Crystal Structure of the α Subunit of Human Translation Initiation Factor 2B

Eukaryotic translation initiation factor 2B (eIF2B) is the heteropentameric guanine-nucleotide exchange factor specific for eukaryotic initiation factor 2 (eIF2). Under stressed conditions, guanine-nucleotide exchange is strongly inhibited by the tight binding of phosphorylated eIF2 to eIF2B. Here, we report the crystal structure of the α subunit of human eIF2B at 2.65 Å resolution. The eIF2Bα structure consists of the N-terminal α-helical domain and the C-terminal Rossmann-fold-like domain. A positively charged pocket, whose entrance is about 15-17 Å in diameter, resides at the boundary between the two domains. A sulfate ion is located at the bottom of the pocket (about 16 Å in depth). The residues comprising the sulfate-ion-binding site are strictly conserved in eIF2Bα. Since this deep, wide pocket with the sulfate-ion-binding site is not conserved in distant homologues, including 5-methylthioribose 1-phosphate isomerases, these characteristics may be distinctive of eIF2Bα. Interestingly, the yeast eIF2Bα missense mutations that reduce the eIF2B sensitivity to phosphorylated eIF2 are mapped on the other side of the pocket. One of the three human eIF2Bα missense mutations that induce the lethal brain disorder vanishing white matter or childhood ataxia with central nervous system hypomyelination is mapped inside the pocket. The β and δ subunits of eIF2B are homologous to eIF2Bα and may have tertiary structures similar to the present eIF2Bα structure. The sulfate-ion-binding residues of eIF2Bα are well conserved in eIF2Bβ/δ. The abovementioned yeast and human missense mutations of eIF2Bβ/δ were also mapped on the eIF2Bα structure, which revealed that the human mutations are clustered on the same side as the pocket, while the yeast mutations reside on the opposite side. As most of the mutated residues are exposed on the surface of the eIF2B subunit structure, these exposed residues are likely to be involved in either the subunit interactions or the interaction with eIF2.     

A Subunit of Eukaryotic Translation Initiation Factor 2α-Phosphatase (CreP/PPP1R15B) Regulates Membrane Traffic

The constitutive reverter of eIF2α phosphorylation (CReP)/PPP1r15B targets the catalytic subunit of protein phosphatase 1 (PP1c) to phosphorylated eIF2α (p-eIF2α) to promote its dephosphorylation and translation initiation. Here, we report a novel role and mode of action of CReP. We found that CReP regulates uptake of the pore-forming Staphylococcus aureus α-toxin by epithelial cells. This function was independent of PP1c and translation, although p-eIF2α was involved. The latter accumulated at sites of toxin attack and appeared conjointly with α-toxin in early endosomes. CReP localized to membranes, interacted with phosphomimetic eIF2α, and, upon overexpression, induced and decorated a population of intracellular vesicles, characterized by accumulation of N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), a lipid marker of exosomes and intralumenal vesicles of multivesicular bodies. By truncation analysis, we delineated the CReP vesicle induction/association region, which comprises an amphipathic α-helix and is distinct from the PP1c interaction domain. CReP was also required for exocytosis from erythroleukemia cells and thus appears to play a broader role in membrane traffic. In summary, the mammalian traffic machinery co-opts p-eIF2α and CReP, regulators of translation initiation.     

Translation Initiation Factor eIF3b Contains a Nine-Bladed β-Propeller and Interacts with the 40S Ribosomal Subunit

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Archaeal aIF2B Interacts with Eukaryotic Translation Initiation Factors eIF2α and eIF2Bα: Implications for aIF2B Function and eIF2B Regulation

