Protein Digestion

Intestinal Processes/Absorption of Amino Acids

Intracellular levels of amino acids are greater than those in blood plasma (total 15‑30 mM vs 2‑4 mM). Blood concentrations are lower of aa's because need to bring them across concentration gradient and aa's need a transporter. The relative proportions of the free amino acids in body fluids do not reflect those in tissue or in dietary protein but are linked to metabolic events. While glutamine and alanine are the most abundant in plasma, alanine, glutamate, glutamine, and glycine are usually the most abundant of the free amino acids inside cells. Absorption from the gut and re-absorption from glomerular filtrate of the kidney are the most active processes, but every cell takes up amino acids. Several mechanisms of amino acid uptake are known; their relative quantitative importance is unknown. Amino acids are usually taken up against concentration gradients with expenditure of energy (active transport). Amino acids can be transported across cell membranes by mechanisms similar to that used for glucose. A gradient of Na+ (outside high, inside low) across cell plasma membranes is created by Na+/K+ ATPase. Transporters in the brush-border membrane simultaneously move Na+ and an amino acid across the membrane, thus using the energy of the Na+ gradient to concentrate amino acids within the cell. Amino acids then enter the bloodstream via the use of a faciliated transporter that does not involve Na+. Several Na+ -coupled transporters have been described which are specific for groups of amino acids rather than for individual ones. Examples are the A-system (for alanine and other small neutral amino acids), the ASC-system (for Ala, Ser, Cys, Thr, Gln, the N-system (for Gln, Asn, His), the system for dibasic (or diamino) amino acids, ornithine (described later), Lys, and Arg, and the system for dicarboxylic amino acids (Glu, Asp). Several Na+ -independent transporters have also been characterized including transporters for neutral amino acids such as leucine, basic amino acids such as lysine and lipophilic amino acids such as phenylalanine. In some tissues, the γ-Glutamyl (Meister) cycle is used for amino acid transport that involves the interaction of the extracellular amino acid with the tripeptide glutathione (γ-glutamyl-cysteinyl-glycine) with a transpeptidase in the cell membrane. This enzyme attaches the amino acid to the glutamyl residue to form a glutamyl-amino acid complex and cysteinylglycine. The amino acid is subsequently released and glutathione is reformed. In this system, the translocated molecule is chemically altered while bound to the transporter. The amino acid derivative is bound less tightly than the amino acid and is released from the transporter inside the cell. Many genetic defects in amino acid transport have been described. Because the transporters are present in kidney to allow reabsorption, several of these abnormalities result in high levels of certain amino acids in urine (aminoaciduria). Cystinuria is one such genetic disorder with a carrier rate of about 1 and 150 (i.e., about 1 in 10,000 have the disease). A defective membrane transporter in the kidney glomeruli in poor absorption of cystine and is associated with excretion of high levels of cysteine. (The cysteine in urine is readily oxidized to the less soluble cystine that can precipitate to form painful kidney stones.) Autosomal recessive. Solute Light Carrier (SLC), some lack these transporters which is important for transporting cystine (oxidized form) and then once in cell it gets reduced to cysteine. Cystine is insoluble so defects in transporter will lead to kidney stones. Defects also lead to high lysine because also uses the same transporter. The proximal convoluted tubule (PCT) region of the kidney just distal to Bowman's capsule receives the glomerular filtrate and functions in a manner analogous to the small intestine in the GI tract: it is the primary site for resorption of useful stuff. The cuboidal cells of the PCT have a rich brush border (apical microvilli) and mitochondria-rich basal infoldings that are essential for rapid resorption. Resorption is a two-step process: active movement of non-waste products at the cells' basal surface and subsequent passive movement into nearby peritubular capillaries leading to the blood. Treatment involves making the urine more alkaline In addition to cysteine, large amounts of arginine, citrulline and lysine are also excreted because this cysteine transporter (a complex of two proteins called SLC7A9 and SLC3A1) also transports basic amino acids. In Hartnup's disease, a single defective transporter in epithelial cells (including the intestine and kidney) results in poor uptake in the intestine and re-uptake in the kidney, the latter resulting in excretion of several neutral amino acids in urine (such as tryptophan). Problems occur with a deficiency of tryptophan (niacin is made from tryptophan). For example, an individual lacking both niacin and tryptophan in their diet may develop pellagra (the 3 symptoms: dermatitis, diarrhea, and dementia).

