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

Circulation ◽  
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.

scholarly journals The proline synthesis enzyme P5CS forms cytoophidia in Drosophila

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.

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

2007 ◽  
Vol 18 (11) ◽  
pp. 4317-4326 ◽  
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.

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

2008 ◽  
Vol 389 (10) ◽  
Author(s):  
Hye Jin Yun ◽  
Jungsun Kwon ◽  
Wongi Seol
Keyword(s):  
Transcriptional Activity ◽  
Constitutive Androstane Receptor ◽  
Splicing Factor ◽  
Yeast Two Hybrid ◽  
Interacting Protein ◽  
Functional Studies ◽  
Reporter Activity ◽  
Inhibitory Function ◽  
Two Hybrid ◽  
Hybrid Screening

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.

scholarly journals Requirement for Bbp1p in the Proper Mitotic Functions of Cdc5p in Saccharomyces cerevisiae

2004 ◽  
Vol 15 (4) ◽  
pp. 1711-1723 ◽  
Author(s):  
Chong J. Park ◽  
Sukgil Song ◽  
Thomas H. Giddings ◽  
Hyeon-Su Ro ◽  
Krisada Sakchaisri ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Spindle Pole ◽  
Stable Complex ◽  
Yeast Two Hybrid ◽  
Binding Studies ◽  
Interacting Protein ◽  
Spindle Pole Bodies ◽  
Vitro Binding ◽  
Two Hybrid

The polo-box domain of the budding yeast polo kinase Cdc5p plays an essential role for targeting the catalytic activity of Cdc5p to spindle pole bodies (SPBs) and cytokinetic neck-filaments. Here, we report the isolation of Bbp1p as a polo-box interacting protein by a yeast two-hybrid screen. Bbp1p localizes to the periphery of the central plaque of the SPB and plays an important role in SPB duplication. Similarly, Cdc5p localized to the cytoplasmic periphery of the SPB. In vitro binding studies showed that Cdc5p interacted with the N-terminal domain of Bbp1p (Bbp1pΔC), but apparently not with Mps2p, a component shown to form a stable complex with Bbp1p. In addition, Bbp1p, but likely not Mps2p, was required for proper localization of Cdc5p to the SPB. The C-terminal coiled-coil domain of Bbp1p (Bbp1p243–385), which is crucial for both the homodimerization and the SPB localization, could target the localization-defective Cdc5pΔC to the SPB and induce the release of Cdc14p from the nucleolus. Consistent with this observation, expression of CDC5ΔC-BBP1243–385 under CDC5 promoter control partially complemented the cdc5Δ defect. These data suggest that Bbp1pΔC interacts with the polo-box domain of Cdc5p, and this interaction is critical for the subcellular localization and mitotic functions of Cdc5p.

Serum and glucocorticoid-regulated kinase Sgk1 inhibits insulin-dependent activation of phosphomannomutase 2 in transfected COS-7 cells

2005 ◽  
Vol 288 (1) ◽  
pp. C148-C155 ◽  
Author(s):  
Miranda Menniti ◽  
Rodolfo Iuliano ◽  
Rosario Amato ◽  
Rosalia Boito ◽  
Monica Corea ◽  
...  
Keyword(s):  
Specific Interaction ◽  
Yeast Two Hybrid ◽  
Intact Cells ◽  
Vitro Assay ◽  
Convergence Point ◽  
Insulin Dependent ◽  
Two Hybrid ◽  
Essential Convergence ◽  
Hybrid Screening

Serum- and glucocorticoid-regulated kinase (Sgk1) is considered to be an essential convergence point for peptide and steroid regulation of ENaC-mediated sodium transport. We tried to identify molecular partners of Sgk1 by yeast two-hybrid screening. Yeast two-hybrid screening showed a specific interaction between Sgk1 and phosphomannomutase (PMM)2, the latter of which is an enzyme involved in the regulation of glycoprotein biosynthesis. The interaction was confirmed in intact cells by coimmunoprecipitation and colocalization detected using confocal microscopy. We were then able to demonstrate that Sgk1 phosphorylated PMM2 in an in vitro assay. In addition, we found that the enzymatic activity of PMM2 is upregulated by insulin treatment and that Sgk1 completely inhibits PMM2 activity both in the absence and in the presence of insulin stimulation. These data provide evidence suggesting that Sgk1 may modulate insulin action on the cotranslational glycosylation of glycoproteins.

Proteome Profiling Identified Amyloid-β Protein Precursor as a Novel Binding Partner and Modulator of VGLUT1

2021 ◽  
pp. 1-58
Author(s):  
Jin-wu Zhou ◽  
Man Zhao ◽  
Wen-liang Rang ◽  
Xiao-yan Zhang ◽  
Zhen-ming Liu ◽  
...  
Keyword(s):  
Yeast Two Hybrid ◽  
In Vivo Models ◽  
Immunoprecipitation Assay ◽  
Protein Partners ◽  
Candidate Protein ◽  
Split Ubiquitin ◽  
Two Hybrid ◽  
Hybrid Screening

