Isokinetic concentric quadriceps and hamstring normative data for elite collegiate American football players participating in the NFL Scouting Combine.

Isokinetic concentric quadriceps and hamstring strength data using a Cybex dynamometer are collected for elite collegiate American football players invited to the annual National Football League Scouting Combine. We constructed a normative (reference) database of the Cybex strength data for the purpose of allowing comparison of an individual's values to his peers. Data reduction was performed to construct frequency distributions of hamstring/quadriceps (H/Q) ratios and side-to-side strength differences. For the cohort (n = 1,252 players), a statistically significant but very small (1.9%) mean quadriceps strength preference existed for dominant side vs. nondominant side. Peak torque (Newton meters, best repetition) for quadriceps and hamstrings was significantly correlated to player body mass (weight) (the same relationship was found for other variables using peak torque in the calculation). Peak torque varied by player position, being greatest for offensive linemen and lowest for kickers (p < 0.0001). Adjusting for body weight overcorrected these differences. The H/Q ratios and frequency distributions were similar across positions, with a mean of 0.6837 ± 0.137 for the cohort dominant side vs. 0.6940 ± 0.145 for the nondominant side (p = 0.021, n = 1,252). Considerable variation was seen for dominant-to-nondominant side difference for peak torque. For quadriceps, 47.2% of players had differences between -10% and +10%, 21.0% had a peak torque dominant-side deficit of 10% or greater compared to nondominant side, and for 31.8% of players, dominant-side peak torque was greater than 10% compared to nondominant side. For hamstrings, 57.0% of players had differences between -10% and +10%, 19.6% had a peak torque dominant-side deficit of 10% or greater compared to nondominant side, and 23.4% of players, dominant-side peak torque was greater than 10% compared to nondominant side. We observed that isokinetic absolute strength variables are dependent on body weight and vary across player position. The H/Q ratios vary only within a relatively narrow range. Side-to-side differences in strength variables >10% are common, not the exception.

Comparative efficiencies of C-terminal signals of native glycophosphatidylinositol (GPI)-anchored proproteins in conferring GPI-anchoring.

Every protein fated to receive the glycophosphatidylinositol (GPI) anchor post-translational modification has a C-terminal GPI-anchor attachment signal sequence. This signal peptide varies with respect to length, content, and hydrophobicity. With the exception of predictions based on an upstream amino acid triplet termed omega-->omega + 2 which designates the site of GPI uptake, there is no information on how the efficiencies of different native signal sequences compare in the transamidation reaction that catalyzes the substitution of the GPI anchor for the C-terminal peptide. In this study we utilized the placental alkaline phosphatase (PLAP) minigene, miniPLAP, and replaced its native 3' end-sequence encoding omega-2 to the C-terminus with the corresponding C-terminal sequences of nine other human GPI-anchored proteins. The resulting chimeras then were fed into an in vitro processing microsomal system where the cleavages leading to mature product from the nascent preproprotein could be followed by resolution on an SDS-PAGE system after immunoprecipitation. The results showed that the native signal of each protein differed markedly with respect to transamidation efficiency, with the signals of three proteins out-performing the others in GPI-anchor addition and those of two proteins being poorer substrates for the GPI transamidase. The data additionally indicated that the hierarchical order of efficiency of transamidation did not depend solely on the combination of permissible residues at omega-->omega + 2.

Modulation of the helicase activity of eIF4A by eIF4B, eIF4H, and eIF4F.

