Editing Ricin (section)

== Biochemistry ==
Ricin is classified as a type 2 [[ribosome-inactivating protein]] (RIP). Whereas type 1 RIPs are composed of a single protein chain that possesses catalytic activity, type 2 RIPs, also known as holotoxins, are composed of two different protein chains that form a [[heterodimeric]] complex. Type 2 RIPs consist of an A chain that is functionally equivalent to a type 1 RIP, covalently connected by a single [[disulfide bond]] to a B chain that is catalytically inactive, but serves to mediate transport of the A-B protein complex from the cell surface, via vesicle carriers, to the lumen of the [[endoplasmic reticulum]] (ER). Both type 1 and type 2 RIPs are functionally active against ribosomes ''in vitro''; however, only type 2 RIPs display [[cytotoxicity]] due to the [[lectin]]-like properties of the B chain. To display its ribosome-inactivating function, the ricin disulfide bond must be [[redox|reductively]] cleaved.<ref name="pmid3606124">{{cite journal | vauthors = Wright HT, Robertus JD | title = The intersubunit disulfide bridge of ricin is essential for cytotoxicity | journal = Archives of Biochemistry and Biophysics | volume = 256 | issue = 1 | pages = 280–284 | date = July 1987 | pmid = 3606124 | doi = 10.1016/0003-9861(87)90447-4 }}</ref>

=== Biosynthesis ===
Ricin is [[Protein biosynthesis|synthesized]] in the [[endosperm]] of castor oil plant seeds.<ref name="Lord_Roberts_2005">{{cite book |veditors=Raffael S, Schmitt M | title = Microbial Protein Toxins | series = Topics in Current Genetics | volume = 11 | publisher = Springer | location = Berlin | year = 2005 | pages = 215–233 | isbn = 978-3-540-23562-0 |vauthors=Lord MJ, Roberts LM | chapter = Ricin: structure, synthesis, and mode of action | doi = 10.1007/b100198 }}</ref> The ricin [[protein precursor|precursor protein]] is 576 [[amino acid residue]]s in length and contains a [[signal peptide]] (residues 1–35), the ricin A chain (36–302), a linker peptide (303–314), and the ricin B chain (315–576).<ref name="urlRicin precursor - Ricinus communis (Castor bean)">{{cite web | url = https://www.uniprot.org/uniprot/P02879 | title = P02879 Ricin precursor – Ricinus communis (Castor bean) | publisher = UniProt Consortium | work = UniProtKB}}</ref> The [[N-terminal]] signal sequence delivers the prepropolypeptide to the [[endoplasmic reticulum]] (ER) and then the signal peptide is cleaved off. Within the [[lumen (anatomy)|lumen]] of the ER the propolypeptide is [[glycosylated]] and a [[protein disulfide isomerase]] catalyzes [[disulfide bond]] formation between [[cysteine]]s 294 and 318. The propolypeptide is further glycosylated within the [[Golgi apparatus]] and transported to protein storage bodies. The propolypeptide is cleaved within protein bodies by an [[endopeptidase]] to produce the mature ricin protein that is composed of a 267 residue A chain and a 262 residue B chain that are covalently linked by a single disulfide bond.<ref name="Lord_Roberts_2005"/>

=== Structure ===
In terms of structure, ricin closely resembles abrin-a, an isomer of [[abrin]]. The [[Protein quaternary structure|quaternary structure]] of ricin is a globular, glycosylated heterodimer of approximately 60–65 [[Dalton (unit)|kDa]].<ref name="pmid4730499"/> Ricin toxin A chain and ricin toxin B chain are of similar molecular weights, approximately 32 kDa and 34 kDa, respectively.[[File:Alignment Abrin Ricin.png|thumb|A comparison of the similar structures of abrin-a (red) and ricin (blue)|alt=|left]]
* '''Ricin toxin A chain''' (RTA) is an ''N''-[[glycoside hydrolase]] composed of 267 amino acids.<ref name="pmid4730499">{{cite journal | vauthors = Olsnes S, Pihl A | title = Different biological properties of the two constituent peptide chains of ricin, a toxic protein inhibiting protein synthesis | journal = Biochemistry | volume = 12 | issue = 16 | pages = 3121–3126 | date = July 1973 | pmid = 4730499 | doi = 10.1021/bi00740a028 }}</ref> It has three structural domains with approximately 50% of the [[polypeptide]] arranged into [[alpha-helix|alpha-helices]] and [[beta-sheet]]s.<ref name="pmid7990130">{{cite journal | vauthors = Weston SA, Tucker AD, Thatcher DR, Derbyshire DJ, Pauptit RA | title = X-ray structure of recombinant ricin A-chain at 1.8 A resolution | journal = Journal of Molecular Biology | volume = 244 | issue = 4 | pages = 410–422 | date = December 1994 | pmid = 7990130 | doi = 10.1006/jmbi.1994.1739 }}</ref> The three domains form a pronounced cleft that is the active site of RTA.
