Introduction

Protein catabolism is the breakdown of proteins into absorbable monomers for further degradation or reassembly. Protein catabolism in the intestinal lumen is important for several reasons, one of which is mobilizing essential amino acids for absorption. Essential amino acids can’t be synthesized in the human body but are needed for the biosynthesis of vital proteins, so their only source is polypeptide breakdown through digestive enzymes. This process begins in the stomach and continues in the small intestine. Large protein chains are disassembled to eventually leave free amino acids that can be taken up into the blood and transported to various cells around the body for further breakdown.

Endopeptidases in zymogen form are released by the stomach mucosa and the exocrine pancreas to cleave the polypeptide chain between particular amino acid residues. Once in a smaller form, exopeptidases remove the last amino acids from the C or N terminus of a di-peptide or tri-peptide one by one, aiding absorption at the microvilli. Cells can use those amino acids to construct vital proteins or as substrates for energy creation. Proteins created intracellularly can also be catabolized for the same reasons. Intracellular proteins that were either misfolded or are no longer functioning in the cell also undergo intracellular protein catabolism in the lysosome, with the help of ubiquitin and proteasome formation. If a cell is in a low energy state, the free amino acids in the cytosol are further degraded to produce citric acid cycle intermediates and are funneled there to produce ATP. While the carbon backbone enters energy-generating pathways, the nitrogen backbone is modified and excreted mostly through the kidneys.

Fundamentals

rThe catabolic process begins when proteolytic enzymes are released in zymogen form from the gastrointestinal mucosa. Once secreted, zymogens are cleaved into active proteases. Pepsin from the gastric mucosa hydrolyzes the larger polypeptide into smaller polypeptides that can later be broken down further by the pancreatic proteases released in the duodenum. Trypsin, chymotrypsin, elastase, and carboxypeptidases synthesized by the exocrine pancreas can cleave the polypeptide at specific cleavage points into amino acids and oligopeptides. Aminopeptidases located on the brush border of intestinal epithelial cells hydrolyze the remaining oligopeptides into amino acids that then absorb into circulation through transmembrane transport systems. Both facilitated and active transport mechanisms exist to transport amino acids into the cells.

Protein catabolism is a vital part of cellular turnover. When cytosolic proteins such as signaling or structural peptides are no longer needed, they must be broken down in lysosomes to create new proteins that can carry out necessary metabolic functions. If the resulting amino acids are not used to synthesize new proteins for vital intracellular functions, they can enter the citric acid cycle for energy generation. When programmed death is the cell's fate, cathepsins and other catabolic enzymes work together in the apoptotic process.

Gastric Peptidases: Chief cells in the gastric mucosa secrete pepsinogen.[1] With the help of hydrochloric acid released by the gastric parietal cells, pepsinogen changes conformation in the strong acid stomach environment and cleaves itself into the active protease, pepsin. The acid also denatures dietary proteins and partially unfolds them for easier proteolysis. Pepsin usually cleaves peptide bonds that contain a carboxyl group from an acidic or aromatic amino acid; however, its broad specificity lets it cleave peptide bonds at other points as well.[2] The smaller peptides, and sometimes free amino acids, then continue into the duodenum to encounter peptidases secreted by the pancreas.

Pancreatic and Intestinal Peptidases: The exocrine pancreas secretes a variety of digestive enzymes, including amylase, lipase, colipase, bicarbonate, and inactive proteases. The bicarbonate released into the intestinal lumen raises the pH and allows the pancreatic proteases to become activated from their zymogen form. One particular protease, trypsin, is the most important to the digestive process because it cleaves proteins on its own and also activates other proteases from their inactive forms. Trypsin is cleaved from its proenzyme form trypsinogen by enteropeptidase. Trypsin then continues on to activate chymotrypsinogen, proelastase, and procarboxypeptidases into chymotrypsin, elastase, and carboxypeptidases, respectively.[3] Trypsin hydrolyzes peptide bonds with the carboxyl group from arginine or lysine. Chymotrypsin, on the other hand, tends to act on peptide bonds between hydrophobic amino acids.[4] Trypsin and chymotrypsin are categorized as serine proteases and have a similar method of proteolysis. They form a catalytic, tetrahedral intermediate by positioning the catalytic triad in a manner that the protons forming the hydrogen bonds can break the peptide bond between two amino acids of the substrate protein.[4] These two enzymes are also very similar in their S1 binding pocket, which has a basic amino acid in trypsin and hydrophobic amino acid in chymotrypsin.[4] Still, the specificities and mechanisms have much more complexity with their dynamic properties, structural flexibility being one of the most important.

