09/23/1440 1 By: Fateme zarei PhD student of animal physiology calpain system & MUSCLE Sunday 26 May 2019 15-16 PM Department of animal science Skeletal Muscle The Three Connective Tissue Layers. Bundles of muscle fibers, called fascicles, are covered by the perimysium. Muscle fibers are covered by the endomysium. 1 SKELETAL MUSCLE FIBERS A skeletal muscle fiber is surrounded by a plasma membrane called the sarcolemma, which contains sarcoplasm, the cytoplasm of muscle cells. A muscle fiber is composed of many fibrils, which give the cell its striated appearance. 2 THE SARCOMERE The sarcomere, the region from one Z-line to the next Z-line, is the functional unit of a skeletal muscle fiber. 3 EXCITATION-CONTRACTION COUPLING Motor End-Plate and Innervation. At the NMJ, the axon terminal releases ACh. The motor end-plate is the location of the ACh-receptors in the muscle fiber sarcolemma. When ACh molecules are released, they diffuse across a minute space called the synaptic cleft and bind to the receptors. 4 Muscle Fiber Contraction A cross-bridge forms between actin and the myosin heads triggering contraction. As long as Ca++ ions remain in the sarcoplasm to bind to troponin, and as long as ATP is available, the muscle fiber will continue to shorten. 5
Transcript
09/23/1440
1
By:Fateme zarei
PhD student of animal physiology
calpain system&
MUSCLE
Sunday 26 May 2019 15-16 PM
Department of animal science
Skeletal Muscle
The Three Connective Tissue Layers. Bundles of muscle fibers, called fascicles, are covered by the perimysium. Muscle fibers are covered by the endomysium.
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SKELETAL MUSCLE FIBERS
A skeletal muscle fiber is surrounded by a plasma membrane called the sarcolemma, which contains sarcoplasm, the cytoplasm of muscle cells. A muscle fiber is composed of many fibrils, which give the cell its striated appearance.
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THE SARCOMEREThe sarcomere, the region from one Z-line to the next Z-line, is the functional unit of a skeletal muscle fiber.
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EXCITATION-CONTRACTION COUPLING
Motor End-Plate and Innervation. At the NMJ, the axon terminal releases ACh. The motor end-plate is the location of the ACh-receptors in the muscle fiber sarcolemma. When ACh molecules are released, they diffuse across a minute space called the synaptic cleft and bind to the receptors.
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Muscle Fiber Contraction
A cross-bridge forms between actinand the myosin heads triggeringcontraction. As long as Ca++ ionsremain in the sarcoplasm to bind totroponin, and as long as ATP isavailable, the muscle fiber willcontinue to shorten.
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Ca++ ions are pumped back into the SR,which causes the tropomyosin to reshieldthe binding sites on the actin strands. Amuscle may also stop contracting when itruns out of ATP and becomes fatigued.
Muscle Fiber Relaxation
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THE SLIDING FILAMENT MODEL OF CONTRACTION
When a sarcomere contracts, the Z lines move closer together, and the I band becomes smaller. The A band stays the same width. At full contraction, the thin and thick filaments overlap.
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The conversion of muscle into meat
After slaughter, the muscle undergoes various biophysico-chemical changes and events that converts it into meat.
This process can be divided into three phases:
1- the pre-rigor phase during which collagen content mainly contributes to the toughness,
2- rigor phase during which further toughening occurs due to muscle shortening,
3- tenderization phase or resolution of rigor during aging during which the muscles undergo a series of changes and observe a remarkable improvement in tenderness
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SOURCES OF ATP IN MUSCLE
• Muscle Metabolism. (a) Some ATP is stored in aresting muscle. As contraction starts, it is used upin seconds. More ATP is generated from creatinephosphate for about 15 seconds. (b) Eachglucose molecule produces two ATP and twomolecules of pyruvic acid, which can be used inaerobic respiration or converted to lactic acid. Ifoxygen is not available, pyruvic acid is convertedto lactic acid, which may contribute to musclefatigue. This occurs during strenuous exercisewhen high amounts of energy are needed butoxygen cannot be sufficiently delivered tomuscle. (c) Aerobic respiration is the breakdownof glucose in the presence of oxygen (O2) toproduce carbon dioxide, water, and ATP.Approximately 95 percent of the ATP required forresting or moderately active muscles is providedby aerobic respiration, which takes place inmitochondria.
