A polypeptide in eukaryotic initiation factors that crosslinks specifically to the 5''-terminal cap in mRNA.

Protein synthesis initiation factors prepared from rabbit reticulocyte and mouse ascites ribosomes were tested for the ability to crosslink to the 5' cap of mRNA. Crosslinking of one polypeptide of apparent molecular weight 24,000 was inhibited by the cap analogs, m7GMP and m7GDP, indicating a specific interaction with the cap. Although specific crosslinking of the 24,000 molecular weight polypeptide was found with eukaryotic initiation factor 3 and to a lesser extent with initiation factor 4B, both of these factors contained less than stoichiometric amounts of this polypeptide. The crosslinking method provides a highly sensitive and specific assay for cap-binding proteins and should facilitate their purification for functional studies.     

Induced gene expression of the hypusine-containing protein eukaryotic initiation factor 5A in activated human T lymphocytes.

The hypusine-containing protein eukaryotic initiation factor 5A (eIF-5A) is a cellular cofactor critically required for the function of the Rev transactivator protein of human immunodeficiency virus type 1 (HIV-1). eIF-5A localizes in the nuclear and cytoplasmic compartments of mammalian cells, suggesting possible activities on the level of regulated mRNA transport and/or protein translation. In this report we show that eIF-5A gene expression is constitutively low but inducible with T-lymphocyte-specific stimuli in human peripheral blood mononuclear cells (PBMCs) of healthy individuals. In contrast, eIF-5A is constitutively expressed at high levels in human cell lines as well as in various human organs. Comparison of eIF-5A levels in the PBMCs of uninfected and HIV-1-infected donors shows a significant upregulation of eIF-5A gene expression in the PBMCs of HIV-1 patients, compatible with a possible role of eIF-5A in HIV-1 replication during T-cell activation.     

Hypusination of eukaryotic initiation factor 5A (eIF5A): a novel therapeutic target in BCR-ABL-positive leukemias identified by a proteomics approach

Inhibition of BCR-ABL tyrosine kinase with imatinib represents a major breakthrough in the treatment of patients with chronic myeloid leukemia (CML). However, resistance to imatinib develops frequently, particularly in late-stage disease. To identify new cellular BCR-ABL downstream targets, we analyzed differences in global protein expression in BCR-ABL-positive K562 cells treated with or without imatinib in vitro. Among the 19 proteins found to be differentially expressed, we detected the down-regulation of eukaryotic initiation factor 5A (eIF5A), a protein essential for cell proliferation. eIF5A represents the only known eukaryotic protein activated by posttranslational hypusination. Hypusination inhibitors (HIs) alone exerted an antiproliferative effect on BCR-ABL-positive and -negative leukemia cell lines in vitro. However, the synergistic dose-response relationship found for the combination of imatinib and HI was restricted to Bcr-Abl-positive cells. Furthermore, this synergistic effect was confirmed by cytotoxicity assays, cell-cycle analysis, and CFSE labeling of primary CD34+ CML cells. Specificity of this effect could be demonstrated by cotreatment of K562 cells with imatinib and siRNA against eIF5. In conclusion, through a comparative proteomics approach and further functional analysis, we identified the inhibition of eIF5A hypusination as a promising new approach for combination therapy in BCR-ABL-positive leukemias.     

Specific binding of eukaryotic initiation factor 2 to satellite tobacco necrosis virus RNA at a 5′-terminal sequence comprising the ribosome binding site

The mRNA-binding property of eukaryotic initiation factor 2 (eIF-2) was examined by studying its interaction with satellite tobacco necrosis virus (STNV) RNA carrying a ³²P-labeled 5′ end. The RNA molecules bound by limiting amounts of eIF-2 were isolated and digested with pancreatic and T1 RNases. Digestion patterns showed that the labeled STNV RNA preparation offered to eIF-2 was heterogeneous, containing more than 30 different 5′ ends; by contrast, the RNA selected by eIF-2 possessed predominantly one 5′ end, pApGpUp..., the 5′-terminal sequence of intact STNV RNA. Binding analysis of individual 5′-terminal fragments generated from isolated, intact, STNV RNA by partial digestion with T1 RNase showed that eIF-2 does not bind detectably to the 32-nucleotide fragment ending with the initiation codon AUG or to shorter ones, but it does bind the 44-nucleotide fragment that contains the ribosome binding site. In addition to the structural features localized at the 5′ end of STNV RNA, eIF-2 appears to recognize a conformation found only in larger molecules, because intact RNA and large 5-′-terminal fragments are bound preferentially over smaller ones. However, binding of short 5′-terminal STNV RNA fragments to eIF-2 is specific, as judged by competition with STNV and ribosomal RNA. Finally, binding of eIF-2 to intact STNV RNA leads to a conformational change in the RNA that greatly facilitates cleavage by T1 and P1 RNases at sites in the vicinity of the initiation region. These results show that eIF-2 interacts specifically with the 5′-terminal region of STNV RNA that contains the ribosome binding site and causes local unfolding of the RNA structure.     

