Release factor regulating termination of complete protein in a eukaryotic organism

Polypeptide release reaction was studied using a protein release factor and a physiological substrate containing a complete polypeptide chain attached to monosomes of the insect Tenebrio molitor. The intermediate substrate used for the release reaction was synthesized using a cell-free protein synthesizing system from Tenebrio capable of polypeptide synthesis but not release of the completed chain. This system synthesized predominantly adult cuticular protein. The released product was characterized by chromatography after tryptic digestion; many of the tryptic peptides corresponded to those of cuticule labeled in vivo. The protein release factor was obtained as microsomal wash and was further purified by ammonium sulfate precipitation and column chromatography. It released about 30% of the monosome-bound peptide in the absence of GTP. The remaining 70% of peptidyl-tRNA was released as peptidyl-puromycin in the absence of release factor, but required transferase II and GTP. The peptidyl-puromycin varied in size from dipeptide to almost complete protein. The puromycin reaction was inhibited by diphtheria toxin and NAD and was dependent on GTP, while the release of completed peptide was independent of GTP and not affected by diphtheria toxin and NAD. The release factor was capable of releasing formylmethionine as formylmethionine-puromycin from ribosomes in response to poly(A₃,U). Hence it is suggested that the release factor is responding to UAA as terminating codon.     

A second SARS-CoV S2 glycoprotein internal membrane-active peptide. Biophysical characterization and membrane interaction

The severe acute respiratory syndrome coronavirus (SARS-CoV) envelope spike (S) glycoprotein, a class I viral fusion protein, is responsible for the fusion between the membranes of the virus and the target cell. The S2 domain of protein S has been suggested to have two fusion peptides, one located at its N-terminus, downstream of the furin cleavage, and another, more internal, located immediately upstream of the HR1. Therefore, we have carried out a study of the binding and interaction with model membranes of a peptide corresponding to segment 873-888 of the SARS-CoV S glycoprotein, peptide SARS IFP, as well as the structural changes taking place in both the phospholipid and the peptide induced by the binding of the peptide to the membrane. We demonstrate that SARS IFP peptide binds to and interacts with phospholipid model membranes and shows a higher affinity for negatively charged phospholipids than for zwitterionic ones. SARS IFP peptide specifically decreases the mobility of the phospholipid acyl chains of negatively charged phospholipids and adopts different conformations in the membrane depending upon their composition. These data support its role in SARS-mediated membrane fusion and suggest that the regions where this peptide resides might assist the fusion peptide and/or the pretransmembrane segment of the SARS-CoV spike glycoprotein in the fusion process.     

Cloning of the Escherichia coli release factor 2 gene.

The protein release factor 2 (RF2) participates in Escherichia coli polypeptide chain termination with codon specificity (UAA or UGA). A colicin E1 recombinant identified in the Carbon and Clarke E. coli bank contains the protein release factor 2 gene. A 1.7-kilobase E. coli fragment has been subcloned into the plasmid pUC9 vector. Bacterial cells, containing the plasmid recombinant, produce elevated levels of protein release factor 2 as detected by an immune precipitation assay and in vitro measurement of UGA-directed peptide chain termination and [3H]UGA codon recognition.     

The inv(16) cooperates with ARF haploinsufficiency to induce acute myeloid leukemia

The inv(16) is one of the most frequent chromosomal translocations associated with acute myeloid leukemia (AML) and creates a chimeric fusion protein consisting of most of the runt-related X1 co-factor, core binding factor beta fused to the smooth muscle myosin heavy chain MYH11. Expression of the ARF tumor suppressor is regulated by runt-related X1, suggesting that the inv(16) fusion protein (IFP) may repress ARF expression. We established a murine bone marrow transplant model of the inv(16) in which wild type, Arf+/-, and Arf-/- bone marrow were engineered to express the IFP. IFP expression was sufficient to induce a myelomonocytic AML even when expressed in wild type bone marrow, yet removal of only a single allele of Arf greatly accelerated the disease, indicating that Arf is haploinsufficient for the induction of AML in the presence of the inv(16).     

Molecular cloning and characterization of an interferon induced human cDNA with sequence homology to a mammalian peptide chain release factor.

