Extraction of Soluble Proteins from Leaves.

Protein biochemistry can provide valuable answers to better understand plant performance and responses to the surrounding environment. In this chapter, we describe the process of extracting proteins from plant leaf samples. We highlight the key aspects to take into consideration to preserve protein integrity, from sample collection to extraction and preparation or storage for subsequent analysis of protein abundance and/or enzymatic activities.

The Use of Antibodies in Small-Molecule Drug Discovery.

Antibodies are powerful research tools that can be used in many areas of biology to probe, measure, and perturb various biological structures. Successful drug discovery is dependent on the correct identification of a target implicated in disease, coupled with the successful selection, optimization, and development of a candidate drug. Because of their specific binding characteristics, with regard to specificity, affinity, and avidity, coupled with their amenability to protein engineering, antibodies have become a key tool in drug discovery, enabling the quantification, localization, and modulation of proteins of interest. This review summarizes the application of antibodies and other protein affinity reagents as specific research tools within the drug discovery process.

The isolation and characterization of temperature-dependent ricin A chain molecules in Saccharomyces cerevisiae.

Ricin is a heterodimeric plant protein that is potently toxic to mammalian cells. Toxicity results from the catalytic depurination of eukaryotic ribosomes by ricin toxin A chain (RTA) that follows toxin endocytosis to, and translocation across, the endoplasmic reticulum membrane. To ultimately identify proteins required for these later steps in the entry process, it will be useful to express the catalytic subunit within the endoplasmic reticulum of yeast cells in a manner that initially permits cell growth. A subsequent switch in conditions to provoke innate toxin action would permit only those strains containing defects in genes normally essential for toxin retro-translocation, refolding or degradation to survive. As a route to such a screen, several RTA mutants with reduced catalytic activity have previously been isolated. Here we report the use of Saccharomyces cerevisiae to isolate temperature-dependent mutants of endoplasmic reticulum-targeted RTA. Two such toxin mutants with opposing phenotypes were isolated. One mutant RTA (RTAF108L/L151P) allowed the yeast cells that express it to grow at 37 degrees C, whereas the same cells did not grow at 23 degrees C. Both mutations were required for temperature-dependent growth. The second toxin mutant (RTAE177D) allowed cells to grow at 23 degrees C but not at 37 degrees C. Interestingly, RTAE177D has been previously reported to have reduced catalytic activity, but this is the first demonstration of a temperature-sensitive phenotype. To provide a more detailed characterization of these mutants we have investigated their N-glycosylation, stability, catalytic activity and, where appropriate, a three-dimensional structure. The potential utility of these mutants is discussed.

The N-terminal ricin propeptide influences the fate of ricin A-chain in tobacco protoplasts.

The plant toxin ricin is synthesized in castor bean seeds as an endoplasmic reticulum (ER)-targeted precursor. Removal of the signal peptide generates proricin in which the mature A- and B-chains are joined by an intervening propeptide and a 9-residue propeptide persists at the N terminus. The two propeptides are ultimately removed in protein storage vacuoles, where ricin accumulates. Here we have demonstrated that the N-terminal propeptide of proricin acts as a nonspecific spacer to ensure efficient ER import and glycosylation. Indeed, when absent from the N terminus of ricin A-chain, the non-imported material remained tethered to the cytosolic face of the ER membrane, presumably by the signal peptide. This species appeared toxic to ribosomes. The propeptide does not, however, influence catalytic activity per se or the vacuolar targeting of proricin or the rate of retrotranslocation/degradation of A-chain in the cytosol. The likely implications of these findings to the survival of the toxin-producing tissue are discussed.

Saporin and ricin A chain follow different intracellular routes to enter the cytosol of intoxicated cells.

