Crystal structure of the NEMO ubiquitin-binding domain in complex with Lys 63-linked di-ubiquitin

NEMO is essential for activation of the NF-κB signaling pathway, which is regulated by ubiquitination of proteins. The C-terminal leucine zipper of NEMO and its adjacent coiled-coil region (CC2-LZ) reportedly bind to linear ubiquitin chains with 1 μM affinity and to Lys 63-linked chains with 100 μM affinity. Here we report the crystal structure of the CC2-LZ region of mouse NEMO in complex with Lys 63-linked di-ubiquitin (K63-Ub₂) at 2.7 Å resolution. The ubiquitin-binding region consists of a 130 Å-long helix and forms a parallel coiled-coil dimer. The Ile 44-centered hydrophobic patch of ubiquitin is recognized in the middle of the NEMO ubiquitin-binding region. NEMO interacts with each K63-Ub₂via a single ubiquitin-binding site, consistent with low affinity binding with K63-Ub₂.

Structured summary

MINT-7262681: NEMO (uniprotkb:O88522) binds (MI:0407) to Ubiquitin (uniprotkb:P62991) by pull down (MI:0096)MINT-7262667: Ubiquitin (uniprotkb:P62991) and NEMO (uniprotkb:O88522) bind (MI:0407) by X-ray crystallography (MI:0114)     

Physical association of the catalytic and helper modules of a family-9 glycoside hydrolase is essential for activity

Clostridium thermocellum cellulase 9I (Cel9I) is a non-cellulosomal tri-modular enzyme, consisting of a family-9 glycoside hydrolase (GH9) catalytic module and two family-3 carbohydrate-binding modules (CBM3c and CBM3b). The presence of CBM3c was previously shown to be essential for activity, however the mechanism by which it functions is unclear. We expressed the three recombinant modules independently in Escherichia coli and examined their interactions. Non-denaturing gel electrophoresis, isothermal titration calorimetry, and affinity purification of the GH9-CBM3c complex revealed a specific non-covalent binding interaction between the GH9 module and CBM3c. Their physical association was shown to recover 60-70% of the intact Cel9I endoglucanase activity.

Structured summary:

MINT-6946626:Cel9I (uniprotkb:Q02934) and Cel9I (uniprotkb:Q02934) bind (MI:0407) by comigration in non-denaturing gel electrophoresis (MI:0404)MINT-6946649:Cel9I (uniprotkb:Q02934) and Cel9I (uniprotkb:Q02934) bind (MI:0407) by molecular sieving (MI:0071)MINT-6946687:Cel9I (uniprotkb:Q02934) and Cel9I (uniprotkb:Q02934) bind (MI:0407) by isothermal titration calorimetry (MI:0065)MINT-6946706:Cel9I (uniprotkb:Q02934) binds (MI:0407) to Cel9I (uniprotkb:Q02934) by pull down (MI:0096)     

Interactions between membrane-bound cellulose synthases involved in the synthesis of the secondary cell wall

It has not yet been reported how the secondary CESA (cellulose synthase) proteins are organized in the rosette structure. A membrane-based yeast two-hybrid (MbYTH) approach was used to analyze the interactions between the CESA proteins involved in secondary cell wall synthesis of Arabidopsis and the findings were confirmed in planta by bimolecular fluorescence complementation (BiFC) assay. Results indicated that although all CESA proteins can interact with each other, only CESA4 is able to form homodimers. A model is proposed for the secondary rosette structure. The RING-motif proved not to be essential for the interaction between the CESA proteins.

Structured summary

MINT-6951243: PIP2-1 (uniprotkb:P43286) physically interacts (MI:0218) with PIP2-1 (uniprotkb:P43286) by bimolecular fluorescence complementation (MI:0809)MINT-6950816: CESA4 (uniprotkb:Q84JA6) physically interacts (MI:0218) withCESA4 (uniprotkb:Q84JA6) by membrane bound complementation assay (MI:0230)MINT-6951056, MINT-6951071, MINT-6951088, MINT-6951103: CESA7 (uniprotkb:Q9SWW6) physically interacts (MI:0218) with CESA4 (uniprotkb:Q84JA6) by bimolecular fluorescence complementation (MI:0809)MINT-6950949, MINT-6950990: CESA4 (uniprotkb:Q84JA6) physically interacts (MI:0218) with CESA8 (uniprotkb:Q8LPK5) by membrane bound complementation assay (MI:0230)MINT-6950909, MINT-6951030: CESA4 (uniprotkb:Q8LPK5) physically interacts (MI:0218) with CESA7 (uniprotkb:Q9SWW6) by membrane bound complementation assay (MI:0230)MINT-6951042: CESA4 (uniprotkb:Q84JA6) physically interacts (MI:0218) with CESA4 (uniprotkb:Q84JA6) by bimolecular fluorescence complementation (MI:0809)MINT-6951004, MINT-6951016: CESA8 (uniprotkb:Q8LPK5) physically interacts (MI:0218) with CESA7 (uniprotkb:Q9SWW6) by membrane bound complementation assay (MI:0230)MINT-6951217, MINT-6951230: CESA4 (uniprotkb:Q84JA6) physically interacts (MI:0218) with CESA8 (uniprotkb:Q8LPK5) by bimolecular fluorescence complementation (MI:0809)MINT-6951120, MINT-6951140, MINT-6951156, MINT-6951170, MINT-6951185: CESA8 (uniprotkb:Q8LPK5) physically interacts (MI:0218) withCESA7 (uniprotkb:Q9SWW6) by bimolecular fluorescence complementation (MI:0809)MINT-6951199: CESA8 (uniprotkb:Q8LPK5) physically interacts (MI:0218) withCESA8 (uniprotkb:Q8LPK5) by bimolecular fluorescence complementation (MI:0809)     