Translation initiation is down-regulated in eukaryotes by phosphorylation of the α-subunit of eIF2 (eukaryotic initiation factor 2), which inhibits its guanine nucleotide exchange factor, eIF2B. The N-terminal S1 domain of phosphorylated eIF2α interacts with a subcomplex of eIF2B formed by the three regulatory subunits α/GCN3, β/GCD7, and δ/GCD2, blocking the GDP-GTP exchange activity of the catalytic ?-subunit of eIF2B. These regulatory subunits have related sequences and have sequences in common with many archaeal proteins, some of which are involved in methionine salvage and CO₂ fixation. Our sequence analyses however predicted that members of one phylogenetically distinct and coherent group of these archaeal proteins [designated aIF2Bs (archaeal initiation factor 2Bs)] are functional homologs of the α, β, and δ subunits of eIF2B. Three of these proteins, from different archaea, have been shown to bind in vitro to the α-subunit of the archaeal aIF2 from the cognate archaeon. In one case, the aIF2B protein was shown further to bind to the S1 domain of the α-subunit of yeast eIF2 in vitro and to interact with eIF2Bα/GCN3 in vivo in yeast. The aIF2B-eIF2α interaction was however independent of eIF2α phosphorylation. Mass spectrometry has identified several proteins that co-purify with aIF2B from Thermococcus kodakaraensis, and these include aIF2α, a sugar-phosphate nucleotidyltransferase with sequence similarity to eIF2B?, and several large-subunit (50S) ribosomal proteins. Based on this evidence that aIF2B has functions in common with eIF2B, the crystal structure established for an aIF2B was used to construct a model of the eIF2B regulatory subcomplex. In this model, the evolutionarily conserved regions and sites of regulatory mutations in the three eIF2B subunits in yeast are juxtaposed in one continuous binding surface for phosphorylated eIF2α.     

Expression and Purification of the α-Subunit of Eukaryotic Initiation Factor eIF2: Use as a Kinase Substrate

The α-subunit of eukaryotic initiation factor eIF2 (eIF2α) plays an important role in the regulation of mRNA translation through modulation of the interaction of eIF2 and a second initiation factor, eIF2B. The interaction of the two proteins is regulatedin vivoby phosphorylation of eIF2α at Ser⁵¹. In the present study, rat eIF2α was expressed in Sf21 cells using the baculovirus expression system. The recombinant protein was purified to >90% homogeneity in a single immunoaffinity chromatographic step. The protein was free of endogenous eIF2α kinase activity and was rapidly phosphorylated by the eIF2α kinases HCR and PKR. A variant of eIF2α in which the phosphorylation site was changed to Ala was also expressed and purified. The variant eIF2α was not phosphorylated by either HCR or PKR, demonstrating that the kinases specifically phosphorylate the correct site in the recombinant protein even in the absence of the other two subunits of the protein. In summary, a rapid and inexpensive method for obtaining eIF2α has been developed. Use of the wildtype and variant forms of eIF2α to measure eIF2α kinase activity in cell and tissue extracts should greatly facilitate examination of the regulation of mRNA translation under a variety of conditions.     

The Role of Nitric-oxide Synthase in the Regulation of UVB Light-induced Phosphorylation of the �� Subunit of Eukaryotic Initiation Factor 2

