Loss of a DNA binding site within the tail of prelamin A contributes to altered heterochromatin anchorage by progerin

Mutations in the lamin A/C (LMNA) gene that cause Hutchinson-Gilford progeria syndrome (HGPS) lead to expression of a protein called progerin with 50 amino acids deleted from the tail of prelamin A. In cells from patients with HGPS, both the amount and distribution of heterochromatin are altered. We designed in vitro assays to ask whether such alterations might reflect changes in chromatin, DNA and/or histone binding properties of progerin compared to wild-type lamin C-terminal tails. We show that progerin tail has a reduced DNA/chromatin binding capacity and modified trimethylated H3K27 binding pattern, offering a molecular mechanism for heterochromatin alterations related to HGPS.

Structured summary

MINT-7893924, MINT-7893941, MINT-7893990, MINT-7894005, MINT-7894023, MINT-7894038: H3 (uniprotkb:Q71DI3) binds (MI:0407) to LaminA (uniprotkb:P02545) by surface plasmon resonance (MI:0107)MINT-7893957, MINT-7893974, MINT-7894055: H3 (uniprotkb:Q71DI3) binds (MI:0407) to progerin (uniprotkb:Q6UYC3) by surface plasmon resonance (MI:0107)     

S100 proteins interact with the N-terminal domain of MDM2

S100 proteins interact with the transactivation domain and the C-terminus of p53. Further, S100B has been shown to interact with MDM2, a central negative regulator of p53. Here, we show that S100B bound directly to the folded N-terminal domain of MDM2 (residues 2-125) by size exclusion chromatography and surface plasmon resonance experiments. This interaction with MDM2 (2-125) is a general feature of S100 proteins; S100A1, S100A2, S100A4 and S100A6 also interact with MDM2 (2-125). These interactions with S100 proteins do not result in a ternary complex with MDM2 (2-125) and p53. Instead, we observe the ability of a subset of S100 proteins to disrupt the extent of MDM2-mediated p53 ubiquitylation in vitro.

Structured summary

MINT-7905256: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100A6 (uniprotkb:P06703) by surface plasmon resonance (MI:0107)MINT-7905063: MDM2 (uniprotkb:Q00987) and s100A1 (uniprotkb:P23297) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905376: s100A4 (uniprotkb:P26447) and MDM2 (uniprotkb:Q00987) physically interact (MI:0915) by competition binding (MI:0405)MINT-7905130: s100A6 (uniprotkb:P06703) and MDM2 (uniprotkb:Q00987) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905207: s100A6 (uniprotkb:P06703) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905043: s100B (uniprotkb:P04271) and MDM2 (uniprotkb:Q00987) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905196: p53 (uniprotkb:P04637) and s100A4 (uniprotkb:P26447) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905358: p53 (uniprotkb:P04637) and s100A4 (uniprotkb:P26447) physically interact (MI:0915) by fluorescence polarization spectroscopy (MI:0053)MINT-7905220: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100B (uniprotkb:P04271) by surface plasmon resonance (MI:0107)MINT-7905104: s100A4 (uniprotkb:P26447) and MDM2 (uniprotkb:Q00987) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905229: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100A1 (uniprotkb:P23297) by surface plasmon resonance (MI:0107)MINT-7905317, MINT-7905162: s100B (uniprotkb:P04271) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905238: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100A2 (uniprotkb:P29034) by surface plasmon resonance (MI:0107)MINT-7905174, MINT-7905308: s100A1 (uniprotkb:P23297) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905247: MDM2 (uniprotkb:Q00987) binds (MI:0407) to s100A4 (uniprotkb:P26447) by surface plasmon resonance (MI:0107)MINT-7905090: s100A2 (uniprotkb:P29034) and MDM2 (uniprotkb:Q00987) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905142, MINT-7905326: MDM2 (uniprotkb:Q00987) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)MINT-7905185, MINT-7905347: s100A2 (uniprotkb:P29034) and p53 (uniprotkb:P04637) bind (MI:0407) by molecular sieving (MI:0071)     

Physical and functional interactions between hnRNP K and PRMT family proteins

The mechanism underlying the protein-protein interaction of hnRNP K and PRMT family proteins is unclear. We examined and confirmed the arginine methylation of hnRNP K protein by PRMT1, not CARM1, via their direct binding. We also studied hnRNP K protein complexes containing CARM1, as well as PRMT1, using co-immunoprecipitation analysis. PRMT family proteins might be involved in the regulation of hnRNP K functions in nuclear receptor coactivator, transactivation, and p21 gene and protein expressions. We believe these observations will help provide insights into the regulation of hnRNP K protein functions via the recruitment of its associated proteins, including its arginine methylation-modifying proteins.

