Improved Stability of Human CGI-58 Induced by Phosphomimetic S237E Mutation.

In lipolysis, the activating function of CGI-58 is regulated by its interaction with perilipin 1 (PLIN1) localized on the lipid droplet (LD), and its release is controlled by phosphorylation. Once lipolysis is stimulated by catecholamines, protein kinase A (PKA)-mediated phosphorylation enables the dissociation of the CGI-58/PLIN1 complex, thereby recruiting adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) to initiate fatty acid release. It has been shown that mouse CGI-58 mutant S239E, which mimics the phosphorylation of this residue, is able to dissociate from the CGI-58/PLIN1 complex and activate ATGL. Here, we analyze the stabilizing effect on human CGI-58 of a triple tryptophan to alanine mutant (3WA) on the LD-binding motif, as well as a quadruple mutant in which the phosphomimetic S237E substitution was introduced to the 3WA construct (3WA/S237E). We found that tryptophan residues promote wild-type (WT) protein aggregation in solution since their substitution for alanine residues favors the presence of the monomer. Our experimental data showed increased thermal stability and solubility of 3WA/S237E protein compared to the 3WA mutant. Moreover, the 3WA/S237E protein showed proper folding and a functional binding site for oleoyl-CoA. The analysis of a bioinformatic three-dimensional (3D) model suggests an intramolecular interaction between the phosphomimetic glutamic acid and a residue of the α/ß hydrolase core. This could explain the increased solubility and stability observed in the 3WA/S237E mutant and evidences the possible role of serine 237 phosphorylation.

Purification and characterization of an FeII- and α-ketoglutarate-dependent xanthine hydroxylase from Aspergillus oryzae.

XanA is an FeII- and α-ketoglutarate-dependent enzyme responsible for the conversion of xanthine to uric acid. It is unique to fungi and it was first described in Aspergillus nidulans. In this work, we present the preliminary characterization of the XanA enzyme from Aspergillus oryzae, a relevant fungus in food production in Japan. The XanA protein (GenBank BAE56701.1) was expressed as a recombinant protein in Escherichia coli BL21 (DE3) Arctic cells. Initial purification assays showed low protein solubility; therefore, the buffer composition was optimized using a fluorescence-based thermal shift assay. The protein was stabilized in solution in the presence of either 600 µM xanthine, 1 M NaCl, 600 µM α-ketoglutarate or 20% glycerol, which increases the melting temperature (Tm) by 2, 4, 5 and 6 °C respectively. The XanA protein was purified by following a three-step purification protocol. The nickel affinity purified protein was subjected to ion-exchange chromatography once the N-terminal 6XHis-tag had been successfully removed, followed by size-exclusion purification. Dynamic light scattering experiments showed that the purified protein was monodisperse and behaved as a monomer in solution. Preliminary activity assays in the presence of xanthine, α-ketoglutarate, and iron suggest that the enzyme is an iron- and α-ketoglutarate-dependent xanthine dioxygenase. Furthermore, the enzyme's optimum activity conditions were determined to be 25 °C, pH of 7.2, HEPES buffer, and 1% of glycerol. In conclusion, we established the conditions to purify the XanA enzyme from A. oryzae in its active form from E. coli bacteria and determined the optimal activity conditions.

Bioinformatic Analysis and Biophysical Characterization Reveal Structural Disorder in G0S2 Protein.

G0S2 is a small protein of 103 residues in length that is involved in multiple cellular processes. To date, several reports have shown that G0S2 functions by making direct protein-protein interactions with key proteins. In lipolysis, G0S2 specifically interacts with adipose triglyceride lipase, inhibiting its activity and resulting in lipolysis being downregulated. In a similar way, G0S2 also participates in the regulation of apoptosis, cell proliferation, and oxidative phosphorylation; however, information regarding G0S2 structural and biophysical properties is limited. In this work, we conducted a comparative structural analysis of human and mouse G0S2 proteins. Bioinformatics suggests the presence of a disordered C-terminal region in human G0S2. Experimental characterization by size-exclusion chromatography and dynamic light scattering showed that human and mouse G0S2 have different hydrodynamic properties. In comparison to the mouse G0S2, which behaves similar to a globular protein, the human G0S2 shows an elongated conformation, most likely by displaying a disordered C-terminal region. Further analysis of hydrodynamic properties under denaturing conditions suggests the presence of a structural element in the mouse protein that undergoes an order to disorder transition at low urea concentration. Structural analysis by circular dichroism revealed that in native conditions, both proteins are mainly unstructured, showing the presence of beta sheet structures. Further analysis of CD data suggests that both proteins belong to the premolten globule family of intrinsically disordered proteins. We suggest that the intrinsic disorder observed in the G0S2 protein may facilitate its interaction with multiple partners in the regulation of cellular metabolism.

Human Gpn1 purified from bacteria binds guanine nucleotides and hydrolyzes GTP as a protein dimer stabilized by its C-terminal tail.

