What are the differences between eukaryotic and prokaryotic mRNA structures and translation mechanisms?

9 Differences Between Eukaryotic and Prokaryotic Protein Synthesis

The overall scheme of protein synthesis is similar in all living cells. However, there are significant differences between bacteria and eukaryotes. These are summarized in Table 13.04 and discussed in the following sections. Note that eukaryotic cells contain mitochondria and chloroplasts, which have their own DNA and their own ribosomes. The ribosomes of these organelles operate similarly to those of bacteria and will be considered separately below. In eukaryotic protein synthesis, it is usually the cytoplasmic ribosomes that translate nuclear genes. Several aspects of eukaryotic protein synthesis are more complex. The ribosomes of eukaryotic cells are larger and contain more rRNA and protein molecules than those of prokaryotes. In addition, eukaryotes have more initiation factors and a more complex initiation procedure.

Table 13.04. Comparison of Protein Synthesis

Eukaryotic ribosomes are larger and more complex than those of prokaryotes.

A few aspects of protein synthesis are actually less complex in eukaryotes. In prokaryotes, mRNA is polycistronic and may carry several genes that are translated to give several proteins. In eukaryotes, each mRNA is monocistronic and carries only a single gene, which is translated into a single protein. In prokaryotes, the genome and the ribosomes are both in the cytoplasm, whereas in eukaryotes the genome is in the nucleus. Consequently, coupled transcription and translation is not possible for eukaryotes (except for their organelles; discussed later).

Both prokaryotes and eukaryotes have a special initiator tRNA that recognizes the start codon and inserts methionine as the first amino acid. In prokaryotes, this first methionine has a formyl group on its amino group (i.e., it is N-formyl-methionine), but in eukaryotes unmodified methionine is used.

9.1 Initiation, Elongation, and Termination of Protein Synthesis in Eukaryotes

Initiation of protein synthesis differs significantly between prokaryotes and eukaryotes. Eukaryotic mRNA has no ribosome-binding site (RBS). Instead recognition and binding to the ribosome rely on a component that is lacking in prokaryotes: The cap structure at the 5′ end, which is added to eukaryotic mRNA before it leaves the nucleus (see Chapter 12: Processing of RNA). Cap-binding protein (one of the subunits of eIF4) binds to the cap of the mRNA.

Eukaryotes also have more initiation factors than prokaryotes and the order of assembly of the initiation complex is different (see Table 13.05). Two different complexes assemble before binding to mRNA. The first is the 43S pre-initiation complex. This is an assembly of the small 40S subunit of the ribosome attached to several eukaryotic initiation factors (eIFs). These include eIF1, eIF1A, eIF3, and eIF5. This binds the charged initiator tRNA, Met-tRNAiMet, plus eIF2. The second complex, the cap-binding complex, contains cap-binding protein (eIF4E), eIF4G, eIF4A, eIF4B, and poly(A)-binding protein (PABP).

Table 13.05. Translation Factors: Prokaryotes vs Eukaryotes

	Prokaryotes	Eukaryotes
Initiation	IF1	eIF1A
IF2	eIF5B (GTPase)
IF3	eIF1
	eIF2 (α, β, γ) (GTPase)
	eIF2B (α, β, γ, δ, ɛ)
	eIF3 (13 subunits)
	eIF4A (RNA helicase)
	eIF4B (activates eIF4A)
	eIF4E (cap-binding protein)
	eIF4G (eIF4 complex scaffold)
	eIF4H
	eIF5
	eIF6
	PABP (Poly(A)-binding protein)
Elongation	EF-Tu	eEF1A
EF-Ts	eEF1B (2–3 subunits)
	SBP2
EF-G	eEF2
Termination	RF1	eRF1
RF2
RF3	eRF3
Recycling	RRF
EF-G
	eIF3
	eIF3j
	eIF1A
	eIF1

Functionally homologous factors are in the same row.

