Evidence of Alternative Splicing as a Regulatory Mechanism for Kissr2 in Pejerrey Fish

Kisspeptin receptors are G-Protein-Coupled Receptors that regulate GnRH synthesis and release in vertebrates. Here, we report the gene structure of two kisspeptin receptors (kissr2 and kissr3) in pejerrey fish. Genomic analysis exposed a gene structure with 5 exons and 4 introns for kissr2 and 6 exons and 5 introns for kissr3. Two alternative variants for both genes, named kissr2_v1 and _v2, and kissr3_v1 and v2, were revealed by gene expression analyses of several tissues. For both receptors, these variants were originated by alternative splicing retaining intron 3 and intron 4 for kissr2_v2 and kissr3_v2, respectively. In the case of kissr2, the intron retention introduced two stop codons leading to a putatively truncated protein whereas for kissr3, the intron retention produced a reading shift leading to a stop codon in exon 5. Modeling and structural analysis of Kissr2 and Kissr3 spliced variants revealed that truncation of the proteins may lead to non-functional proteins, as the structural elements missing are critical for receptor function. To understand the functional significance of splicing variants, the expression pattern for kissr2 was characterized on fish subjected to different diets. Fasting induced an up-regulation of kissr2_v1 in the hypothalamus, a brain region implicated in control of reproduction and food intake, with no expression of kissr2_v2. On the other hand, fasting did not elicit differential expression in testes and habenula. These results suggest that alternative splicing may play a role in regulating Kissr2 function in pejerrey

HAMP structure and function.

<p>(A) Side and top views of the dimeric HAMP four-helix bundle as observed in the trigonal crystal form. Core hydrophobic residues are shown in stick representation. (B) Crick angle deviation plot of the HAMP helices, as calculated with program samCC <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001776#pbio.1001776-DuninHorkawicz1" target="_blank">[52]</a>. (C) The ability of wild-type and mutant <i>cpxA</i> bacteria to respond to the periplasmic overproduction of either the wild-type MalE (open bars) or the folding-defective mutant MalE31 (light gray bars) and to the presence of 0.2% phenethyl alcohol (dark gray bars) was monitored by measuring β-galactosidase activity from a <i>cpxP</i>–<i>lacZ</i> fusion contained in the NS54 strain expressing the different <i>cpxA</i> alleles. The insert shows the cellular levels of CpxA analyzed by immunobloting membrane protein fractions prepared from the NS54 strain transformed by pLCB (lane 1), pLCBA_wt (lane 2), pLCBA₁₉₇ (lane 3), and pLCBA₂₂₈ (lane 4). The additional band observed in the immunoblot is a cross-reacting protein recognized by the antiserum that serves as a loading control. (D) Phosphotransferase activity of CpxA. Both full-length CpxA and CpxA_M228V proteins (10 µM) were first allowed to autophosphorylate for 20 min at 25°C in the presence of 1 mM ATP, and then an equimolar amount of CpxR_N (N-terminal receiver domain) was added to the reactions. Samples were removed at the indicated time points, and phospho-proteins were separated by Phos-tag acrylamide gel electrophoresis. (E) Autokinase activity of full-length CpxA and CpxA_M228V proteins as determined using radioactive ATP. It is worth noting that only a small fraction of the CpxA–Brij35 complex (∼0.1%) is phosphorylated at steady state, as estimated by PhosTag gels run under the same conditions used in the radioactive assays (unpublished data).</p

Identification of intragenic suppressors.

<p>A plasmid encoding CpxA_ΔP, a constitutive kinase CpxA variant lacking the sensor domain, was mutagenized and transformed into a <i>cpxA</i> null P_cpxP–<i>lacZ recA</i> strain background. The table shows the CpxA activity conferred by single mutant <i>cpxA</i> alleles in the NS54 strain. Point mutations were identified from colonies displaying a Lac⁻ phenotype in X-gal containing plates.</p

Mechanical model for HK autophosphorylation control.

<p>The inactive kinase conformation (left) involves a putative symmetric conformation of CpxA [modeled from the structures of HK853 (PDB ID 2C2A) and wild-type Af1503 HAMP-EnvZ DHp chimera (PDB ID 3ZWR)], in which the two CA domains are sequestered in a nonproductive DHp–CA complex. The active kinase state (right, as observed in the trigonal crystal form) displays a highly asymmetric conformation of the HK homodimer. Propagated by conformational changes in the HAMP domain, the input signal induces a stress on the central DHp helices, promoting segmental helical motions that result in a strong dynamical asymmetry: one of the CA domains is highly mobile and can form a competent active site, whereas the second CA domain is retained in an inactive conformation by extended hydrophobic interactions with the DHp domain. The insert on the right shows these segmental helical movements (without the CA domains for clarity), in which each color represents a distinct rigid-body rotational movement. As a consequence of these movements, a gap broadens between two helices (indicated by a black arrow) and allows the partial exposure of core hydrophobic residues that contribute to sequester the second CA domain in an inactive conformation.</p

Conformational and dynamical asymmetry of the homodimer.

