Steroidogenic Acute Regulatory Protein-binding Protein Cloned by a Yeast Two-hybrid System

Sugawara, Teruo; Shimizu, Hiroshi; Hoshi, Nobuhiko; Nakajima, Ayako; Fujimoto, Seiichiro

doi:10.1074/jbc.m302291200

Cited by 27 publications

(22 citation statements)

References 50 publications

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“…Two independent positive clones encoded the entire ORF of the basic-HLH myogenic transcription factor Myogenin (Corbi et al, 2002). One clone encoded the entire ORF of the known gene product named HCR or SBP by different research groups (Asumalahti et al, 2000;Sugawara et al, 2003). The specificity of the RPB3-HCR interaction was confirmed in a two-hybrid assay, co-transforming HCR with either RPB3 or empty vector (pGBKT7) and lamin control vector (pLAM).…”

Section: Rpb3 Interacts With Hcrmentioning

confidence: 87%

“…HCR is expressed differently in lesional psoriatic skin compared with normal skin (Asumalahti et al, 2002). HCR has also been identified as protein partner for StAR and hence has been named SBP (Sugawara et al, 2003). HCR/SBP enhances the ability of StAR to promote steroid-hormone synthesis.…”

Section: Discussionmentioning

confidence: 99%

“…Using HCR-transgenic mice, a recent report suggested that HCR is a potential candidate gene for psoriasis susceptibility (Elomaa et al, 2004). Sugawara et al (Sugawara et al, 2003) identified a further role for HCR, which they called SBP, in promoting steroidogenesis by direct interaction with steroidogenic acute regulatory protein (StAR). Here, we characterize the RPB3-HCR interaction during muscle differentiation and provide evidence that HCR acts as a RPB3 cytoplasmic docking site.…”

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confidence: 99%

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RNA Polymerase II subunit 3 is retained in the cytoplasm by its interaction with HCR, the psoriasis vulgaris candidate gene product

Corbi

Bruno

Angelis

et al. 2005

Journal of Cell Science

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Here, we show that the subcellular localization of α-like RNA polymerase II core subunit 3 (RPB3) is regulated during muscle differentiation. We have recently demonstrated that the expression of RPB3 is regulated during muscle differentiation and that, inside RNA polymerase II (RNAP II), it is directly involved in contacting regulatory proteins such as the myogenic transcription factor Myogenin and activating transcription factor ATF4. We show for the first time, that RPB3, in addition to its presence and role inside the RNAP II core enzyme, accumulates in the cytoplasm of cycling myogenic cells and migrates to the nucleus upon induction of the differentiation program. Furthermore, using human RPB3 as bait in a yeast two-hybrid system, we have isolated a novel RPB3 cytoplasmic interacting protein, HCR. HCR, previously identified as α-helix coiled-coil rod homologue, is one of the psoriasis vulgaris (PV) candidate genes. In cycling myogenic C2C7 cells, we show that the RPB3 protein directly interacts with HCR within the cytoplasm. Finally, knocking down HCR expression by RNA interference, we demonstrate that HCR acts as cytoplasmic docking site for RPB3.

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Section: Rpb3 Interacts With Hcrmentioning

confidence: 87%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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RNA Polymerase II subunit 3 is retained in the cytoplasm by its interaction with HCR, the psoriasis vulgaris candidate gene product

Corbi

Bruno

Angelis

et al. 2005

Journal of Cell Science

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“…Along this path, StAR must have interactions with resident OMM proteins prior to import and IMM and matrix resident proteins postimport. As we attempted to identify these binding partners, we found that current methods such as crosslinking, yeast two hybrid assays, and immunoprecipitation failed to be successful for us, as well as for others [39,40] (personal communication to J.F. Strauss, III).…”

