Reproductive activity in sheep is seasonal, being activated by short-day photoperiods and inhibited by long days. During the nonbreeding season, GnRH secretion is reduced by both steroid-independent and steroid-dependent (increased response to estradiol negative feedback) effects of photoperiod. Kisspeptin (also known as metastin) and gonadotropin-inhibitory hormone (GnIH, or RFRP) are two RFamide neuropeptides that appear critical in the regulation of the reproductive neuroendocrine axis. We hypothesized that expression of kisspeptin and/or RFRP underlies the seasonal change in GnRH secretion. We examined kisspeptin and RFRP (protein and mRNA) expression in the brains of ovariectomized (OVX) ewes treated with estradiol (OVX+E) during the nonbreeding and breeding seasons. In OVX+E ewes, greater expression of kisspeptin and Kiss1 mRNA in the arcuate nucleus and lesser expression of RFRP (protein) in the dorsomedial nucleus of the hypothalamus were concurrent with the breeding season. There was also a greater number of kisspeptin terminal contacts onto GnRH neurons and less RFRP-GnRH contacts during the breeding season (compared with the nonbreeding season) in OVX+E ewes. Comparison of OVX and OVX+E ewes in the breeding and nonbreeding season revealed a greater effect of steroid replacement on inhibition of kisspeptin protein and Kiss1 mRNA expression during the nonbreeding season. Overall, we propose that the two RFamide peptides, kisspeptin and RFRP, act in concert, with opposing effects, to regulate the activity of GnRH neurons across the seasons, leading to the annual change in fertility and the cyclical seasonal transition from nonbreeding to breeding season.

No abstract

Rhomboid peptidases are members of a family of regulated intramembrane peptidases that cleave the transmembrane segments of integral membrane proteins. Rhomboid peptidases have been shown to play a major role in developmental processes in Drosophila and in mitochondrial maintenance in yeast. Most recently, the function of rhomboid peptidases has been directly linked to apoptosis. We have solved the structure of the rhomboid peptidase from Haemophilus influenzae (hiGlpG) to 2.2-Å resolution. The phasing for the crystals of hiGlpG was provided mainly by molecular replacement, by using the coordinates of the Escherichia coli rhomboid (ecGlpG). The structural results on these rhomboid peptidases have allowed us to speculate on the catalytic mechanism of substrate cleavage in a membranous environment. We have identified the relative disposition of the nucleophilic serine to the general base/acid function of the conserved histidine. Modeling a tetrapeptide substrate in the context of the rhomboid structure reveals an oxyanion hole comprising the side chain of a second conserved histidine and the main-chain NH of the nucleophilic serine residue. In both hiGlpG and ecGlpG structures, a water molecule occupies this oxyanion hole.intramembrane peptidase ͉ membrane protein ͉ rhomboid protease ͉ x-ray crystallography