Here we report the molecular cloning of several related human cDNAs from which a full-length sequence can be determined. The cDNAs encode a 2.8 kb mRNA that is strongly induced by interferon (IFN) gamma and the expression of which is not cell-restricted but observed in fibroblasts, macrophages and epithelial cells. The deduced amino acid sequence predicts a protein of 471 amino acids with high sequence similarity to a previously identified rabbit peptide chain release factor. Functional studies to demonstrate release factor activity showed that the protein encoded by this cDNA inhibited the readthrough activity of a yeast UGA suppressor tRNA in an in vitro translation system. The identification of this novel cDNA implies that translational control by IFN induced proteins may not be restricted to the initial steps of protein synthesis but may also act by regulation of peptide chain termination.     

Inactivation of factor VIII by activated protein C and protein S

Factor VIII was inactivated by activated protein C in the presence of calcium and phospholipids. Analysis of the activated protein C-catalyzed cleavage products of factor VIII indicated that inactivation resulted from the cleavage of the heavy chains. The heavy chains appeared to be converted into 93- and 53-kDa peptides. Inactivation of factor VIII that was only composed of the 93-kDa heavy chain and 83-kDa light chain indicated that the 93-kDa polypeptide could be degraded into a 68-kDa peptide that could be subsequently cleaved into 48- and 23-kDa polypeptides. Thus, activated protein C catalyzed a minimum of four cleavages in the heavy chain. Activated protein C did not appear to alter the factor VIII light chain. The addition of protein S accelerated the rate of inactivation and the rate of all of the cleavages. The effect of protein S could be observed on the cleavage of the heavy chains and on secondary cleavages of the smaller products, including the 93-, 68-, and 53-kDa polypeptides. The addition of factor IX to the factor VIII-activated protein C reaction mixture resulted in the inhibition of factor VIII inactivation. The effect of factor IX was dose dependent. Factor VIII was observed to compete with factor Va for activated protein C. The concentration dependence of factor VIII inhibition of factor Va inactivation suggested that factor VIII and factor Va were equivalent substrates for activated protein C.     

Interferon-inducible Myc/STAT-interacting protein Nmi associates with IFP 35 into a high molecular mass complex and inhibits proteasome-mediated degradation of IFP 35

Nmi is an interferon (IFN)-inducible protein homologous to IFN-inducible protein IFP 35. The homology consists of a novel Nmi/IFP 35 domain (NID) of 90-92 amino acids that is repeated in tandem in each protein and mediates Nmi-Nmi protein interactions and subcellular localization. In a yeast two-hybrid screen with a fragment of Nmi protein containing both NIDs, we identified an interaction between Nmi and IFP 35. Deletion derivatives of the proteins indicate that both NIDs are required for the interaction between Nmi and IFP 35. In mammalian cells, Nmi and IFP 35 co-immunoprecipitate and co-localize in large cytoplasmic speckles. Nmi and IFP 35 proteins associate into a high molecular mass complex of 300-400 kDa as determined by native gel electrophoresis and gel filtration. The association of Nmi and IFP 35 into a complex can be demonstrated in multiple cell lines and is not dependent on treatment with IFN. Short term and long term cultures of transfected HEK293 cells suggest that Nmi and IFP 35 proteins stabilize each other through complex formation. IFP 35 appears to be more labile because Nmi was stable in the absence of IFP 35, whereas IFP 35 was degraded in the absence of Nmi. A deletion analysis revealed that Nmi must interact with IFP 35 to prevent its degradation and that the amino terminus of Nmi is required, but not sufficient, for this function. Inhibition of the proteasome, but not other proteases, led to increased levels of IFP 35. Thus, we have shown that Nmi and IFP 35 associate into a protein complex, that IFP 35 is degraded in a proteasome-mediated process, and that a novel function of Nmi is to prevent IFP 35 degradation. The stabilization of IFP 35 by Nmi may serve to amplify the physiologic effects of IFNs.     

