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)     

X-ray crystal structure of Saccharomyces cerevisiae Pdx1 provides insights into the oligomeric nature of PLP synthases

The universal enzymatic cofactor vitamin B6 can be synthesized as pyridoxal 5-phosphate (PLP) by the glutamine amidotransferase Pdx1. We show that Saccharomyces cerevisiae Pdx1 is hexameric by analytical ultracentrifugation and by crystallographic 3D structure determination. Bacterial homologues were previously reported to exist in hexamer:dodecamer equilibrium. A small sequence insertion found in yeast Pdx1 elevates the dodecamer dissociation constant when introduced into Bacillus subtilis Pdx1. Further, we demonstrate that the yeast Pdx1 C-terminus contacts an adjacent subunit, and deletion of this segment decreases enzymatic activity 3.5-fold, suggesting a role in catalysis.

Structured summary

MINT-7147859: PDX1 (uniprotkb:P16451) and PDX1 (uniprotkb:P16451) bind (MI:0407) by cosedimentation in solution (MI:0028)MINT-7147899: PDX1 (uniprotkb:P37528) and PDX1 (uniprotkb:P37528) bind (MI:0407) by cosedimentation in solution (MI:0028)     

An S-locus receptor-like kinase in plasma membrane interacts with calmodulin in Arabidopsis

Calmodulin-regulated protein phosphorylation plays a pivotal role in amplifying and diversifying the action of calcium ion. In this study, we identified a calmodulin-binding receptor-like protein kinase (CBRLK1) that was classified into an S-locus RLK family. The plasma membrane localization was determined by the localization of CBRLK1 tagged with a green fluorescence protein. Calmodulin bound specifically to a Ca²⁺-dependent calmodulin binding domain in the C-terminus of CBRLK1. The bacterially expressed CBRLK1 kinase domain could autophosphorylate and phosphorylates general kinase substrates, such as myelin basic proteins. The autophosphorylation sites of CBRLK1 were identified by mass spectrometric analysis of phosphopeptides.

Structured summary

MINT-6800947:CBRLK1 (uniprotkb:Q9ZT06) and AtCaM2 (uniprotkb:P25069) bind (MI:0407) by electrophoretic mobility shift assay (MI:0413)MINT-6800966:AtCaM2 (uniprotkb:P25069) and CBRLK1 (uniprotkb:Q9ZT06) bind (MI:0407) by competition binding (MI:0405)MINT-6800930:CBRLK1 (uniprotkb:Q9ZT06) binds (MI:0407) to AtCaM2 (uniprotkb:P25069) by far Western blotting (MI:0047)MINT-6800978:AtCaM2 (uniprotkb:P25069) physically interacts (MI:0218) with CBRLK1 (uniprotkb:Q9ZT06) by cytoplasmic complementation assay (MI:0228)     

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)     

HO1 and PcyA proteins involved in phycobilin biosynthesis form a 1:2 complex with ferredoxin-1 required for photosynthesis

The HO1 and PcyA genes, encoding heme oxygenase-1 (HO1) and phycocyanobilin (PCB):ferredoxin (Fd) oxidoreductase (PcyA), respectively, are required for chromophore synthesis in photosynthetic light-harvesting complexes, photoreceptors, and circadian clocks. In the PCB biosynthetic pathway, heme first undergoes cleavage to form biliverdin. I confirmed that Fd1 induced the formation of a stable and functional HO1 complex by the gel mobility shift assay. Furthermore, analysis by a chemical cross-linking technique designed to detect protein-protein interactions revealed that HO1 and PcyA directly interact with Fd in a 1:2 ratio. Thus, Fd1, a one-electron carrier protein in photosynthesis, drives the phycobilin biosynthetic pathway.

