It is composed of individual amino acids that are linked by a covalent bond, called a peptide bond, during the protein translation process.
Proteins are constantly translated and degraded in the cell to ensure adequate concentrations of functional protein to complete various cellular functions.
The process of protein degradation, called proteolysis, breaks down proteins into individual amino acids. Proteolysis is carried out by proteases, a type of enzyme whose mechanism depends on the particular class of protease.
These perform many important functions in the cell, including blood clotting, food digestion, apoptosis and autophagy.
These processes are vital and, therefore, proteases must function efficiently to ensure the survival of organisms.
There are many different types in each cell of each organism, and these enzymes work in different regions of the cell, with different specificities and mechanisms of action.
Proteolysis in the Isolation of proteins
Protease activity is highly regulated in the cell, and this regulation occurs through a variety of mechanisms.
An example of regulated protease activation is during the onset of apoptosis or programmed cell death. Activation of caspase and subsequent cleavage of downstream proteins play an important role in apoptosis.
Most cell caspases exist as a procaspase (an inactive version) that must be cleaved proteolytically to exhibit protease activity.
The proteins in the IAP (inhibitor of apoptosis) block the apoptosis of the family either preventing the excision of a procaspase or directly inhibiting the activity of caspase.
The activity of the proteases can also be controlled by their cellular localization. Some proteases are sequestered within specific organelles in such a way that they can only degrade the proteins that are targeted to those same cellular locations.
For example, cathepsins are proteases predominantly located in lysosomes . These proteases degrade proteins that are targeted to lysosomes for degradation.
In intact cells, proteins not located in lysosomes do not come into contact with these particular proteases and, therefore, can not be proteolyzed by cathepsins.
When the cells are lysed, the regulatory mechanisms are interrupted and the organelles can break down. Proteases that normally do not come into contact with most proteins are no longer sequestered.
In addition, the cellular mechanisms of protease inhibition are altered, so that active proteases could cleave a variety of proteins over which they do not act in intact cells.
Due to the large number and type of proteases in organisms commonly used for protein expression, it is easy to imagine that proteins may be subject to extensive proteolysis following cell lysis.
Therefore, it is necessary to take measures to prevent protein proteolysis during its purification, so that the full-length functional protein is isolated for use in experimental studies.
Protease inhibitors are generally used in these types of studies, and the type of inhibitor that should be used depends on the type of protease that needs to be inhibited.
Proteases are characterized in many different ways. Proteases can be classified by their mechanism of action and substrate specificity.
In general terms, proteases can be characterized as endopeptidases or exopeptidases:
- The endopeptidases cleave bonds within the protein and are generally very specific for particular amino acid sequences.
- Exopeptidases cleave a single amino acid from the end of a protein, and are less specific towards the amino acid that is being cleaved.
Exopeptidases classify themselves as those that bind only from the C-terminus (carboxypeptidases), only from the N-terminus (aminopeptidases), or both terminals (dipeptidases).
Both endopeptidases and exopeptidases can be very problematic during protein purification. The endopeptidases can recognize regions in the protein of interest and perform proteolysis to digest the protein in many smaller pieces that are useless in experimental studies.
Exopeptidases can also be very problematic during purification, and the removal of only a few amino acids is often not detectable by common methods.
Proteases are also characterized by their mechanism of action; that is, the amino acids that are involved in the catalytic site of the enzyme.
The catalytic site contains amino acids that directly play a role in facilitating the hydrolysis of the peptide bond. This process requires a nucleophile to initiate the reaction, which may be an active site amino acid side chain or a water molecule.
During the last stages of proteolysis, the two peptides are released and the active site of the protease returns to its initial state, ready to bind to another substrate for proteolysis.
Proteases are typically grouped into four main classes based on their active site residues:
- Serine proteases.
- Cysteine (thiol) proteases.
- Aspartic proteases.
The serine and cysteine (thiol) proteases have an amino acid within the active site that performs the initial nucleophilic attack, while aspartic proteases and metalloproteases activate a water molecule to perform the initial nucleophilic attack.
The serine proteases act through a catalytic triad composed of a serine, a histidine and an aspartate residue.
The cysteine (thiol) proteases contain a catalytic dyad with a histidine and a cysteine residue. The cysteine performs the nucleophilic attack to initiate the hydrolysis of the peptide bond.
Aspartic proteases contain a catalytic dyad with two aspartate residues.
