A calpain (; , ) is a protein belonging to the family of

calcium Calcium is a chemical element with the symbol Ca and atomic number 20. As an alkaline earth metal, calcium is a reactive metal that forms a dark oxide-nitride layer when exposed to air. Its physical and chemical properties are most similar ...

-dependent, non-lysosomal

cysteine protease Cysteine proteases, also known as thiol proteases, are hydrolase enzymes that degrade proteins. These proteases share a common catalytic mechanism that involves a nucleophilic cysteine thiol in a catalytic triad or dyad. Discovered by Gopal ...

s ( proteolytic enzymes) expressed ubiquitously in mammals and many other organisms. Calpains constitute the C2 family of protease clan CA in the

MEROPS MEROPS is an online database for peptidases (also known as proteases, proteinases and proteolytic enzymes) and their inhibitors. The classification scheme for peptidases was published by Rawlings & Barrett in 1993, and that for protein inhibitors ...

database. The calpain proteolytic system includes the calpain proteases, the small regulatory subunit CAPNS1, also known as CAPN4, and the endogenous calpain-specific inhibitor, calpastatin.

Discovery

The history of calpain's discovery originates in 1964, when calcium-dependent proteolytic activities caused by a "calcium-activated neutral protease" (CANP) were detected in

brain A brain is an organ (biology), organ that serves as the center of the nervous system in all vertebrate and most invertebrate animals. It is located in the head, usually close to the sensory organs for senses such as Visual perception, vision. I ...

, lens of the eye and other tissues. In the late 1960s the enzymes were isolated and characterised independently in both rat brain and

skeletal muscle Skeletal muscles (commonly referred to as muscles) are organs of the vertebrate muscular system and typically are attached by tendons to bones of a skeleton. The muscle cells of skeletal muscles are much longer than in the other types of m ...

. These activities were caused by an intracellular cysteine protease not associated with the

lysosome A lysosome () is a membrane-bound organelle found in many animal cells. They are spherical vesicles that contain hydrolytic enzymes that can break down many kinds of biomolecules. A lysosome has a specific composition, of both its membrane p ...

and having an optimum activity at neutral pH, which clearly distinguished it from the cathepsin family of proteases. The calcium-dependent activity, intracellular localization, and the limited, specific

proteolysis Proteolysis is the breakdown of proteins into smaller polypeptides or amino acids. Uncatalysed, the hydrolysis of peptide bonds is extremely slow, taking hundreds of years. Proteolysis is typically catalysed by cellular enzymes called protease ...

on its substrates, highlighted calpain’s role as a regulatory, rather than a digestive, protease. When the sequence of this enzyme became known, it was given the name "calpain", to recognize its common properties with two well-known proteins at the time, the calcium-regulated signalling protein,

calmodulin Calmodulin (CaM) (an abbreviation for calcium-modulated protein) is a multifunctional intermediate calcium-binding messenger protein expressed in all eukaryotic cells. It is an intracellular target of the secondary messenger Ca2+, and the bin ...

, and the cysteine protease of

papaya The papaya (, ), papaw, () or pawpaw () is the plant species ''Carica papaya'', one of the 21 accepted species in the genus '' Carica'' of the family Caricaceae. It was first domesticated in Mesoamerica, within modern-day southern Mexico and ...

papain Papain, also known as papaya proteinase I, is a cysteine protease () enzyme present in papaya (''Carica papaya'') and mountain papaya (''Vasconcellea cundinamarcensis''). It is the namesake member of the papain-like protease family. It has wide ...

. Shortly thereafter, the activity was found to be attributable to two main isoforms, dubbed μ ("mu")-calpain and m-calpain (or calpain I and II), that differed primarily in their calcium requirements ''in vitro''. Their names reflect the fact that they are activated by micro- and nearly millimolar concentrations of Ca²⁺ within the cell, respectively. To date, these two isoforms remain the best characterised members of the calpain family. Structurally, these two

heterodimeric In biochemistry, a protein dimer is a macromolecular complex formed by two protein monomers, or single proteins, which are usually non-covalently bound. Many macromolecules, such as proteins or nucleic acids, form dimers. The word ''dimer'' has ...

isoforms share an identical small (28 kDa) subunit ( CAPNS1 (formerly CAPN4)), but have distinct large (80 kDa) subunits, known as calpain 1 and calpain 2 (each encoded by the ''

CAPN1 Calpain-1 catalytic subunit (CANP 1) is a protein that in humans is encoded by the ''CAPN1'' gene. Function The calpains, calcium-activated neutral proteases, are nonlysosomal, intracellular cysteine proteases. The mammalian calpains include ...

'' and '' CAPN2'' genes, respectively).

