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Proteolysis is the breakdown ofproteins into smallerpolypeptides oramino acids. Protein degradation is a major regulatory mechanism of gene expression[1] and contributes substantially to shaping mammalian proteomes.[2] Uncatalysed, thehydrolysis ofpeptide bonds is extremely slow, taking hundreds of years. Proteolysis is typicallycatalysed by cellularenzymes calledproteases, but may also occur by intra-molecular digestion.
Proteolysis in organisms serves many purposes; for example,digestive enzymes break down proteins in food to provide amino acids for the organism, while proteolytic processing of a polypeptide chain after its synthesis may be necessary for the production of an active protein. It is also important in the regulation of some physiological and cellular processes includingapoptosis, as well as preventing the accumulation of unwanted or misfolded proteins in cells. Consequently, abnormality in the regulation of proteolysis can cause diseases.
Proteolysis can also be used as an analytical tool for studying proteins in the laboratory, and it may also be used in industry, for example in food processing and stain removal.
Limited proteolysis of a polypeptide during or aftertranslation inprotein synthesis often occurs for many proteins. This may involve removal of theN-terminalmethionine,signal peptide, and/or the conversion of an inactive or non-functional protein to an active one. The precursor to the final functional form of protein is termedproprotein, and these proproteins may be first synthesized as preproprotein. For example,albumin is first synthesized as preproalbumin and contains an uncleaved signal peptide. This forms the proalbumin after the signal peptide is cleaved, and a further processing to remove the N-terminal 6-residue propeptide yields the mature form of the protein.[3]
The initiating methionine (and, in bacteria,fMet) may be removed during translation of the nascent protein. ForE. coli, fMet is efficiently removed if the second residue is small and uncharged, but not if the second residue is bulky and charged.[4] In bothprokaryotes andeukaryotes, the exposed N-terminal residue may determine the half-life of the protein according to theN-end rule.
Proteins that are to be targeted to a particular organelle or for secretion have an N-terminalsignal peptide that directs the protein to its final destination. This signal peptide is removed by proteolysis after their transport through amembrane.
Some proteins and most eukaryotic polypeptide hormones are synthesized as a large precursor polypeptide known as a polyprotein that requires proteolytic cleavage into individual smaller polypeptide chains. The polyproteinpro-opiomelanocortin (POMC) contains many polypeptide hormones. The cleavage pattern of POMC, however, may vary between different tissues, yielding different sets of polypeptide hormones from the same polyprotein.
Manyviruses also produce their proteins initially as a single polypeptide chain that were translated from apolycistronic mRNA. This polypeptide is subsequently cleaved into individual polypeptide chains.[3] Common names for the polyprotein includegag (group-specific antigen) inretroviruses andORF1ab inNidovirales. The latter name refers to the fact that aslippery sequence in the mRNA that codes for the polypeptide causesribosomal frameshifting, leading to two different lengths of peptidic chains (a andab) at an approximately fixed ratio.
Many proteins and hormones are synthesized in the form of their precursors -zymogens,proenzymes, andprehormones. These proteins are cleaved to form their final active structures.Insulin, for example, is synthesized aspreproinsulin, which yieldsproinsulin after the signal peptide has been cleaved. The proinsulin is then cleaved at two positions to yield two polypeptide chains linked by twodisulfide bonds. Removal of two C-terminal residues from the B-chain then yields the mature insulin.Protein folding occurs in the single-chain proinsulin form which facilitates formation of the ultimate inter-peptide disulfide bonds, and the ultimate intra-peptide disulfide bond, found in the native structure of insulin.
Proteases in particular are synthesized in the inactive form so that they may be safely stored in cells, and ready for release in sufficient quantity when required. This is to ensure that the protease is activated only in the correct location or context, as inappropriate activation of these proteases can be very destructive for an organism. Proteolysis of the zymogen yields an active protein; for example, whentrypsinogen is cleaved to formtrypsin, a slight rearrangement of the protein structure that completes the active site of the protease occurs, thereby activating the protein.
Proteolysis can, therefore, be a method of regulating biological processes by turning inactive proteins into active ones. A good example is theblood clotting cascade whereby an initial event triggers a cascade of sequential proteolytic activation of many specific proteases, resulting in blood coagulation. Thecomplement system of theimmune response also involves a complex sequential proteolytic activation and interaction that result in an attack on invading pathogens.