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ATP AND MUSCLE CONTRACTION
Skeletal Muscle Contraction. (a) The active siteon actin is exposed as calcium binds totroponin. (b) The myosin head is attracted toactin, and myosin binds actin at its actin-bindingsite, forming the cross-bridge. (c) During thepower stroke, the phosphate generated in theprevious contraction cycle is released. Thisresults in the myosin head pivoting toward thecenter of the sarcomere, after which theattached ADP and phosphate group arereleased. (d) A new molecule of ATP attaches tothe myosin head, causing the cross-bridge todetach. (e) The myosin head hydrolyzes ATP toADP and phosphate, which returns the myosinto the cocked position.
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Muscle Extensibility and Rigor Mortis
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• Tenderness of meat is the most important quality distinguishing feature of meat in consumer evaluation.
• Various pre-slaughter and post-slaughter factors and their mutual effects influence tenderness of meat.
• The most important pre-slaughter factors include:
• Post-slaughter transformations, including:
rigor mortis
ageing
meat tenderness
AgeSpeciesSexBreedfeeding of animalsdegree of stress prior to slaughter
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Aging
• It was observed that in the ageing process one can distinguish changes in the:
1- ultrastructure of muscle fibers:
- weakenin of myofibrils
- fragmentation
- changes in the area of the Z-line and the I-band
2- degradation of myofibrillar and cytoskeletal proteins:
These changes lead to obtaining the final meat tenderness.
• T troponins
• I troponins
• titins
• desmins
• dystrophins
• nebulins
• Vinculins
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Factors affecting the tenderness during aging
1-Temperature
2-Time of aging
3- pH
4- Fiber-type composition
5- Sarcomere length
6- Proteolysis
Aging
Post-mortem aging results in optimum improvements in the tenderness of meat, however, it does not ensure uniformity in the tenderness as it is influenced by several genetic and environmental factors.
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proteolytic systems present in a muscle which can participate in the postmortem proteolysis and tenderization:
• calpains
• cathepsins
• Proteasomes
• The caspase system
Post-mortem proteolysis and candidate proteolytic systems
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Major intracellular proteolytic systems
The autophagy-lysosome system primarily degrades
nonspecific cell components, including proteins and microorganisms, contained by isolation membranes.
Cathepsins are a group of enzymes comprised of both exo-and endo-peptidases.
categorised into peptidase families:- cysteine (cathepsins B, H, L and X), - aspartic (cathepsins D and E)- serine (cathepsins G)
Lysosome system
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(i) specific receptor-mediated endocytosis
(ii) pinocytosis (non-specific engulfment of cytosolic
droplets containing extracellular fluid)
(iii) phagocytosis (of extracellular particles)
(iv) autophagy (micro- and macro-; of intracellular
proteins and organelles) (with permission from Nature
Publishing Group. Published originally in ref
The four digestive processes mediated by the lysosome:
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Major intracellular proteolytic systems
Proteasomes
- The proteasome is a multicatalytic protease complex.- Proteasomes are ubiquitously expressed in the body and are abundant in skeletal muscle.
- The proteasome (26S) consists of :19S regulatory subunit 20S multicatalytic structure
- The 20S proteasome, also known as the multicatalytic proteinase complex (MCP),
- Proteolysis by the proteasome is an ubiquitin dependent process, - at least four ubiquitin proteins must attach to the lysine residue of the target substra.te. - The polyubiquitinated proteins are subsequently recognised by the proteasome,
which removes the ubiquitin chain and degrades the substrate.
