Editing Proteolysis

{{refimprove|date=December 2024}}
{{Short description|Breakdown of proteins into smaller polypeptides or amino acids}}
[[File:Proteolysis uncat.svg|thumb|300px|The [[hydrolysis]] of a [[protein]] (red) by the [[nucleophilic substitution|nucleophilic attack]] of water (blue). The uncatalysed half-life is several hundred years.]]

'''Proteolysis''' is the breakdown of [[protein]]s into smaller [[polypeptide]]s or [[amino acids]]. Protein degradation is a major regulatory mechanism of gene expression<ref>{{Cite journal |last=McShane |first=Erik |last2=Selbach |first2=Matthias |date=2022-10-06 |title=Physiological Functions of Intracellular Protein Degradation |url=https://www.annualreviews.org/content/journals/10.1146/annurev-cellbio-120420-091943 |journal=Annual Review of Cell and Developmental Biology |language=en |volume=38 |issue=1 |pages=241–262 |doi=10.1146/annurev-cellbio-120420-091943 |issn=1081-0706}}</ref> and contributes substantially to shaping mammalian proteomes.<ref>{{Citation |last=Leduc |first=Andrew |title=Protein degradation and growth dependent dilution substantially shape mammalian proteomes |date=2025-02-12 |url=http://biorxiv.org/lookup/doi/10.1101/2025.02.10.637566 |access-date=2025-02-16 |language=en |doi=10.1101/2025.02.10.637566 |last2=Slavov |first2=Nikolai|pmc=11844506 }}</ref> Uncatalysed, the [[hydrolysis]] of [[peptide bond]]s is extremely slow, taking hundreds of years. Proteolysis is typically [[catalysed]] by cellular [[enzyme]]s called [[protease]]s, 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 including [[apoptosis]], 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.

== Biological functions ==

=== Post-translational proteolytic processing ===
Limited proteolysis of a polypeptide during or after [[translation (biology)|translation]] in [[protein biosynthesis|protein synthesis]] often occurs for many proteins. This may involve removal of the [[N-terminal]] [[methionine]], [[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 termed [[proprotein]], 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.<ref name="creighton">{{cite book |author=Thomas E Creighton |title=Proteins: Structures and Molecular Properties |edition=2nd |pages=[https://archive.org/details/proteinsstructur0000crei/page/78 78–86] |year=1993 |publisher=W H Freeman and Company |isbn=978-0-7167-2317-2 |url=https://archive.org/details/proteinsstructur0000crei/page/78 }}</ref>

==== Removal of N-terminal methionine ====
The initiating methionine (and, in bacteria, [[fMet]]) may be removed during translation of the nascent protein. For ''[[Escherichia coli|E. coli]]'', fMet is efficiently removed if the second residue is small and uncharged, but not if the second residue is bulky and charged.<ref>{{cite journal |author1=P H Hirel |author2=M J Schmitter |author3=P Dessen |author4=G Fayat |author5=S Blanquet |title=Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid |journal=Proc Natl Acad Sci U S A |volume=86|issue=21 |pages=8247–51 |year=1989 |pmid=2682640 |pmc=298257 |doi=10.1073/pnas.86.21.8247|bibcode=1989PNAS...86.8247H |doi-access=free }}</ref> In both [[prokaryotes]] and [[eukaryotes]], the exposed N-terminal residue may determine the half-life of the protein according to the [[N-end rule]].

==== Removal of the signal sequence ====
Proteins that are to be targeted to a particular organelle or for secretion have an N-terminal [[signal peptide]] that directs the protein to its final destination. This signal peptide is removed by proteolysis after their transport through a [[cell membrane|membrane]].

====Cleavage of polyproteins====
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 polyprotein [[pro-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.

Many [[virus (biology)|viruses]] also produce their proteins initially as a single polypeptide chain that were translated from a [[polycistronic]] mRNA. This polypeptide is subsequently cleaved into individual polypeptide chains.<ref name="creighton"/> Common names for the polyprotein include ''gag'' ([[group-specific antigen]]) in [[retrovirus]]es and ''[[ORF1ab]]'' in [[Nidovirales]]. The latter name refers to the fact that a [[slippery sequence]] in the mRNA that codes for the polypeptide causes [[ribosomal frameshift]]ing, leading to two different lengths of peptidic chains (''a'' and ''ab'') at an approximately fixed ratio.

