Abstract. Import of newly synthesized PTS1 proteins into the peroxisome requires the PTS1 receptor (Pex5p), a predominantly cytoplasmic protein that cycles between the cytoplasm and peroxisome. We have identified Pex13p, a novel integral peroxisomal membrane from both yeast and humans that binds the PTS1 receptor via a cytoplasmically oriented SH3 domain. Although only a small amount of Pex5p is bound to peroxisomes at steady state (<5%), loss of Pex13p further reduces the amount of peroxisome-associated Pex5p by ~40-fold. Furthermore, loss of Pex13p eliminates import of peroxisomal matrix proteins that contain either the type-1 or type-2 peroxisomal targeting signal but does not affect targeting and insertion of integral peroxisomal membrane proteins. We conclude that Pexl3p functions as a docking factor for the predominantly cytoplasmic PTS1 receptor.

The role of exosomes in cancer development has become the focus of much research, due to the many emerging roles possessed by exosomes. These micro-vesicles that are ubiquitously released in to the extracellular milieu, have been found to regulate immune system function, particularly in tumorigenesis, as well as conditioning future metastatic sites for the attachment and growth of tumor tissue. Through an interaction with a range of host tissue, exosomes are able to generate a pro-tumor environment that is essential for carcinogenesis. Herein, we discuss the contents of exosomes and their contribution to tumorigenesis, as well as their role in chemotherapeutic resistance and the development of novel cancer treatments and the identification of cancer biomarkers.

Pex5p, a receptor for peroxisomal matrix proteins with a type 1 peroxisome targeting signal (PTS1), has been proposed to cycle from the cytoplasm to the peroxisomal membrane where it docks with Pex14p and Pex13p, the latter an SH3 domain-containing protein.Using in vitro binding assays we have demonstrated that binding of Pex5p to Pex14p is enhanced when Pex5p is loaded with a PTS1-containing peptide. In contrast, Pex5p binding to Pex13p, which involves only the SH3 domain, occurs at 20 -40-fold lower levels and is reduced when Pex5p is preloaded with a PTS1 peptide. Pex14p was also shown to bind weakly to the Pex13p SH3 domain. Site-directed mutagenesis of the Pex13p SH3 domain attenuated binding to Pex5p and Pex14p, consistent with both of these proteins being binding partners for this domain. The SH3 binding site in Pex5p was determined to lie within a 114-residue peptide (Trp 100 -Glu 213 ) in the amino-terminal region of the protein. The interaction between this peptide and the SH3 domain was competitively inhibited by Pex14p. We interpret these data as suggesting that docking of the Pex5p-PTS1 protein complex at the peroxisome membrane occurs at Pex14p and that the Pex13p SH3 domain functions as an associated component possibly involved in sequestering Pex5p after relinquishment of the PTS1 protein cargo to components of the translocation machinery.

hSSB1 is a recently discovered single-stranded DNA binding protein that is essential for efficient repair of DNA double-strand breaks (DSBs) by the homologous recombination pathway. hSSB1 is required for the efficient recruitment of the MRN complex to sites of DSBs and for the efficient initiation of ATM dependent signalling. Here we explore the interplay between hSSB1 and MRN. We demonstrate that hSSB1 binds directly to NBS1, a component of the MRN complex, in a DNA damage independent manner. Consistent with the direct interaction, we observe that hSSB1 greatly stimulates the endo-nuclease activity of the MRN complex, a process that requires the C-terminal tail of hSSB1. Interestingly, analysis of two point mutations in NBS1, associated with Nijmegen breakage syndrome, revealed weaker binding to hSSB1, suggesting a possible disease mechanism.

Historically, two-dimensional (2D) cell culture has been the preferred method of producing disease models in vitro. Recently, there has been a move away from 2D culture in favor of generating three-dimensional (3D) multicellular structures, which are thought to be more representative of the in vivo environment. This transition has brought with it an influx of technologies capable of producing these structures in various ways. However, it is becoming evident that many of these technologies do not perform well in automated in vitro drug discovery units. We believe that this is a result of their incompatibility with high-throughput screening (HTS). In this study, we review a number of technologies, which are currently available for producing in vitro 3D disease models. We assess their amenability with high-content screening and HTS and highlight our own work in attempting to address many of the practical problems that are hampering the successful deployment of 3D cell systems in mainstream research.

