Jorge E. Azevedo

Proteomics characterization of mouse kidney peroxisomes by tandem mass spectrometry and protein correlation profiling.

The peroxisome represents a ubiquitous single membrane-bound key organelle that executes various metabolic pathways such as fatty acid degradation by α- and β-oxidation, ether-phospholipid biosynthesis, metabolism of reactive oxygen species, and detoxification of glyoxylate in mammals. To fulfil this vast array of metabolic functions, peroxisomes accommodate ∼50 different enzymes at least as identified until now. Interest in peroxisomes has been fueled by the discovery of a group of genetic diseases in humans, which are caused by either a defect in peroxisome biogenesis or the deficient activity of a distinct peroxisomal enzyme or transporter. Although this research has greatly improved our understanding of peroxisomes and their role in mammalian metabolism, deeper insight into biochemistry and functions of peroxisomes is required to expand our knowledge of this low abundance but vital organelle. In this work, we used classical subcellular fractionation in combination with MS-based proteomics methodologies to characterize the proteome of mouse kidney peroxisomes. We could identify virtually all known components involved in peroxisomal metabolism and biogenesis. Moreover through protein localization studies by using a quantitative MS screen combined with statistical analyses, we identified 15 new peroxisomal candidates. Of these, we further investigated five candidates by immunocytochemistry, which confirmed their localization in peroxisomes. As a result of this joint effort, we believe to have compiled the so far most comprehensive protein catalogue of mammalian peroxisomes.

Chemical chaperones reduce endoplasmic reticulum stress and prevent mutant HFE aggregate formation.

HFE C282Y, the mutant protein associated with hereditary hemochromatosis (HH), fails to acquire the correct conformation in the endoplasmic reticulum (ER) and is targeted for degradation. We have recently shown that an active unfolded protein response (UPR) is present in the cells of patients with HH. Now, by using HEK 293T cells, we demonstrate that the stability of HFE C282Y is influenced by the UPR signaling pathway that promotes its degradation. Treatment of HFE C282Y-expressing cells with tauroursodeoxycholic acid (TUDCA), a bile acid derivative with chaperone properties, or with the chemical chaperone sodium 4-phenylbutyrate (4PBA) impeded the UPR activation. However, although TUDCA led to an increased stability of the mutant protein, 4PBA contributed to a more efficient disposal of HFE C282Y to the degradation route. Fluorescence microscopy and biochemical analysis of the subcellular localization of HFE revealed that a major portion of the C282Y mutant protein forms intracellular aggregates. Although neither TUDCA nor 4PBA restored the correct folding and intracellular trafficking of HFE C282Y, 4PBA prevented its aggregation. These data suggest that TUDCA hampers the UPR activation by acting directly on its signal transduction pathway, whereas 4PBA suppresses ER stress by chemically enhancing the ER capacity to cope with the expression of misfolded HFE, facilitating its degradation. Together, these data shed light on the molecular mechanisms involved in HFE C282Y-related HH and open new perspectives on the use of orally active chemical chaperones as a therapeutic approach for HH.

Ubiquitination of Mammalian Pex5p, the Peroxisomal Import Receptor

Protein translocation across the peroxisomal membrane requires the concerted action of numerous peroxins. One central component of this machinery is Pex5p, the cycling receptor for matrix proteins. Pex5p recognizes newly synthesized proteins in the cytosol and promotes their translocation across the peroxisomal membrane. After this translocation step, Pex5p is recycled back into the cytosol to start a new protein transport cycle. Here, we show that mammalian Pex5p is ubiquitinated at the peroxisomal membrane. Two different types of ubiquitination were detected, one of which is thiol-sensitive, involves Cys11 of Pex5p, and is necessary for the export of the receptor back into the cytosol. Together with mechanistic data recently described for yeast Pex5p, these findings provide strong evidence for the existence of Pex4p- and Pex22p-like proteins in mammals.

Characterization of peroxisomal Pex5p from rat liver. Pex5p in the Pex5p-Pex14p membrane complex is a transmembrane protein.

Pex5p is the receptor for the vast majority of peroxisomal matrix proteins. Here, we show that about 15% of rat liver Pex5p is found in the peroxisomal fraction representing 0.06% of total peroxisomal protein. This population of Pex5p displays all the characteristics of an intrinsic membrane protein. Protease protection assays indicate that this pool of Pex5p has domains exposed on both sides of the peroxisomal membrane. The strong interaction of Pex5p with the membrane of the organelle is not affected by mild protease treatment of intact organelles, conditions that result in the partial degradation of Pex13p. Cytosolic Pex5p is a monomeric protein. In contrast, virtually all peroxisomal Pex5p was found to be part of a stable 250-kDa protein assembly. This complex was isolated and shown to comprise just two subunits, Pex5p and Pex14p.

