SecB-Mediated Protein Export Need Not Occur via Kinetic Partitioning

In Escherichia coli, the cytosolic chaperone SecB is responsible for the selective entry of a subset of precursor proteins into the Sec pathway. In vitro, SecB binds to a variety of unfolded substrates without apparent sequence specificity, but not native proteins. Selectivity has therefore been suggested to occur by kinetic partitioning of substrates between protein folding and SecB association. Evidence for kinetic partitioning is based on earlier observations that SecB blocks the refolding of the precursor form of maltose-binding protein (preMBP)⁵ and slow-folding maltose-binding protein (MBP) mutants, but not faster-folding mature wild-type MBP. In order to quantitatively validate the kinetic partitioning model, we have independently measured each of the rate constants involved in the interaction of SecB with refolding preMBP (a physiological substrate of SecB) and mature MBP. The measured rate constants correctly predict substrate folding kinetics over a wide range of SecB, MBP, and preMBP concentrations. Analysis of the data reveals that, for many substrates, kinetic partitioning is unlikely to be responsible for SecB-mediated protein export. Instead, the ability of SecB-bound substrates to continue folding while bound to SecB and their ability to interact with other components of the secretory machinery such as SecA may be key opposing determinants that inhibit and promote protein export, respectively.     

Cloning,mechanistic and functional analysis of a fungal sterol C24-methyltransferase implicated in brassicasterol biosynthesis

The first committed step in the formation of 24-alkylsterols in the ascomycetous fungus Paracoccidiodes brasiliensis (Pb) has been shown to involve C24-methylation of lanosterol to eburicol (24(28)-methylene-24,25-dihydro-lanosterol) on the basis of metabolite co-occurrence. A similarity-based cloning strategy was employed to obtain the cDNA clone corresponding to the sterol C24-methyltransferase (SMT) implicated in the C24-methylation reaction. The resulting catalyst, prepared as a recombinant fusion protein (His/Trx/S), was expressed in Escherichia coli BL21(C43) and shown to possess a substrate specificity for lanosterol and to generate a single exocyclic methylene product. The full-length cDNA has an open reading frame of 1131 base pairs and encodes a protein of 377 residues with a calculated molecular mass of 42,502 Da. The enzymatic C24-methylation gave a K_mapp of 38 μM and k_catapp of 0.14 min⁻¹. Quite unexpectedly, “plant” cycloartenol was catalyzed in high yield to 24(28)-methylene cycloartanol consistent with conformational arguments that favor that both cycloartenol and lanosterol are bound pseudoplanar in the ternary complex. Incubation of [27-¹³C]- or [24-²H]cycloartenol with PbSMT and analysis of the enzyme-generated product by a combination of ¹H and ¹³CNMR and mass spectroscopy established the regiospecific conversion of the pro-Z methyl group of the Δ²⁴⁽²⁵⁾-substrate to the pro-R isopropyl methyl group of the product and the migration of H24 to C25 on the Re-face of the original substrate double bond undergoing C24-methylation. Inhibition kinetics and products formed from the substrate analogs 25-azalanosterol (K_i 14 nM) and 26,27-dehydrolanosterol (K_i 54 μM and k_inact of 0.24 min⁻¹) provide direct evidence for distinct reaction channeling capitalized by structural differences in the C24- and C26-sterol acceptors. 25-Azalanosterol was a potent inhibitor of cell growth (IC₅₀, 30 nM) promoting lanosterol accumulation and 24-alkyl sterol depletion. Phylogenetic analysis of PbSMT with related SMTs of diverse origin together with the results of the present study indicate that the enzyme may have a similar complement of active-site amino acid residues compared to related yeast SMTs affording monofunctional C₁-transfer behavior, yet there are sufficient differences in its overall amino acid composition and substrate-dependent partitioning pathways to group PbSMT into a fourth and new class of SMT.     