Pancreatitis

Normally, the contents of the pancreatic duct are flushed into the duodenum. Blockage of the common duct can lead to retention of bile, resulting in the tissue damage and pain of acute pancreatitis (most common form). Other forms of pancreatitis come from genetic defects. A major inherited form arises from a mutation in the trypsin molecule. This mutation prevents binding of the inhibitor to trypsin inside cells and leads to activation of more intracellular trypsin. Complex is so tight trypsin can't digest its peptide inhibitor. Pancreatitis results in >200,000 hospital visits annually

Lysosomal System of Protein Degradation

This system, "autophagy", involves processes that can engulf a variety of particles ranging from bacteria and apoptotic fragments to individual proteins. Extracellular proteins such as ApoB100 are also degraded after pinocytosis by lysosomes. Although some intracellular proteins may be digested in lysosomes this process is not quantitatively important since inhibitors of lysosomal function (like chloroquine) have little effect on the degradation of most intracellular proteins. The lysosomal proteases are collectively known as cathepsins but include several different classes of molecules.

Sources and Uses of Amino Acids

We eat about 70-100g of protein per day and the ONLY significant source of nitrogen in our diet is through protein. Although amino acids are used primarily for synthesis of protein, some are used for synthesis of nitrogenous compounds such as neurotransmitters, heme, and nucleic acids. Amino acids in excess of requirements are degraded to products that are either oxidized for energy or converted into carbohydrates or fats. The body maintains a pool of amino acids in the blood stream and tissues for protein synthesis, to use as fuel, or for conversion into other substances. An increase in the total level of amino acids in the blood amino may be due to: Eclampsia. Fructose intolerance, Ketoacidosis (from diabetes), Kidney failure, Reye syndrome. A decrease in the total level of amino acids in the blood may be due to: Adrenal cortical hyperfunction, Fever, Hartnup disease, Huntington's chorea, Malnutrition, Nephrotic syndrome, Phlebotomus fever, Rheumatoid arthritis. Overall, amino acids are maintained at certain levels in the blood for use by the body and these amino acids come from two sources: our own body breakdown (about ¾) and the digestion of dietary protein (about ¼)

Amino Acid Pools and Essential Amino Acids

Dietary protein usually contributes about 70-100 g/day. It is not a major source of energy; at 4 Kcal/g, it provides only 10-20% of the daily calories. Its importance is to supply certain amino acids that humans cannot synthesize - the essential amino acids and to ensure an adequate level of nitrogen-containing substances in the human body. (Non-essential amino acids can be synthesized in human de novo.) Bacteria and plants can synthesize all of their amino acids. Adults should consume at least 55 g protein per day, otherwise breakdown of body protein will occur. Since amino acids cannot be stored for later use, all twenty must be present in approximately correct proportions for protein synthesis. Even if only one is absent, body protein will be degraded to offset a deficiency. Many specialized tissues, particularly glandular tissue, require specific essential amino acids for hormone synthesis. Because of this drain on the amino acid pool, the essential amino acids are replenished from diet. The essential amino acids are phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, histidine, arginine, leucine and lysine (one mnemonic is Pvt Tim Hall, using non-standard abbreviations for the amino acids). As will be seen later, arginine can be synthesized, but not in sufficient amounts for optimal growth and is considered to be essential. Because dietary proteins vary in their composition and digestibility, their nutritional value differs widely. Some diets may not provide sufficient of the essential amino acids. This is a particular problem for children in developing countries (kwashiorkor) and for vegetarians. Fish, meat and dairy products provide more nutritional protein than most plants. For example, meat contains about 15-20% protein whereas plants may contain only 1-2%. In addition, some plant proteins are difficult to digest. Vegetarians who are strict vegans may need to consume more food than do others to obtain the same amount of protein. To compound matters, some plant proteins have low levels of some essential amino acids and therefore have a lower nutritional value than animal proteins. Such deficiencies can be corrected with an appropriate mixture of plant foods that provide sufficient amounts of all of the essential amino acids. The chemical score of a protein compares the composition of essential amino acids in a protein with that of an "ideal" protein such as egg white which has an assigned value of 100. Results from this convenient method correlate nicely with those from in vivo methods based on nitrogen balance. - Free amino acid levels are low compared to that polymerized in protein). - 95% is replaced every 10 minutes. - Amino acids are transported in plasma (low mM) to replenish intracellular amino acids (about 10x more) - Alanine and glutamine are the most abundant amino acids in plasma , which doesn't reflect their abundance in proteins but more their metabolism.

