Tracy G. Anthony, PhD and Margaret McNurlan, PhD In addition to serving as substrates for protein synthesis, amino acids also are degraded to provide compounds that can enter central pathways of fuel metabolism, and small amounts are converted to nonprotein compounds. For most adults who are in protein balance, the amount of amino acids degraded is essentially equivalent to the amount in the diet. The degradative pathways are also shown schematically in Figure 13-1. Degradation involves the removal of nitrogen, primarily as urea and ammonia, and the catabolism of the carbon skeleton. The end result of the degradation of the carbon skeleton of amino acids is the provision of energy either directly or through the formation of compounds such as glucose and fatty acids, which can then be stored or metabolized to provide energy. The pathways for the oxidative metabolism of amino acids and nitrogen excretion are discussed in detail in Chapter 14, but it is important to understand the integrated nature of protein metabolism that is represented by Figure 13-1. The needs of the body regulate the flux of amino acids through these possible pathways; that is, whether amino acids are used for the synthesis of protein, oxidized for energy, or used to form glucose. The pathways shown in Figure 13-1 can be simplified to focus specifically on the interactions of amino acids with body protein through protein synthesis and protein degradation (Figure 13-2). In this simplified scheme, all the tissue and circulating proteins are considered together, and likewise the free amino acid pool is reduced to a single, homogeneous pool, rather than the complex arrangements of pools in blood, individual tissues, and subcellular compartments that are known to exist. This simplification has proved helpful in conceptualizing and developing methods for measuring the exchange of amino acids between the free amino acid pool and the protein pool. Q, the sum of the rates of either entry or exit from the free amino acid pool, has been termed the flux rate. This is sometimes also known as the rate of appearance, Ra, or rate of disappearance, Rd. In an adult in nitrogen equilibrium or protein balance, nitrogen intake (I) is equal to nitrogen excretion (E), and protein synthesis (S) is equal to protein degradation (D). For an individual to be in positive nitrogen balance, there must be net protein synthesis or accretion (S > D), whereas there must be net protein degradation or loss for an individual to be in negative nitrogen balance (S < D). From the aforementioned relationships, it is clear that protein is retained in the body when synthesis exceeds degradation, and that protein is lost from the body when degradation exceeds synthesis. As shown in Figure 13-3, loss of body protein can occur from a decrease in the synthesis of protein with no change in protein degradation (Figure 13-3, A), an increase in the degradation with no change in protein synthesis (Figure 13-3, B), from either an increase (Figure 13-3, C) or a decrease (Figure 13-3, D) in both synthesis and degradation with protein degradation exceeding protein synthesis, or from an increase in degradation along with a decrease in synthesis (Figure 13-3, E). In a number of pathological conditions, body protein degradation exceeds synthesis, with both protein synthesis and degradation rates elevated over the rates in healthy individuals. In the case of infection in malnourished children, body protein is lost, but both synthesis and degradation rates are depressed. In early starvation, net loss of lean body mass is due to an increase in protein degradation along with a decrease in protein synthesis. Likewise, positive protein balance can be achieved by increases in protein synthesis, by decreases in protein degradation, or with changes in both protein synthesis and degradation, such that synthesis exceeds degradation. For example, in children recovering from burn injury, both the rates of protein synthesis and degradation were increased, but the increase in synthesis was larger than the increase in protein degradation (Borsheim et al., 2010; also see Figure 13-12 later in this chapter). Although the illustration of protein turnover in Figure 13-2 is presented in terms of whole-body protein, the balance between the processes of synthesis and degradation also determines the net protein balance at the level of individual tissues or organs and for individual proteins. Examples of this type of regulation are discussed later in this chapter. TABLE 13-1 Turnover Rates of Enzymes in Rat Liver∗ ∗Turnover rates are expressed as half-lives (t½, the time to replace half the molecules originally present) and fractional turnover rates (kd, percent turned over per day). Data from Waterlow, J. C., Garlick, P. J., & Milward, D. J. (1978). Protein turnover in mammalian tissues and in the whole body (pp. 490–492). Amsterdam: North-Holland Publishing. 