NMDA receptors interact with flotillin-1 and -2, lipid raft-associated proteins

N-methyl-d-aspartate receptors (NMDARs) mediate excitatory synaptic transmission in the brain. Here we demonstrate interactions between the NR2A and NR2B subunits of NMDARs with flotillin-1 (flot-1), a lipid raft-associated protein. When mapped, analogous regions in the far distal C-termini of NR2A and NR2B mediate binding to flot-1, and the prohibitin homology domain of flot-1 contains binding sites for NR2A and NR2B. Although NR2B can also directly bind to flot-2 via a separate region of its distal C-terminus, NMDARs were significantly more colocalized with flot-1 than flot-2 in cultured hippocampal neurons. Overall, this study defines a novel interaction between NMDARs and flotillins.

Structured summary

MINT-7013094: NR2A (uniprotkb:Q00959), NR2B (uniprotkb:Q00960) and Flot2 (uniprotkb:Q9Z2S9) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7013147: Flot1 (uniprotkb:Q9Z1E1) physically interacts (MI:0218) with NR2A (uniprotkb:Q00959) by anti bait coimmunoprecipitation (MI:0006)MINT-7013189: Flot1 (uniprotkb:Q9Z1E1) physically interacts (MI:0218) with Flot2 (uniprotkb:Q9Z2S9) by anti bait coimmunoprecipitation (MI:0006)MINT-7013033: NR2A (uniprotkb:Q00959) physically interacts (MI:0218) with Flot1 (uniprotkb:Q9Z1E1) by two hybrid (MI:0018)MINT-7013178: NR1 (uniprotkb:P35439) physically interacts (MI:0218) with Flot2 (uniprotkb:Q9Z2S9) by anti bait coimmunoprecipitation (MI:0006)MINT-7013197, MINT-7013210: NR2B (uniprotkb:Q00960) and NR2A (uniprotkb:Q00959) physically interact (MI:0218) with Flot2 (uniprotkb:Q9Z2S9) by anti bait coimmunoprecipitation (MI:0006)MINT-7013002: NR2B (uniprotkb:Q00960) physically interacts (MI:0218) with Flot1 (uniprotkb:O08917) by two hybrid (MI:0018)MINT-7013117: Flot1 (uniprotkb:Q9Z1E1), NR2B (uniprotkb:Q00960) and NR2A (uniprotkb:Q00959) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7013171: NR1 (uniprotkb:P35439) physically interacts (MI:0218) with Flot1 (uniprotkb:Q9Z1E1) by anti bait coimmunoprecipitation (MI:0006)MINT-7013017: NR2A (uniprotkb:Q00959) physically interacts (MI:0218) with Flot1 (uniprotkb:O08917) by two hybrid (MI:0018)MINT-7013054: NR2B (uniprotkb:Q00960) physically interacts (MI:0218) with Flot1 (uniprotkb:Q9Z1E1) by two hybrid (MI:0018)MINT-7013129: Flot1 (uniprotkb:Q9Z1E1) physically interacts (MI:0218) with NR2B (uniprotkb:Q00960) by anti bait coimmunoprecipitation (MI:0006)MINT-7013155: NR1 (uniprotkb:P35439) physically interacts (MI:0218) with NR2B (uniprotkb:Q00960) by anti bait coimmunoprecipitation (MI:0006)MINT-7013074: NR2B (uniprotkb:Q00960) physically interacts (MI:0218) with Flot2 (uniprotkb:Q9Z2S9) by two hybrid (MI:0018)MINT-7013162: NR1 (uniprotkb:P35439) physically interacts (MI:0218) with NR2A (uniprotkb:Q00959) by anti bait coimmunoprecipitation (MI:0006)     

Beta-2 adrenergic receptor mediated ERK activation is regulated by interaction with MAGI-3

The beta-2 adrenergic receptor (β2AR) has a carboxyl terminus motif that can interact with PSD-95/discs-large/ZO1 homology (PDZ) domain-containing proteins. In this paper, we identified membrane-associated guanylate kinase inverted-3 (MAGI-3) as a novel binding partner of β2AR. The carboxyl terminus of β2AR binds with high affinity to the fifth PDZ domain of MAGI-3, with the last four amino acids (D-S-L-L) of the receptor being the key determinants of the interaction. In cells, the association of full-length β2AR with MAGI-3 occurs constitutively and is enhanced by agonist stimulation of the receptor. Our data also demonstrated that β2AR-stimulated extracellular signal-regulated kinase-1/2 (ERK1/2) activation was substantially retarded by MAGI-3 expression. These data suggest that MAGI-3 regulates β2AR-mediated ERK activation through the physical interaction between β2AR and MAGI-3.