Origins of Fluorescence in Evolved Bacteriophytochromes

Use of fluorescent proteins to study in vivo processes in mammals requires near-infrared (NIR) biomarkers that exploit the ability of light in this range to penetrate tissue. Bacteriophytochromes (BphPs) are photoreceptors that couple absorbance of NIR light to photoisomerization, protein conformational changes, and signal transduction. BphPs have been engineered to form NIR fluorophores, including IFP1.4, Wi-Phy, and the iRFP series, initially by replacement of Asp-207 by His. This position was suggestive because its main chain carbonyl is within hydrogen-bonding distance to pyrrole ring nitrogens of the biliverdin chromophore, thus potentially functioning as a crucial transient proton sink during photoconversion. To explain the origin of fluorescence in these phytofluors, we solved the crystal structures of IFP1.4 and a comparison non-fluorescent monomeric phytochrome DrCBD_mon. Met-186 and Val-288 in IFP1.4 are responsible for the formation of a tightly packed hydrophobic hub around the biliverdin D ring. Met-186 is also largely responsible for the blue-shifted IFP1.4 excitation maximum relative to the parent BphP. The structure of IFP1.4 revealed decreased structural heterogeneity and a contraction of two surface regions as direct consequences of side chain substitutions. Unexpectedly, IFP1.4 with Asp-207 reinstalled (IFP_rev) has a higher fluorescence quantum yield (∼9%) than most NIR phytofluors published to date. In agreement, fluorescence lifetime measurements confirm the exceptionally long excited state lifetimes, up to 815 ps, in IFP1.4 and IFP_rev. Our research helps delineate the origin of fluorescence in engineered BphPs and will facilitate the wide-spread adoption of phytofluors as biomarkers.     

Two distinct components of release factor function uncovered by nucleophile partitioning analysis

During translation termination, release factor (RF) protein catalyzes a hydrolytic reaction in the large subunit peptidyl transferase center to release the finished polypeptide chain. While the mechanism of catalysis of peptide release remains obscure, important contributing factors have been identified, including conserved active-site nucleotides and a GGQ tripeptide motif in the RF. Here we describe pre-steady-state kinetic and nucleophile competition experiments to examine RF contributions to the rate and specificity of peptide release. We find that while unacylated tRNA stimulates release in a nondiscriminating manner, RF1 is very specific for water. Further analysis reveals that amino acid Q235 of the RF1 GGQ motif is critical for the observed specificity. These data lead to a model where RFs make two distinct contributions to catalysis--a relatively nonspecific activation of the catalytic center and specific selection of water as a nucleophile facilitated by Q235.     

A 130-kDa protein on endothelial cells binds to amino acids 15-42 of the B beta chain of fibrinogen.

Factors which stimulate the release of von Willebrand factor (vWf) from endothelial cell Weibel-Palade bodies and which induce the expression of the leukocyte-binding adhesion molecule P-selectin (PADGEM, GMP-140, CD62) on the endothelial cell surface remain incompletely characterized. Fibrin but not fibrinogen is a potent stimulus for the release of stored von Willebrand factor from endothelial cells. Removal of fibrinopeptides A and B from fibrinogen occurs during the formation of fibrin, and the removal of fibrinopeptide B is a requirement for fibrin to induce vWf secretion. The cleavage of fibrinopeptide A by reptilase enzyme forms a fibrin gel yet it is incapable of stimulating Weibel-Palade body degranulation. As a consequence of removing fibrinopeptide B, B beta 15-42 becomes the new NH2 terminus of the beta chain of fibrin. We have shown that the peptide B beta 15-42 in solution inhibits the release of vWf stimulated by fibrin. In addition, B beta 15-42 coupled to ovalbumin supports the binding and spreading of endothelial cells, while a scrambled form of this peptide coupled to the same carrier does not. We investigated whether these determinants near the amino terminus of the beta chain of fibrin bind to a specific protein on the surface of endothelial cells. A 130-kDa protein was isolated from surface-labeled human umbilical vein endothelial cells by specific binding to B beta 15-42 immobilized on Sepharose. This glycoprotein was eluted with the B beta 15-42 peptide in solution but not with the scrambled form of this peptide. The fibrin-derived peptides B beta 19-26 and B beta 37-56-cysteine were also incapable of eluting the 130-kDa protein bound to immobilized B beta 15-42 as were the arginine-glycine-aspartic acid-serine RGDS tetrapeptide and EDTA. The 130-kDa protein is recognized neither by antibodies to the known integrins found on endothelial cells nor by antibodies to CD31 (endoCAM, PECAM-1), a member of the immunoglobulin family of receptors found on endothelial cells. The beta chain of fibrin thus contains a sequence near its amino terminus which specifically binds to what is likely a novel endothelial cell surface protein. This glycoprotein may promote endothelial cell adhesion to fibrin during the wound healing process and is a candidate for a receptor involved in fibrin-mediated release of Weibel-Palade bodies from endothelial cells.     

