Gentamicin inhibits HSP70-assisted protein folding by interfering with substrate recognition

We previously reported that gentamicin (GM) specifically binds to heat-shock protein with subunit molecular masses of 70 kDa (HSP70). In the present study, we have investigated the effects of GM binding on HSP70-assisted protein folding in vitro. The C-terminal, and not the N-terminal of HSP70 was found to bind to GM. GM significantly suppressed refolding of firefly luciferase in the presence of HSP70 and HSP40, although the ATPase activity of HSP70 was unaffected by GM. A surface plasmon resonance analysis revealed that GM specifically interferes with the binding of HSP70 to a model peptide that mimics the exposed hydrophobic surface of the folding intermediates. These results indicated that GM inhibits the chaperone activity of HSP70 and may suppress protein folding via inhibition of HSP70 in vivo.

Structured summary

MINT-7384283: HSP40 (uniprotkb:P25685) binds (MI:0407) to HSP70 (uniprotkb:P34930) by surface plasmon resonance (MI:0107)MINT-7384430: RNaseA (uniprotkb:P61823) binds (MI:0407) to HSP70 (uniprotkb:P34930) by surface plasmon resonance (MI:0107)     

Structure, dynamics and heparin binding of the C-terminal domain of insulin-like growth factor-binding protein-2 (IGFBP-2)

Insulin-like growth factor-binding protein-2 (IGFBP-2) is the largest member of a family of six proteins (IGFBP-1 to 6) that bind insulin-like growth factors I and II (IGF-I/II) with high affinity. In addition to regulating IGF actions, IGFBPs have IGF-independent functions. The C-terminal domains of IGFBPs contribute to high-affinity IGF binding, and confer binding specificity and have overlapping but variable interactions with many other molecules. Using nuclear magnetic resonance (NMR) spectroscopy, we have determined the solution structure of the C-terminal domain of IGFBP-2 (C-BP-2) and analysed its backbone dynamics based on 15N relaxation parameters. C-BP-2 has a thyroglobulin type 1 fold consisting of an alpha-helix, a three-stranded anti-parallel beta-sheet and three flexible loops. Compared to C-BP-6 and C-BP-1, structural differences that may affect IGF binding and underlie other functional differences were found. C-BP-2 has a longer disordered loop I, and an extended C-terminal tail, which is unstructured and very mobile. The length of the helix is identical with that of C-BP-6 but shorter than that of C-BP-1. Reduced spectral density mapping analysis showed that C-BP-2 possesses significant rapid motion in the loops and termini, and may undergo slower conformational or chemical exchange in the structured core and loop II. An RGD motif is located in a solvent-exposed turn. A pH-dependent heparin-binding site on C-BP-2 has been identified. Protonation of two histidine residues, His271 and His228, seems to be important for this binding, which occurs at slightly acidic pH (6.0) and is more significant at pH 5.5, but is largely suppressed at pH 7.4. Possible preferential binding of IGFBP-2 and its C- domain fragments to glycosaminoglycans in the acidic extracellular matrix (ECM) of tumours may be related to their roles in cancer.     

Characterisation of an alpha-galactosidase with potential relevance to ripening related texture changes

alpha-Galactosidase (EC 3.2.1.22) from ripe papaya (Carica papaya L.) fruit was fractionated by a combination of ion exchange and gel filtration chromatography into three forms, viz., alpha-galactosidase 1, 2 and 3. The predominant isoform, alpha-gal 2, was probably a tetramer with a native molecular mass of about 170 kDa and 52 kDa-sized subunits and an estimated pI of 7.3. The subunit's N-terminal amino acid sequence shared high identity (97%) with the deduced sequence of a papaya cDNA clone encoding a putative alpha-galactosidase PAG2 as well as with an Ajuga reptans L. GGT1 clone encoding a galactan: galactan galactosyltransferase (66%). During ripening, alpha-galactosidase activity increased concomitantly with firmness loss and this increase was largely ascribed to alpha-gal 2. The protein level of alpha-gal 2 as estimated by immunoblot was low in developing fruits and generally increased with ripening. alpha-Galactosidase 2 also had the ability to markedly catalyse increased pectin solubility and depolymerisation while the polymers were still structurally attached to the cell walls mimicking, in part, the changes that occur during ripening. The close correlation between texture changes, alpha-gal 2 activity and protein levels as well as capability to modify intact cell walls suggest that the enzyme might contribute to papaya fruit softening during ripening. The purported mechanism of alpha-gal 2 action as a softening enzyme was discussed in terms of its functional capacity as a glycanase or perhaps, as a transglycosylase.     

Isolation and characterization of a phenol-degrading bacterium from an industrial activated sludge

This paper reports the successful isolation and characterization of a new phenol-degrading bacterium, strain EDP3, from activated sludge. Strain EDP3 is a nonmotile, strictly aerobic, Gram-negative, and short-rod or coccobacillary bacterium, which occurs singly, in pairs, or in clusters. 16S rRNA gene sequence analysis revealed that strain EDP3 belonged to the gamma group of Proteobacteria, with a 97.0% identity to 16S rRNA gene sequences of Acinetobacter calcoaceticus. Strain EDP3 could aerobically grow on a number of aromatic compounds, such as phenol, sodium benzoate, p-hydroxybenzoate, phenylacetate, benzene, ethylbenzene, benzylalcohol, and so on. In particular, it could mineralize up to 1,000 mg l⁻¹ phenol at room temperature (25°C). The growth kinetics of strain EDP3 on phenol as a sole carbon and energy source at 25°C can be described using the Haldane equation. It has a maximal specific growth rate (μ_max) of 0.28 h⁻¹, a half-saturation constant (K _S) of 1,167.1 mg l⁻¹, and a substrate inhibition constant (K _i) of 58.5 mg l⁻¹. Values of yield coefficient (Y _X/S) are between 0.4 and 0.6 mg dry cell (mg phenol)⁻¹. Strain EDP3 has high tolerance to the toxicity of phenol (up to 1,000 mg l⁻¹). It therefore could be an excellent candidate for the biotreatment of high-strength phenol-containing industrial wastewaters and for the in situ bioremediation of phenol-contaminated soils.     

