Editing Ricin (section)

=== 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.