UV light induces phosphorylation of the α subunit of the eukaryotic initiation factor 2 (eIF2α) and inhibits global protein synthesis. Both eIF2 kinases, protein kinase-like endoplasmic reticulum kinase (PERK) and general control of nonderepressible protein kinase 2 (GCN2), have been shown to phosphorylate eIF2α in response to UV irradiation. However, the roles of PERK and GCN2 in UV-induced eIF2α phosphorylation are controversial. The one or more upstream signaling pathways that lead to the activation of PERK or GCN2 remain unknown. In this report we provide data showing that both PERK and GCN2 contribute to UV-induced eIF2α phosphorylation in human keratinocyte (HaCaT) and mouse embryonic fibroblast cells. Reduction of expression of PERK or GCN2 by small interfering RNA decreases phosphorylation of eIF2α after UV irradiation. These data also show that nitric-oxide synthase (NOS)-mediated oxidative stress plays a role in regulation of eIF2α phosphorylation upon UV irradiation. Treating the cells with the broad NOS inhibitor N^G-methyl-l-arginine, the free radical scavenger N-acetyl-l-cysteine, or the NOS substrate l-arginine partially inhibits UV-induced eIF2α phosphorylation. The results presented above led us to propose that NOS mediates UV-induced eIF2α phosphorylation by activation of both PERK and GCN2 via oxidative stress and l-arginine starvation signaling pathways.UV irradiation inhibits translation initiation through activation of kinases that phosphorylate the α-subunit of eukaryotic initiation factor 2 (eIF2α).² Two eIF2α kinases, double strand RNA-dependent protein kinase-like ER kinase (PERK) and general control of amino acid biosynthesis kinase (GCN2), are known to phosphorylate the serine 51 of eIF2α in response to UV irradiation (1–4). However, the one or more upstream pathways that activate eIF2α kinase(s) upon UV irradiation are not known. In this report, we provide evidence that UV-induced nitric-oxide synthase (NOS) activation and nitric oxide (NO^•) production regulate both PERK and GCN2 activation upon UVB irradiation.Expression of inducible nitric-oxide synthase in a mouse macrophage cell line leads to the phosphorylation of eIF2α and inhibition of translation (5). In cultured neuronal and pancreatic cell lines, production of NO^• and peroxynitrite (ONOO⁻) induces endoplasmic reticulum (ER) stress, which activates PERK and results in cell dysfunction and apoptosis (6–9). Cytokine-stimulated inducible nitric-oxide synthase activation in astrocytes depletes l-arginine and activates GCN2, which phosphorylates eIF2α (10). UV irradiation also activates NOS and elevates cellular NO^• (11–13). However, the UV-induced NOS activation and NO^• production have never been shown to be related to the activation of eIF2α kinase(s). Now we demonstrate that UV-induced activation of NOS mediates the activation of both PERK and GCN2, which coordinately regulate the phosphorylation of eIF2α.     

Sequential Eukaryotic Translation Initiation Factor 5 (eIF5) Binding to the Charged Disordered Segments of eIF4G and eIF2β Stabilizes the 48S Preinitiation Complex and Promotes Its Shift to the Initiation Mode

During translation initiation in Saccharomyces cerevisiae, an Arg- and Ser-rich segment (RS1 domain) of eukaryotic translation initiation factor 4G (eIF4G) and the Lys-rich segment (K-boxes) of eIF2β bind three common partners, eIF5, eIF1, and mRNA. Here, we report that both of these segments are involved in mRNA recruitment and AUG recognition by distinct mechanisms. First, the eIF4G-RS1 interaction with the eIF5 C-terminal domain (eIF5-CTD) directly links eIF4G to the preinitiation complex (PIC) and enhances mRNA binding. Second, eIF2β-K-boxes increase mRNA binding to the 40S subunit in vitro in a manner reversed by the eIF5-CTD. Third, mutations altering eIF4G-RS1, eIF2β-K-boxes, and eIF5-CTD restore the accuracy of start codon selection impaired by an eIF2β mutation in vivo, suggesting that the mutual interactions of the eIF segments within the PIC prime the ribosome for initiation in response to start codon selection. We propose that the rearrangement of interactions involving the eIF5-CTD promotes mRNA recruitment through mRNA binding by eIF4G and eIF2β and assists the start codon-induced release of eIF1, the major antagonist of establishing tRNA(i)(Met):mRNA binding to the P site.     

Novel RNA-binding Protein P311 Binds Eukaryotic Translation Initiation Factor 3 Subunit b (eIF3b) to Promote Translation of Transforming Growth Factor β1-3 (TGF-β1-3)

P311, a conserved 8-kDa intracellular protein expressed in brain, smooth muscle, regenerating tissues, and malignant glioblastomas, represents the first documented stimulator of TGF-β1-3 translation in vitro and in vivo. Here we initiated efforts to define the mechanism underlying P311 function. PONDR® (Predictor Of Naturally Disordered Regions) analysis suggested and CD confirmed that P311 is an intrinsically disordered protein, therefore requiring an interacting partner to acquire tertiary structure and function. Immunoprecipitation coupled with mass spectroscopy identified eIF3 subunit b (eIF3b) as a novel P311 binding partner. Immunohistochemical colocalization, GST pulldown, and surface plasmon resonance studies revealed that P311-eIF3b interaction is direct and has a K_d of 1.26 μm. Binding sites were mapped to the non-canonical RNA recognition motif of eIF3b and a central 11-amino acid-long region of P311, here referred to as eIF3b binding motif. Disruption of P311-eIF3b binding inhibited translation of TGF-β1, 2, and 3, as indicated by luciferase reporter assays, polysome fractionation studies, and Western blot analysis. RNA precipitation assays after UV cross-linking and RNA-protein EMSA demonstrated that P311 binds directly to TGF-β 5′UTRs mRNAs through a previously unidentified RNA recognition motif-like motif. Our results demonstrate that P311 is a novel RNA-binding protein that, by interacting with TGF-βs 5′UTRs and eIF3b, stimulates the translation of TGF-β1, 2, and 3.     