Initiation factor 2 crystal structure reveals a different domain organization from eukaryotic initiation factor 5B and mechanism among translational GTPases

The initiation of protein synthesis uses initiation factor 2 (IF2) in prokaryotes and a related protein named eukaryotic initiation factor 5B (eIF5B) in eukaryotes. IF2 is a GTPase that positions the initiator tRNA on the 30S ribosomal initiation complex and stimulates its assembly to the 50S ribosomal subunit to make the 70S ribosome. The 3.1-Å resolution X-ray crystal structures of the full-length Thermus thermophilus apo IF2 and its complex with GDP presented here exhibit two different conformations (all of its domains except C2 domain are visible). Unlike all other translational GTPases, IF2 does not have an effecter domain that stably contacts the switch II region of the GTPase domain. The domain organization of IF2 is inconsistent with the “articulated lever” mechanism of communication between the GTPase and initiator tRNA binding domains that has been proposed for eIF5B. Previous cryo-electron microscopy reconstructions, NMR experiments, and this structure show that IF2 transitions from being flexible in solution to an extended conformation when interacting with ribosomal complexes.The synthesis of proteins in prokaryotes is divided into three distinct processes: initiation, elongation, and termination. The initiation of translation in prokaryotes is directed by three initiation factors (IF1, IF2, and IF3) that govern the binding and positioning of the mRNA, as well as the initiator tRNA, and the joining of ribosomal subunits to form a 70S complex that is ready for the elongation stage of protein synthesis.IF2 is a GTPase that functions to position the initiator tRNA within the 30S ribosomal initiation complex (30S IC) and promotes its joining with the 50S ribosomal subunit to form a 70S ribosome. IF2 is encoded by a single copy of the infB gene and is completely conserved in bacteria (1). The flexible structure of the N terminus has the largest sequence variability among different species (2). Variability also exists within a species; for instance, Escherichia coli IF2 has three isoforms, which vary in the length of their N-terminal domain due to three distinct start sites for its translation initiation (1). The C-terminal part of IF2 (G, II, C1, and C2 domains) contains the highly conserved GTPase domain (G domain) and C2 domain, which interact with the initiator tRNA.The C2 domain recognizes and protects the formylated Met of the initiator tRNA from hydrolysis (3, 4). The formylation of Met results in a fivefold increase in the binding affinity of the tRNA and is made in a G-nucleotide–independent fashion (3, 5). This interaction permits IF2 to assist in positioning the initiator tRNA within a 30S initiation complex and guide the formation of a functional 30S IC on the establishment of the P-site codon–anticodon interaction (2, 6, 7). A functional 30S IC is competent for the 50S ribosomal subunit to join, which is mediated by the formation of the intersubunit salt bridges via an interaction between IF2 and L12 (8). Once the 50S ribosomal subunit joins, IF2 comes into contact with the GTPase activation center, GTP is hydrolyzed by IF2, and IF2 dissociates from the ribosome (2, 9).IF2 is a GTPase homologous to other translational GTPases such as EF-Tu, EF-G, LepA, and RF3 (1). All translational GTPases use a conserved mechanism for GTP hydrolysis in which switch I, switch II and the P-loop are stimulated through interaction with II and VI domains of the 23S rRNA in addition to L11 and L7/L12 of the large ribosomal subunit (1). GTP hydrolysis by IF2 causes it to dissociate from the 70S IC and organizes the 70S ribosome for peptide elongation by a rotation of the ribosomal subunits relative to each other (9–11). Cryo-EM studies of the 30S and 70S particles have shown that the final function of IF2 before it leaves the ribosome is to position the CCA end of the initiator tRNA near the peptidyl transferase center (6, 7, 9, 10).Low-resolution cryo-electron microscopy (cryo-EM) reconstructions have provided structural information about the interactions that govern the initiation process of 70S ribosome assembly in prokaryotes for protein synthesis. No structure from full-length IF2 at atomic resolution has previously been determined. Until recently, the only structural information at atomic resolution on IF2 has been NMR models of four separate domains of IF2 (N, G, C1, and C2) (12–15).The crystal structures of eukaryotic initiation factor 5B (eIF5B) from Methanobacterium thermoautotrophicum have been used to construct a homology model of IF2 to interpret the electron density from cryo-EM reconstructions of the 30S and 70S ICs with IF2 (6, 7, 9, 10). Small angle X-ray scattering (SAXS) studies of E. coli IF2 domains IV–VI (domains G, II, C1, and C2) indicate that its domains are organized differently compared with eIF5B (16). Allen et al. (10) found that the structure of the E. coli IF2 in their cryo-EM reconstruction of a 70S IC differed significantly with that of the crystal structure of M. thermoautotrophicum guanosine 5′-[β,γ-imido]triphosphateeIF5B.Structural information is important for understanding, first how IF2 influences the position of the initiator tRNA in the 30S and 70S ICs, and second how IF2 facilitates joining of the ribosomal subunits to form a 70S ribosome ready for peptide elongation. We address these aspects here and in a companion paper (17).We determined the X-ray crystal structures of Thermus thermophilus IF2 from a full-length protein both with and without GDP bound at 3.09-Å resolution, which improves the model available for interpreting the cryo-EM reconstructions of the 30S and 70S ICs. These two structures exhibit two different conformations of IF2 (in apo and GDP-bound forms), and all of its domains except C2 domain are visible. The initial molecular replacement solution was determined by using a homology model based on an NMR model of the G domain from Geobacillus stearothermophilus (PDB ID code 2LKD) and aligned with the G2 and G3 domains (IF2 G and II domains) of LepA from Aquifex aeolicus (PDB ID codes 2YWE, 2YWF, 2YWG, and 2YWH). While this work was being completed, a crystal structure of the first 363 residues of the T. thermophilus IF2 was determined and that model was then used as an improved search model for molecular replacement (18).This structure of IF2 determined of residues 3–467 exhibits a different 3D organization compared with the homologous domains of eIF5B. We conclude that the organization of the IF2 domains indicates that the communication between the GTPase domain and initiator tRNA binding domain cannot be explained by the “articulated lever” mechanism proposed previously for eIF5B. This difference in domain organization appears to reflect different functions of the two proteins in translation initiation between prokaryotes and eukaryotes. Our crystal structure of isolated IF2 shows that IF2 is unique among crystal structures of isolated translational GTPases because the effecter domains do not directly contact or form a stable interaction with the switch II region of the GTPase domain.     

