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 New insights into in vitro amyloidogenic properties of human serum albumin suggest considerations for therapeutic precautions. New insights into in vitro amyloidogenic properties of human serum albumin suggest considerations for therapeutic precautions. FEBS letters 2015 imex curation Sharma N., Sivalingam V., Maurya S., Prasad A., Khandelwal P., Yadav SC., Patel BK. basantkpatel@iith.ac.in 22-Feb-2016: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro comigration in gel comigration in gel electrophoresis comigration in gel weight identificat confirmation by molecular weight weight identificat Sharma N., Sivalingam V., Maurya S., Prasad A., Khandelwal P., Yadav SC., Patel BK. basantkpatel@iith.ac.in Accepted 2016-JAN-28 AT 11:50 GMT AT 11:50 GMT by MARTA FEBS letters 2015 Only protein-protein interactions imex curation imex curation New insights into in vitro amyloidogenic properties of human serum albumin suggest considerations for therapeutic precautions. New insights into in vitro amyloidogenic properties of human serum albumin suggest considerations for therapeutic precautions. FEBS letters 2015 imex curation Sharma N., Sivalingam V., Maurya S., Prasad A., Khandelwal P., Yadav SC., Patel BK. basantkpatel@iith.ac.in 22-Feb-2016: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column predetermined predetermined participant predetermined Sharma N., Sivalingam V., Maurya S., Prasad A., Khandelwal P., Yadav SC., Patel BK. basantkpatel@iith.ac.in Accepted 2016-JAN-28 AT 11:50 GMT AT 11:50 GMT by MARTA FEBS letters 2015 Only protein-protein interactions imex curation imex curation New insights into in vitro amyloidogenic properties of human serum albumin suggest considerations for therapeutic precautions. New insights into in vitro amyloidogenic properties of human serum albumin suggest considerations for therapeutic precautions. FEBS letters 2015 imex curation Sharma N., Sivalingam V., Maurya S., Prasad A., Khandelwal P., Yadav SC., Patel BK. basantkpatel@iith.ac.in 22-Feb-2016: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro atomic force microsc atomic force microscopy AFM AFM atomic force microsc predetermined predetermined participant predetermined Sharma N., Sivalingam V., Maurya S., Prasad A., Khandelwal P., Yadav SC., Patel BK. basantkpatel@iith.ac.in Accepted 2016-JAN-28 AT 11:50 GMT AT 11:50 GMT by MARTA FEBS letters 2015 Only protein-protein interactions imex curation imex curation New insights into in vitro amyloidogenic properties of human serum albumin suggest considerations for therapeutic precautions. New insights into in vitro amyloidogenic properties of human serum albumin suggest considerations for therapeutic precautions. FEBS letters 2015 imex curation Sharma N., Sivalingam V., Maurya S., Prasad A., Khandelwal P., Yadav SC., Patel BK. basantkpatel@iith.ac.in 22-Feb-2016: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro fluorescence spectr classical fluorescence spectroscopy fluorescence spectr predetermined predetermined participant predetermined Sharma N., Sivalingam V., Maurya S., Prasad A., Khandelwal P., Yadav SC., Patel BK. basantkpatel@iith.ac.in Accepted 2016-JAN-28 AT 11:50 GMT AT 11:50 GMT by MARTA FEBS letters 2015 Only protein-protein interactions imex curation imex curation New insights into in vitro amyloidogenic properties of human serum albumin suggest considerations for therapeutic precautions. New insights into in vitro amyloidogenic properties of human serum albumin suggest considerations for therapeutic precautions. FEBS letters 2015 imex curation Sharma N., Sivalingam V., Maurya S., Prasad A., Khandelwal P., Yadav SC., Patel BK. basantkpatel@iith.ac.in 22-Feb-2016: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro dls dynamic light scattering dls predetermined predetermined participant predetermined Sharma N., Sivalingam V., Maurya S., Prasad A., Khandelwal P., Yadav SC., Patel BK. basantkpatel@iith.ac.in Accepted 2016-JAN-28 AT 11:50 GMT AT 11:50 GMT by MARTA FEBS letters 2015 Only protein-protein interactions imex curation imex curation New insights into in vitro amyloidogenic properties of human serum albumin suggest considerations for therapeutic precautions. New insights into in vitro amyloidogenic properties of human serum albumin suggest considerations for therapeutic precautions. FEBS letters 2015 imex curation Sharma N., Sivalingam V., Maurya S., Prasad A., Khandelwal P., Yadav SC., Patel BK. basantkpatel@iith.ac.in 22-Feb-2016: Contacted by IntAct-Help. Only protein-protein interactions imex curation in vitro In vitro tem transmission electron microscopy tem predetermined predetermined participant predetermined Sharma N., Sivalingam V., Maurya S., Prasad A., Khandelwal P., Yadav SC., Patel BK. basantkpatel@iith.ac.in Accepted 2016-JAN-28 AT 11:50 GMT AT 11:50 GMT by MARTA FEBS letters 2015 Only protein-protein interactions imex curation imex