1. Messenger RNAs (mRNAs), which are used by the translational machinery to determine the order of amino acids incorporated into an elongating polypeptide during the process of mRNA translation. 2. Transfer RNAs (tRNAs), which carry individual amino acids to the mRNA template, thereby allowing correct insertion of amino acids into the growing polypeptide chain. 3. Ribosomal RNAs (rRNAs), which are assembled with numerous ribosomal proteins to form the ribosomes, and which, in eukaryotic cells, include (designated by centrifugal sedimentation size) the 28S, 5S, and 5.8S rRNAs that are associated with the large (60S) ribosomal subunit and the 18S rRNA that is associated with the small (40S) ribosomal subunit. 4. Regulatory RNAs such as microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs), which decrease protein expression by increasing mRNA degradation or reducing mRNA translation. 5. Small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs), which are involved in modifying other RNAs within the nucleus. 6. Other RNAs, including Piwi-interacting RNAs (piRNAs), antisense RNAs, and long noncoding RNAs, all of which can play diverse roles in regulating gene expression. Proteins that bind cis-elements are termed trans-acting factors, or, more commonly, transcription factors. Transcription factors are DNA-binding proteins that can enhance or repress gene expression. Recent estimates suggest there are 1,700 to 1,900 transcription factors in humans (Vaquerizas et al., 2009). Transcription factors bind proximal or distal promoter regions, whereas still others interact with enhancer or repressor elements. For example, a family of proteins identified as TF (for general transcription factors regulating RNAP) interact with the TATA box, and the protein identified as C/EBP (for CCAAT/enhancer binding protein) binds to the CCAAT box element. When binding cis-elements, a transcription factor often pairs up with a second DNA-binding protein. When the second DNA-binding protein is identical, a homodimer is formed; when the second DNA-binding protein is different, a heterodimer is formed. The absolute numbers, ratios, and combinations of transcription factors that interact with regulatory sequences on DNA all impact control of RNA synthesis. This along with the presence of multiple cis-acting elements for each template strand of DNA results in a diverse array of binding sites for different regulatory proteins, revealing substantial combinatorial complexity. It also affords a means of coordinately regulating a number of genes. For example, the pathway for the synthesis of cholesterol (see Chapter 17) involves at least 23 enzymes, and many of the genes for enzymes in this pathway are regulated by a family of transcription factors called sterol regulatory element binding proteins (SREBPs). Another family of transcription factors responsible for coordinate regulation of genes are the retinoic acid (RAR) and 9-cis-retinoic acid (RXR) nuclear receptors that are activated by the binding of vitamin A derivatives and are involved in regulation of differentiation (see Chapter 30). Newly transcribed pre-mRNA undergoes significant posttranscriptional processing as illustrated in Figure 13-6. First, the 5′ end of an eukaryotic mRNA is “capped” with a 7-methylguanosine residue (m7GTP). The covalently attached m7GTP molecule serves to protect the mRNA from exonucleases and more importantly is recognized by specific proteins of the translational machinery (Wilkie et al., 2003). Signals near the end of the template DNA strand denote the site for cleavage of the nascent pre-mRNA strand by an endonuclease and for polyadenylation at the 3′ end of the cut. All mature eukaryotic mRNAs, except histone mRNAs, have a 3′ poly(A) tail, which is a stretch of 20 to 250 adenosine residues added by polyadenylate polymerase. The poly(A) tail protects mRNA from degradation by exonucleases and serves as a binding region for poly(A)-binding protein, which functions in the circularization of mRNA during translation. The pre-mRNA also undergoes a process that excises the introns of the primary transcript and joins the exons to generate a mature mRNA product. This process of intron removal and exon ligation is called RNA splicing. It may begin before transcription of the gene is complete and must be completed before the mature mRNA is exported to the cytoplasm. Except for rare self-splicing introns, splicing requires a specialized RNA–protein complex called a spliceosome. Spliceosomes are multicomponent ribonucleoprotein complexes containing several small nuclear RNAs and more than 100 other proteins. This ribonucleoprotein complex assembles at the splice sites as the