Structural insights into K48-linked ubiquitin chain formation by the Pex4p-Pex22p complex.

Pex4p is a peroxisomal E2 involved in ubiquitinating the conserved cysteine residue of the cycling receptor protein Pex5p. Previously, we demonstrated that Pex4p from the yeast Saccharomyces cerevisiae binds directly to the peroxisomal membrane protein Pex22p and that this interaction is vital for receptor ubiquitination. In addition, Pex22p binding allows Pex4p to specifically produce lysine 48 linked ubiquitin chains in vitro through an unknown mechanism. This activity is likely to play a role in targeting peroxisomal proteins for proteasomal degradation. Here we present the crystal structures of Pex4p alone and in complex with Pex22p from the yeast Hansenula polymorpha. Comparison of the two structures demonstrates significant differences to the active site of Pex4p upon Pex22p binding while molecular dynamics simulations suggest that Pex22p binding facilitates active site remodelling of Pex4p through an allosteric mechanism. Taken together, our data provide insights into how Pex22p binding allows Pex4p to build K48-linked Ub chains.

Giardia lamblia: Identification of peroxisomal-like proteins.

The protozoan parasite Giardia lamblia has traditionally been reported as lacking peroxisomes, organelles involved in fatty acid metabolism and detoxification of reactive oxygen species. We here report the finding with transmission electron microscopy of an oxidase activity in cytoplasmic vesicles of trophozoites and cysts of G. lamblia. These vesicles were positive to 3,3'-diaminobenzidine and to cerium chloride staining. In addition, using bioinformatic tools, two peroxisomal proteins were identified in the G. lamblia proteome: acyl-CoA synthetase long chain family member 4 (ACSL-4) and peroxin-4 (PEX-4). With confocal and immunoelectron microscopy using polyclonal antibodies both proteins were identified in cytoplasmic vesicles of trophozoites. Altogether, our results suggest for the first time the presence of peroxisomal-like proteins in the cytoplasm of G. lamblia.

Regulation of peroxisomal matrix protein import by ubiquitination.

Peroxisomes are organelles that play an important role in many cellular tasks. The functionality of peroxisomes depends on the proper import of their matrix proteins. Peroxisomal matrix proteins are imported posttranslationally in a folded, sometimes even oligomeric state. They harbor a peroxisomal targeting sequence (PTS), which is recognized by dynamic PTS-receptors in the cytosol. The PTS-receptors ferry the cargo to the peroxisomal membrane, where they become part of a transient import pore and then release the cargo into the peroxisomal lumen. Subsequentially, the PTS-receptors are ubiquitinated in order to mark them for the export-machinery, which releases them back to the cytosol. Upon deubiquitination, the PTS-receptors can facilitate further rounds of cargo import. Because the ubiquitination of the receptors is an essential step in the import cycle, it also represents a central regulatory element that governs peroxisomal dynamics. In this review we want to give an introduction to the functional role played by ubiquitination during peroxisomal protein import and highlight the mechanistic concepts that have emerged based on data derived from different species since the discovery of the first ubiquitinated peroxin 15years ago. Moreover, we discuss future tasks and the potential of using advanced technologies for investigating further details of peroxisomal protein transport.

Distinct ubiquitination cascades act on the peroxisomal targeting signal type 2 co-receptor Pex18p.

Peroxisomal matrix protein import is facilitated by cycling receptors that recognize their cargo proteins in the cytosol by a peroxisomal targeting sequence (PTS) and ferry them to the peroxisomal membrane. Subsequently, the cargo is translocated into the peroxisomal lumen, whereas the receptor is released to the cytosol for further rounds of protein import. This cycle is controlled by the ubiquitination status of the receptor, which is best understood for the PTS1-receptor. While polyubiquitination of PTS-receptors results in their proteasomal degradation, the monoubiquitinated PTS-receptors are exported to the cytosol and recycled for further rounds of protein import. Here, we describe the identification of two ubiquitination cascades acting on the PTS2 co-receptor Pex18p. Using in vivo and in vitro approaches, we demonstrate that the polyubiquitination of Pex18p requires the ubiquitin-conjugating enzyme (E2) Ubc4p, which cooperates with the RING (really interesting new gene)-type ubiquitin-protein ligases (E3) Pex2p as well as Pex10p. Monoubiquitination of Pex18p depends on the E2 enzyme Pex4p (Ubc10p), which functions in concert with the E3 enzymes Pex12p and Pex10p. Our findings for the PTS2-pathway complement the data on PTS1-receptor ubiquitination and add up to a unified concept of the ubiquitin-based regulation of peroxisomal import.

Ubiquitination of the glycosomal matrix protein receptor PEX5 in Trypanosoma brucei by PEX4 displays novel features.