Metalloproteases require a zinc ion (or, more rarely, a different divalent metal ion) that coordinates with the protein for three amino acids, whose identity may vary.
The metal ion activates the water molecule, so that it can perform a nucleophilic attack on the carbonyl carbon to break the peptide bond.
Peptide Bond Specificity
Endoproteases specifically recognize certain amino acids or types of amino acids. Not only are the amino acids that make up the peptide bond important, but also the neighboring residues play a role in the specificity.
This recognition is mediated by pockets of specificity, regions within the protease around the active site that bind some amino acid side chains more favorably than others.
The trypsin-like proteases: predominantly cleave proteins on the carboxyl side of arginine or lysine (except when that residue follows a proline).
Proteases similar to chymotrypsin : they prefer to fragment on the carboxyl side of large aromatic residues (tryptophan, tyrosine or phenylalanine).
Caspase-like proteases : are predominantly separated on the carboxyl side of aspartate, but it has been shown that a caspase in Drosophila is also cleaved on the carboxyl side of glutamate.
Elastase type proteases : are predominantly separated on the carboxyl side of the small aliphatic amino acids (glycine, alanine or valine).
Protease inhibitors are molecules that block the activity of proteases and typically work in classes of proteases with similar mechanisms of action.
The protease inhibitors may be in the form of proteins, peptides or small molecules. Naturally occurring protease inhibitors are usually proteins or peptides.
Protease inhibitors used in experimental studies or drug development are often similar to synthetic or small peptide molecules.
Many different protease inhibitors are commercially available for experimental use in both in vitro and in vivo assays and during protein purification.
Mechanism of inhibition of Protease
Protease inhibitors can work in different ways to inhibit the action of proteases. These inhibitors can be classified by the type of protease they inhibit and the mechanism by which they inhibit the enzyme.
While protease inhibitors are sold commercially according to the class of protease they inhibit, understanding the various mechanisms by which inhibitors work is important for a comprehensive understanding of inhibition and for developing protease inhibitors as a therapeutic strategy.
Reversible inhibitors generally bind to the protease with multiple non-covalent interactions, without any reaction of the inhibitor itself. These inhibitors can be removed by dilution or dialysis.
Reversible inhibitors include competitive inhibitors, non-competitive inhibitors and non-competitive inhibitors. Competitive inhibitors bind to the active site of the protease, competing with substrates for access to residues from the active site.
An example of a competitive inhibitor is aprotinin , which inhibits many serine proteases. Competitive inhibitors often have a structure similar to the transition state of natural substrates.
The transition state of the substrate is the structure that binds most closely with the enzyme. Therefore, compounds that mimic this structure bind to the enzyme with a higher resistance than the substrate (in its initial state) can, and the normal enzymatic reaction can not continue.
An example of this type of transition state analogue is LP-130, an HIV-1 protease inhibitor.
Non-competitive inhibitors bind only to the protease when it is already bound to a substrate. Inhibitors have been identified for HIV-1 protease and NS2B-NS3 proteinase from West Nile virus that function noncompetitively.
Non-competitive inhibitors bind to the protease, with or without bound substrate, with similar affinities, and inhibit the activity of the protease through an allosteric mechanism. BBI, a trypsin inhibitor of soy and aminoglycosides, protease inhibitors of the deadly anthrax factor are not competitive.
Irreversible inhibitors work by specifically altering the active site of their specific target through the formation of covalent bonds.
After binding to the inhibitor, the active site of the protease is altered and can no longer perform the hydrolysis of the peptide bond. Some inhibitors do not bind covalently to the protease, but interact with such high affinity that they are not easily eliminated.
An example of a suicide protease inhibitor is the family of serpin proteins, which play a role in the coagulation and inflammation of the blood. A cycle in Serpin serves as an analogous substrate.
The serine residue in the active site of trypsin forms a nucleophilic attack on a carbonyl carbon of the substrate analog, inducing a conformational change in the enzyme which makes the rest of the hydrolysis reaction of the peptide bond unfavorable.
Therefore, the serpin remains covalently bound to the protease so that the enzyme is no longer available to bind to the substrates.
Types of Protease inhibitors
The protease inhibitors can be purchased individually or as a concentrated cocktail containing multiple protease inhibitors in appropriate relative amounts.
Individual protease inhibitors are ideal for situations where a protease inhibitor is needed for use in proteolytic assays of already purified proteins.
Many times, enzymatic assays require a control to show that the enzyme of interest is performing the supervised action; therefore, adding a specific protease inhibitor can adequately serve as a control.