Structured summary

MINT-7716556: beta2AR (uniprotkb:P07550) physically interacts (MI:0915) with MAGI-3 (uniprotkb:Q5TCQ9) by anti tag coimmunoprecipitation (MI:0007)MINT-7716593: beta2AR (uniprotkb:P18762) physically interacts (MI:0915) with MAGI-3 (uniprotkb:Q9EQJ9) by anti bait coimmunoprecipitation (MI:0006)MINT-7716630: MAGI-3 (uniprotkb:Q5TCQ9) and beta2AR (uniprotkb:P07550) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7716382, MINT-7716335: MAGI-3 (uniprotkb:Q5TCQ9) physically interacts (MI:0915) with beta2AR (uniprotkb:P07550) by pull down (MI:0096)MINT-7716320, MINT-7716422, MINT-7716502, MINT-7716450, MINT-7716470: beta2AR (uniprotkb:P07550) binds (MI:0407) to MAGI-3 (uniprotkb:Q5TCQ9) by pull down (MI:0096)     

Interaction of testisin with maspin and its impact on invasion and cell death resistance of cervical cancer cells

Previous studies have shown that testisin promotes malignant transformation in cancer cells. To define the mechanism of testisin-induced carcinogenesis, we performed yeast two-hybrid analysis and identified maspin, a tumor suppressor protein, as a testisin-interacting molecule. The direct interaction and cytoplasmic co-localization of testisin with maspin was confirmed by immunoprecipitation and confocal analysis, respectively. In cervical cancer cells, maspin modulated cell death and invasion; however, these effects were inhibited by testisin in parallel experiments. Of interest, the doxorubicin resistance was dramatically reduced by testisin knockdown (P = 0.016). Moreover, testisin was found to be over-expressed in cervical cancer samples as compared to matched normal cervical tissues. Thus, we postulate that testisin may promote carcinogenesis by inhibiting tumor suppressor activity of maspin.

Structured summary

MINT-7712215, MINT-7712176: Testisin (uniprotkb:Q9Y6M0) binds (MI:0407) to Maspin (uniprotkb:P36952) by pull down (MI:0096)MINT-7712188: Testisin (uniprotkb:Q9Y6M0) and Maspin (uniprotkb:P36952) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7712115: Testisin (uniprotkb:Q9Y6M0) physically interacts (MI:0915) with Maspin (uniprotkb:P36952) by two-hybrid (MI:0018)MINT-7712162, MINT-7712128: Maspin (uniprotkb:P36952) physically interacts (MI:0915) with Testisin (uniprotkb:Q9Y6M0) by anti bait co-immunoprecipitation (MI:0006)MINT-7712147: Testisin (uniprotkb:Q9Y6M0) physically interacts (MI:0915) with Maspin (uniprotkb:P36952) by anti tag co-immunoprecipitation (MI:0007)     

Molecular determinants of the interactions between SRC-1 and LXR/RXR heterodimers

Liver X receptor (LXR)/retinoid X receptor (RXR) heterodimers have been shown to perform critical functions in cholesterol and lipid metabolism. Here, we have conducted a comparative analysis of the contributions of LXR and RXR binding to steroid receptor coactivator-1 (SRC-1), which contains three copies of the NR box. We demonstrated that the coactivator-binding surface of LXR, but not that of RXR, is critically important for physical and functional interactions with SRC-1, thereby confirming that RXR functions as an allosteric activator of SRC-1-LXR interaction. Notably, we identified NR box-2 and -3 as the essential binding targets for the SRC-1-induced stimulation of LXR transactivity, and observed the competitive in vitro binding of NR box-2 and -3 to LXR.

Structured summary

MINT-7986678, MINT-7986639, MINT-7986700, MINT-7986720, MINT-7986736, MINT-7986760, MINT-7986787: LXR (uniprotkb:Q13133) physically interacts (MI:0915) with SRC1 (uniprotkb:Q15788) and RXR (uniprotkb:P19793) by pull down (MI:0096)MINT-7986596, MINT-7986621: SRC1 (uniprotkb:Q15788) physically interacts (MI:0915) with LXR (uniprotkb:Q13133) by pull down (MI:0096)MINT-7986555, MINT-7986575: LXR (uniprotkb:Q13133) physically interacts (MI:0915) with SRC1 (uniprotkb:Q15788) by two hybrid (MI:0018)MINT-7986808, MINT-7986907, MINT-7986890: SRC1 (uniprotkb:Q15788) binds (MI:0407) to LXR (uniprotkb:Q13133) by pull down (MI:0096)MINT-7986822, MINT-7986848, MINT-7986865: SRC1 (uniprotkb:Q15788) binds (MI:0407) to RXR (uniprotkb:P19793) by pull down (MI:0096)     

