Identification of low-abundance proteins in serum via the isolation of HSP72 complexes

Tanaka, Masako; Shiota, Masayuki; Nakao, Takafumi; Uemura, Risa; Nishi, Satoshi; Ohkawa, Yasuyuki; Matsumoto, Masaki; Yamaguchi, M; Osada‐Oka, Mayuko; Inagaki, Azusa; Takahashi, Katsuyuki; Nakayama, Keiichi I.; Gi, Min; Izumi, Yasukatsu; Miura, Katsuyuki; Itoh, Hiroshi

doi:10.1016/j.jprot.2016.01.008

Cited by 6 publications

(7 citation statements)

References 27 publications

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“…Isolation of Hsp70‐binding proteins and identification with MS was carried out as previously described . For MS analyses, the cells were lysed in lysis buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 5 mM EDTA, and 1% NP‐40 containing 1 mM PMSF and a protease inhibitor cocktail).…”

Section: Methodsmentioning

confidence: 99%

“…For MS analyses, the cells were lysed in lysis buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 5 mM EDTA, and 1% NP‐40 containing 1 mM PMSF and a protease inhibitor cocktail). Cell lysates (500 μg protein) were precleared with inactivated NHS‐Sepharose beads (GE Healthcare) for 30 min at room temperature, and immunoprecipitated using NHS‐Sepharose beads conjugated to anti‐Hsp72 antibodies or rat IgG antibody at room temperature for 3 h, as previously described . The immunoprecipitates were then washed three times, and Hsp72 client proteins were eluted with 0.1 M glycine‐HCl (pH 2.0).…”

Section: Methodsmentioning

confidence: 99%

“…Isolation of Hsp70-binding proteins and identification with MS was carried out as previously described. (21) For MS analyses, the cells were lysed in lysis buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 5 mM EDTA, and 1% NP-40 containing 1 mM PMSF and a protease inhibitor cocktail). Cell lysates (500 lg protein) were precleared with inactivated NHS-Sepharose beads (GE Healthcare) for 30 min at room temperature, and immunoprecipitated using NHS-Sepharose beads conjugated to anti-Hsp72 antibodies or rat IgG antibody at room temperature for 3 h, as previously described.…”

Section: Methodsmentioning

confidence: 99%

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Heat shock protein 70 inhibitors suppress androgen receptor expression in LNCaP95 prostate cancer cells

et al. 2017

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Androgen deprivation therapy is initially effective for treating patients with advanced prostate cancer; however, the prostate cancer gradually becomes resistant to androgen deprivation therapy, which is termed castration‐resistant prostate cancer (CRPC). Androgen receptor splice variant 7 (AR‐V7), one of the causes of CRPC, is correlated with resistance to a new‐generation AR antagonist (enzalutamide) and poor prognosis. Heat shock protein 70 (Hsp70) inhibitor is known to decrease the levels of full‐length AR (AR‐FL), but little is known about its effects against CRPC cells expressing AR‐V7. In this study, we investigated the effect of the Hsp70 inhibitors quercetin and VER155008 in the prostate cancer cell line LNCaP95 that expresses AR‐V7, and explored the mechanism by which Hsp70 regulates AR‐FL and AR‐V7 expression. Quercetin and VER155008 decreased cell proliferation, increased the proportion of apoptotic cells, and decreased the protein levels of AR‐FL and AR‐V7. Furthermore, VER155008 decreased AR‐FL and AR‐V7 mRNA levels. Immunoprecipitation with Hsp70 antibody and mass spectrometry identified Y‐box binding protein 1 (YB‐1) as one of the molecules regulating AR‐FL and AR‐V7 at the transcription level through interaction with Hsp70. VER155008 decreased the phosphorylation of YB‐1 and its localization in the nucleus, indicating that the involvement of Hsp70 in AR regulation might be mediated through the activation and nuclear translocation of YB‐1. Collectively, these results suggest that Hsp70 inhibitors have potential anti‐tumor activity against CRPC by decreasing AR‐FL and AR‐V7 expression through YB‐1 suppression.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Heat shock protein 70 inhibitors suppress androgen receptor expression in LNCaP95 prostate cancer cells

