Taxol biosynthesis: Taxane 13α-hydroxylase is a cytochrome P450-dependent monooxygenase

A central feature in the biosynthesis of Taxol is oxygenation at multiple positions of the taxane core structure, reactions that are considered to be mediated by cytochrome P450-dependent monooxygenases. A PCR-based differential display-cloning approach, using Taxus (yew) cells induced for Taxol production, yielded a family of related cytochrome P450 genes, one of which was assigned as a taxane 10 beta-hydroxylase by functional expression in yeast. The acquired clones that did not function in yeast were heterologously expressed by using the Spodoptera fugiperda-baculovirus-based system and were screened for catalytic capability by using taxa-4(20),11(12)-dien-5 alpha-ol and its acetate ester as test substrates. This approach allowed identification of one of the cytochrome P450 clones (which bore 63% deduced sequence identity to the aforementioned taxane 10 beta-hydroxylase) as a taxane 13 alpha-hydroxylase by chromatographic and spectrometric characterization of the corresponding recombinant enzyme product. The demonstration of a second relevant hydroxylase from the induced family of cytochrome P450 genes validates this strategy for elucidating the oxygenation steps of taxane diterpenoid (taxoid) metabolism. Additionally, substrate specificity studies with the available cytochrome P450 hydroxylases now indicate that there is likely more than one biosynthetic route to Taxol in yew species.     

αβ T cell receptor interactions with syngeneic and allogeneic ligands: Affinity measurements and crystallization

Cellular immunity is mediated by the interaction of an αβ T cell receptor (TCR) with a peptide presented within the context of a major histocompatibility complex (MHC) molecule. Alloreactive T cells have αβ TCRs that can recognize both self- and foreign peptide–MHC (pMHC) complexes, implying that the TCR has significant complementarity with different pMHC. To characterize the molecular basis for alloreactive TCR recognition of pMHC, we have produced a soluble, recombinant form of an alloreactive αβ T cell receptor in Drosophila melanogaster cells. This recombinant TCR, 2C, is expressed as a correctly paired αβ heterodimer, with the chains covalently connected via a disulfide bond in the C-terminal region. The native conformation of the 2C TCR was probed by surface plasmon resonance (SPR) analysis by using conformation-specific monoclonal antibodies, as well as syngeneic and allogeneic pMHC ligands. The 2C interaction with H-2K^b-dEV8, H-2K^bm3-dEV8, H-2K^b-SIYR, and H-2L^d-p2Ca spans a range of affinities from K_d = 10⁻⁴ to 10⁻⁶M for the syngeneic (H-2K^b) and allogeneic (H-2K^bm3, H-2L^d) ligands. In general, the syngeneic ligands bind with weaker affinities than the allogeneic ligands, consistent with current threshold models of thymic selection and T cell activation. Crystallization of the 2C TCR required proteolytic trimming of the C-terminal residues of the α and β chains. X-ray quality crystals of complexes of 2C with H-2K^b-dEV8, H-2K^bm3-dEV8 and H-2K^b-SIYR have been grown.     

Reciprocal regulation of Gsα by palmitate and the βγ subunit

Hormonal activation of G_s, the stimulatory regulator of adenylyl cyclase, promotes dissociation of α_s from Gβγ, accelerates removal of covalently attached palmitate from the Gα subunit, and triggers release of a fraction of α_s from the plasma membrane into the cytosol. To elucidate relations among these three events, we assessed biochemical effects in vitro of attached palmitate on recombinant α_s prepared from Sf9 cells. In comparison to the unpalmitoylated protein (obtained from cytosol of Sf9 cells, treated with a palmitoyl esterase, or expressed as a mutant protein lacking the site for palmitoylation), palmitoylated α_s (from Sf9 membranes, 50% palmitoylated) was more hydrophobic, as indicated by partitioning into TX-114, and bound βγ with 5-fold higher affinity. βγ protected GDP-bound α_s, but not α_s· GTP[γS], from depalmitoylation by a recombinant esterase. We conclude that βγ binding and palmitoylation reciprocally potentiate each other in promoting membrane attachment of α_s and that dissociation of α_s·GTP from βγ is likely to mediate receptor-induced α_s depalmitoylation and translocation of the protein to cytosol in intact cells.     