Effects of heat treatment on the protein secondary structure and pigment microenvironment in photosystem 1 complex

The protein secondary structure and pigments' microenvironment in photosystem 1 (PS1) complexes were studied in the temperature range of 25–80 °C using Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy, respectively. Quantitative analysis of the component bands of the amide I band (1 700–1 600 cm⁻¹) showed no significant change below 50 °C. However, apparent conformational changes occurred at 60 °C and further continued at 70 and 80 °C accompanied with transitions of secondary structure mainly from α-helix to the β-sheet structures. CD analysis demonstrated that the regular arrangement, viz. protein microenvironment of pigments of PS1 complexes, was destroyed by heat treatment which might come from the changes of protein secondary structure of PS1. The CD signals at 645 nm contributed by chlorophyll (Chl) b of light-harvesting complex 1 (LHC1) were easily destroyed at the beginning of heat treatment (25–60 °C). When temperature reached 70 and 80 °C, the CD signals at 478 nm contributed mainly by Chl b of LHC1 and 498 nm contributed by carotenoids decreased most rapidly, indicating that LHC1 was more sensitive to high temperature than core complexes. In addition, the oxygen uptake rate decreased by 90.81 % at 70 °C and was lost completely at 80 °C showing that heat treatment damaged the regular function of PS1 complexes. This may be attributed to heat-induced changes of pigment microenvironment and protein secondary structure, especially transmembrane α-helix located in PsaA/B of PS1.     

Methodology of reversible protein labeling for ratiometric fluorescent measurement

The first fluorescent labeling technology, which can induce not only an increase in the fluorescence intensity but also a shift in the fluorescence spectrum, has been developed for "ratiometric" measurements for a protein by utilizing a newly designed "field-sensitive" fluorescent probe and its corresponding unique amino acid tag.     

Reconstitution of Photosystem Ⅱ Reaction Center with Cu-ChlorophyⅡ a

An Isolated photosystem （PS） II reaction center （RC） with altered pigment content was obtained by chemical exchange of native chlorophyll a （Chl） with externally added Cu-Chl a （Cu-Chl）. Pigment composition and spectroscopic properties of the RC exchanged with Cu-Chl were compared with native RC and RC treated with Chl In the same way. High-performance liquid chromatography analysis showed approximately 0.5 Cu-Chl per two pheophytln in the Cu-Chl-reconstltuted RC preparation. Insertion of Cu-Chl resulted in a decrease In absorption at 670 nm and an Increase at 660 nm, suggesting that the peripheral Chl may have been displaced. Fluorescence emission spectra of the Cu-Chl-reconstituted RC displayed a marked decrease In fluorescence yield and a blue shift of the band maximum, accompanied by the appearance of a broad peak at a shorter wavelength, Indicating that energy transfer In the modified RC was disturbed by Cu-Chl, a quencher of the excited state. However, there were few differences in the circular dichrolsm （CD） spectra, suggesting that the arrangement of pigments and proteins responsible for the CD signal was not significantly affected. In addition, no obvious change In peptlde components was found after the exchange procedure.     

Distinct roles for Pax7 and Pax3 in adult regenerative myogenesis

We assessed viable Pax7(-/-) mice in 129Sv/J background and observed reduced growth and marked muscle wasting together with a complete absence of functional satellite cells. Acute injury resulted in an extreme deficit in muscle regeneration. However, a small number of regenerated myofibers were detected, suggesting the presence of residual myogenic cells in Pax7-deficient muscle. Rare Pax3(+)/MyoD+ myoblasts were recovered from Pax7(-/-) muscle homogenates and cultures of myofiber bundles but not from single myofibers free of interstitial tissues. Finally, we identified Pax3+ cells in the muscle interstitial environment and demonstrated that they coexpressed MyoD during regeneration. Sublaminar satellite cells in hind limb muscle did not express detectable levels of Pax3 protein or messenger RNA. Therefore, we conclude that interstitial Pax3+ cells represent a novel myogenic population that is distinct from the sublaminar satellite cell lineage and that Pax7 is essential for the formation of functional myogenic progenitors from sublaminar satellite cells.     

A novel function of Nur77: physical and functional association with protein kinase C

Despite the involvement in diverse physiological process and pleiotropic expression profile, the molecular functions of Nur77 are not likely to be fully elucidated. From the effort to find a novel function of Nur77, we detected molecular interaction between Nur77 and PKC. Details of interaction revealed that C-terminal ligand binding domain (LBD) of Nur77 specifically interacted with highly conserved glycine-rich loop of PKC required for catalytic activity. This molecular interaction resulted in inhibition of catalytic activity of PKCtheta by Nur77. C-terminal LBD of Nur77 is sufficient for inhibiting the phosphorylation of substrate by PKCtheta. Ultimately, inhibition of catalytic activity by Nur77 is deeply associated with repression of PKC-mediated activation of AP-1 and NF-kappaB. Therefore, these findings demonstrate a novel function of Nur77 as a PKC inhibitor and give insights into molecular mechanisms of various Nur77-mediated physiological phenomena.