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From CellBiology

--Mark Hill 18:59, 12 May 2009 (EST) Your individual project (due week 10) is disappointing, in 8 weeks all you have managed is to identify the protein. Please insert any work at any draft stage you have carried out on your individual project before this week's lab.

--Mark Hill 08:33, 28 April 2009 (EST) Please see me ASAP You have not yet selected your individual project protein/method topic and still missing most homework items. 2009_Student#Individual_Projects Individual Projects I had previously left this message on your discussion page.


Calpain

Background

Necrosisis a type of cell death that is cause by cell injury, stress or ischemia. The underline mechanism of necrosis is complicated and it is mediated by certain proteins inside the cell. Calpain 1 and 2 are found to be essential to the necrosis, especially in the brain.

Introduction

Calpains is an intracellular calcium-dependent proteases that requires calcium for its catalytic activity. [1] [2]Calpains exist ubiquitously in cells from humans to microorganisms. More than 10 different types of the calpain are reported in mammals.[3] There are two ubiquitius isoforms of calpain, calpain 1 and calpain 2. These two form of calpains have different sensitivity to Ca2+: calpain 1 being activated at the micromolar Ca2+-concentration while calpain 2 at the millimolar Ca2+-concentration.[3] [4] [5]Researchers suggest that the activation of calpain 1 and 2 in the cell will lead to necrosis in the brain. It is initiated by the loss of Ca2+ homeostasis, which leads to calpain activation, finally to necrotic neruron death.

Structure

Calpain 1 and calpain 2 comprise a highly homologous 80‐kDa large catalytic subunits and a 30‐kDa small regulatory subunit. The large subunits in calpain 1 and 2 are slighty different whereas the small subunits are the same. Both subunit together to from a heterodimer structure and containing multiple calcium-binding sites. [3] [4] [5] [6]The 80 kDa subunit consists of four domains (I - IV) and the 30 kDa unit has 2 domains (V and VI).

3D structure of calpain 2 [7]

Large subunit

  • Domain I is actually not a romain and is removed during autolysis.
  • Domain II is the protease domain and divided into 2 sub-domains with different active sites.
  • Domain III is similar to typical calmodulin binding proteins and interacts with domains IV.
  • Domain IV is a Ca2+-binding domain similar to calmodulin and contain 5 EF-hands.

Small subunit

  • Domain V is a hydrophobic region and interacts with membranes.
  • Domain VI is Ca2+ binding domain similar to domain IV and reported to have six EF-hands [3]

Function

Calpain regulates some major cellular process[8] [9]

  • cell adhesion: calpain cleaves the adhesion complex causing adhesion disassembly
  • cell motility: calpain activation lead to tail retraction of the cells.
  • cell spreading: calpain decrease the rate of cell spreading by decreasing proteolysis of adhesion complex proteins.
  • cell death: increase of intracellular CA2+ intiates the activation of calpain and causing necrosis.

Ischemic Neuronal death

Calpain contributes to the necrotic neuronal death in ischemia and neurodegeneration. [1] [10]Yamashima T. reported that transient global brain ischemia which is a cause of delayed neuronal death of the vulnerable neurons is contributed by necrosis and apoptosis. [11] Calpain mainly play an role in the necrosis part.

After the ischemia of the neuron, there is an increase of Ca2+ concentration in the cell by the influx of the Ca2+ via several channesl in the cell and released by the ER. The raise in the intracellular [Ca2+] signal the activation of the calpain. [10] [11] The activated calpain compromises the integrity of lysosomal membranes which lead to the disruption of the lysosomal membrane. The lysosomal membrane is a physical barrier that prevents hydrolytic enzymes from digesting the cell’s own cytoplasm. The rupture of the lysosomal membrane causes the leakage of the hydrolytic enzymes in to the cytoplasm which then digest the cell constituent proteins and finally to necrotic neuronal death. Cathepsin proteases is one of the enzymes released from the lysosome. So this is called "Calpain-cathepsin hypothesis"[12]Actually, calpain is not killing the cell directly, its role is to mediate the activation of cathepsin that causes cytoskeletal degradation in the cell.


Calpastatin is an inhibitor of calpain which can prevent the activation of calpain in the cell. [2] [4] [6] However, the released of cathepsin from the lysosome mediates the activation of caspase. Caspase then induce the degradation of calpastatin. Finally the activation of calpain is maintained.

Glossary

Autolysis: digestion of cells by enzymes present within them.