The purchase of individual protease inhibitors is also ideal under circumstances in which the protein being purified is a protease. In these cases, the presence of functional protein and purity is often evaluated by enzymatic assays.
For this type of purification, multiple protease inhibitors can be added, excluding those that inhibit the protease of interest. Most offer a broad inhibition of one or more classes of proteases.
Protease inhibitor cocktails are often used for their reliability and reproducibility. The cocktails contain multiple proteases in the appropriate relative amounts, eliminating the need for trial and error to determine the types required and the amounts of inhibitors to be used.
They also minimize the possibility of human error and pipetting, by requiring only one solution versus multiple different solutions. These cocktails come in liquid and solid forms.
Roche cOmplete tablets are one of the most commonly used protease inhibitor cocktails. These tablets are added to a specific volume of buffer to inhibit the most abundant proteases.
It has been shown that Roche cOmplete tablets successfully inhibit a wide range of proteases in lysates of many organisms, including E. coli, yeast, insects and mammals.
The yield of full length protein in the absence of protease inhibitors is less than half that obtained with the addition of protease inhibitors.
The protease inhibitors in tablet form are added to an indicated volume of buffer, and the tablets are dissolved to bring the concentration of each protease inhibitor in the buffer to the appropriate levels.
The addition of protease inhibitors in tablet form is very convenient, since pipetting is not required.
In cases where very small volumes of buffer are required, protease inhibitor cocktails in liquid form may be more suitable to avoid waste of excess buffer, since the tablets require the preparation of a certain volume.
Many companies also sell protease inhibitors individually, such as cocktails, and as sets of multiple individual inhibitors. These sets are useful in determining which protease inhibitors are necessary for a given application.
They can also be useful when it is known that a particular protease inhibitor included in most commercial cocktails interferes with the protein of interest or the downstream application.
Protease inhibitors in action
There are many things to keep in mind when choosing the appropriate protease inhibitor (s).
The most important consideration is the purpose of protease inhibition, whether inhibitors are needed to avoid proteolysis during purification of the protein, to inhibit a purified enzyme as an experimental control or for use in living organisms to affect physiological processes.
The following section includes some important questions and concerns to keep in mind when using protease inhibitors.
What protease inhibitor should be used?
The application plays an important role in the type of protease inhibitor that should be used.
For in vitro and in vivo assays, the choice of protease inhibitor depends on the particular enzyme or physiological process under study. For enzymatic assays, the inhibitor must not interfere with the detection method.
Although if the inhibition is reversible, and another detection method is available (eg, elisa), it may not matter if the target protein is inhibited during purification as long as the inhibitor is absent from the final preparation.
It should be noted that some protease inhibitors are not specific for proteases. For example, PMSF irreversibly inhibits many, but not all, members of the serine hydrolase family.
This family, although it includes proteases such as trypsin and chymotrypsin, also includes many other enzymes that hydrolyse non-protein substrates (eg, acetylcholinesterases, acyl-CoA hydrolases and lipases).
For studies in living organisms, the inhibitor must be permeable to cells, highly specific and non-toxic. For the broad inhibition of proteases to help purify the full-length functional protein, multiple proteases are typically required.
How should Protease Inhibitors be prepared and stored?
Many protease inhibitors are unstable for long periods of time, either as a stock solution or in their working concentration.
It is very important to prepare the solutions according to the supplier’s instructions, since some protease inhibitors are more stable than others under certain conditions.
Typically, if stock solutions have been stored as suggested and working solutions were prepared immediately before use, the inhibitors should retain sufficient function.
Some protease inhibitors, such as PMSF, are highly unstable and must be added several times during lysis and purification to ensure inhibition of the protease.
How can the function of Protease Inhibitors be confirmed?
Several companies sell reagents as a measure to verify the function of the protease, and these can be used to confirm adequate inhibition.
Roche sells a universal protease substrate, which uses an absorbance assay to control the degradation of casein labeled with resorufin. This substrate can be used if there is evidence of suspected inadequate inhibition of the protease.
Typically, it is a better use of time and energy simply to make a fresh protease inhibitor solution if it is suspected that the proteases are still functional.
However, if proteolysis is a recurring problem, these commercial substrates can help determine the most appropriate conditions for protease inhibition.
During cell lysis and protein purification, when should Protease Inhibitors be added?
The protease inhibitors should be added to the lysis buffer so that the inhibition can occur immediately after cell lysis, when the proteases are released from their cell compartments and regulation is interrupted.
