Anti-hepatitis C virus activity and toxicity of type III phosphatidylinositol-4-kinase beta inhibitors.

Type III phosphatidylinositol-4-kinase beta (PI4KIIIß) was previously implicated in hepatitis C virus (HCV) replication by small interfering RNA (siRNA) depletion and was therefore proposed as a novel cellular target for the treatment of hepatitis C. Medicinal chemistry efforts identified highly selective PI4KIIIß inhibitors that potently inhibited the replication of genotype 1a and 1b HCV replicons and genotype 2a virus in vitro. Replicon cells required more than 5 weeks to reach low levels of 3- to 5-fold resistance, suggesting a high resistance barrier to these cellular targets. Extensive in vitro profiling of the compounds revealed a role of PI4KIIIß in lymphocyte proliferation. Previously proposed functions of PI4KIIIß in insulin secretion and the regulation of several ion channels were not perturbed with these inhibitors. Moreover, PI4KIIIß inhibitors were not generally cytotoxic as demonstrated across hundreds of cell lines and primary cells. However, an unexpected antiproliferative effect in lymphocytes precluded their further development for the treatment of hepatitis C.

Evidence for a nuclear passage of nascent polypeptide-associated complex subunits in yeast.

The nascent polypeptide-associated complex (NAC) has been found quantitatively associated with ribosomes in the cytosol by means of cell fractionation or fluorescence microscopy. There have been reports, however, that single NAC subunits may be involved in transcriptional regulation. We reasoned that the cytosolic location might only reflect a steady state equilibrium and therefore investigated the yeast NAC proteins for their ability to enter the nucleus. We found that single subunits of yeast NAC can indeed be transported into the nucleus and that this transport is an active process depending on different nuclear import factors. Translocation into the nucleus was only observed when binding to ribosomes was inhibited. We identified a domain of the ribosome-binding NAC subunit essential for nuclear import via the importin Kapl23p/Pselp-dependent import route. We hypothesize that newly translated NAC proteins travel into the nucleus to bind stoichiometrically to ribosomal subunits and then leave the nucleus together with these subunits to concentrate in the cytosol.

The nascent polypeptide-associated complex (NAC) of yeast functions in the targeting process of ribosomes to the ER membrane.

We study here the binding of ribosomes to the endoplasmic reticulum (ER) membrane and its dependence on nascent polypeptide-associated complex (NAC). For this, we use an in vitro translation system in combination with isolated microsomes. Importantly, all components in the system are derived from a single source, Saccharomyces cerevisiae. Ribosome nascent chains (RNCs) of the two naturally occurring invertase species (secreted or cytosolic) were prepared in wild-type, delta alpha NAC or delta alpha beta 1 beta 3 NAC translation lysates and tested for binding to the corresponding microsomal membranes. We provide evidence that NAC prevents binding of RNCs without a signal sequence to yeast membranes. In the absence of NAC, signal-less RNCs are able to bind to ER membranes. However, following puromycin treatment, only very few nascent chains translocate into the lumen, as detected by glycosylation.

Initial characterization of the nascent polypeptide-associated complex in yeast.

The three subunits of the nascent polypeptide-associated complex (alpha, beta1, beta3) in Saccharomyces cerevisiae are encoded by three genes (EGD2, EGD1, BTT1). We found the complex bound to ribosomes via the beta-subunits in a salt-sensitive manner, in close proximity to nascent polypeptides. Estimation of the molecular weight of the complex of wild-type cells and cells lacking one or two subunits revealed that the composition of the complex is variable and that as yet unknown proteins might be included. Regardless of the variability, a certain balance of the subunits has to be maintained: the deletion of one subunit causes downregulation of the remaining subunits at physiological growth temperature. Cells lacking both beta-subunits are unable to grow at 37 degrees C, most likely due to a toxic effect of the alpha-subunit. Based on in vitro experiments, it has been proposed that the function of mammalian nascent-polypeptide associated complexes (NAC) is to prevent inappropriate targeting of non-secretory nascent polypeptides. In vivo, however, the lack of NAC does not cause secretion of signal-less invertase in yeast. This result and the lack of a drastic phenotype of cells missing one, two or three subunits at optimal conditions (28 degrees C, YPD-medium) suggest either the existence of a substitute for NAC or that cells tolerate or 'repair' the damage caused by the absence of NAC.