Cleavage specificity

No specific

amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha ...

sequence is uniquely recognized by calpains. Amongst protein substrates,

tertiary structure Protein tertiary structure is the three dimensional shape of a protein. The tertiary structure will have a single polypeptide chain "backbone" with one or more protein secondary structures, the protein domains. Amino acid side chains may i ...

elements rather than primary amino acid sequences are likely responsible for directing cleavage to a specific substrate. Amongst

peptide Peptides (, ) are short chains of amino acids linked by peptide bonds. Long chains of amino acids are called proteins. Chains of fewer than twenty amino acids are called oligopeptides, and include dipeptides, tripeptides, and tetrapeptides. ...

and small-molecule substrates, the most consistently reported specificity is for small,

hydrophobic In chemistry, hydrophobicity is the physical property of a molecule that is seemingly repelled from a mass of water (known as a hydrophobe). In contrast, hydrophiles are attracted to water. Hydrophobic molecules tend to be nonpolar and, ...

amino acids (e.g.

leucine Leucine (symbol Leu or L) is an essential amino acid that is used in the biosynthesis of proteins. Leucine is an α-amino acid, meaning it contains an α- amino group (which is in the protonated −NH3+ form under biological conditions), an α- ...

valine Valine (symbol Val or V) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α- amino group (which is in the protonated −NH3+ form under biological conditions), an α- carboxylic acid group (which is in the deprotona ...

and

isoleucine Isoleucine (symbol Ile or I) is an α-amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated −NH form under biological conditions), an α-carboxylic acid group (which is in the depr ...

) at the P2 position, and large hydrophobic amino acids (e.g.

phenylalanine Phenylalanine (symbol Phe or F) is an essential α-amino acid with the formula . It can be viewed as a benzyl group substituted for the methyl group of alanine, or a phenyl group in place of a terminal hydrogen of alanine. This essential amin ...

and

tyrosine -Tyrosine or tyrosine (symbol Tyr or Y) or 4-hydroxyphenylalanine is one of the 20 standard amino acids that are used by cells to synthesize proteins. It is a non-essential amino acid with a polar side group. The word "tyrosine" is from the G ...

) at the P1 position. Arguably, the best currently available fluorogenic calpain substrate is ( EDANS)-Glu-Pro-Leu-Phe=Ala-Glu-Arg-Lys-( DABCYL), with cleavage occurring at the Phe=Ala bond.

Extended family

The Human Genome Project has revealed that more than a dozen other calpain

isoform A protein isoform, or "protein variant", is a member of a set of highly similar proteins that originate from a single gene or gene family and are the result of genetic differences. While many perform the same or similar biological roles, some is ...

s exist, some with multiple splice variants. As the first calpain whose three-dimensional structure was determined, m-calpain is the type-protease for the C2 (calpain) family in the

database.

Function

Although the physiological role of calpains is still poorly understood, they have been shown to be active participants in processes such as cell mobility and

cell cycle The cell cycle, or cell-division cycle, is the series of events that take place in a cell that cause it to divide into two daughter cells. These events include the duplication of its DNA (DNA replication) and some of its organelles, and sub ...

progression, as well as cell-type specific functions such as

long-term potentiation In neuroscience, long-term potentiation (LTP) is a persistent strengthening of synapses based on recent patterns of activity. These are patterns of synaptic activity that produce a long-lasting increase in signal transmission between two neurons ...

neuron A neuron, neurone, or nerve cell is an electrically excitable cell that communicates with other cells via specialized connections called synapses. The neuron is the main component of nervous tissue in all animals except sponges and placozoa ...

s and

cell fusion Cell fusion is an important cellular process in which several uninucleate cells (cells with a single nucleus) combine to form a multinucleate cell, known as a syncytium. Cell fusion occurs during differentiation of myoblasts, osteoclasts and tropho ...

myoblast Myogenesis is the formation of skeletal muscular tissue, particularly during embryonic development. Muscle fibers generally form through the fusion of precursor myoblasts into multinucleated fibers called ''myotubes''. In the early development o ...

s. Under these physiological conditions, a transient and localized influx of calcium into the cell activates a small local population of calpains (for example, those close to Ca²⁺ channels), which then advance the signal transduction pathway by catalyzing the controlled proteolysis of its target proteins. Additionally, phosphorylation by

protein kinase A In cell biology, protein kinase A (PKA) is a family of enzymes whose activity is dependent on cellular levels of cyclic AMP (cAMP). PKA is also known as cAMP-dependent protein kinase (). PKA has several functions in the cell, including regulatio ...