Kwashiorkor Disease

Due to Insufficient Protein or incomplete Protein although total calories are normal. Will sacrifice protein in less essential places such as skin or in albumin in blood, get adema (bloated due to loss of plasma protein & fatty liver) in stomach because of insufficient albumin, thin limbs, and flaky skin. Mainly in children ages 1-3. Different from marasmus, which is a total lack of nutrition.

Celiac Disease

Except in newborns, all proteins are normally digested to amino acids. However, a 33 residue peptide from gluten is not always completely digested. This peptide can be absorbed and precipitate an autoimmune reaction in affected individuals.

Dietary Protein Digestion

Except in specialized cases such as the newborn, dietary protein cannot be absorbed intact and is first hydrolyzed to its constituent amino acids. This proteolysis initiates in the stomach and terminates in the cells of the small intestine. The resulting mixture of amino acids is transported from the intestine to the liver by the portal vein for further processing. Proteolytic enzymes are classified either as endopeptidases, which catalyze the hydrolysis of internal peptide bonds, or exopeptidases, which snip off N or C terminal residues. Protein digestion can be considered in three phases, gastric, pancreatic and intestinal.

Gastric Processes

Gastric Processes - Protein digestion begins with the secretion of gastric juice in response to food. This juice contains pepsin in a 0.1 M solution of hydrochloric acid. The very acidic solution kills bacteria and also denatures and unfolds proteins so that they are more easily attacked by proteases. Pepsin has a pH optimum of about 1.5. Pepsin is synthesized in the gastric mucosa as the inactive zymogen precursor, pepsinogen. It contains a prosegment that blocks the active site at normal cellular pH (around 7). This prosegment also binds and inhibits pepsin above pH 2, but dissociates below this pH in the stomach. The low pH of the gastric juice disrupts electrostatic interactions between the prosegment and the active enzyme moiety, which allows removal of the prosegment with pre-existing pepsin. The resultant conformational change results in active pepsin (where two active site aspartates are brought together in the cleft of a V-shaped structure.) Pepsins give a mixture of large polypeptides that will be further hydrolyzed by proteases from the pancreas.

Intracellular Amino Acid Breakdown

Protein populations in cells are constantly changing in response to physiological need. This continual turnover not only helps to regulate protein populations, but also provides most of the amino acids for the synthesis of new proteins (about 200g daily), even more than the diet (usually 50 to 100g protein). Because of its mass, muscle contributes more than 60% of the total amino acid pool of the blood. Intracellular proteolysis occurs at many levels - from the non-selective degradation of all proteins on the death of a cell or organelle to selective degradation of individual molecules. For example, most hemoglobin molecules survive for three months, the life span of the red blood cell. Senescent cells that have lost their flexibility are engulfed by macrophages where the hemoglobin molecules are broken down to amino acids in lysosomes. Cell death can also come from within by apoptosis. In this process, a variety of cytoplasmic proteases such as the caspases are specifically involved in the recognition and initiation of events leading to cellular fragmentation and lysosomal digestion. However, cell turnover or apoptosis usually contributes only a small part of the amino acid flux in cells. The largest contribution comes from the degradation of proteins in healthy cells by the ubiquitin/proteasome (UPP) pathway. This is a complex and highly coordinated system that allows for selective degradation of specific proteins. Defects in this system are involved in malignancies and neurodegenerative disease. The half-life of many proteins is determined by their function and needs of the cell. For example, the crystallins in lens and the collagen in tendon are very stable and can last a lifetime. In the nucleus the half-life of p53 is only about 0.5 hour whereas that of a histone is over 1 day. In general, structural proteins tend to be long-lived, while proteins involved in time-sensitive transitions such as cell cycle typically have very short half-lives. For many enzymes, life span is determined largely by their function and is subject to substrate and hormonal influences. Multimeric proteins are usually more stable than their component subunits. For example, the aggregation of the subunits of the iron storage protein ferritin is driven by iron that is then stored inside the protein shell. Subunits and shells with little iron are rapidly degraded whereas shells replete with iron have very long half-lives. Thus, the iron ligand affects the stability of both the subunits and the multimeric protein. Ligand binding may therefore change the conformation of ferritin to protect against proteolysis. Other mechanisms of protein degradation include the lysosomal system and the Ubiquitin/Proteasome Pathway (UPP).

