphide cross-linking. EMBO J. (0261-4189) 2012 imex curation Cohen TJ., Hwang AW., Unger T., Trojanowski JQ., Lee VM. Only protein-protein interactions chlae-cos_7 Cercopithecus aethiops simian cells transformed with SV40 Grivet cos_7 cercopithecus aethiops, by transformation with SV40 fluorescence imaging fluorescence microscopy fluorescence imaging immunostaining immunostaining Cohen TJ., Hwang AW., Unger T., Trojanowski JQ., Lee VM. EMBO J. (0261-4189) 2012 Only protein-protein interactions imex curation Redox signalling directly regulates TDP-43 via cysteine oxidation and disulphide cross-linking. Redox signalling directly regulates TDP-43 via cysteine oxidation and disulphide cross-linking. EMBO J. (0261-4189) 2012 imex curation Cohen TJ., Hwang AW., Unger T., Trojanowski JQ., Lee VM. Only protein-protein interactions in vitro In vitro crosslink cross-linking study crosslink western blot western blot Immuno blot Immuno blot Cohen TJ., Hwang AW., Unger T., Trojanowski JQ., Lee VM. EMBO J. (0261-4189) 2012 Only protein-protein interactions imex curation tiar_human Nucleolysin TIAR TIAL1 TIA-1-related protein protein protein human Homo sapiens Human MMEDDGQPRTLYVGNLSRDVTEVLILQLFSQIGPCKSCKMITEHTSNDPYCFVEFYEHRDAAAALAAMNGRKILGKEVKVNWATTPSSQKKDTSNHFHVFVGDLSPEITTEDIKSAFAPFGKISDARVVKDMATGKSKGYGFVSFYNKLDAENAIVHMGGQWLGGRQIRTNWATRKPPAPKSTQENNTKQLRFEDVVNQSSPKNCTVYCGGIASGLTDQLMRQTFSPFGQIMEIRVFPEKGYSFVRFSTHESAAHAIVSVNGTTIEGHVVKCYWGKESPDMTKNFQQVDYSQWGQWSQVYGNPQQYGQYMANGWQVPPYGVYGQPWNQQGFGVDQSPSAAWMGGFGAQPPQGQAPPPVIPPPNQAGYGMASYQTQ 2F8D1C7169A7817E tadbp_human TAR DNA-binding protein 43 TARDBP TDP43 protein protein human Homo sapiens Human MSEYIRVTEDENDEPIEIPSEDDGTVLLSTVTAQFPGACGLRYRNPVSQCMRGVRLVEGILHAPDAGWGNLVYVVNYPKDNKRKMDETDASSAVKVKRAVQKTSDLIVLGLPWKTTEQDLKEYFSTFGEVLMVQVKKDLKTGHSKGFGFVRFTEYETQVKVMSQRHMIDGRWCDCKLPNSKQSQDEPLRSRKVFVGRCTEDMTEDELREFFSQYGDVMDVFIPKPFRAFAFVTFADDQIAQSLCGEDLIIKGISVHISNAEPKHNSNRQLERSGRFGGNPGGFGNQGGFGNSRGGGAGLGNNQGSNMGGGMNFGAFSINPAMMAAAQAALQSSWGMMGMLASQQNQSGPSGNNQNQGNMQREPNQAFGSGNNSYSGSNSGAAIGWGSASNAGSGSGFNGGFGSSMDSKSSGWGM DIP protein Q13148 original sequence version: 147 DIP protein Q13148 original sequence version: 114 DIP protein Q13148-1 original sequence version: 169 DIP protein Q13148-1 original sequence version: 124 8E09A1206FB4EF4A tardbp-tial1 2 TDP43 5 immunostaining immunostaining predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level fixed cell fixed cell TIAR 4 immunostaining immunostaining predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level fixed cell fixed cell colocalization colocalization f1a homomint mint TIAR and TDP43 have been detected as endougenously expressed in green monkey cells (COS-7); since their accession number needs to be updated we have adopted the human isoforms of the protein, because the antibodies also recognizes the human isoforms. tardbp-tardbp 3 TDP43 5 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified Q13148:p.Cys198Ser mutation disrupting interaction mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain C S Tue Jul 12 10:55:53 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation disrupting' Wed May 02 19:40:15 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'cys198ser' Tue Jun 30 18:21:52 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Cys198Ser' Q13148:p.Cys175Ser mutation disrupting interaction mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain C S Tue Jul 12 10:18:30 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation disrupting' Wed May 02 17:24:36 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'cys175ser' Tue Jun 30 18:27:47 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Cys175Ser' Q13148:p.Cys173Ser mutation disrupting interaction mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain C S Tue Jul 12 10:55:57 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation disrupting' Wed May 02 15:08:30 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'cys173ser' Tue Jun 30 17:19:55 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Cys173Ser' Q13148:p.Cys244Ser mutation disrupting interaction mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain C S Tue Jul 12 10:18:35 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation disrupting' Wed May 02 20:05:24 