Eukaryotic initiation factor (eIF) 4A is a DEAD box RNA helicase that works in conjunction with eIF4B, eIF4H, or as a subunit of eIF4F to unwind secondary structure in the 5'-untranslated region of mRNA, which facilitates binding of the mRNA to the 40 S ribosomal subunit. This study demonstrates how the helicase activity of eIF4A is modulated by eIF4B, eIF4H, or as a subunit of eIF4F. Results indicate that a linear relationship exists between the initial rate or amplitude of unwinding and duplex stability for all factor combinations tested. eIF4F, like eIF4A, behaves as a non-processive helicase. Either eIF4B or eIF4H stimulated the initial rate and amplitude of eIF4A-dependent duplex unwinding, and the magnitude of stimulation is dependent on duplex stability. Furthermore, eIF4A (or eIF4F) becomes a slightly processive helicase in the presence of eIF4B or eIF4H. All combinations of factors tested indicate that the rate of duplex unwinding is equivalent in the 5' --> 3' and 3' --> 5' directions. However, the optimal rate of unwinding was dependent on the length of the single-stranded region of the substrate when different combinations of factors were used. The combinations of eIF4A, eIF4A + eIF4B, eIF4A + eIF4H, and eIF4F showed differences in their ability to unwind chemically modified duplexes. A simple model of how eIF4B or eIF4H affects the duplex unwinding mechanism of eIF4A is proposed.

Further characterization of the helicase activity of eIF4A. Substrate specificity.

Eukaryotic initiation factor (eIF) 4A is the archetypal member of the DEAD box family of RNA helicases and is proposed to unwind structures in the 5'-untranslated region of mRNA to facilitate binding of the 40 S ribosomal subunit. The helicase activity of eIF4A has been further characterized with respect to substrate specificity and directionality. Results confirm that the initial rate and amplitude of duplex unwinding by eIF4A is dependent on the overall stability, rather than the length or sequence, of the duplex substrate. eIF4A helicase activity is minimally dependent on the length of the single-stranded region adjacent to the double-stranded region of the substrate. Interestingly, eIF4A is able to unwind blunt-ended duplexes. eIF4A helicase activity is also affected by substitution of 2'-OH (RNA) groups with 2'-H (DNA) or 2'-methoxyethyl groups. These observations, taken together with results from competitive inhibition experiments, suggest that eIF4A may interact directly with double-stranded RNA, and recognition of helicase substrates occurs via chemical and/or structural features of the duplex. These results allow for refinement of a previously proposed model for the mechanism of action of eIF4A helicase activity.

Internal ribosome entry site-mediated translation of a mammalian mRNA is regulated by amino acid availability.

The cationic amino acid transporter, Cat-1, facilitates the uptake of the essential amino acids arginine and lysine. Amino acid starvation causes accumulation and increased translation of cat-1 mRNA, resulting in a 58-fold increase in protein levels and increased arginine uptake. A bicistronic mRNA expression system was used to demonstrate the presence of an internal ribosomal entry sequence (IRES) within the 5'-untranslated region of the cat-1 mRNA. This study shows that IRES-mediated translation of the cat-1 mRNA is regulated by amino acid availability. This IRES causes an increase in translation under conditions of amino acid starvation. In contrast, cap-dependent protein synthesis is inhibited during amino acid starvation, which is well correlated with decreased phosphorylation of the cap-binding protein, eIF4E. These findings reveal a new aspect of mammalian gene expression and regulation that provides a cellular stress response; when the nutrient supply is limited, the activation of IRES-mediated translation of mammalian mRNAs results in the synthesis of proteins essential for cell survival.

Characterization of the two eIF4A-binding sites on human eIF4G-1.

Eukaryotic translation initiation factor 4G-1 (eIF4G) plays a critical role in the recruitment of mRNA to the 43 S preinitiation complex. eIF4G has two binding sites for the RNA helicase eIF4A, one in the central domain and one in the COOH-terminal domain. Recombinant eIF4G fragments that contained each of these sites separately bound eIF4A with a 1:1 stoichiometry, but fragments containing both sites bound eIF4A with a 1:2 stoichiometry. eIF3 did not interfere with eIF4A binding to the central site. Interestingly, at the same concentration of free eIF4A, more eIF4A was bound to an eIF4G fragment containing both eIF4A sites than the sum of binding to fragments containing the single sites, indicating cooperative binding. Binding of eIF4A to an immobilized fragment of eIF4G containing the COOH-terminal site was competed by a soluble eIF4G fragment containing the central site, indicating that a single eIF4A molecule cannot bind simultaneously to both sites. The association rate constant, dissociation rate constant, and dissociation equilibrium constant for each site were determined by surface plasmon resonance and found to be, respectively, 1.2 x 10(5) m(-1) s(-1), 2.1 x 10(-3) s(-1), and 17 nm for the central site and 5.1 x 10(3) m(-1) s(-1), 1.7 x 10(-3) s(-1), and 330 nm for the COOH-terminal site.