Structured summary

MINT-6803853: hnRPK, (uniprotkb:P61978) binds (MI:0407) to PRMT1 (uniprotkb:Q99873) by pull down (MI:0096)MINT-6803884: hnRPK, (uniprotkb:P61978) physically interacts (MI:0218) with CARM1 (uniprotkb:Q86X55) by anti tag coimmunoprecipitation (MI:0007)MINT-6803869: hnRPK, (uniprotkb:P61978) physically interacts (MI:0218) with PRMT1 (uniprotkb:Q99873) by anti tag coimmunoprecipitation (MI:0007)MINT-6803939: hnRPK, (uniprotkb:P61978) binds (MI:0407) to PRMT2 (uniprotkb:P55345) by pull down (MI:0096)MINT-6803929: hnRPK, (uniprotkb:P61978) binds (MI:0407) to RMT (uniprotkb:P38074) by pull down (MI:0096)MINT-6803896: hnRPK, (uniprotkb:P61978) binds (MI:0407) to PRMT3 (uniprotkb:O60678) by pull down (MI:0096)MINT-6803834: PRMT1 (uniprotkb:Q99873) methylates (MI:0213) hnRPK, (uniprotkb:P61978) by methyltransferase assay (MI:0515)     

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)     

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)     

Abi1/Hssh3bp1 pY213 links Abl kinase signaling to p85 regulatory subunit of PI-3 kinase in regulation of macropinocytosis in LNCaP cells

Macropinocytosis is regulated by Abl kinase via an unknown mechanism. We previously demonstrated that Abl kinase activity is, itself, regulated by Abi1 subsequent to Abl kinase phosphorylation of Abi1 tyrosine 213 (pY213) [1]. Here we show that blocking phosphorylation of Y213 abrogated the ability of Abl to regulate macropinocytosis, implicating Abi1 pY213 as a key regulator of macropinocytosis. Results from screening the human SH2 domain library and mapping the interaction site between Abi1 and the p85 regulatory domain of PI-3 kinase, coupled with data from cells transfected with loss-of-function p85 mutants, support the hypothesis that macropinocytosis is regulated by interactions between Abi1 pY213 and the C-terminal SH2 domain of p85—thereby linking Abl kinase signaling to p85-dependent regulation of macropinocytosis.

