Proteolysis is the breakdown of proteins into smaller polypeptides or amino acids. In general,
this occurs by the hydrolysis of the peptide bond, and is most commonly achieved by cellular
enzymes called proteases, but may also occur by intramolecular digestion, as well as by
non-enzymatic methods such as the action of mineral acids and heat.
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 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,
as well as preventing the accumulation of unwanted or abnormal proteins in cells.
Post-translational proteolytic processing Limited proteolysis of a polypeptide during
or after translation in protein synthesis often occur 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. Removal of N-terminal methionine
The initiating methionine may be removed during translation of the nascent protein. For E.
coli, fMet is efficiently removed if the second residue is small and uncharged, but not if
the second residue is bulky and charged. 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 membrane. Cleavage of polyprotein
Some proteins and most eukaryotic polypeptide hormones are synthesized as a large precursor
polypeptide known as polyprotein that require proteolytic cleavage into individual smaller
polypeptide chains. The polyprotein pro-opiomelanocortin 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 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.
Cleavage of precursor proteins Many proteins and hormones are synthesized
in the form of their precursors - zymogens, proenzymes, and prehormones. 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. To form the mature insulin,
the proinsulin is then cleaved at two positions to yield two polypeptide chains linked by
2 disulphide bonds. Proinsulin is necessary for the folding of the polypeptide chain,
as the 2 polypeptide chains of insulin may not correctly assemble into the correct form,
whereas its precursor proinsulin does. 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 down proteins
into smaller peptides and amino acids so that they may be absorbed and used by an organism.
In animals the food may be processed extracellularly in specialized digestive organs or guts, but
in many bacteria the food may be internalized into the cell via phagocytosis.
Proteins in cells are also constantly being broken down into amino acids. This intracellular
degradation of protein serves a number of functions: It removes damaged and abnormal
protein and prevent their accumulation, and 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 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
rate. 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 half-life of a month or more, while, in essence, haemoglobin
lasts for the entire life-time of erythrocyte. 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 have short half-life. 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. The rate of proteolysis may also depend on
the physiological state of the cell, 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 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. Different
enzymes have different specificity for their substrate; trypsin, for example, cleaves the
peptide bond after a positively charged residue; chymotrypsin cleaves the bond after an aromatic
residue; 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, 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. Proteolysis in 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, or the mediation of thrombin signalling through protease-activated receptors.
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 kinases 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. 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
Caspases are an important group of proteases involved in apoptosis. 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 death receptor pathways.
Regulatory domains in proteolysis Protease may have one or more regulatory domains
- Calcium-binding domain - e.g., prothrombin,
factor IX, X, VII, protein C in blood clotting cascade, calpain.
Kringle domain - e.g., in prothrombin it keeps the protease inactive.
Proteolysis and diseases Abnormal proteolytic activity are associated
with many diseases. 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 and generation of peptides that aggregate in cells and their ineffective
removal may result in many age-related neurological diseases such as Alzheimer's.
The imbalance between proteases and antiproteases is likely to have caused 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 serpins such as α1-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 metalloproteinases
and tissue inhibitors of metalloproteinases. Other diseases linked to aberrant proteolysis
include muscular dystrophy, degenerative skin disorders, respiratory and gastrointestinal
diseases, and malignancy. Non-enzymatic proteolysis
Chemicals may be used in laboratory to target specific residues and cleave its peptide bond
so that protein may be broken down into smaller polypeptides for analysis. Cyanogen bromide
is often used to cleave the peptide bond after a methionine. Other methods may be used to
specifically cleave tryptophanyl, aspartyl, cysteinyl, and asparaginyl peptide bonds.
Acids such as trifluoroacetic acid and formic acid may also be used.
Strong mineral acids can readily hydrolyse the peptide bonds in a protein. However, some
proteins are remarkably resistant to hydrolysis. One well-known example is ribonuclease A,
and one method for its purification involves treatment of crude extracts with hot sulphuric
acid so that other proteins become degraded while ribonuclease A is left intact.
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 and 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 SDS, is often
used in the preparation of nucleic acids to remove unwanted nuclease contaminants that
may otherwise degrade the DNA or RNA. 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.
Digestion of proteins in solution for proteome analysis by liquid chromatography-mass spectrometry.
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. Increasing success rate of crystallisation
projects Commonly in microbiology as a source of digested
protein to support bacterial reproduction, and to indirectly culture viruses, e.g. tryptone
in Lysogeny Broth. Venoms
Certain types of venom, such as those produced by venomous snakes, 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, including effects that are:
cytotoxic hemotoxic
myotoxic hemorrhagic
See also Proteolytic enzyme
The Proteolysis Map PROTOMAP a proteomic technology for identifying
proteolytic substrates Proteasome
In-gel digestion References
Further reading Thomas E Creighton. Proteins: Structures and
Molecular Properties. W H Freeman and Company. ISBN 0-7167-2317-4.
External links 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. Proteolysis at eMedicine Dictionary
Proteolysis MAP from Center on Proteolytic Pathways