Current inhibition of human EAG1 potassium channels by the Ca binding protein S100B

Voltage-dependent human ether à go-go (hEAG1) potassium channels are implicated in neuronal signaling as well as in cancer cell proliferation. Unique sensitivity of the channel to intracellular Ca²⁺ is mediated by calmodulin (CaM) binding to the intracellular N- and C-termini of the channel. Here we show that application of the acidic calcium-binding protein S100B to inside-out patches of Xenopus oocytes causes Ca²⁺-dependent inhibition of expressed hEAG1 channels. Protein pull-down assays and fluorescence correlation spectroscopy (FCS) revealed that S100B binds to hEAG1 and shares the same binding sites with CaM. Thus, S100B is a potential alternative calcium sensor for hEAG1 potassium channels.

Structured summary

MINT-7988123: CaM (uniprotkb:P62158) and EAG1 alpha (uniprotkb:O95259) physically interact (MI:0915) by competition binding (MI:0405)MINT-7988019, MINT-7988052: EAG1 alpha (uniprotkb:O95259) binds (MI:0407) to s100B (uniprotkb:P02638) by pull down (MI:0096)MINT-7988074: EAG1 alpha (uniprotkb:O95259) and s100B (uniprotkb:P02638) physically interact (MI:0915) by competition binding (MI:0405)MINT-7988100:CaM (uniprotkb:P62158) and EAG1 alpha (uniprotkb:O95259) bind (MI:0407) by fluorescence correlation spectroscopy (MI:0052).     

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)     

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)     

Regulation of nuclear localization signal-importin α interaction by Ca2+/S100A6

Although the precise intracellular roles of S100 proteins are not fully understood, these proteins are thought to be involved in Ca²⁺-dependent diverse signal transduction pathways. In this report, we identified importin α as a novel target of S100A6. Importin α contains armadillo repeats, essential for binding to nuclear localization signals. Based on the results from GST pull-down assay, gel-shift assay, and co-immunoprecipitation, we demonstrated that S100A6 specifically interacts with the armadillo repeats of importin α in a Ca²⁺-dependent manner, resulting in inhibition of the nuclear localization signal (NLS)-importin α complex formation in vitro and in vivo. These results indicate S100A6 may regulate the nuclear transport of NLS-cargos in response to increasing concentrations of intracellular Ca²⁺.

Structured summary

MINT-8045244: Importin alpha (uniprotkb:P52292) physically interacts (MI:0915) with S100A2 (uniprotkb:P29034) by pull down (MI:0096)MINT-8044928: Importin alpha (uniprotkb:P52292) binds (MI:0407) to S100A6 (uniprotkb:P06703) by pull down (MI:0096)MINT-8044941: Importin alpha (uniprotkb:P52292) and S100A6 (uniprotkb:P06703) bind (MI:0407) by electrophoretic mobility supershift assay (MI:0412)MINT-8044997: Importin alpha (uniprotkb:P52292) physically interacts (MI:0915) with S100A6 (uniprotkb:P06703) by anti bait coimmunoprecipitation (MI:0006)MINT-8045031: Importin beta (uniprotkb:Q14974) physically interacts (MI:0915) with importin alpha (uniprotkb:P52293) and S100A6 (uniprotkb:P06703) by pull down (MI:0096)MINT-8044917: Importin alpha (uniprotkb:P52292) binds (MI:0407) to S100A2 (uniprotkb:P29034) by pull down (MI:0096)MINT-8045257: Importin alpha (uniprotkb:P52292) physically interacts (MI:0915) with S100A6 (uniprotkb:P06703) by pull down (MI:0096)MINT-8045015: Importin beta (uniprotkb:Q14974) physically interacts (MI:0915) with importin alpha (uniprotkb:P52293) and S100A2 (uniprotkb:P29034) by pull down (MI:0096)MINT-8045267: Importin alpha (uniprotkb:P52292) physically interacts (MI:0915) with S100A2 (uniprotkb:P29034) and npm2 (uniprotkb:Q6GQG6) by pull down (MI:0096)MINT-8045316: Importin beta (uniprotkb:Q14974) physically interacts (MI:0915) with importin alpha (uniprotkb:P52293) by pull down (MI:0096)MINT-8045302: Importin alpha (uniprotkb:P52292) physically interacts (MI:0915) with NPM1 (uniprotkb:P06748) and S100A2 (uniprotkb:P29034) by pull down (MI:0096)MINT-8045290: Importin alpha (uniprotkb:P52292) physically interacts (MI:0915) with npm2 (uniprotkb:Q6GQG6) by pull down (MI:0096)MINT-8044963, MINT-8044985: Importin alpha (uniprotkb:P52292) physically interacts (MI:0915) with S100A2 (uniprotkb:P29034) by anti bait coimmunoprecipitation (MI:0006)MINT-8044951: Importin alpha (uniprotkb:P52292) and S100A2 (uniprotkb:P29034) bind (MI:0407) by electrophoretic mobility supershift assay (MI:0412)     