Adapted from Table 1 of Rodnina MV and Wintermeyer W. (2009) Recent mechanistic insights into eukaryotic ribosomes. Curr. Op. Cell Biol. 21: 435–443.

Eukaryotic mRNA is recognized by its cap structure (not by base pairing to rRNA).

During eukaryotic initiation, cap-binding complex first attaches to the mRNA via its cap. Next, the poly(A) tail is bound by PABP so that the mRNA forms a ring. This structure can now bind the 43S assembly. In order to align the Met-tRNAiMet with the correct AUG codon, the two structures work together to scan each codon from the 5′ end. This scanning process uses energy from ATP (Fig. 13.29). Normally, the first AUG is used as the start codon (see Box 13.02 for exceptions), although the sequence surrounding the AUG is important. The consensus is GCCRCCAUGG (R=A or G). If its surrounding sequence is too far from consensus an AUG may be skipped. Once a suitable AUG has been located, eIF5 joins the complex, which in turn allows the 60S subunit to join and the cap-binding protein, eIF2, eIF1, eIF3, and maybe eIF5 to depart. eIF5 uses energy from GTP to accomplish this remodeling of the ribosome.

Figure 13.29. Assembly of the Eukaryotic Initiation Complex

(A) The cap-binding complex includes poly(A)-binding protein (PABP), eIF4A, eIF4B, eIF4E, and eIF4G, which is in an unphosphorylated state when unbound to mRNA. ATP transfers phosphates to the complex to make it competent for binding the mRNA. (B) The 43S initiation complex forms bringing the small ribosomal subunit together with the tRNAimet. This complex uses GTP to attach the tRNA to the 40S subunit via eIF2. In addition, initiation factors eIF1, eIF1A, eIF3, eIF5, and eIF2B guide and make the complex competent to bind to the 5′-UTR of mRNA. (C) The mRNA is recognized by the cap-binding complex via the connections between eIF4E and PABP which bind the 5′ and 3′ ends of the mRNA, respectively. These two connections cause the rest of the mRNA to loop out. When this is established, then the 43S pre-initiation complex can attach and start scanning for the first AUG. After pausing at the first AUG, then the 50S subunit of the ribosome can bind and initiate translation.

Box 13.02

Internal Ribosome Entry Sites

Although most eukaryotic mRNA is scanned by the 40S subunit to find the first AUG, exceptions do occur. Sequences known as internal ribosome entry sites (IRES) are found in a few mRNA molecules. As the name indicates, these allow ribosomes to initiate translation internally, rather than at the 5′ end of the mRNA. IRES sequences were first found in certain viruses that have polycistronic mRNA despite infecting eukaryotic cells. In this case, the presence of IRES sequences in front of each coding sequence allows a single mRNA to be translated to give multiple proteins. The best known examples are members of the Picornavirus family, which includes poliovirus (causative agent of polio) and rhinovirus (one of the agents of common cold).

More recently, it has been found that a few special mRNA molecules encoded by eukaryotic cells themselves also possess IRES sequences. During major stress situations, such as heat shock or energy deficit, synthesis of the majority of proteins is greatly decreased. Much of this regulation occurs at the initiation stage of translation (discussed later). However, a few proteins are exempted from this down-regulation as they are needed under stress conditions. The mRNAs encoding these proteins often contain an IRES sequence. In these cases, the mRNA carries only a single coding sequence and the IRES is located in the 5′-UTR, between the 5′ end of the mRNA and the start of the coding sequence. This allows translation to be initiated at the IRES even in the absence of the standard initiation/scanning procedure.

The next stage is elongation (Fig. 13.30). Of all the stages of translation, elongation in bacteria and eukaryotes is the most similar. As in bacteria, elongation factors work to decode the mRNA and bind the tRNA into the A-site of the ribosome. Rather than EF-Tu and EF-Ts, eukaryotes use eEF1A to deliver the tRNA using GTP hydrolysis for energy and eEF1B to replace the depleted GDP with fresh GTP. The only difference is that eukaryotic elongation factors include more subunits. The remaining steps are the same. The peptidyl transferase activity of the 28S rRNA of the large subunit links the incoming amino acid to the polypeptide chain. Then elongation factor eEF2 (direct counterpart to bacterial EF-G) uses GTP to drive the conformational changes in the ribosome and ratchet the tRNAs from the P- and A-sites into the E- and P-sites. Elongation continues until a stop codon enters the A-site.