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The ubiquitin–proteasome proteolytic system
Ubiquitin is activated by the ubiquitin activating enzyme, E1 (1) followed by its transfer to a ubiquitin-carrier protein (ubiquitin-conjugating enzyme, UBC), E2 (2). E2 transfers the activated ubiquitin moieties to the protein substrate that is bound specifically to a unique ubiquitin ligase E3 (A and B). In the case of RING finger ligases, the transfer is direct (A3). Successive conjugation of ubiquitin moieties to one another generates a polyubiquitin chain (A4) that serves as the binding (A5) signal for the downstream 26S proteasome that degrades the target substrates to peptides (A6). In the case of HECT domain ligases, ubiquitin generates an additional thiol-ester intermediate on the ligase (B3), and only then is transferred to the substrate (B4). Successive conjugation of ubiquitin moieties to one another generates a polyubiquitin chain (B5) that binds to the 26S proteasome (B6) followed by degradation of the substrate to peptides (B7). Free and reusable ubiquitin is released by de ubiquitinating enzymes (DUBs). 19
The calpain system
Calpains differ from other major intracellular
proteolytic components such as proteasomes
and lysosomal proteases functioning in
autophagy; these systems eliminate and recycle
their substrates by degradation. Calpains act by
proteolytic processing, as in the activation of
conventional protein kinase C (PKC).
Calpains are unique in that they directly
recognize substrates, whereas proteasomes and
autophagy rely on other systems ubiquitylation
and autophagosome formation, respectively to
tag their substrates.
The calpain system is the most extensively studied enzyme system involved in meat tenderization
CAPNs act in a limited proteolysis and play a critical role in proteolyticmodulating and processing rather than degradation. Therefore, CAPNs areidentified as the intracellular “modulator” proteases
Major intracellular proteolytic systems
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Comparison of calpain and other intracellular proteolytic systems
proteolysis There are three representative intracellular proteolytic systems, autophagy lysosome system, ubiquitin-proteasome system, and calpain-calpastatin system. In each system, proteases functions rather differently:1- lysosome proteases such as cathepsins randomly and completely degrade substrates to amino acid levels; 2- proteasomes regularly (but not specifically) degrade substrates to 8-12mer oligopeptides;3- calpains proteolyze substrates mainly at the inter domain polypeptide chain. Therefore, unlike substrates degraded by lysosomal proteases or proteasomes, those proteolyzed by calpains are functional in most cases.
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Three known proteolytic systems implicated in muscle atrophy:
The calcium-dependent calpain system (A),
The lysosomal protease system (cathepsins; B),
The ubiquitin (Ub; C)-proteasome system.
Major intracellular proteolytic systems
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• They are a large family of non-lysosomal cysteine proteases which are present in almost all eukaryotes and a few bacteria.
• the first calpain discovered and purified by Dayton et al in 1976 was calpain 2.
• Neutral proteases activated by Ca+2 ions, called calpains, occur universally in animal cells.
• They are unusual proteases in that they require calcium for their activity.
• They are intracellular proteinases with optimum activity at neutral pH.
• The system comprises several isoforms of the proteolytic enzyme calpain and their endogenous inhibitor, calpastatin.
• In mammals, there are 14 large subunit members, one small subunit member, and one endogenous inhibitor.
The calpain systemCalpain family
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calpains, which are classified by two criteria: structure and distribution.
By structure:
Classical and non-classical calpains: (Typical and Atypical calpains)
1-Classical calpains:
Typical calpains contain a penta-EF hand in domain IV that can bind Ca2+, the calpain small subunit (only calpains 1, 2, and 9 have been shown to dimerize), or calpastatin.
The nine human classical calpains CAPN1–3, 8, 9, and 11–14
2- non-classical calpains:
Atypical calpains (5, 6, 7, 10, 13, and 15) lack a penta-EF hand in domain IV and are unable to bind the calpain small subunit or calpastatin.
The calpain system
Calpain homologues
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By distribution:Ubiquitous and tissue-specific calpains:
1- tissue-specific calpains:
Six human calpain genes are tissue-specific;
• calpain 3 (skeletal muscle)
• calpain 6 (placenta)
• calpain 8 (smooth muscle)
• calpain 9 (stomach)
• calpain 11 (testes)
• calpain 12 (skin after birth)
• calpain 13 (testes and lung)
2- Ubiquitous
The calpain system
Calpain homologues
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ubiquitous calpains have basic roles in the cell,
tissue-specific calpains are involved in specific cell functions.