====Cleavage of precursor proteins====
Many proteins and hormones are synthesized in the form of their precursors - [[zymogen]]s, [[proenzyme]]s, and [[prehormone]]s. These proteins are cleaved to form their final active structures. [[Insulin]], for example, is synthesized as [[preproinsulin]], which yields [[proinsulin]] after the signal peptide has been cleaved. The proinsulin is then cleaved at two positions to yield two polypeptide chains linked by two [[disulfide 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, when [[trypsinogen]] is cleaved to form [[trypsin]], 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 the [[blood clotting cascade]] whereby an initial event triggers a cascade of sequential proteolytic activation of many specific proteases, resulting in blood coagulation. The [[complement system]] of the [[immune response]] also involves a complex sequential proteolytic activation and interaction that result in an attack on invading pathogens.

===Protein degradation===
Protein degradation may take place intracellularly or extracellularly. In digestion of food, digestive enzymes may be released into the environment for [[extracellular 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 specialized [[digestive tract|organs]] or [[Gut (anatomy)|gut]]s, but in many bacteria the food may be internalized via [[phagocytosis]]. 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 fungus ''[[Neurospora crassa]]''<ref>Hanson, M.A., Marzluf, G.A., 1975. Control of the synthesis of a single enzyme by multiple regulatory circuits in Neurospora crassa. Proc. Natl. Acad. Sci. U.S.A. 72, 1240–1244.</ref> as well as in of soil organism communities.<ref>Sims, G. K., and M. M. Wander. 2002. Proteolytic activity under nitrogen or sulfur limitation. Appl. Soil Ecol. 568:1-5.</ref>

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.

====Lysosome and proteasome====
[[File:Proteaosome 1fnt side.png|thumb|upright=0.6|Structure of a proteasome. Its active sites are inside the tube (blue) where proteins are degraded.]]

The intracellular degradation of protein may be achieved in two ways—proteolysis in [[lysosome]], or a [[ubiquitin]]-dependent process that targets unwanted proteins to [[proteasome]]. The [[autophagy]]-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 as [[cathepsins]].
 
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.

====Rate of intracellular protein degradation====
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 is [[ornithine decarboxylase]], which has a half-life of 11 minutes. In contrast, other proteins like [[actin]] and [[myosin]] have a half-life of a month or more, while, in essence, [[haemoglobin]] lasts for the entire life-time of an [[erythrocyte]].<ref name="degradation">{{cite book |author=Thomas E Creighton |title=Proteins: Structures and Molecular Properties |edition=2nd |chapter=Chapter 10 - Degradation |pages=[https://archive.org/details/proteinsstructur0000crei/page/463 463–473] |year=1993 |publisher=W H Freeman and Company |isbn=978-0-7167-2317-2 |chapter-url=https://archive.org/details/proteinsstructur0000crei |url=https://archive.org/details/proteinsstructur0000crei/page/463 }}</ref>