Nuclear localization of the pre-mRNA associating protein THOC7 depends upon its direct interaction with Fms tyrosine kinase interacting protein (FMIP)

THOC7 and Fms-interacting protein (FMIP) are members of the THO complex that associate with the mRNA export apparatus. FMIP is a nucleocytoplasmic shuttling protein with a nuclear localization signal (NLS), whereas THOC7 does not contain a typical NLS motif. We show here that THOC7 (50-137, amino acid numbers) binds to the N-terminal portion (1-199) of FMIP directly. FMIP is detected mainly in the nucleus. In the absence of exogenous FMIP, THOC7 resides mainly in the cytoplasm, while in the presence of FMIP, THOC7 is transported into the nucleus with FMIP. Furthermore, THOC7 lacking the FMIP binding site does not co-localize with FMIP, indicating that THOC7/FMIP interaction is required for nuclear localization of THOC7.

Structured summary

MINT-6799962, MINT-6799973, MINT-6800005: THOC7 (uniprotkb:Q6I9Y2) physically interacts (MI:0218) with THOC5 (uniprotkb:Q13769) by pull down (MI:0096)MINT-6800108: FMIP (uniprotkb:Q13769) and THOC7 (uniprotkb: Q6I9Y2) co-localize (MI:0403) by fluorescence microscopy (MI:0416)MINT-6800052: FMIP (uniprotkb:Q13769) physically interacts (MI:0218) with THOC1 (uniprotkb: Q96FV9) by anti tag coimmunoprecipitation (MI:0007)MINT-6800022: THOC7 (uniprotkb:Q6I9Y2) physically interacts (MI:0218) with FMIP (uniprotkb:Q6DFL5) by pull down (MI:0096)MINT-6799989: THOC7 (uniprotkb:Q6I9Y2) binds (MI:0407) to FMIP (uniprotkb:Q13769) by pull down (MI:0096)MINT-6800071, MINT-6800089: FMIP (uniprotkb:Q13769) physically interacts (MI:0218) with THOC7 (uniprotkb:Q6I9Y2) and THOC1 (uniprotkb:Q96FV9) by anti tag coimmunoprecipitation (MI:0007)     

Identification of LRP1B-interacting proteins and inhibition of protein kinase Cα-phosphorylation of LRP1B by association with PICK1

Recent studies show LDL receptor-related protein 1B, LRP1B as a transducer of extracellular signals. Here, we identify six interacting partners of the LRP1B cytoplasmic region by yeast two-hybrid screen and confirmed their in vivo binding by immunoprecipitation. One of the partners, PICK1 recognizes the C-terminus of LRP1B and LRP1. The cytoplasmic domains of LRP1B are phosphorylated by PKCα about 100 times more efficiently than LRP1. Binding of PICK1 inhibits phosphorylation of LRP1B, but does not affect LRP1 phosphorylation.This study presents the possibility that LRP1B participates in signal transduction which PICK1 may regulate by inhibiting PKCα phosphorylation of LRP1B.

Structured summary

MINT-6801075: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with SNTG2 (uniprotkb:Q925E0) by two hybrid (MI:0018)MINT-6801030, MINT-6801468: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with Pick1 (uniprotkb:Q80VC8) by two hybrid (MI:0018)MINT-6801284: LRP1B4 (uniprotkb:Q9JI18) physically interacts (MI:0218) with RanBPM (uniprotkb:P69566) by anti tag coimmunoprecipitation (MI:0007)MINT-6801108: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with Grb7 (uniprotkb:Q03160) by two hybrid (MI:0018)MINT-6801090: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with RanBPM (uniprotkb:P69566) by two hybrid (MI:0018)MINT-6801008: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with Jip-1b (uniprotkb:Q9WVI9-1) by two hybrid (MI:0018)MINT-6801052: Lrp1b (uniprotkb:Q9JI18) physically interacts (MI:0218) with Jip-2 (uniprotkb:Q9ERE9) by two hybrid (MI:0018)MINT-6801258, MINT-6801271: LRP1B4 (uniprotkb:Q9JI18) physically interacts (MI:0218) with Pick1 (uniprotkb:Q80VC8) by anti tag coimmunoprecipitation (MI:0007)MINT-6801244: RanBPM (uniprotkb:P69566) physically interacts (MI:0218) with mLRP4 (uniprotkb:Q8VI56) by anti tag coimmunoprecipitation (MI:0007)MINT-6801131, MINT-6801158: LRP1B4 (uniprotkb:Q9JI18) physically interacts (MI:0218) with Jip-1b (uniprotkb:Q9WVI9-1) by anti tag coimmunoprecipitation (MI:0007)MINT-6801231: PICK1 (uniprotkb:Q80VC8) physically interacts (MI:0218) with mLRP4 (uniprotkb:Q8VI56) by anti tag coimmunoprecipitation (MI:0007)MINT-6801173: Jip-1b (uniprotkb:Q9WVI9-1) physically interacts (MI:0218) with mLRP4 (uniprotkb:Q8VI56) by anti tag coimmunoprecipitation (MI:0007)     