Figure 13.30. Beginning Eukaryotic Translation Elongation

Once the eukaryotic 40S subunit complex finds the first AUG, then the remaining 60S subunit and associated factors combine to form the final 80S ribosome.

Eukaryotic termination differs from prokaryotic termination in two ways. First, rather than having two different release factors (RF1 and RF2) to recognize different stop codons, eukaryotes have a single release factor (eRF1) that recognizes all three stop codons. eRF1 binds the stop codon, but this does not affect peptide bond formation. Instead, eRF3 carrying a GTP molecule binds to eRF1. GTP hydrolysis then rearranges the factors and the final amino acid attaches to the polypeptide. Therefore, eukaryotes require GTP for polypeptide completion, whereas in bacteria, RF1 or RF2 is sufficient.

Finally, as in bacteria, eukaryotic ribosomes are recycled. eIF3 triggers the release of the 60S subunit, and then eIF1 releases the final tRNA. An additional factor, eIF3j, then removes the mRNA. The components are then recycled.

Abstract

Protein synthesis is the process of synthesizing new, or the regeneration of existing, functional peptides. This process is highly regulated, involving a network of upstream and downstream factors that modulate mRNA translation initiation and elongation through the mechanistic target of rapamycin complex 1 (mTORC1) pathway. mTORC1 signaling can be upregulated or inhibited in response to a variety of stimuli that dictate protein balance. This chapter will review the process of protein synthesis, with a focus on the intracellular regulation of mTORC1 signaling in muscle. The regulation of myogenesis, that is, the process of developing new and regeneration of existing muscle cells, will also be detailed in the context of intramuscular regulation of protein synthesis. This chapter will provide the basis for understanding the protein synthetic responses to endogenous and exogenous modulators of human health and function, such as exercise, nutrition, disease, and aging.

Protein Synthesis

Protein synthesis represents the major route of disposal of amino acids. Amino acids are activated by binding to specific molecules of transfer RNA and assembled by ribosomes into a sequence that has been specified by messenger RNA, which in turn has been transcribed from the DNA template. Peptide bonds are then formed between adjacent amino acids. Once the polypeptide chain has been completed the subsequent folding, post-translational amino acid modifications and protein packaging are all determined by the primary sequence of amino acids. The rate of protein synthesis is controlled by the rate of transcription of specific genes, by the number and state of aggregation of ribosomes and by modulation of the rate of initiation of peptide synthesis.

A Mechanism of Action of Inhibitors of Protein Synthesis

Protein synthesis occurs in the cytoplasm on ribonucleoprotein particles, the ribosomes. Messenger RNA, which contains within its nucleotide sequence the code to direct the synthesis of one or several polypeptide chains, is synthesized by RNA polymerase on the DNA template and is transported into the cytoplasm, where it becomes bound to the ribosomes and directs the placement of amino acyl-transfer RNAs in the proper sequence. An amino acid, which has been activated and esterified to a specific species of tRNA, is bound to the ribosomal acceptor site by virtue of codon–anticodon interactions. Peptidyl transferase, an integral part of the ribosome, catalyzes the formation of a peptide bond between the carboxyl group of the nascent peptide (bound as peptidyl-tRNA to the ribosomal donor site) and the amino group of the new amino acid. The resultant peptidyl tRNA is translocated to the donor site by a GTP-requiring enzyme, freeing the acceptor site for the attachment of the next amino acyl-tRNA (Watson, 1970).

