Ricin: A toxic protein
From Proteopedia
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The inactive ricin precursor protein consists of 576 amino acid residues, a signal peptide known as the ricin A-chain and the linker peptide known as the ricin B-chain. The biosynthesis and activation of ricin precursor protein take place in the endosperm of castor oil seeds. The precursor protein is delivered to the endoplasmic reticulum with the help of the N-terminal signal sequence that gets cleaved off once the protein has been delivered. Once in the lumen of the endoplasmic reticulum, the precursor protein gets glycosylated, and a disulfide bond is formed between cysteines 294 and 318 with the help of protein disulfide isomerases causing the precursor protein chains to further fold into globular protein domains. The precursor protein is further glycosylated in the Golgi apparatus before it is transported to different protein bodies through storage vesicles. Once in the protein bodies, the ricin precursor protein is cleaved by an endopeptidase to finally give us the mature Ricin protein made up of a 267 residue A-chain and a 262 residue B-chain that are covalently linked by a disulfide bond. | The inactive ricin precursor protein consists of 576 amino acid residues, a signal peptide known as the ricin A-chain and the linker peptide known as the ricin B-chain. The biosynthesis and activation of ricin precursor protein take place in the endosperm of castor oil seeds. The precursor protein is delivered to the endoplasmic reticulum with the help of the N-terminal signal sequence that gets cleaved off once the protein has been delivered. Once in the lumen of the endoplasmic reticulum, the precursor protein gets glycosylated, and a disulfide bond is formed between cysteines 294 and 318 with the help of protein disulfide isomerases causing the precursor protein chains to further fold into globular protein domains. The precursor protein is further glycosylated in the Golgi apparatus before it is transported to different protein bodies through storage vesicles. Once in the protein bodies, the ricin precursor protein is cleaved by an endopeptidase to finally give us the mature Ricin protein made up of a 267 residue A-chain and a 262 residue B-chain that are covalently linked by a disulfide bond. | ||
| - | == | + | ==Ricin's Mechanism of Action== |
| + | ===Entry into the cytoplasm=== | ||
| + | The B-chain in ricin is responsible for entry into the cell. Ricin B-chain binds to complex carbohydrates on the surface of a eukaryotic cell that contains either the terminal N-acetylgalactosamine or beta-1,4-linked galactose residues. Ricin also contains mannose-type glycans that allow them to bind to cells that contain mannose receptors. Ricin B-chain is seen to be able to bind to cell surfaces in the order of 106 to 108 ricin molecules per cell surface. This abundant binding to surface membranes leads to all types of membrane invagination. This holotoxin can be endocytosed with the help of clathrin-coated pits, or even clathrin-independent pathways such as macropinocytosis and caveolae. Ricin is eventually taken in and delivered to the Golgi apparatus through endosomes. Throughout its journey in the endosomes, the ricin molecule is not affected by the acidic environment because ricin is seen to be stable in a wide pH range. This shows that our cells cannot use degradation by endosomes or lysosomes as a form of protection against ricin. Ricin is transported to the trans-Golgi network, the Golgi apparatus, and finally the lumen of the endoplasmic reticulum through retrograde vesicular transport. Once in the lumen, the A-chain is cleaved from the B-chain in the ricin molecule in order to expose the active site of the A-chain, with the help of protein disulfide isomerases as mentioned before. The resulting free-floating Ricin A-chain partially unfolds and partially buries itself into the endoplasmic reticulum membrane mimicking a misfolded membrane protein. Usually, any misfolded proteins are destroyed or dislocated by arriving in the cytosol, where they are tagged by the molecule ubiquitin by a process known as ubiquitination for degradation, deglycosylated, and finally degraded and dispensed off by proteasomal proteolysis. However, Ricin A-chain avoids this degradation process by avoiding ubiquitination because it has a low content of lysine residues, which are known as the attachment sites for the molecules ubiquitin. This leads to ricin A-chain avoiding the usual fate of misfolded dislocated proteins. At this point, the misfolded ricin A-chain needs to refold in order to regain its catalytic toxicity and become active. This is where chaperone proteins come in, in this case mainly the proteins Hsc70, Hsp90, and their co-chaperones as well as proteasomes. In vitro, the chaperone Hsc70 prevents aggregation of heat-denatured ricin A-chain whereas in vivo it is seen that Hsc70 masks the hydrophobic regions of the A-chain that interact with the membrane thereby stabilizing it, as well as aiding in the A-chain’s solubility allowing it to undergo ribosome-mediated refolding. Once Ricin’s A-chain is folded into a catalytic conformation it proceeds to depurinate ribosomes and halting protein synthesis. | ||
| - | == | + | ===Ribosome inactivation=== |