The [[N-end rule]] may partially determine the half-life of a protein, and proteins with segments rich in [[proline]], [[glutamic acid]], [[serine]], and [[threonine]] (the so-called [[PEST sequence|PEST protein]]s) have short half-life.<ref>{{cite book |author=Voet & Voet |title=Biochemistry |pages=[https://archive.org/details/biochemistry00voet_0/page/1010 1010–1014] |edition=2nd |year=1995 |publisher=John Wiley & Sons |isbn=978-0-471-58651-7 |url=https://archive.org/details/biochemistry00voet_0/page/1010 }}</ref> Other factors suspected to affect degradation rate include the rate deamination of glutamine and [[asparagine]] and oxidation of [[cystein]], [[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.<ref name="degradation"/> Proteins with larger degrees of [[Intrinsically disordered proteins|intrinsic disorder]] also tend to have short cellular half-life,<ref>{{Cite journal|title = Structural disorder serves as a weak signal for intracellular protein degradation|journal = Proteins|date = 2008-05-01|issn = 1097-0134|pmid = 18004785|pages = 903–909|volume = 71|issue = 2|doi = 10.1002/prot.21773|first1 = P.|last1 = Tompa|first2 = J.|last2 = Prilusky|first3 = I.|last3 = Silman|first4 = J. L.|last4 = Sussman|s2cid = 13942948}}</ref> with disordered segments having been proposed to facilitate efficient initiation of degradation by the [[proteasome]].<ref>{{Cite journal|title = Paradigms of protein degradation by the proteasome|journal = Current Opinion in Structural Biology|date = 2014-02-01|issn = 1879-033X|pmc = 4010099|pmid = 24632559|pages = 156–164|volume = 24|doi = 10.1016/j.sbi.2014.02.002|first1 = Tomonao|last1 = Inobe|first2 = Andreas|last2 = Matouschek}}</ref><ref>{{Cite journal|title = Intrinsically Disordered Segments Affect Protein Half-Life in the Cell and during Evolution|journal = Cell Reports| date=25 September 2014| issn = 2211-1247|pmc = 4358326|pmid = 25220455|pages = 1832–1844|volume = 8|issue = 6| doi = 10.1016/j.celrep.2014.07.055|first1 = Robin|last1 = van der Lee|first2 = Benjamin|last2 = Lang|first3 = Kai|last3 = Kruse|first4 = Jörg|last4 = Gsponer|first5 = Natalia|last5 = Sánchez de Groot|first6 = Martijn A.|last6 = Huynen|first7 = Andreas|last7 = Matouschek|first8 = Monika|last8 = Fuxreiter|first9 = M. Madan|last9 = Babu}}</ref> 
 
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.

====Digestion====
In human [[digestion]], proteins in food are broken down into smaller peptide chains by [[digestive enzymes]] such as [[pepsin]], [[trypsin]], [[chymotrypsin]], and [[elastase]], and into amino acids by various enzymes such as [[carboxypeptidase]], [[aminopeptidase]], and [[dipeptidase]]. 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.<ref>{{cite journal |author=Silk DB |title=Progress report. Peptide absorption in man|journal=Gut |volume=15|issue=6|pages=494–501|year=1974|pmid= 4604970 |pmc=1413009 |doi=10.1136/gut.15.6.494 }}</ref> Different enzymes have different specificity for their substrate; trypsin, for example, cleaves the peptide bond after a positively charged residue ([[arginine]] and [[lysine]]); chymotrypsin cleaves the bond after an aromatic residue ([[phenylalanine]], [[tyrosine]], and [[tryptophan]]); 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 causing [[pancreatitis]]), these enzymes are secreted as inactive zymogen. The precursor of [[pepsin]], [[pepsinogen]], is secreted by the stomach, and is activated only in the acidic environment found in stomach. The [[pancreas]] secretes the precursors of a number of proteases such as [[trypsin]] and [[chymotrypsin]]. The zymogen of trypsin is [[trypsinogen]], which is activated by a very specific protease, [[enterokinase]], secreted by the [[mucosa]] of the [[duodenum]]. 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 by ''[[Bacillus subtilis]]'', is produced as preprosubtilisin, and is released only if the signal peptide is cleaved and autocatalytic proteolytic activation has occurred.

===Cellular regulation===
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,<ref>{{cite journal |title=The SREBP Pathway: Regulation of Cholesterol Metabolism by Proteolysis of a Membrane-Bound Transcription Factor|author1=Michael S. Brown |author2=Joseph L. Goldstein |journal=Cell |volume=89 |issue= 3|pages=331–340 |date=May 1997 |pmid=9150132 |doi=10.1016/S0092-8674(00)80213-5|s2cid=17882616 |doi-access=free }}</ref> or the mediation of thrombin signalling through [[protease-activated receptor]]s.<ref>{{cite journal |title=Thrombin signalling and protease-activated receptors |author=Shaun R. Coughlin |journal=Nature |volume=407 |issue=6801 |pages=258–264 |year=2000 |doi=10.1038/35025229 |pmid=11001069 |s2cid=4429634 }}</ref>

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.

