Specific Releasing Factor Research Articles

Origin of the omnipotence of eukaryotic release factor 1

Termination of protein synthesis on the ribosome requires that mRNA stop codons are recognized with high fidelity. This is achieved by specific release factor proteins that are very different in bacteria and eukaryotes. Hence, while there are two release factors with overlapping specificity in bacteria, the single omnipotent eRF1 release factor in eukaryotes is able to read all three stop codons. This is particularly remarkable as it is able to select three out of four combinations of purine bases in the last two codon positions. With recently determined 3D structures of eukaryotic termination complexes, it has become possible to explore the origin of eRF1 specificity by computer simulations. Here, we report molecular dynamics free energy calculations on these termination complexes, where relative eRF1 binding free energies to different cognate and near-cognate codons are evaluated. The simulations show a high and uniform discrimination against the near-cognate codons, that differ from the cognate ones by a single nucleotide, and reveal the structural mechanisms behind the precise decoding by eRF1.

Anti-inflammatory drugs for Duchenne muscular dystrophy: focus on skeletal muscle-releasing factors.

Duchenne muscular dystrophy (DMD), an incurable and a progressive muscle wasting disease, is caused by the absence of dystrophin protein, leading to recurrent muscle fiber damage during contraction. The inflammatory response to fiber damage is a compelling candidate mechanism for disease exacerbation. The only established pharmacological treatment for DMD is corticosteroids to suppress muscle inflammation, however this treatment is limited by its insufficient therapeutic efficacy and considerable side effects. Recent reports show the therapeutic potential of inhibiting or enhancing pro- or anti-inflammatory factors released from DMD skeletal muscles, resulting in significant recovery from muscle atrophy and dysfunction. We discuss and review the recent findings of DMD inflammation and opportunities for drug development targeting specific releasing factors from skeletal muscles. It has been speculated that nonsteroidal anti-inflammatory drugs targeting specific inflammatory factors are more effective and have less side effects for DMD compared with steroidal drugs. For example, calcium channels, reactive oxygen species, and nuclear factor-κB signaling factors are the most promising targets as master regulators of inflammatory response in DMD skeletal muscles. If they are combined with an oligonucleotide-based exon skipping therapy to restore dystrophin expression, the anti-inflammatory drug therapies may address the present therapeutic limitation of low efficiency for DMD.

The Interaction of CCDC104/BARTL1 with Arl3 and Implications for Ciliary Function

SummaryCilia are small antenna-like cellular protrusions critical for many developmental signaling pathways. The ciliary protein Arl3 has been shown to act as a specific release factor for myristoylated and farnesylated ciliary cargo molecules by binding to the effectors Unc119 and PDE6δ. Here we describe a newly identified Arl3 binding partner, CCDC104/CFAP36. Biochemical and structural analyses reveal that the protein contains a BART-like domain and is called BARTL1. It recognizes an LLxILxxL motif at the N-terminal amphipathic helix of Arl3, which is crucial for the interaction with the BART-like domain but also for the ciliary localization of Arl3 itself. These results seem to suggest a ciliary role of BARTL1, and possibly link it to the Arl3 transport network. We thus speculate on a regulatory mechanism whereby BARTL1 aids the presentation of active Arl3 to its GTPase-activating protein RP2 or hinders Arl3 membrane binding in the area of the transition zone.

Effect of Mg 2+ ion in protein secretion by magnesium-resistant strains of Pseudomonas aeruginosa and Vibrio parahaemolyticus isolated from the coastal water of Haldia port

A rapidly growing industrial complex including oil refineries and chemical industries has developed around the coastal area of Haldia port in the district of Midnapore, West Bengal, India. The coastal water is highly polluted with industrial wastes along with petroleum hydrocarbons. The bacteria isolated from the different sites of the coastal waters were Escherichia coli, Alcaligenes, Acinetobacter, Klebsiella spp., Micrococcus spp., Vibrio spp., Pseudomonas aeruginosa and Vibrio parahaemolyticus. The salinity of the water during the time of collection of samples around the port area was 8.2 ppt. Among the isolated organisms, only two isolates, P. aeruginosa and V. parahaemolyticus, showed growth at 300 mM Mg 2+ ion concentration. However, a 3 mM Mg 2+ concentration was detected in the coastal water whereas other metal ion concentrations were less than 3×10 −5 mM. Resistance to Mg 2+ (300 mM) was determined by a 5.5-kb plasmid. A large amount of a 40-kDa outer membrane protein, which was highly soluble in 1 M MgCl 2, was isolated from both V. parahaemolyticus and P. aeruginosa. The secretion of proteins in the culture supernatant of V. parahaemolyticus was highly increased when the cells were grown in the presence of 300 mM Mg 2+, whereas very low secretion was observed in the same concentration of Mg 2+ in the case of P. aeruginosa. Mg 2+ may act as a specific release factor in protein secretion by V. parahaemolyticus strains.

