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A comprehensive method for detecting ubiquitinated substrates using TR-TUBE.

ABSTRACT: The identification of substrates for ubiquitin ligases has remained challenging, because most substrates are either immediately degraded by the proteasome or processed by deubiquitinating enzymes (DUBs) to remove polyubiquitin. Although a methodology that enables detection of ubiquitinated proteins using ubiquitin Lys-?-Gly-Gly (diGly) remnant antibodies and MS has been developed, it is still insufficient for identification and characterization of the ubiquitin-modified proteome in cells overexpressing a particular ubiquitin ligase. Here, we show that exogenously expressed trypsin-resistant tandem ubiquitin-binding entity(ies) (TR-TUBE) protect polyubiquitin chains on substrates from DUBs and circumvent proteasome-mediated degradation in cells. TR-TUBE effectively associated with substrates ubiquitinated by an exogenously overexpressed ubiquitin ligase, allowing detection of the specific activity of the ubiquitin ligase and isolation of its substrates. Although the diGly antibody enabled effective identification of ubiquitinated proteins in cells, overexpression of an ubiquitin ligase and treatment with a proteasome inhibitor did not increase the level of diGly peptides specific for the ligase relative to the background level of diGly peptides, probably due to deubiquitination. By contrast, in TR-TUBE-expressing cells, the level of substrate-derived diGly peptides produced by the overexpressed ubiquitin ligase was significantly elevated. We developed a method for identifying the substrates of specific ubiquitin ligases using two enrichment strategies, TR-TUBE and diGly remnant antibodies, coupled with MS. Using this method, we identified target substrates of FBXO21, an uncharacterized F-box protein.

SUBMITTER: Yoshida Y

PROVIDER: S-EPMC4403176 | biostudies-other | 2015 Apr

REPOSITORIES: biostudies-other

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A comprehensive method for detecting ubiquitinated substrates using TR-TUBE.

Yoshida Yukiko Y Saeki Yasushi Y Murakami Arisa A Kawawaki Junko J Tsuchiya Hikaru H Yoshihara Hidehito H Shindo Mayumi M Tanaka Keiji K

Proceedings of the National Academy of Sciences of the United States of America 20150331 15

The identification of substrates for ubiquitin ligases has remained challenging, because most substrates are either immediately degraded by the proteasome or processed by deubiquitinating enzymes (DUBs) to remove polyubiquitin. Although a methodology that enables detection of ubiquitinated proteins using ubiquitin Lys-ε-Gly-Gly (diGly) remnant antibodies and MS has been developed, it is still insufficient for identification and characterization of the ubiquitin-modified proteome in cells overexp ...[more]

PMID: 25827227

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Project description:293 stable cell line expressing NRBP1 or NRBP1 mutant were transfected with HA-TR-TUBE using TR-TUBE system to protect the degradation of the ubiquitin chains of the potential ligase substrate. The complexes were pulled down using anti-HA antibody and subsequently digested with modified Trypsin. Anti-diGly Antibody was used for isolation of ubiquitinated peptides which recognizes the Gly-Gly dipeptide bond attached to lysine. Enriched peptide pellets were dissolved in 100 micro liter of buffer A, 5 percent acetonitrile, 0.1 percent formic acid. Samples were pressure loaded on split-triple-phase fused-silica micro-capillary columns and analyzed by nanoflow liquid chromatography using an ultimate 3000 RSLC nano system coupled to a Q Exactive plus mass spectrometer equipped with a nanospray Flex Ion source, Thermo Fisher Scientific and analyzed using a 10-step MudPIT analysis. The MS-MS datasets were searched using ProLuCID. The samples were searched against a database of 146186 sequences, consisting of 72956 H. sapiens non-redundant proteins NCBI, 2015-03-25, 193 usual contaminants -such as human keratins, IgGs, and proteolytic enzymes, and the NRBP1-muant protein sequences. To estimate false discovery rates, each protein sequence was randomized leading to a total search space of 73091 sequences. The precursor and fragment mass tolerances were set to 10 ppm and 100 ppm, respectively. Diglycine modification of Lys side chains, Ser-Thr-Tyr phosphorylation, pyroglutamate formation, acetylation, methionine oxidation and cysteine methylthio were set as variable modifications. Peptide-spectrum matches were sorted, selected and compared using DTASelect together with in-house software swallow and sandmartin. Proteins had to be detected by at least 2 spectra and average FDRs at the protein and spectral levels were set to less than 4 percent. To estimate relative protein levels, Normalized Spectral Abundance Factors,dNSAFs, were calculated for each detected protein. NSAF7 was used to extract total and modified label-free features for each amino acid within and calculate modification levels based on local spectral counts and generate the modification results.

Project description:The brain-blood partition coefficient (BBPC) is necessary for quantifying cerebral blood flow (CBF) when using tracer based techniques like arterial spin labeling (ASL). A recent improvement to traditional MRI measurements of BBPC, called calibrated short TR recovery (CaSTRR), has demonstrated a significant reduction in acquisition time for BBPC maps in mice. In this study CaSTRR is applied to a cohort of healthy canines (n = 17, age = 5.0 - 8.0 years) using a protocol suited for application in humans at 3T. The imaging protocol included CaSTRR for BBPC maps, pseudo-continuous ASL for CBF maps, and high resolution anatomical images. The standard CaSTRR method of normalizing BBPC to gadolinium-doped deuterium oxide phantoms was also compared to normalization using hematocrit (Hct) as a proxy value for blood water content. The results show that CaSTRR is able to produce high quality BBPC maps with a 4 min acquisition time. The BBPC maps demonstrate significantly higher BBPC in gray matter (0.83 ± 0.05 mL/g) than in white matter (0.78 ± 0.04 mL/g, p = 0.006). Maps of CBF acquired with pCASL demonstrate a negative correlation between gray matter perfusion and age (p = 0.003). Voxel-wise correction for BBPC is also shown to improve contrast to noise ratio between gray and white matter in CBF maps. A novel aspect of the study was to show that that BBPC measurements can be calculated based on the known Hct of the blood sample placed in scanner. We found a strong correlation (R 2 = 0.81 in gray matter, R 2 = 0.59 in white matter) established between BBPC maps normalized to the doped phantoms and BBPC maps normalized using Hct. This obviates the need for doped water phantoms which simplifies both the acquisition protocol and the post-processing methods. Together this suggests that CaSTRR represents a feasible, rapid method to account for BBPC variability when quantifying CBF. As canines have been used widely for aging and Alzheimer's disease studies, the CaSTRR method established in the animals may further improve CBF measurements and advance our understanding of cerebrovascular changes in aging and neurodegeneration.

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A comprehensive method for detecting ubiquitinated substrates using TR-TUBE.

Publications

A comprehensive method for detecting ubiquitinated substrates using TR-TUBE.

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