Protein tyrosine phosphatase receptor type Z (Ptprz/Ptp/RPTPβ) is a receptor-like protein tyrosine phosphatase (RPTP) which is predominantly expressed in the central nervous system. Tropomyosin-related kinases (Trks) are single-pass transmembrane molecules that are highly expressed in the developing nervous system. Upon the ligand binding of neurotrophins, Trk receptors are activated through autophosphorylation of tyrosine residues; however, the PTPs responsible for the negative regulation of Trk receptors have not been fully elucidated. Here, we identified Ptprz as a specific PTP that efficiently dephosphorylates TrkA as a substrate. Coexpression of Ptprz with Trk receptors in 293T cells showed that Ptprz suppresses the ligand-independent tyrosine phosphorylation of TrkA, but not of TrkB or TrkC, and that Ptprz attenuates TrkA activation induced by nerve growth factor (NGF). Coexpression analyses with TrkA mutants revealed that Ptprz dephosphorylates phosphotyrosine residues in the activation loop of the kinase domain, which are requisite for activation of the TrkA receptor. Consistent with these findings, forced expression of Ptprz in PC12D cells markedly inhibited neurite extension induced by a low dose of NGF. In addition, an increment in the tyrosine phosphorylation of TrkA was observed in the brain of Ptprz-deficient mice. Ptprz thus appears to be one of the PTPs which regulate the activation and signaling of TrkA receptors.

We previously reported that nicotine protected against tunicamycin (Tm)-induced ER stress-mediated apoptosis, but not thapsigargin (Tg)-induced apoptosis in PC12 cells. In the present study, we report that the expression of glucose-regulated protein 78 (GRP78) was suppressed by nicotine in Tm-treated PC12 cells. Interestingly, the GRP78 expression was not changed by nicotine in Tg-treated cells. Moreover, nicotine reduced the activation of caspase-12 in Tm-treated cells, but not in Tg-treated cells. These results suggest that nicotine prevented Tm-induced ER stress-mediated apoptosis by attenuating an early stage of Tm-induced ER stress. It was possible that the suppression of GRP78 expression by nicotine was achieved through the suppression of the Ire1-XBP1 and/or ATF6 pathways. We observed that nicotine suppressed the Tm-induced, but not Tg-induced, splicing of XBP1 mRNA, and also suppressed the Tm-induced, but not Tg-induced, production of cleaved ATF6 in PC12 cells. These results indicate that the suppression of Ire1-XBP1 and ATF6 pathways contributes to the suppression of GRP78 expression by nicotine in Tm-treated PC12 cells, suggesting that nicotine suppresses a common step upstream of both the Ire1-XBP1 and ATF6 pathways which are required for the expression of GRP78 during Tm-induced ER stress.

Protein arginine methylation is a well-known post-translational modification that has been shown to occur in rabbit reticulocyte in vitro translation lysates (RRL); however, it is not known whether this is a general feature of in vitro-produced proteins from other eukaryotic cell-free translation systems, particularly insect-derived lysates (ICL). Because methylation can affect protein localization, RNA binding and protein-protein interactions this may be of great importance as in vitro-produced proteins are often used in assays of protein function. Here, I report the presence of base-stable and base-labile methyltransferase activities in RRL, ICL and wheat germ in vitro extracts (WGE). Indeed, the presence of CARM1 in RRL and ICL and a class II protein arginine methyltransferase activity (PRMT5/7) is documented in all three systems. Additionally, the lysine methyltransferase that modifies eukaryotic elongation factor 1A (eEF-1A) was detected in ICL and WGE. Importantly, using a defined set of substrates under identical conditions I show that all three in vitro systems contain different complements of the various methyltransferases. These data suggest that three systems can be used in a complementary fashion to investigate the effect(s) of post-translational modification on protein function.

Multidimensional NMR was employed to investigate the structural changes in the urea-induced equilibrium unfolding of the dimeric ketosteroid isomerase (KSI) from Pseudomonas putida biotype B. Sequence specific backbone assignments for the native KSI and the protein with 3.5 M urea were carried out using various 3D NMR experiments. Hydrogen exchange measurements indicated that the secondary structures of KSI were not affected significantly by urea up to 3.5 M. However, the chemical shift analysis of ¹H-¹⁵N HSQC spectra at various urea concentrations revealed that the residues in the dimeric interface region, particularly around the β5-strand, were significantly perturbed by urea at low concentrations, while the line-width analysis indicated the possibility of conformational exchange at the interface region around the β6-strand. The results thus suggest that the interface region primarily around the β5- and β6-strands could play an important role as the starting positions in the unfolding process of KSI.

