In vivo studies of translational repression mediated by the granulocyte-macrophage colony-stimulating factor AU-rich element

The AU-rich element (ARE) controls the turnover of many unstable mRNAs and their translation. The granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE is known to be a destabilizing element, but its role in translation remains unclear. Here we studied in vivo the role of the GM-CSF ARE on the mRNA and protein expressions of an enhanced green fluorescent protein reporter gene. The GM-CSF ARE had a repressor effect on translation independently of its effect on mRNA levels. In the context of an internal ribosome entry site, the GM-CSF ARE still repressed translation but was no longer functional as a destabilizing element. Gel retardation assays showed that poly(A)-binding protein is displaced from the poly(A) tail when the ARE is present in the 3'-untranslated region. These data suggest that the GM-CSF ARE controls translation and mRNA decay by interfering with poly(A)-binding protein-mediated mRNA circularization.     

Apomine, a novel hypocholesterolemic agent, accelerates degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and stimulates low density lipoprotein receptor activity

Apomine, a novel 1,1-bisphosphonate ester, has been shown to lower plasma cholesterol concentration in several species. Here we show that Apomine reduced the levels of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the rate-limiting enzyme in the mevalonate pathway, both in rat liver and in cultured cells. Apomine resembles sterols such as 25-hydroxycholesterol in its ability to potently accelerate the rate of HMGR degradation by the ubiquitin-proteasome pathway, a process that depends on the transmembrane domain of the enzyme. The similarity between Apomine and sterols in promoting rapid HMGR degradation extends to its acute requirements for ongoing protein synthesis and mevalonate-derived non-sterol product(s) as a co-regulator. Yet, at suboptimal concentrations, sterols potentiated the effect of Apomine in stimulating HMGR degradation, indicating that these agents act via distinct modes. Furthermore, unlike sterols, Apomine inhibited the activity of acyl-CoA:cholesterol acyltransferase in intact cells but not in cell-free extracts. Apomine stimulated the cleavage of the precursor of sterol-regulatory element-binding protein-2 and increased the activity of low density lipoprotein receptor pathway. This Apomine-enhanced activation of sterol-regulatory element-binding protein-2 was prevented by sterols or mevalonate. Taken together, our results provide a molecular mechanism for the hypocholesterolemic activity of Apomine.     

Hydrophobic helical hairpins: design and packing interactions in membrane environments

Helix-helix interactions within membranes are dominated by van der Waals packing motifs and side chain-side chain hydrogen bond formation, which act in tandem to determine the residues that comprise the interface between two given helices. To explore in a systematic manner the tertiary contacts between transmembrane helices, we have designed and expressed in Escherichia coli highly hydrophobic helix-loop-helix constructs of prototypic sequence K(1)KKKKKKFAIAIAIIAWAX(19)AIIAIAIAIKSPGSKIAIAIAIIAZ(44)AWAIIAIAIAFKKKKKKK(62), where "small" (Ala) and "large" (Ile) residues were used to maximize the tertiary contact area. Evidence that the two transmembrane (TM) segments in the AI construct contain an interface conducive for folding into a hairpin structure was obtained from the results that (i) the single TM AI(pep) peptide derived from the AI hairpin forms SDS-resistant dimers on PAGE gels and (ii) the corresponding sequence forms a strong dimer when examined in vivo in TOXCAT assays. Site-directed mutagenesis of AI hairpins was carried out to incorporate each of the 20 commonly occurring amino acids at X positions. Analysis on Western blots using an oligomerization assay in 12% NuPage-sodium dodecyl sulfate (SDS) indicated that mutants with X = E, D, Q, R, N, H, and K largely formed SDS-resistant dimers-which likely correspond to H-bonded four-helix bundles-while all the others (e.g., X = F, W, L, I, M, V, C, Y, A, T, S, G, and P) remained monomeric. Systematic studies of X/Z double mutants indicated that formation of hairpin dimers is the result of the disruption of stabilizing interactions between the antiparallel helices within the AI construct. The overall results suggest that, in situations where hydrophobic van der Waals packing energy between helices is sufficient to prevent significant rotation about the major axes of interacting helices, intrahairpin side chain-side chain H-bond formation will occur mainly when pairs of polar residues are interfacially located and proximal. Knowledge of the relative contributions of these forces should be of value, for example, in clarifying the context--and the structural consequences--of disease-related mutations.     

