Signal recognition particle causes a transient arrest in the biosynthesis of prepromelittin and mediates its translocation across mammalian endoplasmic reticulum.

The translocation of prepromelittin (pPM) across mammalian endoplasmic reticulum was studied in both wheat germ and reticulocyte lysate. In the wheat germ system, signal recognition particle (SRP) caused a transient arrest in the synthesis of pPM. This was indicated by a slowdown in the rate of synthesis of pPM in the presence of SRP. The arrest was specific, dependent on the concentration of SRP, and more effective at early incubation time. In a tightly synchronized translation system, SRP had no apparent effect on the elongation of pPM, indicating that the effect of SRP on pPM chain synthesis might be at the final stages of chain elongation and release from the ribosome. This was reflected in a transient accumulation of pPM as peptidyl tRNA. Because pPM is composed of only 70 amino acids, arrest by SRP may be very close to chain termination. Arrest at this stage of chain synthesis seems to be unstable and the nascent chain gets terminated and released from the ribosome after a transient delay. The translocation of pPM was shown to be dependent on both SRP and docking protein. The difference in the translocation efficiency of pPM in reticulocyte and wheat germ lysates may reflect a difference in the targeting process in the two systems.

Dithiothreitol and the translocation of preprolactin across mammalian endoplasmic reticulum.

The translocation mode of preprolactin (pPL) across mammalian endoplasmic reticulum was reinvestigated in light of recent findings that nascent secretory polypeptides synthesized in the presence of a highly reducing environment could be translocated posttranslationally and independently of their attachment to the ribosome (Maher, P. A., and S. J. Singer, 1986, Proc. Natl. Acad. Sci. USA, 83:9001-9005). The effects of the reducing agent dithiothreitol (DTT) on pPL synthesis and translocation were studied in this respect. The translocation of pPL was shown to take place only cotranslationally. The apparent posttranslational translocation was due to ongoing chain synthesis irrespective of the presence of high concentrations of DTT. When synthesis was completely blocked, no translocation was observed in the presence or absence of DTT. The synthesis of pPL was retarded by DTT, while its percent translocation was enhanced. The retardation in synthesis was reflected in reduced rates of initiation and elongation. As a consequence of this retardation, which increases the ratio of microsomes to nascent chains, and of possible effects on the conformation of nascent pPL and components of the translocation apparatus, DTT may expand the time and chain length windows for nascent chain translocation competence.

Translocation of proteins across the endoplasmic reticulum. I. Signal recognition protein (SRP) binds to in-vitro-assembled polysomes synthesizing secretory protein.

An 11S protein composed of six polypeptide chains was previously purified from a salt extract of dog pancreas microsomal membranes and shown to be required for translocation of nascent secretory protein across the microsomal membrane (Wistar and Blobel 1980 Proc. Natl. Acad. Sci. U. S. A. 77:7112-7116). This 11S protein, termed signal recognition protein (SRP), has been shown here (a) to inhibit translation in the wheat germ cell-free system selectively of mRNA for secretory protein (bovine preprolactin) but not of mRNA for cytoplasmic protein (alpha and beta chain of rabbit globin); (b) to bind with relatively low affinity (apparent KD less than 5 x 10(-5)) to monomeric wheat germ ribosomes; and (c) to bind selectively and with 6,000-fold higher affinity (apparent KD less than 8 x 10(-9)) to wheat germ ribosomes engaged in the synthesis of secretory protein but not to those engaged in the synthesis of cytoplasmic protein. Low- and high-affinity binding as well as the selective translation-inhibitory effect were abolished after modification of SRP by N-ethyl maleimide. High-affinity binding and the selective translation-inhibitory effect of SRP were largely abolished when the leucine (Leu) analogue beta-hydroxy leucine was incorporated into the nascent secretory polypeptide.

Cryopyrin and pyrin activate caspase-1, but not NF-kappaB, via ASC oligomerization.

Mutations in cryopyrin and pyrin proteins are responsible for several autoinflammatory disorders in humans, suggesting that these proteins play important roles in regulating inflammation. Using a HEK293 cell-based reconstitution system that stably expresses ASC and procaspase-1 we demonstrated that neither cryopyrin nor pyrin or their corresponding disease-associated mutants could significantly activate NF-kappaB in this system. However, both cryopyrin and two disease-associated cryopyrin mutants induced ASC oligomerization and ASC-dependent caspase-1 activation, with the disease-associated mutants being more potent than the wild-type (WT) cryopyrin, because of increased self-oligomerization. Contrary to the proposed anti-inflammatory activity of WT pyrin, our results demonstrated that pyrin, like cryopyrin, can also assemble an inflammasome complex with ASC and procaspase-1 leading to ASC oligomerization, caspase-1 activation and interleukin-1beta processing. Thus, we propose that pyrin could function as a proinflammatory molecule.