Complex formation of gramicidin (GA) and desformylgramicidin (des-GA) with sterols was investigated by measuring the intrinsic Trp fluorescence. In organic solvents, the Trp fluorescence of momeric GA was quenched upon binding either cholesterol or ergosterol, but that of monomeric des-GA was not quenched by adding cholesterol. Both dimeric GA and des-GA bound highly to ergosterol, but not to cholesterol, determined by quenching of Trp fluorescence. Furthermore, GA- and des-GA-loaded lysophosphatidylcholine micelles were incubated with phosphatidylcholine vesicles containing cholesterol or ergosterol. The results showed that both monomeric and dimeric peptides hardly bound to cholesterol incorporated into phospholipid vesicles, but markedly bound to ergosterol incorporated into the bilayer membranes. Interestingly, des-GA bound more specifically to the two sterols than GA. In addition, fluorescence resonance energy transfer analysis showed that des-GA bound more specifically to the two sterol than GA.

The aim of this work was to investigate the putative modulation of glucose uptake in trophoblast cells by several dietary compounds and by drugs of abuse. For this, the acute (26 min) and chronic (48 h) effect of these substances on the apical uptake of ³H-2-deoxy-D-glucose (³H-DG) by a human choriocarcinoma cell line (BeWo) was determined. ³H-DG apical uptake by BeWo cells was time-dependent, displayed saturable kinetics (V_max=1210±29 nmol mg protein^-1 6 min^-1 and K_m=13.4±0.5 mM) and was insulin-insensitive and cytochalasin B-sensitive (by up to 60 per cent). Acutely, acetaldehyde (30-100 mM), resveratrol, xanthohumol, epigallocatechin-3-gallate (100 µM), chrysin and quercetin (10-100 µM) decreased ³H-DG apical uptake, whereas rutin, catechin (10-100 µM), epicatechin (100 µM) and ethanol (10 mM) increased it. Quercetin and xanthohumol seem to be non-competitive inhibitors of ³H-DG apical uptake, whereas both epigallocatechin-3-gallate and acetaldehyde decreased both the K_m and V_max values. Chronically, rutin and myricetin increased the apical uptake of ³H-DG both isolated (0.1-1 µM) and in combination (both at 1 µM), whereas theophylline (0.1-1 µM) and amphetamine, 3,4-methylenedioxymethamphetamine (0.25-1 µM) and ⁹-tetrahydrocannabinol (1 nM) decreased it. In conclusion, ³H-DG apical uptake by BeWo cells is differentially modulated by different compounds present in drinks and by drugs of abuse.

The backbone dynamics of Y14F mutant of ⁵-3-ketosteroid isomerase (KSI) from Comamonas testosteroni has been studied in free enzyme and its complex with a steroid analogue, 19-nortestosterone hemisuccinate (19-NTHS), by ¹⁵N NMR relaxation measurements. Model-free analysis of the relaxation data showed that the single point mutation induced a substantial decrease in the order parameters (S²) in free Y14F KSI, indicating that the backbone structures of Y14F KSI became significantly mobile by mutation, while the chemical shift analysis indicated that the structural perturbations of Y14F KSI were more profound than those of wild-type (WT) KSI upon 19-NTHS binding. In the 19-NTHS complexed Y14F KSI, however, the key active site residues including Tyr14, Asp38, and Asp99 or the regions around them remained flexible with significantly reduced S² values, whereas the S² values for many of the residues in Y14F KSI became even greater than those of WT KSI upon 19-NTHS binding. The results thus suggest that the hydrogen bond network in the active site might be disrupted by the Y14F mutation, resulting in a loss of the direct interactions between the catalytic residues and 19-NTHS.

4C8 is a new mouse anti-human CD34 monoclonal antibody (mAb), which recognizes class II CD34 epitopes and can be used for clinical hematopoietic stem/progenitor cell selection. In an attempt to improve its safety profiles, we have developed a humanized antibody of 4C8 by complementarity-determining region (CDR) grafting method in this study. Using a molecular model of 4C8 built by computer-assisted homology modeling, framework region (FR) residues of potential importance to the antigen binding were identified. A humanized version of 4C8, denoted as h4C8, was generated by transferring these key murine FR residues onto a human antibody framework that was selected based on homology to the mouse antibody framework, together with the mouse CDR residues. The resultant humanized antibody was shown to possess antigen-binding affinity and specificity similar to that of the original murine antibody, suggesting that it might be an alternative to mouse anti-CD34 antibodies routinely used clinically.