Thermodynamic and biophysical characterization of cytochrome P450 BioI from Bacillus subtilis

Cytochrome P450 BioI (CYP107H1) from Bacillus subtilis is involved in the early stages of biotin synthesis. Previous studies have indicated that BioI can hydroxylate fatty acids and may also perform an acyl bond cleavage reaction [Green, A. J., Rivers, S. L., Cheesman, M., Reid, G. A., Quaroni, L. G., Macdonald, I. D. G., Chapman, S. K., and Munro, A. W. (2001) J. Biol. Inorg. Chem. 6, 523-533. Stok, J. E., and De Voss, J. J. (2000) Arch. Biochem. Biophys. 384, 351-360]. Here we show novel binding features of P450 BioI--specifically that it binds steroids (including testosterone and progesterone) and polycyclic azole drugs with similar affinity to that for fatty acids (K(d) values in the range 0.1-160 microM). Sigmoidal binding curves for titration of BioI with azole drugs suggests a cooperative process in this case. BioI as isolated from Escherichia coli is in a mixed heme iron spin state. Alteration of the pH of the buffer system affects the heme iron spin-state equilibrium (higher pH increasing the low-spin content). Steroids containing a carbonyl group at the C(3) position induce a shift in heme iron spin-state equilibrium toward the low-spin form, whereas fatty acids produce a shift toward the high-spin form. Electron paramagnetic resonance (EPR) studies confirm the heme iron spin-state perturbation inferred from optical titrations with steroids and fatty acids. Potentiometric studies demonstrate that the heme iron reduction potential becomes progressively more positive as the proportion of high-spin heme iron increases (potential for low-spin BioI = -330 +/- 1 mV; for BioI as purified from E. coli (mixed-spin) = 228 +/- 2 mV; for palmitoleic acid-bound BioI = -199 +/- 2 mV). Extraction of bound substrate-like molecule from purified BioI indicates palmitic acid to be bound. Differential scanning calorimetry studies indicate that the BioI protein structure is stabilized by binding of steroids and bulky azole drugs, a result confirmed by resonance Raman studies and by analysis of disruption of BioI secondary and tertiary structure by the chaotrope guanidinium chloride. Molecular modeling of the BioI structure indicates that a disulfide bond is present between Cys250 and Cys275. Calorimetry shows that structural stability of the protein was altered by addition of the reductant dithiothreitol, suggesting that the disulfide is important to integrity of BioI structure.     

G Protein-coupled receptor kinase 2 regulator of G protein signaling homology domain binds to both metabotropic glutamate receptor 1a and Galphaq to attenuate signaling

Heterotrimeric guanine nucleotide-binding (G) protein-coupled receptor kinases (GRKs) are cytosolic proteins that contribute to the adaptation of G protein-coupled receptor signaling. The canonical model for GRK-dependent receptor desensitization involves GRK-mediated receptor phosphorylation to promote the binding of arrestin proteins that sterically block receptor coupling to G proteins. However, GRK-mediated desensitization, in the absence of phosphorylation and arrestin binding, has been reported for metabotropic glutamate receptor 1 (mGluR1) and gamma-aminobutyric acid B receptors. Here we show that GRK2 mutants impaired in Galphaq/11 binding (R106A, D110A, and M114A), bind effectively to mGluR1a, but do not mediate mGluR1a adaptation. Galphaq/11 is immunoprecipitated as a complex with mGluR1a in the absence of agonist, and either agonist treatment or GRK2 overexpression promotes the dissociation of the receptor/Galphaq/11 complex. However, these mGluR1a/Galphaq/11 interactions are not antagonized by the overexpression of either GRK2 mutants defective in Galphaq/11 binding or RGS4. We have also identified a GRK2-D527A mutant that binds Galphaq/11 in an AlF4(-)-dependent manner but is unable to either bind mGluR1a or attenuate mGluR1a signaling. We conclude that the mechanism underlying GRK2 phosphorylation-independent attenuation of mGluR1a signaling is RH domain-dependent, requiring the binding of GRK2 to both Galphaq/11 and mGluR1a. This serves to coordinate GRK2 interactions with Galphaq/11 and to disrupt receptor/Galphaq/11 complexes. Our findings indicate that GRK2 regulates receptor/G protein interactions, in addition to its traditional role as a receptor kinase.     

Targeted deletion of protein kinase C lambda reveals a distribution of functions between the two atypical protein kinase C isoforms

Protein kinase C lambda (PKClambda) is an atypical member of the PKC family of serine/threonine kinases with high similarity to the other atypical family member, PKCzeta. This similarity has made it difficult to determine specific roles for the individual atypical isoforms. Both PKClambda and PKCzeta have been implicated in the signal transduction, initiated by mediators of innate immunity, that culminates in the activation of MAPKs and NF-kappaB. In addition, work from invertebrates shows that atypical PKC molecules play a role in embryo development and cell polarity. To determine the unique functions of PKClambda, mice deficient for PKClambda were generated by gene targeting. The ablation of PKClambda results in abnormalities early in gestation with lethality occurring by embryonic day 9. The role of PKClambda in cytokine-mediated cellular activation was studied by making mouse chimeras from PKClambda-deficient embryonic stem cells and C57BL/6 or Rag2-deficient blastocysts. Cell lines derived from these chimeric animals were then used to dissect the role of PKClambda in cytokine responses. Although the mutant cells exhibited alterations in actin stress fibers and focal adhesions, no other phenotypic differences were noted. Contrary to experiments using dominant interfering forms of PKClambda, mutant cells responded normally to TNF, serum, epidermal growth factor, IL-1, and LPS. In addition, no abnormalities were found in T cell development or T cell activation. These data establish that, in vertebrates, the two disparate functions of atypical PKC molecules have been segregated such that PKCzeta mediates signal transduction of the innate immune system and PKClambda is essential for early embryogenesis.