Conformational control of the interaction of eukaryotic elongation factors EF-1 and EF-2 with ribosomes

As in the case with prokaryotic systems, Artemia salina elongation factors EF-1 and EF-2 interact with a common site or with closely overlapping sites on the Artemia ribosome. This feature of ribosomal design must restrict interaction with the ribosome to only one of the factors at alternating steps of chain elongation. In support of this view we find that EF-1, but not EF-2, interacts with the post-translocation ribosome, whereas the reverse is true of the pre-translocation ribosome. Conformational changes probably account for the alternating selectivity of the translating ribosome for each elongation factor.     

NQO1单核苷酸多态性和环境因素的交互作用与肝细胞癌的关联研究

目的 研究NQO1基因多态性和环境因素的交互作用与肝细胞癌易感性的关系. 方法 采用以医院为基础的病例对照研究方法,运用TaqMan MGB荧光定量实时PCR分析方法对病例组400例肝细胞癌患者和对照组400例非肿瘤患者进行NQO1基因C609T位点的基因型分析.以非条件logistic回归模型分析比较各基因型在两组中分布频率的差异,以及基因多态性和环境因素的交互作用.结果 NQO1基因C609T位点CC、CT和TT各基因型频率分别为23.75％、50.25％和28.00％,对照组中3种基因型频率分别为37.55％、43.75％和18.25％,差异有统计学意义(P＜0.05).与CC基因型相比,CT或者TT基因型的个体罹患HCC的风险OR分别为2.106(95％CI:1.137～3.110)和2.564(95％CI:1.357～4.744).T等位基因携带者患肝细胞癌的危险性是C等位基因携带者的1.86倍(OR=1.86,95％ CI:1.235 ～ 2.980).交互作用分析结果表明NQO1基因多态性与肿瘤家族史、HBsAg阳性之间在肝细胞癌发生中存在交互作用,交互作用的OR值分别为2.431、8.359. 结论 NQO1C609T基因型可能是广西南部地区人群患肝细胞癌的危险因素之一,NQO1基因多态性与肿瘤家族史、HBsAg阳性之间在肝细胞癌发生中存在交互作用,能增加罹患肝细胞癌的风险.     

