Accumulation of a novel spliceosomal complex on pre-mRNAs containing branch site mutations.

Pre-mRNA assembles into spliceosomal complexes in the stepwise pathway E-->A-->B-->C. We show that mutations in the metazoan branchpoint sequence (BPS) have no apparent effect on E complex formation but block the assembly of the A complex and the UV cross-linking of U2 small nuclear ribonucleoprotein particle (snRNP) proteins. Unexpectedly, a novel complex, designated E*, assembles on pre-mRNAs containing BPS mutations. Unlike the E complex, the E* complex accumulates in the presence of ATP. U1 snRNP and U2AF, which are tightly bound to pre-mRNA in the E complex, are not tightly bound in the E* complex. Significantly, previous work showed that U1 snRNP and U2AF become destabilized from pre-mRNA after E complex assembly on normal pre-mRNAs. Thus, our data are consistent with a model in which there are two steps in the transition from the E complex to the A complex (E-->E*-->A). In the first step, U1 snRNP and U2AF are destabilized in an ATP-dependent, BPS-independent reaction. In the second step, the stable binding of U2 snRNP occurs in a BPS-dependent reaction.     

Structure and E3-ligase activity of the Ring-Ring complex of polycomb proteins Bmi1 and Ring1b

Polycomb group proteins Ring1b and Bmi1 (B-cell-specific Moloney murine leukaemia virus integration site 1) are critical components of the chromatin modulating PRC1 complex. Histone H2A ubiquitination by the PRC1 complex strongly depends on the Ring1b protein. Here we show that the E3-ligase activity of Ring1b on histone H2A is enhanced by Bmi1 in vitro. The N-terminal Ring-domains are sufficient for this activity and Ring1a can replace Ring1b. E2 enzymes UbcH5a, b, c or UbcH6 support this activity with varying processivity and selectivity. All four E2s promote autoubiquitination of Ring1b without affecting E3-ligase activity. We solved the crystal structure of the Ring-Ring heterodimeric complex of Ring1b and Bmi1. In the structure the arrangement of the Ring-domains is similar to another H2A E3 ligase, the BRCA1/BARD1 complex, but complex formation depends on an N-terminal arm of Ring1b that embraces the Bmi1 Ring-domain. Mutation of a critical residue in the E2/E3 interface shows that catalytic activity resides in Ring1b and not in Bmi1. These data provide a foundation for understanding the critical enzymatic activity at the core of the PRC1 polycomb complex, which is implicated in stem cell maintenance and cancer.     

Hepatitis C virus envelope glycoproteins complementation patterns and the role of the ecto- and transmembrane domains

We separated E1 and E2 of hepatitis C virus (HCV) genotypes 1a, 1b, and 2a into two individual expression plasmids and replaced the transmembrane domains of 1b and 2a E1 and E2 with that of genotype 1a. The complementation features of E1 and E2 as well as the contributions of both the ecto- and transmembrane domains to the formation of the E1E2 complex were evaluated using the HCV pseudoparticle(s) (HCVpp(s)) system.We demonstrated that 1aE2 could not only complement its native 1aE1, but could also complement 1bE1 as well; in genotype 1b, glycoprotein complex formation is primarily dependent on the overall biological characteristics of the intact native E1 and E2; in genotype 2a, although the interaction of intact native E1 and E2 is critical for the formation of the glycoprotein complex, the ectodomain made a greater contribution than that of the transmembrane domain.Our study provides valuable findings regarding HCV E1 and E2 biology and will be of use in both anti-HCV strategy and understanding on the mechanisms of coinfection of different HCV strains.     

Pyruvate dehydrogenase kinase activity of pig heart pyruvate dehydrogenase (E1 component of pyruvate dehydrogenase complex).