====Cell cycle regulation====
[[Cyclins]] are a group of proteins that activate [[kinase]]s involved in cell division. The degradation of cyclins is the key step that governs the exit from [[mitosis]] and progress into the next [[cell cycle]].<ref>{{cite journal |vauthors=Glotzer M, Murray AW, Kirschner MW |journal=Nature |volume=349 |issue=6305|pages=132–8 |year=1991 |pmid=1846030 |doi=10.1038/349132a0 |title=Cyclin is degraded by the ubiquitin pathway|bibcode=1991Natur.349..132G |s2cid=205003883 }}</ref> Cyclins accumulate in the course the cell cycle, then abruptly disappear just before the [[anaphase]] of mitosis. The cyclins are removed via a ubiquitin-mediated proteolytic pathway.

====Apoptosis====
[[Caspase]]s are an important group of proteases involved in [[apoptosis]] or [[programmed cell death]]. The precursors of caspase, procaspase, may be activated by proteolysis through its association with a protein complex that forms [[apoptosome]], or by [[granzyme B]], or via the [[Tumor necrosis factor receptor|death receptor]] pathways.

==Autoproteolysis==

Autoproteolysis takes place in some proteins, whereby the [[peptide bond]] is cleaved in a self-catalyzed [[intramolecular reaction]]. Unlike [[zymogen]]s, 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 of [[von Willebrand factor]] type D (VWD) domains<ref>{{Cite journal|last1=Lidell|first1=Martin E.|last2=Johansson|first2=Malin E. V.|last3=Hansson|first3=Gunnar C.|date=2003-04-18|title=An autocatalytic cleavage in the C terminus of the human MUC2 mucin occurs at the low pH of the late secretory pathway|journal=The Journal of Biological Chemistry|volume=278|issue=16|pages=13944–13951|doi=10.1074/jbc.M210069200|issn=0021-9258|pmid=12582180|doi-access=free}}</ref><ref>{{Cite journal|last1=Bi|first1=Ming|last2=Hickox|first2=John R|last3=Winfrey|first3=Virginia P|last4=Olson|first4=Gary E|last5=Hardy|first5=Daniel M|date=2003-10-15|title=Processing, localization and binding activity of zonadhesin suggest a function in sperm adhesion to the zona pellucida during exocytosis of the acrosome.|journal=Biochemical Journal|volume=375|issue=Pt 2|pages=477–488|doi=10.1042/BJ20030753|issn=0264-6021|pmc=1223699|pmid=12882646}}</ref> and ''[[Neisseria meningitidis]]'' FrpC self-processing domain,<ref>{{Cite journal|last1=Sadilkova|first1=Lenka|last2=Osicka|first2=Radim|last3=Sulc|first3=Miroslav|last4=Linhartova|first4=Irena|last5=Novak|first5=Petr|last6=Sebo|first6=Peter|date=October 2008|title=Single-step affinity purification of recombinant proteins using a self-excising module from Neisseria meningitidis FrpC|journal=Protein Science|language=en|volume=17|issue=10|pages=1834–1843|doi=10.1110/ps.035733.108|pmc=2548358|pmid=18662906}}</ref> cleavage of the Asn-Pro bond in ''[[Salmonella]]'' FlhB protein,<ref>{{Cite journal|last1=Minamino|first1=Tohru|last2=Macnab|first2=Robert M.|date=2000-09-01|title=Domain Structure of Salmonella FlhB, a Flagellar Export Component Responsible for Substrate Specificity Switching|journal=Journal of Bacteriology|language=en|volume=182|issue=17|pages=4906–4914|doi=10.1128/JB.182.17.4906-4914.2000|issn=1098-5530|pmc=111371|pmid=10940035}}</ref> ''[[Yersinia]]'' YscU protein,<ref>{{Cite journal|last1=Björnfot|first1=Ann-Catrin|last2=Lavander|first2=Moa|last3=Forsberg|first3=Åke|last4=Wolf-Watz|first4=Hans|date=2009-07-01|title=Autoproteolysis of YscU of Yersinia pseudotuberculosis Is Important for Regulation of Expression and Secretion of Yop Proteins|journal=Journal of Bacteriology|language=en|volume=191|issue=13|pages=4259–4267|doi=10.1128/JB.01730-08|issn=0021-9193|pmc=2698497|pmid=19395493}}</ref> as well as cleavage of the Gly-Ser bond in a subset of sea urchin sperm protein, enterokinase, and agrin (SEA) domains.<ref name="Johansson 1130–1143">{{Cite journal|last1=Johansson|first1=Denny G. A.|last2=Macao|first2=Bertil|last3=Sandberg|first3=Anders|last4=Härd|first4=Torleif|date=2008-04-04|title=SEA domain autoproteolysis accelerated by conformational strain: mechanistic aspects|journal=Journal of Molecular Biology|volume=377|issue=4|pages=1130–1143|doi=10.1016/j.jmb.2008.01.050|issn=1089-8638|pmid=18314133}}</ref> In some cases, the autoproteolytic cleavage is promoted by conformational strain of the peptide bond.<ref name="Johansson 1130–1143"/>