Isolation and characterization of factors from wheat germ that exhibit eukaryotic initiation factor 4B activity and overcome 7-methylguanosine 5''-triphosphate inhibition of polypeptide synthesis.

Three highly purified preparations (preparations I, II-1, and II-2) have been obtained from wheat germ and shown to support in vitro polypeptide synthesis directed by capped or uncapped mRNAs in a eukaryotic initiation factor 4B (eIF-4B)-deficient system. The three preparations differ, however, in polypeptide composition and in the ability to overcome the inhibitory effect of 7-methylguanosine 5'-triphosphate (m7GTP) on in vitro polypeptide synthesis. Preparation I contains two polypeptides (Mr = 80,000 and 28,000), which are present in a 1:1 molar ratio and are associated in a high molecular weight complex. Preparation II-1 contains two major polypeptides (Mr = 220,000 and 26,000) and preparation II-2 also contains two major polypeptides (Mr = 110,000 and 26,000). Preparations II-1 and II-2 are high molecular weight complexes; neither contains detectable amounts of a Mr 80,000 or a Mr 50,000 component. Preparations II-1 and II-2 both overcome m7GTP inhibition, whereas preparation I does not. These findings raise several questions with regard to the identity of eIF-4B and its relationship to cap recognition factors.     

The observation unit: a new interface between inpatient and outpatient care

PURPOSE: Observation units for patients who present to emergency departments with chest pain have become common. We describe our 3-year experience with a multipurpose observation unit in which chest pain accounts for only a minority of patients' presenting clinical syndromes. SUBJECTS AND METHODS: We analyzed the effects of a 12-bed observation unit on inpatient admissions for common clinical syndromes, as well as its overall effects on inpatient medical admissions during its first 3 years of operation (1996 to 1998) compared with the 3 years preceding its creation (1993 to 1995). RESULTS: Among 7,507 patients admitted to the observation unit in 1996 to 1998, 6,334 (85%) were discharged home within 23 hours. Total inpatient medical admissions fell by a similar number (n = 5,366) during the 3 years of operation of the observation unit when compared with the 3 preceding years (39,569 admissions in 1996 to1998 versus 44,935 in 1993 to 1995). Analysis of local area trends suggested that the use of the observation unit contributed to reduced hospital admissions, rather than vice versa. CONCLUSION: Observation units can serve patients with diverse clinical syndromes and may reduce inpatient admissions. This novel "point of care" deserves further evaluation.     

A generalized seizure following initiation of nelfinavir in a patient with human immunodeficiency virus type 1 infection, suspected due to interaction between nelfinavir and phenytoin.

Nelfinavir, one of human immunodeficiency virus (HIV) specific protease inhibitors(PIs), is widely used for the treatment of HIV infection. Nelfinavir, which is metabolized with the cytochrome p450 isoforms, elevate the phenytoin level theoretically because nelfinavir acts as an inhibitor of phenytoin metabolism through the enzyme. However, we encountered a case of seizure recurrence caused by a lowered phenytoin level after initiation of nelfinavir. We should be aware of the change in the phenytoin level in concomitant use of nelfinavir.     

Inhibition of the interactions between eukaryotic initiation factors 4E and 4G impairs long-term associative memory consolidation but not reconsolidation

Considerable evidence indicates that the general blockade of protein synthesis prevents both the initial consolidation and the postretrieval reconsolidation of long-term memories. These findings come largely from studies of drugs that block ribosomal function, so as to globally interfere with both cap-dependent and -independent forms of translation. Here we show that intra-amygdala microinfusions of 4EGI-1, a small molecule inhibitor of cap-dependent translation that selectively disrupts the interaction between eukaryotic initiation factors (eIF) 4E and 4G, attenuates fear memory consolidation but not reconsolidation. Using a combination of behavioral and biochemical techniques, we provide both in vitro and in vivo evidence that the eIF4E-eIF4G complex is more stringently required for plasticity induced by initial learning than for that triggered by reactivation of an existing memory.     