The pyruvate dehydrogenase (E1) and acetyltransferase (E2) components of pig heart and ox kidney pyruvate dehydrogenase (PDH) complex were separated and purified. The E1 component was phosphorylated (alpha-chain) and inactivated by MgATP. Phosphorylation was mainly confined to site 1. Addition of E2 accelerated phosphorylation of all three sites in E1 alpha and inactivation of E1. On the basis of histone H1 phosphorylation, E2 is presumed to contain PDH kinase, which was removed (greater than 98%) by treatment with p-hydroxymercuriphenylsulphonate. Stimulation of ATP-dependent inactivation of E1 by E2 was independent of histone H1 kinase activity of E2. The effect of E2 is attributed to conformational change(s) induced in E1 and/or E1-associated PDH kinase. PDH kinase activity associated with E1 could not be separated from it be gel filtration or DEAE-cellulose chromatography. Subunits of PDH kinase were not detected on sodium dodecyl sulphate/polyacrylamide gels of E1 or E2, presumably because of low concentration. The activity of pig heart PDH complex was increased by E2, but not by E1, indicating that E2 is rate-limiting in the holocomplex reaction. ATP-dependent inactivation of PDH complex was accelerated by E1 or by phosphorylated E1 plus associated PDH kinase, but not by E2 plus presumed PDH kinase. It is suggested that a substantial proportion of PDH kinase may accompany E1 when PDH complex is dissociated into its component enzymes. The possibility that E1 may possess intrinsic PDH kinase activity is considered unlikely, but may not have been fully excluded.     

Disruption and mutagenesis of the Saccharomyces cerevisiae PDX1 gene encoding the protein X component of the pyruvate dehydrogenase complex

Disruption of the PDX1 gene encoding the protein X component of the mitochondrial pyruvate dehydrogenase (PDH) complex in Saccharomyces cerevisiae did not affect viability of the cells. However, extracts of mitochondria from the mutant, in contrast to extracts of wild-type mitochondria, did not catalyze a CoA- and NAD(+)-linked oxidation of pyruvate. The PDH complex isolated from the mutant cells contained pyruvate dehydrogenase (E1 alpha + E1 beta) and dihydrolipoamide acetyltransferase (E2) but lacked protein X and dihydrolipoamide dehydrogenase (E3). Mutant cells transformed with the gene for protein X on a unit-copy plasmid produced a PDH complex that contained protein X and E3, as well as E1 alpha, E1 beta, and E2, and exhibited overall activity similar to that of the wild-type PDH complex. These observations indicate that protein X is not involved in assembly of the E2 core nor is it an integral part of the E2 core. Rather, protein X apparently plays a structural role in the PDH complex; i.e., it binds and positions E3 to the E2 core, and this specific binding is essential for a functional PDH complex. Additional evidence for this conclusion was obtained with deletion mutations. Deletion of most of the lipoyl domain (residues 6-80) of protein X had little effect on the overall activity of the PDH complex. This observation indicates that the lipoyl domain, and its covalently bound lipoyl moiety, is not essential for protein X function. However, deletion of the putative subunit binding domain (residues approximately 144-180) of protein X resulted in loss of high-affinity binding of E3 and concomitant loss of overall activity of the PDH complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ligand-induced conformational changes and a reaction intermediate in branched-chain 2-oxo acid dehydrogenase (E1) from Thermus thermophilus HB8, as revealed by X-ray crystallography