A novel association of mGluR1a with the PDZ scaffold protein CAL modulates receptor activity

Metabotropic glutamate receptor subtype 1a (mGluR1a) associates with the proteins mediating its receptor activity, suggesting a complex-controlled function of mGluR1a. Here, using glutathione-S-transferase pull-down, co-immnoprecipitation and immnoflurescence assays in vitro and in vivo, we have found CFTR-associated ligand (CAL) to be a novel binding partner of mGluR1a, through its PSD95/discslarge/ZO1homology domain. Deletion of mGluR1a-carboxyl terminus (CT) or mutation of Leu to Ala in the CT of mGluR1a reduces the association, indicating the essential binding region of mGluR1a for CAL. Functionally, the interaction of mGluR1a with CAL was shown to inhibit mGluR1a-mediated ERK1/2 activation, without an apparent effect, via the C-terminal-truncated receptor. These findings might provide a novel mechanism for the regulation of mGluR1a-mediated signaling through the interaction with CAL.

Structured summary

MINT-6797987, MINT-6798009:

NHERF-2 (uniprotkb:Q15599) binds (MI:0407) to mGluR1a (uniprotkb:Q9R0W0) by proteinarray (MI:0089)

MINT-6798026, MINT-6798048, MINT-6798066:

mGluR1a (uniprotkb:Q9R0W0) physically interacts (MI:0218) with CAL (uniprotkb:Q9HD26) by pull down (MI:0096)

MINT-6797953, MINT-6797970:

NHERF-1 (uniprotkb:O14745) binds (MI:0407) to mGluR1a (uniprotkb:Q9R0W0) by protein array (MI:0089)

MINT-6797935:

CAL (uniprotkb:Q9HD26) binds (MI:0407) to mGluR1a (uniprotkb:Q9R0W0) by protein array (MI:0089)

MINT-6798084:

CAL (uniprotkb:Q9HD26) binds (MI:0407) to mGluR1a (uniprotkb:Q9R0W0) by filter binding (MI:0049)

MINT-6798134:

mGluR1a (uniprotkb:Q9R0W0) physically interacts (MI:0218) with CAL (uniprotkb:Q9HD26) by anti tag coimmunoprecipitation (MI:0007)

MINT-6798158:

CAL (uniprotkb:B4F775) physically interacts (MI:0218) with mGluR1a (uniprotkb:Q9R0W0) by anti bait coimmunoprecipitation (MI:0006)

MINT-6798233:

CAL (uniprotkb:Q9HD26) colocalizes (MI:0403) with mGluR1a (uniprotkb:Q9R0W0) by fluorescence microscopy (MI:0416)

     

14-3-3 Binding to Pim-phosphorylated Ser166 and Ser186 of human Mdm2 - Potential interplay with the PKB/Akt pathway and p14

Here we show that 14-3-3 proteins bind to Pim kinase-phosphorylated Ser166 and Ser186 on the human E3 ubiquitin ligase mouse double minute 2 (Mdm2), but not protein kinase B (PKB)/Akt-phosphorylated Ser166 and Ser188. Pim-mediated phosphorylation of Ser186 blocks phosphorylation of Ser188 by PKB, indicating potential interplay between the Pim and PKB signaling pathways in regulating Mdm2. In cells, expression of Pim kinases promoted phosphorylation of Ser166 and Ser186, interaction of Mdm2 with endogenous 14-3-3s and p14^ARF, and also increased the amount of Mdm2 protein by a mechanism that does not require Pim kinase activities. The implications of these findings for regulation of the p53 pathway, oncogenesis and drug discovery are discussed.

Structured summary

MINT-6823587:PIM3 (uniprotkb:Q86V86) phosphorylates (MI:0217) MDM2 (uniprotkb:Q00987) by protein kinase assay (MI:0424)MINT-6823623:MDM2 (uniprotkb:Q00987) physically interacts (MI:0218) with p14ARF (uniprotkb:Q8N7268N726) by coimmunoprecipitation (MI:0019)MINT-6823537:PKB (uniprotkb:P31749) phosphorylates (MI:0217) MDM2 (uniprotkb:Q00987) by protein kinase assay (MI:0424)MINT-6823574:PIM2 (uniprotkb:QP1W9) phosphorylates (MI:0217) MDM2 (uniprotkb:Q00987) by protein kinase assay (MI:0424)MINT-6823555:PIM1 (uniprotkb:P11309)P phosphorylates (MI:0217) MDM2 (uniprotkb:Q00987) by protein kinase assay (MI:0424)     

Transcriptional activation of hypoxia-inducible factor-1α by HDAC4 and HDAC5 involves differential recruitment of p300 and FIH-1

The interplay between hypoxia-inducible factor-1α (HIF-1α) and histone deacetylase (HDACs) have been well studied; however, the mechanism of cross-talk is unclear. Here, we investigated the roles of HDAC4 and HDAC5 in the regulation of HIF-1α function and its associated mechanisms. HDAC4 and HDAC5 enhanced transactivation by HIF-1α without stabilizing HIF-1α. HDAC4 and HDAC5 physically associated with HIF-1α through the inhibitory domain (ID) that is the binding site for factor inhibiting HIF-1 (FIH-1). In the presence of these HDACs, binding of HIF-1α to FIH-1 decreased, whereas binding to p300 increased. These results indicate that HDAC4 and HDAC5 increase the transactivation function of HIF-1α by promoting dissociation of HIF-1α from FIH-1 and association with p300.

