Allosteric and electrostatic protein-protein interactions regulate the assembly of the heterohexameric Tim9-Tim10 complex

Protein-protein interactions are crucial processes in virtually all cellular events. The heterohexameric Tim9-Tim10 complex of the mitochondrial intermembrane space plays an important role during import of mitochondrial membrane proteins. It consists of three molecules of each subunit arranged alternately in a ring-shaped structure. While the individual protein Tim9 forms a homodimer, Tim10 is a monomer. Further to our previous investigation on the complex formation pathway, in this study, the assembly mechanism of Tim9-Tim10 was investigated using a stopped-flow technique coupled with mutagenesis. We show that while the initial velocity of the assembly depends on Tim9 concentration linearly, it presents a sigmoid curve on Tim10. In addition, the overall rate of assembly depends on the pH level in a bell-shaped profile, and two pK_a values that are in good agreement with the respective isoelectric points of Tim9 and Tim10 were determined. Using a Tim10F70W mutant, we were able to show that there was clear salt concentration dependence in the rate of assembly at the early stages. Taken together, the results of pH and salt concentration dependence indicate that electrostatic interactions are important and provide an initial driving force for the complex formation. Thus, this study not only demonstrates that allosteric and electrostatic interactions are two key regulators for the assembly of the Tim9-Tim10 complex but also has important implications for our understanding of how proteins interact with their partners.     

Estimating treatment coverage for people with substance use disorders: an analysis of data from the World Mental Health Surveys

Substance use is a major cause of disability globally. This has been recognized in the recent United Nations Sustainable Development Goals (SDGs), in which treatment coverage for substance use disorders is identified as one of the indicators. There have been no estimates of this treatment coverage cross‐nationally, making it difficult to know what is the baseline for that SDG target. Here we report data from the World Health Organization (WHO)'s World Mental Health Surveys (WMHS), based on representative community household surveys in 26 countries. We assessed the 12‐month prevalence of substance use disorders (alcohol or drug abuse/dependence); the proportion of people with these disorders who were aware that they needed treatment and who wished to receive care; the proportion of those seeking care who received it; and the proportion of such treatment that met minimal standards for treatment quality (“minimally adequate treatment”). Among the 70,880 participants, 2.6% met 12‐month criteria for substance use disorders; the prevalence was higher in upper‐middle income (3.3%) than in high‐income (2.6%) and low/lower‐middle income (2.0%) countries. Overall, 39.1% of those with 12‐month substance use disorders recognized a treatment need; this recognition was more common in high‐income (43.1%) than in upper‐middle (35.6%) and low/lower‐middle income (31.5%) countries. Among those who recognized treatment need, 61.3% made at least one visit to a service provider, and 29.5% of the latter received minimally adequate treatment exposure (35.3% in high, 20.3% in upper‐middle, and 8.6% in low/lower‐middle income countries). Overall, only 7.1% of those with past‐year substance use disorders received minimally adequate treatment: 10.3% in high income, 4.3% in upper‐middle income and 1.0% in low/lower‐middle income countries. These data suggest that only a small minority of people with substance use disorders receive even minimally adequate treatment. At least three barriers are involved: awareness/perceived treatment need, accessing treatment once a need is recognized, and compliance (on the part of both provider and client) to obtain adequate treatment. Various factors are likely to be involved in each of these three barriers, all of which need to be addressed to improve treatment coverage of substance use disorders. These data provide a baseline for the global monitoring of progress of treatment coverage for these disorders as an indicator within the SDGs.     

A key enzyme of animal steroidogenesis can function in plants enhancing their immunity and accelerating the processes of growth and development

Background

The initial stage of the biosynthesis of steroid hormones in animals occurs in the mitochondria of steroidogenic tissues, where cytochrome P450_SCC (CYP11A1) encoded by the CYP11A1 gene catalyzes the conversion of cholesterol into pregnenolone – the general precursor of all the steroid hormones, starting with progesterone. This stage is missing in plants where mitochondrial cytochromes P450 (the mito CYP clan) have not been found. Generating transgenic plants with a mitochondrial type P450 from animals would offer an interesting option to verify whether plant mitochondria could serve as another site of P450 monooxygenase reaction for the steroid hormones biosynthesis.

Results

For a more detailed comparison of steroidogenic systems of Plantae and Animalia, we have created and studied transgenic tobacco and tomato plants efficiently expressing mammalian CYP11A1 cDNA. The detailed phenotypic characterization of plants obtained has shown that through four generations studied, the transgenic tobacco plants have reduced a period of vegetative development (early flowering and maturation of bolls), enlarged biomass and increased productivity (quantity and quality of seeds) as compared to the only empty-vector containing or wild type plants. Moreover, the CYP11A1 transgenic plants show resistance to such fungal pathogen as Botrytis cinerea. Similar valuable phenotypes (the accelerated course of ontogenesis and/or stress resistance) are also visible in two clearly distinct transgenic tomato lines expressing CYP11A1 cDNA: one line (No. 4) has an accelerated rate of vegetative development, while the other (No. 7) has enhanced immunity to abiotic and biotic stresses. The progesterone level in transgenic tobacco and tomato leaves is 3–5 times higher than in the control plants of the wild type.

