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Background: We investigated the different modes of calreticulin-substrate binding. Results: Calreticulin binds glycosylated and nonglycosylated proteins with similar affinities but distinct kinetics and P-domain conformations. Conclusion: Successful substrate recruitment by calreticulin requires glycan and P-domain-dependent interactions. Significance: Elucidation of the distinct modes of calreticulin binding to substrate glycan and polypeptide components and their combined contributions to substrate recruitment in cells. [ABSTRACT FROM AUTHOR]

Calreticulin is a calcium-binding chaperone that has several functions in the immune response. In the endoplasmic reticulum (ER), calreticulin facilitates the folding of major histocompatibility complex (MHC) class I molecules and their assembly factor tapasin, thereby influencing antigen presentation to cytotoxic T cells. Although calreticulin is normally ER-resident, it is found at the cell surface of living cancer cells and dying cells. Here, calreticulin promotes cellular phagocytic uptake. In tumor vaccine models, drugs that induce cell surface calreticulin confer enhanced tumor protection in an extracellular calreticulin-dependent manner. Much remains to be understood about the roles of calreticulin in these distinct functions. Further investigations are important towards advancing basic knowledge of glycoprotein-folding pathways, and towards developing new cancer therapeutic strategies. [Copyright &y& Elsevier]

Calreticulin is a soluble calcium-binding chaperone of the endoplasmic reticulum (ER) that is also detected on the cell surface and in the cytosol. Calreticulin contains a single high affinity calcium-binding site within a globular domain and multiple low affinity sites within a C-terminal acidic region. We show that the secondary structure of calreticulin is remarkably thermostable at a given calcium concentration. Rather than corresponding to complete unfolding events, heat-induced structural transitions observed for calreticulin relate to tertiary structural changes that expose hydrophobic residues and reduce protein rigidity. The thermostability and the overall secondary structure content of calreticulin are impacted by the divalent cation environment, with the ER range of calcium concentrations enhancing stability, and calcium-depleting or high calcium environments reducing stability. Furthermore, magnesium competes with calcium for binding to calreticulin and reduces thermostability. The acidic domain of calreticulin is an important mediator of calcium-dependent changes in secondary structure content and thermostability. Together, these studies indicate interactions between the globular and acidic domains of calreticulin that are impacted by divalent cations. These interactions influence the structure and stability of calreticulin, and are likely to determine the multiple functional activities of calreticulin in different subcellular environments. [ABSTRACT FROM AUTHOR]

The Cramer classification scheme has emerged as one of the most extensively-adopted predictive toxicology tools, owing in part to its employment for chemical categorisation within threshold of toxicological concern evaluation. The characteristics of several of its rules have contributed to inconsistencies with respect to degree of hazard attributed to common (particularly food-relevant) substances. This investigation examines these discrepancies, and their origins, raising awareness of such issues amongst users seeking to apply and/or adapt the rule-set. A dataset of over 3000 compounds was assembled, each with Cramer class assignments issued by up to four groups of industry and academic experts. These were complemented by corresponding outputs from in silico implementations of the scheme present within Toxtree and OECD QSAR Toolbox software, including a working of a "Revised Cramer Decision Tree". Consistency between judgments was assessed, revealing that although the extent of inter-expert agreement was very high (≥97%), general concordance between expert and in silico calls was more modest (∼70%). In particular, 22 chemical groupings were identified to serve as prominent sources of disagreement, the origins of which could be attributed either to differences in subjective interpretation, to software coding anomalies, or to reforms introduced by authors of the revised rules. • Cramer Classes (CC) represent a structural categorisation scheme. • CC are relied upon in safety assessments based on the TTC approach. • Concordance between different CC assignment approaches was modest to very high. • A dataset of >3000 compounds was used to identify reasons for inconsistencies. [ABSTRACT FROM AUTHOR]

Abstract: Neuropathy target esterase (NTE) was discovered by M.K. Johnson in his quest for the entity responsible for the striking and mysterious paralysis brought about by certain organophosphorus (OP) esters. His pioneering work on OP neuropathy led to the view that the biochemical lesion consisted of NTE that had undergone OP inhibition and aging. Indeed, nonaging NTE inhibitors failed to produce disease but protected against neuropathy from subsequently administered aging inhibitors. Thus, inhibition of NTE activity was not the culprit; rather, formation of an abnormal protein was the agent of the disorder. More recently, however, Paul Glynn and colleagues showed that whereas conventional knockout of the NTE gene was embryonic lethal, conditional knockout of central nervous system NTE produced neurodegeneration, suggesting to these authors that the absence of NTE rather than its presence in some altered form caused disease. We now know that NTE is the 6th member of a 9-protein family called patatin-like phospholipase domain-containing proteins, PNPLA1–9. Mutations in the catalytic domain of NTE (PNPLA6) are associated with a slowly developing disease akin to OP neuropathy and hereditary spastic paraplegia called NTE-related motor neuron disorder (NTE-MND). Furthermore, the NTE protein from affected individuals has altered enzymological characteristics. Moreover, closely related PNPLA7 is regulated by insulin and glucose. These seemingly disparate findings are not necessarily mutually exclusive, but we need to reconcile recent genetic findings with the historical body of toxicological data indicating that inhibition and aging of NTE are both necessary in order to produce neuropathy from exposure to certain OP compounds. Solving this mystery will be satisfying in itself, but it is also an enterprise likely to pay dividends by enhancing our understanding of the physiological and pathogenic roles of the PNPLA family of proteins in neurological health and disease, including a potential role for NTE in diabetic neuropathy. [Copyright &y& Elsevier]