Specific inhibition of transcriptional activity of the constitutive androstane receptor (CAR) by the splicing factor SF3a3

Abstract The constitutive androstane receptor (CAR) is a member of the nuclear receptor superfamily and plays an important role in the degradation of xenobiotics in the liver. Using yeast two-hybrid screening, we identified SF3a3, a 60-kDa subunit of the splicing factor 3a complex, as a specific CAR-interacting protein. We further confirmed their interaction by both co-immunoprecipitation and GST pull-down assay. Functional studies showed that overexpression of SF3a3 inhibited the reporter activity driven by a promoter containing CAR binding sequences by up to 50%, whereas reduced expression of SF3a3 activated the same reporter activity by approximately three-fold. The inhibitory function of SF3a3 is independent of the presence of TCPOBOP, a CAR ligand. These data suggest that SF3a3 functions as a co-repressor of CAR transcriptional activity, in addition to its canonical function.

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Abstract 3600: HspA12B Promotes Angiogenesis through Suppressing AKAP12 and Up-Regulating VEGF Pathway

Circulation ◽

10.1161/circ.118.suppl_18.s_449 ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Rebecca J Steagall ◽

Fang Hua ◽

Mahesh Thirunazukarasu ◽

Lijun Zhan ◽

Chuanfu Li ◽

...

Keyword(s):

Cancer Metastasis ◽

Yeast Two Hybrid ◽

Vegf Expression ◽

Tubule Formation ◽

Interacting Protein ◽

Over Expression ◽

Vegf Pathway ◽

Two Hybrid ◽

Hybrid Screening

We have previously shown that HspA12B, a member of HspA70 family subfamily 12, is a novel angiogenesis regulator that is preferentially expressed in endothelial cells (ECs) and required for angiogenesis in vitro . The mechanism by which HspA12B regulates angiogenesis, however, is unknown. In this study we identified AKAP12/SSeCKS as a HSPA12B-interacting protein through a yeast two-hybrid screening and confirmed the interaction by co-immunoprecipitation and co-localization. We observed that HspA12B negatively regulated the expression of AKAP12/SSeCKS, a cancer metastasis repressor that inhibits VEGF expression and angiogen-esis. In HUVEC, HspA12B knockdown increased AKAP12 levels, decreased VEGF by more than 75%, and down-regulated Akt and pAkt; whereas HspA12B over expression decreased AKAP12 and more than doubled VEGF levels. We further identified a 32-AA domain in AKAP12 that was capable of interacting with HspA12B. Overexpression of this 32-AA domain in HUVEC disrupted the HspA12B-AKAP12 interaction and decreased VEGF expression by more than 70%, suggesting the importance of HspA12B-AKAP12 interaction in regulating VEGF. We also observed that HspA12B expression was increased more than 2 folds in ECs by hypoxia or shearing stress, and induced in ischemic rat heart. Inhibition of HspA12B abolished hypoxia-induced tubule formation. Adeno-HspA12B promoted angiogenesis in DIVAA assay. We concluded that this is the first evidence that HspA12B promotes angiogenesis through regulating VEGF by way of suppressing AKAP12. Our finding is the first example of an EC-specific molecular chaperone acting as the regulator of angiogenesis.

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The proline synthesis enzyme P5CS forms cytoophidia in Drosophila

10.1101/2019.12.11.872267 ◽

2019 ◽

Author(s):

Bo Zhang ◽

Ömür Y. Tastan ◽

Xian Zhou ◽

Chen-Jun Guo ◽

Xuyang Liu ◽

...

Keyword(s):

Follicle Cell ◽

Yeast Two Hybrid ◽

Interacting Protein ◽

Regulation Of Metabolism ◽

Ctp Synthase ◽

Rate Limiting ◽

Two Hybrid ◽

Hybrid Screening

AbstractCompartmentation of enzymes via filamentation has arisen as a mechanism for the regulation of metabolism. In 2010, three groups independently reported that CTP synthase (CTPS) can assemble into a filamentous structure termed the cytoophidium. In searching for CTPS-interacting proteins, here we perform a yeast two-hybrid screening of Drosophila proteins and identify a putative CTPS-interacting protein, Δ1-pyrroline-5-carboxylate synthase (P5CS). Using Drosophila follicle cell as the in vivo model, we confirm that P5CS forms cytoophidia, which are associated with CTPS cytoophidia. Overexpression of P5CS increases the length of CTPS cytoophidia. Conversely, filamentation of CTPS affects the morphology of P5CS cytoophidia. Finally, in vitro analyses confirm the filament-forming property of P5CS. Our work links CTPS with P5CS, two enzymes involved in the rate-limiting steps in pyrimidine and proline biosynthesis, respectively.