Several protein toxins, such as the potent plant toxin ricin, enter mammalian cells by endocytosis and undergo retrograde transport via the Golgi complex to reach the endoplasmic reticulum (ER). In this compartment the catalytic moieties exploit the ER-associated degradation (ERAD) pathway to reach their cytosolic targets. Bacterial toxins such as cholera toxin or Pseudomonas exotoxin A carry KDEL or KDEL-like C-terminal tetrapeptides for efficient delivery to the ER. Chimeric toxins containing monomeric plant ribosome-inactivating proteins linked to various targeting moieties are highly cytotoxic, but it remains unclear how these molecules travel within the target cell to reach cytosolic ribosomes. We investigated the intracellular pathways of saporin, a monomeric plant ribosome-inactivating protein that can enter cells by receptor-mediated endocytosis. Saporin toxicity was not affected by treatment with Brefeldin A or chloroquine, indicating that this toxin follows a Golgi-independent pathway to the cytosol and does not require a low pH for membrane translocation. In intoxicated Vero or HeLa cells, ricin but not saporin could be clearly visualized in the Golgi complex using immunofluorescence. The saporin signal was not evident in the Golgi, but was found to partially overlap with that of a late endosome/lysosome marker. Consistently, the toxicities of saporin or saporin-based targeted chimeric polypeptides were not enhanced by the addition of ER retrieval sequences. Thus, the intracellular movement of saporin differs from that followed by ricin and other protein toxins that rely on Golgi-mediated retrograde transport to reach their retrotranslocation site.

Ricin: current understanding and prospects for an antiricin vaccine.

Ricin is a potent cytotoxin that can be rapidly internalized into mammalian cells leading to cell death. The ease in obtaining the toxin and its deadly nature combine to implicate ricin as a convenient agent for bioterrorism. Research into the mechanism of toxicity, as well as strategies for treatment and protection from the toxin has been widely undertaken for a number of years. This article reviews the current understanding of the mechanism of action of the toxin, the clinical effects of ricin intoxication and how these relate to current and continuing prospects for vaccine development.

Protein disulphide-isomerase reduces ricin to its A and B chains in the endoplasmic reticulum.

Cells expressing ricin B chain within the secretory pathway are significantly more resistant to intoxication by ricin holotoxin but not to other cytotoxins that exploit similar endocytic routes to the cytosol. Furthermore, cells expressing the related B chain of abrin are protected against both incoming abrin and ricin. These phenotypes can be correlated with the abilities of the respective B chains to form disulphide-linked A-B holotoxins, since abrin B chain forms heterodimers with either abrin or ricin A chains, whereas ricin B chain forms heterodimers with ricin A chain only. In the ricin B-expressing cells, this newly made lectin disappears with biphasic kinetics comprising a retention phase followed by slow turnover and disposal after disengagement from calnexin cycle components. Interference with ricin cytotoxicity occurs during the early retention phase when ricin B chain is associated with PDI (protein disulphide-isomerase). The data show that retrotranslocation of incoming toxin is impeded by PDI-catalysed formation of heterodimers between endogenous B and A chains derived from reduced holotoxin, thus proving that reduction of ricin occurs in the endoplasmic reticulum. In contrast with other toxins, ricin does not appear to require either proteolytic cleavage or unfolding for PDI-catalysed reduction.

Insertional mutagenesis of ricin A chain: a novel route to an anti-ricin vaccine.

The insertion of a specific 25-residue internal peptide into ricin toxin A chain (RTA) reduced the catalytic activity of this protein approximately 300-fold. Directed proteolytic cleavage of the peptide insert essentially restored catalytic activity of the resulting two peptide A chain to normal levels. Ricin holotoxin containing unprocessed mutant A chain was not toxic to cultured mammalian cells, due to enhanced proteasomal degradation, nor was it toxic when injected into rats at a concentration that is lethal in the case of native ricin. Rats treated in this way were completely resistant to native ricin when subsequently challenged with a potentially lethal dose of the toxin. These ricin-resistant animals had a significant anti-ricin antibody titer, indicating that this approach has potential for developing an effective vaccine against this toxin.

The effect of mutations surrounding and within the active site on the catalytic activity of ricin A chain.