Structured summary

MINT-7908602: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to SHIP2 (uniprotkb:O15357) by array technology (MI:0008)MINT-7908362: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Emt (uniprotkb:Q08881) by array technology (MI:0008)MINT-7908235: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Lyn (uniprotkb:P07948) by array technology (MI:0008)MINT-7908075: Abi1 (uniprotkb:Q8IZP0)binds (MI:0407) to Fgr (uniprotkb:P09769) by array technology (MI:0008)MINT-7908330, MINT-7908522: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Vav1 (uniprotkb:P15498) by array technology (MI:0008)MINT-7907962: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Fyn (uniprotkb:P06241) by array technology (MI:0008)MINT-7908203: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Src (uniprotkb:P12931) by array technology (MI:0008)MINT-7908570: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to SHP-2 (uniprotkb:P35235) by array technology (MI:0008)MINT-7908187, MINT-7908586: Abi1(uniprotkb:Q8IZP0) binds (MI:0407) to Gap (uniprotkb:P20936) by array technology (MI:0008)MINT-7907981, MINT-7907995: Abi1 (uniprotkb:Q8IZP0) physically interacts (MI:0915) with p85a (uniprotkb:P26450) by anti tag coimmunoprecipitation (MI:0007)MINT-7908251: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to PLCG1 (uniprotkb:P19174) by array technology (MI:0008)MINT-7908346: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Grb2 (uniprotkb:P62993) by array technology (MI:0008)MINT-7907945: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Abl (uniprotkb:P00519) by array technology (MI:0008)MINT-7908474: Abi1 (uniprotkb:Q8IZP0)binds (MI:0407) to p85b (uniprotkb:O00459) by array technology (MI:0008)MINT-7908107: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Hck (uniprotkb:P08631) by array technology (MI:0008)MINT-7908011: p85a (uniprotkb:P26450) physically interacts (MI:0915) with Abi1 (uniprotkb:Q8IZP0) by pull down (MI:0096)MINT-7908155: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to FynT (uniprotkb:P06241-2) by array technology (MI:0008)MINT-7908283, MINT-7908490: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to p55g (uniprotkb:Q92569) by array technology (MI:0008)MINT-7907929, MINT-7907815, MINT-7907832, MINT-7907865, MINT-7907897, MINT-7907913, MINT-7907881, MINT-7907848: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to p85a (uniprotkb:P27986) by array technology (MI:0008)MINT-7908059: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Frk (uniprotkb:P42685) by array technology (MI:0008)MINT-7908378: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to CblC (uniprotkb:Q9ULV8) by array technology (MI:0008)MINT-7908618: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to CblA (uniprotkb:B5MC15) by array technology (MI:0008)MINT-7908139, MINT-7908538: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Nap4 (uniprotkb:O14512) by array technology (MI:0008)MINT-7908426: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to CblB (uniprotkb:Q13191) by array technology (MI:0008)MINT-7908506: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Crk (uniprotkb:P46108) by array technology (MI:0008)MINT-7908554: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to mAbl (uniprotkb:P00520) by array technology (MI:0008)MINT-7908043, MINT-7908394: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Vav2 (uniprotkb:P52735) by array technology (MI:0008)MINT-7908458: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to mSck/ShcB (uniprotkb:Q8BMC3) by array technology (MI:0008)MINT-7908091: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Yes (uniprotkb:P07947) by array technology (MI:0008)MINT-7908219: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Src (uniprotkb:P00523) by array technology (MI:0008)MINT-7908123: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Fer (uniprotkb:P16591) by array technology (MI:0008)MINT-7908410: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to CrkL (uniprotkb:P46109) by array technology (MI:0008)MINT-7908314, MINT-7908442: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Arg (uniprotkb:P42684) by array technology (MI:0008)MINT-7908299: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to PLCG1 (uniprotkb:P10686) by array technology (MI:0008)MINT-7908171: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Fes (uniprotkb:P07332) by array technology (MI:0008)MINT-7908027: Abi1 (uniprotkb:Q8IZP0) binds (MI:0407) to Lck (uniprotkb:P06239) by array technology (MI:0008)     

The interaction between EAP30 and ELL is modulated by MCM2

ELL-associated protein 30 (EAP30) was initially characterized as a component of the Holo-ELL complex, which contains the elongation factor ELL. Both ELL and Holo-ELL stimulate RNA pol II elongation in vitro. However, ELL and not Holo-ELL inhibits RNA pol II initiation. It is not clear how these two discrete functions of ELL are regulated. Here we report that mini-chromosome maintenance 2 (MCM2) binds to EAP30 and show that MCM2 competes with ELL for binding to EAP30 thus potentially modulating the stability of Holo-ELL.

Structured summary

MINT-7277033: EAP30 (uniprotkb:Q96H20) physically interacts (MI:0915) with RPB1 (uniprotkb:P24928) by anti tag coimmunoprecipitation (MI:0007)MINT-7277085: EAP30 (uniprotkb:Q96H20) binds (MI:0407) to ELL (uniprotkb:P55199) by pull down (MI:0096)MINT-7277072: EAP30 (uniprotkb:Q96H20) physically interacts (MI:0915) with ELL (uniprotkb:P55199) by anti tag coimmunoprecipitation (MI:0007)MINT-7277100: EAP30 (uniprotkb:Q96H20) physically interacts (MI:0915) with ELL (uniprotkb:P55199) by competition binding (MI:0405)MINT-7277153: MCM2 (uniprotkb:P49736) binds (MI:0407) to ELL (uniprotkb:P55199) by pull down (MI:0096)MINT-7276989: EAP30 (uniprotkb:Q96H20) physically interacts (MI:0915) with MCM2 (uniprotkb:P49736) by pull down (MI:0096)MINT-7277005: EAP30 (uniprotkb:Q96H20) physically interacts (MI:0915) with RPB1 (uniprotkb:P24928) by pull down (MI:0096)MINT-7276960, MINT-7277168: MCM2 (uniprotkb:P49736) physically interacts (MI:0915) with EAP30 (uniprotkb:Q96H20) by two hybrid (MI:0018)MINT-7276971, MINT-7277121, MINT-7277137: MCM2 (uniprotkb:P49736) binds (MI:0407) to EAP30 (uniprotkb:Q96H20) by pull down (MI:0096)MINT-7277018, MINT-7277061: EAP30 (uniprotkb:Q96H20) physically interacts (MI:0915) with MCM2 (uniprotkb:P49736) by anti tag coimmunoprecipitation (MI:0007)     

