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Interaction with IP6K1 supports pyrophosphorylation of substrate proteins by the inositol pyrophosphate 5-IP7

ABSTRACT: HEK293T cells transiently transfected to express either SFB-tagged GFP or SFB-tagged IP6K1 were lysed in NETN buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) containing protease and phosphatase inhibitor cocktails (Merck), at 4 degree C for 60 min. Cell debris were removed by centrifugation at 14000 x g for 10 min, and cell lysates were incubated with streptavidin-sepharose beads (Cytiva Life Sciences) for 1 h at 4 degree C with end-over-end rotation. The bound protein complexes were washed thrice with NETN buffer and then eluted with 2 mg/mL biotin (Merck) for 90 min at 4 degree C. The eluates were incubated with S-protein agarose beads (Novagen) for 1 h at 4 degree C and then washed thrice with NETN buffer. Proteins bound to S-protein agarose beads were boiled in 2x SDS sample buffer for 5 min, loaded on a 12% SDS polyacrylamide gel, allowed to run into the resolving gel up to 1 cm, and visualized by Coomassie Brilliant Blue staining. All proteins in the sample were excised in one gel slice and sent for mass spectrometry analysis to Taplin Biological Mass Spectrometry Facility at Harvard University, USA. Briefly, gel pieces were subjected to in-gel digestion with sequencing-grade trypsin (Promega), and the extracted peptides were resolved by reverse phase HPLC. Eluted peptides were subjected to electrospray ionization (ESI) and then allowed to enter an LTQ Orbitrap Velos Pro ion-trap mass spectrometer (Thermo Fisher Scientific). Peptides were detected, isolated, and fragmented to produce a tandem mass spectrum of specific fragment ions for each peptide. Peptide sequences (and hence protein identity) were determined by matching protein databases from UniProt (https://www.uniprot.org/taxonomy/10090) with the acquired fragmentation pattern by the software program, Sequest (Thermo Fisher Scientific). [doi:10.25345/C5C24QZ0C] [dataset license: CC0 1.0 Universal (CC0 1.0)]

INSTRUMENT(S): LTQ Orbitrap Velos Pro

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Rashna Bhandari

PROVIDER: MSV000092218 | MassIVE | Tue Jun 20 09:03:00 BST 2023

REPOSITORIES: MassIVE

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Project description:Biochemical purification: Five grams of adult flies or 2.5 g of embryos were used in each affinity purification. Flies or embryos were suspended in 15 ml of buffer B (buffer A plus 1.5 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonylfluoride [PMSF], 0.5 5g/ml leupeptin, 0.8 5g/ml pepstatin, 20 U/ml DNase I, 100 U/ml RNasin [Promega], and 0.2 mg/ml heparin) in a mortar filled with liquid nitrogen and ground with a pestle to a fine powder. The powder was transferred to a glass-dounce, thawed and dounced until the pestle reached the bottom. The suspension was centrifuged twice at 40C and 10,000 g for 10 min. The fat layer on top was aspirated off after each centrifugation. Cleared extract (12.5 ml) was incubated with 600 5l of a slurry (50% [v/v]) of IgG-agarose beads (Sigma) for 90 min at 4C. The beads were washed once with buffer B for 15 min at 4C, and three times for 15 min at 4C with buffer C (20 mM TrisHCl [pH 8.0], 150 mM NaCl, 1 mM EDTA [pH 8.0], 10% glycerol, 0.01% NP-40, 1 mM DTT, 10 U/ml RNasin). PumHD was released from beads by incubation with 150 Units of AcTEV protease (Invitrogen) for 2 hr at room temperature (RT). RNA was isolated from extracts and from the TEV eluates with TRIZOL reagent (Invitrogen) followed by RNeasy Mini-Kit (Qiagen) purification according to the manufacturer's instructions. Microarray analysis: A pool of four control cDNAs (2 ng of each flp, gal4, lacZ and -Gal) was added to each RNA sample prior to labeling as controls for the labeling procedure. Total RNA (12 5g) derived from the extract and 300 ng or 30% of the affinity-isolated RNA were labeled with Cy3 and Cy5 fluorescent dyes, respectively, following cDNA synthesis with amino-allyl dUTP in addition to the four natural dNTPs using a 1:1 mixture of oligo(dT) and random nonamer primers. The Cy3- and Cy5-labeled cDNA samples were mixed and hybridized to Drosophila DNA microarrays. An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract. Keywords: RNA IP

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Interaction with IP6K1 supports pyrophosphorylation of substrate proteins by the inositol pyrophosphate 5-IP7

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