Structured summary

MINT-7014657: Fd1 (uniprotkb:P0A3C9) and HO1 (uniprotkb:Q8DLW1) bind (MI:0407) by comigration in non-denaturing gel electrophoresis (MI:0404)MINT-7014666: HO1 (uniprotkb:Q8DLW1 and Fd1 (uniprotkb:P0A3C9) bind (MI:0407) by cross-linking studies (MI:0030)MINT-7014675: PcyA (uniprotkb:P59288) and Fd1 (uniprotkb:P0A3C9) bind (MI:0407) by cross-linking studies (MI:0030)     

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)     

Defining the structural requirements for ribose 5-phosphate-binding and intersubunit cross-talk of the malarial pyridoxal 5-phosphate synthase

Most organisms synthesise the B₆ vitamer pyridoxal 5-phosphate (PLP) via the glutamine amidotransferase PLP synthase, a large enzyme complex of 12 Pdx1 synthase subunits with up to 12 Pdx2 glutaminase subunits attached. Deletion analysis revealed that the C-terminus has four distinct functionalities: assembly of the Pdx1 monomers, binding of the pentose substrate (ribose 5-phosphate), formation of the reaction intermediate I₃₂₀, and finally PLP synthesis. Deletions of distinct C-terminal regions distinguish between these individual functions. PLP formation is the only function that is conferred to the enzyme by the C-terminus acting in trans, explaining the cooperative nature of the complex.

Structured summary

MINT-7994448: PfPdx1 (uniprotkb:C6KT50) and PfPdx1 (uniprotkb:C6KT50) bind (MI:0407) by molecular sieving (MI:0071)MINT-7994425, MINT-7994413, MINT-7994435: PfPdx1 (uniprotkb:C6KT50) and PfPdx1 (uniprotkb:C6KT50) bind (MI:0407) by cosedimentation in solution (MI:0028).     

Oligomeric structure of LOV domains in Arabidopsis phototropin

Oligomeric structures of the four LOV domains in Arabidopsis phototropin1 (phot1) and 2 (phot2) were studied using crosslinking. Both LOV1 domains of phot1 and phot2 form a dimer independently on the light conditions, suggesting that the LOV1 domain can be a stable dimerization site of phot in vivo. In contrast, phot1-LOV2 is in a monomer-dimer equilibrium and phot2-LOV2 exists as a monomer in the dark. Blue light-induced a slight increase in the monomer population in phot1-LOV2, suggesting a possible blue light-inducible dissociation of dimers. Furthermore, blue light caused a band shift of the phot2-LOV2 monomer. CD spectra revealed the unfolding of helices and the formation of strand structures. Both light-induced changes were reversible in the dark.

Structured summary

MINT-6823377, MINT-6823391:PHOT1 (uniprotkb:O48963) and PHOT1 (uniprotkb: O48963) bind (MI:0407) by cross-linking studies (MI:0030)MINT-6823495, MINT-6823508:PHOT2 (uniprotkb:P93025) and PHOT2 (uniprotkb:P93025) bind (MI:0407) by cross-linking studies (MI:0030)     

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)     

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)     

Interaction between the C-terminal domains of N and P proteins of measles virus investigated by NMR

In this paper we investigate the interaction between the C-terminal domains of the measles virus phosphoprotein (XD) and nucleoprotein (N_TAIL) by using nuclear magnetic resonance chemical shift perturbation experiments. Using both N_TAIL constructs and peptides, we show that contrary to the conserved Box2 region (N^489-506), the C-terminal region of N_TAIL (N^513-525) does not directly interact with XD, and yet affects binding to XD. We tentatively propose a model where the C-terminus of N_TAIL would stabilize the N_TAIL-XD complex either via a functional coupling with N^489-506 or by reducing the entropic penalty associated to the binding-coupled-to-folding process.

Structured summary

MINT-7009780, MINT-7009793, MINT-7009808: N-tail (uniprotkb:Q89933) and P (uniprotkb:P03422) bind (MI:0407) by nuclear magnetic resonance (MI:0077)     

Phosphorylation of more than one site is required for tight interaction of human tau protein with 14-3-3ζ

Serine residues phosphorylated by protein kinase A (PKA) in the shortest isoform of human tau protein (τ3) were sequentially replaced by alanine and interaction of phosphorylated τ3 and its mutants with 14-3-3 was investigated. Mutation S156A slightly decreased interaction of phosphorylated τ3 with 14-3-3. Double mutations S156A/S267A and especially S156A/S235A, strongly inhibited interaction of phosphorylated τ3 with 14-3-3. Thus, two sites located in the Pro-rich region and in the pseudo repeats of τ3 are involved in phosphorylation-dependent interaction of τ3 with 14-3-3. The state of τ3 phosphorylation affects the mode of 14-3-3 binding and by this means might modify tau filament formation.