Effect of Mg(2+) ion in protein secretion by magnesium-resistant strains of Pseudomonas aeruginosa and Vibrio parahaemolyticus isolated from the coastal water of Haldia port.

A rapidly growing industrial complex including oil refineries and chemical industries has developed around the coastal area of Haldia port in the district of Midnapore, West Bengal, India. The coastal water is highly polluted with industrial wastes along with petroleum hydrocarbons. The bacteria isolated from the different sites of the coastal waters were Escherichia coli, Alcaligenes, Acinetobacter, Klebsiella spp., Micrococcus spp., Vibrio spp., Pseudomonas aeruginosa and Vibrio parahaemolyticus. The salinity of the water during the time of collection of samples around the port area was 8. 2 ppt. Among the isolated organisms, only two isolates, P. aeruginosa and V. parahaemolyticus, showed growth at 300 mM Mg(2+) ion concentration. However, a 3 mM Mg(2+) concentration was detected in the coastal water whereas other metal ion concentrations were less than 3x10(-5) mM. Resistance to Mg(2+) (300 mM) was determined by a 5.5-kb plasmid. A large amount of a 40-kDa outer membrane protein, which was highly soluble in 1 M MgCl(2), was isolated from both V. parahaemolyticus and P. aeruginosa. The secretion of proteins in the culture supernatant of V. parahaemolyticus was highly increased when the cells were grown in the presence of 300 mM Mg(2+), whereas very low secretion was observed in the same concentration of Mg(2+) in the case of P. aeruginosa. Mg(2+) may act as a specific release factor in protein secretion by V. parahaemolyticus strains.

Cellular localization of prolactin-releasing peptide messenger RNA in the rat brain

Prolactin-releasing peptide (PrRP), a novel peptide identified as the endogenous ligand for an orphan receptor isolated from the pituitary, is a potent stimulator of prolactin release. To get a clue of the functional roles of the peptide, we performed in situ hybridization histochemistry for PrRP mRNA to define the cellular localization of PrRP-producing cells in the brain of the cycling adult female rat during diestrus. The PrRP mRNA-containing cells were located in the caudal part of the dorsomedial nucleus of the hypothalamus. In the brainstem, the cells were found in the caudal part of the solitary tract nucleus and in the caudal ventrolateral medulla (ventrolateral intermediate reticular field). Specific signals for PrRP mRNA were not detected in other brain regions. Although PrRP is a candidate for being a hypophysiotropic specific releasing factor, the discrete distribution of PrRP in the extrahypothalamic area suggests that the peptide has other physiological functions in the central nervous system.

Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone.

The endocrine system displays highly complex interactions among its components. Excesses or deficiencies of hormone production in one gland may alter the production of hormones by others. Several physiological functions are affected by a balance among hormones acting either together or in sequence. For example, FSH secretion has been demonstrated to be affected by hypothalamic influences upon the anterior pituitary through a specific releasing factor, the decapeptide LRF. This decapeptide stimulates the release of both LH and FSH by the pituitary, and these gonadotropins cause the production of steroids by the testes and the ovaries. Gonadal steroids in the blood act directly upon the anterior pituitary to regulate the output of gonadotropins as originally proposed by Moore and Price in 1932 (3), or act indirectly upon the hypothalamus to adjust the output of pituitary hormones in accordance with the needs of the reproductive system. However, such a simple negative feedback of steroids on the hypothalamic-hypophysial axis cannot account for the differential secretion of FSH observed during the estrus cycle. Therefore, the concept that a gonadal protein, inhibin, specifically regulates FSH secretion was proposed. This concept has now been validated by the isolation and characterization of two forms of inhibin that exert their effects on the pituitary to suppress FSH secretion both in vitro and probably in vivo. Furthermore, the production of inhibin is stimulated by FSH, thus establishing a reciprocal relationship between the release of FSH and inhibin. Since hormones in the body are controlled through interlocking complexes of factors, a variety of secondary factors, in one way or another, may also exert influence on the regulation of FSH secretion. As an example, TGF beta, a protein growth factor found in all tissues, promotes the basal secretion of FSH by the pituitary and enhances FSH-mediated estrogen production by the granulosa cells. It is therefore not surprising that two forms of a novel protein, activin and activin A, isolated from the same FF from which inhibins were isolated, show bioactivities similar to those of TGF beta. These activins are formed as dimers of the two beta-subunits of inhibin, probably as a result of the rearrangement of the gene products. This novel observation that different arrangements of gene products can result in opposite biological activities may thus reflect a wholly different level of control of FSH secretion. If such a phenomenon occurs in other biosystems, it would represent an important form of homeostatic mechanism for controlling biologically active substances.(ABSTRACT TRUNCATED AT 400 WORDS)

Increased responsiveness of the hypothalamic-pituitary axis after neurotoxin-induced hypothalamic denervation.