Electrophoretic polymorphism in rabbit tear lysozyme.

Rabbit tears were found to contain two lysozymes which differed in their electrophoretic mobility and were designated tear lysozymes 1 and 2. Rabbit tear lysozyme 1 was purified to homogeneity by conventional purification methods. It was found to be distinct from other known mammalian c-type lysozymes, rabbit tear lysozyme 2 and the major rabbit gastrointestinal lysozyme. The activity profile is centered around the neutral region with an optimum of 7 which is slightly lower than that for chicken lysozyme. The thermal stability as well as inhibition profiles by the substrate analogues, N-acetylglucosamine (NAG) and chitotetraose (NAG)4 are comparable to those of chicken lysozyme. Based on its molecular weight and catalytic properties this isozyme is classified as a c-type lysozyme.

Presence of Fragmented QRS Complexes in Patients with Obstructive Sleep Apnea Syndrome / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Obstructive sleep apnea syndrome (OSAS) is a disease with increasing prevalence, which is mainly characterized by increased cardiopulmonary mortality and morbidity. It is well-known that OSAS patients have increased prevalence of cardiovascular diseases including coronary heart disease, heart failure, and arrhythmias. The aim of this study was to evaluate the presence of prolonged and fragmented QRS complexes, which have previously been associated with cardiovascular mortality, in OSAS patients.</p><p><b>METHODS</b>Our study included 51 patients (mean age 41.6 ± 10.1 years) who were recently diagnosed with OSAS (apnea-hypopnea index [AHI] ≥5 events/h) and never received therapy. The control group consisted of 34 volunteers (mean age 43.1 ± 11.6 years) in whom OSAS was excluded (AHI <5 events/h). The longest QRS complexes was measured in the 12-lead electrocardiogram (ECG) and the presence of fragmentation in QRS complexes was investigated.</p><p><b>RESULTS</b>Fragmented QRS frequency was significantly higher in patients with OSAS (n = 31 [61%] vs. n = 12 [35%], P = 0.021). QRS and QTc durations were also significantly longer in OSAS patients than controls (99.8 ± 13.9 ms vs. 84.7 ± 14.3 ms, P < 0.001; 411.4 ± 26.9 ms vs. 390.1 ± 32.2 ms, P = 0.001, respectively). Analysis of the patient and controls groups combined revealed a weak-moderate correlation between AHI and QRS duration (r = 0.292, P = 0.070). OSAS group had no correlation between AHI and QRS duration (r = -0.231, P = 0.203).</p><p><b>CONCLUSIONS</b>In our study fragmented QRS frequency and QRS duration were found to increase in OSAS patients. Both parameters are related with increased cardiovascular mortality. Considering the prognostic importance of ECG parameters, it may be reasonable to recommend more detailed evaluation of OSAS patients with fragmented or prolonged QRS complexes with respect to presence of cardiovascular diseases.</p>

GSK3β: A plausible molecular target in the cytokinemodulating effect of exogenous insulin in a murine model of malarial infection

@# Malaria is a life-threatening disease caused by the Plasmodium sp. parasite. Infection results in heightened pro-inflammatory response which contributes to the pathophysiology of the disease. To mitigate the overwhelming cytokine response, host-directed therapy is a plausible approach. Glycogen synthase kinase-3β (GSK3β), a serine/threonine kinase plays a pivotal role in the regulation of inflammatory response during pathogenic infections. The present study was conducted to investigate the chemo-suppressive and cytokine-modulating effects of insulin administration in malaria-infected mice and the involvement of GSK3β. Intraperitoneal administrations of 0.3 and 0.5 U/kg body weight insulin each for four consecutive days into Plasmodium berghei NK65 (PbN)-infected mice resulted in chemo-suppression exceeding 60% and improved median survival time of infected mice (20.5 days and 19 days respectively compared to 15.5 days for non-treated control). Western analysis revealed that pGSK3β (Ser9) intensity in brain samples from insulin-treated (0.3 and 0.5 U/kg body weight) infected mice each were 0.6 and 2.2 times respectively than that in control. In liver samples, pGSK3β (Ser9) intensity from insulin-treated infected mice were significantly higher (4.8 and 16.1 fold for 0.3 and 0.5 U/kg bw respectively) than that in control. Insulin administration decreased both brain and liver pNF-κB p65 (Ser536) intensities (to 0.8 and 0.6 times for 0.3 U/kg bw insulin; and to 0.2 and 0.1 times for 0.5 U/kg bw insulin respectively compared to control). Insulin treatment (0.5 U/kg bw) also significantly decreased the serum levels of pro-inflammatory cytokines (TNF-α (3.3 times) and IFN-γ (4.9 times)) whilst significantly increasing the levels of anti-inflammatory cytokines (IL-4 (4.9 fold) and IL-10 (2.1 fold)) in PbN-infected mice. Results from this study demonstrated that the cytokinemodulating effects of insulin at least in part involve inhibition of GSK3β and consequent inhibition of the activation of NF-κB p65 suggesting insulin as a potential adjunctive therapeutic for malaria.

