Spliceosomal intron insertions in genome compacted ray-finned fishes as evident from phylogeny of MC receptors, also supported by a few other GPCRs

Background

Insertions of spliceosomal introns are very rare events during evolution of vertebrates and the mechanisms governing creation of novel intron(s) remain obscure. Largely, gene structures of melanocortin (MC) receptors are characterized by intron-less architecture. However, recently a few exceptions have been reported in some fishes. This warrants a systematic survey of MC receptors for understanding intron insertion events during vertebrate evolution.

Methodology/Principal Findings

We have compiled an extended list of MC receptors from different vertebrate genomes with variations in fishes. Notably, the closely linked MC2Rs and MC5Rs from a group of ray-finned fishes have three and one intron insertion(s), respectively, with conserved positions and intron phase. In both genes, one novel insertion was in the highly conserved DRY motif at the end of helix TM3. Further, the proto-splice site MAG↑R is maintained at intron insertion sites in these two genes. However, the orthologs of these receptors from zebrafish and tetrapods are intron-less, suggesting these introns are simultaneously created in selected fishes. Surprisingly, these novel introns are traceable only in four fish genomes. We found that these fish genomes are severely compacted after the separation from zebrafish. Furthermore, we also report novel intron insertions in P2Y receptors and in CHRM3. Finally, we report ultrasmall introns in MC2R genes from selected fishes.

Conclusions/Significance

The current repository of MC receptors illustrates that fishes have no MC3R ortholog. MC2R, MC5R, P2Y receptors and CHRM3 have novel intron insertions only in ray-finned fishes that underwent genome compaction. These receptors share one intron at an identical position suggestive of being inserted contemporaneously. In addition to repetitive elements, genome compaction is now believed to be a new hallmark that promotes intron insertions, as it requires rapid DNA breakage and subsequent repair processes to gain back normal functionality.     

A correlation between membrane cholesterol level, cell adhesion and tumourigenicity of polyoma virus transformed cells

We have investigated the implications of the rise in membrane cholesterol levels on several in vitro and in vivo properties of polyoma virus transformed rat fibroblasts (PyF), with a special emphasis on alpha5beta1 integrin functions. We show that increased membrane cholesterol causes the PyF cells to change their shape and become more bipolar in appearance. These cells also show significantly higher adhesion to the cell-binding domain of fibronectin, increased localization of alpha5beta1 integrin and talin molecules in focal adhesions and a more robust actin cytoskeleton organization. PyF cells with increased membrane cholesterol show reduced growth in vitro and tumours caused by these cells in nude mice are slow growing. These changes in the growth properties of PyF cells are reversible when the cholesterol levels of PyF cells become normal. Our results suggest that changes in membrane cholesterol levels influence the growth and morphological properties of transformed cells, which can be exploited in controlling the growth of tumours in vivo.     

The fatty acid chain elongase,Elovl1, is required for kidney and swim bladder development during zebrafish embryogenesis

Very long chain fatty acids are required for sphingolipid synthesis, lipid homeostasis, myelin formation, epidermal permeability, and retinal function. Seven different enzymes are known to be involved in the elongation cycle of fatty acids, with different chain-length specificities. Elovl1 is one of those enzymes whose function has been linked mainly to the synthesis of sphingolipids and the epidermal barrier. However, the role of Elovl1 in organogenesis is not clear. In zebrafish, 2 Elovl1 genes, elovl1a and elovl1b, are highly expressed in the swim bladder, and elovl1b is also expressed in the kidney. We found that both elovl1 knockdown embryos contain increased levels of long chain fatty acids from carbon number 14 to 20 as compared to control embryos. Oil-Red-O staining shows that yolk lipid consumption is greatly reduced, whereas lipid droplets accumulate within the swim bladder. Notably, knockdown of either elovl1a or elovl1b affects the expression of genes involved in swim bladder development and impairs inflation of the swim bladder. Consistent with its expression in the pronephros, knockdown of elovl1b alone affects the expression of genes required for kidney development and reduces renal clearance. Our findings strongly suggest that both elovl1 genes are a key determinant of swim bladder and kidney development in zebrafish, which may be comparatively applicable to lung and kidney development in humans.     