The Herpes Simplex Virus US11 Protein Effectively Compensates for the γ134.5 Gene if Present before Activation of Protein Kinase R by Precluding Its Phosphorylation and That of the α Subunit of Eukaryotic Translation Initiation Factor 2

In herpes simplex virus-infected cells, viral γ₁34.5 protein blocks the shutoff of protein synthesis by activated protein kinase R (PKR) by directing the protein phosphatase 1α to dephosphorylate the α subunit of eukaryotic translation initiation factor 2 (eIF-2α). The amino acid sequence of the γ₁34.5 protein which interacts with the phosphatase has high homology to a domain of the eukaryotic protein GADD34. A class of compensatory mutants characterized by a deletion which results in the juxtaposition of the α47 promoter next to U_S11, a γ₂ (late) gene in wild-type virus-infected cells, has been described. In cells infected with these mutants, protein synthesis continues even in the absence of the γ₁34.5 gene. In these cells, PKR is activated but eIF-2α is not phosphorylated, and the phosphatase is not redirected to dephosphorylate eIF-2α. We report the following: (i) in cells infected with these mutants, U_S11 protein was made early in infection; (ii) U_S11 protein bound PKR and was phosphorylated; (iii) in in vitro assays, U_S11 blocked the phosphorylation of eIF-2α by PKR activated by poly(I-C); and (iv) U_S11 was more effective if present in the reaction mixture during the activation of PKR than if added after PKR had been activated by poly(I-C). We conclude the following: (i) in cells infected with the compensatory mutants, U_S11 made early in infection binds to PKR and precludes the phosphorylation of eIF-2α, whereas U_S11 driven by its natural promoter and expressed late in infection is ineffective; and (ii) activation of PKR by double-stranded RNA is a common impediment countered by most viruses by different mechanisms. The γ₁34.5 gene is not highly conserved among herpesviruses. A likely scenario is that acquisition by a progenitor of herpes simplex virus of a portion of the cellular GADD34 gene resulted in a more potent and reliable means of curbing the effects of activated PKR. U_S11 was retained as a γ₂ gene because, like many viral proteins, it has multiple functions.The herpes simplex virus 1 (HSV-1) genome encodes two sets of functions. The first and paramount are functions related to viral gene expression, replication of viral DNA, synthesis of virion proteins, assembly, packaging, and egress of the virus from the infected cell. The second set of functions, no less important in the survival of the virus in the human population, is creation of the environment necessary to maximize the yield and spread of virus from cell to cell and from infected to uninfected individuals (reviewed in reference 38). Of these known genes, several play a significant role in abating or delaying a host response to infection. The earliest to be expressed is the U_L41 gene which encodes a protein that is introduced into the cell in virions during infection (26, 27). This protein reduces the synthesis of host proteins by causing the destruction of mRNA in a rather nonspecific manner and therefore could be expected to reduce the synthesis of cellular proteins deleterious to viral replication (26, 27, 44).A second and very different approach to blocking host defense mechanisms is exemplified by infected cell protein 47 (ICP47). Proteosomal degradation of viral proteins could be expected to produce antigenic peptides which, if presented on the cell surface, could provoke a cytotoxic cell response early in infection and thus reduce viral yield. ICP47, an α protein made immediately after infection, blocks the presentation of antigenic peptides on the surface of the infected cells (20).The focus of this laboratory has been on a third viral pathway designed to block cellular response to infection. In cells infected with most viruses, the synthesis of complementary mRNA leads to activation of double-stranded RNA-dependent protein kinase R (PKR). This enzyme phosphorylates the α subunit of eukaryotic translation initiation factor 2 (eIF-2α) (23). A consequence of this phosphorylation is total shutoff of protein synthesis. This would be an example of a noble sacrifice of the infected cell for the sake of survival of the organism were it not for the fact that viruses, while activating the PKR kinase pathway by making double-stranded RNA, also express functions which block this host defense system (2–4, 6, 7, 10, 28, 30, 34). In the case of HSV-1, more than 50% of the viral DNA is represented late in infection in the form of cRNA (21, 25), and the gene whose product blocks the consequences of activation of PKR is