Analysis of eukaryotic translation in purified and semipurified systems.

Much of the current understanding of the sequential steps involved in translation initiation has been obtained using sucrose gradients to isolate ribosomes and ribosomal subunits, as described here. These purified components are combined with purified translation factors to analyze the formation of intermediates in translation initiation and the roles of the translation factors in vitro.

A new pathway of translational regulation mediated by eukaryotic initiation factor 3.

We report a new pathway of translation regulation that may operate in interferon-treated or virus-infected mammalian cells. This pathway is activated by P56, a protein whose synthesis is strongly induced by interferons or double-stranded RNA. Using a yeast two-hybrid screen, we identified the P48 subunit of the mammalian translation initiation factor eIF-3 as a protein that interacts with P56. The P56-P48 interaction was confirmed in human cells by co-immunoprecipitation assays and confocal microscopy. Gel filtration assays revealed that P56 binds to the large eIF-3 complex that contains P48. Purified recombinant P56 inhibited in vitro translation of reporter mRNAs in a dose-dependent fashion, and that inhibition was reversed by the addition of purified eIF-3. In vivo, expression of transfected P56 or induction of the endogenous P56 by interferon caused an inhibition of overall cellular protein synthesis and the synthesis of a transfected reporter protein. As expected, a P56 mutant that does not interact with P48 and eIF-3 failed to inhibit protein synthesis in vitro and in vivo.

Further biochemical and kinetic characterization of human eukaryotic initiation factor 4H.

A cDNA encoding human eukaryotic initiation factor (eIF) 4H was subcloned into a bacterial expression plasmid for purification of recombinant protein. Recombinant human eIF4H (heIF4H) was purified to greater than 95% homogeneity and shown to have similar physical characteristics to eIF4H purified from rabbit reticulocyte lysate as described previously. Functional studies have revealed that recombinant heIF4H functions identically to rabbit eIF4H in stimulating protein synthesis, and the ATP hydrolysis and helicase activities of eIF4A. More detailed enzymatic studies revealed that eIF4H increases the affinity of eIF4A for RNA by 2-fold, but has no effect on the binding of ATP by eIF4A. eIF4H stimulates the helicase activity of eIF4A at least 4-fold, and it is postulated that this stimulation occurs through increasing the processivity of eIF4A. Northern blot analysis shows that eIF4H is expressed ubiquitously in human tissues, and displays different levels of expression in given tissues relative to eIF4B. Secondary structure analysis of heIF4H by circular dichroism suggest that eIF4H has a mostly beta-sheet structure, which appears similar to other RNA recognition motif-containing proteins. Finally, it is suggested that eIF4H functions in translation initiation through protein-protein interactions that possibly stabilize conformational changes that occur in eIF4A during RNA binding, ATP hydrolysis, and RNA duplex unwinding.

Eukaryotic translation initiation factor 4AIII (eIF4AIII) is functionally distinct from eIF4AI and eIF4AII.

Eukaryotic initiation factor 4A (eIF4A) is an RNA-dependent ATPase and ATP-dependent RNA helicase that is thought to melt the 5' proximal secondary structure of eukaryotic mRNAs to facilitate attachment of the 40S ribosomal subunit. eIF4A functions in a complex termed eIF4F with two other initiation factors (eIF4E and eIF4G). Two isoforms of eIF4A, eIF4AI and eIF4AII, which are encoded by two different genes, are functionally indistinguishable. A third member of the eIF4A family, eIF4AIII, whose human homolog exhibits 65% amino acid identity to human eIF4AI, has also been cloned from Xenopus and tobacco, but its function in translation has not been characterized. In this study, human eIF4AIII was characterized biochemically. While eIF4AIII, like eIF4AI, exhibits RNA-dependent ATPase activity and ATP-dependent RNA helicase activity, it fails to substitute for eIF4AI in an in vitro-reconstituted 40S ribosome binding assay. Instead, eIF4AIII inhibits translation in a reticulocyte lysate system. In addition, whereas eIF4AI binds independently to the middle and carboxy-terminal fragments of eIF4G, eIF4AIII binds to the middle fragment only. These functional differences between eIF4AI and eIF4AIII suggest that eIF4AIII might play an inhibitory role in translation under physiological conditions.