The essential GTPase Gpn1 mediates RNA polymerase II nuclear targeting and controls microtubule dynamics in yeast and human cells by molecular mechanisms still under investigation. Here, we purified human HisGpn1 expressed as a recombinant protein in bacteria E. coli BL-21 (DE3). Affinity purified HisGpn1 eluted from a size exclusion column as a protein dimer, a state conserved after removing the hexa-histidine tail and confirmed by separating HisGpn1 in native gels, and in dynamic light scattering experiments. Human HisGpn1 purity was higher than 95%, molecularly monodisperse and could be concentrated to more than 10 mg/mL without aggregating. Circular dichroism spectra showed that human HisGpn1 was properly folded and displayed a secondary structure rich in alpha helices. HisGpn1 effectively bound GDP and the non-hydrolyzable GTP analogue GMPPCP, and hydrolyzed GTP. We next tested the importance of the C-terminal tail, present in eukaryotic Gpn1 but not in the ancestral archaeal Gpn protein, on HisGpn1 dimer formation. C-terminal deleted human HisGpn1 (HisGpn1ΔC) was also purified as a protein dimer, indicating that the N-terminal GTPase domain contains the interaction surface needed for dimer formation. In contrast to HisGpn1, however, HisGpn1ΔC dimer spontaneously dissociated into monomers. In conclusion, we have developed a method to purify properly folded and functionally active human HisGpn1 from bacteria, and showed that the C-terminal tail, universally conserved in all eukaryotic Gpn1 orthologues, stabilizes the GTPase domain-mediated Gpn1 protein dimer. The availability of recombinant human Gpn1 will open new research avenues to unveil the molecular and pharmacological properties of this essential GTPase.

Biochemical and Molecular Characterization of a Novel Cu/Zn Superoxide Dismutase from Amaranthus hypochondriacus L.: an Intrinsically Disordered Protein.

A novel Cu/ZnSOD from Amaranthus hypochondriacus was cloned, expressed, and characterized. Nucleotide sequence analysis showed an open reading frame (ORF) of 456 bp, which was predicted to encode a 15.6-kDa molecular weight protein with a pI of 5.4. Structural analysis showed highly conserved amino acid residues involved in Cu/Zn binding. Recombinant amaranth superoxide dismutase (rAhSOD) displayed more than 50 % of catalytic activity after incubation at 100 °C for 30 min. In silico analysis of Amaranthus hypochondriacus SOD (AhSOD) amino acid sequence for globularity and disorder suggested that this protein is mainly disordered; this was confirmed by circular dichroism, which showed the lack of secondary structure. Intrinsic fluorescence studies showed that rAhSOD undergoes conformational changes in two steps by the presence of Cu/Zn, which indicates the presence of two binding sites displaying different affinities for metals ions. Our results show that AhSOD could be classified as an intrinsically disordered protein (IDP) that is folded when metals are bound and with high thermal stability.

Characterization of antidiabetic and antihypertensive properties of canary seed (Phalaris canariensis L.) peptides.

Canary grass is used as traditional food for diabetes and hypertension treatment. The aim of this work is to characterize the biological activity of encrypted peptides released after gastrointestinal digestion of canary seed proteins. Canary peptides showed 43.5% inhibition of dipeptidyl peptidase IV (DPPIV) and 73.5% inhibition of angiotensin-converting enzyme (ACE) activity. An isolated perfused rat heart system was used to evaluate the canary seed vasoactive effect. Nitric oxide (NO), a major vasodilator agent, was evaluated in the venous effluent from isolated perfused rat heart. Canary seed peptides (1 µg/mL) were able to induce the production of NO (12.24 µM) in amounts similar to those induced by captopril (CPT) and bradykinin (BK). These results show that encrypted peptides in canary seed have inhibitory activity against DPPIV and ACE, enzymes that are targets for diabetes and hypertension treatments.

In vitro inhibition of dipeptidyl peptidase IV by peptides derived from the hydrolysis of amaranth (Amaranthus hypochondriacus L.) proteins.

Bioactive compounds present in foods could potentially have beneficial effects on human health. In this study we report the in vitro inhibitory capacity of peptides released from amaranth seed proteins after enzymatic digestion, against dipeptidyl peptidase IV (DPPIV); an enzyme known to deactivate incretins, hormones involved in insulin secretion. Other seeds, such as soybean, black bean, and wheat were also tested. The highest inhibition of DPPIV was observed with amaranth peptides released after simulated gastrointestinal digestion, showing an IC(50) of 1.1mg/mL in a dose-dependent manner. In silico tryptic digestion of amaranth globulins was carried out releasing peptides larger than 13 residues. Some of these peptides were used for the in silico prediction of their binding modes with DPPIV. Docking models showed that the possible mechanism of globulin peptides to inhibit DPPIV was through blocking the active dimer formation. Peptides were also found inside the major cavity where the natural substrates reach the catalytic site of the enzyme. This is the first report of the identification of inhibitory DPPIV peptides from amaranth hydrolysates and the prediction of their binding modes at the molecular level, leading to their possible use as functional food ingredients in the prevention of diabetes.

A nuclear export sequence in GPN-loop GTPase 1, an essential protein for nuclear targeting of RNA polymerase II, is necessary and sufficient for nuclear export.