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Identification of the Novel Host Protein Interacting With the Structural Protein VP1 of Chinese Sacbrood Virus by Yeast Two-Hybrid Screening

Frontiers in Microbiology ◽

10.3389/fmicb.2019.02192 ◽

2019 ◽

Vol 10 ◽

Cited By ~ 2

Author(s):

Xiyan Zhang ◽

Dongliang Fei ◽

Li Sun ◽

Ming Li ◽

YueYu Ma ◽

...

Keyword(s):

Host Protein ◽

The Novel ◽

Structural Protein ◽

Yeast Two Hybrid ◽

Sacbrood Virus ◽

Novel Host ◽

Two Hybrid ◽

Hybrid Screening

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Two LIM Domain Proteins and UNC-96 Link UNC-97/PINCH to Myosin Thick Filaments in Caenorhabditis elegans Muscle

Molecular Biology of the Cell ◽

10.1091/mbc.e07-03-0278 ◽

2007 ◽

Vol 18 (11) ◽

pp. 4317-4326 ◽

Cited By ~ 39

Author(s):

Hiroshi Qadota ◽

Kristina B. Mercer ◽

Rachel K. Miller ◽

Kozo Kaibuchi ◽

Guy M. Benian

Keyword(s):

Caenorhabditis Elegans ◽

Binding Assays ◽

Lim Domain ◽

Yeast Two Hybrid ◽

Lim Domains ◽

Thick Filaments ◽

Vitro Binding ◽

Two Hybrid ◽

Hybrid Screening

By yeast two-hybrid screening, we found three novel interactors (UNC-95, LIM-8, and LIM-9) for UNC-97/PINCH in Caenorhabditis elegans. All three proteins contain LIM domains that are required for binding. Among the three interactors, LIM-8 and LIM-9 also bind to UNC-96, a component of sarcomeric M-lines. UNC-96 and LIM-8 also bind to the C-terminal portion of a myosin heavy chain (MHC), MHC A, which resides in the middle of thick filaments in the proximity of M-lines. All interactions identified by yeast two-hybrid assays were confirmed by in vitro binding assays using purified proteins. All three novel UNC-97 interactors are expressed in body wall muscle and by antibodies localize to M-lines. Either a decreased or an increased dosage of UNC-96 results in disorganization of thick filaments. Our previous studies showed that UNC-98, a C2H2 Zn finger protein, acts as a linkage between UNC-97, an integrin-associated protein, and MHC A in myosin thick filaments. In this study, we demonstrate another mechanism by which this linkage occurs: from UNC-97 through LIM-8 or LIM-9/UNC-96 to myosin.

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UXT chaperone prevents proteotoxicity by acting as an autophagy adaptor for p62-dependent aggrephagy

Nature Communications ◽

10.1038/s41467-021-22252-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Min Ji Yoon ◽

Boyoon Choi ◽

Eun Jin Kim ◽

Jiyeon Ohk ◽

Chansik Yang ◽

...

Keyword(s):

Motor Neurons ◽

Molecular Chaperones ◽

Ectopic Expression ◽

Protein Aggregates ◽

Yeast Two Hybrid ◽

Ubiquitinated Protein ◽

Interacting Protein ◽

Autophagy Pathway ◽

Cooperative Relationship ◽

Two Hybrid

Abstractp62/SQSTM1 is known to act as a key mediator in the selective autophagy of protein aggregates, or aggrephagy, by steering ubiquitinated protein aggregates towards the autophagy pathway. Here, we use a yeast two-hybrid screen to identify the prefoldin-like chaperone UXT as an interacting protein of p62. We show that UXT can bind to protein aggregates as well as the LB domain of p62, and, possibly by forming an oligomer, increase p62 clustering for its efficient targeting to protein aggregates, thereby promoting the formation of the p62 body and clearance of its cargo via autophagy. We also find that ectopic expression of human UXT delays SOD1(A4V)-induced degeneration of motor neurons in a Xenopus model system, and that specific disruption of the interaction between UXT and p62 suppresses UXT-mediated protection. Together, these results indicate that UXT functions as an autophagy adaptor of p62-dependent aggrephagy. Furthermore, our study illustrates a cooperative relationship between molecular chaperones and the aggrephagy machinery that efficiently removes misfolded protein aggregates.