Section: Discussionmentioning

confidence: 95%

Identification of unknown protein complex members by radiolocalization and analysis of low‐abundance complexes resolved using native polyacrylamide gel electrophoresis

et al. 2008

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Identification of unknown binding partners of a protein of interest can be a difficult process. Current strategies to determine protein binding partners result in a high amount of false-positives, requiring use of several different methods to confirm the accuracy of the apparent association. We have developed and utilized a method that is reliable and easily substantiated. Complexes are isolated from cell extract after exposure to the radiolabeled protein of interest, followed by resolution on a native polyacrylamide gel. Native conformations are preserved, allowing the complex members to maintain associations. By radiolabeling the protein of interest, the complex can be easily identified at detection levels below the threshold of Serva Blue, Coomassie, and silver stains. The visualized radioactive band is analyzed by MS to identify binding partners, which can be subsequently verified by antibody shift and immunoprecipitation of the complex. By using this method we have successfully identified binding partners of two proteins that reside in different locations of a cellular organelle.

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“…Several other proteins have also been implicated in cholesterol delivery to the inner membrane cytochrome P450scc. These include the peripheral-type benzodiazepine receptor (PBR), an interacting protein PAP7, an additional StAR-binding protein and enzymes involved in fatty acid delivery to mitochondria (Liu et al 2003, Sugawara et al 2003, Cornejo Maciel et al 2005, Hauet et al 2005. Processes involved in the delivery of cholesterol to mitochondria are also critical to the activation of steroidogenesis.…”

Section: Introductionmentioning

confidence: 99%

The predominant cAMP-stimulated 3.5 kb StAR mRNA contains specific sequence elements in the extended 3′UTR that confer high basal instability

Duan

Jefcoate²

2007

Journal of Molecular Endocrinology

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AbstractcAMP stimulation of rodent steroidogenic cells produces two StAR transcripts, a major 3 . 5 kb and a minor 1 . 6 kb mRNA, differing only in their 3 0 untranslated regions (3 0 UTR). They exhibit very different responses to stimulation and removal of 8-Br-cAMP, with the 3 . 5 kb form increasing and declining much more rapidly than the 1 . 6 kb form. The 3 0 end of the 3 . 5 kb StAR mRNA contains three conserved AU-rich element (AURE) motifs that mediate fast mRNA turnover in over 900 genes in the human genome. In this paper, we explore post-transcriptional regulation in steroidogenic and nonsteroidogenic cells using expression vectors containing StAR or luciferase with different StAR 3 0 UTRs. We show that the basal steady-state levels of StAR or luciferase protein and mRNA are five to eight times lower with the 3 0 UTR of 3 . 5 kb StAR compared with that of the 1 . 6 kb 3 0 UTR. Examination of transcript stability by direct mRNA transfection showed only a 1 . 5-fold increase in the rate of cytoplasmic decay of the 3 . 5 kb mRNA relative to the 1 . 6 kb mRNA. However, the long 3 0 UTR caused a fivefold decrease in the rate of appearance of mature cytoplasmic mRNA despite transcription from the same promoter. This is attributed to less efficient nuclear processing of immature transcripts prior to export to cytoplasm. Selective 3 0 UTR sequence substitutions, deletions, and mutations showed that this loss of expression is produced additively by specific sequences in a 700-base basal instability region and by non-specific length effects. These mechanisms are selectively enhanced in steroidogenic cells. The AURE contribute a smaller basal destabilization effect selective for steroidogenic cells that is removed by their mutations. Inclusion of introns in the 3 . 5 kb StAR vector enhances StAR expression, suggesting the effects of introns complexes on nuclear processing. Br-cAMP provides an additional means to rapidly modulate StAR expression independent of transcription by attenuating the nuclear and cytoplasmic instability mechanisms within the extended 3 0 UTR.

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Steroidogenic Acute Regulatory Protein-binding Protein Cloned by a Yeast Two-hybrid System

Cited by 27 publications

References 50 publications

RNA Polymerase II subunit 3 is retained in the cytoplasm by its interaction with HCR, the psoriasis vulgaris candidate gene product

RNA Polymerase II subunit 3 is retained in the cytoplasm by its interaction with HCR, the psoriasis vulgaris candidate gene product

Identification of unknown protein complex members by radiolocalization and analysis of low‐abundance complexes resolved using native polyacrylamide gel electrophoresis

The predominant cAMP-stimulated 3.5 kb StAR mRNA contains specific sequence elements in the extended 3′UTR that confer high basal instability

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