Signaling signatures and functional properties of anti-human CD28 superagonistic antibodies

Superagonistic CD28 antibodies (CD28SAs) activate T lymphocytes without concomitant perturbation of the TCR/CD3-complex. In rodents these reagents induce the preferential expansion of regulatory T cells and can be used for the treatment of autoimmune diseases. Unexpectedly, the humanized CD28 superagonist TGN1412 caused severe and life threatening adverse effects during a recently conducted phase I clinical trail. The underlying molecular mechanisms are as yet unclear. We show that TGN1412 as well as the commercially available CD28 superagonist ANC28.1 induce a delayed but extremely sustained calcium response in human naïve and memory CD4⁺ T cells but not in cynomolgus T lymphocytes. The sustained Ca⁺⁺-signal was associated with the activation of multiple intracellular signaling pathways and together these events culminated in the rapid de novo synthesis of high amounts of pro-inflammatory cytokines, most notably IFN-γ and TNF-α. Importantly, sustained transmembranous calcium flux, activation of Src-kinases as well as activation of PI3K were found to be absolutely required for CD28SA-mediated production of IFN-γ and IL-2. Collectively, our data suggest a molecular basis for the severe side effects caused by TGN1412 and impinge upon the relevance of non-human primates as preclinical models for reagents that are supposed to modify the function of human T cells.     

Comparison of the PTS1- and Rab8b-binding properties of Pex5p and Pex5Rp/TRIP8b

Tetratricopeptide (TPR)-domain proteins are involved in various cellular processes. The TPR domain is known to be responsible for interaction with other proteins commonly recognizing sequence motifs at the C-termini. One such TPR-protein, TRIP8b, was originally identified in rat as an interaction partner of Rab8b, and its human orthologue as a protein related to the peroxisomal targeting signal 1 (PTS1) receptor Pex5p (Pex5Rp). Somewhat later, the mouse orthologue was reported to bind the hyperpolarization-activated, cyclic nucleotide-regulated HCN channels, and, very recently, the rat orthologue was shown to interact with latrophilin 1, the calcium-independent receptor of alpha-latrotoxin. Here we employed various methodological approaches to investigate and compare the binding specificities of the human PTS1 receptor Pex5p and the related protein Pex5Rp/TRIP8b towards a subset of targets, including Rab8b and various C-termini resembling PTS1. The results show that the TPR domains of Pex5p and Pex5Rp/TRIP8b have distinct but overlapping substrate specificities. This suggests that selectivity in the recognition of substrates by the TPR domains of Pex5p and Pex5Rp/TRIP8b is a matter of considerable complexity, and that no single determinant appears to be sufficient in unambiguously defining a binding target for either protein. This idea is further corroborated by our observations that changes in the surrounding residues or the conformational state of one of the binding partners can profoundly alter their binding activities. The implications of these findings for the possible peroxisome-related functions of Pex5Rp/TRIP8b are discussed.     

Targeting of malate synthase 1 to the peroxisomes of Saccharomyces cerevisiae cells depends on growth on oleic acid medium.

The eukaryotic glyoxylate cycle has been previously hypothesized to occur in the peroxisomal compartment, which in the yeast Saccharomyces cerevisiae additionally represents the sole site for fatty acid beta-oxidation. The subcellular location of the key glyoxylate-cycle enzyme malate synthase 1 (Mls1p), an SKL-terminated protein, was examined in yeast cells grown on different carbon sources. Immunoelectron microscopy in combination with cell fractionation showed that Mls1p was abundant in the peroxisomes of cells grown on oleic acid, whereas in ethanol-grown cells Mls1p was primarily cytosolic. This was reinforced using a green fluorescent protein (GFP)-Mls1p reporter, which entered peroxisomes solely in cells grown under oleic acid-medium conditions. Although growth of cells devoid of Mls1p on ethanol or acetate could be fully restored using a cytosolic Mls1p devoid of SKL, this construct could only partially alleviate the requirement for native Mls1p in cells grown on oleic acid. The combined results indicated that Mls1p remained in the cytosol of cells grown on ethanol, and that targeting of Mls1p to the peroxisomes was advantageous to cells grown on oleic acid as a sole carbon source.     

Evidence for diet partitioning among three coexisting native freshwater fishes in South Africa's Cape Fold Ecoregion

The partitioning of limited resources commonly explains how different species can coexist within the same ecological community. In this 2010 study, the diets of three coexisting freshwater fishes (Cape galaxias Galaxias zebratus, n = 27; Cape kurper Sandelia capensis, n = 60; Breede River redfin Pseudobarbus burchelli, n = 77) were characterised and compared in three headwater streams in South Africa's Cape Fold Ecoregion using gut contents and stable isotope analyses. These data were analysed to ascertain whether the three species exploit distinct trophic niches. Both approaches provided evidence that these species occupy different trophic niches, though with some overlap. However, dietary differences among sites were not consistent and were probably influenced by site-specific factors like resource availability. Pseudobarbus burchelli had a broader niche breadth at Tierkloof Stream than the other two species, but not at Waaihoek or Tierstel Streams. Our results also suggest that P. burchelli consumed a more omnivorous diet than do the other two species, whereas S. capensis occupied a higher trophic position than the other two species and consumed vertebrates. Our findings suggest that these species occupy non-equivalent feeding niches in Cape Fold Ecoregion headwater streams, and that diet partitioning might facilitate their coexistence in these systems.     