Mutations in a GTP-binding motif of eukaryotic elongation factor 1A reduce both translational fidelity and the requirement for nucleotide exchange.

A series of mutations in the highly conserved N(153)KMD(156)GTP-binding motif of the Saccharomyces cerevisiae translation elongation factor 1A (eEF1A) affect the GTP-dependent functions of the protein and increase misincorporation of amino acids in vitro. Two critical regulatory processes of translation elongation, guanine nucleotide exchange and translational fidelity, were analyzed in strains with the N153T, D156N, and N153T/D156E mutations. These strains are omnipotent suppressors of nonsense mutations, indicating reduced A site fidelity, which correlates with changes either in total translation rates in vivo or in GTPase activity in vitro. All three mutant proteins also show an increase in the K(m) for GTP. An in vivo system lacking the guanine nucleotide exchange factor eukaryotic elongation factor 1Balpha (eEF1Balpha) and supported for growth by excess eEF1A was used to show the two mutations with the highest K(m) for GTP restore most but not all growth defects found in these eEF1Balpha deficient-strains to near wild type. An increase in K(m) alone, however, is not sufficient for suppression and may indicate eEF1Balpha performs additional functions. Additionally, eEF1A mutations that suppress the requirement for guanine nucleotide exchange may not effectively perform all the functions of eEF1A in vivo.

Biochemical and kinetic characterization of the RNA helicase activity of eukaryotic initiation factor 4A.

Eukaryotic initiation factor (eIF) 4A is the prototypic member of the DEAD box family of proteins and has been proposed to act as an RNA helicase to unwind secondary structure in the 5'-untranslated region of eukaryotic mRNAs. Previous studies have shown that the RNA helicase activity of eIF4A is dependent on the presence of a second initiation factor, eIF4B. In this report, eIF4A has been demonstrated to function independently of eIF4B as an ATP-dependent RNA helicase. The biochemical and kinetic properties of this activity were examined. By using a family of RNA duplexes with an unstructured single-stranded region followed by a duplex region of increasing length and stability, it was observed that the initial rate of duplex unwinding decreased with increasing stability of the duplex. Furthermore, the maximum amount of duplex unwound also decreased with increasing stability. Results suggest that eIF4A acts in a non-processive manner. eIF4B and eIF4H were shown to stimulate the helicase activity of eIF4A, allowing eIF4A to unwind longer, more stable duplexes with both an increase in initial rate and maximum amount of duplex unwound. A simple kinetic model is proposed to explain the mechanism by which eIF4A unwinds RNA duplex structures in an ATP-dependent manner.

Site-directed mutagenesis of yeast eEF1A. Viable mutants with altered nucleotide specificity.

Site-directed mutants of eEF1A (formerly eEF-1alpha) were generated using a modification of a highly versatile yeast shuttle vector (Cavallius, J., Popkie, A. P., and Merrick, W. C. (1997) Biochim. Biophys. Acta 1350, 345-358). The nucleotide specificity sequence NKMD (residues number 153-156) was targeted for mutagenesis, and the following mutants were obtained: N153D (DKMD), N153T (TKMD), D156N (NKMN), D156W (NKMW), and the double mutant N153T,D156E (TKNE). All of the yeast strains containing the mutant eEF1As as the sole source of eEF1A were viable except for the N153D mutant. Most of the purified mutant eEF1As had specific activities in the poly(U)-directed synthesis of polyphenylalanine similar to wild type, although with a Km for GTP increased by 1-2 orders of magnitude. The mutants showed a reduced rate of GTP hydrolysis, and most displayed misincorporation rates greater than wild type. The mutant NKMW eEF1A showed unusual properties. The yeast strain was temperature sensitive for growth, although the purified protein was not. Second, this form of eEF1A was 10-fold more accurate in protein synthesis, and its rate of GTP hydrolysis was about 20% of wild type. In total, the wild-type protein contains the most optimal nucleotide specificity sequence, NKMD, and even subtle changes in this sequence have drastic consequences on eEF1A function in vitro or yeast viability.