and dephosphorylation by

alkaline phosphatase The enzyme alkaline phosphatase (EC 3.1.3.1, alkaline phosphomonoesterase; phosphomonoesterase; glycerophosphatase; alkaline phosphohydrolase; alkaline phenyl phosphatase; orthophosphoric-monoester phosphohydrolase (alkaline optimum), systematic ...

have been found to positively regulate the activity of μ-calpains by increasing random coils and decreasing β-sheets in its structure. Phosphorylation improves proteolytic activity and stimulates auto-activation of μ-calpains. However, increased calcium concentration overruns the effects of phosphorylation and dephosphorylation on calpain activity, and thus calpain activity ultimately depends on the presence of calcium. Other reported roles of calpains are in

cell Cell most often refers to: * Cell (biology), the functional basic unit of life Cell may also refer to: Locations * Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery ...

function, helping to regulate

clotting Coagulation, also known as clotting, is the process by which blood changes from a liquid to a gel, forming a blood clot. It potentially results in hemostasis, the cessation of blood loss from a damaged vessel, followed by repair. The mechanism o ...

and the diameter of

blood vessel The blood vessels are the components of the circulatory system that transport blood throughout the human body. These vessels transport blood cells, nutrients, and oxygen to the tissues of the body. They also take waste and carbon dioxide awa ...

s, and playing a role in

memory Memory is the faculty of the mind by which data or information is encoded, stored, and retrieved when needed. It is the retention of information over time for the purpose of influencing future action. If past events could not be remember ...

. Calpains have been implicated in apoptotic cell death, and appear to be an essential component of

necrosis Necrosis () is a form of cell injury which results in the premature death of cells in living tissue by autolysis. Necrosis is caused by factors external to the cell or tissue, such as infection, or trauma which result in the unregulated diges ...

. Detergent fractionation revealed the cytosolic localization of calpain. Enhanced calpain activity, regulated by CAPNS1, significantly contributes to platelet hyperreactivity under hypoxic environment. In the brain, while μ-calpain is mainly located in the

cell body The soma (pl. ''somata'' or ''somas''), perikaryon (pl. ''perikarya''), neurocyton, or cell body is the bulbous, non-process portion of a neuron or other brain cell type, containing the cell nucleus. The word 'soma' comes from the Greek '' σῶμ ...

and

dendrite Dendrites (from Greek δένδρον ''déndron'', "tree"), also dendrons, are branched protoplasmic extensions of a nerve cell that propagate the electrochemical stimulation received from other neural cells to the cell body, or soma, of the ...

s of

s and to a lesser extent in

axon An axon (from Greek ἄξων ''áxōn'', axis), or nerve fiber (or nerve fibre: see spelling differences), is a long, slender projection of a nerve cell, or neuron, in vertebrates, that typically conducts electrical impulses known as action p ...

s and

glial cell Glia, also called glial cells (gliocytes) or neuroglia, are non-neuronal cells in the central nervous system (brain and spinal cord) and the peripheral nervous system that do not produce electrical impulses. They maintain homeostasis, form myel ...

s, m-calpain is found in glia and a small number in axons. Calpain is also involved in skeletal muscle protein breakdown due to exercise and altered nutritional states.

Clinical significance

Pathology

The structural and functional diversity of calpains in the cell is reflected in their involvement in the pathogenesis of a wide range of disorders. At least two well known genetic disorders and one form of cancer have been linked to tissue-specific calpains. When defective, the mammalian calpain 3 (also known as p94) is the gene product responsible for limb-girdle muscular dystrophy type 2A, calpain 10 has been identified as a susceptibility gene for type II diabetes mellitus, and calpain 9 has been identified as a tumour suppressor for gastric cancer. Moreover, the hyperactivation of calpains is implicated in a number of pathologies associated with altered calcium homeostasis such as

Alzheimer's disease Alzheimer's disease (AD) is a neurodegenerative disease that usually starts slowly and progressively worsens. It is the cause of 60–70% of cases of dementia. The most common early symptom is difficulty in remembering recent events. As ...

, and

cataract A cataract is a cloudy area in the lens of the eye that leads to a decrease in vision. Cataracts often develop slowly and can affect one or both eyes. Symptoms may include faded colors, blurry or double vision, halos around light, trouble ...

formation, as well as secondary degeneration resulting from acute cellular stress following myocardial ischemia, cerebral (neuronal) ischemia, traumatic brain injury and spinal cord injury. Excessive amounts of calpain can be activated due to Ca²⁺ influx after

cerebrovascular accident A stroke is a medical condition in which poor blood flow to the brain causes cell death. There are two main types of stroke: ischemic, due to lack of blood flow, and hemorrhagic, due to bleeding. Both cause parts of the brain to stop funct ...

