De Novo Protein Biosynthesis

Inhibition of protein synthesis by aminoglycoside antibiotics, especially by streptomycin, is bactericidal (rev.46)). The antibiotic binds to the smaller ribosomal subunit and leads to the formation of abortive initiation complexes of ribosomes, streptomycin and amino acyl tRNA which progressively trap ribosomes in the form of such biologically irreversible complexes. When protein synthesis is prematurely terminated by puromycin and ribosomes are thus made available for reinitiation of de novo protein biosynthesis, the bactericidal action of streptomycin is accelerated47). Destruction of ribosomes under the influence of primaquine operationally also results in non-occurrence of protein synthesis and in a marked bactericidal effect48, 49 ... 

In breast cancer cells ZR-75-1 and T-47D, which also express only low levels of endogenous PXR, treatment with rifampin transiently induces the expression of SLCO1A2 mRNA in a dose- and time-dependent manner [114], Induction by rifampin was shown to be independent of de novo protein biosynthesis. In contrast to SLC01A2, MDR1 was not induced [114], The physiological relevance of these findings has to await further clarification. 

For C. roseus suspension-cultured cells, elicitation with fungal elicitors results in the induction of TDC activity (99,186,202,203,284,329-331). This is due to the induction of expression of the Tdc gene. Similarly, SSS activity is induced (202,203,284,329,330). The induction by the Pythium aphanider-matum or yeast elicitor of the transcription of both genes is not affected by cycloheximide that is, the induction is independent of de novo protein biosynthesis, and thus follows an already available signal-transduction chain. The response is quite fast, for the enhanced transcription can already be measured 15 min after elicitation (202,203). Also, the NADPH cytochrome P-450 reductase mRNA level is induced by elicitation with fungal elicitors (113). Moreno et al (99,151) measured activities of a number of enzymes involved in secondary metabolism in C. roseus before and after elicitation with a P. aphanidermatum preparation. GlOH activity was found to be slightly decreased by elicitation and IPP-isomerase showed similar behavior. The pattern of terpenoids formed by the crude enzyme extracts from elicited and nonelicited cells was different. The total incorporation decreased, that is, the activities of the enzymes of the terpenoid pathway were lower. The relative incorporation decreased particularly for squalene. 

This lack of success seemed even more difficult to explain when the biochemical details behind the aspirin inhibition of platelet function were unravelled. Aspirin was found to irreversibly inhibit the cyclo-oxygenase by acetylation of a serine residue at the active site [398-400]. Since platelets lack a nucleus, they have very little capacity for de novo protein biosynthesis, and thus the inhibition of the cyclo-oxygenase lasts for the entire life span of the platelet, i.e. about 7-10 days [398], and the effects of aspirin are overcome only by the gradual replacement of these damaged cells by new ones. It seemed likely that the use of such an efficient drug should give more pronounced clinical results than was actually the case. 

The hypE proteins are 302-376 residues long and appear to consist of three domains. Domain 1 shows sequence identity to a domain from phosphoribosyl-aminoimida-zole synthetase which is involved in the fifth step in de novo purine biosynthesis and to a domain in thiamine phosphate kinase which is involved in the synthesis of the cofactor thiamine diphosphate (TDP). TDP is required by enzymes which cleave the bond adjacent to carbonyl groups, e.g. phosphoketolase, transketolase or pyruvate decarboxylase. Domain 2 also shows identity to a domain found in thiamine phosphate kinase. Domain 3 appears to be unique to the HypF proteins. 

Mnltifunctional protein which initiates and regnlates de novo pyrimidine biosynthesis caspases-activated DNAse Cdk-activating kinase... 

In 1995, Bonnarme et al. [110] used the analytical techniques that combine isotopic tracing, nuclear magnetic resonance spectroscopy, and mass spectroscopy to compare the enzyme systems of intact cells of high- and low-producing strains of A. terreus. Results show that itaconate formation requires de novo protein synthesis. During acid formation, TCA cycle intermediates increase in both strains. Furthermore, data showed that both the BMP pathway and the TCA cycle are involved in itaconate biosynthesis. Based on the biosynthetic pathway (Fig. 15), one itaconate molecule is produced from one hexose molecule with a theoretical weight yield of 72.2%. The actual yield should be lower due to the loss of carbon to biomass accumulation and cell maintenance. 