 Elastase, also a serine protease, breaks down elastin and also proteins with small hydrophobic side chains such as glycine or alanine. Pancreatic acinar cells release two forms of elastase, with elastase II having a broader specificity than elastase I.[5] The nucleophilic amino acid at the active site of the endopeptidase hydrolyzes the peptide bond into smaller peptide chains that need further degradation; this can occur through exopeptidases that remove one amino acid from the end of a protein chain. An example of an exopeptidase is the trypsin-activated pancreatic protease carboxypeptidase. There are two forms of this proteolytic enzyme, A and B. Carboxypeptidase A tends to cleave hydrophobic amino acids from the peptide chain, and carboxypeptidase B cleaves basic amino acids off the peptide. Intestinal epithelial cells also contain exopeptidases on the brush border, called aminopeptidases, that release single amino acids that are transportable into cells.[6]

Intracellular protein turnover: proteins within the cell also undergo catabolism to replenish the intracellular amino acid pool. While all proteins have a half-life, some degrade within minutes and some within hours or days. Some examples of heavily synthesized and degraded proteins are muscle cell proteins, hemoglobin, gastrointestinal epithelial cells, and proteolytic enzymes themselves. Many of these proteins are broken down by lysosomal proteases so that their amino acid constituents are usable for rebuilding proteins whose needs depend on the current physiological status of the cell. For example, in starvation periods, the amino acids released from muscle protein catabolism can funnel into gluconeogenesis and act as an energy source. In a fed state or during metabolic acidosis, Glutamine can be a fuel for the renal system. Skeletal muscle can absorb alanine, aspartate, and glutamate, and through transamination, form useful Krebs cycle intermediates.

Cellular Level

Transmembrane Transport: The work of endopeptidases, chymotrypsin, elastase and carboxypeptidase and exopeptidases, carboxypeptidase and aminopeptidase, ultimately yields free amino acids. Facilitated diffusion and active transport mechanisms exist to absorb these compounds from the intestinal lumen through secondary active transport[7]. Amino acids cross the intestinal cell brush border through a sodium-dependent transport system, in which the low intracellular sodium concentration drives the co-entry of sodium and amino acid. A sodium-potassium ATPase on the basolateral membrane maintains the low sodium concentration inside the cell. Once amino acids enter the cell on the apical membrane, they are transported down their gradient on the basal membrane into the bloodstream, typically into the portal circulation. The six different sodium-dependent amino acid carriers overlap in their specificity for the amino acids they transport across the apical membrane of the brush border; however, carriers show preferences for the types of amino acids transported.[7] The preferences include neutral amino acids, acidic amino acids, basic amino acids, cystine, proline, and hydroxyproline. The sodium-dependent amino acid carriers on the luminal intestinal cells also exist on the renal epithelium in different isozyme forms.[8] On the other hand, the facilitated transport systems on the basal side are comparable to those found in other tissue types. Amino acids can also be absorbed from the blood through these facilitated transporters and used as substrates for the citric acid cycle for energy generation—the bidirectionality of the facilitated transporters aids in the survival of the intestinal epithelium in times of starvation.

The transporters on the apical surface of the muscle, liver, and other tissues absorb amino acids from the blood and concentrate them. Although similar to the luminal sodium-dependent amino acid transporters on the intestinal epithelium, these transporters differ in their genetic bases, protein composition, and specificities.[8] Many tissues express A-system, ASC-system, and L-system for amino acid transport into the cell; however, of these, the L-system is the only one without a sodium-dependent mechanism. Intestinal and kidney cells express B-system, X-system, and imino- system.[9] A particular tissue type can contain a specific type of amino acid transporter not seen in other tissues, for example, the liver’s expression of the N-system and its specificity for glutamine absorption. An isoform of N-system for glutamine uptake may express in another tissue type. However, it will have different properties and composition than the one present in the liver. Each system differs in its specificity for amino acids and has implications in numerous different pathologies.