| + | As mentioned previously, Ricin’s A-chain has an rRNA N-glycosylase activity. This activity causes the cleavage of a glycosylic bond of the 60S ribosomes within the rRNA. The A-chain specifically and reversibly hydrolyzes the N-glycosidic bond of Adenine at position 4324 in the 28S rRNA however it leaves the backbone of the RNA intact. This target adenine at position 4324 (A4324) is in a highly conserved sequence of 12 universal nucleotides found in eukaryotic ribosomes. This highly conserved sequence is referred to as the Sarcin-ricin loop (SRL), it exists as an autonomous unit on the ribosome and exposed to the solvent. It has this unique feature in order for the loop to be accessible to external factors. The Sarcin-ricin loop is structured like a hairpin, it has basic organized elements including the GAGA loop which is where the ricin target adenine resides. Studies have shown that SRL is required for recruiting different elements responsible for translation elongation (translational GTPases) however it is seen that the loop has also been a target for many toxins. After the cleavage of the adenine residue from the loop, the RNA chain is left open to hydrolysis or even cleavage by other cellular lyases. Due to the function of the loop, any small disruption could lead to major effects on protein synthesis like inhibition, and eventually, when protein synthesis is inhibited, cell death. It is seen that a single molecule of ricin could inactivate over 1000 ribosomes per minute. | ||
| - | == Relevance == | ||
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| - | == Structural highlights == | ||
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| - | This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes. | ||
</StructureSection> | </StructureSection> | ||
== References == | == References == | ||
<references/> | <references/> | ||
Revision as of 18:27, 29 April 2021
Contents |
Background
Ricin is a toxin protein found in castor beans and the seeds of the castor oil plant. This heterodimeric protein is produced as a part of the waste that comes from the production of castor oil. Ricin was discovered more than a century ago when the protein was isolated from seeds by Stillmark. The protein is considered toxic due to its observed ability to clump up red blood cells. Later studies showed that ricin was a mixture of the potent cytotoxin and hemagglutinin. Such mechanism of the ricin was discovered on 28S ribosomal RNA. Since then, several functionally related proteins have been discovered from different plants. Such proteins are referred to as ribosome-inactivating proteins due to their ability to irreversibly inactivate eukaryotic ribosomes and terminating protein synthesis. Certain types of ricin known as ‘type I Ricins’ have toxic characteristics however it is seen that they do not have the capability to enter cells in order to reach the ribosomes. Other types are known as ‘type II Ricin’ however, have the capability to enter the cells due to their differences in structures.
Structure
only consists of the A-chain whereas is comprised of the and the which are folded peptide chains, with the two chains linked by . Ricin has a molecular weight of 64-63 kDa, with the A-chain being 32 kDa and the B-chain being 36 kDa. The A-chain has the conformation of a globular protein domain with 267 amino acids consisting of 8 alpha-helices and 8 beta-sheets with the active site as a long cleft on its surface. The active site consists of a key catalytic residue Glutamic acid 177 which is deprotonated to glutamate reducing the activity immensely. Whereas the B-chain has the conformation of a barbell structure consisting of 26 amino acids, with a sugar-binding site at each end which allows it to hydrogen bond to galactose and N-acetyl galactosamine that is found on cell surfaces. The B-chain and the A-chain respectively do not cause the ricin to be toxic, Ricin’s toxicity is due to the presence of both chains because of their crucial roles together. The B-chain’s role is to acquire entry into eukaryotic cells, meanwhile, the A-chain is responsible for the toxicity because of its RNA N-glycosidase activity. Once the molecule enters the cell, the B-chain dissociates from the A-chain leaving it to exert its toxicity.
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This is a default text for your page Ricin: A toxic protein. Click above on edit this page to modify. Be careful with the < and > signs. You may include any references to papers as in: the use of JSmol in Proteopedia [1] or to the article describing Jmol [2] to the rescue.
Biosynthesis
The inactive ricin precursor protein consists of 576 amino acid residues, a signal peptide known as the ricin A-chain and the linker peptide known as the ricin B-chain. The biosynthesis and activation of ricin precursor protein take place in the endosperm of castor oil seeds. The precursor protein is delivered to the endoplasmic reticulum with the help of the N-terminal signal sequence that gets cleaved off once the protein has been delivered. Once in the lumen of the endoplasmic reticulum, the precursor protein gets glycosylated, and a disulfide bond is formed between cysteines 294 and 318 with the help of protein disulfide isomerases causing the precursor protein chains to further fold into globular protein domains. The precursor protein is further glycosylated in the Golgi apparatus before it is transported to different protein bodies through storage vesicles. Once in the protein bodies, the ricin precursor protein is cleaved by an endopeptidase to finally give us the mature Ricin protein made up of a 267 residue A-chain and a 262 residue B-chain that are covalently linked by a disulfide bond.