Conclusions

For the first time, we could show the compatibility in vivo of even the most specific components of the systems of biosynthesis of steroid hormones in Plantae and Animalia. The hypothesis is proposed and substantiated that the formation of the above-noted special phenotypes of transgenic plants expressing mammalian CYP11A1 cDNA is due to the increased biosynthesis of progesterone that can be considered as a very ancient bioregulator of plant cells and the first real hormone common to plants and animals.

     

Applications of genetically encoded photosensitizer miniSOG: from correlative light electron microscopy to immunophotosensitizing

Genetically encoded photosensitizers (PSs), e.g. ROS generating proteins, correspond to a novel class of PSs that are highly desirable for biological and medical applications since they can be used in combination with a variety of genetic engineering manipulations allowing for precise spatio‐temporal control of ROS production within living cells and organisms. In contrast to the commonly used chemical PSs, they can be modified using genetic engineering approaches and targeted to particular cellular compartments and cell types. Mini Singlet Oxygen Generator (miniSOG), a small flavoprotein capable of singlet oxygen production upon blue light irradiation, was initially reported as a high contrast probe for correlative light electron microscopy (CLEM) without the need of exogenous ligands, probes or destructive permeabilizing detergents. Further miniSOG was successfully applied for chromophore‐assisted light inactivation (CALI) of proteins, as well as for photo‐induced cell ablation in tissue cultures and in Caenorhabditis elegans. Finally, a novel approach of immunophotosensitizing has been developed, exploiting the specificity of mini‐antibodies or selective scaffold proteins and photo‐induced cytotoxicity of miniSOG, which is particularly promising for selective non‐invasive photodynamic therapy of cancer (PDT) due to the spatial selectivity and locality of destructive action compared to other methods of oncotherapy.

     

Key elements in maintenance of the tRNA L-shape

Based on in vivo selection of effective suppressor tRNAs from two different combinatorial gene libraries in which several nucleotides in the D and T-loops were randomized, we show that the position of the reverse-Hoogsteen base-pair in the T-loop, normally formed between nucleotides 54-58, co-varies with the length of the D-domain. When the D-domain has the normal length, the position of the reverse-Hoogsteen base-pair in the T-loop is always such that it allocates two unpaired nucleotides 59-60 for the bulge that fills the space between the D and T-domains. However, when the D-domain becomes shorter, the position of the reverse-Hoogsteen base-pair changes in the way that more nucleotides are now allocated to the T-loop bulge, so that the total length of the D-domain and of the bulge remains the same. Such compensation guarantees that in all tRNAs, the D and T-domains are always juxtaposed in the standard way. It also demonstrates the major role of the two T-loop elements, the bulge and the reverse-Hoogsteen base-pair, in the formation of the canonical tRNA L-shape.     

Retinoids and TIMP1 prevent radiation-induced apoptosis of capillary endothelial cells

Radiation-induced changes in capillaries constitute a basic injury in the pathogenesis of chronic radiation damage to the heart, lung, liver, kidney and brain. It is important to identify new radioprotectors for capillary endothelial cells for use during radiotherapy to minimize normal tissue damage and possibly to increase the deliverable dose. Previously we demonstrated that exposure to ionizing radiation (10 Gy) results in death of bovine adrenal capillary endothelial cells in confluent monolayers by apoptosis. We also showed that retinoids inhibit the growth of endothelial cells, induce their differentiation, down-regulate matrix metalloproteinase (MMP) production, and up-regulate tissue inhibitors of matrix metalloproteinases (TIMPs). In the present studies, we demonstrated that radiation (10 Gy) induced an immediate increase in the amounts and activation of MMP1 and MMP2 in the cell fraction and medium of bovine capillary endothelial cells followed by an incidence of apoptosis. We also obtained data indicating that radiation-induced apoptosis can be inhibited by exposing bovine capillary endothelial cells to all-trans-retinol or all-trans-retinoic acid for 6 days before irradiation, even when the vitamins were removed 24 h before irradiation. Finally, we determined that inhibition of MMPs by TIMP was sufficient to block radiation-induced apoptosis, suggesting that the mechanism of protection by retinoids is through the alteration of levels of MMPs and TIMPs produced by the cells.     