* '''Ricin toxin B chain''' (RTB) is a [[lectin]] composed of 262 amino acids that is able to bind terminal [[galactose]] residues on cell surfaces.<ref name="pmid1717462">{{cite journal | vauthors = Wales R, Richardson PT, Roberts LM, Woodland HR, Lord JM | title = Mutational analysis of the galactose binding ability of recombinant ricin B chain | journal = The Journal of Biological Chemistry | volume = 266 | issue = 29 | pages = 19172–19179 | date = October 1991 | pmid = 1717462 | doi = 10.1016/S0021-9258(18)54978-4 | doi-access = free }}</ref> RTB forms a bilobal, barbell-like structure lacking [[alpha helix|alpha-helices]] or [[beta sheet|beta-sheets]] where individual lobes contain three [[protein domain|subdomains]]. At least one of these three subdomains in each homologous lobe possesses a sugar-binding pocket that gives RTB its functional character.
While other plants contain the protein chains found in ricin, both protein chains must be present to produce toxic effects. For example, plants that contain only protein chain A, such as [[barley]], are not toxic because without the link to protein chain B, protein chain A cannot enter the cell and do damage to ribosomes.<ref name="Harkup-2015">{{Cite book|title = A is For Arsenic: The poisons of Agatha Christie| author = [[Kathryn Harkup]] |publisher = Bloomsbury Sigma|year = 2015|isbn = 978-1-4729-1130-8|location = London|pages = [https://archive.org/details/isforarsenicpois0000hark/page/222 222–236]|url = https://archive.org/details/isforarsenicpois0000hark/page/222}}</ref>

=== Entry into the cytoplasm ===
Ricin B chain binds complex carbohydrates on the surface of [[eukaryotic]] cells containing either terminal [[N-acetylgalactosamine|''N''-acetylgalactosamine]] or beta-1,4-linked galactose residues. In addition, the [[mannose]]-type [[glycan]]s of ricin are able to bind to cells that express [[mannose receptor]]s.<ref name="pmid8453986">{{cite journal | vauthors = Magnusson S, Kjeken R, Berg T | title = Characterization of two distinct pathways of endocytosis of ricin by rat liver endothelial cells | journal = Experimental Cell Research | volume = 205 | issue = 1 | pages = 118–125 | date = March 1993 | pmid = 8453986 | doi = 10.1006/excr.1993.1065 }}</ref> RTB has been shown to bind to the cell surface on the order of 10<sup>6</sup>–10<sup>8</sup> ricin molecules per cell surface.<ref name="pmid7657599">{{cite journal | vauthors = Sphyris N, Lord JM, Wales R, Roberts LM | title = Mutational analysis of the Ricinus lectin B-chains. Galactose-binding ability of the 2 gamma subdomain of Ricinus communis agglutinin B-chain | journal = The Journal of Biological Chemistry | volume = 270 | issue = 35 | pages = 20292–20297 | date = September 1995 | pmid = 7657599 | doi = 10.1074/jbc.270.35.20292 | doi-access = free }}</ref>

The profuse binding of ricin to surface membranes allows internalization with all types of membrane [[invagination]]s. The holotoxin can be taken up by [[clathrin]]-coated pits, as well as by clathrin-independent pathways including [[caveolae]] and [[macropinocytosis]].