γ₁34.5 (7). In the absence of the gene, eIF-2α is phosphorylated and protein synthesis is impaired beginning approximately 5 h after infection (7, 9). In its presence, protein synthesis continues unabated even though PKR is activated (9). Recent studies have shown that the carboxyl terminus of the γ₁34.5 gene binds to the protein phosphatase 1α (PP1) and redirects it to dephosphorylate eIF-2α (19). The effectiveness of the γ₁34.5-PP1 complex is apparent from the observation that the rate of dephosphorylation of eIF-2α in cells infected with wild-type virus is more than 1000 times that of uninfected cells or cells infected with the γ₁34.5⁻ virus (5, 19).The studies described in this report concern another aspect of virus-induced block of the consequence of activation of PKR. Briefly, Mohr and Gluzman reported that serial passage of a γ₁34.5⁻ mutant resulted in the selection of a compensatory mutation capable of sustained protein synthesis (35). A characteristic of the compensatory mutants isolated by Mohr and Gluzman is a deletion in the α47 gene resulting in the juxtaposition of the promoter of the α47 gene next to the 5′ end of U_S11, a late (γ₂) viral gene. Preliminary studies of those mutants revealed that PKR was activated in cells infected with either the wild-type parent or the γ₁34.5⁻ virus, but protein synthesis was unaffected in cells infected with wild-type virus or the mutant carrying the compensatory mutations (5, 18).In an attempt to define the phenotype of the virus carrying the compensatory mutation, we constructed a mutant lacking the γ₁34.5 and the U_S8 to -12 genes. This mutant, designated R5103, activated PKR and caused a shutoff of protein synthesis (5). We then inserted into the R5103 genome a DNA fragment consisting of the intact U_S10 gene and the U_S11 open reading frame fused to the α47 promoter. This virus, designated R5104, activated PKR but did not induce the shutoff of protein synthesis. Consistent with the conclusion of Mohr and Gluzman (35), the mutation maps in the domain inserted into the R5104 virus (5). Further studies yielded two significant observations. First, in stark contrast to lysates of cells infected with R5103 and other γ₁34.5⁻ mutants, the lysates of R5104 virus failed to phosphorylate the α subunit of eIF-2 (5). Second, in striking contrast to lysates of wild-type virus-infected cells, the phosphatase activity of lysates of R5104 virus-infected cells specific for eIF-2α could not be differentiated from that of mock-infected cells or those of cells infected with other γ₁34.5⁻ mutants (5). These results indicated that the compensatory mutation blocks PKR from phosphorylating eIF-2α.The studies summarized in this report focused on U_S11 protein. We report that in cells infected with the R5104 recombinant the U_S11 protein is made early in infection, that U_S11 protein interacts with PKR and blocks the phosphorylation of eIF-2α by activated PKR in in vitro assays, and that the effectiveness of the U_S11 protein is greater if the protein is present in the reaction before activation of PKR than if it is after PKR has been activated by the addition of poly(I-C). We also found that U_S11 is phosphorylated in the presence of activated PKR but not in its absence. We conclude that U_S11 may have been an ancient mechanism for blocking the effects of activated PKR and that it has been supplanted by acquisition of the carboxyl-terminal domain of the γ₁34.5 protein from a cellular gene. We also note that U_S11 protein made late in infection, after PKR has been activated, is ineffective.Relevant to this report are some of the properties of the U_S11 protein. U_S11 is one of the most abundant viral proteins expressed at late times in viral infection (22, 31). It binds mRNA in a sequence- and conformation-specific fashion (39–41). In HSV-1-infected cells, U_S11 suppresses the synthesis of a truncated RNA colinear with the 5′ domain of the U_L34 mRNA (40). The protein accumulates in nucleoli, in the cytoplasm in association with the 60S ribosomal subunit, and it is also packaged in virions (31, 37, 41). In newly infected cells, the U_S11 protein has been found associated with ribosomes (41).Recently a plethora of reports suggested that U_S11 may have novel functions not readily apparent from its localization in the infected cell. Thus, U_S11 protein has been reported to have functions similar to those of human immunodeficiency Tat and Rev proteins and has also been reported to complement Rev function in a Rev⁻ human immunodeficiency virus mutant (11). The U_S11 protein has been reported to confer thermotolerance and help restore protein synthesis in HeLa cells subjected to thermal injury (12).     