Abnormalities in the UPP in Disease

Several diseases have been associated with defects in the UPP system that can be characterized as loss of function mutations resulting in increased stability of substrates or gain of function with decreased stability or half-life. Cancers can arise from stabilization of oncogenes or, as with E6 protein of HPV, from destabilization of tumor suppressors such as p53. Several neurodegenerative disorders are characterized by accumulations of ubiquitin conjugates. These include Lewy bodies in Parkinson's disease, neurofibrillary tangles of Alzheimer's disease, and nuclear inclusions in disorders involving CAG repeats such as Huntington's disease. The Lewy bodies cause a loss of neuronal cells & loss of dopamine, a neural transmitter. The most widely used form of treatment is L-dopa in various forms. However, it has not been determined whether such events are the underlying defect or some secondary event. In some types of Parkinson's disease mutations have been found in the protein Parkin which is a ubiquitin conjugating ligase while other forms are associated with a malfunctioning deubiquitinase. Parkin mutation in E3-type ubiquitin ligase, don't get degradation of protein, accumulates and forms protein based aggregate in neurons, leading to loss of neurons and dopamine NT. Other mutation in UCH-L1 deubiquitinase, single ubiquitin remains and leads to formation of different protein aggregate leading to Parkinson's. One form of Alzheimer's is associated with a mutated ubiquitin. In CAG repeat diseases such as Huntington's disease, the accumulation of abnormal proteins with the polyglutamine tract may be linked to the inability of the proteasome to cleave within the polyglutamine tract.

Ubiquitin/Proteasome Pathway (UPP) of Protein Degradation

The UPP is the major pathway for the degradation of intracellular proteins. Proteins are targeted for degradation by conjugation with a marker polypeptide called ubiquitin and subsequently degraded in large complex structures called proteasomes. Selectivity of proteins destined for degradation is conferred by presentation of ubiquitinated substrates to the regulatory portion (RP) of the proteasome. The barrel-like core of proteasomes (CP) contain proteases that degrade a wide variety of proteins. The lid of the RP has multiple roles in selecting substrates and translocating them into the CP. The ubiquitinated protein is subsequently transferred to the proteasome where it is digested to small peptides. During this process, ubiquitin is released from the complex and is recycled to target other proteins. One type of subunit in the RP recognizes the ubiquitinated substrate while another is involved in removing the ubiquitin before the protein enters the CP. A third subunit type has an editing function to remove ubiquitin from inappropriately tagged proteins. Details remain fuzzy, such as how the door-keeping and editing functions work and how ubiquitin escapes degradation in the proteasome. At least three different types of protease line the inside of the core particle. The size of the digestion products is 7 to 10 residues (range of 3 to 23). Some of these peptides can be transported through the ER via a Tap transporter protein for presentation to the immune system by class I HLA molecules (MHC). Others are degraded to amino acids by other cytosolic proteases and aminopeptidases. Selection for ubiquitination - The recognition and ubiquitination of the protein by a member of the E3 ubiquitin-conjugating family confers remarkable specificity and diversity of the degradation of intracellular proteins. (Ubiquitin itself is first activated by E1 and E2 enzymes.) The ubiquitin chains are then extended, also by E3 enzymes. Conjugation usually occurs through the carboxyl group of activated ubiquitin with free amino groups in the target protein. How this specificity is achieved is not fully understood. Damaged or mutant proteins are particularly susceptible but little is known of how the cell distinguishes these from normal proteins. Specific sequences, covalent modification or interaction with other protein facilitators are also important. N-end Rule - This was the first system to be recognized as a determinant for ubiquitination and applies to some, but not all, proteins. From analyses of turnover rates of natural proteins and of genetically engineered proteins, it was observed that the half-life of a protein varied with its N-terminus composition because of differences in binding to a particular E3 ligase. Methionine has been shown to be slowest degraded, proteins with longer half-lives have methionine at N-term. Phosphorylation of the substrate or E3 - Covalent modification of a substrate or its E3 can profoundly affect the half-life of a protein. Many substrates or their cognate E3s require to be phosphorylated before they can bind for ubiquitination. These include regulators of cell cycle such as cyclins and cyclin-dependent kinase. In other cases, such as the protooncogene c-mos or the anti-apoptotic protein Bcl-2, phosphorylation can prevent interaction with an E3 and thereby stabilize the protein. Destruction boxes, PEST sequences, damaged proteins -Some E3s recognize internal sequences in the substrate. For example, a short basic peptide near the N terminus of a family of cell cycle regulators called geminin proteins is responsible for their rapid ubiquitination and short half lives (destruction boxes). Point mutations in this region result in reduced ubiquitination and increased half-lives. PEST sequences: sequences enriched in proline (P), glutamic acid (E), serine (S) and threonine (T) sometimes target proteins for rapid destruction. Some proteins are recognized if they show oxidative damage (typically on thiols, amines, and methionine). Facilitators and Chaperones - Interaction with other proteins can also determine the likelihood that a particular protein will be ubiquitinated. In such cases, the E3 ubiquitin ligase does not bind the target protein itself but binds to a protein complexed to the target. This strategy is exploited by viruses to destroy proteins that interfere with their propagation. For example, the E6 protein of human papillomavirus (HPV) binds to the tumor suppressor p53 and to E6AP—a particular E3 ligase. This interaction results in the ubiquitination and degradation of p53 and contributes to the oncogenicity of the virus.

