Protein Turnover – Protein and Amino Acids in
2.1 Nitrogen b alance and p rotein r equirements
2.1.5 Tissue p rotein c atabolism
There are two main routes for the normal catabolism of tissue proteins: a more or less random process involving uptake into lysosomes (section 2.1.5.1 ),
which is the main mechanism for catabolism of proteins with a relatively long half - life; and targeting of proteins with a short half - life for catabolism by either the proteasome (section 2.1.5.2 ) or lysosomes.
Caspases are proteases with cysteine at the catalytic site that cleave peptide bonds to the carboxyl side of aspartate residues. In response to signals for apoptosis, the synthesis of caspases is increased and inactive caspase zymogens are activated. When activated, they cleave a wide variety of structural and other proteins in the cell, so initiating the programmed events of apoptosis (Earnshaw et al. , 1999 ).
The calcium activated neutral cysteine proteases, the calpains, are non lysosomal proteases that are normally inhibited by the associated inhibitory protein calpastatin, and they are activated by an increase in intracellular (or cytoplasmic) calcium ion concentration. Their physiological function is unclear, but they produce relatively large, sometimes catalytically active, peptide fragments from their target proteins, suggesting that they may have a role in cell signalling.
In muscle cells, calpains are associated with myofi brils, and they cleave tropomyosin and myosin, suggesting that they may have a role in muscle atrophy in response to trauma and prolonged bed rest. In non - muscle cells, they are associated with the cell membrane (again suggesting a role in signal transduction) and the cytoskeleton, suggesting a role in remodelling of the cytoskeleton (Croall & DeMartino, 1991 ; Goll et al. , 2003 ).
2.1.5.1 Lysosomal a utophagy A primary response to nutrient limitation in the cell is the formation of autophagosomes, whereby cytoplasm and organelles are non - specifi cally sequestered in a double membrane vesicle that then fuses with a lysosome. The various cathepsins (proteases) and other hydrolytic enzymes in the lysosome then hydrolyze the macromolecules in the autophagosome and return the products to the cytosol for re - utilization.
This autophagy is thus the intra - cellular equivalent of phagocytosis. Because the capture of proteins in autophagosomes is a random process, it is likely that most of the proteins catabolized in this way will be those with a relatively long half - life.
The formation of autophagosomes is regulated by insulin and glucagon. In response to insulin, mToR kinase is activated, and phosphorylated mToR (mammalian target of rapamycin) inhibits autophagosome formation. In response to nutrient depletion, there is dephosphorylation and, hence, down-regulation of mToR, activating the formation of autophagosomes. Intracel-lular free amino acids also activate the mToR kinase pathway, so inhibiting autophagy (Kilberg et al. , 2005 ; Mizushima & Klionsky, 2007 ).
Some proteins are transported into lysosomes for catabolism, either because they have a lysosomal targeting sequence (Lys - Phe - Glu - Arg - Gly), or because they have been ubiquitinated (section 2.1.5.2 ). Proteins with the
lysosomal targeting sequence are transported into lysosomes by a chaperone protein.
2.1.5.2 Ubiquitin and the p roteasome Ubiquitin is a small (76 amino acid), highly conserved protein that is present in all eukaryotic cells. It is a globular protein with a protruding carboxy - terminal region, and it is highly resistant to proteolysis. It forms a peptide bond from its carboxy - terminal glycine to theε - amino group of a lysine residue in the target protein. Like lysosomal autophagy (section 2.1.5.1 ), ubiquitination of proteins occurs in response to nutritional deprivation and stress, or the need for repair. However, ubiquitination is additionally important in controlling the intracellular con-centrations of regulatory enzymes with a short half - life, and targeting abnor-mal proteins or proteins that have undergone oxidative or other damage for catabolism. In addition to this role in intracellular protein catabolism, ubiquitination is involved in the DNA repair mechanism, remodelling of chromatin, antigen processing and regulation of the cell cycle and endocytosis (Reyes- Turcu et al. , 2009 ).
Three genes code for ubiquitin. Classes I and II encode a fusion protein between ubiquitin and a zinc - fi nger protein that is required for the formation of ribosomes. These genes are expressed constitutively and, since one gene product is involved in protein catabolism and the other in protein synthesis, they presumably act mainly to balance the rates of protein catabolism and synthesis under normal conditions. The class III ubiquitin gene encodes head to - tail repeats of ubiquitin, which yield free ubiquitin after post - synthetic cleavage. Expression of this gene is induced in response to metabolic and other stress.
Three enzymes are involved in the ubiquitination of target proteins:
• Ubiquitin activating enzyme catalyzes adenylation of ubiquitin and the formation of a thio - ester bond between the carboxy - terminal glycine of ubiquitin adenylate and a cysteine residue in the enzyme.
• The next stage is the transfer of ubiquitin adenylate onto a cysteine residue in one of the ubiquitin carrier proteins. Most of these are small proteins that require ubiquitin ligase to transfer ubiquitin onto the target protein, but some have amino - and carboxy - terminal extensions and cata-lyze transfer of the ubiquitin onto the target protein themselves.