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'cys244ser' Tue Jun 30 19:15:30 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Cys244Ser' TDP43 5 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified Q13148:p.Cys173Ser mutation disrupting interaction mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain C S Tue Jul 12 10:18:25 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation disrupting' Wed May 02 14:48:35 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'cys173ser' Tue Jun 30 20:17:57 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Cys173Ser' Q13148:p.Cys175Ser mutation disrupting interaction mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain C S Tue Jul 12 10:55:52 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation disrupting' Wed May 02 14:41:08 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'cys175ser' Tue Jun 30 18:48:12 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Cys175Ser' Q13148:p.Cys198Ser mutation disrupting interaction mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain C S Tue Jul 12 10:18:30 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation disrupting' Wed May 02 17:46:48 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'cys198ser' Tue Jun 30 16:30:00 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Cys198Ser' Q13148:p.Cys244Ser mutation disrupting interaction mutation disrupting mutation disrupting interaction mutation disrupting mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain C S Tue Jul 12 10:18:37 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation disrupting' Wed May 02 20:13:30 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'cys244ser' Tue Jun 30 18:01:03 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Cys244Ser' direct interaction direct interaction homomint mint Given that oxidized cysteines can readily form disulphide bonds, we asked if H2O2 induced TDP-43 disulphide crosslinking in vitro. A dramatic accumulation of high molecular weight (HMW) TDP-43 immunoreactive bands was observed following H2O2 treatment using a panel of TDP-43 antibodies (Figure 3B, see HMW species). HMW disulphide bond formation was also observed using the sensitive cross-linking agent ethylene glycol disuccinate (EGS) (Figure 3C). Notably, in addition to HMW TDP-43 species, peroxide treatment led to a significant mobility shift in monomeric TDP-43, consistent with intra-molecular cysteine interactions (Figure 3C, lane 2). To further confirm the formation of intra- and inter-molecular interactions, oxidized TDP-43 was subsequently treated with the reducing agent dithiothreitol (DTT), which resulted in the complete collapse of all TDP- 43 species into a distinct 43 kDa protein band (Figure 3C, compare lanes 2 and 4). To test this hypothesis, we generated recombinant cysteine-deficient mutants, TDP-2CS (C173/175S) and TDP-4CS (C173/175/ 198/244S), and these proteins were analysed by immunoblotting. WTand 2CS proteins readily formed HMW cross-linked species in response to peroxide treatment (Figure 6B, see lanes 4 and 5 as well as lanes 7 and 8), which were reduced with DTT treatment even following 10mM peroxide treatment (Figure 6B, lanes 10 and 11). However, the formation of peroxide-induced HMW species was almost completely impaired in the absence of all four cysteine residues (Figure 6B, compare WT and 2CS to 4CS in lanes 6 and 9), indicating that Cys173, 175, 198, and 244 are likely critical for TDP-43 cross-linking. In addition to the fact that the 4CS mutant abrogated formation of HMW TDP-43 species, the gel mobility of monomeric 4CS was more compact in the absence of reducing agents compared with the more diffusely migrating WT or 2CS protein bands (Figure 6C, lane 3), suggesting impaired formation of intra-molecular disulphides. f3b f3c f6b tardbp-tardbp-1 3 TDP43 5 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified TDP43 5 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified disulfide bond disulfide bond disulfide bridge SS-bond homomint mint Peroxidetreated recombinant TDP-43 protein, including both HMW and monomeric species (Figure 5A, TDP-43ox protein bands identified with bracket and arrows), were excised from a nonreducing gel and subjected to mass spectrometry analysis. The peptide WCDCKLPNSK (mass 1190.52) was