Alternative splicing of RGS8 gene changes the binding property to the M1 muscarinic receptor to confer receptor type-specific Gq regulation

RGS proteins constitute a large family that modulates heterotrimeric G-protein signaling. We previously showed that RGS8 suppressed Gq signaling in a receptor type-specific manner. To elucidate molecular mechanisms underlying receptor-specific attenuation by RGS8, we examined whether RGS8 can interact with certain G-protein-coupled receptors. By pull-down assay, we showed that RGS8 directly binds to the third intracellular (i3) loop of M1 and M3 muscarinic acetylcholine receptors (mAChRs). The binding of RGS8S, a splice variant with a different N-terminus, was weaker. RGS8 could bind specifically to the C-terminal part of M1i3 (containing amino acids of 304-353 of i3 of human M1-mAChR), but RGS8S could not. Moreover, deletion of the N-terminal 9 amino acids and substitution of both Arg-8 and Arg-9 of RGS8 with Ala resulted in reduced binding to M1i3. BRET experiments revealed that RGS8 actually interacts with M1-mAChR, but RGS8S does not interact in the living cells. The RGS8 mutant, which had less binding ability to M1i3, showed a reduced inhibitory function of Gq signaling through M1-mAChR. These results demonstrated that RGS8 can directly interact with M1-mAChR via its N-terminus and the i3 loop of the receptor, and this binding must play an essential role in receptor-specific suppression by RGS8.     

Direct interaction and functional coupling between voltage-gated CaV1.3 Ca channel and GABAB receptor subunit 2

Although Ca_V1.2 and Ca_V1.3 are two subtypes of L-type Ca²⁺ channels expressed in the CNS, functions of Ca_V1.3 have not been well elucidated compared to Ca_V1.2. Here, we found that Ca_V1.3-NT associates with GABA_BR2-CT using yeast two-hybrid, GST pull-down and co-immunoprecipitation assays. We also demonstrated co-localization of Ca_V1.3 and GABA_BR2 in HEK293 cells and cultured hippocampal neurons. Whole-cell patch-clamp and Ca²⁺-imaging experiments revealed that activation of GABA_BR increases Ca_V1.3 currents and intracellular Ca²⁺ via Ca_V1.3, but not Ca_V1.2. These results show a physical and functional interaction between Ca_V1.3 and GABA_BR, suggesting the potential pivotal roles of Ca_V1.3 in the CNS.

Structured summary

MINT-7975667: Cav1.3 (uniprotkb:P27732) physically interacts (MI:0915) with GABABR2 (uniprotkb:O88871) by two hybrid (MI:0018)MINT-7975740: Cav1.3 (uniprotkb:P27732) and GABABR2 (uniprotkb:O75899) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7966007, MINT-7966016: Cav1.3 (uniprotkb:P27732) physically interacts (MI:0915) with GABABR2 (uniprotkb:O88871) by anti bait coimmunoprecipitation (MI:0006)MINT-7975712, MINT-7975691: Cav1.3 (uniprotkb:P27732) physically interacts (MI:0915) with GABABR2 (uniprotkb:O88871) by pull down (MI:0096)MINT-7966026: GABABR2 (uniprotkb:O88871) and Cav1.3 (uniprotkb:P27732) colocalize (MI:0403) by fluorescence microscopy (MI:0416)     

Measurement of heterotrimeric G-protein and regulators of G-protein signaling interactions by time-resolved fluorescence resonance energy transfer