Cell surface tubulin in leukemic cells: molecular structure, surface binding, turnover, cell cycle expression, and origin

We report here new characteristics of cell surface tubulin from a human leukemia cell line. These cells (CEM cells) possess tubulin that is readily iodinated on the surface of living cells, turns over at a rate identical to that of other surface proteins, and is present throughout the cell cycle. When removed with trypsin, it rapidly returns to the surface. Peptide mapping of iodinated surface tubulin indicates that it possesses a similar, but not identical, primary structure to total CEM and rat brain tubulin. Living CEM cells are able to bind specifically a subfraction of CEM tubulin from metabolically labeled high speed supernatants of lysed CEM cells. Surface tubulin is more basic than the total tubulin pool. The binding, which is saturable, is inhibited by unlabeled CEM high speed supernatants but not by excess thrice-cycled rat or bovine brain tubulin. Surface tubulin is also shown to bind to living nontransformed normal rat kidney cells but not to normal, circulating, mononuclear white cells. Activated lymphocytes produce a tubulin that binds to CEM cells. Since CEM tubulin was detected in the media of 6-h cultures of CEM cells, we must conclude that at least some of the surface tubulin comes from the media. We further conclude that these leukemic cells produce an unusual tubulin that may bind specifically to any membrane. The presence of iodinatable surface tubulin, however, appears to require both the production of a unique tubulin and the presence of a "receptor-like" surface binding component.     

Presenilin 1 is required for maturation and cell surface accumulation of nicastrin

Proteolytic processing of amyloid precursor protein generates beta-amyloid (Abeta) peptides that are deposited in senile plaques in brains of aged individuals and patients with Alzheimer's disease. Presenilins (PS1 and PS2) facilitate the final step in Abeta production, the intramembranous gamma-secretase cleavage of amyloid precursor protein. Biochemical and pharmacological evidence support a catalytic or accessory role for PS1 in gamma-secretase cleavage, as well as a regulatory role in select membrane protein trafficking. In this report, we demonstrate that PS1 is required for maturation and cell surface accumulation of nicastrin, an integral component of the multimeric gamma-secretase complex. Using kinetic labeling studies we show that in PS1(-/-)/PS2(-/-) cells nicastrin fails to reach the medial Golgi compartment, and as a consequence, is incompletely glycosylated. Stable expression of human PS1 restores these deficiencies in PS1(-/-) fibroblasts. Moreover, membrane fractionation studies show co-localization of PS1 fragments with mature nicastrin. These results indicate a novel chaperone-type role for PS1 and PS2 in facilitating nicastrin maturation and transport in the early biosynthetic compartments. Our findings are consistent with PS1 influencing gamma-secretase processing at multiple steps, including maturation and intracellular trafficking of substrates and component(s) of the gamma-secretase complex.     

Regulation of cyclic adenosine 3',5'- monophosphate signaling and pulsatile neurosecretion by Gi-coupled plasma membrane estrogen receptors in immortalized gonadotrophin-releasing hormone neurons

Immortalized GnRH neurons (GT1-7) express receptors for estrogen [estrogen receptor-alpha and-13(ERa and ERI3)] and progesterone (progesterone receptor A) and exhibit positive immunostaining for both intracellular and plasma membrane ERs. Exposure of GT1-7 cells to picomolar estradiol concentrations for 5-60 min caused rapid, sustained,and dose-dependent inhibition of cAMP production. In contrast, treatment with nanomolar estradiol concentrations for 60 min increased cAMP production. The inhibitory and stimulatory actions of estradiol on cAMP formation were abolished by the ER antagonist, ICI 182,780. The estradiol-induced inhibition of cAMP production was prevented by treatment with pertussis toxin, consistent with coupling of the plasma membrane ER to an inhibitory G protein. Coimmunoprecipitation studies demonstrated an estradiol-regulated stimulatory interaction between ERa and G,3 that was prevented by the ER antagonist, ICI 182,780. Exposure of perifused GT1-7 cells and hypothalamic neurons to picomolar estradiol levels increased the GnRH peak interval, shortened peak duration, and increased peak amplitude. These findings indicate that occupancy of the plasma membrane-associated ERs expressed in GT1-7 neurons by physio-logical estradiol levels causes activation of a G, protein and modulates cAMP signaling and neuropeptide secretion.     