Calmodulin: a ubiquitous, calcium-binding protein that can bind to and regulate a multitude of different protein targets.

EF-hand: a helix-loop-helix structural domain found in a large family of calcium-binding proteins.

Heterodimer: a protein composed of two polypeptide chains differing in composition in the order, number, or kind of their amino acid residues

Ischemia: block of blood flow causing insufficient oxygen supply to cells.

Proteolysis: protein degradation by proteases or by intramolecular digestion

References

  1. 1.0 1.1 Croall D. E. & Ersfeld K. (2007), The calpains: modular designs and functional diversity, Genome Biology 8:218-229 PMID 17608959
  2. 2.0 2.1 Wang K. K. (2000), Calpain and caspase: can you tell the difference?, Trends in neucrosciences, 23(1): 20-26 PMID 10631785
  3. 3.0 3.1 3.2 3.3 Sorimachi H, Ishiura S & Suzuki K. (1997), Structure and physiological function of calpains, The biochemical journal, 328(3): 721-732 PMID 9396712
  4. 4.0 4.1 4.2 Croall D. E. and DeMartino G. N. (1991), Calcium-activated neutral protease (calpain) system: structure, function, and regulation, Physiological reviews, 71(3): 813-847 PMID 2057527
  5. 5.0 5.1 Sorimachi H and Suzuki K. (2001), The structure of calpain., Journal of biochemistry, 129(5): 653-654 PMID 11328585
  6. 6.0 6.1 Carafoli E., Molinari M. (1998), Calpain: a protease in search of a function?, Biochemical and biophysical communication, 247(2): 193-203 PMID 9642102
  7. Strobl S, Fernandez-Catalan C, Braun M, Huber R, Masumoto H, Nakagawa K et al. (2008), The crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium, PNAS, 97(2): 588-592 PMID 10639123
  8. Wells A., Huttenlocher A. & Lauffenburger D. A. (2005), Calpain Proteases in Cell Adhesion and Motility, International review of cytology, 245:1-16 PMID 16125543
  9. Franco S. J. and Huttenlocher A. (2005), Regulating cell migration: calpains make the cut, Journal of cell science, 118:3829-3838 PMID 16129881
  10. 10.0 10.1 Syntichaki P., Xu K., Driscoll M.& Tavernarakis N.(2002), Specific aspartyl and calpain proteases are required for neurodegeneration in C. elegans, Nature, 419(6910): 939-944PMID 12410314
  11. 11.0 11.1 Yamashima T. (2004), Ca2+-dependent proteases in ischemic neuronal death A conserved ‘calpain-cathepsin cascade’from nematodes to primates, Cell Calcium 36(3-4):285–293 PMID 15261484
  12. Syntichaki P, Tavernarakis N (2002),Death by necrosis. Uncontrollable catastrophe, or is there order behind the chaos?, EMOB Report, 3:7 604-609 PMID 12101090

Lecture 4

What did you find interesting and did not know about the nucleus?

The 2 layers of membranes surrounding the necleus which are the outer membrane and the nuclear lamina. And there are intermediate filaments at the neclear lamina that i didn't know before. The membranes also contain pores for both passive and active passive. The concept of Chromosome Territories is so interesting, different chromsome occupying a specify region within the nucleus.


Lecture 5

What did you find difficult to understand about exocytosis

The structures and functions oftThe 4 components involved in exocytosis is easy to understander. But I found that the concept of secretory vesicles is hard to understand and how does it related to membrane turnover?


Lecture 7

What types of cellular processes require lots of energy from the mitochondria?

Cellular Respiration

Cell growth

Cell repair

Active transport

Apoptosis


Lecture 8

What do the acronyms (N-CAM) N-CAM, Ng-CAM L-CAM, I-CAM mean?

N-CAM -- Neural Cell Adhesion Molecule

Ng-CAM-- Neuron-glia cell adhesion molecule

L-CAM -- Liver CAM

I-CAM -- Intercellular adhesion molecule


Lecture 10

What is the name of the epidermal layer between the basal and granulosa layer and how does it relate to intermediate filaments?

Stratum spinosum. It synthesizes a type of intermediate filament known as Cytokeratin, which are composed of keratin.


Lecture 14

What are the 2 main forms of generating confocal microscopy?

Confocal laser scanning microscopes

Spinning-disk confocal microscopes


Lecture 15

What does "S" stand for in the S phase?

S phase stand for "Synthesis"




--Mark Hill 18:34, 19 March 2009 (EST)You still need to provide feedback for Lecture 4 - Nucleus.