Background: The toxicity of excessive glutamate release has been implicated in various acute and chronic neurodegenerative conditions. Vesicular glutamate transporters (VGLUTs) are the major mediators for the uptake of glutamate into synaptic vesicles. However, the dynamics and mechanism of this process in glutamatergic neurons are still largely unknown. Objective: This study aimed to investigate the candidate protein partners of VGLUT1 and their regulatory roles in the vesicles in rat brain. Methods: Pull down assay, co-immunoprecipitation assay, or split-ubiquitin membrane yeast two hybrid screening coupled with nanoRPLC-MS/MS were used to identify the candidate protein partners of VGLUT1 in the vesicles in rat brain. The in vitro and in vivo models were used to test effects of AβPP, Atp6ap2, Gja1, and Synataxin on VGLUT1 expression. Results: A total of 255 and 225 proteins and 172 known genes were identified in the pull down assay, co-immunoprecipitation assay, or split-ubiquitin yeast two-hybrid screening respectively. The physiological interactions of SV2A, Syntaxin 12, Gja1, AβPP, and Atp6ap2 to VGLUT1 were further confirmed. Knockdown of Atp6ap2, Gja1, and Synataxin increased VGLUT1 mRNA expression and only knockdown of AβPP increased both mRNA and protein levels of VGLUT1 in PC12 cells. The regulatory function of AβPP on VGLUT1 expression was further confirmed in the in vitro and in vivo models. Conclusion: These results elucidate that the AβPP and VGLUT1 interacts at vesicular level and AβPP plays a role in the regulation of VGLUT1 expression which is essential for maintaining vesicular activities.

Calsenilin and CALP interact with the cytoplasmic tail of UDP-Gal:GA2/GM2/GD2 β-1,3-galactosyltransferase

2008 ◽  
Vol 412 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Cristián A. Quintero ◽  
Javier Valdez-Taubas ◽  
Mariana L. Ferrari ◽  
Sergio D. Haedo ◽  
Hugo J. F. Maccioni
Keyword(s):  
Calcium Binding ◽  
Chinese Hamster ◽  
Cytoplasmic Tail ◽  
Yeast Two Hybrid ◽  
Molecular Determinants ◽  
Vesicular Stomatitis ◽  
Two Hybrid ◽  
Hybrid Screening

GalT2 (UDP-Gal:GA2/GM2/GD2 β-1,3-galactosyltransferase) is a Golgi-resident type II membrane protein that participates in the synthesis of glycosphingolipids. The molecular determinants for traffic and localization of this and other glycosyltransferases are still poorly characterized. Considering the possibility that interactions with other proteins may influence these processes, in the present study we carried out a yeast two-hybrid screening using elements of the N-terminal domain of GalT2 as bait. In this screening, we identified calsenilin and its close homologue CALP (calsenilin-like protein), both members of the recoverin-NCS (neuronal calcium sensor) family of calcium-binding proteins. In vitro, GalT2 binds to immobilized recombinant CALP, and CALP binds to immobilized peptides with the GalT2 cytoplasmic tail sequence. GalT2 and calsenilin interact physically when co-expressed in CHO (Chinese-hamster ovary)-K1 cells. The expression of CALP or calsenilin affect Golgi localization of GalT2, and of two other glycosyltransferases, SialT2 (CMP-NeuAc:GM3 sialyltransferase) and GalNAcT (UDP-GalNAc:lactosylceramide/GM3/GD3 β1-4 N-acetylgalactosaminyltransferase), by redistributing them from the Golgi to the ER (endoplasmic reticulum), whereas the localization of the VSV-G (G-protein of the vesicular stomatitis virus) or the Golgin GM130 was essentially unaffected. Conversely, the expression of GalT2 affects the localization of calsenilin and CALP by shifting a fraction of the molecules from being mostly diffuse in the cytosol, to clustered structures in the perinuclear region. These combined in vivo and in vitro results suggest that CALP and calsenilin are involved in the trafficking of Golgi glycosyltransferases.

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

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.

scholarly journals A Novel Human Ada2 Homologue Functions with Gcn5 or Brg1 To Coactivate Transcription

2003 ◽  
Vol 23 (19) ◽  
pp. 6944-6957 ◽  
Author(s):  
Nickolai A. Barlev ◽  
Alexander V. Emelyanov ◽  
Paola Castagnino ◽  
Philip Zegerman ◽  
Andrew J. Bannister ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Chromatin Remodeling ◽  
Adaptor Protein ◽  
Saga Complex ◽  
Yeast Two Hybrid ◽  
Functional Assays ◽  
Two Hybrid ◽  
Histone Acetyltransferase Activity

ABSTRACT In yeast, the transcriptional adaptor yeast Ada2 (yAda2) is a part of the multicomponent SAGA complex, which possesses histone acetyltransferase activity through action of the yGcn5 catalytic enzyme. yAda2, among several SAGA proteins, serves to recruit SAGA to genes via interactions with promoter-bound transcription factors. Here we report identification of a new human Ada2 homologue, hAda2β. Ada2β differs both biochemically and functionally from the previously characterized hAda2α, which is a stable component of the human PCAF (human Gcn5 homologue) acetylase complex. Ada2β, relative to Ada2α, interacted selectively, although not stably, with the Gcn5-containing histone acetylation complex TFTC/STAGA. In addition, Ada2β interacted with Baf57 (a component of the human Swi/Snf complex) in a yeast two-hybrid screen and associated with human Swi/Snf in vitro. In functional assays, hAda2β (but not Ada2α), working in concert with Gcn5 (but not PCAF) or Brg1 (the catalytic component of hSwi/Snf complex), increased transcription via the B-cell-specific transcription factor Pax5/BSAP. These findings support the view that Gcn5 and PCAF have distinct roles in vivo and suggest a new mechanism of coactivator function, in which a single adaptor protein (Ada2β) can coordinate targeting of both histone acetylation and chromatin remodeling activities.

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