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)     

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)     

IbpA the small heat shock protein from Escherichia coli forms fibrils in the absence of its cochaperone IbpB

Small heat shock proteins (sHsps) associate with aggregated proteins, changing their physical properties in such a way that chaperone mediated disaggregation becomes much more efficient. In Escherichia coli two small Hsps, IbpA and IbpB, exist. They are 48% identical at the amino acid level, yet their roles in stabilisation of protein aggregates are quite distinct. Here we analysed the biochemical properties of IbpA. We found that IbpA assembles into protofilaments which in turn form mature fibrils. Such fibrils are atypical for sHsps. Interaction of IbpA with either its cochaperone IbpB or an aggregated substrate blocks IbpA fibril formation.

Structured summary

MINT-7876715: ibpA (uniprotkb:P0C054) and ibpA (uniprotkb:P0C054) bind (MI:0407) by molecular sieving (MI:0071)MINT-7888427: ibpB (uniprotkb:P0C058) and ibpB (uniprotkb:P0C058) bind (MI:0407) by molecular sieving (MI:0071)MINT-7888448: ibpA (uniprotkb:P0C054) and ibpA (uniprotkb:P0C054) bind (MI:0407) by electron microscopy (MI:0040)MINT-7888434: ibpB (uniprotkb:P0C058) and ibpB (uniprotkb:P0C058) bind (MI:0407) by electron microscopy (MI:0040)MINT-7888459: ibpA (uniprotkb:P0C054) and ibpA (uniprotkb:P0C054) bind (MI:0407) by fluorescence microscopy (MI:0416)     

POSH2 is a RING finger E3 ligase with Rac1 binding activity through a partial CRIB domain

The Plenty of SH3 domains protein (POSH) is an E3 ligase and a scaffold in the JNK mediated apoptosis, linking Rac1 to downstream components.We here describe POSH2 which was identified from a p21-activated kinase 2 (PAK2) interactor screen. POSH2 is highly homologous with other members of the POSH family; it contains four Src homology 3 (SH3) domains and a RING finger domain which confers E3 ligase activity to the protein. In addition POSH2 contains an N-terminal extension which is conserved among its mammalian counterparts. POSH2 interacts with GTP-loaded Rac1. We have mapped this interaction to a previously unrecognized partial Cdc42/Rac1-interactive binding domain.

Structured summary

MINT-7987761: POSH1 (uniprotkb:Q9HAM2) physically interacts (MI:0915) with Ubiquitin (uniprotkb:P62988) by anti tag coimmunoprecipitation (MI:0007)MINT-7987932: PAK2 (uniprotkb:Q13177) binds (MI:0407) to CDC42 (uniprotkb:Q07912) by solid phase assay (MI:0892)MINT-7987908: POSH1 (uniprotkb:Q9HAM2) binds (MI:0407) to Rac1 (uniprotkb:P63000) by solid phase assay (MI:0892)MINT-7987880: POSH2 (uniprotkb:Q8TEJ3) binds (MI:0407) to Rac1 (uniprotkb:P63000) by solid phase assay (MI:0892)MINT-7987734: POSH2 (uniprotkb:Q8TEJ3) physically interacts (MI:0915) with Ubiquitin (uniprotkb:P62988) by anti tag coimmunoprecipitation (MI:0007)MINT-7987779, MINT-7987804, MINT-7987824, MINT-7987838, MINT-7987853: Rac1 (uniprotkb:P63000) physically interacts (MI:0915) with POSH2 (uniprotkb:Q8TEJ3) by anti tag coimmunoprecipitation (MI:0007)MINT-7987920: PAK2 (uniprotkb:Q13177) binds (MI:0407) to Rac1 (uniprotkb:P63000) by solid phase assay (MI:0892)     