Protein degradation may take place intracellularly or extracellularly. In digestion of food, digestive enzymes may be released into the environment forextracellular digestion whereby proteolytic cleavage breaks proteins into smaller peptides and amino acids so that they may be absorbed and used. In animals the food may be processed extracellularly in specializedorgans orguts, but in many bacteria the food may be internalized viaphagocytosis. Microbial degradation of protein in the environment can be regulated by nutrient availability. For example, limitation for major elements in proteins (carbon, nitrogen, and sulfur) induces proteolytic activity in the fungusNeurospora crassa[5] as well as in of soil organism communities.[6]
Proteins in cells are broken into amino acids. This intracellular degradation of protein serves multiple functions: It removes damaged and abnormal proteins and prevents their accumulation. It also serves to regulate cellular processes by removing enzymes and regulatory proteins that are no longer needed. The amino acids may then be reused for protein synthesis.
The intracellular degradation of protein may be achieved in two ways—proteolysis inlysosome, or aubiquitin-dependent process that targets unwanted proteins toproteasome. Theautophagy-lysosomal pathway is normally a non-selective process, but it may become selective upon starvation whereby proteins with peptide sequence KFERQ or similar are selectively broken down. The lysosome contains a large number of proteases such ascathepsins.
The ubiquitin-mediated process is selective. Proteins marked for degradation are covalently linked to ubiquitin. Many molecules of ubiquitin may be linked in tandem to a protein destined for degradation. The polyubiquinated protein is targeted to an ATP-dependent protease complex, the proteasome. The ubiquitin is released and reused, while the targeted protein is degraded.
Different proteins are degraded at different rates. Abnormal proteins are quickly degraded, whereas the rate of degradation of normal proteins may vary widely depending on their functions. Enzymes at important metabolic control points may be degraded much faster than those enzymes whose activity is largely constant under all physiological conditions. One of the most rapidly degraded proteins isornithine decarboxylase, which has a half-life of 11 minutes. In contrast, other proteins likeactin andmyosin have a half-life of a month or more, while, in essence,haemoglobin lasts for the entire life-time of anerythrocyte.[7]
TheN-end rule may partially determine the half-life of a protein, and proteins with segments rich inproline,glutamic acid,serine, andthreonine (the so-calledPEST proteins) have short half-life.[8] Other factors suspected to affect degradation rate include the rate deamination of glutamine andasparagine and oxidation ofcystein,histidine, and methionine, the absence of stabilizing ligands, the presence of attached carbohydrate or phosphate groups, the presence of free α-amino group, the negative charge of protein, and the flexibility and stability of the protein.[7] Proteins with larger degrees ofintrinsic disorder also tend to have short cellular half-life,[9] with disordered segments having been proposed to facilitate efficient initiation of degradation by theproteasome.[10][11]
The rate of proteolysis may also depend on the physiological state of the organism, such as its hormonal state as well as nutritional status. In time of starvation, the rate of protein degradation increases.
In humandigestion, proteins in food are broken down into smaller peptide chains bydigestive enzymes such aspepsin,trypsin,chymotrypsin, andelastase, and into amino acids by various enzymes such ascarboxypeptidase,aminopeptidase, anddipeptidase. It is necessary to break down proteins into small peptides (tripeptides and dipeptides) and amino acids so they can be absorbed by the intestines, and the absorbed tripeptides and dipeptides are also further broken into amino acids intracellularly before they enter the bloodstream.[12] Different enzymes have different specificity for their substrate; trypsin, for example, cleaves the peptide bond after a positively charged residue (arginine andlysine); chymotrypsin cleaves the bond after an aromatic residue (phenylalanine,tyrosine, andtryptophan); elastase cleaves the bond after a small non-polar residue such as alanine or glycine.
In order to prevent inappropriate or premature activation of the digestive enzymes (they may, for example, trigger pancreatic self-digestion causingpancreatitis), these enzymes are secreted as inactive zymogen. The precursor ofpepsin,pepsinogen, is secreted by the stomach, and is activated only in the acidic environment found in stomach. Thepancreas secretes the precursors of a number of proteases such astrypsin andchymotrypsin. The zymogen of trypsin istrypsinogen, which is activated by a very specific protease,enterokinase, secreted by themucosa of theduodenum. The trypsin, once activated, can also cleave other trypsinogens as well as the precursors of other proteases such as chymotrypsin and carboxypeptidase to activate them.
In bacteria, a similar strategy of employing an inactive zymogen or prezymogen is used.Subtilisin, which is produced byBacillus subtilis, is produced as preprosubtilisin, and is released only if the signal peptide is cleaved and autocatalytic proteolytic activation has occurred.
Proteolysis is also involved in the regulation of many cellular processes by activating or deactivating enzymes, transcription factors, and receptors, for example in the biosynthesis of cholesterol,[13] or the mediation of thrombin signalling throughprotease-activated receptors.[14]
Some enzymes at important metabolic control points such as ornithine decarboxylase is regulated entirely by its rate of synthesis and its rate of degradation. Other rapidly degraded proteins include the protein products of proto-oncogenes, which play central roles in the regulation of cell growth.