Models for the binding of the sarcin-ricin loop (SRL) of 28S ribosomal RNA to ricin A chain (RTA) suggest that several surface exposed arginine residues surrounding the active site cleft make important interactions with the RNA substrate. The data presented in this study suggest differing roles for these arginyl residues. Substitution of Arg48 or Arg213 with Ala lowered the activity of RTA 10-fold. Furthermore, substitution of Arg213 with Asp lowered the activity of RTA 100-fold. The crystal structure of this RTA variant showed it to have an unaltered tertiary structure, suggesting that the positively charged state of Arg213 is crucial for activity. Substitution of Arg258 with Ala had no effect on activity, although substitution with Asp lowered activity 10-fold. Substitution of Arg134 prevented expression of folded protein, suggesting a structural role for this residue. Several models have been proposed for the binding of the SRL to the active site of RTA in which the principal difference lies in the conformation of the second 'G' in the target GAGA motif in the 28S rRNA substrate. In one model, the sidechain of Asn122 is proposed to make interactions with this G, whereas another model proposes interactions with Asp75 and Asn78. Site-directed mutagenesis of these residues of RTA favours the first of these models, as substitution of Asn78 with Ser yielded an RTA variant whose activity was essentially wild-type, whereas substitution of Asn122 reduced activity 37.5-fold. Substitution of Asp75 failed to yield significant folded protein, suggesting a structural role for this residue.

Ricin. Mechanisms of cytotoxicity.

Ricin is a heterodimeric protein produced in the seeds of the castor oil plant (Ricinus communis). It is exquisitely potent to mammalian cells, being able to fatally disrupt protein synthesis by attacking the Achilles heel of the ribosome. For this enzyme to reach its substrate, it must not only negotiate the endomembrane system but it must also cross an internal membrane and avoid complete degradation without compromising its activity in any way. Cell entry by ricin involves a series of steps: (i) binding, via the ricin B chain (RTB), to a range of cell surface glycolipids or glycoproteins having beta-1,4-linked galactose residues; (ii) uptake into the cell by endocytosis; (iii) entry of the toxin into early endosomes; (iv) transfer, by vesicular transport, of ricin from early endosomes to the trans-Golgi network; (v) retrograde vesicular transport through the Golgi complex to reach the endoplasmic reticulum; (vi) reduction of the disulphide bond connecting the ricin A chain (RTA) and the RTB; (vii) partial unfolding of the RTA to render it translocationally-competent to cross the endoplasmic reticulum (ER) membrane via the Sec61p translocon in a manner similar to that followed by misfolded ER proteins that, once recognised, are targeted to the ER-associated protein degradation (ERAD) machinery; (viii) avoiding, at least in part, ubiquitination that would lead to rapid degradation by cytosolic proteasomes immediately after membrane translocation when it is still partially unfolded; (ix) refolding into its protease-resistant, biologically active conformation; and (x) interaction with the ribosome to catalyse the depurination reaction. It is clear that ricin can take advantage of many target cell molecules, pathways and processes. It has been reported that a single molecule of ricin reaching the cytosol can kill that cell as a consequence of protein synthesis inhibition. The ready availability of ricin, coupled to its extreme potency when administered intravenously or if inhaled, has identified this protein toxin as a potential biological warfare agent. Therapeutically, its cytotoxicity has encouraged the use of ricin in 'magic bullets' to specifically target and destroy cancer cells, and the unusual intracellular trafficking properties of ricin potentially permit its development as a vaccine vector. Combining our understanding of the ricin structure with ways to cripple its unwanted properties (its enzymatic activity and promotion of vascular leak whilst retaining protein stability and important immunodominant epitopes), will also be crucial in the development of a long awaited protective vaccine against this toxin.

Expression, Purification and Characterization of Ricin vectors used for exogenous antigen delivery into the MHC Class I presentation pathway.

Disarmed versions of the cytotoxin ricin can deliver fused peptides into target cells leading to MHC class I-restricted antigen presentation [Smith et al. J Immunol 2002; 169:99-107]. The ricin delivery vector must contain an attenuated catalytic domain to prevent target cell death, and the fused peptide epitope must remain intact for delivery and functional loading to MHC class I molecules. Expression in E. coli and purification by cation exchange chromatography of the fusion protein is described. Before used for delivery, the activity of the vector must be characterized in vitro, via an N-glycosidase assay, and in vivo, by a cytotoxicity assay. The presence of an intact epitope must be confirmed using mass spectrometry by comparing the actual mass with the predicted mass.