The relationship between protein synthesis and the physiological expression of radiation damage has been explored primarily with the use of inhibitors of protein synthesis. The conclusions drawn from these studies are based on two assumptions: first, that the inhibition affects one and only one biochemical reaction, and second, that this specific biochemical reaction has no rapid indirect effects on the general metabolism of the cell.

Puromycin, which functions as an analog of amino acyl-tRNA (Morris and Schweet, 1961; Rabinovitz and Fisher, 1962), appears to inhibit protein synthesis in prokaryotic and eukaryotic cells by releasing incomplete polypeptide chains from the ribosome (Allen and Zamecnik, 1962; Nathans, 1964). Cycloheximide can inhibit the initiation, elongation, or termination of protein synthesis in eukaryotic cells by blocking translocation, thereby preventing further movement of the ribosome along the messenger RNA (Obrig et al., 1971; Rajalakshmi et al., 1971). Chloramphenicol inhibits the synthesis of protein in bacteria and selectively inhibits protein synthesis in the mitochondria and chloroplasts of the eukaryotic cells that have been studied (Sager, 1972). This antibiotic binds to the large ribosomal subunit (Vazquez, 1965) and interferes with peptide bond formation (e.g., Traut and Monro, 1964). Streptomycin specifically inhibits microbial and mitochondrial protein synthesis by binding to the small ribosomal subunit (Davies, 1964; Cox et al., 1964) and causing misreading of the genetic code (Davies et al., 1964).

Introductory Comments

Protein synthesis in mitochondria follows the same basic steps seen in bacterial and eukaryotic cytoplasmic translational systems. The process is divided into four major stages – initiation, elongation, termination and ribosome recycling. Given the presumed prokaryotic origin of mitochondria, it is expected that the process of protein synthesis in this organelle will be more closely related to that of bacteria than to that of the eukaryotic cell cytoplasm. This idea is borne out by studies of the translational machinery; however, there are a number of interesting and fundamental differences between mitochondrial and bacterial translation. Further, there are clearly distinct differences in how this process takes place in the mitochondria of different organisms. The most detailed studies have been carried out with the mammalian and yeast mitochondrial systems as summarized below.

Low nutritional intake and low protein intake

Muscle protein synthesis rate is reported to be reduced 30% in the elderly, but there is controversy as to the extent to which this reduction is due to nutrition, disease, or physical inactivity rather than aging.82,83 It is recognized by some that protein intake in elders should exceed the 0.8 g/kg per day recommend intake.84 Muscle protein synthesis is also decreased in fasting elderly subjects, especially in specific muscle fractions like mitochondrial proteins,85 and thus, the anorexia of aging and its underlying mechanisms contribute to sarcopenia by reducing protein intake.

Muscle protein synthesis is directly stimulated by amino acid and essential amino acids intake,86 and protein supplementation has been explored in the prevention of sarcopenia. However, many interventional studies have not reported a significant increase muscle mass or protein synthesis with a high protein diet even when accompanied by resistance training.87–89 The lack of effect of protein intake on protein synthesis stimulation may have several explanations.38 A higher splanchnic extraction of dietary amino acids has been already reported.90 This could limit the delivery of dietary amino acids to the peripheral skeletal muscle.

1 Theory

Protein synthesis is a highly regulated process that is controlled by a complex network of proteins. Many of these proteins are essential for viability and mutations are not well tolerated, often affecting the fidelity or rates of protein synthesis that can dramatically affect growth (Carr-Schmid et al., 1999; Hinnebusch, 1985). Several forms of cellular stress can also trigger the repression of protein synthesis. Moreover, many bacterial and viral pathogens target the host translation machinery (Gradi et al., 1998; Honjo et al., 1968; Shenton et al., 2006). For these reasons, it is essential to have quantitative methods for measuring protein synthesis in vivo under a variety of conditions. One commonly used method is the incorporation of [35 S]-methionine into total cellular proteins during an interval of time that allows protein synthesis to be measured. This method has the advantages of being performed in vivo and without modification of the yeast strains to be studied unless the strain is auxotrophic for methionine biosysnthesis. It cannot, however distinguish between the inhibition of translation at different stages of translation (initiation, elongation or termination). This requires polyribosomal analysis to be performed in addition to [35 S]-methionine incorporation.