• Ubiquitin ligase transfers ubiquitin adenylate from the thio - ester on the carrier protein onto theε - amino group of a lysine residue on the target protein. There are at least 600 ubiquitin ligase genes in the human genome, and this stage obviously provides the main specifi city for the proteins to
be targeted for catabolism. At least four ubiquitin molecules have to be attached to a target protein (as a poly - ubiquitin chain with each successive ubiquitin forming a peptide bond between its carboxy - terminal glycine and lysine - 48 of that already attached) in order for proteins to be targeted for catabolism (Deshaies & Joazeiro, 2009 ; Hershko, 1991 ).
It is the amino - terminal amino acid of the target protein that confers spe-cifi city for binding to, and ubiquitination by, ubiquitin ligase. In a number of cases, the target protein has undergone post - synthetic modifi cation of an acidic amino - terminal amino acid by addition of arginine from arginyl tRNA Arg . The arginine tRNA synthase involved in this reaction is distinct from that involved in charging tRNA for ribosomal protein synthesis (Ciech-anover & Schwartz, 1989 ; Morris, 2009 ; Tasaki & Kwon, 2007 ).
After the degradation of poly - ubiquitinated proteins in either the protea-some or lysoprotea-somes, poly - ubiquitin is released, and this is cleaved to free ubiquitin by stepwise removal of ubiquitin molecules by a ubiquitin - specifi c protease that hydrolyzes the glycine -ε - amino lysine peptide bond.
There are at least one hundred genes for ubiquitin - specifi c proteases in the human genome, suggesting that there is a considerable degree of specifi city for the de ubiquitination of proteins. It is likely that proteins are mono ubiquitinated more or less at random. Only if the target protein is damaged, sensed by exposure of a hydrophobic region that causes a conformational change in ubiquitin, is the side - chain of lysine - 48 of ubiquitin exposed for further ubiquitination. Otherwise, the single ubiquitin molecule is removed.
Up to 40 per cent of newly synthesized protein is catabolized within minutes because of errors in translation or folding. Some of the peptides formed by partial proteolysis in the proteasome can bind to MHC (major histocompat-ability complex) class I molecules and establish cell immunogenicity (Kloet-zel, 2001 ; Reyes - Turcu et al. , 2009 ; Wilkinson, 1995 ).
The eukaryotic proteasome is a 2.5 MDa multi - enzyme complex with 33 subunits, 28 of which form a hollow barrel - like core. Proteasomes occur free in the cytosol, attached to the endoplasmic reticulum and in the nucleus.
Similar multi enzyme complexes are found in archaea and prokaryotes. Poly ubiquitinated proteins bind to the regulatory subunit, which catalyzes the ATP - dependent removal of ubiquitin and ATP - dependent unfolding of the protein. When a ubiquitinated protein is bound, a conformational change in the regulatory subunit causes the opening of a channel into the core protein.
This permits the unfolded target protein to enter the central chamber of the core protein. This central chamber is lined with proteases that have a catalytic threonine residue at the amino - terminal. There are three proteases, one each with specifi city for esters of acidic, basic and aromatic amino acids, and the end product is a mixture of small (8 – 10 amino acid) peptides (Finley, 2009 ).
A number of cell surface receptors, including the GABA receptor, are mono- ubiquitinated, internalized and either sequestered in intracellular vesicles for recycling to the cell surface, or catabolized in lysosomes. Mono ubiquitination is also involved in endocytosis, histone regulation and the budding of retroviruses from the cell membrane (Barriere et al. , 2007 ; Weiss-man, 2001 ).
The proteasome normally requires ubiquitination of target proteins, but the protein antizyme targets ornithine decarboxylase to the proteasome without ubiquitination. As will be discussed in section 5.8.1 , ornithine decar-boxylase is the regulatory enzyme of polyamine synthesis; it has a very short half - life, of the order of 11 minutes.
2.1.5.3 Active s ite p roteolysis of a po - e nzymes In most pyridoxal phos-phate- dependent enzymes there is a highly conserved sequence of amino acids around the lysine that forms the Schiff base with the cofactor (Figure 3.2 ). There is a specifi c protease in small intestine and skeletal muscle that targets this sequence and initiates the hydrolysis of the apo - enzymes of pyri-doxal phosphate - dependent enzymes. It does not hydrolyze other proteins and also does not hydrolyze the holo - enzymes, in which this conserved sequence is hidden by the coenzyme. In experimental animals, the activity of this enzyme increases 10 – 20 fold in vitamin B 6 defi ciency, so reducing the competition between different apo - enzymes for the scarce cofactor.
A similar, but distinct, protease has been isolated from tissues of niacin defi cient rats that catalyzes hydrolysis of the conserved NAD binding site of a number of NAD - dependent enzymes when it is not occupied (Katunuma et al. , 1971a , 1971b ).