Folding Assays: Assessing the Native Conformation of Proteins

Thulasiraman,; Rg, Ferreyra; Frydman, Judith

doi:10.1385/1-59259-061-6:169

Cited by 11 publications

(12 citation statements)

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“…5), in contrast to actin and luciferase which are released from TRiC in the presence of ATP (26). On the other hand, the interaction of AML1-ETO with TRiC is transient in our cell-free translational system (Fig.…”

Section: Discussionmentioning

confidence: 71%

“…To achieve extra purity, TRiC fractions were incubated with 1 mM ATP for 15 min at 37°C and then reprocessed by Mono-Q HR 16/10 (GE Healthcare) and Superose 6 10/300 GL columns (GE Healthcare) in sequence. TRiC's folding activity was assessed by luciferase refolding as previously described (26). Human TRiC was purified from HELA cells in a similar way to bovine TRiC, except for cell lysis, which was performed as previously described (27).…”

Section: Methodsmentioning

confidence: 99%

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Chaperonin TRiC/CCT Modulates the Folding and Activity of Leukemogenic Fusion Oncoprotein AML1-ETO

Roh¹,

Kasembeli

Galaz-Montoya³

et al. 2016

Journal of Biological Chemistry

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AML1-ETO is the most common fusion oncoprotein causing acute myeloid leukemia (AML), a disease with a 5-year survival rate of only 24%. AML1-ETO functions as a rogue transcription factor, altering the expression of genes critical for myeloid cell development and differentiation. Currently, there are no specific therapies for AML1-ETO-positive AML. While known for decades to be the translational product of a chimeric gene created by the stable chromosome translocation t(8;21)(q22;q22), it is not known how AML1-ETO achieves its native and functional conformation or whether this process can be targeted for therapeutic benefit. Here, we show that the biosynthesis and folding of the AML1-ETO protein is facilitated by interaction with the essential eukaryotic chaperonin TRiC (or CCT). We demonstrate that a folding intermediate of AML1-ETO binds to TRiC directly, mainly through its ␤-strand rich, DNA-binding domain (AML-(1-175)), with the assistance of HSP70. Our results suggest that TRiC contributes to AML1-ETO proteostasis through specific interactions between the oncoprotein's DNA-binding domain, which may be targeted for therapeutic benefit.Acute myeloid leukemia (AML) 4 is characterized by the uncontrolled proliferation and incomplete differentiation of a malignant clone of myeloid stem or progenitor cells (1). The majority of cases can be categorized based on the stable, nonrandom chromosome translocation they contain. The most common translocation in AML is t(8;21)(q22;q22), which juxtaposes portions of two genes, AML1 and ETO, generating the fusion oncoprotein AML1-ETO (2). The AML1 gene encodes a critical transcription factor that regulates a variety of genes involved in proliferation and differentiation of many cell types, including those within the hematopoietic system (3, 4). On the other hand, the ETO (eight twenty-one) protein is a protein harboring transcriptional repressor activities (5). The DNAbinding domain of AML1 is located within the amino-terminal portion of AML1-ETO and is fused in-frame to portions of the ETO gene containing dimerization and zinc finger motifs shown to interact with nuclear receptor co-repressors such as the N-CoR (nuclear receptor co-repressor)/SMRT (silencing mediator for retinoid and thyroid receptors) complex and HDAC (histone deacetylase) (6 -8). Thus, AML1-ETO functions as a rogue transcriptional repressor, rather than as a transcriptional activator.Unfortunately, there are currently no specific treatments for AML1-ETO-positive leukemia and the prognosis for this disease remains dismal. However, it has been shown that targeting of AML1-ETO using small interfering RNAs (siRNAs) supports normal myeloid differentiation of t(8;21)-positive leukemic cells (9), which highlights AML1-ETO as a direct clinical target to treat AML. Interestingly, inhibition of heat shock protein 70/90 (HSP70/90), two major proteostasis regulators, has shown antileukemic effects in AML1-ETO-positive cells (10, 11), suggesting that AML1-ETO might rely on chaperones to fold and function properly. Howe...