Requirement of Local Protein Synthesis for Sema3A Signaling.

Inhibition of protein synthesis prevented Sema3A-induced growth cone turning or collapse. Upon Sema3A stimulation, local protein synthesis in growth cones was rapidly increased by inactivation of the translation repressor eIF-4EBP1 and subsequent activation of the translation initiation factor eIF-4E. Although not shown directly, local protein synthesis may alter the receptor composition on the growth cone surface, a mechanism similarly found in ephrin signaling (see Modulation of Ephrin/Eph Signaling earlier). In contrast, inhibition of the ubiquitin-proteasome system did not alter the chemotropic response to Sema3A.48 Therefore, local protein synthesis but not degradation seems to be required for Sema3A signaling.

3.9 Roles of Protein Molecules in Heat Tolerance

Ubiquitination serves as a versatile posttranslational modification that mediates growth and development of all eukaryotic species. Ubiquitin is a stable, highly conserved, and universally expressed protein. The covalent attachment of ubiquitin to a lysine residue of select proteins can regulate stability, activity, and trafficking. Genome sequencing has revealed the extent to which plants rely on protein ubiquitination to regulate organismal processes. For example, over 6% of A. thaliana protein-coding genes are dedicated to the ubiquitin 26S proteasome system (UPS) (Vierstra, 2009). In plant species, the UPS regulates fundamental processes such as embryogenesis, photomorphogenesis, and organ development (Thomann et al., 2005; Sonoda et al., 2009; Pokhilko et al., 2011). In addition to regulating these fundamental processes, the UPS has recently emerged as a major player in plant responses to abiotic stresses.

The UPS functions within the cytoplasm and nucleus to modulate the levels of regulatory proteins and to remove misfolded or damaged proteins that may accumulate as a result of exposure to abiotic stress. One of the first indications that the UPS was involved in regulating plant stress tolerance was the observation that expression of polyubiquitin genes is stress-regulated (Christensen et al., 1992; Genschik et al., 1992; Sun and Callis, 1997). Ubiquitin is encoded by multiple polyubiquitin genes (UBQ3, UBQ4, UBQIO, UBQ11, and UBQ14) that contain three to six ubiquitin-coding regions in tandem (Callis et al., 1995). Following translation, nascent polyubiquitin proteins are proteolytically processed into ubiquitin monomers (Vierstra, 1996). The pool of free ubiquitin molecules is regulated through differential expression of the polyubiquitin genes (Christensen et al., 1992; Genschik et al., 1992; Sun and Callis, 1997). Specifically, transcript abundance of Arabidopsis UBQ14 is increased during heat stress (Sun and Callis, 1997). Similarly, high temperatures also induce the expression of multiple polyubiquitin genes in tobacco, potato, and maize (Christensen et al., 1992; Garbarino et al., 1992; Genschik et al., 1992). In fact, overexpression of a single mono-ubiquitin gene enhances tolerance to multiple stresses without adversely affecting growth and development under favorable conditions (Guo et al., 2008). Transgenic tobacco overexpressing a wheat polyubiquitin gene, containing a single ubiquitin repeat, were more tolerant of cold, high salinity, and drought conditions compared with control plants. The stress-induced expression of polyubiquitin genes is consistent with the role of the UPS in turning over damaged proteins to mitigate the negative effects of environmental stress (Lyzenga and Stone, 2012). Defects in 26S proteasome function also alter plant tolerance to various environmental stresses. The 26S proteasome is an ATP-dependent protease complex consisting of a proteolytic 20S complex capped on one or both ends by a 19S regulatory particle. Access to the active sites of the 20S complex is regulated by the regulatory particle that mediates substrate recruiting, unfolding, translocation into the proteolytic chamber of the 20S, and recycling of ubiquitin molecules (Strickland et al., 2000; Navon and Goldberg, 2001).