Structured summary

MINT-7233358, MINT-7233372, MINT-7233384: 14-3-3 zeta (uniprotkb:P63104) and Tau 3 (uniprotkb:P10636-3) bind (MI:0407) by molecular sieving (MI:0071)MINT-7233323, MINT-7233334, MINT-7233346: Tau 3 (uniprotkb:P10636-3) and 14-3-3 zeta (uniprotkb:P63104) bind (MI:0407) by crosslinking studies (MI:0030)MINT-7233285, MINT-7233297, MINT-7233310: 14-3-3 zeta (uniprotkb:P63104) and Tau 3 (uniprotkb:P10636-3) bind (MI:0407) by comigration in non-denaturing gel electrophoresis (MI:0404)     

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)     

Interaction between separated consecutive complement control modules of human C1r: implications for dimerization of the full-length protease

Complement control protein modules (CCP) typically mediate protein:protein interaction during immune response in vertebrates. Using NMR chemical shift perturbation mapping, we present previously lacking experimental evidence for intermolecular interactions between the CCP1 and CCP2 modules of the human C1r serine protease (SP). The identified interface is clearly distinct from that observed in the covalently linked CCP1-CCP2 pair. Structural models of the CCP1-CCP2-SP segments of two C1r molecules built on the basis of shift perturbation data are fully consistent with an extended interaction interface and suggests the possibility of a structural rearrangement as a switch between functional states of human C1r.

Structured summary

MINT-8045767: CCP1 (uniprotkb:P00736) and CCP2 (uniprotkb:P00736) bind (MI:0407) by nuclear magnetic resonance (MI:0077)     

A disulfide driven domain swap switches off the activity of Shigella IpaH9.8 E3 ligase

We show that the monomeric form of Shigella IpaH9.8 E3 ligase catalyses the ubiquitination of human U2AF35 in vitro, providing a molecular mechanism for the observed in vivo effect. We further discover that under non-reducing conditions IpaH9.8 undergoes a domain swap driven by the formation of a disulfide bridge involving the catalytic cysteine and that this dimer is unable to catalyse the ubiquitination of U2AF35. The crystal structure of the domain-swapped dimer is presented. The redox inactivation of IpaH9.8 could be a mechanism of regulating the activity of the IpaH9.8 E3 ligase in response to cell damage so that the host cell in which the bacteria resides is maintained in a benign state suitable for bacterial survival.

Structured summary

MINT-7993779: ipaH9.8 (uniprotkb:Q8VSC3) and ipaH9.8 (uniprotkb:Q8VSC3) bind (MI:0408) by X-ray crystallography (MI:0114) MINT-7993812: ipaH9.8 (uniprotkb:Q8VSC3) and ipaH9.8 (uniprotkb:Q8VSC3) bind (MI:0407) by affinity chromatography technology (MI:0004) MINT-7993790: ipaH9.8 (uniprotkb:Q8VSC3) and ipaH9.8 (uniprotkb:Q8VSC3) bind (MI:0407) by blue native page (MI:0276)     

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)     

Binding of epigallocatechin-3-gallate to transthyretin modulates its amyloidogenicity

More than 100 transthyretin (TTR) variants are associated with hereditary amyloidosis. Approaches for TTR amyloidosis that interfere with any step of the cascade of events leading to fibril formation have therapeutic potential. In this study we tested (−)-epigallocatechin-3-gallate (EGCG), the most abundant catechin of green tea, as an inhibitor of TTR amyloid formation. We demonstrate that EGCG binds to TTR “in vitro” and “ex vivo” and that EGCG inhibits TTR aggregation “in vitro” and in a cell culture system. These findings together with the low toxicity of the compound raise the possibility of using EGCG in a therapeutic approach for familial amyloidotic polyneuropathy, the most frequent form of hereditary TTR amyloidosis.

Structured summary

MINT-7294529: TTR (uniprotkb:P02766) and TTR (uniprotkb:P02766) bind (MI:0407) by comigration in non-denaturing gel electrophoresis (MI:0404)     

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)     

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)