nascent pre-mRNA is transcribed. Mature mRNAs in association with ribonucleoproteins are exported through the nuclear pores into the cytoplasm. Another common mechanism regulating mRNA stability is through the base pairing of miRNAs to the 3′ UTR of mRNA. Currently there are over 500 known mammalian genes that encode miRNAs, and each miRNA is capable of repressing hundreds of genes (Williams, 2008). Transcription of miRNA genes in the nucleus results in formation of primary miRNA, which are relatively short transcripts (~1 kb) that fold to form short hairpin structures. The primary miRNA is first processed in the nucleus by Drosha (a double-stranded RNA-specific ribonuclease) into a short hairpin structure called pre-miRNA. Following transport into the cytoplasm, pre-miRNA is further processed by a protein complex called Dicer, resulting in formation of short (20- to 30-nucleotide) RNA duplexes. After Dicer processing, the miRNA duplex is unwound and the mature miRNA strand binds to an argonaute protein to form the core component of the effector complex that mediates miRNA function. This complex is known as the RNA-induced silencing complex (RISC) (Pratt and MacRae, 2009). Usually only one strand of the mi-RNA duplex is loaded into the RISC complex; this tends to be the strand in which the 5′ end is less stably base-paired to its complement. The base pairing of miRNA within RISC to the 3′ UTR of a target mRNA promotes cleavage of the mRNA by ribonucleases, resulting in mRNA degradation. One of the four argonaute proteins in humans is an active endonuclease and can cleave mRNAs to which it binds with extensive complementarity. However, most miRNAs form base pairs with their mRNA targets with imperfect complementarity and repress the translation of their mRNA targets without endonucleolytic cleavage. Translational repression, however, commonly leads to mRNA destabilization and mRNA degradation by other machinery of the cell. Ribosome biogenesis describes the making and assembling of ribosomal proteins and rRNAs into the 40S and 60S ribosomal subunits. Expression of the genes encoding the numerous constituents of ribosomes requires transcription by all three classes of RNAPs (Mayer and Grummt, 2006). A signaling network in yeast named target of rapamycin (TOR) is identified as critical in controlling ribosomal protein gene expression and coordinating the relative activity of all three RNAPs to achieve the proper stoichiometry of ribosomal components. Both nutrients and stress can influence mammalian TOR (mTOR) signaling, linking ribosomal capacity to nutrient availability and other environmental cues. Conditions of rapid growth require enhanced ribosome production, and increased levels of ribosomes have been observed in growing tumors (Belin et al., 2009). Ribosomal protein mRNAs all contain a cis-regulatory element consisting of several pyrimidines at the 5′ end. This terminal oligopyrimidine (TOP) tract is also present in mRNA translation elongation factors and poly(A)-binding protein. Feeding a protein-containing meal maximally enhances TOP mRNA translation, whereas amino acid deficiency completely abrogates translation of TOP mRNAs (Anthony et al., 2001). This exaggerated “all-or-none” binary control mechanism suggests that in the repressed state, translation is blocked. A number of studies have implicated the phosphatidylinositol 3-kinase (PtdIns3K) and mTOR signaling pathways in the activation of TOP mRNAs during high growth conditions, but consensus on the mechanism underlying this regulatory process has not been reached. The regulation of translational capacity provides the organism with an ability to adapt to chronic or sustained conditions of change. The majority of translational control lies at the initiation step, which is summarized in Figure 13-7. This step can be further subdivided into three events that determine overall initiation activity. The first event involves assembly of a ternary complex (TC) consisting of the initiating tRNA (specifically, a particular initiator methionyl-tRNA, or Met-tRNAi) bound to the protein factor eIF2 in association with GTP (guanosine 5′-triphosphate). eIF2 is a guanine nucleotide-binding protein (i.e., G-protein) made of α, β, and γ subunits. eIF2 exists either in an active GTP-bound state or in an inactive GDP (guanosine 5′-diphosphate)-bound form. Only when eIF2 is in the GTP-bound form can the TC bind the small ribosomal subunit. Following TC formation, the TC and other protein factors (e.g., eIF1, eIF3, eIF5) bind to the 40S ribosomal subunit to form the 43S preinitiation complex and eIF2–GDP is released (Lorsch and Dever, 2010). Regeneration of the eIF2–GTP from eIF2–GDP is catalyzed by eIF2B, a guanine nucleotide exchange factor (GEF). The GEF activity of eIF2B is regulated primarily