A functional interaction between the signal peptide and the translation apparatus is detected by the use of a single point mutation which blocks translocation across mammalian endoplasmic reticulum.

A functional interaction between the signal sequence and the translation apparatus which may serve as a first step in chain targeting to the membrane is described. To this end, we exploited the powerful technique of molecular cloning in a procaryotic system and the well characterized translocation system of mammalian endoplasmic reticulum. The signal peptide of subunit B of the heat labile enterotoxin of Escherichia coli (EltB) was fused to several proteins. Single base substitutions were introduced in the signal peptide and their effect on protein synthesis and translocation was studied. We sought a single amino acid substitution which may define certain steps in the coordinated regulation of chain synthesis and targeting to the membrane. The substitution of proline for leucine at residue -8 in the signal peptide abolished all known functions of the signal peptide. In contrast to wild type signal peptide, the mutant signal peptide did not lead to arrest of nascent chain synthesis by signal recognition particle or translocation of the precursor protein across the membrane of the endoplasmic reticulum. Furthermore, the mutant signal peptide was not cleaved by purified E. coli signal peptidase. Interestingly, the mutation resulted in about a 2-fold increase in the rate of synthesis of the precursor protein, suggesting a role for the signal peptide in regulating the synthesis of the nascent secretory chain as a means of ensuring early and efficient targeting of this chain to the membrane. This role might involve interaction of the signal peptide with components of the translation apparatus and/or endogenous signal recognition particle. These results were obtained with three different fusion proteins carrying the signal peptide of EltB thus leading to the conclusion that the effect of the mutation on the structure and function of the signal peptide is independent of the succeeding sequence to which the signal peptide is attached.

Nascent secretory polypeptides synthesized on Escherichia coli ribosomes are not translocated across mammalian endoplasmic reticulum.

Cell-free protein-synthesizing systems from Escherichia coli and wheat germ were compared for their capacity to support the translocation of secretory proteins across microsomal membranes derived from mammalian endoplasmic reticulum. Three different secretory proteins, two of bacterial and one of eucaryotic origin, were tested in this respect. In all three cases a contrast between the results in the eucaryotic and procaryotic protein-synthesizing systems was revealed. Whereas the eucaryotic system, as expected, supported the translocation of nascent secretory proteins across the microsomal membranes, the procaryotic system failed to do so. This failure was not due to the absence of a translocation-promoting activity or the presence of a translocation-blocking activity in the procaryotic system. These results demonstrate a specificity in the requirement of components of the protein-synthesizing machinery for protein translocation. These components might participate in forming a functional ribosome-membrane junction during protein translocation. The nascent secretory chain alone is not sufficient for making this junction, which might involve the postulated binding of the ribosome to the signal recognition particle or another component of the membrane.

Determinants for protein translocation across mammalian endoplasmic reticulum. Membrane insertion of truncated and full-length prelysozyme molecules.

The translocation of fragments of prelysozyme lacking varying portions of the COOH terminus of the protein is studied in comparison to full-length prelysozyme using transcription-coupled capping of RNA and subsequent translation in a wheat germ cell-free system. The fragments are generated by restricting cloned lysozyme cDNA at selected sites. We found that fragments of 102 and 74 amino acid residues could still be translocated by mammalian endoplasmic reticulum. Addition of signal-recognition particles (SRP) to the cell-free system blocked the nascent chain synthesis. The SRP-depleted membrane by itself could neither process nor translocate the prepolypeptide chain. The presence of both components was essential for processing and translocation as well as the release of the nascent chain arrest induced by SRP. However, when the size of the fragment was limited to 51 amino acids, the SRP-induced arrest, the translocation and processing failed to take place. These results define minimum length and structural requirements for translocation of the nascent chain across mammalian endoplasmic reticulum.

Amino acid sequence of California quail lysozyme. Effect of evolutionary substitutions on the antigenic structure of lysozyme.