• Accordingly, defects in ubiquitous calpains can be lethal,
• whereas defects in tissue-specific calpains may cause tissue-specific phenotypes, such as the:
• muscular dystrophy caused by CAPN3 mutations
• cardiomyopathy
• traumatic ischaemia
The calpain system
Calpain homologues
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The calpain system
Schematic structures of calpain superfamily members.
PC1 (protease core domain 1), PC2 (protease core domain 2), CBSW(calpain-type beta-sandwich), PEF (penta-EF-hand) etc. Symbols: N, N-terminal region; PEF(L) and PEF(S), PEF domains of large catalytic and small regulatory subunits, respectively; GR, glycine-rich hydrophobic domain
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The calpain systemPhylogenetic tree and schematic structures of human calpains.
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The calpain system
Deregulation of its activity has been implicated in various pathological conditions such as:
Alzheimer, Parkinson’s and Huntington’s Diseases
Type 2 Diabetes Mellitus
Neuronal degeneration
Muscular Dystrophy
Metastasis
Cataract
Cancer
Functional roles of calpain
Although its physiological function is still not fully understood, it is implicated in a variety of calcium-regulated cellular processes such as:
- Adhesion Modulation – Spreading and Motility- Cell Death, apoptosis and Destruction- cell proliferation- signal transduction pathways- cell cycle progression- Cell differentiation - membrane fusion- regulation of the cytoskeleton- platelet activation
Studies using knock-out and inactiveknowk-in mice of CAPN8 and CAPN9shwed that both calpains are required forstomach mucosal defence mechanismsagainst stress. Loss of either CAPN8 or 9leads to significant stomach ulcer uponalcoholic stress. Furthermore, CAPN8 andCAPN9 form 1:1 complex, and functiontogether in the stomach.G-calpain (G:gastric)
Single nucleotide polymorphisms (SNPs) reported for human CAPN8 and CAPN9 includes non-sense mutations and mis-sense
mutations that changes amino acid residues highly conserved among calpain family members. These must perturbe functions of
CAPN8 and CAPN9.Actually, our in vitro assay showed that these mutant CAPN8 and CAPN9did not have activity. 30
CAPN3 and muscular dystrophy
pathogenic mutations of human CAPN3 areresponsible for limb-girdle muscular dystrophytype 2A (LGMD2A, also called "calpainopathy").CAPN3 is essential for skeletal muscle functions,and, since then, studies on CAPN3 has beendeveloping in relation with LGMD2A pathogenicmechanisms. We use biochemical and geneticanalyses on mice genetically modified in calpain-related genes (transgenic, knock-out, andprotease-inactive (active site Cys129 changed toSer [CAPN3:C129S]) knock-in mice) to elucidatephysiological and patho-physiological functions ofCAPN3. Using CAPN3:C129S knock-in mice, wehave shown that a loss of protease activity ofCAPN3 is responsible for LGMD2A. Moreover,CAPN3 dynamically changes its cellular localizationits activity-dependently, and this movement isimportant for stress-response of skeletal muscle
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Calpain 10 cleaves electron transport chain proteins, calpain 1 cleaves BH3-interacting domain death agonist (Bid),AIF,and NCX,calpain 2 cleaves voltage-dependent anion channel (VDAC).
calpains and mitochondria
in endothelial cells, Ca2+ overload causes mitochondrial calpain 1 cleavage of the Na+/Ca2+
exchanger leading to mitochondrial Ca2+
accumulation. Also, activated calpain 1 cleaves Bid, inducing cytochrome c release and apoptosis. In renal cells, calpains 1 and 2 promote apoptosis and necrosis by cleaving cytoskeletal proteins, which increases plasma membrane permeability and cleavage of caspases.
Calpain 10 cleaves electron transport chain proteins, causing decreased mitochondrial respiration and excessive activation, or inhibition of calpain 10 activity induces mitochondrial dysfunction and apoptosis.