The alpha(2)beta(2) tetrameric E1 component of the branched-chain 2-oxo acid (BCOA) dehydrogenase multienzyme complex is a thiamin diphosphate (ThDP)-dependent enzyme. E1 catalyzes the decarboxylation of a BCOA concomitant with the formation of the alpha-carbanion/enamine intermediate, 2-(1-hydroxyalkyl)-ThDP, followed by transfer of the 1-hydroxyalkyl group to the distal sulfur atom on the lipoamide of the E2 component. In order to elucidate the catalytic mechanism of E1, the alpha- and beta-subunits of E1 from Thermus thermophilus HB8 have been co-expressed in Escherichia coli, purified and crystallized as a stable complex, and the following crystal structures have been analyzed: the apoenzyme (E1(apo)), the holoenzyme (E1(holo)), E1(holo) in complex with the substrate analogue 4-methylpentanoate (MPA) as an ES complex model, and E1(holo) in complex with 4-methyl-2-oxopentanoate (MOPA) as the alpha-carbanion/enamine intermediate (E1(ceim)). Binding of cofactors to E1(apo) induces a disorder-order transition in two loops adjacent to the active site. Furthermore, upon binding of MPA to E1(holo), the loop comprised of Gly121beta-Gln131beta moves close to the active site and interacts with MPA. The carboxylate group of MPA is recognized mainly by Tyr86beta and N4' of ThDP. The hydrophobic moiety of MPA is recognized by Phe66alpha, Tyr95alpha, Met128alpha and His131alpha. As an intermediate, MOPA is decarboxylated and covalently linked to ThDP, and the conformation of the protein loop is almost the same as in the substrate-free (holoenzyme) form. These results suggest that E1 undergoes an open-closed conformational change upon formation of the ES complex with a BCOA, and the mobile region participates in the recognition of the carboxylate group of the BCOA. ES complex models of E1(holo).MOPA and of E1(ceim).lipoamide built from the above structures suggest that His273alpha and His129beta' are potential proton donors to the carbonyl group of a BCOA and to the proximal sulfur atom on the lipoamide, respectively.     

Self-assembly and catalytic activity of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus.

The pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus was reconstituted in vitro from recombinant proteins derived from genes over-expressed in Escherichia coli. Titrations of the icosahedral (60-mer) dihydrolipoyl acetyltransferase (E2) core component with the pyruvate decarboxylase (E1, alpha2beta2) and dihydrolipoyl dehydrogenase (E3, alpha2) peripheral components indicated a variable composition defined predominantly by tight and mutually exclusive binding of E1 and E3 with the peripheral subunit-binding domain of each E2 chain. However, both analysis of the polypeptide chain ratios in complexes generated from various mixtures of E1 and E3, and displacement of E1 or E3 from E1-E2 or E3-E2 subcomplexes by E3 or E1, respectively, showed that the multienzyme complex does not behave as a simple competitive binding system. This implies the existence of secondary interactions between the E1 and E3 subunits and E2 that only become apparent on assembly. Exact geometrical distribution of E1 and E3 is unlikely and the results are best explained by preferential arrangements of E1 and E3 on the surface of the E2 core, superimposed on their mutually exclusive binding to the peripheral subunit-binding domain of the E2 chain. Correlation of the subunit composition with the overall catalytic activity of the enzyme complex confirmed the lack of any requirement for precise stoichiometry or strict geometric arrangement of the three catalytic sites and emphasized the crucial importance of the flexibility associated with the lipoyl domains and intramolecular acetyl group transfer in the mechanism of active-site coupling.     

Molecular architecture and mechanism of an icosahedral pyruvate dehydrogenase complex: a multifunctional catalytic machine

Electron cryo-microscopy of 'single particles' is a powerful method to determine the three-dimensional (3D) architectures of complex cellular assemblies. The pyruvate dehydrogenase multi-enzyme complex couples the activity of three component enzymes (E1, E2 and E3) in the oxidative decarboxylation of pyruvate to generate acetyl-CoA, linking glycolysis and the tricarboxylic acid cycle. We report here a 3D model for an 11 MDa, icosahedral pyruvate dehydrogenase sub-complex, obtained by combining a 28 A structure derived from electron cryo-microscopy with previously determined atomic coordinates of the individual E1 and E2 components. A key feature is that the E1 molecules are located on the periphery of the assembly in an orientation that allows each of the 60 mobile lipoyl domains tethered to the inner E2 core to access multiple E1 and E2 active sites from inside the icosahedral complex. This unexpected architecture provides a highly efficient mechanism for active site coupling and catalytic rate enhancement by the motion of the lipoyl domains in the restricted annular region between the inner core and outer shell of the complex.     