To examine the effect of amino acid substitutions in lysozyme on the binding of antibodies to lysozyme, we purified lysozyme from the egg whites of California quail and Gambel quail. Tryptic peptides were isolated from digests of the reduced and carboxymethylated lysozymes and subjected to quantitative analysis of their amino acid compositions. The two proteins were identical by this criterion. Each peptide from the California quail lysozyme was then sequenced by quantitative Edman degradation, and the peptides were ordered by homology with other bird lysozymes. California quail lysozyme is most similar in amino acid sequence to bobwhite quail lysozyme, from which it differs by two substitutions: arginine for lysine at position 68 and histidine for glutamine at position 121. California and bobwhite quail lysozymes were antigenically distinct from each other in quantitative microcomplement fixation tests, indicating that substitutions at one or both of these positions can alter the antigenic structure of lysozyme. Yet neither of these positions is among those claimed to account for the precise and entire antigenic structure of lysozyme [Atassi, M. Z., & Lee, C.-L. (1978) Biochem. J. 171, 429--434]. Two possible explanations for this discrepancy are discussed.

A bacterial secretory protein requires signal recognition particle for translocation across mammalian endoplasmic reticulum.

In vitro transcription of DNA from plasmid pBR322 was coupled to cell-free translation in a wheat germ system. The major translation product was pre-beta-lactamase. Upon addition of dog pancreas microsomes, the precursor was processed to authentic beta-lactamase as shown by partial NH2-terminal sequence analysis. Processing was linked to translocation into the microsomal vesicles. Salt-extracted microsomes did not process pre-beta lactamase but could be reactivated by purified signal recognition particle, which is the functional component of the salt wash (Walter, P., and Blobel, G. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 7112-7116). Signal recognition particle alone caused a drastic translation arrest that could be released by salt-depleted membranes. These data are consistent with those obtained for eukaryotic proteins and suggest that co-translational translocation of both bacterial and eukaryotic secretory proteins across the endoplasmic reticulum require identical components.

A novel in vitro transcription-translation system: accurate and efficient synthesis of single proteins from cloned DNA sequences.

A system is described which permits the efficient synthesis of single proteins in vitro. The essential element in this expression system is a strong promoter derived from coliphage T5 which produces, with high efficiency, specific RNAs in capped or uncapped form, depending upon the experimental conditions used. The transcription-coupled capping of RNA allows the direct translation of the RNA in eukaryotic extracts from wheat germ as well as from HeLa cells. The synthesis of three different proteins is reported, including lysozyme, which is shown to be translocated across membranes when appropriate assay conditions are used. The simplicity of the experimental procedure, the high purity and specific activity of the [35S]methionine-labelled proteins produced offer a number of possibilities for the study of structure-function relationships of proteins.

Association of degradation and secretion of three chimeric polypeptides in Escherichia coli.

We investigated the stability of fusion proteins composed of the signal peptide of the heat-labile enterotoxin of Escherichia coli and three polypeptides: the bacterial cytoplasmic chloramphenicol acetyltransferase, the mouse dihydrofolate reductase, and human immune interferon. We demonstrate that these proteins are rapidly degraded as a result of being targeted to the secretion apparatus of E. coli, with the extent of degradation varying among the three fusion proteins. Four lines of experimental evidence are presented in support of this suggestion. First, the chimeric polypeptides containing a functional signal peptide were detected in low amounts in vivo. When a mutation was introduced in the signal peptide, resulting in lack of recognition by the secretion apparatus, the chimeric proteins accumulated at high levels in the cytoplasm of the cell. Second, both the wild-type and mutant polypeptides accumulated in a purified and reconstituted in vitro translation system from E. coli and were equally susceptible to digestion by an exogenous protease. Third, the chimeric polypeptides lacking the signal peptide accumulated in a stable form in vivo. Fourth, the precursors of the proteins containing a functional signal peptide accumulated in a secA ts mutant at the restrictive temperature when secretion was blocked, suggesting that degradation is tightly linked to the secretion apparatus.

Amino acid sequences of stomach and nonstomach lysozymes of ruminants.

Complete amino acid sequences are presented for lysozymes c from camel and goat stomachs and compared to sequences of other lysozymes c. Tree analysis suggests that the rate of amino acid replacement went up as soon as lysozyme was recruited for the stomach function in early ruminants. The two lysozymes from goat stomach are the products of a gene duplication that probably took place before the divergence of cow, goat, and deer about 25 million years ago. Partial sequences of three lysozymes from goat tears indicated that (a) the goat tear family of lysozymes may have diverged from the stomach lysozyme family by an ancient duplication and (b) later duplications are probably responsible for the multiple forms of tear and milk lysozymes in ruminants.