G-protein-coupled receptors transduce their signals through G-protein subunits which in turn are subject to modulation by other intracellular proteins such as the regulators of G-protein signaling (RGS) proteins. We have developed a cell-free, homogeneous (mix and read format), time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor heterotrimeric G-protein subunit interactions and the interaction of the G alpha subunit with RGS4. The assay uses a FRET pair consisting of a terbium cryptate chelate donor spectrally matched to an Alexa546 fluor acceptor, each of which is conjugated to separate protein binding partners, these being G alpha(i1):beta4gamma2 or G alpha(i1):RGS4. Under conditions favoring specific binding between labeled partners, high-affinity interactions were observed as a rapid increase (>fivefold) in the FRET signal. The specificity of these interactions was demonstrated using denaturing or competitive conditions which caused significant reductions in fluorescence (50-85%) indicating that labeled proteins were no longer in close proximity. We also report differential binding effects as a result of altered activation state of the G alpha(i1) protein. This assay confirms that interactions between G-protein subunits and RGS4 can be measured using TR-FRET in a cell- and receptor-free environment.     

Inhibition of human initiator caspase 8 and effector caspase 3 by cross-class inhibitory bovSERPINA3-1 and A3-3

Serpins are a superfamily of structurally conserved proteins. Inhibitory serpins use a suicide substrate-like mechanism. Some are able to inhibit cysteine proteases in cross-class inhibition. Here, we demonstrate for the first time the strong inhibition of initiator and effector caspases 3 and 8 by two purified bovine SERPINA3s. SERPINA 3-1 (uniprotkb:Q9TTE1) binds tighly to human CASP3 (uniprotkb:P42574) and CASP8 (uniprotkb:Q14790) with k_ass of 4.2 × 10⁵ and 1.4 × 10⁶ M⁻¹ s⁻¹, respectively. A wholly similar inhibition of human CASP3 and CASP8 by SERPINA3-3 (uniprotkb:Q3ZEJ6) was also observed with k_ass of 1.5 × 10⁵ and 2.7 × 10⁶ M⁻¹ s⁻¹, respectively and form SDS-stable complexes with both caspases. By site-directed mutagenesis of bovSERPINA3-3, we identified Asp³⁷¹ as the potential P1 residue for caspases. The ability of other members of this family to inhibit trypsin and caspases was analysed and discussed.

Structured summary

MINT-7234656: CASP8 (uniprotkb:Q14790) and SERPINA3-1 (uniprotkb:Q9TTE1) bind (MI:0407) by biochemical (MI:0401)MINT-7234634: SERPINA3-3 (uniprotkb:Q3ZEJ6) and CASP3 (uniprotkb:P42574) bind (MI:0407) by biochemical (MI:0401)MINT-7234663: CASP8 (uniprotkb:Q14790) and SERPINA3-3 (uniprotkb:Q3ZEJ6) bind (MI:0407) by biochemical (MI:0401)MINT-7234625: SERPINA3-1 (uniprotkb:Q9TTE1) and CASP3 (uniprotkb:P42574) bind (MI:0407) by biochemical (MI:0401)     

A scanning peptide array approach uncovers association sites within the JNK/βarrestin signalling complex

βarrestins are molecular scaffolds that can bring together three-component mitogen-activated protein kinase signalling modules to promote signal compartmentalisation. We use peptide array technology to define novel interfaces between components within the c-Jun N-terminal kinase (JNK)/βarrestin signalling complex. We show that βarrestin 1 and βarrestin 2 associate with JNK3 via the kinase N-terminal domain in a region that, surprisingly, does not harbour a known ‘common docking’ motif. In the N-domain and C-terminus of βarrestin 1 and βarrestin 2 we identify two novel apoptosis signal-regulating kinase 1 binding sites and in the N-domain of the βarrestin 1 and βarrestin 2 we identify a novel MKK4 docking site.

Structured summary

MINT-7263196, MINT-7263175: Arrestin beta-2 (uniprotkb:P32121) binds (MI:0407) to ASK1 (uniprotkb:Q99683) by peptide array (MI:0081)MINT-7263136: JNK3 (uniprotkb:P53779) binds (MI:0407) to Arrestin beta-1 (uniprotkb:P49407) by peptide array (MI:0081)MINT-7263161: JNK3 (uniprotkb:P53779) binds (MI:0407) to Arrestin beta-2 (uniprotkb:P32121) by peptide array (MI:0081)MINT-7263304: Arrestin beta-1 (uniprotkb:P49407) physically interacts (MI:0915) with ASK1 (uniprotkb:Q99683) by anti tag coimmunoprecipitation (MI:0007)MINT-7263286: Arrestin beta-2 (uniprotkb:P32121) binds (MI:0407) to MKK4 (uniprotkb:P45985) by peptide array (MI:0081)MINT-7263231, MINT-7263254: Arrestin beta-1 (uniprotkb:P49407) binds (MI:0407) to ASK1 (uniprotkb:Q99683) by peptide array (MI:0081)MINT-7263269: Arrestin beta-1 (uniprotkb:P49407) binds (MI:0407) to MKK4 (uniprotkb:P45985) by peptide array (MI:0081)     