<ref name="pmid2862151">{{cite journal | vauthors = Moya M, Dautry-Varsat A, Goud B, Louvard D, Boquet P | title = Inhibition of coated pit formation in Hep2 cells blocks the cytotoxicity of diphtheria toxin but not that of ricin toxin | journal = The Journal of Cell Biology | volume = 101 | issue = 2 | pages = 548–559 | date = August 1985 | pmid = 2862151 | pmc = 2113662 | doi = 10.1083/jcb.101.2.548 }}</ref><ref name="pmid11567873">{{cite journal | vauthors = Nichols BJ, Lippincott-Schwartz J | title = Endocytosis without clathrin coats | journal = Trends in Cell Biology | volume = 11 | issue = 10 | pages = 406–412 | date = October 2001 | pmid = 11567873 | doi = 10.1016/S0962-8924(01)02107-9 }}</ref> Intracellular [[vesicle (biology)|vesicles]] shuttle ricin to [[endosome]]s that are delivered to the [[Golgi apparatus]]. The active acidification of endosomes is thought to have little effect on the functional properties of ricin. Because ricin is stable over a wide pH range, degradation in endosomes or [[lysosome]]s offers little or no protection against ricin.<ref name="pmid14579547">{{cite journal | vauthors = Lord MJ, Jolliffe NA, Marsden CJ, Pateman CS, Smith DC, Spooner RA, Watson PD, Roberts LM | title = Ricin. Mechanisms of cytotoxicity | journal = Toxicological Reviews | volume = 22 | issue = 1 | pages = 53–64 | year = 2003 | pmid = 14579547 | doi = 10.2165/00139709-200322010-00006 }}</ref> Ricin molecules are thought to follow [[retrograde transport]] via early endosomes, the trans-Golgi network, and the Golgi to enter the [[Lumen (anatomy)|lumen]] of the [[endoplasmic reticulum]] (ER).<ref name="pmid16603059">{{cite journal | vauthors = Spooner RA, Smith DC, Easton AJ, Roberts LM, Lord JM | title = Retrograde transport pathways utilised by viruses and protein toxins | journal = Virology Journal | volume = 3 | pages = 26 | date = April 2006 | pmid = 16603059 | pmc = 1524934 | doi = 10.1186/1743-422X-3-26 | doi-access = free }}</ref>

For ricin to function cytotoxically, RTA must be reductively cleaved from RTB to release a [[steric]] block of the RTA active site. This process is catalysed by the protein PDI ([[protein disulphide isomerase]]) that resides in the lumen of the ER.<ref name="pmid15225124">{{cite journal | vauthors = Spooner RA, Watson PD, Marsden CJ, Smith DC, Moore KA, Cook JP, Lord JM, Roberts LM | title = Protein disulphide-isomerase reduces ricin to its A and B chains in the endoplasmic reticulum | journal = The Biochemical Journal | volume = 383 | issue = Pt 2 | pages = 285–293 | date = October 2004 | pmid = 15225124 | pmc = 1134069 | doi = 10.1042/BJ20040742 }}</ref><ref name="pmid15081871">{{cite journal | vauthors = Bellisola G, Fracasso G, Ippoliti R, Menestrina G, Rosén A, Soldà S, Udali S, Tomazzolli R, Tridente G, Colombatti M | title = Reductive activation of ricin and ricin A-chain immunotoxins by protein disulfide isomerase and thioredoxin reductase | journal = Biochemical Pharmacology | volume = 67 | issue = 9 | pages = 1721–1731 | date = May 2004 | pmid = 15081871 | doi = 10.1016/j.bcp.2004.01.013 }}</ref> Free RTA in the ER lumen then partially unfolds and partially buries into the ER membrane, where it is thought to mimic a misfolded membrane-associated protein.<ref name="pmid19211561">{{cite journal | vauthors = Mayerhofer PU, Cook JP, Wahlman J, Pinheiro TT, Moore KA, Lord JM, Johnson AE, Roberts LM | title = Ricin A chain insertion into endoplasmic reticulum membranes is triggered by a temperature increase to 37 {degrees}C | journal = The Journal of Biological Chemistry | volume = 284 | issue = 15 | pages = 10232–10242 | date = April 2009 | pmid = 19211561 | pmc = 2665077 | doi = 10.1074/jbc.M808387200 | doi-access = free }}</ref> Roles for the ER chaperones [[GRP94]],<ref name="Spooner">{{cite journal | vauthors = Spooner RA, Hart PJ, Cook JP, Pietroni P, Rogon C, Höhfeld J, Roberts LM, Lord JM | title = Cytosolic