Purification, characterization and inhibition of sterol C24-methyltransferase from Candida albicans

Solubilized sterol C24-methyltransferase (24-SMT) was purified to homogeneity from a cell extract of the yeast Candida albicans (Ca) by anion exchange chromatography, gel permeation chromatography and fast performance liquid chromatography using a Mono Q column. The purified enzyme has an apparent molecular mass of 178 kDa on gel permeation chromatography and 43 kDa on SDS/PAGE, indicating that it is composed of four identical subunits. The substrate requirement of the native enzyme has an optimal specificity for zymosterol with associated kinetic constants of K_m 50 μM and k_cat of 0.01 s⁻¹. The product of the enzyme incubated with zymosterol was fecosterol. Inhibition of the catalyst was observed with substrate analogs designed as transition state analogs (25-azalanosterol, K_i = 54 nM and 24 (R,S),25-epiminolanosterol, K_i = 11 nM) or as mechanism-based inactivators (26,27-dehydrozymosterol, K_i 9 μM) and k_inact = 0.03 min⁻¹) of the C24-methylation reaction. Product analogs ergosterol and fecosterol, but neither cholesterol nor sitosterol, inhibited activity affording K_i values of 20 and 72 μM, respectively. Ammonium and thia analogs of the intermediates of the sterol C24-methyl reaction sequence were effective growth inhibitors exhibiting IC₅₀ values that ranged from 3 to 20 μM.     

Selective determination of mimosine and its dihydroxypyridinyl derivative in plant systems

Summary. Our observations on the growth stimulatory nature of mimosine, (β-(3-hydroxy-4-pyridon-1-yl)-L-alanine), the toxic non-protein plant amino acid, in some model experimental systems, warranted sensitive and selective routine estimations. For the determination of both mimosine and DHP, an indirect spectrophotometric method was developed based on their individual reaction with known excess of DZSAM and by estimating the remaining DZSAM with N-(1-naphthyl)ethylene-diamine (NEDA). The resultant decrease in the secondary coupled product was measured at 540 nm. On equimolar basis, DHP had 40% of the reactivity of mimosine while interference from other relevant compounds was 15–35%. The determination of mimosine and DHP in tissue samples under different physiological conditions was effected after paper chromatographic separation of mimosine and DHP with distinctly differing R_f, from other compounds. The indirect method is superior in terms of absolute selectivity, sensitivity and ease of applicability with linear decreases in absorbance, proportional to increasing concentrations of mimosine from 0.1 to 0.75 μM or DHP from 0.2 to 1.5 μM and with recoveries of 99.2 to 100.5%.     

Ligand-modulated parallel mechanical unfolding pathways of maltose-binding proteins

Protein folding and unfolding are complex phenomena, and it is accepted that multidomain proteins generally follow multiple pathways. Maltose-binding protein (MBP) is a large (a two-domain, 370-amino acid residue) bacterial periplasmic protein involved in maltose uptake. Despite the large size, it has been shown to exhibit an apparent two-state equilibrium unfolding in bulk experiments. Single-molecule studies can uncover rare events that are masked by averaging in bulk studies. Here, we use single-molecule force spectroscopy to study the mechanical unfolding pathways of MBP and its precursor protein (preMBP) in the presence and absence of ligands. Our results show that MBP exhibits kinetic partitioning on mechanical stretching and unfolds via two parallel pathways: one of them involves a mechanically stable intermediate (path I) whereas the other is devoid of it (path II). The apoMBP unfolds via path I in 62% of the mechanical unfolding events, and the remaining 38% follow path II. In the case of maltose-bound MBP, the protein unfolds via the intermediate in 79% of the cases, the remaining 21% via path II. Similarly, on binding to maltotriose, a ligand whose binding strength with the polyprotein is similar to that of maltose, the occurrence of the intermediate is comparable (82% via path I) with that of maltose. The precursor protein preMBP also shows a similar behavior upon mechanical unfolding. The percentages of molecules unfolding via path I are 53% in the apo form and 68% and 72% upon binding to maltose and maltotriose, respectively, for preMBP. These observations demonstrate that ligand binding can modulate the mechanical unfolding pathways of proteins by a kinetic partitioning mechanism. This could be a general mechanism in the unfolding of other large two-domain ligand-binding proteins of the bacterial periplasmic space.     