The PH domain containing protein CKIP-1 binds to IFP35 and Nmi and is involved in cytokine signaling

The pleckstrin homology domain-containing protein CKIP-1 is implicated in regulation of cell differentiation, apoptosis, cytoskeleton as well as recruitment of CK2 and ATM kinases to plasma membrane. Protein-protein interactions of CKIP-1 were required for these functions. Here we identify the IFN-induced protein IFP35 and its homologue Nmi as two novel CKIP-1 interacting partners. The NID domains of IFP35 and Nmi are required for the interactions. Similar to IFP35 and Nmi, CKIP-1 can be up-regulated dramatically by IFN-gamma and IL-2 and form homodimer and homotrimer in vivo. Nmi stabilizes IFP35, whereas CKIP-1 destabilizes IFP35 via inhibiting IFP35-Nmi interaction. The ratio of Nmi to CKIP-1 determines the stability of IFP35 and control cytokine signaling in a novel mechanism. Importantly, similar to Nmi and contrast to IFP35, CKIP-1 inhibits tumor cell growth and Akt-mediated cell survival. Thus, our results provide a novel role of CKIP-1 in cytokine signaling response and the biochemical mechanism, by which two previously identified modulators IFP35 and Nmi are involved via interactions.     

Hydratation of the rubredoxin polypeptide chain from data on the spatial structure

The hydration water distribution around the main chain protein rubredoxin has been analysed using the crystal data at high resolution obtained earlier. The analysis was based on the consideration of all nearest neighbour atoms around the N and O atoms of peptide groups. The atoms which can form hydrogen binds were the subject of final analysis. The nitrogen atom of a peptide NH group has only one vacancy for neighbours. The oxygen atom of a peptide CO group has one, two or more neighbours, some of them are oxygen-water atoms. About 27% of NH and 53% of CO peptide groups are hydrated, that corresponds to 0.12 H2O per gram of protein. A detailed analysis shows that NH and CO groups of the main chain are hydrated according to the principle of maximum possible in situ saturation of hydrogen bonds. Thus the peptide groups incorporated in the peptide hydrogen bond network were not hydrated as a rule. Consequently, for rubredoxin a pleated sheet region, some regions for the large and small main chain loops, and Fe-containing pocket are not hydrated. A method for evaluation of the main chain hydration is proposed when the coordinates of protein atoms are available.     

The selective blocking of the polymerization reaction of striated muscle actin leading to a derivative suitable for crystallization. Modification of Tyr-53 by 5-diazonium-(1H)tetrazole.

The polymerization reaction of rabbit muscle actin was completely inhibited by reaction of one amino acid side chain per protein monomer with 5-diazonium-(1H)[14C]tetrazole. A tryptic peptide fingerprint showed a single peptide labeled by the reagent. The peptide was isolated and the labeled amino acid identified by amino acid analysis as Tyr-53. This side chain is not accessible to the reagent in F-actin. The modification is compared to similar inhibitions by other reagents.     

The accumulation of lens-specific protein and mRNA in cultures of neural retina from 312-day chick embryos

A detergent insoluble 58 000 D polypeptide is phosphorylated at an increased rate during mitotic arrest in cultured Chinese hamster ovary cells. Using one-dimensional peptide mapping, this polypeptide was identified as a phosphorylated form of intermediate filament protein (IFP). The increased rate of phosphorylation is observed with either colcemid- or vinblastine-treated mitotic cells, but is not seen with similarly treated interphase cells.     

Increased phosphorylation rate of intermediate filaments during mitotic arrest

A detergent insoluble 58 000 D polypeptide is phosphorylated at an increased rate during mitotic arrest in cultured Chinese hamster ovary cells. Using one-dimensional peptide mapping, this polypeptide was identified as a phosphorylated form of intermediate filament protein (IFP). The increased rate of phosphorylation is observed with either colcemid- or vinblastine-treated mitotic cells, but is not seen with similarly treated interphase cells.     

Structure and membrane interaction of the internal fusion peptide of avian sarcoma leukosis virus.

