Dissecting the Conformational Dynamics of the Bile Acid Transporter Homologue ASBTNM

Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na⁺ as the driving force. The crystal structures of two bacterial homologues ASBT_NM and ASBT_Yf have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na⁺-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na⁺ and substrate in the conformational isomerization remains unclear. In this study, we utilized site-directed alkylation monitored by in-gel fluorescence (SDAF) to probe the solvent accessibility of the residues lining the substrate permeation pathway of ASBT_NM under different Na⁺ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBT_NM is a monomer protein, unlike the other elevator-type transporters, usually forming a homodimer or a homotrimer. The conformational dynamics observed by the biochemical experiments were further validated using DEER measuring the distance between the spin-labelled pairs. Our results revealed that Na⁺ ions shift the conformational equilibrium of ASBT_NM toward the inward-facing state thereby facilitating cytoplasmic uptake of substrate. The current findings provide a novel perspective on the conformational equilibrium of secondary active transporters.     

Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection

1. Download : Download high-res image (212KB)
2. Download : Download full-size image

     

Stem-like intestinal Th17 cells give rise to pathogenic effector T cells during autoimmunity

1. Download : Download high-res image (193KB)
2. Download : Download full-size image

     

A Toxoplasma type 2C serine-threonine phosphatase is involved in parasite growth in the mammalian host cell

Toxoplasma gondii is a human protozoan parasite that belongs to the phylum of Apicomplexa and causes toxoplasmosis. As the other members of this phylum, T. gondii obligatory multiplies within a host cell by a peculiar type of mitosis that leads to daughter cell assembly within a mother cell. Although parasite growth and virulence have been linked for years, few molecules controlling mitosis have been yet identified and they include a couple of kinases but not the counteracting phosphatases. Here, we report that in contrast to other animal cells, type 2C is by far the major type of serine threonine phosphatase activity both in extracellular and in intracellular dividing parasites. Using wild type and transgenic parasites, we characterized the 37 kDa TgPP2C molecule as an abundant cytoplasmic and nuclear enzyme with activity being under tight regulation. In addition, we showed that the increase in TgPP2C activity significantly affected parasite growth by impairing cytokinesis while nuclear division still occurred. This study supports for the first time that type 2C protein phosphatase is an important regulator of cell growth in T. gondii.     

Study on the enzymatic 5′-deiodination of 3′,5′-diiodothyronine using a radioimmunoassay for 3′-iodothyronine

A radioimmunoassay for 3′-iodothyronine has been developed. All iodothyronine analogues (except 3,3′-diiodothyronine) showed very little (0.02% at most) cross-reactivity, and the assay was sensitive to 1 pg 3′-iodothyronine/ tube. We have studied the 5′-deiodination of 3′,5′-diiodothyronine by rat liver microsomal fraction in the presence of dithiothreitol. Production of 3′-iodothyronine at 37°C was found to be linear with time of incubation up to 30 min and with concentration of microsomal protein up to 100 μg/ml. The reaction rate reached a limit on increasing 3′,5′-diiodothyronine concentration to 10 μM. The effect of pH on 3′-iodothyronine production was found to depend on 3′,5′-diiodothyronine concentration. Increasing 3′,5′-diiodothyronine concentration from 0.1 to 10 μM resulted in a shift of the pH optimum from 6–6.5 to 7.5. Similar effects on the 5′-deiodination of 3,3′,5′-triiodothyronine were observed, supporting the hypothesis that these reactions are catalysed by a single enzyme (iodothyronine 5′-deiodinase).     

Gradients in maize roots: local elongation and pH

The elongation rate, the gradient of the local elongation rate and the surface pH of maize roots were measured over 12 h. A data bank was constituted by storing these values. By sorting these results on the basis of different elongation rates, different classes of root were obtained. Two classes were chosen: the low-growth roots and the high-growth roots. The mean growth of these two root classes was stable with time and differed significantly from one another. The surface pH of the elongation zone was the same for the roots of these two classes, but the roots selected for their higher growth rate had a larger acid efflux in this zone.     