Isolation of a point-mutated p47 lacking binding affinity to p97ATPase

p47, a p97-binding protein, functions in Golgi membrane fusion together with p97 and VCIP135, another p97-binding protein. We have succeeded in creating p47 with a point mutation, F253S, which lacks p97-binding affinity. p47 mapping experiments revealed that p47 had two p97-binding regions and the F253S mutation occurred in the first p97-binding site. p47(F253S) could not form a complex with p97 and did not caused any cisternal regrowth in an in vitro Golgi reassembly assay. In addition, mutation corresponding to the p47 F253S mutation in p37 and ufd1 also abolished their binding ability to p97.

Structured summary

MINT-7987189, MINT-7987207, MINT-7987303: p47 (uniprotkb:O35987) binds (MI:0407) to p97 (uniprotkb:Q01853) by pull down (MI:0096)MINT-7987226: p97 (uniprotkb:P46462) binds (MI:0407) to p47 (uniprotkb:O35987) by pull down (MI:0096)MINT-7987348: p97 (uniprotkb:P46462) physically interacts (MI:0915) with Ufd1 (uniprotkb:P70362) by pull down (MI:0096)MINT-7987264: p97 (uniprotkb:P46462) and p47 (uniprotkb:O35987) bind (MI:0407) by competition binding (MI:0405)MINT-7987326: p97 (uniprotkb:P46462) binds (MI:0407) to p37 (uniprotkb:Q0KL01) by pull down (MI:0096)     

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)     

The scaffold protein JIP3 functions as a downstream effector of the small GTPase ARF6 to regulate neurite morphogenesis of cortical neurons

The small GTPase ADP-ribosylation factor 6 (ARF6) plays crucial roles in a wide variety of cell functions. To better understand the molecular mechanisms of ARF6-mediated signaling and cellular functions, we sought new ARF6-binding proteins in the mouse brain. We identified the signaling scaffold protein JNK-interacting protein 3 (JIP3), which is exclusively expressed in neurons, as a downstream effector of ARF6. Overexpression of a unique dominant negative mutant of ARF6, which was unable to interact with JIP3, and knockdown of JIP3 in mouse cortical neurons stimulated the elongation and branching of neurites. These results provide evidence that ARF6/JIP3 signaling regulates neurite morphogenesis.

Structured summary

MINT-7892698: PIP5K gamma 661 (uniprotkb:O70161) physically interacts (MI:0915) with Arf6 (uniprotkb:P62331) by anti tag coimmunoprecipitation (MI:0007)MINT-7892333, MINT-7892573, MINT-7892594, MINT-7892629, MINT-7892644, MINT-7892522, MINT-7892716: Arf6 (uniprotkb:P62331) physically interacts (MI:0915) with JLP (uniprotkb:Q58A65) by anti tag coimmunoprecipitation (MI:0007)MINT-7892509: Arf6 (uniprotkb:P62331) physically interacts (MI:0915) with JIP3 (uniprotkb:Q9ESN9) by pull down (MI:0096)MINT-7892770: Arf6 (uniprotkb:P62331) binds (MI:0407) to JIP3 (uniprotkb:Q9ESN9) by pull down (MI:0096)MINT-7892755: Arf6 (uniprotkb:P62331) binds (MI:0407) to JLP (uniprotkb:Q58A65) by pull down (MI:0096)MINT-7892289, MINT-7892314: Arf6 (uniprotkb:P62331) physically interacts (MI:0915) with JLP (uniprotkb:Q58A65) by pull down (MI:0096)MINT-7892353, MINT-7892615, MINT-7892657, MINT-7892672, MINT-7892549, MINT-7892738: Arf6 (uniprotkb:P62331) physically interacts (MI:0915) with JIP3 (uniprotkb:Q9ESN9) by anti tag coimmunoprecipitation (MI:0007)     

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).     

DnaK-mediated association of ClpB to protein aggregates. A bichaperone network at the aggregate surface

Intracellular protein aggregates formed under severe thermal stress can be reactivated by the concerted action of the Hsp70 system and Hsp100 chaperones. We analyzed here the interaction of DnaJ/DnaK and ClpB with protein aggregates. We show that aggregate properties modulate chaperone binding, which in turn determines aggregate reactivation efficiency. ClpB binding strictly depends on previous DnaK association with the aggregate. The affinity of ClpB for the aggregate-DnaK complex is low (K_d = 5-10 μM), indicating a weak interaction. Therefore, formation of the DnaK-ClpB bichaperone network is a three step process. After initial DnaJ binding, the cochaperone drives association of DnaK to aggregates, and in the third step, as shown here, DnaK mediates ClpB interaction with the aggregate surface.