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calpains and Motility
- Calpain 2 can cleave adhesion complex proteins such as
FAK, paxillin and talin 1, possibly resulting in integrin
activation, adhesion complex turnover or detachment of
the cell rear.
- Proteolysis of the actin-regulating protein cortactin
might lead to inhibition of membrane protrusion.
- Cleavage of integrin β-tails might be important for the
formation of small integrin clusters during the early
stages of cell spreading, whereas proteolysis of the small
GTPase RhoA negatively regulates cell spreading.
- Interaction of αPIX with calpain small subunit 1 (CSS1)
can also mediate cell spreading.
- Proteolysis of the adaptor protein MARCKS might also
regulate cell migration in myoblasts, possibly by
promoting adhesion formation.
- The isoforms required for proteolysis of integrins, RhoA
and MARCKS remain to be determined, as do the
processes affected by proteolysis of nearly 100 other
calpain substrates.
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Calpain substrates
• Among the >100 proteins identified as calpain substrates are:
• transcription factors
• transmembrane receptors
• Signaling enzymes
• cytoskeletal proteins
The calpain system
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• Calpains are a superfamily of 14 cysteine proteases,
• the system of calpains in a skeletal muscle consists of at least 3 proteases:
• calpain I (μ)
• calpain II (m)
• calpain 3 (p94)
• calpastatin – being a calpain inhibitor.
• The µ-calpain is mostly bound to myofibrils (70%)
• The m-calpain is located in the cytosol whereas the location of their inhibitor, calpastatin.
• The µ-calpain and m-calpain require different Ca2+ levels for their activation.
• µ- and m-calpain are calcium-activated proteases, requiring micro- and millimolar concentrations of Ca2+ for activation, respectively.
• m-calpain needs 400–800 µM Ca2+ for half-maximal activity,
• µ-calpain needs only 3–50 µM Ca2+ for half-maximal activity.
µ-calpain and m-calpain are heterodimers composed of a similar
80 kDa catalytic subunit
28 kDa small subunit
The small subunit has two domains (V, VI) :
V: Domain V is rich in glycine and is the site for phospholipid binding.
VI: Domain VI contains five Ca2+-binding sites also known as EF-hand motif
The calpain system
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The large 80 kDa subunit of each calpain is composed of four (I, II, III, IV) domains
I: The N-terminal domain of 80 kDa subunit, domain I, has no sequence homology to any known polypeptide and its removal modulates the proteolytic activity
II: The catalytic domain, domain II, contains a cysteine residue (sub-domain IIA) as well as a histidine residue (sub-domain IIB) that are in relative positions that are conserved in all cysteine proteinases
III: A Ca2+-binding domain, domain III, is not homologous to any other known protein and is linked to the catalytic domain II. It has sequences that predict EF hand calcium binding sites and may regulate the activity of calpain through binding of phospholipids and critical electrostatic interactions.
Structure of calpains
IV: A calmodulin-like domain, domain IV, is also known as penta-EF domain and has five EF-hand calcium binding sites. The first four EF-hands in domain VI also contain Ca2+-binding sites.
The calpain system
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Domains within calpains
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• Calpastatin, an endogenous inhibitor of µ- and m-calpain,
• is a 70–80 kDa protein
• and has an N-terminal L domain
• and four repeating domains (I, II, III, IV).
• Each of the four repeating domains is able to inhibit one calpain molecule.
• calpastatin must bind domain II and domain IV or VI to inhibit calpains
Structure of Calpastatin
Originally referred to as p94, calpain 3 is a 94 kDa calpain isoform bearing sequence homology of approximately 50% with the large domain of µ - and m-calpain.It may form homodimers in vivo as it lacks the 28 kDa small subunit .
This calpain protease is specific to skeletal muscle is also found in an 82 kDa form in the retina and lens .
Structure of Calpain 3 The calpain system
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Location :
The calpain system
calpain 3 in the largest degree is situated in a sarcomere near the Z- and M-line.
calpastatine localized in the muscle cell is similar to that of calpains.
- Z-line 66%
- I-band (20%), the rest
- A-band (14%).
- Z-line 52%
- I-band 27%
- A-band 21%
calpain I is in 70% bonded to myofibrils.