A protein complex required for signal-sequence-specific sorting and translocation.

We have purified a nascent-polypeptide-associated complex (NAC) which prevents short ribosome-associated nascent polypeptides from inappropriate interactions with proteins in the cytosol. NAC binds nascent-polypeptide domains emerging from ribosomes unless a signal peptide is fully exposed. Depletion of cytosolic proteins (including NAC) from ribosomes carrying nascent polypeptides allows the signal recognition particle (SRP) to crosslink to polypeptides irrespective of whether or not they contain signal peptides. In the absence of cytosol, proteins lacking signal peptides can be mistranslocated into the endoplasmic reticulum in vitro, albeit with low efficiency. Readdition of NAC restores the specificity of SRP and fidelity of translocation.

Overexpression of the ER-membrane protein P-450 CYP52A3 mimics sec mutant characteristics in Saccharomyces cerevisiae.

High expression of microsomal cytochrome P-450 CYP52A3 from Candida maltosa induces the formation of membrane stacks in Saccharomyces cerevisiae. Membrane proliferation is accompanied by coinduction of the ER proteins KAR2p and SEC61p and accumulation of precursor forms of proteins that have to translocate across the ER membrane (KAR2p, alpha factor). Cytosolic proteins (SSA1p and 2p) and mitochondrial proteins (CYT c1p and F1 beta p) are not affected. N-terminal truncated P-450 proteins remain in the cytoplasm and fail to induce membrane proliferation, KAR2p/SEC61p expression, and precursor accumulation. Membrane and precursor protein accumulation are typical features of sec mutants. We assume that the high amounts of P-450p block one or more factor(s) of the transport machinery and thereby cause the observed phenomena.

Molecular cloning and characterization of the primary structure of the alkane hydroxylating cytochrome P-450 from the yeast Candida maltosa.

A cDNA library was established starting from poly(A) RNA of n-alkane-grown Candida maltosa cells and cDNA clones were isolated containing the entire coding sequence for the alkane hydroxylating cytochrome P-450. The deduced protein consists of 521 amino acids, contains two putative transmembrane segments in the N-terminal region and has a characteristic heme-binding sequence in the C-terminal part. Sequence alignments with members of 11 reported cytochrome P-450 families revealed a strong homology to an alkane-inducible cytochrome P-450 from Candida tropicalis.

Post-translational transport of proteins into microsomal membranes of Candida maltosa.

We have isolated from the yeast Candida maltosa microsomal membranes that are active in the translocation of proteins synthesized in cell-free systems derived from C. maltosa, Saccharomyces cerevisiae or wheat germ. Translocation and core glycosylation of prepro-alpha-factor, a secretory protein, were observed with yeast microsomes added during or after translation. The signal peptide is cleaved off. Cytochrome P-450 from C. maltosa, the first integral membrane protein studied in a yeast system, is also inserted both co- and post-translationally into Candida microsomal membranes. Its insertion into canine microsomes occurs efficiently only in a co-translational manner and is dependent on the function of the signal recognition particle.

Oxygen limitation induces indirectly the synthesis of cytochrome P-450 mRNA in alkane-growing Candida maltosa.

Candida maltosa cells grown on hexadecane under oxygen limitation have an up to 6-fold higher cytochrome P-450 content in comparison to cells cultivated at oxygen saturation. We show by mRNA quantification using an in vitro translation system and subsequent specific immunoprecipitation that the cytochrome P-450 induction occurs mainly on the transcriptional level. The signal for induction may be the enhanced intracellular hexadecane concentration owing to a reduced hydroxylation capacity of the cytochrome P-450 at oxygen limitation.

N-alkanes induce the synthesis of cytochrome P-450 mRNA in Candida maltosa.

In the yeast Candida maltosa the level of cytochrome P-450 was 100-300-fold higher in alkane-grown cells than in glucose-grown ones (detected by a radioimmunoassay). It was shown immunochemically (1) that this was not the result of an assembly of preexisting apoenzyme with the prosthetic heme group. By cell-free translation of total poly(A)RNA in a wheat germ system and subsequent immunoprecipitation it was shown that the amount of mRNA coding for cytochrome P-450 paralleled its concentration in the cell.