BackgroundPremature aging syndromes recapitulate many aspects of natural aging and provide an insight into this phenomenon at a molecular and cellular level. The progeria syndromes appear to cause rapid aging through disruption of normal nuclear structure. Recently, a coding mutation (c.34G > A [p.A12T]) in the Barrier to Autointegration Factor 1 (BANF1) gene was identified as the genetic basis of Néstor-Guillermo Progeria syndrome (NGPS). This mutation was described to cause instability in the BANF1 protein, causing a disruption of the nuclear envelope structure.ResultsHere we demonstrate that the BANF1 A12T protein is indeed correctly folded, stable and that the observed phenotype, is likely due to the disruption of the DNA binding surface of the A12T mutant. We demonstrate, using biochemical assays, that the BANF1 A12T protein is impaired in its ability to bind DNA while its interaction with nuclear envelope proteins is unperturbed. Consistent with this, we demonstrate that ectopic expression of the mutant protein induces the NGPS cellular phenotype, while the protein localizes normally to the nuclear envelope.ConclusionsOur study clarifies the role of the A12T mutation in NGPS patients, which will be of importance for understanding the development of the disease.

The peroxisome-biogenesis disorders (PBDs) are a genetically and phenotypically diverse group of diseases caused by defects in peroxisome assembly. One of the milder clinical variants within the PBDs is neonatal adrenoleukodystrophy (NALD), a disease that is usually associated with partial defects in the import of peroxisomal matrix proteins that carry the type 1 or type 2 peroxisomal targeting signals. Here, we characterize the sole representative of complementation group 13 of the PBDs, a patient with NALD (patient PBD222). Skin fibroblasts from patient PBD222 display defects in the import of multiple peroxisomal matrix proteins. However, residual matrix-protein import can be detected in cells from patient PBD222, consistent with the relatively mild phenotypes of the patient. PEX13 encodes a peroxisomal membrane protein with a cytoplasmically exposed SH3 domain, and we find that expression of human PEX13 restores peroxisomal matrix-protein import in cells from patient PBD222. Furthermore, these cells are homozygous for a missense mutation at a conserved position in the PEX13 SH3 domain. This mutation attenuated the activity of human PEX13, and an analogous mutation in yeast PEX13 also reduced its activity. The mutation was absent in >100 control alleles, indicating that it is not a common polymorphism. Previous studies have demonstrated extragenic suppression in the PBDs, but the phenotypes of patient PBD222 cells could not be rescued by expression of any other human PEX genes. Taken together, these results provide strong evidence that mutations in PEX13 are responsible for disease in patient PBD222 and, by extension, in complementation group 13 of the PBDs.

The proper function of the spindle is crucial to the high fidelity of chromosome segregation and is indispensable for tumor suppression in humans. Centrobin is a recently identified centrosomal protein that has a role in stabilizing the microtubule structure. Here we functionally characterize the defects in centrosome integrity and spindle assembly in Centrobin-depleted cells. Centrobin-depleted cells show a range of spindle abnormalities including unfocused poles that are not associated with centrosomes, S-shaped spindles and mini spindles. These cells undergo mitotic arrest and subsequently often die by apoptosis, as determined by live cell imaging. Co-depletion of Mad2 relieves the mitotic arrest, indicating that cells arrest due to a failure to silence the spindle checkpoint in metaphase. Consistent with this, Centrobin-depleted metaphase cells stained positive for BubR1 and BubR1 S676. Staining with a panel of centrosome markers showed a loss of centrosome anchoring to the mitotic spindle. Furthermore, these cells show less cold-stable microtubules and a shorter distance between kinetochore pairs. These results show a requirement of Centrobin in maintaining centrosome integrity, which in turn promotes anchoring of mitotic spindle to the centrosomes. Furthermore, this anchoring is required for the stability of microtubulekinetochore attachments and biogenesis of tension-ridden and properly functioning mitotic spindle.