IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ http://www.ebi.ac.uk/ wang-2010-7 in vitro In vitro pull down pull down tag visualisation tag visualisation Wang ML., Motamed M., Infante RE., Abi-Mosleh L., Kwon HJ., Brown MS., Goldstein JL. Cholesterol binding Assay:Medium containing His tagged NPC2 or NCP1 was incubated with [3H]cholesterol, and the NPC-bound sterol was isolated by nickel chromatography. mike.brown@utsouthwestern.edu,joe.goldstein@utsouthwestern.edu Cell metabolism 2010 imex curation Only protein-protein interactions wang-2010-8 in vitro In vitro liposome binding assay liposome binding assay tag visualisation tag visualisation Wang ML., Motamed M., Infante RE., Abi-Mosleh L., Kwon HJ., Brown MS., Goldstein JL. Liposome Transfer Assay:Radiolabelled NPC1(NTD) was incubated with liposomes made from phosphatidylcholine and incorporating Texas Red dye. Components were separated on a Nickel-agarose column and amount of cholesterol transferred to liposomes was quantified by scintillation counting. The red dye shows the location of the liposomes. "PC" was prepared from egg yolk L-alpha--phosphatidyl-choline; it has fatty acid contents of approximately 33% 16:0 (palmitic), 13% 18:0 (stearic), 31% 18:1(oleic), and 15% 18:2 (linoleic) (other fatty acids being minor contributors). mike.brown@utsouthwestern.edu,joe.goldstein@utsouthwestern.edu Cell metabolism 2010 imex curation Only protein-protein interactions wang-2010-9 in vitro In vitro pull down pull down tag visualisation tag visualisation Wang ML., Motamed M., Infante RE., Abi-Mosleh L., Kwon HJ., Brown MS., Goldstein JL. Cholesterol Dissociation Assay:Cholesterol:NPC2-His10 was diluted 9-fold with buffer containing unlabelled cholesterol. After incubation the sample was applied to Ni-Agarose beads, centifuged and the supernatant assyed for radioactivity. mike.brown@utsouthwestern.edu,joe.goldstein@utsouthwestern.edu Cell metabolism 2010 imex curation Only protein-protein interactions wang-2010-10 in vitro In vitro anti tag coip anti tag coimmunoprecipitation anti tag coip tag visualisation tag visualisation Wang ML., Motamed M., Infante RE., Abi-Mosleh L., Kwon HJ., Brown MS., Goldstein JL. Cholesterol Transfer Assays Type "B": [3H]- cholesterol is pre-bound to a donor protein (NCP2) : it is transferred to acceptor NPC1(NTD)-LVPRGS-His8-FLAG. Anti-FLAG M2-agarose beads were used to isolate NCP1, and associated radioactivity counted. mike.brown@utsouthwestern.edu,joe.goldstein@utsouthwestern.edu Cell metabolism 2010 imex curation Only protein-protein interactions wang-2010-11 in vitro In vitro pull down pull down tag visualisation tag visualisation Wang ML., Motamed M., Infante RE., Abi-Mosleh L., Kwon HJ., Brown MS., Goldstein JL. Cholesterol Transfer Assays:- Type "A": Assay contained [3H] lipid pre-bound to a donor protein. Acceptor protein is His-tagged and subsequently isolated by Ni-resin pulldown. Protein-bound [3H]lipid was eluted and quantified by scintillation counting. mike.brown@utsouthwestern.edu,joe.goldstein@utsouthwestern.edu Cell metabolism 2010 imex curation Only protein-protein interactions cholesterol cholesterol small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) npc2_human NPC intracellular cholesterol transporter 2 NPC2 Niemann-Pick disease type C2 protein HE1 Human epididymis-specific protein 1 Epididymal secretory protein E1 protein protein human Homo sapiens Human MRFLAATFLLLALSTAAQAEPVQFKDCGSVDGVIKEVNVSPCPTQPCQLSKGQSYSVNVTFTSNIQSKSSKAVVHGILMGVPVPFPIPEPDGCKSGINCPIQKDKTYSYLNKLPVKSEYPSIKLVVEWQLQDDKNQSLFCWEIPVQIVSHL npc1_human NPC intracellular cholesterol transporter 1 NPC1 Niemann-Pick C1 protein protein protein human Homo sapiens Human MTARGLALGLLLLLLCPAQVFSQSCVWYGECGIAYGDKRYNCEYSGPPKPLPKDGYDLVQELCPGFFFGNVSLCCDVRQLQTLKDNLQLPLQFLSRCPSCFYNLLNLFCELTCSPRQSQFLNVTATEDYVDPVTNQTKTNVKELQYYVGQSFANAMYNACRDVEAPSSNDKALGLLCGKDADACNATNWIEYMFNKDNGQAPFTITPVFSDFPVHGMEPMNNATKGCDESVDEVTAPCSCQDCSIVCGPKPQPPPPPAPWTILGLDAMYVIMWITYMAFLLVFFGAFFAVWCYRKRYFVSEYTPIDSNIAFSVNASDKGEASCCDPVSAAFEGCLRRLFTRWGSFCVRNPGCVIFFSLVFITACSSGLVFVRVTTNPVDLWSAPSSQARLEKEYFDQHFGPFFRTEQLIIRAPLTDKHIYQPYPSGADVPFGPPLDIQILHQVLDLQIAIENITASYDNETVTLQDICLAPLSPYNTNCTILSVLNYFQNSHSVLDHKKGDDFFVYADYHTHFLYCVRAPASLNDTSLLHDPCLGTFGGPVFPWLVLGGYDDQNYNNATALVITFPVNNYYNDTEKLQRAQAWEKEFINFVKNYKNPNLTISFTAERSIEDELNRESDSDVFTVVISYAIMFLYISLALGHMKSCRRLLVDSKVSLGIAGILIVLSSVACSLGVFSYIGLPLTLIVIEVIPFLVLAVGVDNIFILVQAYQRDERLQGETLDQQLGRVLGEVAPSMFLSSFSETVAFFLGALSVMPAVHTFSLFAGLAVFIDFLLQITCFVSLLGLDIKRQEKNRLDIFCCVRGAEDGTSVQASESCLFRFFKNSYSPLLLKDWMRPIVIAIFVGVLSFSIAVLNKVDIGLDQSLSMPDDSYMVDYFKSISQYLHAGPPVYFVLEEGHDYTSSKGQNMVCGGMGCNNDSLVQQIFNAAQLDNYTRIGFAPSSWIDDYFDWVKPQSSCCRVDNITDQFCNASVVDPACVRCRPLTPEGKQRPQGGDFMRFLPMFLSDNPNPKCGKGGHAAYSSAVNILLGHGTRVGATYFMTYHTVLQTSADFIDALKKARLIASNVTETMGINGSAYRVFPYSVFYVFYEQYLTIIDDTIFNLGVSLGAIFLVTMVLLGCELWSAVIMCATIAMVLVNMFGVMWLWGISLNAVSLVNLVMSCGISVEFCSHITRAFTVSMKGSRVERAEEALAHMGSSVFSGITLTKFGGIVVLAFAKSQIFQIFYFRMYLAMVLLGATHGLIFLPVLLSYIGPSVNKAKSCATEERYKGTERERLLNF