Adenovirus E4 34k and E1b 55k oncoproteins target host DNA ligase IV for proteasomal degradation

Cells infected by adenovirus E4 mutants accumulate end-to-end concatemers of the viral genome that are assembled from unit-length viral DNAs by nonhomologous end joining (NHEJ). Genome concatenation can be prevented by expression either of E4 11k (product of E4orf3) or of the complex of E4 34k (product of E4orf6) and E1b 55k. Both E4 11k and the E4 34k/E1b 55k complex prevent concatenation at least in part by inactivation of the host protein Mre11: E4 11k sequesters Mre11 in aggresomes, while the E4 34k/E1b 55k complex participates in a virus-specific E3 ubiquitin ligase that mediates ubiquitination and proteasomal degradation. The E4 34k/E1b 55k complex, but not E4 11k, also inhibits NHEJ activity on internal breaks in the viral genome and on V(D)J recombination substrate plasmids, suggesting that it may interfere with NHEJ independently of its effect on Mre11. We show here that DNA ligase IV, which performs the joining step of NHEJ, is degraded as a consequence of adenovirus infection. Degradation is dependent upon E4 34k and E1b 55k, functional proteasomes, and the activity of cellular cullin 5, a component of the adenoviral ubiquitin ligase. DNA ligase IV also interacts physically with E1b 55k. The data demonstrate that DNA ligase IV, like Mre11, is a substrate for the adenovirus-specific E3 ubiquitin ligase; identify an additional viral approach to prevention of genome concatenation; and provide a mechanism for the general inhibition of NHEJ by adenoviruses.     

An Integrin Binding-defective Mutant of Insulin-like Growth Factor-1 (R36E/R37E IGF1) Acts as a Dominant-negative Antagonist of the IGF1 Receptor (IGF1R) and Suppresses Tumorigenesis but Still Binds to IGF1R

Insulin-like growth factor-1 (IGF1) is a major therapeutic target for cancer. We recently reported that IGF1 directly binds to integrins (αvβ3 and α6β4) and induces ternary complex formation (integrin-IGF1-IGF1 receptor (IGF1R)) and that the integrin binding-defective mutant of IGF1 (R36E/R37E) is defective in signaling and ternary complex formation. These findings predict that R36E/R37E competes with WT IGF1 for binding to IGF1R and inhibits IGF signaling. Here, we described that excess R36E/R37E suppressed cell viability increased by WT IGF1 in vitro in non-transformed cells. We studied the effect of R36E/R37E on viability and tumorigenesis in cancer cell lines. We did not detect an effect of WT IGF1 or R36E/R37E in cancer cells under anchorage-dependent conditions. However, under anchorage-independent conditions, WT IGF1 enhanced cell viability and induced signals, whereas R36E/R37E did not. Notably, excess R36E/R37E suppressed cell viability and signaling induced by WT IGF1 under anchorage-independent conditions. Using cancer cells stably expressing WT IGF1 or R36E/R37E, we determined that R36E/R37E suppressed tumorigenesis in vivo, whereas WT IGF1 markedly enhanced it. R36E/R37E suppressed the binding of WT IGF1 to the cell surface and the subsequent ternary complex formation induced by WT IGF1. R36E/R37E suppressed activation of IGF1R by insulin. WT IGF1, but not R36E/R37E, induced ternary complex formation with the IGF1R/insulin receptor hybrid. These findings suggest that 1) IGF1 induces signals under anchorage-independent conditions and that 2) R36E/R37E acts as a dominant-negative inhibitor of IGF1R (IGF1 decoy). Our results are consistent with a model in which ternary complex formation is critical for IGF signaling.     

Crystal structure of the DNA binding domain of the replication initiation protein E1 from papillomavirus

Papillomaviral infection causes both benign and malignant lesions and is a necessary cause of cervical carcinoma. Replication of this virus requires the replication initiation proteins E1 and E2, which bind cooperatively at the origin of replication (ori) as an (E1)2-(E2)2-DNA complex. This is a precursor to larger E1 complexes that distort and unwind the ori. We present the crystal structure of the E1 DNA binding domain refined to 1.9 A resolution. Residues critical for DNA binding are located on an extended loop and an alpha helix. We identify the E1 dimerization surface by selective mutations at an E1/E1 interface observed in the crystal and propose a model for the (E1)2-DNA complex. These and other observations suggest how the E1 DNA binding domain orchestrates assembly of the hexameric helicase on the ori.