Calpain II is in majority located in the cytosol
In a muscle, calpains are located in the cytoplasm and cell membranes
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Mechanisms of action in calpains
• Calpain exists in the cytosol as an inactive enzyme
• translocates to membranes in response to increases in the cellular Ca2 level.
• At the membrane, calpain is activated in the presence of Ca2 and phospholipids.
• Autocatalytic hydrolysis of domain I takes place during activation,
• dissociation of 30K from 80K occurs as a result.
• Activated calpain or 80K hydrolyzes substrate proteins at membranes or in cytosol after release from membranes.
• In the absence of Ca2, two protease subdomains Ila and IIb are separated by structural constraints imposed by domain interaction. Ca2-induced structural changes that release the constraints are prerequisite for activation to form a functional catalytic site.
• There are at least three different Ca2-binding sites in m-calpain:
• two calmodulin-like domains IV and VI
• an acidic loop region in C2-like domain III
• and a protease domain II
The calpain system
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Activation mechanism of calpain by Ca
Binding of Ca2 and phospholipids (PL) to m-calpain induces conformational changes, which brings IIa and IIb closer together to
form a functional catalytic site and causes dissociation of 30K from 80K, resulting in 30K homodimer formation. There are atleast three different calcium-binding sites in m-calpain, two calmodulin-like EF-hand structures in domains IV and VI, an acidic
loop in domain III, and two non–EF-hand calcium-binding sites in IIa and IIb. C105 and H262 are catalytic residues. K7 and
D154 form a salt bridge in the absence of calcium ions. Nt, NH2-terminal residue.
The calpain system
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The calpain system
Schematic of the 3D structure of inactive and active m-calpain.
Surface-type schematic 3D structures of inactive (Ca2+ free) and active (Ca2+-and calpastatin-bound) forms of m-calpain using PDB data, The oligopeptides represented by the yellow ribbon + ball-and-stick indicate portions of calpastatin bound to active m-calpain. The dotted lines indicate portions that were too mobile for the 3D structure to be determined. The active protease domain (CysPc) is formed by the fusion of core domains PC1 and PC2 upon the binding of one Ca2+ to each of the core domains. The active site is circled in black; red balls represent Ca2+ (not all are visible).
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calpain autolyzes
• Calcium is required for calpain activation,
• both µ- calpain and m-calpain will also autolyze when exposed to the calcium levels necessary for their activation,
• thus proteolytic ability is accompanied with autolysis.
• The autolysis results in progressive degradation of subunit:
- µ- calpain: 80 kDa to 78 kDa and eventually to 76 kDa
- m-calpain: 80 kDa reduces to 78 kDa
- 28 kDa subunit: reduced to 18 kDa after losing its glycine-rich domain.
• The calcium requirement of µ-calpain and m-calpain for their activity is reduced by brief autolysis
• whereas extended autolysis results in inactivation of these enzymes
• This reduction of the calcium requirement explains how these enzymes are active in cellular conditions which rarely, if ever, acquire the calcium levels needed for their activity .
• The autolyzed forms of both µ-calpain (76 kDa) and m-calpain (78 kDa) requires %92 and %82 less calcium, respectively, than their unautolyzed (80 kDa) forms.
The calpain system
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Regulation of calpain activity
In the absence of Ca2+ (or at low Ca2+ concentrations), calpastatin binds to the inactive calpain in the cytosol.
An increase in cytosolic Ca2+ has been suggested to lead to calpain autolysis in the cytosol or to cause its translocation to the membrane where it undergoes autoproteolysis and can bind to phospholipids which increase the sensitivity of calpainto Ca2+.
A further increase in cytosolic Ca2+ enhances calpainautoproteolysis and activation and leads to calpastatincleavage.
The activity of calpains (particularly m-calpain) can also be regulated by kinases such as :
- protein kinase C (PKC)
- extracellular regulated kinases (ERK)
- tyrosine kinases (TK)
- protein phosphatase 2A (PP2A)
Phosphorylation by protein kinase A (PKA) results in m-calpaininactivation.