The β1 subunit of Na/K-ATPase interacts with BKCa channels and affects their steady-state expression on the cell surface

Large conductance Ca²⁺-activated K⁺ channels (BK_Ca) encoded by the Slo1 gene play a role in the physiological regulation of many cell types. Here, we show that the β1 subunit of Na⁺/K⁺-ATPase (NKβ1) interacts with the cytoplasmic COOH-terminal region of Slo1 proteins. Reduced expression of endogenous NKβ1 markedly inhibits evoked BK_Ca currents with no apparent effect on their gating. In addition, NKβ1 down-regulated cells show decreased density of Slo1 subunits on the cell surface.

Structured summary

MINT-7260438, MINT-7260555: Slo1 (uniprotkb:Q8AYS8) physically interacts (MI:0915) with NKbeta1 (uniprotkb:P08251) by anti bait coimmunoprecipitation (MI:0006)MINT-7260587, MINT-7260606, MINT-7260619, MINT-7260632: Slo1 (uniprotkb:Q08460) physically interacts (MI:0915) with NKbeta 1 (uniprotkb:P08251) by pull down (MI:0416)MINT-7260570: NKbeta1 (uniprotkb:P08251) and Slo1 (uniprotkb:Q8AYS8) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7260414: Slo1 (uniprotkb:Q08460) physically interacts (MI:0915) with NKbeta1 (uniprotkb:P08251) by two hybrid (MI:0018)     

Dynamin-1 co-associates with native mouse brain BKCa channels: Proteomics analysis of synaptic protein complexes

In every synapse, a large number of proteins interact with other proteins in order to carry out signaling and transmission in the central nervous system. In this study, we used interaction proteomics to identify novel synaptic protein interactions in mouse cortical membranes under native conditions. Using immunoprecipitation, immunoblotting, and mass spectrometry, we identified a number of novel synaptic protein interactions involving soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), calcium-activated potassium channel (BK_Ca) alpha subunits, and dynamin-1. These novel interactions offer valuable insight into the protein-protein interaction network in intact synapses that could advance understanding of vesicle trafficking, release, and recycling.