identified in both monomeric and HMW TDP-43 species, in which Cys173 and Cys175 formed intra-molecular disulphide bonds, as indicated by the following: (1) specific de-hydrogenation of the indicated cysteine residues within the peptide, (2) impaired peptide fragmentation peaks surrounding the disulphide bond, and (3) impaired ability to alkylate these modified cysteine residues (see Figure 5C and Supplementary Figure S3). f5a sf3 tardbp-1 3 TDP43 5 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified Q13148:p.Gly348Cys mutation disrupting interaction mutation increasing mutation increasing interaction mutation increasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain G C Tue Jul 12 10:17:48 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation disrupting' Wed May 02 18:43:24 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'gly348cys' Tue Jun 30 16:01:22 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Gly348Cys' Stoichiometry: 2.0 direct interaction direct interaction mint f8a f8b Interestingly, two of these mutations, G348C and S379C, introduce an additional cysteine residue that could enhance TDP-43 crosslinking via generation of additional disulphide bonds. To examine this possibility in vitro, recombinant WT TDP-43 or a panel of ALS mutant TDP-43 proteins were oxidized and analysed by Coomassie staining and immunoblotting. Interestingly, peroxide-induced oxidation of G348C and S379C mutants generated a distinct TDP-43 banding pattern characterized by a prominent B90kDa species, a putative TDP-43 dimer, that was absent in WT as well as G294A and R361S mutant samples, as detected by Coomassie staining (Figure 8A) and anti-TDP-43 antibodies (Figure 8B, see bands marked with asterisks). tardbp-1 3 TDP43 5 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified Q13148:p.Ser379Cys mutation disrupting interaction mutation increasing mutation increasing interaction mutation increasing mutation analysis mutation analysis certain certain sequence position certain certain certain sequence position certain S C Tue Jul 12 10:17:49 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'mutation disrupting' Wed May 02 20:31:44 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser379cys' Tue Jun 30 17:50:51 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.Ser379Cys' Stoichiometry: 2.0 direct interaction direct interaction mint f8a f8b Interestingly, two of these mutations, G348C and S379C, introduce an additional cysteine residue that could enhance TDP-43 crosslinking via generation of additional disulphide bonds. To examine this possibility in vitro, recombinant WT TDP-43 or a panel of ALS mutant TDP-43 proteins were oxidized and analysed by Coomassie staining and immunoblotting. Interestingly, peroxide-induced oxidation of G348C and S379C mutants generated a distinct TDP-43 banding pattern characterized by a prominent B90kDa species, a putative TDP-43 dimer, that was absent in WT as well as G294A and R361S mutant samples, as detected by Coomassie staining (Figure 8A) and anti-TDP-43 antibodies (Figure 8B, see bands marked with asterisks). tardbp-tardbp-2 1 TDP43 5 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell TDP43 5 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey over-expressed over expressed level over-expressed living cell living cell disulfide bond disulfide bond disulfide bridge SS-bond homomint mint Mass spectrometry analysis of all samples in the presence of DTT did not detect disulphide-bonded peptides. To confirm these results using a cell-based approach, overexpressed TDP-43 was immunoprecipitated from insoluble fractions of peroxide or arsenite-treated cells, and the TDP-43 protein bands were analysed by gel electrophoresis and Coomassie staining (Figure 5B). TDP-43 immunoprecipitates from insoluble fractions were excised for mass spectrometry analysis (Figure 5B, lanes 2 and 3). Similar to the in vitro analysis, the identical Cys173/Cys175 cross-linked peptide, WCDCKLPNSK, was observed in either peroxide or arsenitetreated cells, further confirming the presence of this intramolecular interaction in cells. Thus, an intra-molecular Cys173/Cys175 interaction contributes, in part, to the fully cross-linked TDP-43 species generated in response to oxidative stress. We were f5b sf3