Protein phosphatase-1 (PP1) plays a key role in dephosphorylation in numerous biological processes such as glycogen metabolism, cell cycle regulation, smooth muscle contraction, and protein synthesis. Microorganisms produce a variety of inhibitors of PP1, which include the microcystin class of inhibitors and okadaic acid, the latter being the major cause of diarrhetic shellfish poisoning and a powerful tumor promoter. We have determined the crystal structure of the molecular complex of okadaic acid bound to PP1 to a resolution of 1.9 Å. This structure reveals that the acid binds in a hydrophobic groove adjacent to the active site of the protein and interacts with basic residues within the active site. Okadaic acid exhibits a cyclic structure, which is maintained via an intramolecular hydrogen bond. This is reminiscent of other macrocyclic protein phosphatase inhibitors. The inhibitor-bound enzyme shows very little conformational change when compared with two other PP1 structures, except in the inhibitor-sensitive ␤12-␤13 loop region. The selectivity of okadaic acid for protein phosphatases-1 and -2A but not PP-2B (calcineurin) may be reassessed in light of this study.The phosphorylation and dephosphorylation of proteins is vital to the regulation of many cellular pathways and processes. Two classes of enzymes in the cell that catalyze cellular dephosphorylation activity are tyrosine phosphatases and serine/threonine phosphatases (1). Classification of serine/threonine phosphatases can be subdivided into four categories: protein phosphatase-1 (PP1), 1 -2A (PP2A), -2B(PP2B) and -2C (PP2C) (2). The first three of these categories comprise what is known as the PPP family of protein phosphatases since they contain extensive sequence similarity in their catalytic domains and little or no sequence homology to PP2C or to tyrosine phosphatases. There are several natural toxin inhibitors of the PPP family of enzymes. These include microcystins, calyculins, tautomycin and okadaic acid (OA) ( Fig. 1) (1).OA is a tumor-promoting C 38 polyether fatty acid produced by marine dinoflagellates (1, 3-6). OA contains acidic and hydrophobic moieties and is cyclic (via an intramolecular hydrogen bond) (6). This toxin can accumulate in filter-feeding organisms and is the principle cause of diarrhetic shellfish poisoning worldwide (4).There have been many biochemical and modeling studies on the inhibition of the PPP family of phosphatases by the natural toxins, but the lone crystal structure is of microcystin-LR (MCLR) bound to PP1 (␣ isoform) (8). Here we describe the crystal structure of OA bound to the recombinant catalytic subunit of PP1 (␥ isoform). EXPERIMENTAL PROCEDURESCrystallization-The catalytic subunit of protein phosphatase-1 ␥ isoform was purified as described previously (9, 10). OA was purified from Prorocentrum lima (9, 10). Crystals were obtained by the hanging drop vapor diffusion method at room temperature. The enzyme and inhibitor were mixed in a 1:2 molar ratio with the concentration of protein being ϳ0.4 mM. The P...

ATP-citrate lyase (ACLY, EC 2.3.3.8) 3 catalyzes the reaction, citrate ϩ CoA ϩ ATP 3 acetyl-CoA ϩ oxaloacetate ϩ ADP ϩ P i , in the presence of magnesium ions (1). ACLY is the cytoplasmic enzyme linking energy metabolism from carbohydrates to the production of fatty acids. Acetyl-CoA produced in the mitochondria cannot be exported to the cytoplasm. Instead, acetylCoA in mitochondria is transformed to citrate by citrate synthase, and citrate is exported to the cytoplasm where ACLY regenerates acetyl-CoA. This acetyl-CoA is an important precursor for fatty acid synthesis. In addition, ACLY has been shown to signal the metabolic state of cells, likely by providing acetyl-CoA for histone acetyltransferases in the cells' nuclei (2). The physiological importance of the enzyme is supported by knock-out experiments in which mice embryos lacking ATPcitrate lyase could not be obtained (3).Our understanding of the reaction mechanism of ACLY has been based on studies of this enzyme and of two enzymes with sequence similarity to ACLY. The first is succinyl-CoA synthetase (SCS), which catalyzes the formation of succinyl-CoA from succinate and CoA using ATP (4). The second is citrate synthase, the enzyme that generates citrate from acetyl-CoA and oxaloacetate in mitochondria. The reaction catalyzed by ACLY is thought to occur in four steps (Reactions 1-4):where E represents ACLY. Like SCS, ACLY is phosphorylated by ATP on an active site histidine residue to give E-P in step 1 (Reaction 1) (5, 6). The phosphoryl group is transferred to citrate in step 2 (Reaction 2). Citryl-phosphate is thought to remain bound to the enzyme, symbolized by E⅐citryl-P. Phosphate is released in the attack by CoA to form the citryl-CoA thioester bond in step 3 (Reaction 3) (7). In the last step (Reaction 4), citryl-CoA is cleaved to give acetyl-CoA and oxaloacetate, the reverse reaction to that catalyzed by citrate synthase.