Structured summary

MINT-7258957: G6PDH (uniprotkb:P0AC53) and G6PDH (uniprotkb:P0AC53) bind (MI:0407) by dynamic light scattering (MI:0038)MINT-7258951: alpha glucosidase (uniprotkb:P21517) and alpha glucosidase (uniprotkb:P21517) bind (MI:0407) by dynamic light scattering (MI:0038)MINT-7258903: AdhE (uniprotkb:P0A9Q7) and AdhE (uniprotkb:P0A9Q7) bind (MI:0407) by dynamic light scattering (MI:0038)MINT-7258900: G6PDH (uniprotkb:P0AC53) and G6PDH (uniprotkb:P0AC53) bind (MI:0407) by biophysical (MI:0013)MINT-7258974: DnaK (uniprotkb:P0A6Y8), ClpB (uniprotkb:P63284), DnaJ (uniprotkb:P08622) and G6PDH (uniprotkb:P0AC53) physically interact (MI:0914) by cosedimentation (MI:0027)     

Reconstitution of the mitochondrial Hsp70 (mortalin)-p53 interaction using purified proteins - Identification of additional interacting regions

Previous studies have shown that the mammalian mitochondrial 70 kDa heat-shock protein (mortalin) can also be detected in the cytosol. Cytosolic mortalin binds p53 and by doing so, prevents translocation of the tumor suppressor into the nucleus. In this study, we developed a novel binding assay, using purified proteins, for tracking the interaction between p53 and mortalin. Our results reveal that: (i) P53 binds to the peptide-binding site of mortalin which enhances the ability of the former to bind DNA. (ii) An additional previously unknown binding site for mortalin exists within the C-terminal domain of p53.

Structured summary

MINT-7557591: p53 (uniprotkb:P04637) binds (MI:0407) to DnaK (uniprotkb:P0A6Y8) by affinity chromatography technology (MI:0004)MINT-7557644: mortalin (uniprotkb:P38646) binds (MI:0407) to p53 (uniprotkb:P04637) by pull down (MI:0096)MINT-7557580, MINT-7557611: p53 (uniprotkb:P04637) binds (MI:0407) to mortalin (uniprotkb:P38646) by affinity chromatography technology (MI:0004)     

Stoichiometric protein complex formation and over-expression using the prokaryotic native operon structure

In prokaryotes, operon encoded proteins often form protein-protein complexes. Here, we show that the native structure of operons can be used to efficiently overexpress protein complexes. This study focuses on operons from mycobacteria and the use of Mycobacterium smegmatis as an expression host. We demonstrate robust and correct stoichiometric expression of dimers to higher oligomers. The expression efficacy was found to be largely independent of the intergenic distances. The strategy was successfully extended to express mycobacterial protein complexes in Escherichia coli, showing that the operon structure of gram-positive bacteria is also functional in gram-negative bacteria. The presented strategy could become a general tool for the expression of large quantities of pure prokaryotic protein complexes for biochemical and structural studies.

Structured summary

MINT-7542207: ESAT-6 (uniprotkb:Q50206) and CFP-10 (uniprotkb:O33084) bind (MI:0407) by blue native page (MI:0276)MINT-7542534: ESAT-6 (uniprotkb:P0A564) and CFP-10 (uniprotkb:P0A566) bind (MI:0407) by X-ray crystallography (MI:0114)MINT-7542187: CFP-10 (uniprotkb:P0A566) and ESAT-6 (uniprotkb:P0A564) bind (MI:0407) by blue native page (MI:0276)MINT-7542652: CFP-10 (uniprotkb:P0A566) and ESAT-6 (uniprotkb:P0A564) bind (MI:0407) by molecular sieving (MI:0071)MINT-7542474, MINT-7542303: CFP-10 (uniprotkb:P0A566) physically interacts (MI:0915) with ESAT-6 (uniprotkb:P0A564) by pull down (MI:0096)