Characterization of the interaction between polymerized human albumin and hepatitis B surface antigen

The interaction of polymerized human albumin with hepatitis B surface antigen was characterized utilizing a solid-phase radioimmunoassay. The interaction was specific as shown by quantitative inhibition of binding by soluble polymerized human albumin. The interaction was species restricted in that hepatitis B surface antigen did not bind nonhuman polymerized albumins. The albumin polymer size was critical to the binding reaction as was the concentration of hepatitis B surface antigen; the interaction was also temperature, ionic strength, and pH dependent. Hepatitis B e antigen positive sera possessed greater polyalbumin binding reactivity than hepatitis B e antigen negative sera. This interaction was shown to be inhibited by normal human serum which suggested that the polyalbumin receptor on hepatitis B surface antigen may be a host rather than viral component. In view of our previous observation that polymerized human albumin binds human C1q, the interactions between polymerized human albumin, hepatitis B surface antigen, and human C1q may be relevant to hepatitis B virus-host receptor mechanisms.     

Archaeal translation initiation revisited: The initiation factor 2 and eukaryotic initiation factor 2B α-β-δ subunit families

As the amount of available sequence data increases, it becomes apparent that our understanding of translation initiation is far from comprehensive and that prior conclusions concerning the origin of the process are wrong. Contrary to earlier conclusions, key elements of translation initiation originated at the Universal Ancestor stage, for homologous counterparts exist in all three primary taxa. Herein, we explore the evolutionary relationships among the components of bacterial initiation factor 2 (IF-2) and eukaryotic IF-2 (eIF-2)/eIF-2B, i.e., the initiation factors involved in introducing the initiator tRNA into the translation mechanism and performing the first step in the peptide chain elongation cycle. All Archaea appear to posses a fully functional eIF-2 molecule, but they lack the associated GTP recycling function, eIF-2B (a five-subunit molecule). Yet, the Archaea do posses members of the gene family defined by the (related) eIF-2B subunits α, β, and δ, although these are not specifically related to any of the three eukaryotic subunits. Additional members of this family also occur in some (but by no means all) Bacteria and even in some eukaryotes. The functional significance of the other members of this family is unclear and requires experimental resolution. Similarly, the occurrence of bacterial IF-2-like molecules in all Archaea and in some eukaryotes further complicates the picture of translation initiation. Overall, these data lend further support to the suggestion that the rudiments of translation initiation were present at the Universal Ancestor stage.     

Relationship between structure of the 5'' noncoding region of viral mRNA and efficiency in the initiation step of protein synthesis in a eukaryotic system.

To determine whether the rate of protein synthesis is controlled by the structure of mRNA near its 5' terminus, protein-synthesizing ability, especially in its initial stage, was compared among RNAs of plant viruses. Those viruses used here contain several definite pieces of single-stranded RNA. Each of these RNAs acts as a messenger. Cucumber mosaic virus (CMV) RNA 5 synthesizes a small amount of a protein, Mr 7000, in an in vitro protein-synthesizing system from wheat germ or reticulocyte. Brome mosaic virus (BMV) RNA 4 synthesizes a large amount of a coat protein under the same conditions. Both RNAs have the same 5'-cap structure and a short noncoding region (10 nucleotides in CMV RNA 5 and 9 in BMV RNA 4) between the 5' terminus and the initiation codon AUG. A sequence complementary to the 3' terminal of 18S ribosomal RNA is contained in BMV RNA 4 but is not apparent in CMV RNA 5. Formation of the initiation complex for protein synthesis by the 5'-terminal-labeled mRNA of cytoplasmic polyhedrosis virus was inhibited by the addition of unlabeled BMV RNA 4 whereas it was only slightly inhibited by unlabeled CMV RNA 5. BMV RNA 4, which has a sequence complementary to rRNA, can form the initiation complex more easily than CMV RNA5. It is concluded that an apparent complementary sequence in the 3' terminal of 18S rRNA in the 5' noncoding region of eukaryotic mRNA and the 5'-cap structure enhance the rate of initiation complex formation in protein synthesis.     

Mapping of human microtubule-associated protein 1B in proximity to the spinal muscular atrophy locus at 5q13.

A polyclonal antiserum directed against the C-terminal domain of dystrophin was used to isolate a cDNA clone encoding an antigenically cross-reactive protein, microtubule-associated protein 1B (MAP-1B). Physical mapping of the human MAP-1B locus places its chromosomal location at 5q13, in proximity to the spinal muscular atrophy (SMA) locus. SMA is a degenerative disorder primarily affecting motor neurons. Genetic linkage analysis of SMA families using a human dinucleotide repeat polymorphism just 3' of the MAP-1B gene has shown tight linkage to SMA mutations. These mapping data together with the postulated role of MAP-1B in neuronal morphogenesis and its localization in anterior horn motor neurons suggest a possible association with SMA.