==Proteolysis and diseases==
Abnormal proteolytic activity is associated with many diseases.<ref>{{cite journal |url=http://pt7mdv.ceingebi.unam.mx/computo/pdfs/ubiquita/enfermedades.pdf |title=Ubiquitin-dependent proteolysis: its role in human diseases and the design of therapeutic strategies |author=Kathleen M. Sakamoto |journal=Molecular Genetics and Metabolism |volume=77 |year=2002 |pages=44–56 |pmid=12359129 |doi=10.1016/S1096-7192(02)00146-4 |issue=1–2 |access-date=2012-06-30 |archive-date=2016-03-04 |archive-url=https://web.archive.org/web/20160304070429/http://pt7mdv.ceingebi.unam.mx/computo/pdfs/ubiquita/enfermedades.pdf |url-status=dead }}</ref> In [[pancreatitis]], leakage of proteases and their premature activation in the pancreas results in the self-digestion of the [[pancreas]]. People with [[diabetes mellitus]] may have increased lysosomal activity and the degradation of some proteins can increase significantly. Chronic inflammatory diseases such as [[rheumatoid 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 as [[Alzheimer]]'s due to the generation and ineffective removal of peptides that aggregate in cells.<ref>{{Cite journal |title=Proteases and proteolysis in Alzheimer disease: a multifactorial view on the disease process |author=De Strooper B. |journal=Physiological Reviews |year=2010 |volume=90|issue=2 |pages=465–94 |pmid=20393191 |doi=10.1152/physrev.00023.2009 }}</ref>

Proteases may be regulated by [[antiprotease]]s or [[Protease inhibitor (biology)|protease inhibitors]], and imbalance between proteases and antiproteases can result in diseases, for example, in the destruction of lung tissues in [[emphysema]] brought on by [[smoking]] tobacco. Smoking is thought to increase the [[neutrophils]] and [[macrophages]] in the lung which release excessive amount of proteolytic enzymes such as [[elastase]], such that they can no longer be inhibited by [[serpin]]s such as [[alpha 1-antitrypsin|α<sub>1</sub>-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 example [[matrix metalloproteinase]]s (MMPs) and [[tissue inhibitors of metalloproteinases]] (TIMPs).<ref>{{cite journal |journal=International Journal of Tuberculosis and Lung Disease |year=2008 |volume=12 |issue=4 |pages=361–7 |title=Pathogenesis of COPD. Part I. The role of protease-antiprotease imbalance in emphysema |author=Abboud RT1, Vimalanathan S |pmid=18371259 }}</ref>

Other diseases linked to aberrant proteolysis include [[muscular dystrophy]], degenerative skin disorders, respiratory and gastrointestinal diseases, and [[malignancy]].