Structured summary

MINT-7543319: Snap-25 (uniprotkb:P60879) physically interacts (MI:0914) with Tubulin beta-5 chain (uniprotkb:P99024), V-type proton ATPase subunit d 1 (uniprotkb:P51863), Zinc finger homeobox protein 3 (uniprotkb:Q61329), Tubulin beta-2A chain (uniprotkb:Q7TMM9), Synaptophysin (uniprotkb:Q62277), Gapdh (uniprotkb:P16858), Basement membrane-specific heparan sulfate proteoglycan core protein (uniprotkb:Q05793), Tubulin alpha-4A chain (uniprotkb:P68368), Tubulin alpha-1A chain (uniprotkb:P68369), Microtubule-associated protein 6 (uniprotkb:Q7TSJ2), AP-2 complex subunit beta (uniprotkb:Q9DBG3), Phosphofurin acidic cluster sorting protein 1 (uniprotkb:Q8K212), AP-2 complex subunit alpha-1 (uniprotkb:P17426), Kinesin-1 heavy chain (uniprotkb:Q617r68), Kinesin heavy chain isoform 5C (uniprotkb:P28738), Sodium/potassium-transporting ATPase subunit alpha-1 (uniprotkb:Q8VDN2) and Nck-associated protein 1 (uniprotkb:P28660) by anti bait co-immunoprecipitation (MI:0006)MINT-7543636: Calcium-activated potassium channel subunit alpha-1 (uniprotkb:Q08460) physically interacts (MI:0914) with AMP deaminase 2 (uniprotkb:Q9DBT5), Gamma-tubulin complex component 4 (uniprotkb:Q9D4F8), Gamma-tubulin complex component 2 (uniprotkb:Q921G8), Sodium/potassium-transporting ATPase subunit alpha-1 (uniprotkb:Q8VDN2), Phosphoinositide 3-kinase regulatory subunit 4 (uniprotkb:Q8VD65), Beta-centractin (uniprotkb:Q8R5C5), KIAA1107 (uniprotkb:Q80TK0), Sodium/potassium-transporting ATPase subunit alpha-2 (uniprotkb:Q6PIE5), Sodium/potassium-transporting ATPase subunit alpha-3 (uniprotkb:Q6PIC6), Phosphatidylinositol 3-kinase catalytic subunit type 3 (uniprotkb:Q6PF93), KH domain-containing, RNA-binding, signal transduction-associated protein 1 (uniprotkb:Q60749), Tubulin gamma-1 chain (uniprotkb:P83887), Heat shock cognate 71 kDa protein (uniprotkb:P63017), Alpha-centractin (uniprotkb:P61164), Gamma-tubulin complex component 3 (uniprotkb:P58854), Dynamin-1 (uniprotkb:P39053), Kinesin heavy chain isoform 5C (uniprotkb:P28738), Elongation factor 1-alpha 1 (uniprotkb:P10126), Kinesin light chain 2 (uniprotkb:O88448), Activated CDC42 kinase 1 (uniprotkb:O54967) and Syntaxin-binding protein 1 (uniprotkb:O08599) by anti bait co-immunoprecipitation (MI:0006)MINT-7544031: Calcium-activated potassium channel subunit alpha-1 (uniprotkb:Q08460) physically interacts (MI:0914) with Syntaxin-binding protein 1 (uniprotkb:O08599), Syntaxin-1A (uniprotkb:O35526) and Dynamin-1 (uniprotkb:P39053) by anti bait co-immunoprecipitation (MI:0006)MINT-7543287: Syntaxin-1A (uniprotkb:O35526) physically interacts (MI:0914) with Vamp2 (uniprotkb:P63044), Snap-25 (uniprotkb:P60879), munc-18 (uniprotkb:O08599) and BK_Ca alpha subunit (uniprotkb:Q08460) by anti bait co-immunoprecipitation (MI:0006)MINT-7543972: Vamp-2 (uniprotkb:P63044) physically interacts (MI:0914) with Dynamin-1 (uniprotkb:P39053), Snap-25 (uniprotkb:P60879), Syntaxin-1A (uniprotkb:O35526) and Synaptophysin (uniprotkb:Q62277) by anti bait co-immunoprecipitation (MI:0006)MINT-7543728: Dynamin-1 (uniprotkb:P39053) physically interacts (MI:0914) with Clathrin heavy chain 1 (uniprotkb:Q68FD5) and Calcium-activated potassium channel subunit alpha-1 (uniprotkb:Q08460) by anti bait co-immunoprecipitation (MI:0006)MINT-7543905: Snap-25 (uniprotkb:P60879) physically interacts (MI:0914) with Syntaxin-1A (uniprotkb:O35526) and Vamp-2 (uniprotkb:P63044) by anti bait co-immunoprecipitation (MI:0006)MINT-7543476: Vamp-2 (uniprotkb:P63044) physically interacts (MI:0914) with Syntaxin-7 (uniprotkb:O70439), Neuronal membrane glycoprotein M6-a (uniprotkb:P35802), Syntaxin-1B (uniprotkb:P61264), Beta-soluble NSF attachment protein (uniprotkb:P28663), Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-3 (uniprotkb:Q61011), Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 (uniprotkb:P62874), Guanine nucleotide-binding protein G(o) subunit alpha (uniprotkb:P18872), V-type proton ATPase subunit d 1 (uniprotkb:P51863), Zinc transporter 3 (uniprotkb:P97441), Sodium/potassium-transporting ATPase subunit alpha-2 (uniprotkb:Q6PIE5), Sodium/potassium-transporting ATPase subunit alpha-3 (uniprotkb:Q6PIC6), Sodium/potassium-transporting ATPase subunit alpha-1 (uniprotkb:Q8VDN2), Potassium-transporting ATPase alpha chain 1 (uniprotkb:Q64436), Synaptophysin (uniprotkb:Q62277), Syntaxin-1A (uniprotkb:O35526) and Dynamin-1 (uniprotkb:P39053) by anti bait co-immunoprecipitation (MI:0006)     

Biochemical characterization of glutaredoxins from Chlamydomonas reinhardtii: Kinetics and specificity in deglutathionylation reactions

Protein deglutathionylation is mainly catalyzed by glutaredoxins (GRXs). We have analyzed the biochemical properties of four of the six different GRXs of Chlamydomonas reinhardtii. Kinetic parameters were determined for disulfide and dehydroascorbate reduction but also for deglutathionylation of artificial and protein substrates. The results indicate that GRXs exhibit striking differences in their catalytic properties, mainly linked to the class of GRX considered but also to the pK_a of the N-terminal catalytic cysteine. Furthermore, glutathionylated proteins were found to exhibit distinct reactivities with GRXs. These results suggest that glutathionylation may allow a fine tuning of cell metabolism under stress conditions.