Cyclins are a group of proteins that activatekinases involved in cell division. The degradation of cyclins is the key step that governs the exit frommitosis and progress into the nextcell cycle.[15] Cyclins accumulate in the course the cell cycle, then abruptly disappear just before theanaphase of mitosis. The cyclins are removed via a ubiquitin-mediated proteolytic pathway.
Caspases are an important group of proteases involved inapoptosis orprogrammed cell death. The precursors of caspase, procaspase, may be activated by proteolysis through its association with a protein complex that formsapoptosome, or bygranzyme B, or via thedeath receptor pathways.
Autoproteolysis takes place in some proteins, whereby thepeptide bond is cleaved in a self-catalyzedintramolecular reaction. Unlikezymogens, these autoproteolytic proteins participate in a "single turnover" reaction and do not catalyze further reactions post-cleavage. Examples include cleavage of the Asp-Pro bond in a subset ofvon Willebrand factor type D (VWD) domains[16][17] andNeisseria meningitidis FrpC self-processing domain,[18] cleavage of the Asn-Pro bond inSalmonella FlhB protein,[19]Yersinia YscU protein,[20] as well as cleavage of the Gly-Ser bond in a subset of sea urchin sperm protein, enterokinase, and agrin (SEA) domains.[21] In some cases, the autoproteolytic cleavage is promoted by conformational strain of the peptide bond.[21]
Abnormal proteolytic activity is associated with many diseases.[22] Inpancreatitis, leakage of proteases and their premature activation in the pancreas results in the self-digestion of thepancreas. People withdiabetes mellitus may have increased lysosomal activity and the degradation of some proteins can increase significantly. Chronic inflammatory diseases such asrheumatoid arthritis may involve the release of lysosomal enzymes into extracellular space that break down surrounding tissues. Abnormal proteolysis may result in age-related neurological diseases such asAlzheimer's due to the generation and ineffective removal of peptides that aggregate in cells.[23]
Proteases may be regulated byantiproteases orprotease inhibitors, and imbalance between proteases and antiproteases can result in diseases, for example, in the destruction of lung tissues inemphysema brought on bysmoking tobacco. Smoking is thought to increase theneutrophils andmacrophages in the lung which release excessive amount of proteolytic enzymes such aselastase, such that they can no longer be inhibited byserpins such asα1-antitrypsin, thereby resulting in the breaking down of connective tissues in the lung. Other proteases and their inhibitors may also be involved in this disease, for examplematrix metalloproteinases (MMPs) andtissue inhibitors of metalloproteinases (TIMPs).[24]
Other diseases linked to aberrant proteolysis includemuscular dystrophy, degenerative skin disorders, respiratory and gastrointestinal diseases, andmalignancy.
Protein backbones are very stable in water at neutral pH and room temperature, although the rate of hydrolysis of different peptide bonds can vary. The half-life of a peptide bond under normal conditions can range from 7 years to 350 years, even higher for peptides protected by modified terminus or within the protein interior.[25][26][27] The rate of hydrolysis however can be significantly increased by extremes of pH and heat. Spontaneous cleavage of proteins may also involve catalysis by zinc on serine and threonine.[28]
Strongmineral acids can readily hydrolyse the peptide bonds in a protein (acid hydrolysis). The standard way to hydrolyze a protein or peptide into its constituent amino acids for analysis is to heat it to 105 °C for around 24 hours in 6Mhydrochloric acid.[29] However, some proteins are resistant to acid hydrolysis. One well-known example isribonuclease A, which can be purified by treating crude extracts with hotsulfuric acid so that other proteins become degraded while ribonuclease A is left intact.[30]
Certain chemicals cause proteolysis only after specific residues, and these can be used to selectively break down a protein into smaller polypeptides for laboratory analysis.[31] For example,cyanogen bromide cleaves the peptide bond after amethionine. Similar methods may be used to specifically cleavetryptophanyl,aspartyl,cysteinyl, andasparaginyl peptide bonds. Acids such astrifluoroacetic acid andformic acid may be used for cleavage.
Like other biomolecules, proteins can also be broken down by high heat alone. At 250 °C, the peptide bond may be easily hydrolyzed, with its half-life dropping to about a minute.[29][32] Protein may also be broken down without hydrolysis throughpyrolysis; smallheterocyclic compounds may start to form upon degradation. Above 500 °C,polycyclic aromatic hydrocarbons may also form,[33][34] which is of interest in the study of generation ofcarcinogens in tobacco smoke and cooking at high heat.[35][36]
Proteolysis is also used in research and diagnostic applications:
Proteases may be classified according to the catalytic group involved in its active site.[41]
Certain types of venom, such as those produced by venomoussnakes, can also cause proteolysis. These venoms are, in fact, complex digestive fluids that begin their work outside of the body. Proteolytic venoms cause a wide range of toxic effects,[42] including effects that are:
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