chaperones influence the fate of a toxin dislocated from the endoplasmic reticulum | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 105 | issue = 45 | pages = 17408–17413 | date = November 2008 | pmid = 18988734 | pmc = 2580750 | doi = 10.1073/pnas.0809013105 | doi-access = free | bibcode = 2008PNAS..10517408S | jstor = 25465291 }}</ref> [[EDEM1|EDEM]]<ref name="pmid16452630">{{cite journal | vauthors = Slominska-Wojewodzka M, Gregers TF, Wälchli S, Sandvig K | title = EDEM is involved in retrotranslocation of ricin from the endoplasmic reticulum to the cytosol | journal = Molecular Biology of the Cell | volume = 17 | issue = 4 | pages = 1664–1675 | date = April 2006 | pmid = 16452630 | pmc = 1415288 | doi = 10.1091/mbc.E05-10-0961 }}</ref> and [[Binding immunoglobulin protein|BiP]]<ref name="pmid23666197">{{cite journal | vauthors = Gregers TF, Skånland SS, Wälchli S, Bakke O, Sandvig K | title = BiP negatively affects ricin transport | journal = Toxins | volume = 5 | issue = 5 | pages = 969–982 | date = May 2013 | pmid = 23666197 | pmc = 3709273 | doi = 10.3390/toxins5050969 | doi-access = free }}</ref> have been proposed prior to the 'dislocation' of RTA from the ER lumen to the cytosol in a manner that uses components of the endoplasmic reticulum-associated protein degradation ([[ERAD]]) pathway. ERAD normally removes misfolded ER proteins to the cytosol for their destruction by cytosolic proteasomes. Dislocation of RTA requires ER membrane-integral E3 [[ubiquitin ligase]] complexes,<ref name="pmid20519439">{{cite journal | vauthors = Li S, Spooner RA, Allen SC, Guise CP, Ladds G, Schnöder T, Schmitt MJ, Lord JM, Roberts LM | title = Folding-competent and folding-defective forms of ricin A chain have different fates after retrotranslocation from the endoplasmic reticulum | journal = Molecular Biology of the Cell | volume = 21 | issue = 15 | pages = 2543–2554 | date = August 2010 | pmid = 20519439 | pmc = 2912342 | doi = 10.1091/mbc.E09-08-0743 }}</ref> but RTA avoids the [[ubiquitination]] that usually occurs with ERAD substrates because of its low content of [[lysine]] residues, which are the usual attachment sites for [[ubiquitin]].<ref name="pmid11876649">{{cite journal | vauthors = Deeks ED, Cook JP, Day PJ, Smith DC, Roberts LM, Lord JM | title = The low lysine content of ricin A chain reduces the risk of proteolytic degradation after translocation from the endoplasmic reticulum to the cytosol | journal = Biochemistry | volume = 41 | issue = 10 | pages = 3405–3413 | date = March 2002 | pmid = 11876649 | doi = 10.1021/bi011580v }}</ref> Thus, RTA avoids the usual fate of dislocated proteins (destruction that is mediated by targeting ubiquitinylated proteins to the cytosolic proteasomes). In the mammalian cell cytosol, RTA then undergoes triage by the cytosolic molecular chaperones [[Hsc70]] and [[Hsp90]] and their co-chaperones, as well as by one subunit (RPT5) of the [[proteasome]] itself, that results in its folding to a catalytic conformation,<ref name="Spooner" /><ref name="pmid23617410">{{cite journal | vauthors = Pietroni P, Vasisht N, Cook JP, Roberts DM, Lord JM, Hartmann-Petersen R, Roberts LM, Spooner RA | title = The proteasome cap RPT5/Rpt5p subunit prevents aggregation of unfolded ricin A chain | journal = The Biochemical Journal | volume = 453 | issue = 3 | pages = 435–445 | date = August 2013 | pmid = 23617410 | pmc = 3778710 | doi = 10.1042/BJ20130133 }}</ref> which de-purinates [[ribosome]]s, thus halting protein synthesis.