Trypanosomatids contain peroxisome-like organelles called glycosomes. Peroxisomal biogenesis involves a cytosolic receptor, PEX5, which, after its insertion into the organellar membrane, delivers proteins to the matrix. In yeasts and mammalian cells, transient PEX5 monoubiquitination at the membrane serves as the signal for its retrieval from the organelle for re-use. When its recycling is impaired, PEX5 is polyubiquitinated for proteasomal degradation. Stably monoubiquitinated TbPEX5 was detected in cytosolic fractions of Trypanosoma brucei, indicative for its role as physiological intermediate in receptor recycling. This modification's resistance to dithiothreitol suggests ubiquitin conjugation of a lysine residue. T. brucei PEX4, the functional homologue of the ubiquitin-conjugating (UBC) enzyme responsible for PEX5 monoubiquitination in yeast, was identified. It is associated with the cytosolic face of the glycosomal membrane, probably anchored by an identified putative TbPEX22. The involvement of TbPEX4 in TbPEX5 ubiquitination was demonstrated using procyclic ∆PEX4 trypanosomes. Surprisingly, glycosomal matrix protein import was only mildly affected in this mutant. Since other UBC homologues were upregulated, it might be possible that these have partially rescued PEX4's function in PEX5 ubiquitination. In addition, the altered expression of UBCs, notably of candidates involved in cell-cycle control, could be responsible for observed morphological and motility defects of the ∆PEX4 mutant.

Association of TP53 polymorphisms on the risk of Wilms tumor.

BACKGROUND: Molecular factors influencing Wilms tumor (WT) development remain largely unknown. TP53 mutations seem to be restricted to the anaplastic WT subtype. However, TP53 polymorphisms do not have a defined role in the disease. PROCEDURE: To assess the impact of TP53 mutations and polymorphisms (PIN2, PIN3, and PEX4) on risk of development, age at diagnosis, and survival in WT, we analyzed 46 blood DNA samples and 31 fresh tumor DNA samples from 52 patients with WT. Sequencing of TP53 exons 2-11 was performed. RESULTS: Tumor DNA analysis revealed TP53 pathogenic missense mutations (p.V197M, p.R213Q, p.R248W, and p.R337C) in four samples (12.9%). Blood DNA samples revealed a novel intronic mutation, IVS2 + 37C > T, in one patient (2.2%). Bilaterality was associated with a twofold decrease in survival (P = 0.00037). Diffuse anaplasia also presented a lower survival probability compared to patients with non-anaplastic tumors, or with focal anaplasia (P = 0.045). Patients with a TP53 somatic mutation showed survival probability of 37.5% versus 85.0% for patients with no somatic mutations, although the difference was not statistically significant (P = 0.0706). PIN3 duplicated allele was associated with a 20-month later mean age at diagnosis (P = 0.0084). TP53 PEX4 C allele showed an increased risk for WT development (P = 0.0379). No relationship was found between survival and gender, age at diagnosis, or the less frequent alleles of PIN2, PIN3, and PEX4. CONCLUSIONS: Our results demonstrate an association between PIN3 and age at diagnosis, as well as an association of PEX4 and risk of development of WT.

The Pex4p-Pex22p complex from Hansenula polymorpha: biophysical analysis, crystallization and X-ray diffraction characterization.

Peroxisomes are a major cellular compartment of eukaryotic cells, and are involved in a variety of metabolic functions and pathways according to species, cell type and environmental conditions. Their biogenesis relies on conserved genes known as PEX genes that encode peroxin proteins. Peroxisomal membrane proteins and peroxisomal matrix proteins are generated in the cytosol and are subsequently imported into the peroxisome post-translationally. Matrix proteins containing a peroxisomal targeting signal type 1 (PTS1) are recognized by the cycling receptor Pex5p and transported to the peroxisomal lumen. Pex5p docking, release of the cargo into the lumen and recycling involve a number of peroxins, but a key player is the Pex4p-Pex22p complex described in this manuscript. Pex4p from the yeast Saccharomyces cerevisiae is a ubiquitin-conjugating enzyme that is anchored on the cytosolic side of the peroxisomal membrane through its binding partner Pex22p, which acts as both a docking site and a co-activator of Pex4p. As Pex5p undergoes recycling and release, the Pex4p-Pex22p complex is essential for monoubiquitination at the conserved cysteine residue of Pex5p. The absence of Pex4p-Pex22p inhibits Pex5p recycling and hence PTS1 protein import. This article reports the crystallization of Pex4p and of the Pex4p-Pex22p complex from the yeast Hansenula polymorpha, and data collection from their crystals to 2.0 and 2.85 Šresolution, respectively. The resulting structures are likely to provide important insights to understand the molecular mechanism of the Pex4p-Pex22p complex and its role in peroxisome biogenesis.