XAB1/Gpn1 is a GTPase that associates with RNA polymerase II (RNAPII) in a GTP-dependent manner. Although XAB1/Gpn1 is essential for nuclear accumulation of RNAPII, the underlying mechanism is not known. A XAB1/Gpn1-EYFP fluorescent protein, like endogenous XAB1/Gpn1, localized to the cytoplasm but it rapidly accumulated in the cell nucleus in the presence of leptomycin B, a chemical inhibitor of the nuclear transport receptor Crm1. Crm1 recognizes short peptides in substrate proteins called nuclear export sequences (NES). Here, we employed site-directed mutagenesis and fluorescence microscopy to assess the functionality of all six putative NESs in XAB1/Gpn1. Mutating five of the six putative NESs did not alter the cytoplasmic localization of XAB1/Gpn1-EYFP. However, a V302A/L304A double mutant XAB1/Gpn1-EYFP protein was clearly accumulated in the cell nucleus, indicating the disruption of a functional NES. This functional XAB1/Gpn1 NES displays all features present in most common and potent NESs, including, in addition to Φ1-Φ4, a critical fifth hydrophobic amino acid Φ0. Therefore, in human Gpn1 this NES spans amino acids 292-LERLRKDMGSVAL-304. XAB1/Gpn1 NES is remarkably conserved during evolution. XAB1/Gpn1 NES was sufficient for nuclear export activity, as it caused a complete exclusion of EYFP from the cell nucleus. Molecular modeling of XAB1/Gpn1 provided a mechanistic reason for NES selection, as functionality correlated with accessibility, and it also suggested a mechanism for NES inhibition by intramolecular masking. In conclusion, we have identified a highly active, evolutionarily conserved NES in XAB1/Gpn1 that is critical for nucleo-cytoplasmic shuttling and steady-state cytoplasmic localization of XAB1/Gpn1.

Purification and characterization of the FeII- and alpha-ketoglutarate-dependent xanthine hydroxylase from Aspergillus nidulans.

His6-tagged xanthine/alpha-ketoglutarate (alphaKG) dioxygenase (XanA) of Aspergillus nidulans was purified from both the fungal mycelium and recombinant Escherichia coli cells, and the properties of the two forms of the protein were compared. Evidence was obtained for both N- and O-linked glycosylation on the fungus-derived XanA, which aggregates into an apparent dodecamer, while bacterium-derived XanA is free of glycosylation and behaves as a monomer. Immunological methods identify phosphothreonine in both forms of XanA, with phosphoserine also detected in the bacterium-derived protein. Mass spectrometric analysis confirms glycosylation and phosphorylation of the fungus-derived sample, which also undergoes extensive truncation at its amino terminus. Despite the major differences in the properties of these proteins, their kinetic parameters are similar (kcat = 30-70 s-1, Km of alphaKG = 31-50 muM, Km of xanthine approximately 45 muM, and pH optima at 7.0-7.4). The enzyme exhibits no significant isotope effect when [8-2H]xanthine is used; however, it demonstrates a 2-fold solvent deuterium isotope effect. CuII and ZnII potently inhibit the FeII-specific enzyme, whereas CoII, MnII, and NiII are weaker inhibitors. NaCl decreases the kcat and increases the Km of both alphaKG and xanthine. The alphaKG cosubstrate can be substituted with alpha-ketoadipate (9-fold decrease in kcat and 5-fold increase in the Km compared to those of the normal alpha-keto acid), while the alphaKG analogue N-oxalylglycine is a competitive inhibitor (Ki = 0.12 muM). No alternative purines effectively substitute for xanthine as a substrate, and only one purine analogue (6,8-dihydroxypurine) results in significant inhibition. Quenching of the endogenous fluorescence of the two enzyme forms by xanthine, alphaKG, and DHP was used to characterize their binding properties. A XanA homology model was generated on the basis of the structure of the related enzyme TauD (PDB entry 1OS7) and provided insights into the sites of posttranslational modification and substrate binding. These studies represent the first biochemical characterization of purified xanthine/alphaKG dioxygenase.

Inversion of the allosteric response of Escherichia coli glucosamine-6-P deaminase to N-acetylglucosamine 6-P, by single amino acid replacements.

Amino acid replacements in the active site of glucosamine-6-P deaminase from Escherichia coli (GlcN6P deaminase, EC 3.5.99.6) involving the residues D141 and E148 produce atypical allosteric kinetics. These residues are located in the chain segment 139-156 which is part of the active site and which also forms several intersubunit contacts close to the allosteric site. In the D141N and E148Q mutant forms of this deaminase, there is an inversion of the effect of its physiological allosteric effector, N-acetylglucosamine 6-P, which becomes an inhibitor at substrate concentrations above a critical value. For both mutants, this particular point appears at low substrate concentration and the inhibition by the allosteric activator is the dominant effect in velocity versus substrate curves. These effects are analyzed as a particular case of the concerted allosteric model, assuming that the R state, the conformer displaying the higher affinity for the substrate, is the less catalytic state, thus producing an inverted allosteric response.