=== Ribosome inactivation ===
RTA has [[rRNA N-glycosylase|rRNA ''N''-glycosylase]] activity that is responsible for the cleavage of a [[glycosidic bond]] within the large [[ribosomal RNA|rRNA]] of the [[60S]] subunit of eukaryotic ribosomes.<ref name="pmid3036799">{{cite journal | vauthors = Endo Y, Tsurugi K | title = RNA N-glycosidase activity of ricin A-chain. Mechanism of action of the toxic lectin ricin on eukaryotic ribosomes | journal = The Journal of Biological Chemistry | volume = 262 | issue = 17 | pages = 8128–8130 | date = June 1987 | pmid = 3036799 | doi = 10.1016/S0021-9258(18)47538-2 | doi-access = free }}</ref> RTA specifically and irreversibly [[hydrolyses]] the ''N''-glycosidic bond of the [[adenine]] residue at position 4324 (A4324) within the [[28S ribosomal RNA|28S]] rRNA, but leaves the [[phosphodiester]] backbone of the RNA intact.<ref name="pmid3288622">{{cite journal | vauthors = Endo Y, Tsurugi K | title = The RNA N-glycosidase activity of ricin A-chain. The characteristics of the enzymatic activity of ricin A-chain with ribosomes and with rRNA | journal = The Journal of Biological Chemistry | volume = 263 | issue = 18 | pages = 8735–8739 | date = June 1988 | pmid = 3288622 | doi = 10.1016/S0021-9258(18)68367-X | doi-access = free }}</ref> The ricin targets A4324 that is contained in a highly [[conserved sequence]] of 12 [[nucleotide]]s universally found in eukaryotic ribosomes. The sequence, 5'-AGUACGAGAGGA-3', termed the sarcin-ricin loop, is important in binding [[elongation factor]]s during protein synthesis.<ref name="pmid4360718">{{cite journal | vauthors = Sperti S, Montanaro L, Mattioli A, Stirpe F | title = Inhibition by ricin of protein synthesis in vitro: 60 S ribosomal subunit as the target of the toxin | journal = The Biochemical Journal | volume = 136 | issue = 3 | pages = 813–815 | date = November 1973 | pmid = 4360718 | pmc = 1166019 | doi = 10.1042/bj1360813 }}</ref> The depurination event rapidly and completely inactivates the ribosome, resulting in toxicity from inhibited protein synthesis. A single RTA molecule in the [[cytosol]] is capable of depurinating approximately 1500 [[ribosomes]] per minute.

=== Depurination reaction ===
Within the active site of RTA, there exist several invariant amino acid residues involved in the [[depurination]] of ribosomal RNA.<ref name=pmid14579547/> Although the exact mechanism of the event is unknown, key amino acid residues identified include [[tyrosine]] at positions 80 and 123, [[glutamic acid]] at position 177, and [[arginine]] at position 180. In particular, Arg180 and Glu177 have been shown to be involved in the [[catalytic]] mechanism, and not substrate binding, with [[enzyme kinetics|enzyme kinetic]] studies involving RTA mutants. The model proposed by Mozingo and Robertus,<ref name=pmid7990130/> based on X-ray structures, is as follows:
# Sarcin-ricin loop substrate binds RTA active site with target adenine stacking against tyr80 and tyr123.
# Arg180 is positioned such that it can [[Protonation|protonate]] ''N''-3 of adenine and break the bond between ''N''-9 of the adenine ring and ''C''-1' of the [[ribose]].
# [[Bond cleavage]] results in an [[oxycarbonium]] ion on the ribose, stabilized by Glu177.
# ''N''-3 protonation of adenine by Arg180 allows [[deprotonation]] of a nearby water molecule.
# Resulting [[hydroxyl]] attacks ribose [[carbonium ion]].
# Depurination of adenine results in a neutral ribose on an intact phosphodiester RNA backbone.