Biomedical subjects

Peter D Watson

Publications and source records attributed to Peter D Watson.

4 recordsLinked to original sources

PubMed2006

Internalized Pseudomonas exotoxin A can exploit multiple pathways to reach the endoplasmic reticulum.

Receptor-mediated internalization to the endoplasmic reticulum (ER) and subsequent retro-translocation to the cytosol are essential sequential processes required for the intoxication of mammalian cells by Pseudomonas exotoxin A (PEx). The toxin binds the alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein. Here, we show that in HeLa cells, PEx recruits a proportion of this receptor to detergent-resistant microdomains (DRMs). Uptake of receptor-bound PEx involves transport steps both directly from early endosomes to the trans-Golgi network (TGN) independently of Rab9 function and from late endosomes to the TGN in a Rab9-dependent manner. Furthermore, treatments that simultaneously perturb both Arf1-dependent and Rab6-dependent retrograde pathways show that PEx can use multiple routes to reach the ER. The Rab6-dependent route has only been described previously for cargo with lipid-sorting signals. These findings suggest that partial localization of PEx within DRM permits a choice of trafficking routes consistent with a model that DRM-associated toxins reach the ER on a lipid-dependent sorting pathway whilst non-DRM-associated PEx exploits the previously characterized KDEL receptor-mediated uptake pathway. Thus, unexpectedly, an ER-directed toxin with a proteinaceous receptor shows promiscuity in its intracellular trafficking pathways, exploiting routes controlled by both lipid- and protein-sorting signals.

3T3 Cells↗

PubMed2005

The Reynolds Adolescent Depression Scale in New Zealand adolescents.

OBJECTIVE: To examine aspects of the reliability and validity of the Reynolds Adolescent Depression Scale (RADS) in measuring depression in New Zealand adolescents of all major ethnic groups. METHOD: A sample of 9699 randomly selected New Zealand secondary school students participated in the Youth2000 Health and Wellbeing Survey which included the RADS. Data from this survey have been used to assess some aspects of the reliability and validity of the RADS in the New Zealand context across different ethnic groups. Cronbach's alpha, item-total score correlations, correlation to other questions and a factor analysis were done in order to examine the internal reliability, content validity, convergent validity and construct validity of the data and compare to the original Reynolds validation study. RESULTS: Tests of the scale resulted in scores over 0.90 on Cronbach's alpha and high item-total score correlations, with a median correlation of 0.62 and 25 of the 30 correlations measuring more than 0.5. The scores were found to have similar factor structure to the original scale and the correlations to other depression related questions indicate acceptable concurrent validity. CONCLUSIONS: On all of the tests conducted, the RADS was found to have acceptable reliability and validity for New Zealand adolescents across the major different ethnic groups, indicating that it is a valid and appropriate instrument to use with New Zealand adolescents.

Adolescent↗

PubMed2004

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.

Abrin↗

PubMed2003

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.

Animals↗