Structured summary

MINT-7761120: GRX6 (uniprotkb:A8HN52) and GRX6 (uniprotkb:A8HN52) bind (MI:0408) by comigration in non denaturing gel electrophoresis (MI:0404)MINT-7761098:GRX5 (uniprotkb:A8I7Q4) and GRX5 (uniprotkb:A8I7Q4) bind (MI:0408) by comigration in non denaturing gel electrophoresis (MI:0404)     

Mcl-1 fragment induces apoptosis through direct interaction with Bax

Mcl-1 full-length (Mcl-1^1-350), a tightly regulated protein, plays an important role in protecting cells against apoptosis. Cleavage of Mcl-1 at Asp127 by caspase (Mcl-1^C1) contributes to the regulation of Mcl-1 expression, but its pro-apoptotic function remains controversial. Here, we reported that Mcl-1^128-350 expression induced caspase-dependent apoptosis. We demonstrated that Mcl-1^128-350 but not Mcl-1^1-350 interacts with Bax. This interaction required an intact BH3 Mcl-1^128-350 domain and leads to Bax activation and translocation to mitochondria. The silencing of Bax, but not of Bak, prevented Mcl-1^128-350 induced apoptosis. In conclusion, Mcl-1^128-350 exerts a pro-apoptotic function governed by its capacity to interact with Bax.

Structured summary

MINT-7306752: Mcl-1 (uniprotkb:Q07820) physically interacts (MI:0915) with BAK (uniprotkb:Q16611) by anti tag coimmunoprecipitation (MI:0007)MINT-7306728: Mcl-1 (uniprotkb:Q07820) physically interacts (MI:0914) with BAX (uniprotkb:Q07812) and BAK (uniprotkb:Q16611) by anti tag coimmunoprecipitation (MI:0007)MINT-7307171: F1 ATPase (uniprotkb:Q5TC12), Mcl-1 (uniprotkb:Q07820) and BAX (uniprotkb:Q07812) colocalize (MI:0403) by cosedimentation through density gradients (MI:0029)     

Regulation of HOXA2 gene expression by the ATP-dependent chromatin remodeling enzyme CHD8

Chromodomain, helicase, DNA-binding protein 8 (CHD8) is an ATP-dependent chromatin remodeling enzyme that has been demonstrated to exist within a large protein complex which includes WDR5, Ash2L, and RbBP5, members of the Mixed Lineage Leukemia (MLL) histone modifying complexes. Here we show that CHD8 relocalizes to the promoter of the MLL regulated gene HOXA2 upon gene activation. Depletion of CHD8 enhances HOXA2 expression under activating conditions. Furthermore, depletion of CHD8 results in a loss of the WDR5/Ash2L/RbBP5 subcomplex, and consequently H3K4 trimethylation, at the HOXA2 promoter. These studies suggest that CHD8 alters HOXA2 gene expression and regulates the recruitment of chromatin modifying enzymes.

Structured summary

MINT-7542810: CHD8 (uniprotkb:Q9HCK8) physically interacts (MI:0915) with RbBP5 (uniprotkb:Q15291) by anti tag coimmunoprecipitation (MI:0007)MINT-7542794: CHD8 (uniprotkb:Q9HCK8) physically interacts (MI:0915) with WDR5 (uniprotkb:P61964) by anti tag coimmunoprecipitation (MI:0007)MINT-7542820: CHD8 (uniprotkb:Q9HCK8) physically interacts (MI:0915) with ASH2L (uniprotkb:Q9UBL3) by anti tag coimmunoprecipitation (MI:0007)MINT-7542769: CHD8 (uniprotkb:Q9HCK8) physically interacts (MI:0914) with RbBP5 (uniprotkb:Q15291), ASH2L (uniprotkb:Q9UBL3) and WDR5 (uniprotkb:P61964) by anti tag coimmunoprecipitation (MI:0007)     

S100 proteins regulate the interaction of Hsp90 with Cyclophilin 40 and FKBP52 through their tetratricopeptide repeats