phosphatidylcholine phosphatidylcholine 1,2-diacyl-glycero-3-phosphocholine a phosphatidylcholine (0) 2,3-bis(acyloxy)propyl 2-(trimethylammonio)ethyl phosphate small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) npc2-cholesterol-1 2876177 N/A 2876182 unspecified role unspecified role prey prey region 3h radiolabel 3h radiolabel 3H tritium H3 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 2876183 unspecified role unspecified role bait bait purified purified P61916:p.Val81Ala mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false P61916:p.Pro114Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false P61916:p.Lys116Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false P61916:p.Tyr119Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false P61916:p.Ile122Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false P61916:p.Leu124Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false P61916:p.Val81Asp mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false P61916:p.Gln45_Pro46delinsAlaAla mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false P61916:p.Gly76Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false P61916:p.Leu78Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false P61916:p.[His75Ala;Ile77Ala] mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false certain certain sequence position certain certain certain sequence position certain false P61916:p.Phe85Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false P61916:p.Ile87Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false P61916:p.Pro88Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false P61916:p.Pro100Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false c_terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false crigr-cho_k1 Cricetulus griseus ovary K1 cho_k1 Chinese Hamster Ovary K1 direct interaction direct interaction false false false 2876177 1 A/C, 2 B/C Mutations reducing binding >85% have been entered as shown by the blue bands in Fig 1C. npc1-cholesterol-3 2876177 N/A 2876182 unspecified role unspecified role prey prey region 3h radiolabel 3h radiolabel 3H tritium H3 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 2876184 unspecified role unspecified role bait bait purified purified ntd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false c_terminus flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged c-term range C-terminal range c-term range c-term range C-terminal range c-term range false c_terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false O15118:p.Leu175_Leu176delinsGlnGln mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false direct interaction direct interaction false false false 4A npc1-npc2-1 2876178 N/A 2876184 unspecified role unspecified role neutral component neutral component purified purified ntd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false c_terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false c_terminus flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged c-term range C-terminal range c-term range c-term range C-terminal range c-term range false crigr-cho_k1 Cricetulus griseus ovary K1 cho_k1 Chinese Hamster Ovary K1 N/A 2876185 unspecified role unspecified role neutral component neutral component N/A 2876182 unspecified role unspecified role neutral component neutral component whole_molecule sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false region 3h radiolabel 3h radiolabel 3H tritium H3 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 2876183 stimulator stimulator neutral component neutral component P61916:p.Met79Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false P61916:p.Val81Ala mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false P61916:p.Val83Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false crigr-cho_k1 Cricetulus griseus ovary K1 cho_k1 Chinese Hamster Ovary K1 2876221 2876216 physical association physical association false false false 1 B/D Mutants showing transfer activity less than 40% of WT have been entered. It is not stated if NCP2 is tagged or not, in Figure 1 Liposome Transfer Assays. cholesterol-npc1-1 2876178 N/A 2876184 unspecified role unspecified role neutral component neutral component purified purified ntd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false c_terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false c_terminus flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged c-term range C-terminal range c-term range c-term range C-terminal range c-term range false crigr-cho_k1 Cricetulus griseus ovary K1 