The elongation factor 1 A-2 isoform from rabbit: cloning of the cDNA and characterization of the protein.

Eukaryotic elongation factor 1 A (eEF1A, formerly elongation factor-1 alpha) is an important component of the protein synthesis apparatus. Here we report the isolation and characterization of the cDNA sequence encoding rabbit eEF1A-2, an isoform of eEF1A, as well as a structural and functional comparison of the two rabbit isoforms. Northern analysis of the expression pattern of eEF1A-2 showed that this isoform is expressed in skeletal muscle, heart, brain and aorta, while transcripts are not detected in liver, kidney, spleen and lung. In contrast, the previously characterized eEF1A-1 isoform is expressed in all tissues examined except skeletal muscle. We have recently purified eEF1A-2 from rabbit skeletal muscle. By partial amino acid sequencing and determination of the post-translational modifications of eEF1A-2 we found that both of the glycerylphosphorylethanolamine modifications observed in eEF1A-1 appear to be present in eEF1A-2. However, two of the residues found dimethylated in eEF1A-1 appeared to be trimethylated in eEF1A-2. A comparison of the enzymatic activity showed that eEF1A-1 and eEF1A-2 have indistinguishable activity in an in vitro translation system. In contrast, the GDP dissociation rate constant is approximately 7 times higher for eEF1A-1 than for eEF1A-2. The nucleotide preference ratio (GDP/GTP) for eEF1A-1 was 0.82, while the preference ratio for eEF1A-2 was 1.50.

Promotion of met-tRNAiMet binding to ribosomes by yIF2, a bacterial IF2 homolog in yeast.

Delivery of the initiator methionine transfer RNA (Met-tRNAiMet) to the ribosome is a key step in the initiation of protein synthesis. Previous results have indicated that this step is catalyzed by the structurally dissimilar translation factors in prokaryotes and eukaryotes-initiation factor 2 (IF2) and eukaryotic initiation factor 2 (eIF2), respectively. A bacterial IF2 homolog has been identified in both eukaryotes and archaea. By using a combination of molecular genetic and biochemical studies, the Saccharomyces cerevisiae IF2 homolog is shown to function in general translation initiation by promoting Met-tRNAiMet binding to ribosomes. Thus, the mechanism of protein synthesis in eukaryotes and prokaryotes is more similar than was previously realized.

Purification and characterisation of a tissue specific elongation factor 1 alpha (EF-1 alpha 2) from rabbit muscle.

The peptide elongation factor 1 alpha (EF-1 alpha) has been isolated and characterised from a number of species. Recently we and others have reported the existence of an isoform of the ubiquitously expressed EF-1 alpha mRNA in higher eukaryotes, including human cells. This isoform has a tissue specific expression pattern, confining it primarily to muscle, heart, and brain. In the present study we have purified the isoform of EF-1 alpha from rabbit muscle. Using partial amino acid analysis, we can conclude that in rabbit muscle essentially only the isoform of elongation factor 1 alpha, designated EF-1 alpha 2, is translated. Preliminary activity assays show that the isoform has the same functional activities as the normal EF-1 alpha, designated EF-1 alpha 1, in relation to protein synthesis, but may behave differently in the ability to bind nucleotides. Based on the availability of the isoforms of EF-1 alpha purified from a mammalian species, it will be possible to conduct further comparative studies in order to elucidate the different functions of EF-1 alpha 1 and EF-1 alpha 2 proteins.

Purification and characterization of a new eukaryotic protein translation factor. Eukaryotic initiation factor 4H.