S100 proteins are a subfamily of the EF-hand type calcium sensing proteins, the exact biological functions of which have not been clarified yet. In this work, we have identified Cyclophilin 40 (CyP40) and FKBP52 (called immunophilins) as novel targets of S100 proteins. These immunophilins contain a tetratricopeptide repeat (TPR) domain for Hsp90 binding. Using glutathione-S transferase pull-down assays and immunoprecipitation, we have demonstrated that S100A1 and S100A2 specifically interact with the TPR domains of FKBP52 and CyP40 in a Ca²⁺-dependent manner, and lead to inhibition of the CyP40-Hsp90 and FKBP52-Hsp90 interactions. These findings have suggested that the Ca²⁺/S100 proteins are TPR-targeting regulators of the immunophilins-Hsp90 complex formations.

Structured summary

MINT-7710442: FKBP52 (uniprotkb:Q02790) physically interacts (MI:0915) with S100A6 (uniprotkb:P06703) by competition binding (MI:0405)MINT-7710192: Cyp40 (uniprotkb:P26882) binds (MI:0407) to S100A1 (uniprotkb:P35467) by pull down (MI:0096)MINT-7710412: Cyp40 (uniprotkb:P26882) physically interacts (MI:0915) with S100A2 (uniprotkb:P29034) by competition binding (MI:0405)MINT-7710374: FKBP52 (uniprotkb:Q02790) binds (MI:0407) to S100A2 (uniprotkb:P29034) by pull down (MI:0096)MINT-7710452: Cyp40 (uniprotkb:P26882) physically interacts (MI:0914) with S100A2 (uniprotkb:P29034) and Hsp90 (uniprotkb:P07900) by anti tag coimmunoprecipitation (MI:0007)MINT-7710387: FKBP52 (uniprotkb:Q02790) binds (MI:0407) to S100A6 (uniprotkb:P06703) by pull down (MI:0096)MINT-7710279: FKBP52 (uniprotkb:Q02790) physically interacts (MI:0915) with S100A1 (uniprotkb:P35467) by competition binding (MI:0405)MINT-7710224: FKBP52 (uniprotkb:Q02790) binds (MI:0407) to Hsp90 (uniprotkb:P07900) by pull down (MI:0096)MINT-7710464: Cyp40 (uniprotkb:P26882) physically interacts (MI:0914) with S100A6 (uniprotkb:P06703) and Hsp90 (uniprotkb:P07900) by anti tag coimmunoprecipitation (MI:0007)MINT-7710249: Cyp40 (uniprotkb:P26882) binds (MI:0407) to Hsp90 (uniprotkb:P07900) by pull down (MI:0096)MINT-7710422: Cyp40 (uniprotkb:P26882) physically interacts (MI:0915) with S100A6 (uniprotkb:P06703) by competition binding (MI:0405)MINT-7710348: Cyp40 (uniprotkb:P26882) binds (MI:0407) to S100A2 (uniprotkb:P29034) by pull down (MI:0096)MINT-7710208: FKBP52 (uniprotkb:Q02790) binds (MI:0407) to S100A1 (uniprotkb:P35467) by pull down (MI:0096)MINT-7710265: Cyp40 (uniprotkb:P26882) physically interacts (MI:0915) with S100A1 (uniprotkb:P35467) by competition binding (MI:0405)MINT-7710361: Cyp40 (uniprotkb:P26882) binds (MI:0407) to S100A6 (uniprotkb:P06703) by pull down (MI:0096)MINT-7710476: FKBP52 (uniprotkb:Q02790) physically interacts (MI:0914) with S100A2 (uniprotkb:P29034) and Hsp90 (uniprotkb:P07900) by anti tag coimmunoprecipitation (MI:0007)MINT-7710316: FKBP52 (uniprotkb:Q02790) physically interacts (MI:0914) with S100A1 (uniprotkb:P35467) and Hsp90 (uniprotkb:P07900) by anti tag coimmunoprecipitation (MI:0007)MINT-7710432: FKBP52 (uniprotkb:Q02790) physically interacts (MI:0915) with S100A2 (uniprotkb:P29034) by competition binding (MI:0405)MINT-7710488: FKBP52 (uniprotkb:Q02790) physically interacts (MI:0914) with S100A6 (uniprotkb:P06703) and Hsp90 (uniprotkb:P07900) by anti tag coimmunoprecipitation (MI:0007)MINT-7710329: S100A6 (uniprotkb:P14069) physically interacts (MI:0914) with FKBP52 (uniprotkb:P30416) and Cyp40 (uniprotkb:Q08752) by anti bait coimmunoprecipitation (MI:0006)MINT-7710295: Cyp40 (uniprotkb:P26882) physically interacts (MI:0914) with Hsp90 (uniprotkb:P07900) and S100A1 (uniprotkb:P35467) by anti tag coimmunoprecipitation (MI:0007)     