These protease inhibitors should remain in the early buffers used in the purification scheme until it can be assumed that most of the contaminating proteases have been sufficiently separated from the protein of interest.
Typically, if the first chromatographic step is stringent (eg, affinity chromatography), the protease inhibitors need only be present through the washing step of this chromatographic process.
The elution buffer and the remaining purification buffers usually do not require protease inhibitors.
This is an important financial consideration for large-scale protein purification efforts where large amounts of buffers may be required.
Even for a well-funded industrial laboratory, the inclusion of high concentrations of protease inhibitors throughout large-scale purifications can be prohibitively expensive.
However, many proteases are highly efficient catalysts with high turnover rates and, therefore, if even a small amount of a protease is co-purified with the target protein, it can cause problems.
This may present as an obvious degradation of the target protein or may not be apparent until N-terminal or mass spectroscopic analysis reveals that the purified protein preparation contains a variety of species with different N-terminal ends.
For many applications, such microheterogeneity may not be a problem, while for others it may cause significant problems.
For example, in a structural biology context, such N-terminal heterogeneity may affect the ability to crystallize a protein and / or the quality of any crystal obtained.
Therefore, in some cases it may be necessary to include inhibitors against one or more protease classes in the later stages of the purification protocol.
The use of protease assays (as discussed above) can be used to identify the class of protease to be blocked.
What else can be done to stop Proteolysis?
Cell lysis and purification should be performed at low temperature. Typically, lysis and purification are carried out at 4 ° C. This not only helps with the folding and stability of the proteins, but also slows down the rate of proteolysis by contaminating the proteases.
In addition, the faster contaminating proteases are removed from the protein of interest, the less time they have to interact and possibly degrade the protein. Do not allow cell lysates to sit around, even on ice, for long periods of time.
Continue the purification steps as quickly as possible and store the purified protein appropriately.
If the proteolysis of a recombinant protein is a particular problem, a number of approaches can be considered. For the expression of E. coli, it is possible that the decrease in growth temperature reduces proteolysis of the target protein that occurs before harvest and cell lysis.
It may also be possible to reduce the intracellular proteolysis of the target protein by directing expression to the periplasm and thereby reducing exposure to intracellular proteases.
Similarly, to avoid proteolysis by intracellular mammalian proteases, it may be possible to use an expression system that directs the secretion of the recombinant protein into the culture medium.
Considering the different range of proteases produced by different organisms, it may also be possible to reduce, or even eliminate, proteolysis problems by changing the expression to a different host organism (eg, E. coli to baculovirus / insect cells).
If I do not know which protease inhibitors I should use, where should I start?
The best place to start in choosing the right combination of inhibitors for use during cell lysis and protein purification is to try a cocktail of commonly used inhibitors. For most lysates and applications, these are sufficient.
However, if full-length protein is not obtained or if problems are experienced in subsequent applications, individual protease inhibitors or pools of protease inhibitors may be used to determine the most appropriate combination of protease inhibitors.
Protease inhibitors in clinical practice
The inhibitors were tested in several clinical trials.
In particular, several structural subtypes of tryptase inhibitors (TI) show promising therapeutic effects for lung diseases. The mechanisms of their actions include the covalent attachment of serine residues in the active domains or the binding aspartate and the formation of hydrogen bonds.
It was demonstrated that TI-based benzene sulfenamide effectively suppresses allergic pulmonary reactions, such as acute bronchospasm, in animal models.
TIs containing dipeptides were effective in reducing both asthma-mediated pathology and liver fibrosis.
In addition, guanidino-based IT was tested as an additional treatment for ulcerative colitis. In addition, TIS containing beta-lactam can be used to decrease lung inflammation.
With respect to cardiovascular diseases, subtilsin-kexin convertase type 9 suppressants were shown to decrease low density lipoproteins and were approved for the treatment of patients with severe hyperlipidemia.
In addition, dipeptidyl peptidase-4 (DPP-4) suppressors can effectively lower glucose levels and are used for the treatment of patients with type 2 diabetes.
In addition, it was shown that the DPP-4 inhibitor linagliptin suppresses the development of diabetic retinopathy in an experimental model.
In addition, inhibitors of cathepsin K cysteine protease increase bone mineral density in individuals with postmenopausal osteoporosis. In addition, the sudator cathepsin K odanacatib decreased bone resorption in male and female patients with osteoporosis.