Genetic Analysis of B2t, the Structural Gene for a Testis-Specific β-Tubulin Subunit in DROSOPHILA MELANOGASTER

Genetic analysis of the B2t locus has resulted in the recovery of four recessive mutations in the B2t structural gene and a deficiency that deletes the locus. Two of the mutations were recovered as suppressors of B2t^D, a dominant male sterile mutation at the locus, and two were induced on wild-type chromosomes. All four mutant genes encode β₂-tubulin subunits that are synthesized at normal rates but do not accumulate. All mutants are completely male sterile as homozygotes.     

Coordination of Eukaryotic Translation Elongation Factor 1A (eEF1A) Function in Actin Organization and Translation Elongation by the Guanine Nucleotide Exchange Factor eEF1B��

Eukaryotic translation elongation factor 1A (eEF1A) both shuttles aminoacyl-tRNA (aa-tRNA) to the ribosome and binds and bundles actin. A single domain of eEF1A is proposed to bind actin, aa-tRNA and the guanine nucleotide exchange factor eEF1Bα. We show that eEF1Bα has the ability to disrupt eEF1A-induced actin organization. Mutational analysis of eEF1Bα F163, which binds in this domain, demonstrates effects on growth, eEF1A binding, nucleotide exchange activity, and cell morphology. These phenotypes can be partially restored by an intragenic W130A mutation. Furthermore, the combination of F163A with the lethal K205A mutation restores viability by drastically reducing eEF1Bα affinity for eEF1A. This also results in a consistent increase in actin bundling and partially corrected morphology. The consequences of the overlapping functions in this eEF1A domain and its unique differences from the bacterial homologs provide a novel function for eEF1Bα to balance the dual roles in actin bundling and protein synthesis.The final step of gene expression takes place at the ribosome as mRNA is translated into protein. In the yeast Saccharomyces cerevisiae, elongation of the polypeptide chain requires the orchestrated action of three soluble factors. The eukaryotic elongation factor 1 (eEF1)² complex delivers aminoacyl-tRNA (aa-tRNA) to the empty A-site of the elongating ribosome (1). The eEF1A subunit is a classic G-protein that acts as a “molecular switch” for the active and inactive states based on whether GTP or GDP is bound, respectively (2). Once an anticodon-codon match occurs, the ribosome acts as a GTPase-activating factor to stimulate GTP hydrolysis resulting in the release of inactive GDP-bound eEF1A from the ribosome. Because the intrinsic rate of GDP release from eEF1A is extremely slow (3, 4), a guanine nucleotide exchange factor (GEF) complex, eEF1B, is required (5, 6). The yeast S. cerevisiae eEF1B complex contains two subunits, the essential catalytic subunit eEF1Bα (5) and the non-essential subunit eEF1Bγ (7).The co-crystal structures of eEF1A:eEF1Bα C terminus:GDP: Mg²⁺ and eEF1A:eEF1Bα C terminus:GDPNP (8, 9) demonstrated a surprising structural divergence from the bacterial EF-Tu-EF-Ts (10) and mammalian mitochondrial EF-Tu_mt-EF-Ts_mt (11). While the G-proteins have a similar topology and consist of three well-defined domains, a striking difference was observed in binding sites for their GEFs. The C terminus of eEF1Bα interacts with domain I and a distinct pocket of domain II eEF1A, creating two binding interfaces. In contrast, the bacterial counterpart EF-Ts and mammalian mitochondrial EF-Ts_mt, make extensive contacts with domain I and III of EF-Tu and EF-Tu_mt, respectively. The altered binding interface of eEF1Bα to domain II of eEF1A is