Pex14p is a member of the protein linkage map of Pex5p.

To identify members of the translocation machinery for peroxisomal proteins, we made use of the two-hybrid system to establish a protein linkage map centered around Pex5p from Saccharomyces cerevisiae, the receptor for the C-terminal peroxisomal targeting signal (PTS1). Among the five interaction partners identified, Pex14p was found to be induced under conditions allowing peroxisome proliferation. Deletion of the corresponding gene resulted in the inability of yeast cells to grow on oleate as well as the absence of peroxisomal structures. The PEX14 gene product of approximately 38 kDa was biochemically and ultrastructurally demonstrated to be a peroxisomal membrane protein, despite the lack of a membrane-spanning domain. This protein was shown to interact with itself, with Pex13p and with both PTS receptors, Pex5p and Pex7p, indicating a central function for the import of peroxisomal matrix proteins, either as a docking protein or as a releasing factor at the organellar membrane.     

Alternatives to the isomerase-dependent pathway for the beta-oxidation of oleic acid are dispensable in Saccharomyces cerevisiae. Identification of YOR180c/DCI1 encoding peroxisomal delta(3,5)-delta(2,4)-dienoyl-CoA isomerase.

Fatty acids with double bonds at odd-numbered positions such as oleic acid can enter beta-oxidation via a pathway relying solely on the auxiliary enzyme Delta(3)-Delta(2)-enoyl-CoA isomerase, termed the isomerase-dependent pathway. Two novel alternative pathways have recently been postulated to exist in mammals, and these additionally depend on Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase (di-isomerase-dependent) or on Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase and 2,4-dienoyl-CoA reductase (reductase-dependent). We report the identification of the Saccharomyces cerevisiae oleic acid-inducible DCI1 (YOR180c) gene encoding peroxisomal di-isomerase. Enzyme assays conducted on soluble extracts derived from yeast cells overproducing Dci1p using 3,5,8,11,14-eicosapentenoyl-CoA as substrate demonstrated a specific di-isomerase activity of 6 nmol x min(-1) per mg of protein. Similarly enriched extracts from eci1Delta cells lacking peroxisomal 3,2-isomerase additionally contained an intrinsic 3,2-isomerase activity that could generate 3, 5,8,11,14-eicosapentenoyl-CoA from 2,5,8,11,14-eicosapentenoyl-CoA but not metabolize trans-3-hexenoyl-CoA. Amplification of this intrinsic activity replaced Eci1p since it restored growth of the eci1Delta strain on petroselinic acid for which di-isomerase is not required whereas Eci1p is. Heterologous expression in yeast of rat di-isomerase resulted in a peroxisomal protein that was enzymatically active but did not re-establish growth of the eci1Delta mutant on oleic acid. A strain devoid of Dci1p grew on oleic acid to wild-type levels, whereas one lacking both Eci1p and Dci1p grew as poorly as the eci1Delta mutant. Hence, we reasoned that yeast di-isomerase does not additionally represent a physiological 3,2-isomerase and that Dci1p and the postulated alternative pathways in which it is entrained are dispensable for degrading oleic acid.     

Functional flower traits and their diversity drive pollinator visitation

Recent studies have shown that the diversity of flowering plants can enhance pollinator richness and visitation frequency and thereby increase the resilience of pollination. It is assumed that flower traits explain these effects, but it is still unclear which flower traits are responsible, and knowing that, if pollinator richness and visitation frequency are more driven by mass‐ratio effects (mean trait values) or by trait diversity. Here, we analyse a three‐year data set of pollinator observations collected in a European grassland plant diversity experiment (The Jena experiment). The data entail comprehensive flower trait measurements, including reward traits (nectar and pollen amount), morphological traits (height, symmetry, area, colour spectra) and chemical traits (nectar‐amino acid and nectar‐sugar concentration). We test if pollinator species richness and visitation frequency of flower communities depend on overall functional diversity combining all flower traits within a community, single trait diversities (within trait variation) and community‐weighted means of the single traits, using Bayesian inference. Overall functional diversity did not affect pollinator species richness, but reduced visitation frequency. When looking at individual flower traits separately, we found that single trait diversity of flower reflectance and flower morphology were important predictors of pollinator visitation frequency. Moreover, independent of total flower abundance, community‐weighted means of flower height, area, reflectance, nectar‐sugar concentration and nectar‐amino acid concentration strongly affected both pollinator species richness and visitation frequency. Our results, challenge the idea that functional diversity always positively affects ecosystem functions. Nonetheless, we demonstrate that both single trait diversity and mass‐ratio effects of flower traits play an important role for diverse and frequent flower visits, which underlines the functionality of flower traits for pollination services.