Structured summary:

MINT-6802187:HIF1 alpha (uniprotkb:Q16665) physically interacts (MI:0218) with FIH1 (uniprotkb:Q9NWT6) by anti bait coimmunoprecipitation (MI:0006)MINT-6802058:HIF1 alpha (uniprotkb:Q16665) physically interacts (MI:0218) with HDAC4 (uniprotkb:P56524) by pull down (MI:0096)MINT-6802021:HIF1 alpha (uniprotkb:Q61221) physically interacts (MI:0218) with HDAC4 (uniprotkb:P56524) by anti bait coimmunoprecipitation (MI:0006)MINT-6802036:HIF1 alpha (uniprotkb:Q61221) physically interacts (MI:0218) with HDAC5 (uniprotkb:Q9UQL6) by anti bait coimmunoprecipitation (MI:0006)MINT-6802102:HIF1 alpha (uniprotkb:Q16665) physically interacts (MI:0218) with HDAC5 (uniprotkb:Q9UQL6) by pull down (MI:0096)MINT-6802121, MINT-6802156:P300 (uniprotkb:Q09472) physically interacts (MI:0218) with HIF1 alpha (uniprotkb:Q16665) by anti bait coimmunoprecipitation (MI:0006)     

Bag-1M inhibits the transactivation of the glucocorticoid receptor via recruitment of corepressors

The Bcl-2 associated athanogene 1M (Bag-1M) is known to repress the transactivation of the glucocorticoid receptor (GR). We report here that Bag-1M inhibits the action of GR via recruitment of corepressors, including nuclear receptor corepressor (NcoR) and silencing mediator for retinoic acid and thyroid hormone receptor (SMRT), and histone deacetylase (HDAC)3 to the genomic response element of a glucocorticoid-regulated human metallothionein IIa (hMTIIa) gene. A mutant GR lacking the interaction with BAG-1M fails to recruit the corepressors NcoR and SMRT. RNAi-mediated knock down of corepressors and the use of HDAC inhibitor relieved Bag-1M-induced repression on the transactivation of the GR. In addition, Bag-1M is not involved in the degradation of the receptor. These findings indicate a novel mechanism by which Bag-1M acts as a corepressor and downregulates the activity of the GR.

Structured summary

MINT-7216164: HDAC3 (uniprotkb:O15379) physically interacts (MI:0914) with Bag1 (uniprotkb:Q99933) by anti bait coimmunoprecipitation (MI:0006)MINT-7216183: NCOR (uniprotkb:O75376) physically interacts (MI:0914) with Bag1 (uniprotkb:Q99933) by anti bait coimmunoprecipitation (MI:0006)MINT-7216175: SMRT (uniprotkb:Q9Y618) physically interacts (MI:0914) with Bag1 (uniprotkb:Q99933) by anti bait coimmunoprecipitation (MI:0006)     

Interaction of the human somatostatin receptor 3 with the multiple PDZ domain protein MUPP1 enables somatostatin to control permeability of epithelial tight junctions

The presence of heterotrimeric G-proteins at epithelial tight junctions suggests that these cellular junctions are regulated by so far unknown G-protein coupled receptors. We identify here an interaction between the human somatostatin receptor 3 (hSSTR3) and the multiple PDZ protein MUPP1. MUPP1 is a tight junction scaffold protein in epithelial cells, and as a result of the interaction with MUPP1 the hSSTR3 is targeted to tight junctions. Interaction with MUPP1 enables the receptor to regulate transepithelial permeability in a pertussis toxin sensitive manner, suggesting that hSSTR3 can activate G-proteins locally at tight junctions.