particularly unexpected given the functions associated with domain II of eEF1A and EF-Tu. The crystal structure of the EF-Tu:GDPNP:Phe-tRNA^Phe complex reveals aa-tRNA binding to EF-Tu requires only minor parts of both domain II and tRNA to sustain stable contacts (12). That eEF1A employs the same aa-tRNA binding site is supported by genetic and biochemical data (13-15). Interestingly, eEF1Bα contacts many domain II eEF1A residues in the region hypothesized to be involved in the binding of the aa-tRNA CCA end (8). Because, the shared binding site of eEF1Bα and aa-tRNA on domain II of eEF1A is significantly different between the eukaryotic and bacterial/mitochondrial systems, eEF1Bα may play a unique function aside from guanine nucleotide release in eukaryotes.In eukaroytes, eEF1A is also an actin-binding and -bundling protein. This noncanonical function of eEF1A was initially observed in Dictyostelium amoebae (16). It is estimated that greater than 60% of Dictyostelium eEF1A is associated with the actin cytoskeleton (17). The eEF1A-actin interaction is conserved among species from yeast to mammals, suggesting the importance of eEF1A for cytoskeleton integrity. Using a unique genetic approach, multiple eEF1A mutations were identified that altered cell growth and morphology, and are deficient in bundling actin in vitro (18, 19). Intriguingly, most mutations localized to domain II, the shared aa-tRNA and eEF1Bα binding site. Previous studies have demonstrated that actin bundling by eEF1A is significantly reduced in the presence of aa-tRNA while eEF1A bound to actin filaments is not in complex with aa-tRNA (20). Therefore, actin and aa-tRNA binding to eEF1A is mutually exclusive. In addition, overexpression of yeast eEF1A or actin-bundling deficient mutants do not affect translation elongation (18, 19, 21), suggesting eEF1A-dependent cytoskeletal organization is independent of its translation elongation function (18, 20). Thus, while aa-tRNA binding to domain II is conserved between EF-Tu and eEF1A, this actin bundling function associated with eEF1A domain II places greater importance on its relationship with the “novel” binding interface between eEF1A domain II and eEF1Bα.Based on this support for an overlapping actin bundling and eEF1Bα binding site in eEF1A domain II, we hypothesize that eEF1Bα modulates the equilibrium between actin and translation functions of eEF1A and is perhaps the result of evolutionary selective pressure to balance the eukaryotic-specific role of eEF1A in actin organization. Here, we present kinetic and biochemical evidence using a F163A mutant of eEF1Bα for the importance of the interactions between domain II of eEF1A and eEF1Bα to prevent eEF1A-dependent actin bundling as well as promoting guanine nucleotide exchange. Furthermore, altered affinities of eEF1Bα mutants for eEF1A support that this complex formation is a determining factor for eEF1A-induced actin organization. Interestingly, the F163A that reduces eEF1A affinity is an intragenic suppressor of the lethal K205A eEF1Bα mutant that displays increased affinity for eEF1A. This, along with a consistent change in the actin bundling correlated with the affinity of eEF1Bα for eEF1A, indicates that eEF1Bα is a balancer, directing eEF1A to translation elongation and away from actin, and alterations in this balance result in detrimental effects on cell growth and eEF1A function.     

BIG1, a Brefeldin A-Inhibited Guanine Nucleotide-Exchange Factor,Is Required for GABA-Gated Cl– Influx Through Regulation of GABAA Receptor Trafficking