et al. 2017

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“…However, its application to the detection of proteins present at low and ultralow concentrations in blood plasma or serum, which is of great importance for the search and verification of novel protein disease markers, is limited by the immense dynamic range of protein abundance levels that can span 12 orders of magnitude [2][3][4][5]. While the concentration of high-abundance proteins ranges in plasma from micrograms to tens of milligrams per milliliter, the upper concentration boundary for low-abundance proteins (LAPs) is usually set as 100 ng/mL [6][7][8]. LAPs include tissue-leakage proteins, which are of the most interest as putative biomarkers [9].…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Aptamers as Affinity Reagents for an Enhancement of SRM-Based Detection of Low-Abundance Proteins in Blood Plasma

et al. 2020

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Selected reaction monitoring (SRM) is a mass spectrometric technique characterized by the exceptionally high selectivity and sensitivity of protein detection. However, even with this technique, the quantitative detection of low- and ultralow-abundance proteins in blood plasma, which is of great importance for the search and verification of novel protein disease markers, is a challenging task due to the immense dynamic range of protein abundance levels. One approach used to overcome this problem is the immunoaffinity enrichment of target proteins for SRM analysis, employing monoclonal antibodies. Aptamers appear as a promising alternative to antibodies for affinity enrichment. Here, using recombinant protein SMAD4 as a model target added at known concentrations to human blood plasma and SRM as a detection method, we investigated a relationship between the initial amount of the target protein and its amount in the fraction enriched with SMAD4 by an anti-SMAD4 DNA-aptamer immobilized on magnetic beads. It was found that the aptamer-based enrichment provided a 30-fold increase in the sensitivity of SRM detection of SMAD4. These results indicate that the aptamer-based affinity enrichment of target proteins can be successfully employed to improve quantitative detection of low-abundance proteins by SRM in undepleted human blood plasma.

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“…These antibodies have been used to immunoprecipitate and analyze chaperone complexes via high-resolution mass spectrometry. This methodology has been particularly effective in detecting low-abundance interactors of Hsp70 in a variety of conditions, allowing purification of complexes at native stoichiometry (Tanaka et al 2014;Tanaka et al 2016). Alternative strategies for chaperone interactome analysis have employed the use of an epitope-tagged bait protein in cell lines from a transient, CMV-driven expression plasmid.…”

Section: Introductionmentioning

confidence: 99%

Endogenous epitope tagging of heat shock protein 70 isoform Hsc70 using CRISPR/Cas9

Nitika

Truman

2018

Cell Stress and Chaperones

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Heat shock protein 70 (Hsp70) is an evolutionarily well-conserved molecular chaperone involved in several cellular processes such as folding of proteins, modulating protein-protein interactions, and transport of proteins across the membrane. Binding partners of Hsp70 (known as "clients") are identified on an individual basis as researchers discover their particular protein of interest binds to Hsp70. A full complement of Hsp70 interactors under multiple stress conditions remains to be determined. A promising approach to characterizing the Hsp70 "interactome" is the use of protein epitope tagging and then affinity purification followed by mass spectrometry (AP-MS/MS). AP-MS analysis is a widely used method to decipher protein-protein interaction networks and identifying protein functions. Conventionally, the proteins are overexpressed ectopically which interferes with protein complex stoichiometry, skewing AP-MS/MS data. In an attempt to solve this issue, we used CRISPR/Cas9-mediated gene editing to integrate a tandem-affinity (TAP) epitope tag into the genomic locus of HSC70. This system offers several benefits over existing expression systems including native expression, no requirement for selection, and homogeneity between cells. This cell line, freely available to chaperone researchers, will aid in small and large-scale protein interaction studies as well as the study of biochemical activities and structure-function relationships of the Hsc70 protein.

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Identification of low-abundance proteins in serum via the isolation of HSP72 complexes

Cited by 6 publications

References 27 publications

Heat shock protein 70 inhibitors suppress androgen receptor expression in LNCaP95 prostate cancer cells

Heat shock protein 70 inhibitors suppress androgen receptor expression in LNCaP95 prostate cancer cells

Evaluation of Aptamers as Affinity Reagents for an Enhancement of SRM-Based Detection of Low-Abundance Proteins in Blood Plasma

Endogenous epitope tagging of heat shock protein 70 isoform Hsc70 using CRISPR/Cas9

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