Hyperresponsive febrile reactions to interleukin (IL) 1α and IL-1β, and altered brain cytokine mRNA and serum cytokine levels, in IL-1β-deficient mice

IL-1β is an endogenous pyrogen that is induced during systemic lipopolysaccharide (LPS)- or IL-1-induced fever. We have examined the fever and cytokine responses following i.p. injection of IL-1 agonists, IL-1α and IL-1β, and compared these with response to LPS (i.p.) in wild-type and IL-1β-deficient mice. The IL-1β deficient mice appear to have elevated body temperature but exhibit a normal circadian temperature cycle. Exogenously injected IL-1β, IL-1α, or LPS induced hyperresponsive fevers in the IL-1β-deficient mice. We also observed phenotypic differences between wild-type and IL-1β-deficient mice in hypothalamic basal mRNA levels for IL-1α and IL-6, but not for IL-1β-converting enzyme or IL-1 receptor type I or type II. The IL-1α mRNA levels were down-regulated, whereas the IL-6 mRNA levels were up-regulated in the hypothalamus of IL-1β-deficient mice as compared with wild-type mice. The IL-1β-deficient mice also responded to LPS challenge with significantly higher serum corticosterone and with lower serum tumor necrosis factor type α levels than the wild-type mice. The data suggest that, in the redundant cascade of proinflammatory cytokines, IL-1β plays an important but not obligatory role in fever induction by LPS or IL-1α, as well as in the induction of serum tumor necrosis factor type α and corticosterone responses either by LPS or by IL-1α or IL-1β.     

Molecular picture of folding of a small α/β protein

We characterize, at the atomic level, the mechanism and thermodynamics of folding of a small α/β protein. The thermodynamically significant states of segment B1 of streptococcal protein G (GB1) are probed by using the statistical mechanical methods of importance sampling and molecular dynamics. From a thermodynamic standpoint, folding commences with overall collapse, accompanied by formation of ~35% of the native structure. Specific contacts form at the loci experimentally inferred to be structured early in folding kinetics studies. Our study reveals that these initially structured regions are not spatially adjacent. As folding progresses, fluid-like nonlocal native contacts form, with many contacts forming and breaking as the structure searches for the native conformation. Although the α-helix forms early, the β-sheet forms concomitantly with the overall topology. Water is present in the protein core up to a late stage of folding, lubricating conformational transitions during the search process. Once 80% of the native contacts have formed, water is squeezed from the protein interior and the structure descends into the native manifold. Examination of the onset of side-chain mobility within our model indicates side-chain motion is most closely linked to the overall volume of the protein and no sharp order–disorder transition appears to occur. Exploration of models for hydrogen deuterium exchange show qualitative agreement with equilibrium measurement of hydrogen/deuterium protection factors.     

The amino-terminal region of Tyk2 sustains the level of interferon α receptor 1, a component of the interferon α/β receptor

Tyk2 belongs to the Janus kinase (JAK) family of receptor associated tyrosine kinases, characterized by a large N-terminal region, a kinase-like domain and a tyrosine kinase domain. It was previously shown that Tyk2 contributes to interferon-α (IFN-α) signaling not only catalytically, but also as an essential intracellular component of the receptor complex, being required for high affinity binding of IFN-α. For this function the tyrosine kinase domain was found to be dispensable. Here, it is shown that mutant cells lacking Tyk2 have significantly reduced IFN-α receptor 1 (IFNAR1) protein level, whereas the mRNA level is unaltered. Expression of the N-terminal region of Tyk2 in these cells reconstituted wild-type IFNAR1 level, but did not restore the binding activity of the receptor. Studies of mutant Tyk2 forms deleted at the N terminus indicated that the integrity of the N-terminal region is required to sustain IFNAR1. These studies also showed that the N-terminal region does not directly modulate the basal autophosphorylation activity of Tyk2, but it is required for efficient in vitro IFNAR1 phosphorylation and for rendering the enzyme activatable by IFN-α. Overall, these results indicate that distinct Tyk2 domains provide different functions to the receptor complex: the N-terminal region sustains IFNAR1 level, whereas the kinase-like domain provides a function toward high affinity ligand binding.     

A synthetic cryIC gene, encoding a Bacillus thuringiensis δ-endotoxin, confers Spodoptera resistance in alfalfa and tobacco

Spodoptera species, representing widespread polyphagous insect pests, are resistant to Bacillus thuringiensis δ-endotoxins used thus far as insecticides in transgenic plants. Here we describe the chemical synthesis of a cryIC gene by a novel template directed ligation–PCR method. This simple and economical method to construct large synthetic genes can be used when routine resynthesis of genes is required. Chemically phosphorylated adjacent oligonucleotides of the gene to be synthesized are assembled and ligated on a single-stranded, partially homologous template derived from a wild-type gene (cryIC in our case) by a thermostable Pfu DNA ligase using repeated cycles of melting, annealing, and ligation. The resulting synthetic DNA strands are selectively amplified by PCR with short specific flanking primers that are complementary only to the new synthetic DNA. Optimized expression of the synthetic cryIC gene in alfalfa and tobacco results in the production of 0.01–0.2% of total soluble proteins as CryIC toxin and provides protection against the Egyptian cotton leafworm (Spodoptera littoralis) and the beet armyworm (Spodoptera exigua). To facilitate selection and breeding of Spodoptera-resistant plants, the cryIC gene was linked to a pat gene, conferring resistance to the herbicide BASTA.     