Lysosomal Degradation: Whether proteins are no longer needed or synthesized incorrectly, intracellular proteins are broken down using lysosomes and the proteasome complex. Proteins enter the lysosome through autophagy, a highly regulated process in which vesicles fuse with the lysosome membrane.[10] Autophagy can be promoted by low energy states such as low ATP or high AMP levels within the cell. Because AMP-activated protein kinase becomes activated with high AMP levels, it can phosphorylate tuberous sclerosis complex 1 and 2, leading to the activation of Rheb-GTPase.[11] This activation is important because it inactivates Rheb and, thus, also inactivating mTOR, a major inhibitor of autophagy; this is how low energy states favor protein catabolism. Conversely, activation of the tuberous sclerosis complex 1 and 2 by insulin through Akt kinase leads to active mTOR.[11] Activated mTOR favors protein synthesis rather than degradation.

Ubiquitination is the process in which a ubiquitin molecule becomes covalently attached to the proteins to be degraded and interacts with a proteasome within the lysosome to break down said protein using an ATP-dependent process.[12] Ubiquitin must first become activated through E1, E2, and E3, before becoming attached covalently to an epsilon amino group of lysine side chains through an enzyme complex.[13] Typically, a protein obtains more ubiquitin molecules at the terminal lysine, forming a tail. Once the tail forms, the ubiquitinated protein dissociates from the ligating enzyme complex and moves to the proteasome. The proteasome is a cylindrical 26S protease complex with several catalytic sites on the inside. The 26S proteasome complex has two alpha and two beta rings containing three internal catalytic sites similar to trypsin, chymotrypsin, and post-glutamyl peptidyl hydrolase. The design of the four-ring structure is such that the alpha rings play a stabilizing role, whereas the beta subunits are actively catalytic portions of the complex.[13] It conserves the ubiquitin molecules but uses ATP hydrolysis to unfold and move the protein deeper into the complex and release free amino acids.[10] Proteins hydrolyzed by this process are known as PEST sequences, so named after the common amino acids included in the chain. Amino acids proline, glutamate, serine, and threonine are in high concentrations in PEST sequence proteins.[14] 

Once inside a lysosome, lysosomal proteases called cathepsins can also degrade cytosolic proteins labeled for catabolism. Cathepsins are another type of cysteine protease present within pericellular environments, cytosol nuclei, and mitochondria.[15] Tumor necrosis factor signaling, cell stress, and caspase can trigger the lysosomal release of cathepsin, cleaving intracellular proteins like Bid, leading to downstream activation of the apoptotic caspase cascade.[16]

Molecular Level

The fate of the intracellular amino acid pool strongly depends on the metabolic state of the individual tissue and body in general. In a fed physiologic state, the amino acids can be degraded further to their carbon and nitrogen skeletons. While the carbon backbone can undergo oxidation for energy, the nitrogen component is eliminated from the body through nitrogenous waste products. Initially, the nitrogen is present in the form of ammonia, a toxic substance to the human body. The liver converts toxic ammonia into urea, through the urea cycle, into a non-toxic, water-soluble form of nitrogen that can be eliminated easily through the kidneys. 

As the nitrogen backbone becomes discarded, the carbon skeleton of amino acids can be used for energy production by oxidation into pyruvate (tryptophan, glycine, alanine, serine, cysteine) and acetyl-CoA. Pyruvate and acetyl Coenzyme A are substrates for the citric acid cycle to ultimately produce ATP and carbon dioxide. Acetyl CoA can also form ketone bodies released into the bloodstream for use as an energy source by specific tissues. Amino acids that become acetyl CoA or acetoacetate are named ketogenic because of their propensity to become ketone bodies.[17] For example, lysine and leucine are both ketogenic and also essential amino acids.

Another possible fate is for the carbon skeleton to convert into citric acid cycle intermediates alpha-ketoglutarate (arginine, histidine, glutamine, proline, glutamate), succinyl CoA (valine, threonine, isoleucine, methionine), fumarate (aspartate, tyrosine, phenylalanine), and oxaloacetate (aspartate, asparagine).[18] It is worth noting that malate, a citric acid cycle intermediate, can travel through the circulation to the liver and participate in gluconeogenesis.[19] If an amino acid can become a precursor to glucose, it is called glucogenic. Due to the various intermediates and entry points into the Krebs cycle, amino acids can be both glucogenic and ketogenic.

