1st class ticket to class I: protein toxins as pathfinders for antigen presentation

A number of bacterial toxins have evolved diverse strategies for crossing membrane barriers in order to reach their substrates in the mammalian cytosol. Recent studies show that this property can be exploited for the delivery of fused antigens into the major histocompatibility complex class I-restricted presentation pathway, with the goal of eliciting a specific immune response. Here we discuss the peculiarities of the trafficking pathways of a variety of toxins, and how these may allow the toxins to be used as delivery vehicles for therapeutic and diagnostic purposes.     

Transforming acidic coiled-coil-containing protein 4 interacts with centrosomal AKAP350 and the mitotic spindle apparatus

AKAP350 is a multiply spliced family of 350-450-kDa protein kinase A-anchoring proteins localized to the centrosomes and the Golgi apparatus. Using AKAP350A as bait in a yeast two-hybrid screen of a rabbit parietal cell library, we have identified a novel AKAP350-interacting protein, transforming acidic coiled-coil-containing protein 4 (TACC4). Two-hybrid binary assays demonstrate interaction of both TACC3 and TACC4 with AKAP350A and AKAP350B. Antibodies raised to a TACC4-specific peptide sequence colocalize TACC4 with AKAP350 at the centrosome in interphase Jurkat cells. Mitotic cell staining reveals translocation of TACC4 from the centrosome to the spindle apparatus with the majority of TACC4 at the spindle poles. Truncated TACC4 proteins lacking the AKAP350 minimal binding domain found in the carboxyl coiled-coil region of TACC4 could no longer target to the centrosome. Amino-truncated TACC4 proteins could no longer target to the spindle apparatus. Further, overexpression of TACC4 fusion proteins that retained spindle localization in mitotic cells resulted in an increased proportion of cells present in prometaphase. We propose that AKAP350 is responsible for sequestration of TACC4 to the centrosome in interphase, whereas a separate TACC4 domain results in functional localization of TACC4 to the spindle apparatus in mitotic cells.     

The low lysine content of ricin A chain reduces the risk of proteolytic degradation after translocation from the endoplasmic reticulum to the cytosol

Several protein toxins, including the A chain of ricin (RTA), enter mammalian cells by endocytosis and subsequently reach their cytosolic substrates by translocation across the endoplasmic reticulum (ER) membrane. To achieve this export, such toxins exploit the ER-associated protein degradation (ERAD) pathway but must escape, at least in part, the normal degradative fate of ERAD substrates. Toxins that translocate from the ER have an unusually low lysine content. Since lysyl residues are potential ubiquitination sites, it has been proposed that this paucity of lysines reduces the chance of ubiquitination and subsequent ubiquitin-mediated proteasomal degradation [Hazes, B., and Read, R. J. (1997) Biochemistry 36, 11051-11054]. Here we provide experimental support for this hypothesis. The two lysyl residues within RTA were changed to arginyl residues. Their replacement in RTA did not have a significant stabilizing effect, suggesting that the endogenous lysyl residues are not the usual sites for ubiquitin attachment. However, when four additional lysines were introduced into RTA in a way that did not compromise the activity, structure, or stability of the toxin, degradation was significantly enhanced. Enhanced degradation resulted from ubiquitination that predisposed the toxin to proteasomal degradation. Treatment with the proteasome inhibitor clasto-lactacystin beta-lactone increased the cytotoxicity of the lysine-rich RTA to a level approaching that of wild-type ricin. The introduction of four additional lysyl residues into a second ribosome-inactivating protein, abrin A chain, also dramatically decreased the cytotoxicity of the holotoxin compared to wild-type abrin. This effect could also be reversed by proteasomal inhibition. Our data support the hypothesis that the evolution of a low lysine content is a degradation-avoidance strategy for toxins that retrotranslocate from the ER.     

Effect of complexing agents on reduction of Cr(VI) by Desulfovibrio vulgaris ATCC 29579

The reduction of Cr(VI) at the expense of molecular hydrogen was studied using resting cells of Desulfovibrio vulgaris ATCC 29579 in anaerobic resting cell suspensions in MOPS buffer. Bioreduction occurred only in the presence of ligands or chelating agents (CO32-, citrate, NTA, EDTA, DTPA). The stimulatory effect of these ligands on the rate of Cr(VI) reduction was correlated (r = 0.988) with the strength of the ligand/chelate complex of Cr(III). The data are examined with respect to likely solution and redox equilibria in the ionic matrix of the carrier solution, and with respect to the potential for bioremediation of Cr(VI).     

Design, synthesis and in vitro evaluation of potent, novel, small molecule inhibitors of plasminogen activator inhibitor-1

We have synthesized and evaluated a series of tetramic acid-based and hydroxyquinolinone-based inhibitors of plasminogen activator inhibitor-1 (PAI-1). These studies resulted in the identification of several compounds which showed excellent potency against PAI-1. The design, synthesis and SAR of these compounds are described.     

Structure-activity relationships of non-imidazole H(3) receptor ligands. Part 2: binding preference for D-amino acids motifs

Structure-activity relationship studies on novel non-imidazole, D-amino acid containing ligands of histamine 3 receptors are presented. A-304121 is a D-alanine piperazine amide with high affinity at the rat H(3) receptor.     

Outer egg coats of the marsupial conceptus: secretion and protein composition

Little is known of the composition of the outer egg coats. We aimed to quantify secretion during embryonic development, identify precursor secreting cells and investigate protein composition. The study was based on 259 egg coats and 14 reproductive tracts of 104 T. vulpecula undergoing natural and induced cycles and 341 coats from 35 Sminthopsis macroura undergoing natural cycles. Following PAGE, Western blotting, and amino acid sequencing of egg coats, the short peptide sequences obtained from T. vulpecula and S. macroura coats were found to be dissimilar to each other and to any known protein. However, in T. vulpecula, S. macroura coat polyclonal antibody cross-reacted with coat precursors, suggesting similar epitopes, and showed mucoid precursors in secretory cells in oviduct epithelia and shell precursors in glands in the utero-tubal junction and uterus. Immuno-electron microscopy located shell coat precursors in various previously unidentified cell types, including pre-ovulatory apoptotic cells, early post-ovulatory holocrine cells, and milk-producing cells, found at blastocyst stages. Ultrastructural and quantitative volumetric analysis of the intact shell coat suggested a second wave of secretion at the blastocyst stages in T. vulpecula. Despite differences in protein composition, it was concluded that marsupial egg coats are homologous to each other because of similarities in ultrastructure and time and location of secretion.