(during the

ischemic cascade The ischemic (ischaemic) cascade is a series of biochemical reactions that are initiated in the brain and other aerobic tissues after seconds to minutes of ischemia (inadequate blood supply). This is typically secondary to stroke, injury, or cardi ...

) or some types of

traumatic brain injury A traumatic brain injury (TBI), also known as an intracranial injury, is an injury to the brain caused by an external force. TBI can be classified based on severity (ranging from mild traumatic brain injury TBI/concussionto severe traumatic br ...

such as diffuse axonal injury. Increase in concentration of calcium in the cell results in calpain activation, which leads to unregulated proteolysis of both target and non-target proteins and consequent irreversible tissue damage. Excessively active calpain breaks down molecules in the

cytoskeleton The cytoskeleton is a complex, dynamic network of interlinking protein filaments present in the cytoplasm of all cells, including those of bacteria and archaea. In eukaryotes, it extends from the cell nucleus to the cell membrane and is co ...

such as spectrin,

microtubule Microtubules are polymers of tubulin that form part of the cytoskeleton and provide structure and shape to eukaryotic cells. Microtubules can be as long as 50 micrometres, as wide as 23 to 27 nm and have an inner diameter between 1 ...

subunits,

microtubule-associated protein In cell biology, microtubule-associated proteins (MAPs) are proteins that interact with the microtubules of the cellular cytoskeleton. MAPs are integral to: the stability of the cell and its internal structures and the transport of components within ...

s, and

neurofilament Neurofilaments (NF) are classed as type IV intermediate filaments found in the cytoplasm of neurons. They are protein polymers measuring 10 nm in diameter and many micrometers in length. Together with microtubules (~25 nm) and mi ...

s. It may also damage

ion channel Ion channels are pore-forming membrane proteins that allow ions to pass through the channel pore. Their functions include establishing a resting membrane potential, shaping action potentials and other electrical signals by gating the flow of ...

s, other enzymes,

cell adhesion molecule Cell adhesion molecules (CAMs) are a subset of cell surface proteins that are involved in the binding of cells with other cells or with the extracellular matrix (ECM), in a process called cell adhesion. In essence, CAMs help cells stick to each ...

s, and

cell surface receptors Cell surface receptors (membrane receptors, transmembrane receptors) are receptors that are embedded in the plasma membrane of cells. They act in cell signaling by receiving (binding to) extracellular molecules. They are specialized integral m ...

. This can lead to degradation of the cytoskeleton and

plasma membrane The cell membrane (also known as the plasma membrane (PM) or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of all cells from the outside environment (t ...

. Calpain may also break down

sodium channel Sodium channels are integral membrane proteins that form ion channels, conducting sodium ions (Na+) through a cell's membrane. They belong to the superfamily of cation channels and can be classified according to the trigger that opens the chan ...

s that have been damaged due to axonal stretch injury, leading to an influx of

sodium Sodium is a chemical element with the symbol Na (from Latin ''natrium'') and atomic number 11. It is a soft, silvery-white, highly reactive metal. Sodium is an alkali metal, being in group 1 of the periodic table. Its only stable ...

into the cell. This, in turn, leads to the neuron's

depolarization In biology, depolarization or hypopolarization is a change within a cell, during which the cell undergoes a shift in electric charge distribution, resulting in less negative charge inside the cell compared to the outside. Depolarization is ess ...

and the influx of more Ca²⁺. A significant consequence of calpain activation is the development of cardiac contractile dysfunction that follows ischemic insult to the heart. Upon reperfusion of the ischemic myocardium, there is development of calcium overload or excess in the heart cell (cardiomyocytes). This increase in calcium leads to activation of calpain. Recently calpain has been implicated in promoting high altitude induced venous thrombosis by mediating platelet hyperactivation.

Therapeutic inhibitors

The exogenous regulation of calpain activity is therefore of interest for the development of therapeutics in a wide array of pathological states. As a few of the many examples supporting the therapeutic potential of calpain inhibition in ischemia, calpain inhibitor AK275 protected against focal ischemic brain damage in rats when administered after ischemia, and MDL28170 significantly reduced the size of damaged infarct tissue in a rat focal ischemia model. Also, calpain inhibitors are known to have neuroprotective effects: PD150606, SJA6017, ABT-705253, and SNJ-1945. Calpain may be released in the brain for up to a month after a head injury, and may be responsible for a shrinkage of the brain sometimes found after such injuries. However, calpain may also be involved in a "resculpting" process that helps repair damage after injury.

References

-animation. {{Portal bar, Biology, border=no EC 3.4.22 Proteases Protein families Penta-EF-hand proteins