Pancreatic Processes

The acid chyme containing the partially digested protein passes into the duodenum where most proteolysis occurs. The partially digested protein triggers the release of the peptide hormones, secretin and cholecystokinin, from specialized mucosal endocrine cells into the circulation. These hormones in turn cause the contraction of the gall bladder and the release of an alkaline secretion from the pancreas containing bicarbonate and a mixture of zymogen forms into the lumen of the duodenum (the first section of the small intestine). These include the endopeptidases (trypsin, chymotrypsin and elastase) and the exopeptidases, carboxypeptidase A and B. The bicarbonate neutralizes the hydrochloric acid to give the duodenum a slightly alkaline pH favorable for activity of these proteases. Like pepsin, the pancreatic proteases are also secreted as inactive precursors and their activation involves removal of specific peptides by an activator secreted from another cell type. Although trypsin, chymotrypsin and elastase have different specificities, all are structurally and functionally related. All depend on a serine residue for activity and are therefore collectively known as serine proteases. Carboxypeptidases contain a functional zinc ion and are metalloproteases. Their concerted actions break the large peptides into a mixture of di and tripeptides. These are mostly hydrolyzed to amino acids in the lumen by aminopeptidases and dipeptidases or within the intestinal epithelial cells. The key event is the activation of trypsinogen by the enzyme enteropeptidase (formerly enterokinase) which is secreted from the luminal surface of the small intestine under the influence of cholecystokinin (CCK). Enteropeptidase clips a hexapeptide from the N terminus of trypsinogen, allowing for the generation of trypsin activity through a conformational change. In this rearrangement, key aspartate, serine and histidine residues are brought into close proximity to generate the catalytic triad. The newly formed trypsin now acts autocatalytically by hydrolyzing the same bond as enteropeptidase. Trypsin plays a central role in regulating protein digestion since it activates other pancreatic zymogens as well as proaminopeptidase and procarboxypeptidase from the intestinal mucosa. Trypsin turns more trypsinogen into trypsin; also more chymotrypsinogen into chymotrypsin; more proelastase into elastase & more procarboxypeptidase into carboxypeptidase. Special safeguards are required to avoid internal liquifaction of cells. As noted, one mechanism is the secretion of zymogen and activator from different cell types (e.g trypsinogen secreted from the pancreas and enteropeptidase from the small intestine). The proteolytic enzymes are themselves degraded. The amount of enzyme protein required to digest dietary protein is large and approaches the protein levels of the diet - the breakdown of enzymes typically contributes about the same amount of amino acids as diet! **Five isoforms are found in humans-pepsins A, B and F, gastricsin and chymosin. Pepsins A and B and gastricsin are the predominant forms in normal adults and pepsin F and chymosin predominate in infants. There are three isoforms of trypsinogen, encoded by the protease, serine (PRSS) genes 1, 2 and 3. On the basis of their relative electrophoretic mobility, the three are commonly known as cationic, anionic and meso-trypsinogens. In normal human pancreatic juice the cationic isoform constitutes about 2/3 of the total trypsinogen content, the anionic form about 1/3 and mesotrypsinogen less than 5%. These isoforms have different sensitivities to inhibitors and this may be advantageous for digesting foods containing natural inhibitors of trypsin. **Serious problems arise from aberrant expression or premature activation of proteases. Similarly, pancreatic cells synthesize a peptide that inhibits trypsin activity in the pancreatic cells or ducts.