Protein synthesis elongation factor Tu (EF-Tu) is a protein that plays a central role in the elongation phase of protein synthesis in bacteria and organelles including mitochondria and plastids in plants. The cytosolic homolog of EF-Tu in plants is EF-1α. The polypeptide elongation cycle proceeds in three steps:

EF-Tu binds GTP and aminoacyl-tRNA, which leads to the codon-dependent placement of this aminoacyl-tRNA at the A site of the ribosome, GTP hydrolysis, and release of EF-Tu-GDP from the ribosome;

EF-Ts (elongation factor Ts) facilitates the exchange of EF-Tu-bound GDP for GTP;

Upon the peptide bond formation, EF-G (elongation factor G) translocates the mRNA one codon to allow for the arrival of the new aminoacyl-tRNA in the A site (Riis et al., 1990).

EF-Tu and EF-Ts were first isolated as components of so-called factor T (transfer), and labeled as thermounstable (Tu) and thermostable (Ts) fractions (Lucas-Lenard, 1971), respectively. This comparison of thermostability is questionable because EF-Tu was later proved to endure high-temperature treatments (Rao et al., 2004), especially when complexed with nucleotide factors, GTP or GDP (Caldas et al., 1998). The effect of EF-Tu overexpression on development of organismal heat tolerance was first examined in E. coli (Moriarty et al., 2002). A gene for maize plastid EF-Tu (Zmeftu1) was isolated from a cDNA library constructed using mRNA from aerial tissues of maize line B73 exposed first to drought stress and then to 45°C heat stress (Bhadula et al., 2001). The pTrcHis2A vector carrying Zmeftu1 was used to transform competent E. coli cells of the strain DH5α. E. coli transformed with a maize EF-Tu expression construct (pTrcHis2A-Zmeftu1) was exposed to a high temperature of 55°C and viability assessed at 37°C. Analysis of the E. coli protein extracts showed that the maize EF-Tu protein was produced at a high level, and the EF-Tu proteins were in the soluble form in the bacterial cells. Significantly, much more E. coli cells induced to produce the recombinant EF-Tu survived high-temperature exposure than their noninduced counterparts and nontransformed control cells, demonstrating that the maize EF-Tu was involved in the development of heat tolerance.

EF-Tu plays an important role in heat tolerance. Fu et al. (2008) hypothesized that overexpression of an EF-Tu gene may enhance heat tolerance in crop plants and tested this hypothesis by introducing a maize plastid EF-Tu gene (Zmeftu1) into two cultivars of hexaploid wheat: Bobwhite (BW) and Xinchun 9 (XC9). Twenty-four transgenic cell lines have been regenerated and grown in a greenhouse, and 23 lines produced T1 seeds. Molecular analyses (PCR and genomic DNA blotting) of transgenic plants have confirmed the stable and inheritable insertion of the transgene in the wheat genome. The transgenic cell lines are independent events. Also, wheat genome appears to harbor three plastid EF-Tu genes (copies) as genomic DNA blots of two nontransgenic controls (BW and XC9), probed with maize EF-Tu cDNA, have showed three hybridization bands. These hybridization bands also indicate high similarity between maize and wheat EF-Tu gene sequences. Indeed, an alignment of a wheat plastid EF-Tu cDNA sequence (TC264979) from wheat cDNA (EST) database and the maize EF-Tu cDNA probe sequence shows 88% identity. At the protein level, these two plastid EF-Tu sequences show 88% identity and 93% similarity (BLAST 2 Sequences Program); RNA blotting analysis has demonstrated that maize EF-Tu mRNA is accumulated at a high level in transgenic wheat. One-dimensional immunoblotting has shown that several transgenic events display significantly higher levels of EF-Tu proteins than nontrans-control XC9. Two-dimensional immunoblotting has shown that the maize EF-Tu protein in transgenic wheat appears to be posttranslationally modified because several maize EF-Tu protein spots with different pI are detected. The overexpression of maize EF-Tu in transgenic wheat does not have any noticeable adverse effects on plant growth and development and does not compromise agronomic performance in nonstress conditions. Thermal aggregation assays have showed that transgenic events with increased expression of plastid EF-Tu display reduced thermal aggregation of leaf proteins. The transgenic plants with increased levels of the plastid EF-Tu displayed reduced injuries to photosynthetic membranes (thylakoids), enhanced rate of CO2 fixation, and fewer visible signs of heat injuries following exposure to heat stress (Fu et al., 2012). The reduced injury and enhanced CO2 fixation are probably contributable to the protection of photosynthetic membranes and photosynthesis-related enzymes under heat stress. Several high-performance transgenic lines have been advanced to the homozygous stage (Fu et al., 2008; Fu and Ristic, 2010). Field trials for testing the performance of these transgenic wheat lines in adverse natural environments are currently underway. A screening of transposon-mutagenized maize library TUSC (Trait Utility System for Corn) has identified and led to the isolation of a maize plastid EF-Tu null mutant in which a 5-kb transposon Mu9 is inserted in one of the two plastid EF-Tu gene sequences. The mutant has reduced EF-Tu protein level and reduced heat tolerance (Ristic et al., 2004).