==Non-enzymatic processes==
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.<ref>{{cite journal |author1=Daniel. Kahne |author2=W. Clark Still |journal=J. Am. Chem. Soc. |title=Hydrolysis of a peptide bond in neutral water|year= 1988 |volume= 110 |issue=22|pages= 7529–7534|doi= 10.1021/ja00230a041 |bibcode=1988JAChS.110.7529K }}</ref><ref>{{cite journal|last1=Radzicka|first1=Anna|last2=Wolfenden|first2=Richard|title=Rates of Uncatalyzed Peptide Bond Hydrolysis in Neutral Solution and the Transition State Affinities of Proteases|journal=Journal of the American Chemical Society|date=January 1996|volume=118|issue=26|pages=6105–6109|doi=10.1021/ja954077c|bibcode=1996JAChS.118.6105R }}</ref><ref>{{cite book |url=https://books.google.com/books?id=U-PDqHikphYC&pg=PA270 |title=Hydrolysis in Drug and Prodrug Metabolism|author=Bernard Testa |author2=Joachim M. Mayer |pages=270–288 |publisher=Wiley VCH|date=1 July 2003 |isbn=978-3-906390-25-3 }}</ref>  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.<ref>{{cite journal |title= Spontaneous cleavage of proteins at serine and threonine is facilitated by zinc |author1=Brian Lyons |author2=Ann H. Kwan |author3=Roger J.W. Truscott |journal=Aging Cell|date=April 2016 |volume= 15|issue=2|pages= 237–244|doi= 10.1111/acel.12428 |pmid= 26751411 |pmc= 4783340 }}</ref>

Strong [[mineral 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&nbsp;°C for around 24 hours in 6M [[hydrochloric acid]].<ref name=creighton2>{{cite book |author=Thomas E Creighton |title=Proteins: Structures and Molecular Properties |edition=2nd |page=[https://archive.org/details/proteinsstructur0000crei/page/6 6] |year=1993 |publisher=W H Freeman and Company |isbn=978-0-7167-2317-2 |url=https://archive.org/details/proteinsstructur0000crei/page/6 }}</ref>  However, some proteins are resistant to acid hydrolysis. One well-known example is [[ribonuclease A]], which can be purified by treating crude extracts with hot [[sulfuric acid]] so that other proteins become degraded while ribonuclease A is left intact.<ref>{{cite web |url= http://www.rcsb.org/pdb/101/motm.do?momID=105 |title=Ribonuclease A |publisher= Protein Data Bank }}</ref>

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.<ref>{{cite book |title=The Protein Protocols Handbook |url=https://archive.org/details/proteinprotocols00walk_410 |url-access=limited |author=Bryan John Smith |editor=John M. Walker |chapter=Chapter 71-75 |edition=2 |publisher=Humana Press |year=2002 |pages= [https://archive.org/details/proteinprotocols00walk_410/page/n477 485]–510 |doi=10.1385/1592591698 |isbn=978-0-89603-940-7 |s2cid=3692961 }}</ref> For example, [[cyanogen bromide]] cleaves the peptide bond after a [[methionine]]. Similar methods may be used to specifically cleave [[tryptophan]]yl, [[aspartyl]], [[cysteinyl]], and [[asparagin]]yl peptide bonds. Acids such as [[trifluoroacetic acid]] and [[formic acid]] may be used for cleavage.