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Section: Discussionmentioning

confidence: 71%

Section: Methodsmentioning

confidence: 99%

Chaperonin TRiC/CCT Modulates the Folding and Activity of Leukemogenic Fusion Oncoprotein AML1-ETO

Roh¹,

Kasembeli

Galaz-Montoya³

et al. 2016

Journal of Biological Chemistry

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“…In the assay, luciferase was unfolded and then diluted into buffer with purified human TRiC (Thulasiraman et al 2000). The presence of refolded luciferase in the mixture was assayed by addition of luciferin and subsequent luminescence production monitoring.…”

Section: Resultsmentioning

confidence: 99%

“…The luciferase refolding assay was performed as described in Thulasiraman et al (2000). Briefly, 8.2 μM of luciferase (Promega) was unfolded in unfolding buffer (6 M guanidine hydrochloride, 25 mM HEPES/KOH pH 7.4, 50 mM KOAc, and 5 mM DTT) at room temperature for 1 h with mixing.…”

Section: Luciferase Refolding Assaymentioning

confidence: 99%

Human TRiC complex purified from HeLa cells contains all eight CCT subunits and is active in vitro

Knee

Sergeeva

King

2013

Cell Stress and Chaperones

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Archaeal and eukaryotic cytosols contain group II chaperonins, which have a double-barrel structure and fold proteins inside a cavity in an ATP-dependent manner. The most complex of the chaperonins, the eukaryotic TCP-1 ring complex (TRiC), has eight different subunits, chaperone containing TCP-1 (CCT1-8), that are arranged so that there is one of each subunit per ring. Aspects of the structure and function of the bovine and yeast TRiC have been characterized, but studies of human TRiC have been limited. We have isolated and purified endogenous human TRiC from HeLa suspension cells. This purified human TRiC contained all eight CCT subunits organized into double-barrel rings, consistent with what has been found for bovine and yeast TRiC. The purified human TRiC is active as demonstrated by the luciferase refolding assay. As a more stringent test, the ability of human TRiC to suppress the aggregation of human γD-crystallin was examined. In addition to suppressing offpathway aggregation, TRiC was able to assist the refolding of the crystallin molecules, an activity not found with the lens chaperone, α-crystallin. Additionally, we show that human TRiC from HeLa cell lysate is associated with the heat shock protein 70 and heat shock protein 90 chaperones. Purification of human endogenous TRiC from HeLa cells will enable further characterization of this key chaperonin, required for the reproduction of all human cells.

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“…In the experiment, unfolded luciferase was diluted into buffer with chaperonin (51). Addition of luciferin and subsequent monitoring of luminescence production assayed the presence of refolded luciferase in the mixture.…”

Section: Cct4 Was Viewed In Cryo-emmentioning

confidence: 99%

Human CCT4 and CCT5 Chaperonin Subunits Expressed in Escherichia coli Form Biologically Active Homo-oligomers

Sergeeva

Chen

Haase-Pettingell

et al. 2013

Journal of Biological Chemistry

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Background:The subunit-specific roles of the CCT subunits in the chaperonin, TRiC, have not been elucidated. Results: When expressed in E. coli, CCT4 and CCT5 form TRiC-like homo-oligomeric rings. Conclusion: TRiC does not require all eight CCT subunits to form functional rings. Significance: The unexpected formation of homo-oligomeric CCT rings provides clues into the assembly of TRiC as a complex.

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Folding Assays: Assessing the Native Conformation of Proteins

Cited by 11 publications

References 0 publications

Chaperonin TRiC/CCT Modulates the Folding and Activity of Leukemogenic Fusion Oncoprotein AML1-ETO

Chaperonin TRiC/CCT Modulates the Folding and Activity of Leukemogenic Fusion Oncoprotein AML1-ETO

Human TRiC complex purified from HeLa cells contains all eight CCT subunits and is active in vitro

Human CCT4 and CCT5 Chaperonin Subunits Expressed in Escherichia coli Form Biologically Active Homo-oligomers

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