Novel sterol metabolic network of Trypanosoma brucei procyclic and bloodstream forms

Trypanosoma brucei is the protozoan parasite that causes African trypanosomiasis, a neglected disease of people and animals. Co-metabolite analysis, labelling studies using [methyl-2H3]-methionine and substrate/product specificities of the cloned 24-SMT (sterol C24-methyltransferase) and 14-SDM (sterol C14demethylase) from T. brucei afforded an uncommon sterol metabolic network that proceeds from lanosterol and 31-norlanosterol to ETO [ergosta-5,7,25(27)-trien-3β-ol], 24-DTO [dimethyl ergosta-5,7,25(27)-trienol] and ergosterol [ergosta-5,7,22(23)-trienol]. To assess the possible carbon sources of ergosterol biosynthesis, specifically 13C-labelled specimens of lanosterol, acetate, leucine and glucose were administered to T. brucei and the 13C distributions found were in accord with the operation of the acetate-mevalonate pathway, with leucine as an alternative precursor, to ergostenols in either the insect or bloodstream form. In searching for metabolic signatures of procyclic cells, we observed that the 13C-labelling treatments induce fluctuations between the acetyl-CoA (mitochondrial) and sterol (cytosolic) synthetic pathways detected by the progressive increase in 13C-ergosterol production (control<[2-(13)C]leucine<[2-(13)C]acetate<[1-(13)C]glucose) and corresponding depletion of cholesta-5,7,24-trienol. We conclude that anabolic fluxes originating in mitochondrial metabolism constitute a flexible part of sterol synthesis that is further fluctuated in the cytosol, yielding distinct sterol profiles in relation to cell demands on growth.     

Molecular probing of the Saccharomyces cerevisiae sterol 24-C methyltransferase reveals multiple amino acid residues involved with C2-transfer activity

Two families of sterol C24-methyltransferase (SMT) are responsible for the formation of the ergostane (C(1)-transfer activity; SMT1) and stigmastane (C(2)-transfer activity: SMT2) sterol side chains, respectively. The fungal Saccharomyces cerevisiae SMT1 (Erg6p) operates the first C(1)-transfer in concerted fashion to form a single product whereas the protozoan and plant SMTs are bifunctional capable of catalyzing two sequential, mechanistically distinct C-methylation activities in the conversion of a Delta(24)-sterol acceptor to diverse doubly alkylated products. Previous mutation of the amino acids of Erg6p at D79, Y81 and E82 afforded C(1) or C(2)-transfer activities typical of the protozoan and plant SMT. In this study, scanning mutagenesis experiments involving a leucine replacement of 52 amino acids in Erg6p followed by substitution of key residues with functionally or structurally similar amino acids indicated that 5 new residues at positions Y192, G217, G218, T219 and Y223 can switch the course of C(1)-transfer activity to include plant-like C(2)-transfer activity. The data support a model in which several conserved and non-conserved amino acids located in distinct regions of the Erg6p regulate the course of the C-methylation reaction toward product differences.     

Mimosine Mitigates Oxidative Stress in Selenium Deficient Seedlings of <Emphasis Type="Italic">Vigna radiata</Emphasis>-Part I: Restoration of Mitochondrial Function

Mimosine, a non-protein plant amino acid found in Mimosa pudica and certain species of Leucaena, was beneficial for the growth of seedlings of Vigna radiata germinated under selenium-deficient stressed condition (−Se stressed) despite the recognized toxicity of the allelochemical. Exposure of mimosine at 0.1 mM (Mim-0.1) promoted the growth of the seedlings and significantly enhanced mitochondrial functional efficiency. Growth-related parameters including root and shoot lengths and dry weight were increased by 44–58% in the Mim-0.1 group compared to that of the −Se-stressed group. Oxygen uptake by mitochondria of Mim-0.1 group, studied with different substrates, revealed enhanced State 3 respiratory rates with regulated State 4 rates, resulting in high respiratory control ratio (RCR) of 3.4 to 3.9 indicative of a high degree of oxidative coupling. Specific activities of mitochondrial electron transport enzymes, nicotinamide adenine dinucleotide (reduced form) (NADH)–cytochrome (cyt) c oxidoreductase, succinate dehydrogenase, and cyt c oxidase in the Mim-0.1 group were enhanced by 53% to threefold over those of the Se-stressed group. Marked decreases in the extent of mitochondrial lipid peroxidation ensued upon mimosine exposure, indicative of its antioxidant function. Mitochondrial ⁴⁵Ca²⁺ uptake was notably augmented twofold in the Mim-0.1 group, compared to the Se-stressed group. Detailed kinetic analyses of Ca²⁺ uptake revealed positive cooperative interactions in both −Se-stressed group and Mim-0.1 groups with Hill coefficient (nH) values of 1.7 and 2, respectively. The present study establishes the beneficial effects of mimosine exposure at 0.1 mM on the growth and mitochondrial function of the seedlings grown under selenium-deficient stressed condition and a significant physiological role can be ascribed to mimosine.     