Like other biomolecules, proteins can also be broken down by high heat alone. At 250&nbsp;°C, the peptide bond may be easily hydrolyzed, with its half-life dropping to about a minute.<ref name=creighton2 /><ref>{{cite journal |journal=Nature |year= 1984 |volume=310 |issue=5976|pages=430–2|title=Hydrolytic stability of biomolecules at high temperatures and its implication for life at 250 degrees C |author=White RH |pmid= 6462230 |doi=10.1038/310430a0|s2cid= 4315057 }}</ref> Protein may also be broken down without hydrolysis through [[pyrolysis]]; small [[heterocyclic compound]]s may start to form upon degradation. Above 500&nbsp;°C, [[polycyclic aromatic hydrocarbon]]s may also form,<ref>{{cite journal |title=Formation of low molecular weight heterocycles and polycyclic aromatic compounds (PACs) in the pyrolysis of α-amino acids|author1=Ramesh K. Sharmaa |author2=W.Geoffrey Chana |author3=Jeffrey I. Seemanb |author4=Mohammad R. Hajaligola |journal=Journal of Analytical and Applied Pyrolysis|volume= 66 |issue= 1–2 |date=January 2003| pages =97–121 |doi=10.1016/S0165-2370(02)00108-0 |bibcode=2003JAAP...66...97S }}</ref><ref>{{cite journal |journal=Anal Bioanal Chem |year= 2010 |volume=397|issue=1|pages=309–17| doi= 10.1007/s00216-010-3563-5|title=GC-MS determination of polycyclic aromatic hydrocarbons evolved from pyrolysis of biomass|vauthors=Fabbri D, Adamiano A, Torri C |pmid= 20213167 |s2cid= 33835929 }}</ref> which is of interest in the study of generation of [[carcinogen]]s in tobacco smoke and cooking at high heat.<ref>{{cite journal |journal= Food Chem Toxicol |date= May 2001 |volume=39|issue=5|pages=499–505|title=Effect of pyrolysis temperature on the mutagenicity of tobacco smoke condensate|vauthors=White JL, Conner BT, Perfetti TA, Bombick BR, Avalos JT, Fowler KW, Smith CJ, Doolittle DJ |pmid=11313117 |doi=10.1016/s0278-6915(00)00155-1}}</ref><ref>{{cite web |url=http://www.cancer.gov/about-cancer/causes-prevention/risk/diet/cooked-meats-fact-sheet |title=Chemicals in Meat Cooked at High Temperatures and Cancer Risk |work=National Cancer Institute |date=2 April 2018 }}</ref>

==Laboratory applications==
Proteolysis is also used in research and diagnostic applications: 
* Cleavage of [[fusion protein]] so that the fusion partner and [[protein tag]] used in [[Protein expression (biotechnology)|protein expression]] and [[Protein purification|purification]] may be removed. The proteases used have high degree of specificity, such as [[thrombin]], [[enterokinase]], and [[TEV protease]], so that only the targeted sequence may be cleaved.
* Complete inactivation of undesirable enzymatic activity or removal of unwanted proteins. For example, [[proteinase K]], a broad-spectrum proteinase stable in [[urea]] and [[Sodium dodecyl sulfate|SDS]], is often used in the preparation of [[nucleic acids]] to remove unwanted [[nuclease]] contaminants that may otherwise degrade the DNA or RNA.<ref>{{cite journal |vauthors=Hilz H, Wiegers U, Adamietz P |title=Stimulation of Proteinase K action by denaturing agents: application to the isolation of nucleic acids and the degradation of 'masked' proteins|journal=European Journal of Biochemistry|volume=56|issue=1|pages=103–108|year=1975|pmid=1236799|doi=10.1111/j.1432-1033.1975.tb02211.x|doi-access=free}}</ref>
* Partial inactivation, or changing the functionality, of specific protein. For example, treatment of [[DNA polymerase I]] with [[subtilisin]] yields the [[Klenow fragment]], which retains its polymerase function but lacks 5'-exonuclease activity.<ref>{{cite journal|vauthors=Klenow H, Henningsen I| title=Selective Elimination of the Exonuclease Activity of the Deoxyribonucleic Acid Polymerase from Escherichia coli B by Limited Proteolysis| journal= Proc. Natl. Acad. Sci. USA| volume= 65 |pages=168–175 | year=1970 | pmid=4905667 | doi = 10.1073/pnas.65.1.168 | issue=1| pmc=286206| bibcode=1970PNAS...65..168K| doi-access=free}}</ref>
* Digestion of proteins in solution for [[proteomics|proteome analysis]] by [[liquid chromatography-mass spectrometry]] (LC-MS). This may also be done by [[in-gel digestion]] of [[proteins]] after separation by [[gel electrophoresis]] for the identification by [[mass spectrometry]].
* Analysis of the stability of folded domain under a wide range of conditions.<ref>{{Cite journal|author= Minde DP|title= Determining biophysical protein stability in lysates by a fast proteolysis assay, FASTpp |journal= PLOS ONE |volume= 7 |pages= e46147 |year= 2012 |doi= 10.1371/journal.pone.0046147|issue= 10|editor1-last= Uversky|editor1-first= Vladimir N|last2= Maurice|first2= Madelon M.|last3= Rüdiger|first3= Stefan G. D.|pmid= 23056252|pmc= 3463568|bibcode= 2012PLoSO...746147M |doi-access= free }}</ref>
* Increasing success rate of crystallisation projects<ref>{{cite journal|pmid=19352432|year=2009|last1=Wernimont|first1=A|last2=Edwards|first2=A|title=In situ proteolysis to generate crystals for structure determination: An update|volume=4|issue=4|pages=e5094|doi=10.1371/journal.pone.0005094|pmc=2661377|journal=PLOS ONE|bibcode=2009PLoSO...4.5094W|editor1-last=Song|editor1-first=Haiwei|doi-access=free}}</ref>
* Production of digested protein used in growth media to culture bacteria and other organisms, e.g. [[tryptone]] in [[Lysogeny Broth]].