Site-specific cleavage of the 40S ribosomal subunit reveals eukaryote-specific ribosomal protein S28 in the subunit head

After resolving the crystal structure of the prokaryotic ribosome, mapping the proteins in the eukaryotic ribosome is a challenging task. We applied RNase H digestion to split the human 40S ribosomal subunit into head and body parts. Mass spectrometry of the proteins in the 40S subunit head revealed the presence of eukaryote-specific ribosomal protein S28e. Recombinant S28e was capable of specific binding to the 3′ major domain of the 18S rRNA (K_a = 8.0 ± 0.5 × 10⁹ M⁻¹). We conclude that S28e has a binding site on the 18S rRNA within the 40S subunit head.

Structured summary

MINT-8044084: S8 (uniprotkb:P62241) and S19 (uniprotkb:P39019) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044095: S8 (uniprotkb:P62241), S19 (uniprotkb:P39019) and S13 (uniprotkb:P62277) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044024: S29 (uniprotkb:P62273), S28 (uniprotkb:P62857), S21 (uniprotkb:P63220), S20 (uniprotkb:P60866), S26 (uniprotkb:P62854), S25 (uniprotkb:P62851), S12 (uniprotkb:P25398), S17 (uniprotkb:P08708), S19 (uniprotkb:P39019), S14 (uniprotkb:P62263), S16 (uniprotkb:P62249) and S11 (uniprotkb:P62280) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044065: S29 (uniprotkb:P62273), S28 (uniprotkb:P62857), S19 (uniprotkb:P39019), S14 (uniprotkb:P62263) and S16 (uniprotkb:P62249) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)     

Characterization of GmCaMK1, a member of a soybean calmodulin-binding receptor-like kinase family

Calmodulin(CaM)-regulated protein phosphorylation forms an important component of Ca²⁺ signaling in animals but is less understood in plants. We have identified a CaM-binding receptor-like kinase from soybean nodules, GmCaMK1, a homolog of Arabidopsis CRLK1. We delineated the CaM-binding domain (CaMBD) of GmCaMK1 to a 24-residue region near the C-terminus, which overlaps with the kinase domain. We have demonstrated that GmCaMK1 binds CaM with high affinity in a Ca²⁺-dependent manner. We showed that GmCaMK1 is expressed broadly across tissues and is enriched in roots and developing nodules. Finally, we examined the CaMBDs of the five-member GmCaMK family in soybean, and orthologs present across taxa.

Structured summary

MINT-8051564: AtCRLK2 (uniprotkb:Q9LFV3) binds (MI:0407) to CaM (uniprotkb:P62199) by filter binding (MI:0049)MINT-8051416: GmCaMK3 (uniprotkb:C6ZRS6) binds (MI:0407) to CaM (uniprotkb:P62199) by filter binding (MI:0049)MINT-8051258: CaM (uniprotkb:P62199) and GmCaMK1 (genbank_protein_gi:223452504) bind (MI:0407) by isothermal titration calorimetry (MI:0065)MINT-8051400: GmCaMK2 (uniprotkb: C6ZRY5) binds (MI:0407) to CaM (uniprotkb:P62199) by filter binding (MI:0049)MINT-8051242, MINT-8051295, MINT-8051313, MINT-8051327, MINT-8051341, MINT-8051355: GmCaMK1 (genbank_protein_gi:223452504) binds (MI:0407) to CaM (uniprotkb:P62199) by filter binding (MI:0049)MINT-8051467: GmCaMK4 (uniprotkb: C6TIQ0) binds (MI:0407) to CaM (uniprotkb:P62199) by filter binding (MI:0049)MINT-8051276: CaM (uniprotkb:P62199) and GmCaMK1 (genbank_protein_gi:223452504) bind (MI:0407) by comigration in non denaturing gel electrophoresis (MI:0404)MINT-8051374: CaM (uniprotkb:P62199) and GmCaMK1 (genbank_protein_gi:223452504) bind (MI:0407) by mass spectrometry studies of complexes (MI:0069)