Plasmodium induces swelling-activated ClC-2 anion channels in the host erythrocyte

Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum depends on delivery of nutrients. Moreover, infection challenges cell volume constancy of the host erythrocyte requiring enhanced activity of cell volume regulatory mechanisms. Patch clamp recording demonstrated inwardly and outwardly rectifying anion channels in infected but not in control erythrocytes. The molecular identity of those channels remained elusive. We show here for one channel type that voltage dependence, cell volume sensitivity, and activation by oxidation are identical to ClC-2. Moreover, Western blots and FACS analysis showed protein and functional ClC-2 expression in human erythrocytes and erythrocytes from wild type (Clcn2(+/+)) but not from Clcn2(-/-) mice. Finally, patch clamp recording revealed activation of volume-sensitive inwardly rectifying channels in Plasmodium berghei-infected Clcn2(+/+) but not Clcn2(-/-) erythrocytes. Erythrocytes from infected mice of both genotypes differed in cell volume and inhibition of ClC-2 by ZnCl(2) (1 mm) induced an increase of cell volume only in parasitized Clcn2(+/+) erythrocytes. Lack of ClC-2 did not inhibit P. berghei development in vivo nor substantially affect the mortality of infected mice. In conclusion, activation of host ClC-2 channels participates in the altered permeability of Plasmodium-infected erythrocytes but is not required for intraerythrocytic parasite survival.     

The HD domain of the Escherichia coli tRNA nucleotidyltransferase has 2',3'-cyclic phosphodiesterase, 2'-nucleotidase, and phosphatase activities

In all mature tRNAs, the 3'-terminal CCA sequence is synthesized or repaired by a template-independent nucleotidyltransferase (ATP(CTP):tRNA nucleotidyltransferase; EC 2.7.7.25). The Escherichia coli enzyme comprises two domains: an N-terminal domain containing the nucleotidyltransferase activity and an uncharacterized C-terminal HD domain. The HD motif defines a superfamily of metal-dependent phosphohydrolases that includes a variety of uncharacterized proteins and domains associated with nucleotidyltransferases and helicases from bacteria, archaea, and eukaryotes. The C-terminal HD domain in E. coli tRNA nucleotidyltransferase demonstrated Ni(2+)-dependent phosphatase activity toward pyrophosphate, canonical 5'-nucleoside tri- and diphosphates, NADP, and 2'-AMP. Assays with phosphodiesterase substrates revealed surprising metal-independent phosphodiesterase activity toward 2',3'-cAMP, -cGMP, and -cCMP. Without metal or in the presence of Mg(2+), the tRNA nucleotidyltransferase hydrolyzed 2',3'-cyclic substrates with the formation of 2'-nucleotides, whereas in the presence of Ni(2+), the protein also produced some 3'-nucleotides. Mutations at the conserved His-255 and Asp-256 residues comprising the C-terminal HD domain of this protein inactivated both phosphodiesterase and phosphatase activities, indicating that these activities are associated with the HD domain. Low concentrations of the E. coli tRNA (10 nm) had a strong inhibiting effect on both phosphatase and phosphodiesterase activities. The competitive character of inhibition by tRNA suggests that it might be a natural substrate for these activities. This inhibition was completely abolished by the addition of Mg(2+), Mn(2+), or Ca(2+), but not Ni(2+). The data suggest that the phosphohydrolase activities of the HD domain of the E. coli tRNA nucleotidyltransferase are involved in the repair of the 3'-CCA end of tRNA.     

Halogenated diterpenoids from the red alga Laurencia nipponica

Chemical compositions of three collections of the red alga Laurencia nipponica from the western part of the Sea of Japan were studied. One of them contained a series of the previously known sesquiterpenoids. Another one gave C15 bromoallene ethers, predominantly. Finally, two new halogenated diterpenes, 15-bromoparguer-9(11)-ene-16-ol and 15-bromoparguer-7-ene-16-ol, were isolated from the third collection of the same species. Structures of these diterpenoids were established by 1D and 2D NMR (1H-1H COSY, DEPT, HMQC, HMBC and NOESY) along with molecular calculations for conformations having lowest energies and mass spectroscopy. Diversity and variability of halogenated secondary metabolites in L. nipponica were discussed.     

GFP-like proteins as ubiquitous metazoan superfamily: evolution of functional features and structural complexity

Homologs of the green fluorescent protein (GFP), including the recently described GFP-like domains of certain extracellular matrix proteins in Bilaterian organisms, are remarkably similar at the protein structure level, yet they often perform totally unrelated functions, thereby warranting recognition as a superfamily. Here we describe diverse GFP-like proteins from previously undersampled and completely new sources, including hydromedusae and planktonic Copepoda. In hydromedusae, yellow and nonfluorescent purple proteins were found in addition to greens. Notably, the new yellow protein seems to follow exactly the same structural solution to achieving the yellow color of fluorescence as YFP, an engineered yellow-emitting mutant variant of GFP. The addition of these new sequences made it possible to resolve deep-level phylogenetic relationships within the superfamily. Fluorescence (most likely green) must have already existed in the common ancestor of Cnidaria and Bilateria, and therefore GFP-like proteins may be responsible for fluorescence and/or coloration in virtually any animal. At least 15 color diversification events can be inferred following the maximum parsimony principle in Cnidaria. Origination of red fluorescence and nonfluorescent purple-blue colors on several independent occasions provides a remarkable example of convergent evolution of complex features at the molecular level.