Ricin's Mechanism of Action
Entry into the cytoplasm
The B-chain in ricin is responsible for entry into the cell. Ricin B-chain binds to complex carbohydrates on the surface of a eukaryotic cell that contains either the terminal N-acetylgalactosamine or beta-1,4-linked galactose residues. Ricin also contains mannose-type glycans that allow them to bind to cells that contain mannose receptors. Ricin B-chain is seen to be able to bind to cell surfaces in the order of 106 to 108 ricin molecules per cell surface. This abundant binding to surface membranes leads to all types of membrane invagination. This holotoxin can be endocytosed with the help of clathrin-coated pits, or even clathrin-independent pathways such as macropinocytosis and caveolae. Ricin is eventually taken in and delivered to the Golgi apparatus through endosomes. Throughout its journey in the endosomes, the ricin molecule is not affected by the acidic environment because ricin is seen to be stable in a wide pH range. This shows that our cells cannot use degradation by endosomes or lysosomes as a form of protection against ricin. Ricin is transported to the trans-Golgi network, the Golgi apparatus, and finally the lumen of the endoplasmic reticulum through retrograde vesicular transport. Once in the lumen, the A-chain is cleaved from the B-chain in the ricin molecule in order to expose the active site of the A-chain, with the help of protein disulfide isomerases as mentioned before. The resulting free-floating Ricin A-chain partially unfolds and partially buries itself into the endoplasmic reticulum membrane mimicking a misfolded membrane protein. Usually, any misfolded proteins are destroyed or dislocated by arriving in the cytosol, where they are tagged by the molecule ubiquitin by a process known as ubiquitination for degradation, deglycosylated, and finally degraded and dispensed off by proteasomal proteolysis. However, Ricin A-chain avoids this degradation process by avoiding ubiquitination because it has a low content of lysine residues, which are known as the attachment sites for the molecules ubiquitin. This leads to ricin A-chain avoiding the usual fate of misfolded dislocated proteins. At this point, the misfolded ricin A-chain needs to refold in order to regain its catalytic toxicity and become active. This is where chaperone proteins come in, in this case mainly the proteins Hsc70, Hsp90, and their co-chaperones as well as proteasomes. In vitro, the chaperone Hsc70 prevents aggregation of heat-denatured ricin A-chain whereas in vivo it is seen that Hsc70 masks the hydrophobic regions of the A-chain that interact with the membrane thereby stabilizing it, as well as aiding in the A-chain’s solubility allowing it to undergo ribosome-mediated refolding. Once Ricin’s A-chain is folded into a catalytic conformation it proceeds to depurinate ribosomes and halting protein synthesis.
Ribosome inactivation
As mentioned previously, Ricin’s A-chain has an rRNA N-glycosylase activity. This activity causes the cleavage of a glycosylic bond of the 60S ribosomes within the rRNA. The A-chain specifically and reversibly hydrolyzes the N-glycosidic bond of Adenine at position 4324 in the 28S rRNA however it leaves the backbone of the RNA intact. This target adenine at position 4324 (A4324) is in a highly conserved sequence of 12 universal nucleotides found in eukaryotic ribosomes. This highly conserved sequence is referred to as the Sarcin-ricin loop (SRL), it exists as an autonomous unit on the ribosome and exposed to the solvent. It has this unique feature in order for the loop to be accessible to external factors. The Sarcin-ricin loop is structured like a hairpin, it has basic organized elements including the GAGA loop which is where the ricin target adenine resides. Studies have shown that SRL is required for recruiting different elements responsible for translation elongation (translational GTPases) however it is seen that the loop has also been a target for many toxins. After the cleavage of the adenine residue from the loop, the RNA chain is left open to hydrolysis or even cleavage by other cellular lyases. Due to the function of the loop, any small disruption could lead to major effects on protein synthesis like inhibition, and eventually, when protein synthesis is inhibited, cell death. It is seen that a single molecule of ricin could inactivate over 1000 ribosomes per minute.
</StructureSection>
References
- ↑ Hanson, R. M., Prilusky, J., Renjian, Z., Nakane, T. and Sussman, J. L. (2013), JSmol and the Next-Generation Web-Based Representation of 3D Molecular Structure as Applied to Proteopedia. Isr. J. Chem., 53:207-216. doi:http://dx.doi.org/10.1002/ijch.201300024
- ↑ Herraez A. Biomolecules in the computer: Jmol to the rescue. Biochem Mol Biol Educ. 2006 Jul;34(4):255-61. doi: 10.1002/bmb.2006.494034042644. PMID:21638687 doi:10.1002/bmb.2006.494034042644
