Discovery and barcoding by analysis of spliced leader RNA gene sequences of new isolates of Trypanosomatidae from Heteroptera in Costa Rica and Ecuador

Trypanosomatid diversity in Heteroptera was sampled using a culture-independent approach based on amplification and sequencing of Spliced Leader RNA gene repeats from environmental samples. By combining the data collected herein with that of previous work, the prevalence of parasites was found to be 22%-23%. Out of approximately 170 host species investigated nearly 60 were found to harbor trypanosomatids. The parasites found were grouped by cluster analysis into 48 typing units. Most of these were well separated from the known groups and, therefore, likely represent new trypanosomatid species. The sequences for each typing unit serve as barcodes to facilitate their recognition in the future. As the sampled host species represent a minor fraction of potential hosts, the entire trypanosomatid diversity is far greater than described thus far. Investigations of trypanosomatid diversity, host-specificity, and biogeography have become feasible using the approach described herein.     

Solution structure of selenoprotein W and NMR analysis of its interaction with 14-3-3 proteins

Selenium is a trace element with significant biomedical potential. It is essential in mammals due to its occurrence in several proteins in the form of selenocysteine (Sec). One of the most abundant mammalian Sec-containing proteins is selenoprotein W (SelW). This protein of unknown function has a broad expression pattern and contains a candidate CXXU (where U represents Sec) redox motif. Here, we report the solution structure of the Sec13-->Cys variant of mouse SelW determined through high resolution NMR spectroscopy. The protein has a thioredoxin-like fold with the CXXU motif located in an exposed loop similarly to the redox-active site in thioredoxin. Protein dynamics studies revealed the rigidity of the protein backbone and mobility of two external loops and suggested a role of these loops in interaction with SelW partners. Molecular modeling of structures of other members of the Rdx family based on the SelW structure identified new conserved features in these proteins, including an aromatic cluster and interacting loops. Our previous study suggested an interaction between SelW and 14-3-3 proteins. In the present work, with the aid of NMR spectroscopy, we demonstrated specificity of this interaction and identified mobile loops in SelW as interacting surfaces. This finding suggests that 14-3-3 are redox-regulated proteins.     

The structure of a full-length response regulator from Mycobacterium tuberculosis in a stabilized three-dimensional domain-swapped, activated state

The full-length, two-domain response regulator RegX3 from Mycobacterium tuberculosis is a dimer stabilized by three-dimensional domain swapping. Dimerization is known to occur in the OmpR/PhoB subfamily of response regulators upon activation but has previously only been structurally characterized for isolated receiver domains. The RegX3 dimer has a bipartite intermolecular interface, which buries 2357 A(2) per monomer. The two parts of the interface are between the two receiver domains (dimerization interface) and between a composite receiver domain and the effector domain of the second molecule (interdomain interface). The structure provides support for the importance of threonine and tyrosine residues in the signal transduction mechanism. These residues occur in an active-like conformation stabilized by lanthanum ions. In solution, RegX3 exists as both a monomer and a dimer in a concentration-dependent equilibrium. The dimer in solution differs from the active form observed in the crystal, resembling instead the model of the inactive full-length response regulator PhoB.     

Regulation of activity of transient receptor potential melastatin 8 (TRPM8) channel by its short isoforms

One important mechanism of the regulation of membrane ion channels involves their nonfunctional isoforms generated by alternative splicing. However, knowledge of such isoforms for the members of the transient receptor potential (TRP) superfamily of ion channels remains quite limited. This study focuses on the TRPM8, which functions as a cold receptor in sensory neurons but is also expressed in tissues not exposed to ambient temperatures, as well as in cancer tissues. We report the cloning from prostate cancer cells of new short splice variants of TRPM8, termed short TRPM8α and short TRPM8β. Our results show that both variants are in a closed configuration with the C-terminal tail of the full-length TRPM8 channel, resulting in stabilization of its closed state and thus reducing both its cold sensitivity and activity. Our findings therefore uncover a new mode of regulation of the TRPM8 channel by its splice variants.     

Respective roles of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMP) in cell surface expression of CRLR/RAMP heterodimeric receptors

Receptor activity modifying proteins RAMP1, RAMP2, and RAMP3 are responsible for defining affinity to ligands of the calcitonin receptor-like receptor (CRLR). It has also been proposed that receptor activity-modifying proteins (RAMP) are molecular chaperones required for CRLR transport to the cell surface. Here, we have studied the respective roles of CRLR and RAMP in transporting CRLR/RAMP heterodimers to the plasma membrane by using a highly specific binding assay that allows quantitative detection of cell surface-expressed CRLR or RAMP in the Xenopus oocytes expression system. We show that: (i) heterodimer assembly is not a prerequisite for efficient cell surface expression of CRLR, (ii) N-glycosylated RAMP2 and RAMP3 are expressed at the cell surface and their transport to the plasma membrane requires N-glycans, (iii) RAMP1 is not N-glycosylated and is transported to the plasma membrane only upon formation of heterodimers with CRLR, and (iv) introduction of N-glycosylation sites in the RAMP1 sequence (D58N/G60S, Y71N, and K103N/P105S) allows cell surface expression of these mutants at levels similar to that of wild-type RAMP1 co-expressed with CRLR. Our data argue against a chaperone function for RAMP and identify the role of N-glycosylation in targeting these molecules to the cell surface.     