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• The inhibition of the activity of µ-calpain and m-calpain by calpastatin isalso a calcium dependent event as calpain-calpastatin binding requirescalcium.
• Calpastatin inhibits calpain by:
- preventing the calpain proteolytic activation,
- membrane binding
- and the expression of catalytic activity
• Conformational changes are induced in the calpains due to the binding ofcalcium ions, specif-ically in domains I-IV and VI.
• The region A and region C of the calpastatin molecule binds to domain IVand VI of calpain molecule, respectively. This causes the region B of thecalpastatin inhibitory domain to make various contacts with regions ofdomains I to III of calpains,
• thus blocking the active site of calpains and inhibiting its activity.
Mechanism of action in calpastatin
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Model for Capn3 activation
Capn3 is mainly present in a full-length inactive state inskeletal muscle, presumably through its binding to titin.Upon receiving an activation signal, a subset of Capn3molecules undergoes intramolecular autolysis in S1. Thisfirst event allows the complete autoprocessing of thesemolecules, consisting of cleavage of S2 and S3. These fullyactivated Capn3 molecules can thereafter intermolecularlyautolyze other Capn3 molecules which have not receivedthe activation signal. This ultimate step generates anamplification cascade leading to global activation of theCapn3 pool.
The calpain system
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Calpain 3 may function in conjunction with ubiquitin ligases to mediate sarcomere remodeling
Calpain 3 (red) is anchored to the sarcomere at the N2 line and M line, through its association with titin (not shown). The contractile proteins are highly organized and entwined and cannot be degraded in the proteasome without the initiating step of proteolytic dissociation of this complex (for simplicity, only actin and myosin are shown). Data suggest that calpain3 is one protein that performs the initial proteolytic cleavage that allows E3 ubiquitin ligases to ubiquitinate the peptides and target them for degradation in the proteasome
The calpain system
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The caspase system
Major intracellular proteolytic systems
Caspases are a family of cysteine aspartate-specific proteases to date 14 members of the caspase family have been identified These highly specialized enzymes create the intracellular network signaling proteolysis. Proteolysis activation of caspases may occur with the participation of enzymes such as: 1- granzyme B (a strong activator of procaspases 3 and 7) 2- cathepsin G 3- calpains4- cathepsin D
Apoptosis is the organised dismantling of the cell, characterised by:- cell shrinkage, - DNA fragmentation, - chromatin condensation, - membrane blebbing- the formation of apoptotic bodies without inducing an inflammatory response
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The caspase system
Major intracellular proteolytic systems
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The caspase system
Schematic diagram of the intrinsic, extrinsic and ER-mediated
apoptosis pathways showing the caspases involved in each pathway
Major intracellular proteolytic systems
1- The cell death pathway or extrinsic pathway istriggered by cell surface receptors and initiator.caspases 8 and 10 are activated via this pathway2- The intrinsic pathway involves caspase 9 and isactivated in response to environmental stress suchas hypoxia and ischaemia.3- The ER mediated pathway is activated via stressdirectly upon the ER, for example disruption inCa2+ homeostasis, which in turn activates initiatorcaspase 12.Effector caspases are activated by initiator caspases upstream and once activated target and cleave specific substrates, resulting in cell disassembly.To date more than 280 caspase targets have been identified including myofibrillar and cytoskeletal proteins
Caspases are activated via three main pathways:
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The caspase system
Major intracellular proteolytic systems
- Caspases can be activated early in pathological events associated with hypoxia/ischaemia- which is not that dissimilar to the hypoxic conditions in muscle after slaughter.- In meat animals the process of exsanguination occurs after slaughter, depriving all cells and tissues ofnutrients and oxygen.- After death muscle continues to metabolise and therefore muscle cells will presumably engaged in the process ofcell death, with apoptosis rather necrosis considered to be the most likely process of cell death.- Therefore it has been hypothesised that the process of slaughter and exsanguination could initiate the apoptoticpathways and caspase activity may contribute to early post-mortem proteolysis and meat tenderisation.
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Schematic representation of conventional calpain substrates and major events of tumor cell pathophysiology