<p>(A) Superposition of all crystallographically independent CpxA_HDC dimers present in the five different crystal forms reported in this study. The main conformational difference between the distinct CpxA_HDC dimers consists in the position of one CA domain (left in the figure) relative to an invariant region containing both DHp domains and the second CA domain. (B) Phos-tag gel retardation autophosphorylation assay. The assay was performed with 10 µM CpxA_HDC and 1 mM ATP in 20 mM Hepes buffer (pH 7.6), 100 mM NaCl, 50 mM KCl, and 5 mM MgCl₂ at 25°C. At the indicated time points, 15 µl aliquots were removed and mixed with SDS loading buffer. Phospho-proteins were separated by Phos-tag acrylamide gel electrophoresis. (C) Total amount of CpxA_HDC-P and CpxA_HDC in each band was determined by densitometry analysis. The continuous lines were the best fits of the data to a single exponential term.</p

Overall structure of CpxA.

<p>(A) Linear representation of the prototypical CpxA domain organization. CpxA is an integral membrane receptor with a periplasmic sensor region (residues 29 to 163) flanked by two transmembrane helices (TM1 and TM2). TM2 connects the sensor domain to the cytosolic transmitter core (residues 188 to 457) formed by three domains: HAMP, DHp, and CA, rainbow colored from N-terminus–C-terminus (blue-red). (B) Cartoon representation of the CpxA_HDC homodimer in the trigonal crystal form. The homodimer is highly asymmetric due to helical kinks nearby Ser238 and Pro253 in helix α₂ (shown in stick representation) and large differences in the positioning and orientation of the two CA domains (shown in surface representation) with respect to the central DHp helical core.</p

Autophosphorylating Michaelis complex.

<p>(A) Close view of the active site showing the residues directly involved in catalysis, as revealed by the hexagonal crystal structure of the CpxA_{HDC_M228V}–AMPPNP complex (a very similar active site architecture was observed for the trigonal and hexagonal crystal structures of wild-type CpxA_HDC in complex with ATP, obtained at lower resolution). The regions corresponding to the conserved N and H boxes are highlighted in dark green and yellow, respectively. (B) Activation of His248 for phosphoryl transfer. Hydrogen bonding interactions between the imidazole ring of His248, the adjacent acidic residue in the CA domain (Glu249) acting as a general base, and a polar residue from the DHp domain (Asn356) contribute to activate His248 for nucleophilic attack to γ-P of ATP. (C) Schematic representation of the CpxA homodimer illustrating the <i>trans</i>-autophosphorylation reaction. (D) Anchoring of Phe403 and Leu419 to the DHp four-helix bundle represented by its electrostatic surface. The DHp-sequestered (yellow) and mobile (green) CA domains are shown in cartoon representation.</p

Crystallographic data collection and refinement statistics.

<p>Values for the highest resolution shell are shown in parentheses.</p

Potent and specific inhibition of glycosidases by small artificial binding proteins (affitins).

Glycosidases are associated with various human diseases. The development of efficient and specific inhibitors may provide powerful tools to modulate their activity. However, achieving high selectivity is a major challenge given that glycosidases with different functions can have similar enzymatic mechanisms and active-site architectures. As an alternative approach to small-chemical compounds, proteinaceous inhibitors might provide a better specificity by involving a larger surface area of interaction. We report here the design and characterization of proteinaceous inhibitors that specifically target endoglycosidases representative of the two major mechanistic classes; retaining and inverting glycosidases. These inhibitors consist of artificial affinity proteins, Affitins, selected against the thermophilic CelD from Clostridium thermocellum and lysozyme from hen egg. They were obtained from libraries of Sac7d variants, which involve either the randomization of a surface or the randomization of a surface and an artificially-extended loop. Glycosidase binders exhibited affinities in the nanomolar range with no cross-recognition, with efficient inhibition of lysozyme (Ki = 45 nM) and CelD (Ki = 95 and 111 nM), high expression yields in Escherichia coli, solubility, and thermal stabilities up to 81.1°C. The crystal structures of glycosidase-Affitin complexes validate our library designs. We observed that Affitins prevented substrate access by two modes of binding; covering or penetrating the catalytic site via the extended loop. In addition, Affitins formed salt-bridges with residues essential for enzymatic activity. These results lead us to propose the use of Affitins as versatile selective glycosidase inhibitors and, potentially, as enzymatic inhibitors in general