Maize plastid EF-Tu promoters are enriched in abiotic stress response elements (REs), including heat, low temperature, drought, and salinity REs. This may explain the findings that the maize plastid EF-Tu protein is upregulated by heat and by the combination of heat and dehydration (Ristic et al., 1991). The possibilities if the EF-Tu genes are upregulated by salinity and low temperature have not been tested in maize. The existence of low-temperature REs in the promoter is somewhat unexpected due to the fact that EF-Tu protein is accumulated by high temperatures. The presence of diverse abiotic elements in the promoters of the EF-Tu indicates the involvement of this gene in diverse abiotic responses. In fact, any abiotic stress, if sufficiently intense, will result in nonnative conformations (denaturation) of proteins (Feder and Hofman, 1999). Thus, molecular chaperones, such as plastid EF-Tu, may be required in facilitating the recovery of denatured proteins.

7.14.6.2.4 Fungal protein biosynthesis inhibitors

Protein synthesis has long been considered as an attractive target in the development of antimicrobial agents, in light of the widespread use of antibacterial antibiotics that target the specific areas of this process. However, application of this idea to the field of antifungal therapy is not an easy task, due to the eukaryotic rather than prokaryotic nature of fungi, and therefore the great degree of similarity between the fungal and mammalian protein synthesis machineries. Two soluble elongation factors show some fungal specificity: EF3, a factor that is required by fungal ribosomes only, and EF2, which has been demonstrated to possess at least one functional distinction from its mammalian counterpart. The sordarins are the most important family of antifungal agents acting at the protein synthesis level. Compounds in this class inhibit in vitro translation in C. albicans, C. tropicalis, C. kefyr, and C. neoformans, to varying degrees.117 The lack of activity of the sordarins against C. krusei, C. glabrata, and C. parapsilosis, in comparison with their extremely high levels of potency against C. albicans, suggests that these compounds have a highly specific binding site, which may also be the basis for the greater selectivity of these compounds in inhibiting fungal, but not the mammalian, protein synthesis. The most advanced inhibitors of fungal protein biosynthesis are analogs of the natural product lead sordarin lead, GR135402 (see Figure 13).146,147 Its spectrum of activity includes C. albicans, C. tropicalis, and C. neoformans, where impressive antifungal activity has been observed in vitro (MIC<1 μg mL−1 in many cases), but not in other Candida or Aspergillus species, which severely restricts its potential as a quality lead. Efficacy has been seen in animal models,147 although at high dose and predominantly via nonoral routes, reflecting the potential for rapid clearance and limited oral bioavailability.