Mimosine Mitigates Oxidative Stress in Selenium Deficient Seedlings of Vigna radiata. Part II: Mitochondrial Uptake of 75Selenium and Mimosine

During the growth of selenium (Se)-deficient seedlings of Vigna radiata, exposure to mimosine [2-amino-3-(3-hydroxy-4-oxo-1H-pyridin-1-yl)-propanoic acid], a nonprotein plant amino acid, effectively mitigated stress at 0.1 mM, as reflected in enhancement of growth and efficiency of mitochondrial functions. Since the changes in the seedlings elicited by exposure to mimosine were similar to those effected by Se at an optimal exposure level of 0.75 ppm (Sreekala et al., Biol Trace Elem Res 70:193–207, 1999), the uptake of Se and that of mimosine itself was individually studied in the respiring mitochondria of Se-deficient seedlings (−Se-stressed group) in comparison with those exposed to mimosine during growth at 0.1 mM (Mim 0.1 group). In both groups, the mitochondrial uptake of ⁷⁵Se at 10 μM added increased linearly up to 2 min, attaining steady-state levels thereafter. Uptake levels were 2.3-fold higher in the Mim 0.1 group than in the −Se-stressed group. Double-reciprocal plots of mitochondrial ⁷⁵Se uptake against 2–20 μM in the medium were nonlinear and negative cooperative effects during the uptake were confirmed by Scatchard plots, whereas Hill coefficients were 0.8 and 0.85 for the two groups. Mitochondrial uptake of mimosine, at added levels of 25 or 50 μM, increased linearly up to 1 min and decelerated thereafter. Initial uptake levels of mimosine at 1 min were higher by 6.5-fold at 25 μM and 4-fold at 50 μM in the Mim 0.1 group than those in the −Se-stressed group. Initial uptake levels with added mimosine up to 50 or 100 μM yielded nonlinear double-reciprocal plots; and kinetic analyses at 5 to 50 μM revealed the prevalence of positive cooperativity in the −Se-stressed group and negative cooperativity in the Mim 0.1 group. Involvement of active thiol groups in the uptake of both Se and mimosine were indicated by inhibition studies. Evidence presented for mimosine mediated increase in mitochondrial Se uptake and cooperative interactions thereof underscores the metabolic significance of mimosine.     

Impairment of Colour Vision in Diabetes with No Retinopathy: Sankara Nethralaya Diabetic Retinopathy Epidemiology and Molecular Genetics Study (SNDREAMS- II,Report 3)

Purpose

To assess impairment of colour vision in type 2 diabetics with no diabetic retinopathy and elucidate associated risk factors in a population-based cross-sectional study.

Methods

This is part of Sankara Nethralaya Diabetic Retinopathy Epidemiology and Molecular-genetics Study (SN-DREAMS II) which was conducted between 2007–2010. FM 100 hue-test was performed in 253 subjects with no clinical evidence of diabetic retinopathy. All subjects underwent detailed ophthalmic evaluation including cataract grading using LOCS III and 45° 4-field stereoscopic fundus photography. Various ocular and systemic risk factors for impairment of colour vision (ICV) were assessed in subjects with diabetes but no retinopathy. P value of < 0.05 was considered statistically significant.

Results

The mean age of the study sample was 57.08 ± 9.21 (range: 44–86 years). Gender adjusted prevalence of ICV among subjects with diabetes with no retinopathy was 39.5% (CI: 33.5–45.5). The mean total error score in the study sample was 197.77 ± 100 (range: 19–583). The risk factors for ICV in the study were women OR: 1.79 (1.00–3.18), increased resting heart rate OR: 1.04 (1.01–1.07) and increased intraocular pressure OR: 1.12 (1.00–1.24). Significant protective factor was serum high-density lipoprotein OR: 0.96 (0.93–0.99).

Conclusions

Acquired ICV is an early indicator of neurodegenerative changes in the retina. ICV found in diabetic subjects without retinopathy may be of non-vascular etiology.