Differential vasoconstrictions induced by angiotensin II: role of AT1 and AT2 receptors in isolated C57BL/6J mouse blood vessels

Genetically altered mice are increasingly used as experimental models. However, ANG II responses in mouse blood vessels have not been well defined. Therefore, the aim of this study was to determine the role of ANG II in regulating major blood vessels in C57/BL6J mice with isometric force measurements. Our results showed that in mouse abdominal aorta ANG II induced a concentration-dependent contraction (EC50 4.6 nM) with a maximum contraction of 75.1 +/- 4.9% at 100 nM compared with that of 60 mM K+. Similarly, femoral artery also exhibited a contractile response of 76.0 +/- 3.4% to the maximum concentration of ANG II (100 nM). In contrast, ANG II (100 nM)-induced contraction was significantly less in carotid artery (24.5 +/- 6.6%) and only minimal (3.5 +/- 0.31%) in thoracic aorta. The nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester and the AT2 antagonist PD-123319 failed to enhance ANG II-induced contractions. However, an AT1 antagonist, losartan (10 microM), completely inhibited ANG II (100 nM) response in abdominal aorta and carotid artery. An AT1 agonist, [Sar1]-ANG II (100 nM), behaved similarly to ANG II (100 nM) in abdominal aorta and carotid artery. RT-PCR analyses showed that mouse thoracic aorta has a significantly lower AT1 mRNA level than abdominal aorta. These results demonstrate that major mouse vessels exhibit differential contractions to ANG II, possibly because of varied AT1 receptor levels.     

Design and synthesis of 3-(azol-1-yl)phenylpropanes as microbicidal spermicides for prophylactic contraception

We designed a series of 25 3-(azol-1-yl)phenylpropanes which yielded 10 compounds (3, 4, 7, 8, 13, 14, 19, 21, 23, 26) that irreversibly immobilized 100% human sperm at 1% (w/v) concentration in 60 s; 12 compounds (8, 9, 15, 16, 19-21, 23-25, 27, 28) that showed potent microbicidal activity at 12.5-50 μg/mL against Trichomonas vaginalis; and 17 compounds (3-11, 13, 15, 19, 21, 23, 26, 28, 30) that exhibited potent anticandida activity with minimum inhibitory concentration (MIC) of 12.5-50 μg/mL. Almost all the compounds exhibited high level of safety towards normal vaginal flora (Lactobacillus) and human cervical (HeLa) cells in comparison to the marketed spermicide nonoxynol-9 (N-9). All the biological activities were evaluated in vitro. Two compounds (4, 8) with good safety profile exhibited multiple (spermicidal, antitrichomonas and anticandida) activities, warranting further lead optimization for furnishing a prophylactic vaginal contraceptive.     

A three-hybrid system to probe in vivo protein-protein interactions: application to the essential proteins of the RD1 complex of M. tuberculosis

Background

Protein-protein interactions play a crucial role in enabling a pathogen to survive within a host. In many cases the interactions involve a complex of proteins rather than just two given proteins. This is especially true for pathogens like M. tuberculosis that are able to successfully survive the inhospitable environment of the macrophage. Studying such interactions in detail may help in developing small molecules that either disrupt or augment the interactions. Here, we describe the development of an E. coli based bacterial three-hybrid system that can be used effectively to study ternary protein complexes.

Methodology/Principal Findings

The protein-protein interactions involved in M. tuberculosis pathogenesis have been used as a model for the validation of the three-hybrid system. Using the M. tuberculosis RD1 encoded proteins CFP10, ESAT6 and Rv3871 for our proof-of-concept studies, we show that the interaction between the proteins CFP10 and Rv3871 is strengthened and stabilized in the presence of ESAT6, the known heterodimeric partner of CFP10. Isolating peptide candidates that can disrupt crucial protein-protein interactions is another application that the system offers. We demonstrate this by using CFP10 protein as a disruptor of a previously established interaction between ESAT6 and a small peptide HCL1; at the same time we also show that CFP10 is not able to disrupt the strong interaction between ESAT6 and another peptide SL3.

Conclusions/Significance

The validation of the three-hybrid system paves the way for finding new peptides that are stronger binders of ESAT6 compared even to its natural partner CFP10. Additionally, we believe that the system offers an opportunity to study tri-protein complexes and also perform a screening of protein/peptide binders to known interacting proteins so as to elucidate novel tri-protein complexes.