cho_k1 Chinese Hamster Ovary K1 N/A 2876185 unspecified role unspecified role neutral component neutral component N/A 2876182 unspecified role unspecified role neutral component neutral component whole_molecule sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false region 3h radiolabel 3h radiolabel 3H tritium H3 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 2876183 stimulator stimulator neutral component neutral component purified purified P61916:p.Val81Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false P61916:p.Val81Asp mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false c_terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false crigr-cho_k1 Cricetulus griseus ovary K1 cho_k1 Chinese Hamster Ovary K1 2876234 2876229 physical association physical association false false false 2 A npc2-cholesterol-2 2876179 N/A 2876182 unspecified role unspecified role prey prey whole_molecule sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false region 3h radiolabel 3h radiolabel 3H tritium H3 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false N/A 2876183 unspecified role unspecified role bait bait purified purified P61916:p.Val81Asp mutation with no effect mutation with no effect mutation with no effect mutation not affecting interaction certain certain sequence position certain certain certain sequence position certain false c_terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false whole_molecule sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false crigr-cho_k1 Cricetulus griseus ovary K1 cho_k1 Chinese Hamster Ovary K1 2876247 2876242 physical association physical association false false false 2876179 2D cholesterol-npc1-2 2876180 N/A 2876183 unspecified role unspecified role unspecified role unspecified role purified purified c_terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false P61916:p.Val81Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false P61916:p.Val81Asp mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false whole_molecule sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false crigr-cho_k1 Cricetulus griseus ovary K1 cho_k1 Chinese Hamster Ovary K1 N/A 2876184 unspecified role unspecified role bait bait purified purified ntd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false c_terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false c_terminus flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged c-term range C-terminal range c-term range c-term range C-terminal range c-term range false crigr-cho_k1 Cricetulus griseus ovary K1 cho_k1 Chinese Hamster Ovary K1 N/A 2876182 unspecified role unspecified role prey prey whole_molecule sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false region 3h radiolabel 3h radiolabel 3H tritium H3 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false 2876259 2876253 physical association physical association false false false 2E cholesterol-npc1-3 2876181 N/A 2876183 unspecified role unspecified role unspecified role unspecified role purified purified c_terminus flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged c-term range C-terminal range c-term range c-term range C-terminal range c-term range false P61916:p.Val81Ala mutation decreasing strength mutation decreasing interaction strength mutation decreasing strength certain certain sequence position certain certain certain sequence position certain false P61916:p.Val81Asp mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false whole_molecule sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false crigr-cho_k1 Cricetulus griseus ovary K1 cho_k1 Chinese Hamster Ovary K1 N/A 2876184 unspecified role unspecified role bait bait purified purified ntd sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain false c_terminus his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag c-term range C-terminal range c-term range c-term range C-terminal range c-term range false c_terminus flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged c-term range C-terminal range c-term range c-term range C-terminal range c-term range false O15118:p.Leu175_Leu176delinsGlnGln mutation disrupting strength mutation disrupting interaction strength mutation disrupting strength certain certain sequence position certain certain certain sequence position certain false crigr-cho_k1 Cricetulus griseus ovary K1 cho_k1 Chinese Hamster Ovary K1 N/A 2876182 unspecified role unspecified role prey prey whole_molecule sufficient to bind sufficient binding region sufficient to bind undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false region 3h radiolabel 3h radiolabel 3H tritium H3 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined false 2876273 2876266 physical association physical association false false false 4B