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During the protein translation process, it is composed of individual amino acids linked by a covalent bond, called a peptide bond.
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, an enzyme whose mechanism depends on the particular protease class.
These perform many vital 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 other 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 essential 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.
Their cellular localization can also control the activity of the proteases. Some proteases are sequestered within specific organelles so that they can only degrade the proteins targeted to those exact 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 generally 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 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 kind of Protease that needs to be inhibited.
Protease Classification
Proteases are characterized in many different ways. Proteases can be classified by their mechanism of action and substrate specificity.
General ranking
In general terms, proteases can be characterized as endopeptidases or exopeptidases:
- The endopeptidases cleave proteins’ bonds and are 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 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 standard methods.
Proteolysis Mechanism
Proteases are also characterized by their mechanism of action: the amino acids involved in the enzyme’s catalytic site.
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. 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.
- Metaloprotezas.
The serine and cysteine (thiol) proteases have an amino acid that performs the initial nucleophilic attack within the active site. In contrast, aspartic proteases and metalloproteases activate a water molecule to complete 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 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 to 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 necessary, 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 are similar to chymotrypsin: they prefer to fragment on the carboxyl side of significant aromatic residues (tryptophan, tyrosine, or phenylalanine).
Caspase-like proteases: are predominantly separated on the carboxyl side of aspartate. Still, 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
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 practical use in vitro and in vivo assays and 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 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 essential 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), and the normal enzymatic reaction can not continue.
An example of this type of transition state analog is LP-130, an HIV-1 protease inhibitor.
Non-competitive inhibitors bind only to the Protease when it is already attached to a substrate. Inhibitors have been identified for HIV-1 Protease and NS2B-NS3 proteinase from West Nile virus that functions non-competitively.
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, and the deadly anthrax factor protease inhibitors are not competitive.
Irreversible inhibitors work by explicitly 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 attach to the substrates.
Types of Protease inhibitors
The protease inhibitors can be purchased individually or as a concentrated cocktail containing multiple protease inhibitors inappropriate relative amounts.
Individual protease inhibitors are ideal for situations where a protease inhibitor is needed in proteolytic assays of already purified proteins.
Enzymatic assays often require 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 trial and error to determine the required types and the inhibitors’ quantities.
They also minimize the possibility of human error and pipetting by requiring only one solution versus multiple different answers. 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 buffer volume to inhibit the most abundant proteases.
It has been shown that Roche cOmplete tablets successfully inhibit a wide range of proteases in the lysates of many organisms, including E. coli, yeast, insects, and mammals.
The yield of complete 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. 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 tiny 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 specific volume.
Many companies also sell protease inhibitors individually, such as cocktails and assets of multiple individual inhibitors. These sets help determine which protease inhibitors are necessary for a given application.
They can also be helpful 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
When choosing the appropriate protease inhibitor (s), many things to consider.
The most important consideration is the purpose of protease inhibition, whether inhibitors are needed to avoid Proteolysis during purification of the protein, inhibit a purified enzyme as an experimental control, or 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 a vital role in the type of protease inhibitor 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 (e.g., 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.
Although it includes proteases such as trypsin and chymotrypsin, this family also has many other enzymes that hydrolyze non-protein substrates (e.g., acetylcholinesterases, acyl-CoA hydrolases, and lipases).
The inhibitor must be permeable to cells for studies in living organisms, particular and non-toxic. Multiple proteases are typically required for broad inhibition of proteases to help purify the full-length functional protein.
How should Protease Inhibitors be prepared and stored?
Many protease inhibitors are unstable for long periods, either as a stock solution or their working concentration.
It is essential to prepare the solutions according to the supplier’s instructions since some protease inhibitors are more stable than others under certain conditions.
Typically, the inhibitors should retain sufficient function if stock solutions have been stored as suggested and working solutions were prepared immediately before use.
Some protease inhibitors, such as PMSF, are volatile 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 to make a new 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. 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 (e.g., 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 do not require protease inhibitors.
This is a significant financial consideration for large-scale protein purification efforts where large buffers may be required.
Even for a well-funded industrial laboratory, including high concentrations of protease inhibitors throughout large-scale purifications can be prohibitively expensive.
However, many proteases are highly efficient catalysts with high turnover rates. Therefore, if even a tiny amount of a protease is co-purified with the target protein, it can cause problems.
This may present as a noticeable 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.
Such microheterogeneity may not be a problem for many applications, while it may cause significant issues for others.
For example, in a structural biology context, such N-terminal heterogeneity may affect the ability to crystallize a protein and 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 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 temperatures. Typically, lysis and purification are carried out at 4 ° C. This helps with the folding and stability of the proteins and 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.
Continue the purification steps as quickly as possible and store the purified protein appropriately.
Several approaches can be considered if the Proteolysis of a recombinant protein is a particular problem. For the expression of E. coli, the decrease in growth temperature may reduce the Proteolysis of the target protein 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 mammalian intracellular 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 other organisms, it may also be possible to reduce, or even eliminate, proteolysis problems by changing the expression to a different host organism (e.g., 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 the full-length protein is not obtained or 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.
In animal models, it was demonstrated that Ti-based benzene sulfenamide effectively suppresses pulmonary allergic reactions, such as acute bronchospasm.
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.
Concerning cardiovascular diseases, subtilisin-Kexin convertase type 9 suppressants were shown to decrease low-density lipoproteins and were approved to treat patients with severe hyperlipidemia.
In addition, dipeptidyl peptidase-4 (DPP-4) suppressors can effectively lower glucose levels and are used to treat 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, cathepsin K cysteine protease inhibitors increase bone mineral density in individuals with postmenopausal osteoporosis. In addition, the auditor cathepsin K odanacatib decreased bone resorption in male and female patients with osteoporosis.