An enhancer-blocking element between α and δ gene segments within the human T cell receptor α/δ locus

T cell receptor (TCR) α and δ gene segments are organized within a single genetic locus but are differentially regulated during T cell development. An enhancer-blocking element (BEAD-1, for blocking element alpha/delta 1) was localized to a 2.0-kb region 3′ of TCR δ gene segments and 5′ of TCR α joining gene segments within this locus. BEAD-1 blocked the ability of the TCR δ enhancer (E_δ) to activate a promoter when located between the two in a chromatin-integrated construct. We propose that BEAD-1 functions as a boundary that separates the TCR α/δ locus into distinct regulatory domains controlled by E_δ and the TCR α enhancer, and that it prevents E_δ from opening the chromatin of the TCR α joining gene segments for VDJ recombination at an early stage of T cell development.     

MIC-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF-β superfamily

Macrophages play a key role in both normal and pathological processes involving immune and inflammatory responses, to a large extent through their capacity to secrete a wide range of biologically active molecules. To identify some of these as yet not characterized molecules, we have used a subtraction cloning approach designed to identify genes expressed in association with macrophage activation. One of these genes, designated macrophage inhibitory cytokine 1 (MIC-1), encodes a protein that bears the structural characteristics of a transforming growth factor β (TGF-β) superfamily cytokine. Although it belongs to this superfamily, it has no strong homology to existing families, indicating that it is a divergent member that may represent the first of a new family within this grouping. Expression of MIC-1 mRNA in monocytoid cells is up-regulated by a variety of stimuli associated with activation, including interleukin 1β, tumor necrosis factor α (TNF-α), interleukin 2, and macrophage colony-stimulating factor but not interferon γ, or lipopolysaccharide (LPS). Its expression is also increased by TGF-β. Expression of MIC-1 in CHO cells results in the proteolytic cleavage of the propeptide and secretion of a cysteine-rich dimeric protein of M_r 25 kDa. Purified recombinant MIC-1 is able to inhibit lipopolysaccharide -induced macrophage TNF-α production, suggesting that MIC-1 acts in macrophages as an autocrine regulatory molecule. Its production in response to secreted proinflammatory cytokines and TGF-β may serve to limit the later phases of macrophage activation.     

Rescue of cardiac α-actin-deficient mice by enteric smooth muscle γ-actin

The muscle actins in higher vertebrates display highly conserved amino acid sequences, yet they show distinct expression patterns. Thus, cardiac α-actin, skeletal α-actin, vascular smooth muscle α-actin, and enteric smooth muscle γ-actin comprise the major actins in their respective tissues. To assess the functional and developmental significance of cardiac α-actin, the murine (129/SvJ) cardiac α-actin gene was disrupted by homologous recombination. The majority (≈56%) of the mice lacking cardiac α-actin do not survive to term, and the remainder generally die within 2 weeks of birth. Increased expression of vascular smooth muscle and skeletal α-actins is observed in the hearts of newborn homozygous mutants and also heterozygotes but apparently is insufficient to maintain myofibrillar integrity in the homozygous mutants. Mice lacking cardiac α-actin can be rescued to adulthood by the ectopic expression of enteric smooth muscle γ-actin using the cardiac α-myosin heavy chain promoter. However, the hearts of such rescued cardiac α-actin-deficient mice are extremely hypodynamic, considerably enlarged, and hypertrophied. Furthermore, the transgenically expressed enteric smooth muscle γ-actin reduces cardiac contractility in wild-type and heterozygous mice. These results demonstrate that alterations in actin composition in the fetal and adult heart are associated with severe structural and functional perturbations.     

Expression of αv and β3 integrin chains on murine lymphocytes

The vitronectin receptor is a member of the integrin family of adhesion protein receptors and binds a broad spectrum of ligands, including fibronectin and fibrinogen in addition to vitronectin. We have generated four mAbs that recognize the murine αvβ3 vitronectin receptor. Biochemical and expression analyses showed that two of the mAbs are specific for the αv chain, and two are specific for the β3 chain. The mAbs are effective blocking reagents and inhibited cell adhesion to vitronectin, fibrinogen, and fibronectin. Staining analysis revealed expression of αv and β3 on certain populations of murine thymocytes, splenocytes, and bone marrow cells. The expression of αv and β3 appeared to be modulated at specific stages of thymocyte development, suggesting a possible function for the αvβ3 vitronectin receptor in T cell development.     