GABA_A receptors (GABA_ARs) mediate the majority of fast synaptic inhibition. Trafficking regulation and protein–protein interactions that maintain the appropriate number of GABA_ARs at the cell surface are considered to be important mechanisms for controlling the strength of synaptic inhibition. Here, we report that BIG1, a brefeldin A (BFA)-inhibited guanine nucleotide-exchange factor (GEF) which has a known role in vesicle trafficking, is a new binding partner of GABA_ARs. Treatment of neurons with BFA, an uncompetitive inhibitor of BIG1 GEF activity, or depletion of BIG1 by small RNA interference (siRNA) significantly decreased GABA_ARs at the neuronal surface and suppressed GABA-gated influx of chloride ions. Over-expression of HA-tagged BIG1-E793K, a dominant-negative mutant, also significantly decreased GABA_ARs at the neuronal surface, but had no effect on the total amount of GABA_ARs. Inhibition of GABA_AR endocytosis by muscimol increased both GABA_ARs and BIG1 at the neuronal surface in a time-dependent fashion, and this increase could be abolished by bicuculline. Finally, depletion of BIG1 by siRNA inhibited the muscimol-stimulated increase of GABA_ARs. Those data suggest an important function of BIG1 in trafficking of GABA_ARs to the cell surface through its GEF activity. Thus, we identify an important role of BIG1 in modulating GABA-gated Cl^? influx through the regulation of cell surface expression of GABA_ARs.     

Guanine Nucleotide Exchange Factor αPIX Leads to Activation of the Rac 1 GTPase/Glycogen Phosphorylase Pathway in Interleukin (IL)-2-stimulated T Cells

Recently, we have reported that the active form of Rac 1 GTPase binds to the glycogen phosphorylase muscle isoform (PYGM) and modulates its enzymatic activity leading to T cell proliferation. In the lymphoid system, Rac 1 and in general other small GTPases of the Rho family participate in the signaling cascades that are activated after engagement of the T cell antigen receptor. However, little is known about the IL-2-dependent Rac 1 activator molecules. For the first time, a signaling pathway leading to the activation of Rac 1/PYGM in response to IL-2-stimulated T cell proliferation is described. More specifically, αPIX, a known guanine nucleotide exchange factor for the small GTPases of the Rho family, preferentially Rac 1, mediates PYGM activation in Kit 225 T cells stimulated with IL-2. Using directed mutagenesis, phosphorylation of αPIX Rho-GEF serines 225 and 488 is required for activation of the Rac 1/PYGM pathway. IL-2-stimulated serine phosphorylation was corroborated in Kit 225 T cells cultures. A parallel pharmacological and genetic approach identified PKCθ as the serine/threonine kinase responsible for αPIX serine phosphorylation. The phosphorylated state of αPIX was required to activate first Rac 1 and subsequently PYGM. These results demonstrate that the IL-2 receptor activation, among other early events, leads to activation of PKCθ. To activate Rac 1 and consequently PYGM, PKCθ phosphorylates αPIX in T cells. The biological significance of this PKCθ/αPIX/Rac 1 GTPase/PYGM signaling pathway seems to be the control of different cellular responses such as migration and proliferation.     

The Risk Factors for Failure of an Upper Extremity Replantation: Is the Use of Cigarettes/Tobacco a Significant Factor?

Background

The purpose of this study was to explore the potential risk factors associated with the failure of an upper extremity replantation with a focus on cigarette or tobacco use.

Patients and Methods

A cohort of 102 patients with 149 replants (6 extremities, 143 digits) and a mean age of 41 years (range 5 to 72 years) was enrolled in this study. The data collected included age, gender, tobacco/cigarettes use, trauma mechanism, underlying disease (e.g., hypertension (HTN), diabetes mellitus (DM), etc.), and vein graft use. An analysis with a multivariable regression was conducted to identify the risk factors of replant failure and their respective odds ratios (ORs).

Results

Multilevel generalized linear mixed models (GLMMs) with a binomial distribution and logit link showed that smoking did not increase the risk of replant failure (p = 0.234). In addition, the survival of replants was not affected by DM or HTN (p = 0.285 and 0.938, respectively). However, the replantation results were significantly affected by the age of the patients and the mechanism of injury. Patients older than 50 years and those with avulsion or crush injuries tended to have a higher risk of replant failure (OR = 2.29, 6.45, and 5.42, respectively; p = 0.047, 0.028, and 0.032, respectively).

Conclusions

This study showed that the use of cigarettes/tobacco did not affect the replantation outcome. The main risks for replant failure included being older than 50 years and the trauma mechanism (avulsion or crush injuries).