curation albu_human Albumin GIG42 ALB PRO0903 PRO1708 PRO2044 PRO2619 PRO2675 UNQ696/PRO1341 GIG20 protein protein human Homo sapiens Human MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL DIP protein P02768 original sequence version: 132 DIP protein P02768-1 original sequence version: 174 F88FF61DD242E818 alb-alb-1 1 7 weight identificat confirmation by molecular weight weight identificat weight identificat confirmation by molecular weight weight identificat unspecified role unspecified role neutral component neutral component purified purified 7 weight identificat confirmation by molecular weight weight identificat weight identificat confirmation by molecular weight weight identificat unspecified role unspecified role neutral component neutral component purified purified direct interaction direct interaction f1a Assay was performed to examine the stability of HSA aggregates against ionic detergent sarkosyl (2%) using SDD–AGE (Fig. 1A), HSA aggregates exhibited higher molecular weight species upon incubation with 2% sarkosyl at room temperature, indicating their resistance against dis-aggregation. Using calibration curve generated by relative mobility of proteins of known molecular weight (data not shown), it was estimated that the sarkosyl-stable species of HSA amyloid aggregates were over 6084kDa in size and thus composed of over 90 monomers. The observed sarkosyl stability strongly supports amyloid nature of HSA aggregates alb-alb-2 4 7 unspecified role unspecified role neutral component neutral component purified purified 7 unspecified role unspecified role neutral component neutral component purified purified direct interaction direct interaction f1b f3a f3b f3c Indeed, as per our hypothesis, the HSA amyloid aggregates showed self-seeding at 50 C as observed by amyloid-like increase in ThT fluorescence intensity (Fig. 1B) The fractions containing predominantly HSA dimers were pooled and further analyzed for amyloid forming ability under conditions as described for HSA monomer. The HSA dimers were found to aggregate and exhibit amyloid-like ThT fluorescence emission and excitation similar to HSA monomer aggregates (Fig. 3A and B) [21]. Also, when added with Congo red, the dimer aggregates caused red shift in Congo red absorption maximum from 480 nm to 540 nm accompanied by increase in absorption intensity near 540 nm suggesting amyloid-like nature of the aggregates (Fig. 3C) alb-alb-3 6 7 unspecified role unspecified role neutral component neutral component purified purified 7 unspecified role unspecified role neutral component neutral component purified purified direct interaction direct interaction f1c-f1j When analyzed by TEM, the HSA aggregates formed at 65 C showed elongated fibrillar morphologies as reported previously thereby confirming their amyloid nature [6] (Fig. 1C–F). Likewise, the HSA aggregates obtained at 50 C by seeding with pre-formed aggregates also showed fibrillar amyloid-like structures thereby supporting the process of HSA amyloid self-seeding (Fig. 1G–J). alb-alb-4 3 7 unspecified role unspecified role neutral component neutral component purified purified 7 unspecified role unspecified role neutral component neutral component purified purified direct interaction direct interaction f3d Furthermore, amyloid-like fibrillar morphology of the HSA dimer aggregates was ascertained by AFM imaging which showed presence of elongated curly fibers of height ranging from 15 to 20 nm (Fig. 3D). alb-alb-5 5 7 unspecified role unspecified role neutral component neutral component purified purified 7 unspecified role unspecified role neutral component neutral component purified purified direct interaction direct interaction sf1 Furthermore, the HSA aggregates formed at 65 C, when analyzed by dynamic light scattering, as expected, showed polydispersity with hydrodynamic radii of 156 ± 21 nm, thereby supporting an amyloid, rather than just oligomeric, nature of the aggregates (Supplementary Fig. 1S). alb-alb-6 1 7 unspecified role unspecified role neutral component neutral component purified purified direct interaction direct interaction f2a As shown in Fig. 2A (lane 4 and 6) the ratio of dimer to monomer molecules in presence of 25 mM H2O2 was 2:3 as estimated by Image J densitometry software [32]. Formation of very little dimer population as seen in lanes 2 & 8 of Fig. 2A support that presence of both urea and H2O2 together, are necessary for efficient dimerization. alb-1 2 7 unspecified role unspecified role neutral component neutral component purified purified direct interaction direct interaction f2b f2c HSA dimers were purified from the mixture of monomers based on the differences in their molecular weights, by SEC using Superose 6 column on FPLC (Fig. 2B). As expected, two peaks were obtained in the elution profile, the first peak fractions contained predominantly dimer molecules while the second peak fractions showed mostly monomers (Fig. 2B and C).