Oxidative regeneration pathways of RNase A, which has four SS linkages, were studied at 25 °C and pH 8.0 by using trans-3,4-dihydroxy-1-selenolane oxide (DHS^ox), a new selenoxide reagent with strong oxidation power. The short-term folding study using a quench-flow instrument (~1 min) revealed that early intermediates (1S, 2S, 3S, and 4S) are formed stochastically and irreversibly from the reduced protein (R) and do not have any stable structures. In the long-term folding study (~300 min), on the other hand, slow generation of the key intermediates (des[65–72] and des[40–95]) through SS rearrangement from the 3S intermediate ensemble was observed, followed by slight formation of native RNase A (N). The parallel UV and CD measurements demonstrated that formation of the key intermediates is accompanied with the formation of the native-like structures. Thus, DHS^ox allowed facile identification of the conformational folding steps coupled with SS rearrangement on the major oxidative folding pathways.

Bacillus subtilis (Bs) DivIVA comprises coiled-coil structures and self-associates forming a 10-12 mer complex in vitro. Using bioinformatic approaches, we determined that Enterococcus faecalis (Ef) DivIVA comprises four coiled-coil domains, one at the N-terminus, the second and the third in the central region of the protein and the fourth at the C-terminus. We determined that DivIVA_Ef self-interacts and forms a 10-12 multimeric complex. Point mutations or deletions of the central regions predicted bioinformatically to disrupt the coiled-coil structures either eliminated or weakened DivIVA_Ef self-interaction and reduced oligomerization. Mutations disrupting the N- and C-terminal coiled-coils of DivIVA_Ef did not affect DivIVA_Ef oligomerization. The introduction of DivIVA_Ef mutations to both the N-terminal and the central coiled-coil domains were lethal unless rescued by expressing wild type DivIVA_Ef in trans. E. faecalis cells expressing these mutations displayed aberrant cell morphology, indicating disruption of the normal cell division phenotype. The results in E. faecalis also indicate that both the N-terminal and the central coiled-coil structures of DivIVA_Ef are indispensable for proper biological function. Overexpression of wild type DivIVA_Ef in both rod-shaped and round Escherichia coli cells resulted in morphological changes, while the overexpression of DivIVA_Ef mutations failed to induce such alterations.

To obtain lectins without tedious purification steps, we developed a convenient method for a one-step purification of lectins using Sugar-immobilized Gold Nano-Particles (SGNPs). Proteins in crude extracts from plant materials were precipitated with 60% ammonium sulfate, and the precipitate was re-dissolved in a small volume of phosphate buffer. The resultant solution was then mixed with appropriate SGNPs under an optimized condition. After incubating overnight at 4^oC, lectins in the mixture formed aggregate with SGNPs, which was visually detected and easily sedimented by centrifugation. The aggregate was dissolved by adding inhibitory sugars, which were identical to the non-reducing sugar moieties on the SGNPs. According to SDS-PAGE and MS of thus obtained proteins, it was found that SGNPs isolated lectins with a high purity. For example, a protein isolated from banana using Glc-GNP (-glucose immobilized Gold Nano-Particle) was identified as banana lectin by trypsin-digested peptide-MS finger printing method.

A-crystallin is abundant in the lens of the eye and acts as a molecular chaperone by preventing aggregation of denaturing proteins. We previously found that chemical modification of the guanidino group of selected arginine residues by a metabolic -dicarbonyl compound, methylglyoxal (MGO), makes human A-crystallin a better chaperone. Here, we examined how the introduction of additional guanidino groups and modification by MGO influence the structure and chaperone function of A-crystallin. A-crystallin lysine residues were converted to homoarginine by guanidination with o-methylisourea (OMIU) and then modified with MGO. LC-ESI-mass spectrometry identified homoargpyrimidine and homohydroimidazolone adducts after OMIU and MGO treatment. Treatment with 0.25 M OMIU abolished most of the chaperone function. However, subsequent treatment with 1.0 mM MGO not only restored the chaperone function but increased it by ~ 40% and ~ 60% beyond that of unmodified A-crystallin, as measured with citrate synthase and insulin aggregation assays, respectively. OMIU reduced the surface hydrophobicity but after MGO treatment, it was ~39% higher than control. FRET analysis revealed that A-crystallin subunit exchange rate was markedly retarded by OMIU modification, but was enhanced after MGO modification. These results indicate a pattern of loss and gain of chaperone function within the same protein that is associated with introduction of guanidino groups and their neutralization. These findings support our hypothesis that positively charged guanidino group on arginine residues keeps the chaperone function of A-crystallin in check and that a metabolic -dicarbonyl compound neutralizes this charge to restore and enhance chaperone function.