Testing

Ubiquitination is the beginning of intracellular protein catabolism. Testing the function of this process to understand specific pathologies involves combining cultured cells with plasmids containing a protein with epitope-tagged ubiquitin. The next steps include the addition of stabilizing buffers, lysing, and sonicating the cells, followed by rounds of incubation. Because the ultimate form of analysis involves immunoblotting with SDS-PAGE gel, conjugated antibodies against the protein must be formed[20]. There are also ways to analyze the ubiquitination process in vitro rather than in vivo.[20]

There are ways to test for the enzymes released by the exocrine pancreas, trypsin, chymotrypsin, and elastase. One test is the fecal elastase test, in which the stool is analyzed for the concentration of elastase, thus confirming patency of the pancreatic duct, the secretive function of the exocrine pancreas, and the presence of activating trypsin.[21] This assay is the most widely used test due to its high specificity and non-invasive method. A more invasive test is an endoscopic pancreatic function test and is only available at select medical facilities. In this test, a patient is sedated and is given secretin before the endoscopic procedure.[22] The endoscope suctions out the other fluid in the upper gastrointestinal tract on its way to the duodenum. Then the pancreatic secretion samples are obtained at the major papilla and analyzed for bicarbonate in the lab.[22] Typically, samples are collected at 15, 30, 45, and 60-minute intervals. Evaluating the secretion and activity of these enzymes is important for assessing the pathologies of protein malabsorption.

Pathophysiology

Cystic Fibrosis is an autosomal recessive mutation in the CFTR gene, which codes the proteins that compose the chloride channel pore.[23] The severity of the disease varies, but some features are common to all forms, for example, the formation of thick mucus plugs in the pancreatic duct, the lungs, and the male genitourinary system. Because proteins must break down to be absorbed, a mucus plug blocking the zymogen release from the exocrine pancreas would lead to the absence of vital protein catabolism in the intestinal lumen.[23] Patients with cystic fibrosis have severe protein deficiency and must receive exogenous pancreatic enzyme supplementation, though it bears mention that side effects of long-term pancreatic enzyme supplementation are unknown at this time.[24]

Kwashiorkor is another severe protein deficiency; however, it is due to a lack of protein intake rather than a genetic disorder. The digestive enzymes are present in kwashiorkor; however, because there is little protein ingested, symptoms of protein deficiency are present. In malnourished individuals with kwashiorkor, the vital reactions in the body are sustained through intracellular protein catabolism. The need for available essential amino acids leads to an extensive reduction in peripheral muscle mass from muscle breakdown.[10] Protein deficiency also leads to decreased serum levels of albumin, thus decreasing intravascular colloid pressure, which leads to edema and abdominal distention. Severe kwashiorkor quickly deteriorates because digestive enzymes are no longer produced, and the small intestinal epithelium is not regenerated.

 A defect in facilitated and active transport mechanisms can lead to pathological malabsorptive states. Cystinuria and Hartnup disease are both genetic disorders involving the membrane amino acid transporters but differ in the groups of amino acids transported and so present different clinically.[9] Cystinuria is a defect in transporting basic amino acids through the membranes of the renal and gastrointestinal systems. The hallmark presentation of this disorder is the formation of renal calculi due to the inability to resorb the basic amino acid cystine from the glomerular filtrate. Because the transporter also exists on the small intestine epithelium, cystine and other basic amino acids are not well absorbed from the intestinal lumen. A mechanistically similar disorder, Hartnup disease is a defect in transporting neutral amino acids across the renal and intestinal system. Because tryptophan is one of the neutral amino acids poorly absorbed, a pellagra-like presentation of rash, diarrhea, and psychiatric disturbances are present on physical exam. Unlike pellagra, supplementation with niacin has little resolution of symptoms and should reveal the need for genetic testing.

Clinical Significance

Proper functioning of protein catabolism is of utmost importance to sustain the metabolic needs of the human body. The breakdown of large polypeptide chains to unleash free essential and non-essential amino acids provides cells with the needed substrates for protein synthesis or energy creation. Intracellular protein breakdown is important for normal turnover as well as in malnutrition and malnourished physiological states to mobilize amino acids for export to support the energetic demands of vital organs. Protein catabolism defects can lead to a variety of clinical presentations affecting almost every organ system.