==Protease enzymes==
{{main|Protease}}
Proteases may be classified according to the catalytic group involved in its active site.<ref>{{cite journal |title=New families of carboxyl peptidases: serine-carboxyl peptidases and glutamic peptidases |author=Kohei Oda |journal=Journal of Biochemistry |year= 2012 |volume=151 |issue=1|pages=13–25 | doi= 10.1093/jb/mvr129 |pmid= 22016395 |doi-access=free }}</ref>
*[[Cysteine protease]]
*[[Serine protease]]
*[[Threonine protease]]
*[[Aspartic protease]]
*[[Glutamic protease]]
*[[Metalloprotease]]
*[[Asparagine peptide lyase]]

===Venoms===
Certain types of venom, such as those produced by venomous [[snake]]s, 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,<ref>Hayes WK. 2005. [http://www.llu.edu/llu/grad/natsci/hayes/research-c-venom.html?PHPSESSID=55842bf3eeb83dcdfec66c45b91925fc Research on Biological Roles and Variation of Snake Venoms.] {{Webarchive|url=https://web.archive.org/web/20190915094718/https://behavioralhealth.llu.edu/?PHPSESSID=55842bf3eeb83dcdfec66c45b91925fc |date=2019-09-15 }} Loma Linda University.</ref> including effects that are:
* [[Cytotoxin|cytotoxic]] (cell-destroying)
* [[Hemotoxin|hemotoxic]] (blood-destroying)
* [[Myotoxin|myotoxic]] (muscle-destroying)
* [[Hemorrhage|hemorrhagic]] (bleeding)

==See also==
{{Portal|Biology}}

* [[The Proteolysis Map]]
* [[Protomap (proteomics)|PROTOMAP]] a proteomic technology for identifying proteolytic substrates
* [[Proteasome]]
* [[In-gel digestion]]
* [[Ubiquitin]]

==References==
{{Reflist}}

==Further reading==
*{{cite book |author=Thomas E Creighton |title=Proteins: Structures and Molecular Properties |edition=2nd |year=1993 |publisher=W H Freeman and Company |isbn=978-0-7167-2317-2 |url=https://archive.org/details/proteinsstructur0000crei }}

==External links==
* [http://www.jproteolysis.com The Journal of Proteolysis] is an open access journal that provides an international forum for the electronic publication of the whole spectrum of high-quality articles and reviews in all areas of proteolysis and proteolytic pathways. 
* [https://web.archive.org/web/20081121051237/http://www.proteolysis.org/ Proteolysis MAP from Center on Proteolytic Pathways]

{{Protein posttranslational modification}}
{{Proteases}}
{{Enzymes}}
{{Authority control}}

[[Category:Post-translational modification]]
[[Category:Metabolism]]
[[Category:EC 3.4]]