A few other less studied biochemical approaches such as purine and pyrimidine metabolism protein biosynthesis and lipid metabohsm in helminths also provide targets for antiparasitic drug design [83]. Like protozoal parasites, some helminths such as S. mansoni (adult and larval forms) lack de novo purine biosynthesis and, therefore, depend entirely on the salvage mechanism for their purine requirements. Similarly amino acid metabolism and biosynthesis of proteins has also been not worked out in many parasites [83a]. Although the helminths meet their requirements of amino acids by absorbing freely from the host, they may also synthesize some amino acids. For example. Fasciola hepatica, schistosomes and other trematodes produce proline by a reaction sequence given in Chart 8. Similarly H. diminuta can... 

All of the enzyme activities involved in de novo UMP biosynthesis have been identified in the trematodes Schistosoma mansoni (81,106,107) and S. japonicum (108). Carbamoyl phosphate synthetase II, aspartate transcarbamoylase and dihydro-orotase have been partially purified from the cytosol and appear to exist, as in mammalian cells, as a multienzyme protein (81,106). Dihydro-orotate oxidase is membrane bound (81), but its electron acceptor has not been identified. Orotate PRTase and OMP decarboxylase are also cytosolic. In mammalian cells these last two enzymes exist as part of a multienzyme protein that efficiently channels orotate to UMP since little free OMP is present in the cell. In S. mansoni they also exist as a multienzyme protein but apparently the channeling is less efiBcient since levels of free OMP are significantly higher than those of UMP (107). 

Most of the de novo lipid biosynthesis in birds occurs in the liver (see Section 4.3), and this is transported to peripheral tissues (induding the developing oocytes) in the form of VLDL. VLDL comprises about 13% protein and the rest is triglyceride, phospholipid. 

Morrison, A.R., Moritz, H. and Needleman, P. (1978). Mechanism of enhanced renal prostaglandin biosynthesis in ureter obstruction. Role of de novo protein synthesis. J. Biol Chem., 253, 8210-12... 

CAD is a 1.5 Mda complex that catalyzes the first three steps in de novo pyrimidine biosynthesis in mammalian cells. The protein consists of six copies of a 243 kDa polypeptide that is organized into 15 domains, subdomains and linkers each with a specific catalytic or regulatory function. Most of these domains have been subcloned and expressed in E. coli where they fold into stable, fully functional proteins. While each domain functions autonomously, interdomain signaling modulates the reactions occurring on different domains to ensure that biosynthesis proceeds in a coordinated fashion. Insights into the signaling mechanism have been provided by the analysis of several hybrid and chimeric molecules constructed by combining domains and subdomains of the mammalian, yeast and E. coli proteins in novel ways. 

Adipose tissue obtained from diabetic animals shows a decrease of the incorporation of labeled amino acid into protein and in the activity of a number of enzymes hexokinase II, glucose-6-P04 dehydrogenase, phosphofructokinase and pyruvate kinase. Amino acid incorporation and enzyme activities return to normal after injection of insulin. The effect of insulin is, at least in the case of hexokinase II and glucose-6-PO4 dehydrogenase, impaired by the injection of actinomycin D indicating that insulin stimulates de novo protein synthesis by triggering the biosynthesis of new messenger RNA. 

It has been proposed that in mammalian cells (1), as in the dase of E, coli (2), de novo purine biosynthesis is controlled by both feedback inhibition by the end products of the pathway and by repression. In procaryotes, repression appears to involve an interaction between a specific nucleotide (ATP or GTP) and a repressor protein binding to the operator region of the early purine genes (2). We have recently isolated a series of PurR" mutants in E. coli, in which repression of the de novo pathway is abolished (3). However such mutants are still subject to feedback inhibition. 

The mechanism of rubber formation was studied by in vitro rubber biosynthesis, by the rubber transferase tightly bound to rubber particles, enzymatically active rubber particles. At least two mechanisms have been postulated for the conversion of IPP to the in vitro synthesized rubber. First is the initiation of new rubber molecules on the surface of the rubber particles and the chain extension of pre-existing rubber molecules, the rubber molecules from the rubber particles used for the rubber biosynthesis. In case of de novo rubber biosynthesis, the new rubber molecules are initiated by the added APP initiators and/or the APP initiators synthesized by the soluble proteins, i.e. IPP isomerase and trowi -prenyltransferase remained in the system. The second is... 

One example of a naturally occurring diazirine, duazomycin A (137 Scheme 11.20), has been reported, isolated in 1985 from a Streptomyces species during a screen for herbicidal compounds [196], It was fotind to inhibit de novo starch synthesis and it was suggested that this is due to direct inhibition of protein synthesis. Duazomycin A is structurally related to 6-diazo-5-oxo-L-norleucine (138), also reported as a natural product from Streptomyces [197], which acts as a glutamine antagonist and inhibits purine biosynthesis [198],... 

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