Structured summary:

MINT-6800756, MINT-6800770: MUPP1 (uniprotkb:O75970) and hSSTR3 (uniprotkb:P32745) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-6800587:hSSTR3 (uniprotkb:P32745) physically interacts (MI:0218) with MUPP1 (uniprotkb:O55164) by pull down (MI:0096)MINT-6800562:hSSTR3 (uniprotkb:P32745) physically interacts (MI:0218) with MUPP1 (uniprotkb:O75970) by two hybrid (MI:0018)MINT-6800622:hSSTR3 (uniprotkb:P32745) physically interacts (MI:0218) with PIST (uniprotkb: Q9HD26), Hsp70 (uniprotkb:P08107), Maguk p55 (uniprotkb: Q8N3R9), MAGI3 (uniprotkb:Q5TCQ9), ZO-2 (uniprotkb:Q9UDY2), ZO-1 (uniprotkb:Q07157) and MUPP1 (uniprotkb:O55164) by pull down (MI:0096)MINT-6800607, MINT-6801122:hSSTR3 (uniprotkb:P32745) physically interacts (MI:0218) with MUPP1 (uniprotkb:O75970) by anti bait coimmunoprecipitation (MI:0006)     

Interactions with LC3 and polyubiquitin chains link nbr1 to autophagic protein turnover

Nbr1, a ubiquitous kinase scaffold protein, contains a PB1, and a ubiquitin-associated (UBA) domain. We show here that the nbr1 UBA domain binds to lysine-48 and -63 linked polyubiquitin-B chains. Nbr1 also binds to the autophagic effector protein LC3-A via a novel binding site. Ubiquitin-binding, but not PB1-mediated p62/SQSTM1 interaction, is required to target nbr1 to LC3 and polyubiquitin-positive bodies. Nbr1 binds additionally to proteins implicated in ubiquitin-mediated protein turnover and vesicle trafficking: ubiquitin-specific peptidases USP8, and the endosomal transport regulator p14/Robld3. Nbr1 thus contributes to specific steps in protein turnover regulation disrupted in several hereditary human diseases.

Structured summary

MINT-7034452: USP8 (uniprotkb:P40818) physically interacts (MI:0218) with NBR1 (uniprotkb:Q14596) by pull down (MI:0096)MINT-7034438: SQSTM1 (uniprotkb:Q13501) and LC3 (uniprotkb:Q9H492) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7034309: NBR1 (uniprotkb:Q14596) physically interacts (MI:0218) with Ubiquitin (uniprotkb:P62988) by pull down (MI:0096)MINT-7034323: NBR1 (uniprotkb:P97432) physically interacts (MI:0218) with Ubiquitin (uniprotkb:P62988) by pull down (MI:0096)MINT-7034233: NBR1 (uniprotkb:Q14596) physically interacts (MI:0218) with USP8 (uniprotkb:P40818) by two hybrid (MI:0018)MINT-7034207: NBR1 (uniprotkb:Q14596) physically interacts (MI:0218) with Robld3 (uniprotkb:Q9JHS3) by two hybrid (MI:0018)MINT-7034400, MINT-7034418: NBR1 (uniprotkb:Q14596) and LC3 (uniprotkb:Q9H492) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7034167: NBR1 (uniprotkb:Q14596) physically interacts (MI:0218) with Ubiquitin B (uniprotkb:Q78XY9) by two hybrid (MI:0018)MINT-7034470: NBR1 (uniprotkb:Q14596) and USP8 (uniprotkb:P40818) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7034194: NBR1 (uniprotkb:Q14596) physically interacts (MI:0218) with LC3-A (uniprotkb:Q91VR7) by two hybrid (MI:0018)MINT-7034336: SQSTM1 (uniprotkb:Q13501) physically interacts (MI:0218) with Ubiquitin (uniprotkb:P62988) by pull down (MI:0096)MINT-7034375: NBR1 (uniprotkb:Q14596) physically interacts (MI:0218) with LC3 (uniprotkb:Q9H492) by pull down (MI:0096)MINT-7034350: NBR1 (uniprotkb:Q14596) and Ubiquitin (uniprotkb:P62988) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7034181: NBR1 (uniprotkb:Q14596) physically interacts (MI:0218) with Tmed10 (uniprotkb:Q9D1D4) by two hybrid (MI:0018)MINT-7034220: NBR1 (uniprotkb:Q14596) physically interacts (MI:0218) with ube2o (uniprotkb:Q6ZPJ3) by two hybrid (MI:0018)     

BS69 cooperates with TRAF3 in the regulation of Epstein-Barr virus-derived LMP1/CTAR1-induced NF-κB activation

Epstein-Barr virus latent membrane protein 1 (LMP1) activates NF-κB signaling pathways through two C-terminal regions, CTAR1 and CTAR2. Previous studies have demonstrated that BS69, a multidomain cellular protein, regulates LMP1/CTAR2-mediated NF-κB activation by interfering with the complex formation between TRADD and LMP1/CTAR2. Here, we found that BS69 directly interacted with the LMP1/CTAR1 domain and regulated LMP1/CTAR1-mediated NF-κB activation and subsequent IL-6 production. Regarding the mechanisms involved, we found that BS69 directly interacted with TRAF3, a negative regulator of NF-κB activation. Furthermore, small-interfering RNA-mediated knockdown experiments revealed that TRAF3 was involved in the BS69-mediated suppression of LMP1/CTAR1-induced NF-κB activation.