A new protein with translational activity has been identified on the basis of its ability to stimulate translation in an in vitro globin synthesis assay deficient in eukaryotic initiation factor (eIF) 4B and eIF4F. This protein has been purified to greater than 80% homogeneity from rabbit reticulocyte lysate and has been given the name eIF4H. eIF4H was shown to stimulate the in vitro activities of eIF4B and eIF4F in globin synthesis, as well as the in vitro RNA-dependent ATPase activities of eIF4A, eIF4B, and eIF4F. Three tryptic fragments of eIF4H yielded amino acid sequences that were 100% identical to a human sequence found in the GeneBankTM that codes for a previously uncharacterized protein (HUMORFU_1). The calculated molecular weight of the protein encoded by this sequence, its predicted cyanogen bromide fragmentation, and calculated isoelectric point are all consistent with those determined experimentally for eIF4H. Also, the presence of an RNA recognition motif within HUMORFU_1 suggests that eIF4H may interact with mRNA. We conclude that this newly characterized protein, eIF4H, functions to stimulate the initiation of protein synthesis at the level of mRNA utilization, and is encoded by the gene for HUMORFU_1.

Structure of cDNAs encoding human eukaryotic initiation factor 3 subunits. Possible roles in RNA binding and macromolecular assembly.

The mammalian translation initiation factor 3 (eIF3), is a multiprotein complex of approximately 600 kDa that binds to the 40 S ribosome and promotes the binding of methionyl-tRNAi and mRNA. cDNAs encoding 5 of the 10 subunits, namely eIF3-p170, -p116, -p110, -p48, and -p36, have been isolated previously. Here we report the cloning and characterization of human cDNAs encoding the major RNA binding subunit, eIF3-p66, and two additional subunits, eIF3-p47 and eIF3-p40. Each of these proteins is present in immunoprecipitates formed with affinity-purified anti-eIF3-p170 antibodies. Human eIF3-p66 shares 64% sequence identity with a hypothetical Caenorhabditis elegans protein, presumably the p66 homolog. Deletion analyses of recombinant derivatives of eIF3-p66 show that the RNA-binding domain lies within an N-terminal 71-amino acid region rich in lysine and arginine. The N-terminal regions of human eIF3-p40 and eIF3-p47 are related to each other and to 17 other eukaryotic proteins, including murine Mov-34, a subunit of the 26 S proteasome. Phylogenetic analyses of the 19 related protein sequences, called the Mov-34 family, distinguish five major subgroups, where eIF3-p40, eIF3-p47, and Mov-34 are each found in a different subgroup. The subunit composition of eIF3 appears to be highly conserved in Drosophila melanogaster, C. elegans, and Arabidopsis thaliana, whereas only 5 homologs of the 10 subunits of mammalian eIF3 are encoded in S. cerevisiae.

The translation initiation factor eIF3-p48 subunit is encoded by int-6, a site of frequent integration by the mouse mammary tumor virus genome.

Translation initiation factor eIF3 is a large, multisubunit protein complex that plays a central role in the pathway of initiation by promoting the binding of both methionyl-tRNAi and mRNA to the 40S ribosomal subunit. As part of a broad effort to elucidate the structure of eIF3, we have cloned and sequenced the human cDNA encoding the 48-kDa subunit, eIF3-p48. The recombinant protein comigrates with the authentic p48 subunit in purified eIF3 and coprecipitates with affinity-purified antibodies to the p170 subunit of eIF3. A search of the data base indicates that the mouse gene encoding eIF3-p48 had previously been identified and characterized by others as int-6. The int-6 gene is the site of frequent integration of mouse mammary tumor virus DNA into chromosomes, implicating the gene in the regulation of cell proliferation. In addition, it was shown elsewhere that the homologous human int-6 gene product binds to the human T-cell leukemia virus type I Tax protein, leading to the translocation of Int-6 to the cytoplasm. We discuss how the cytosolic function of eIF3-p48 (Int-6) in protein synthesis may account for oncogenesis caused by these two viruses.