Identification of a GM1-Binding Protein on the Surface of Murine Neuroblastoma Cells

S20Y murine neuroblastoma cells appear to express a protein component(s) able to adhere specifically to the oligosaccharide portion of GM1 (oligo-GM1). To identify proteins with which the oligo-GM1 becomes closely associated, a radiolabeled (125I), photoactivatable derivative of oligo-GM1 was prepared. This was accomplished by reductive amination of the glucosyl moiety of oligo-GM1 to 1-deoxy-1-aminoglucitol, followed by reaction of the amine with sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD). Crosslinking studies using the photoactivatable probe indicated that it came in close proximity to a protein with an apparent molecular mass of approximately 71 kDa. In competition experiments, as little as a 10-fold molar excess of oligo-GM1 resulted in a selective reduction in labeling of this protein; preincubation with a 200-fold molar excess of siayllactose was necessary to observe the same change in the labeling pattern, lending additional support to the hypothesis that the approximately 71-kDa protein specifically associates with oligo-GM1. Cell surface location of the oligo-GM1 binding protein was confirmed using subcellular fractionation and morphological analyses.     

Regulatory T Cells Condition Lymphatic Endothelia for Enhanced Transendothelial Migration

1. Download : Download high-res image (154KB)
2. Download : Download full-size image

     

Reactivity and binding of benzo(a)pyrene diol epoxide to poly(dG-dC).(dG-dC) and poly(dG-m5dC).(dG-m5dC) in the B and Z forms

The physical and covalent binding of the carcinogen benzo(a)pyrene-7,8-diol-9,10-oxide (BaPDE) to poly(dG-dC).(dG-dC) and poly(dG-m5dC).(dG-m5dC) in the B and Z forms were studied utilizing absorbance, fluorescence and linear dichroism techniques. In the case of poly(dG-dC).(dG-dC) the decrease in the covalent binding of BaPDE with increasing NaCl concentration (0.1-4 M) as the B form is transformed to the Z form is attributed to the effects of high ionic strengths on the reactivity and physical binding of BaPDE to the polynucleotides; these effects tend to obscure differences in reactivities with the B and Z forms of the nucleic acids. In the case of poly(dG-m5dC).(dG-m5dC) the B-to-Z transition is induced at low ionic strength (2 mM NaCl + 10 microM Co(NH3)6Cl3) and the covalent binding is found to be 2-3-times lower to the Z form than to the B form. Physical binding of BaPDE by intercalation, which precedes the covalent binding reaction, is significantly lower in the Z form than in the B form, thus accounting, in part, for the lower covalent binding. The linear dichroism characteristics of BaPDE covalently bound to the Z and B forms of poly(dG-m5dC).(dG-m5dC) are consistent with nonintercalative, probably external conformations of the aromatic pyrenyl residues.     

Expression level of enzymes related to in situ estrogen synthesis and clinicopathological parameters in breast cancer patients

In order to evaluate the importance of estrogen production in tumor and surrounding tissues, we measured mRNA expression levels of 5 enzymes participating to estrogen synthesis in situ and 4 breast cancer-related proteins in 27 pairs of tumor and non-malignant tissues. Steroid sulfatase (STS) mRNA was more frequently detected in tumor tissues rather than in their non-malignant counterparts. Estrogen sulfotransferase (EST) was constantly expressed with high level not only in tumor tissues but also in their surrounding non-malignant counterparts. In contrast, mRNA expression levels of aromatase, and 17β-hydroxysteroid dehydrogenase type I and II were relatively low and detected only in small proportion of the patients. We also measured the mRNA expression levels of the same nine genes in tumor tissues of 197 breast cancer patients, and analyzed relationship between the mRNA expression level and the clinicopathological parameters. The mRNA expression levels of STS, aromatase and erbB2 in tumor tissues increased as breast cancer progressed. The tumoral mRNA expression levels of STS, estrogen receptor β, and erbB2 in patients with recurrence were higher than those in patients without recurrence. Upregulation of STS expression plays an important role in tumor progression of human breast cancer and is considered to be responsible for estrogen production in tumor and surrounding tissues.