Rad59-Facilitated Acquisition of Y′ Elements by Short Telomeres Delays the Onset of Senescence

Telomerase-negative yeasts survive via one of the two Rad52-dependent recombination pathways, which have distinct genetic requirements. Although the telomere pattern of type I and type II survivors is well characterized, the mechanistic details of short telomere rearrangement into highly evolved pattern observed in survivors are still missing. Here, we analyze immediate events taking place at the abruptly shortened VII-L and native telomeres. We show that short telomeres engage in pairing with internal Rap1-bound TG_1–3-like tracts present between subtelomeric X and Y′ elements, which is followed by BIR-mediated non-reciprocal translocation of Y′ element and terminal TG_1–3 repeats from the donor end onto the shortened telomere. We found that choice of the Y′ donor was not random, since both engineered telomere VII-L and native VI-R acquired Y′ elements from partially overlapping sets of specific chromosome ends. Although short telomere repair was associated with transient delay in cell divisions, Y′ translocation on native telomeres did not require Mec1-dependent checkpoint. Furthermore, the homeologous pairing between the terminal TG_1–3 repeats at VII-L and internal repeats on other chromosome ends was largely independent of Rad51, but instead it was facilitated by Rad59 that stimulates Rad52 strand annealing activity. Therefore, Y′ translocation events taking place during presenescence are genetically separable from Rad51-dependent Y′ amplification process that occurs later during type I survivor formation. We show that Rad59-facilitated Y′ translocations on X-only telomeres delay the onset of senescence while preparing ground for type I survivor formation.     

Probing dimerization and structural flexibility of mammalian lipoxygenases by small-angle X-ray scattering

Human lipoxygenases (LOXs) and their metabolites have a great impact on human homeostasis and are of interest for targeted drug design. This goal requires detailed knowledge of their structures and an understanding of structure-function relationship. At the moment, there are two complete crystal structures for mammalian LOX [rabbit 12/15LOX (r-12/15LOX) and human 5LOX (h-5LOX)] and a fragment of human 12LOX. The low-resolution structures in solution for various LOX isoforms have brought about controversial results. Here we explored the behavior of r-12/15LOX in aqueous solution under different conditions (salt and pH) by small-angle X-ray scattering (SAXS) and compared it with human platelet-type 12S-LOX (hp-12LOX) and h-5LOX. Thermodynamic calculations concerning the stability of molecular assemblies, thermal motion analysis [TLSMD (translation, libration, and screw rotation motion detection based on crystallographic temperature factor B_j)], and results of SAXS analyses brought about the following conclusions: (i) in contrast to its crystal structure, r-12/15LOX functions as a monomer that dominates in solution; (ii) it dimerizes at higher protein concentrations in the presence of salt and with increasing degree of motional freedom of the N-terminal PLAT domain, as suggested by the Y98,614 → R double mutant; (iii) in aqueous solutions, hp-12LOX is stable as a dimer, in contrast to h-5LOX and r-12/15LOX, which are monomeric; and (iv) all three mammalian isozymes show a high level of flexibility not only for the PLAT domain but also for other subdomains of the catalytic part in TLS (translation, libration, and screw rotation) analysis and hp-12LOX in SAXS.     

The active conformation of glutamate synthase and its binding to ferredoxin

Glutamate synthases (GltS) are crucial enzymes in ammonia assimilation in plants and bacteria, where they catalyze the formation of two molecules of L-glutamate from L-glutamine and 2-oxoglutarate. The plant-type ferredoxin-dependent GltS and the functionally homologous alpha subunit of the bacterial NADPH-dependent GltS are complex four-domain monomeric enzymes of 140-165 kDa belonging to the NH(2)-terminal nucleophile family of amidotransferases. The enzymes function through the channeling of ammonia from the N-terminal amidotransferase domain to the FMN-binding domain. Here, we report the X-ray structure of the Synechocystis ferredoxin-dependent GltS with the substrate 2-oxoglutarate and the covalent inhibitor 5-oxo-L-norleucine bound in their physically distinct active sites solved using a new crystal form. The covalent Cys1-5-oxo-L-norleucine adduct mimics the glutamyl-thioester intermediate formed during L-glutamine hydrolysis. Moreover, we determined a high resolution structure of the GltS:2-oxoglutarate complex. These structures represent the enzyme in the active conformation. By comparing these structures with that of GltS alpha subunit and of related enzymes we propose a mechanism for enzyme self-regulation and ammonia channeling between the active sites. X-ray small-angle scattering experiments were performed on solutions containing GltS and its physiological electron donor ferredoxin (Fd). Using the structure of GltS and the newly determined crystal structure of Synechocystis Fd, the scattering experiments clearly showed that GltS forms an equimolar (1:1) complex with Fd. A fundamental consequence of this result is that two Fd molecules bind consecutively to Fd-GltS to yield the reduced FMN cofactor during catalysis.