by phosphorylation of eIF2 on its α subunit, which increases its binding affinity to several eIF2B subunits, stalling eIF2B GEF activity. Phosphorylation of eIF2 is catalyzed by a family of four kinases, each responsive to a distinct set of environmental stressors. The mammalian eIF2α kinases are heme-regulated inhibitor (HRI), which is sensitive to heme deprivation; double-stranded RNA-dependent protein kinase (PKR), which is activated by a viral infection; PKR-like endoplasmic reticulum resident kinase (PERK), which is activated by misfolded proteins or other stress in the ER; and general control nonderepressible kinase 2 (GCN2), which is activated by conditions of amino acid deprivation (Wek et al., 2006). Another way in which the GEF activity of eIF2B is modulated is by the binding of the protein factor eIF5 to eIF2, sequestering eIF2 away from eIF2B, thus preventing guanine nucleotide exchange (Singh et al., 2006). The second event in translation initiation subject to major regulation involves the binding of the 43S preinitiation complex to the selected mRNA. This event requires eIF3 and several other initiation factors collectively called eIF4 (or eIF4F). One of the proteins in this group, named eIF4E, selects the mRNA to be translated by binding its 5′-m7GTP cap structure. A second member of the eIF4 group, called eIF4G, functions as a scaffold to bring the small ribosomal subunit and the mRNA close to each other. eIF4G accomplishes this task by binding both eIF4E, which is bound to the mRNA cap, and eIF3, which is associated with the 40S ribosomal subunit in the preinitiation complex. A family of repressor proteins known as the eIF4E-binding proteins (4E-BPs) can prevent the interaction of eIF4G and eIF4E and thereby inhibit the 40S ribosome from binding mRNA. The repressor activity of the 4E-BP is regulated by phosphorylation, with increased phosphorylation reducing its affinity to associate with eIF4E. A second function of eIF4G is to associate with poly(A)-binding protein, which results in 5′,3′-circularization of mRNA, as shown in the lower part of Figure 13-7. Circularization of mRNA is believed to be important for stabilizing recruited 40S ribosomal subunits and for efficient recycling of terminating ribosomes for another round of translation of the same mRNA (Wilkie et al., 2003). After the eIF4 complex has brought the 43S preinitiation complex and mRNA together, the small ribosomal subunit moves along the mRNA toward the 3′ end scanning for the start codon. Scanning is facilitated by eukaryotic initiation factor eIF4A, which functions as an ATP-dependent helicase to unwind mRNA secondary structure in the 5′ UTR. All three eEFs are subject to phosphorylation in mammalian cells. Phosphorylation of eEF1A stimulates elongation activity, whereas phosphorylation of its GEF, eEF1B, on several subunits has no reported influence on elongation rates. On the other hand, eEF2 is inactivated by phosphorylation. Phosphorylation of eEF2 does not impact the ability of the ribosome to hydrolyze GTP but instead reduces the affinity of eEF2 for the ribosome (Carlberg et al., 1990). Phosphorylation of eEF2 occurs in response to stimuli that either increase energy demand or reduce its supply. This likely serves to slow down protein synthesis and thus conserve energy under such circumstances (Andersen et al., 2003). Examples of conditions that increase eEF2 phosphorylation include intense exercise, alcohol intake, ischemia, and denervation.
Protein Synthesis and Degradation
Protein Turnover
Overview of Protein Turnover
Synthesis and Degradation of Protein in Relation to Protein Balance
Protein Turnover and Adaptation
ENZYME
CELLULAR COMPARTMENT
t½
kd (% per day)
Ornithine decarboxylase
Cytosol
11 minutes
91
5-Aminolevulinate synthetase
Cytosol
20 minutes
50
5-Aminolevulinate synthetase
Mitochondria
72 minutes
14
Hydroxymethylglutaryl CoA reductase
Endoplasmic reticulum
4.0 hours
4.2
Phosphoenolpyruvate carboxykinase
Cytosol
5.0 hours
3.3
Alanine-glyoxylate aminotransferase
Cytosol
3.5 days
0.20
Arginase
Cytosol
4.0 days
0.17
NAD+ nucleosidase
Endoplasmic reticulum
16 days
0.04
Protein Synthesis
DNA Transcription
Regulation of Transcription by Transcription Factors
Pre-mRNA Processing and Export
Stability of mRNA
Regulation of mRNA Stability by miRNA
Translation of mRNA
Regulation of Translation by Regulation of Ribosome Biogenesis
Translational Efficiency
Control of Translation Initiation: Formation of Preinitiation Complex and Its Regulation by eIF2α Phosphorylation
Control of Translation Initiation: Association of the 40S Ribosomal Subunit with mRNA by Cap-Dependent Recognition and Its Regulation by eIF4E Binding Protein Phosphorylation
Polypeptide Synthesis: Elongation and Its Regulation
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