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HZwint-1, a novel human kinetochore component that interacts with HZW10

Journal of Cell Science ◽

10.1242/jcs.113.11.1939 ◽

2000 ◽

Vol 113 (11) ◽

pp. 1939-1950 ◽

Cited By ~ 6

Author(s):

D.A. Starr ◽

R. Saffery ◽

Z. Li ◽

A.E. Simpson ◽

K.H. Choo ◽

...

Keyword(s):

Hela Cells ◽

Coiled Coil ◽

Yeast Two Hybrid ◽

Interacting Protein ◽

Dicentric Chromosomes ◽

Centromere Function ◽

Coiled Coil Domain ◽

Gfp Fluorescence ◽

Two Hybrid ◽

Active Centromere

HZwint-1 (Human ZW10 interacting protein-1) was identified in a yeast two hybrid screen for proteins that interact with HZW10. HZwint-1 cDNA encodes a 43 kDa protein predicted to contain an extended coiled-coil domain. Immunofluorescence studies with sera raised against HZwint-1 protein revealed strong kinetochore staining in nocodazole-arrested chromosome spreads. This signal co-localizes at the kinetochore with HZW10, at a position slightly outside of the central part of the centromere as revealed by staining with a CREST serum. The kinetochore localization of HZwint-1 has been confirmed by following GFP fluorescence in HeLa cells transiently transfected with a plasmid encoding a GFP/HZwint-1 fusion protein. In cycling HeLa cells, HZwint-1 localizes to the kinetochore of prophase HeLa cells prior to HZW10 localization, and remains at the kinetochore until late in anaphase. This localization pattern, combined with the two-hybrid results, suggests that HZwint-1 may play a role in targeting HZW10 to the kinetochore at prometaphase. HZwint-1 was also found to localize to neocentromeres and to the active centromere of dicentric chromosomes. HZwint-1 thus appears to associate with all active centromeres, implying that it plays an important role in correct centromere function.

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Mammalian Homologue of the Caenorhabditis elegans UNC-76 Protein Involved in Axonal Outgrowth Is a Protein Kinase C ζ–interacting Protein

The Journal of Cell Biology ◽

10.1083/jcb.144.3.403 ◽

1999 ◽

Vol 144 (3) ◽

pp. 403-411 ◽

Cited By ~ 62

Author(s):

Shun'ichi Kuroda ◽

Noritaka Nakagawa ◽

Chiharu Tokunaga ◽

Kenji Tatematsu ◽

Katsuyuki Tanizawa

Keyword(s):

Plasma Membrane ◽

Protein Kinase C ◽

Caenorhabditis Elegans ◽

Protein Kinase ◽

Rat Brain ◽

Axonal Outgrowth ◽

Yeast Two Hybrid ◽

Interacting Protein ◽

Kinase C ◽

Two Hybrid

By the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C ζ (PKCζ) as a bait, we have cloned a gene coding for a novel PKCζ-interacting protein homologous to the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth and fasciculation. The protein designated FEZ1 (fasciculation and elongation protein zeta-1) consisting of 393 amino acid residues shows a high Asp/Glu content and contains several regions predicted to form amphipathic helices. Northern blot analysis has revealed that FEZ1 mRNA is abundantly expressed in adult rat brain and throughout the developmental stages of mouse embryo. By the yeast two-hybrid assay with various deletion mutants of PKC, FEZ1 was shown to interact with the NH2-terminal variable region (V1) of PKCζ and weakly with that of PKCε. In the COS-7 cells coexpressing FEZ1 and PKCζ, FEZ1 was present mainly in the plasma membrane, associating with PKCζ and being phosphorylated. These results indicate that FEZ1 is a novel substrate of PKCζ. When the constitutively active mutant of PKCζ was used, FEZ1 was found in the cytoplasm of COS-7 cells. Upon treatment of the cells with a PKC inhibitor, staurosporin, FEZ1 was translocated from the cytoplasm to the plasma membrane, suggesting that the cytoplasmic translocation of FEZ1 is directly regulated by the PKCζ activity. Although expression of FEZ1 alone had no effect on PC12 cells, coexpression of FEZ1 and constitutively active PKCζ stimulated the neuronal differentiation of PC12 cells. Combined with the recent finding that a human FEZ1 protein is able to complement the function of UNC-76 necessary for normal axonal bundling and elongation within axon bundles in the nematode, these results suggest that FEZ1 plays a crucial role in the axon guidance machinery in mammals by interacting with PKCζ.

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