Neonatal treatment with monosodium glutamate (MSG) induces severe neuronal damage in selected brain areas, which in turn results in a number of neuroendocrine abnormalities during adult life. The present study was designed to determine what effects this partial and selective denervation of the hypothalamic-pituitary axis may have on the sensitivity of two key components of that axis, the median eminence (ME) and the anterior pituitary (AP). In order to test any changes in response that may occur after MSG treatment, the release of several peptide hormones from either the ME or the AP was evaluated in vitro, employing specific or general secretagogues. Male newborn pups of the Holtzman strain were injected with MSG every other day for 5 days, starting on day 2 of life; littermate controls received an injection of 10% NaCl. All animals were used when adult, at about 5-7 months of age. After decapitation, ME and AP tissues from control and MSG-treated rats were dissected out and incubated in vitro. Release of LHRH, SRIF, arginine vasopressin, and oxytocin from the ME was measured by direct RIA, under basal conditions and after stimulation with high K+ medium (28 mM). The results clearly indicate a marked hyperresponse of the release of each of the four neuropeptides by ME fragments from MSG-lesioned animals. The altered release was not attributable to changes in ME peptide content. In the case of the AP, the release of ACTH, LH, PRL, and GH in response to high K+ or, in some cases, to specific releasing factors, was evaluated in a dispersed cell preparation. The release of all four protein hormones was increased by high K+, and again the MSG-lesioned rats showed a pronounced hyperresponse. Corticotropin-releasing factor, at concentrations of 10(-9) and 10(-8) M enhanced ACTH release from control and MSG-lesioned rats, but the latter presented a marked hyperresponse. A similar observation on LH release was seen after stimulation with LHRH (10(-9) M). The results indicate that the neuronal damage induced by neonatal MSG treatment results in a generalized hyperresponsiveness in vitro to either specific or general secretagogues by ME or AP tissues, which may suggest the development of a denervation-type supersensitivity in the hypothalamic-pituitary axis. Since the release of these peptide hormones is sluggish in MSG-lesioned rats under in vivo conditions, it seems plausible to conclude that the major defect after the neurotoxin damage may reside in the loss of neurotransmitter systems normally innervating and regulating the activity of the peptidergic neurons.

Immunologic evidence for structural homology between the release factors of Escherichia coli

Immune sera of high antibody titer were raised against the release factors RF1 and RF2, which are required for codon-directed termination of protein biosynthesis in Escherichia coli. The antigens were of high purity and contained no detectable crosscontamination. Each serum inhibited both RF1 and RF2 activity, but exhibited greater recognition for the specific release factor used for immunization. RF2 neutralized the inhibition of RF1 by anti-RF2 and blocked the binding of RF1 to anti-RF2-Sepharose columns. Therefore, we conclude that RF1 and RF2 share antigenic determinants, thus indicating structural homology between the release factors. Partially purified preparations of RF1 and RF2 were purified extensively by immunoaffinity chromatography. Each was eluted from anti-RF2-Sepharose columns with 6 m guanidine-HCl. Following dialysis, 30–50% of the initial release factor activity was recovered. Molecular weight values of 49,000 for RF1 and 50,000 for RF2 have been calculated from multiple sodium dodecyl sulfate-polyacrylamide gel electrophoresis studies.

Sequential translation of trinucleotide codons for peptide bond formation, translocation, and termination.

The addition of GUA, valyl-tRNA, T factor, GTP, and ethanol to reactions containing fMet-tRNA.AUG.ribosome intermediate results in the synthesis of fMet-valyl-tRNA which can be translocated if G factor is also present. The translocated peptidyl-tRNA can interact with puromycin to form fMet-Val-puromycin. Release of fMet-valine can also take place after translocation if a terminator trinucleotide and its specific release factor are present.

Requirement of Ca++ and Mg++ ions for the in vitro release of follicle-stimulating hormone from rat pituitary glands and in its subsequent biosynthesis.

The in vitro release of FSH from rat pituitary glands, when stimulated by a specific releasing factor, viz., follicle-stimulating hormone releasing factor (FRF), or nonspecific factors such as cyclic 3′,5′-AMP (cAMP), or high [K+] medium, required the presence of Ca++ in the external medium. Ca++ together with cAMP probably plays an active intracellular role in the processes leading to the release of FSH. No definite conclusion can be made as to the requirement for Mg++ in these processes. The synthesis (or activation) of FSH, observed during incubation in the presence of FRF, was also Ca++ dependent. However, further experiments are necessary in order to find out whether the presence of Ca++ is required directly for the synthesis of FSH, or whether the synthesis is only a secondary phenomenon arising from the release of FSH. In the latter case, the lack of Ca++, which inhibited the release of FSH, would consequently inhibit its synthesis. (Endocrinology 86: 761, 1970)