The structure and membrane interaction of the internal fusion peptide (IFP) fragment of the avian sarcoma and leucosis virus (ASLV) envelope glycoprotein was studied by an array of biophysical methods. The peptide was found to induce lipid mixing of vesicles more strongly than the fusion peptide derived from the N-terminal fusion peptide of influenza virus (HA2-FP). It was observed that the helical structure was enhanced in association with the model membranes, particularly in the N-terminal portion of the peptide. According to the infrared study, the peptide inserted into the membrane in an oblique orientation, but less deeply than the influenza HA2-FP. Analysis of NMR data in sodium dodecyl sulfate micelle suspension revealed that Pro13 of the peptide was located near the micelle-water interface. A type II beta-turn was deduced from NMR data for the peptide in aqueous medium, demonstrating a conformational flexibility of the IFP in analogy to the N-terminal FP such as that of gp41. A loose and multimodal self-assembly was deduced from the rhodamine fluorescence self-quenching experiments for the peptide bound to the membrane bilayer. Oligomerization of the peptide and its variants can also be observed in the electrophoretic experiments, suggesting a property in common with other N-terminal FP of class I fusion proteins.     

Serine-53 at the tip of the glycine-rich loop of cAMP-dependent protein kinase: role in catalysis, P-site specificity, and interaction with inhibitors

The glycine-rich loop, one of the most important motifs in the conserved protein kinase catalytic core, embraces the entire nucleotide, is very mobile, and is exquisitely sensitive to what occupies the active site cleft. Of the three conserved glycines [G(50)TG(52)SFG(55) in cAMP-dependent protein kinase (cAPK)], Gly(52) is the most important for catalysis because it allows the backbone amide of Ser(53) at the tip of the loop to hydrogen bond to the gamma-phosphate of ATP [Grant, B. D. et al. (1998) Biochemistry 37, 7708]. The structural model of the catalytic subunit:ATP:PKI((5)(-)(24)) (heat-stable protein kinase inhibitor) ternary complex in the closed conformation suggests that Ser(53) also might be essential for stabilization of the peptide substrate-enzyme complex via a hydrogen bond between the P-site carbonyl in PKI and the Ser(53) side-chain hydroxyl [Bossemeyer, D. et al. (1993) EMBO J. 12, 849]. To address the importance of the Ser(53) side chain in catalysis, inhibition, and P-site specificity, Ser(53) was replaced with threonine, glycine, and proline. Removal of the side chain (i.e., mutation to glycine) had no effect on the steady-state phosphorylation of a peptide substrate (LRRASLG) or on the interaction with physiological inhibitors, including the type-I and -II regulatory subunits and PKI. However, this mutation did affect the P-site specificity; the glycine mutant can more readily phosphorylate a P-site threonine in a peptide substrate (5-6-fold better than wild-type). The proline mutant is compromised catalytically with altered k(cat) and K(m) for both peptide and ATP and with altered sensitivity to both regulatory subunits and PKI. Steric constraints as well as restricted flexibility could account for these effects. These combined results demonstrate that while the backbone amide of Ser(53) may be required for efficient catalysis, the side chain is not.     

鼻咽癌细胞中p53相互作用蛋白质的分离和鉴定

鼻咽癌中p53基因突变罕见,但绝大部分鼻咽癌中存在p53蛋白过表达/聚集且功能失活.然而,到目前为止p53蛋白失活的机制仍然不清楚.为揭示鼻咽癌中p53蛋白功能失活的机制,采用免疫共沉淀技术分别富集鼻咽癌细胞系HNE1和HNE2的p53结合蛋白,SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）对免疫沉淀复合物进行分离,从胶中切取p53结合蛋白条带,胶内酶解后进行电喷雾串联质谱（LC-ESI-MS/MS）分析,得到相应的肽序列标签（peptide sequence tags, PST）,通过搜索数据库在鼻咽癌细胞系中鉴定了9个p53结合蛋白.分别是热休克蛋白70（HSP70）家族成员GRP-78和GRP-75、HSP90家族成员GRP-94、核纤层蛋白A/C （Lamin A/C）、α-actinin 4、Ezrin/Cytovillin、DNA复制准许因子/MCM3蛋白(DNA replication licensing factor/minichromosome maintenance 3 protein, MCM3)、CD98/4F2 heavy chain和蛋白激酶C（PKC）.并用免疫共沉淀和蛋白质印迹分析技术对HNE₁细胞蛋白条带3鉴定的p53相互作用蛋白之一HSP78进行了验证.首次在鼻咽癌细胞中鉴定了9个p53结合蛋白,为阐明鼻咽癌中p53蛋白聚集及失活的机制提供了重要依据和线索.