Structured summary

MINT-7556591: lmp1 (uniprotkb:P03230) physically interacts (MI:0915) with BS69 (uniprotkb:Q15326) by anti tag coimmunoprecipitation (MI:0007)MINT-7556646: TRAF6 (uniprotkb:Q9Y4K3) physically interacts (MI:0915) with BS69 (uniprotkb:Q15326) by anti tag coimmunoprecipitation (MI:0007)MINT-7556658, MINT-7556670: TRAF3 (uniprotkb:Q13114) physically interacts (MI:0915) with BS69 (uniprotkb:Q15326) by anti tag coimmunoprecipitation (MI:0007)MINT-7556607: TRAF1 (uniprotkb:Q13077) physically interacts (MI:0915) with BS69 (uniprotkb:Q15326) by anti tag coimmunoprecipitation (MI:0007)MINT-7556634: TRAF5 (uniprotkb:O00463) physically interacts (MI:0915) with BS69 (uniprotkb:Q15326) by anti tag coimmunoprecipitation (MI:0007)MINT-7556622: TRAF2 (uniprotkb:Q12933) physically interacts (MI:0915) with BS69 (uniprotkb:Q15326) by anti tag coimmunoprecipitation (MI:0007)     

The fast-mobility isoform of mouse Mcl-1 is a mitochondrial matrix-localized protein with attenuated anti-apoptotic activity

The full-length pro-survival protein Mcl-1 predominantly resides on the outer membrane of mitochondria. Here, we identified a mitochondrial matrix-localized isoform of Mcl-1 that lacks 33 amino acid residues at the N-terminus which serve both as a mitochondrial targeting and processing signal. Ectopically-expressed Mcl-1 without the N-terminal 33 residues failed to enter the mitochondrial matrix but retained wt-like activities both for interaction with BH3-only proteins and anti-apoptosis. In contrast, the mitochondrial matrix-localized isoform failed to interact with BH3-only proteins and manifested an attenuated anti-apoptotic activity. This study reveals that import of Mcl-1 into the mitochondrial matrix results in the attenuation of Mcl-1’s anti-apoptotic function.

Structured summary

MINT-7965637: NOXA (uniprotkb:Q9JM54) physically interacts (MI:0915) with Mcl-1 (uniprotkb:P97287) by anti tag coimmunoprecipitation (MI:0007)MINT-7965699: Mcl-1 (uniprotkb:P97287) physically interacts (MI:0915) with Bim (uniprotkb:O43521) by anti bait coimmunoprecipitation (MI:0006)MINT-7965655: Mcl-1 (uniprotkb:P97287) physically interacts (MI:0915) with NOXA (uniprotkb:Q9JM54) by anti bait coimmunoprecipitation (MI:0006)MINT-7965711: Bim (uniprotkb:O43521) physically interacts (MI:0915) with Mcl-1 (uniprotkb:P97287) by anti tag coimmunoprecipitation (MI:0007)MINT-7965673: PUMA (uniprotkb:Q9BXH1) physically interacts (MI:0915) with Mcl-1 (uniprotkb:P97287) by anti tag coimmunoprecipitation (MI:0007)MINT-7965685: Mcl-1 (uniprotkb:P97287) physically interacts (MI:0915) with PUMA (uniprotkb:Q9BXH1) by anti bait coimmunoprecipitation (MI:0006)     

FKBP25, a novel regulator of the p53 pathway, induces the degradation of MDM2 and activation of p53

The p53 tumour suppressor protein is tightly controlled by the E3 ubiquitin ligase, mouse double minute 2 (MDM2), but maintains MDM2 expression as part of a negative feedback loop. We have identified the immunophilin, 25 kDa FK506-binding protein (FKBP25), previously shown to be regulated by p53-mediated repression, as an MDM2-interacting partner. We show that FKBP25 stimulates auto-ubiquitylation and proteasomal degradation of MDM2, leading to the induction of p53. Depletion of FKBP25 by siRNA leads to increased levels of MDM2 and a corresponding reduction in p53 and p21 levels. These data are consistent with the idea that FKBP25 contributes to regulation of the p53-MDM2 negative feedback loop.

Structured summary

MINT-6823686:MDM2 (uniprotkb:Q00987) physically interacts (MI:0218) with FKBP25 (uniprotkb:Q00688) by anti bait coimmunoprecipitation (MI:0006)MINT-6823707, MINT-6823722:MDM2 (uniprotkb:Q00987) physically interacts (MI:0218) with FKBP25 (uniprotkb:Q62446) by pull down (MI:0096)MINT-6823775:P53 (uniprotkb:Q04637) physically interacts (MI:0218) with MDM2 (uniprotkb:Q00987) by anti bait coimmunoprecipitation (MI:0006)MINT-6823735, MINT-6823749:FKBP25 (uniprotkb:Q62446) binds (MI:0407) to MDM2 (uniprotkb:Q00987) by pull down (MI:0096)MINT-6823761:Ubiquitin (UNIPROTKB:62988)P physically interacts (MI:0218) with MDM2 (uniprotkb:Q00987) by pull down (MI:0096)MINT-6823669:MDM2 (uniprotkb:Q00987) physically interacts (MI:0218) with FKBP25 (uniprotkb:Q00688) by two hybrid (MI:0018)