Modulation of the chaperone-like activity of bovine α-crystallin

The effects of pantethine, glutathione, and selected chemical reagents on the anti-aggregation activity of α-crystallin was evaluated. Protein aggregation was monitored by light scattering of solutions of denatured β_L-crystallin or alcohol dehydrogenase (ADH). The ratios of β_L-crystallin/α-crystallin and ADH/α-crystallin were adjusted so that partial inhibition of protein aggregation at 60°C or 37°C, respectively, was observed and modulation of the chaperone action of α-crystallin could be evaluated easily with selected endogenous metabolites. Enhancement of the anti-aggregation activity in the β_L-crystallin assay was strongest with pantethine, which appeared to interact with α-crystallin. Enhancement of the anti-aggregation activity in the ADH assay was strongest with glutathione which appeared to interact with ADH. The results indicated that the products of common metabolic pathways can modulate the chaperone-like effects of α-crystallin on protein aggregation.     

Hydrophobic sequence minimization of the α-lactalbumin molten globule

The molten globule, a widespread protein-folding intermediate, can attain a native-like backbone topology, even in the apparent absence of rigid side-chain packing. Nonetheless, mutagenesis studies suggest that molten globules are stabilized by some degree of side-chain packing among specific hydrophobic residues. Here we investigate the importance of hydrophobic side-chain diversity in determining the overall fold of the α-lactalbumin molten globule. We have replaced all of the hydrophobic amino acids in the sequence of the helical domain with a representative amino acid, leucine. Remarkably, the minimized molecule forms a molten globule that retains many structural features characteristic of a native α-lactalbumin fold. Thus, nonspecific hydrophobic interactions may be sufficient to determine the global fold of a protein.     

Cloning and characterization of hSRP1γ, a tissue-specific nuclear transport factor

Nuclear import of proteins containing a nuclear localization signal (NLS) is dependent on the presence of a cytoplasmic NLS receptor, the GTPase Ran, and p10/NTF2. The NLS receptor is a heterodimeric protein consisting of subunits of approximately 60 and 97 kDa, which have been termed importin α/β, karyopherin α/β, or PTAC 58/97. Members of the 60-kDa/importin α subunit family directly bind to the NLS motif and have been shown to function as adaptors that tether NLS-containing proteins to the p97/importin β subunit and to the downstream transport machinery. Herein we report the identification and characterization of hSRP1γ, a human importin α homologue. The hSRP1γ protein is around 45% identical to the two previously identified human importin α homologues hSRP1α/Rch1 and NPI/hSRP1. hSRP1γ can form a complex with importin β and is able to mediate import of a BSA-NLS substrate in an in vitro nuclear import system. Interestingly, hSRP1γ shows a very selective expression pattern and is most abundantly expressed in skeletal muscle, representing more than 1% of the total protein in this tissue. A potential role for hSRP1γ in tissue-specific transport events is discussed.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Folding of the N-terminal, ligand-binding region of integrin α-subunits into a β-propeller domain

The N-terminal ≈440 aa of integrin α subunits contain seven sequence repeats. These are predicted here to fold into a β-propeller domain. A homologous domain from the enzyme phosphatidylinositol phospholipase D is predicted to have the same fold. The domains contain seven four-stranded β-sheets arranged in a torus around a pseudosymmetry axis. The trimeric G-protein β subunit (G beta) appears to be the most closely related β-propeller. Integrin ligands and a putative Mg²⁺ ion are predicted to bind to the upper face of the β-propeller. This face binds substrates in β-propeller enzymes and is used by the G protein β subunit to bind the G protein α subunit. The integrin α subunit I domain, which is structurally homologous to the G protein α subunit, is tethered to the top of the β-propeller domain by a hinge that may allow movement of the domains relative to one another. The Ca²⁺-binding motifs in integrin α subunits are on the lower face of the β-propeller.     

Targeted disruption of the mouse αA-crystallin gene induces cataract and cytoplasmic inclusion bodies containing the small heat shock protein αB-crystallin

αA-crystallin (αA) and αB-crystallin (αB) are among the predominant proteins of the vertebrate eye lens. In vitro, the α-crystallins, which are isolated together as a high molecular mass aggregate, exhibit a number of properties, the most interesting of which is their ability to function as molecular chaperones for other proteins. Here we begin to examine the in vivo functions of α-crystallin by generating mice with a targeted disruption of the αA gene. Mice that are homozygous for the disrupted allele produce no detectable αA in their lenses, based on protein gel electrophoresis and immunoblot analysis. Initially, the αA-deficient lenses appear structurally normal, but they are smaller than the lenses of wild-type littermates. αA^−/− lenses develop an opacification that starts in the nucleus and progresses to a general opacification with age. Light and transmission electron microscopy reveal the presence of dense inclusion bodies in the central lens fiber cells. The inclusions react strongly with antibodies to αB but not significantly with antibodies to β- or γ-crystallins. In addition, immunoblot analyses demonstrate that a significant portion of the αB in αA^−/− lenses shifts into the insoluble fraction. These studies suggest that αA is essential for maintaining lens transparency, possibly by ensuring that αB or proteins closely associated with this small heat shock protein remain soluble.