Members of the E2D (UbcH5) Family Mediate the Ubiquitination of the Conserved Cysteine of Pex5p, the Peroxisomal Import Receptor

According to current models of peroxisomal biogenesis, newly synthesized peroxisomal matrix proteins are transported into the organelle by Pex5p. Pex5p recognizes these proteins in the cytosol, mediates their membrane translocation, and is exported back into the cytosol in an ATP-dependent manner. We have previously shown that export of Pex5p is preceded by (and requires) monoubiquitination of a conserved cysteine residue present at its N terminus. In yeasts, and probably also in plants, ubiquitination of Pex5p is mediated by a specialized ubiquitin-conjugating enzyme, Pex4p. In mammals, the identity of this enzyme has remained unknown for many years. Here, we provide evidence suggesting that E2D1/2/3 (UbcH5a/b/c) are the mammalian functional counterparts of yeast/plant Pex4p. The mechanistic implications of these findings are discussed.

A nonsense mutation in the LIMP-2 gene associated with progressive myoclonic epilepsy and nephrotic syndrome

The main clinical features of two siblings from a consanguineous marriage were progressive myoclonic epilepsy without intellectual impairment and a nephrotic syndrome with a strong accumulation of C1q in capillary loops and mesangium of kidney. The biochemical analysis of one of the patients revealed a normal beta-glucocerebrosidase activity in leukocytes, but a severe enzymatic deficiency in cultured skin fibroblasts. This deficiency suggested a defect in the intracellular sorting pathway of this enzyme. The sequence analysis of the gene encoding LIMP-2 (SCARB2), the sorting receptor for beta-glucocerebrosidase, confirmed this hypothesis. A homozygous nonsense mutation in codon 178 of SCARB2 was found in the patient, whereas her healthy parents were heterozygous for the mutation. Besides lacking immunodetectable LIMP-2, patient fibroblasts also had decreased amounts of beta-glucocerebrosidase, which was mainly located in the endoplasmic reticulum, as assessed by its sensitivity to Endo H. This is the first report of a mutation in the SCARB2 gene associated with a human disease, which, contrary to earlier proposals, shares no features with Charcot-Marie-Tooth disease both at the clinical and neurophysiological levels.

The Energetics of Pex5p-mediated Peroxisomal Protein Import

Most newly synthesized peroxisomal matrix proteins are targeted to the organelle by Pex5p, the peroxisomal cycling receptor. According to current models of peroxisomal biogenesis, Pex5p interacts with cargo proteins in the cytosol and transports them to the peroxisomal membrane. After delivering the passenger protein into the peroxisomal matrix, Pex5p returns to the cytosol to catalyze additional rounds of transportation. Obviously, such cyclic pathway must require energy, and indeed, data confirming this need are already available. However, the exact step(s) of this cycle where energy input is necessary remains unclear. Here, we present data suggesting that insertion of Pex5p into the peroxisomal membrane does not require ATP hydrolysis. This observation raises the possibility that at the peroxisomal membrane ATP is needed predominantly (if not exclusively) downstream of the protein translocation step to reset the Pex5p-mediated transport system.

Insertion of Pex5p into the Peroxisomal Membrane Is Cargo Protein-dependent

It is now generally accepted that Pex5p, the receptor for most peroxisomal matrix proteins, cycles between the cytosol and the peroxisomal compartment. According to current models of peroxisomal biogenesis, this intracellular trafficking of Pex5p is coupled to the transport of newly synthesized peroxisomal proteins into the organelle matrix. However, direct evidence supporting this hypothesis was never provided. Here, using an in vitroperoxisomal import system, we show that insertion of Pex5p into the peroxisomal membrane requires the presence of cargo proteins. Strikingly the peroxisomal docking/translocation machinery is also able to catalyze the membrane insertion of a Pex5p truncated molecule lacking any known cargo-binding domain. These results suggest that the cytosol/peroxisomal cycle in which Pex5p is involved is directly or indirectly regulated by Pex5p itself and not by the peroxisomal docking/translocation machinery.

Properties of the Ubiquitin-Pex5p Thiol Ester Conjugate

Pex5p, the peroxisomal protein cycling receptor, binds newly synthesized peroxisomal matrix proteins in the cytosol and promotes their translocation across the organelle membrane. During its transient passage through the membrane, Pex5p is monoubiquitinated at a conserved cysteine residue, a requisite for its subsequent ATP-dependent export back into the cytosol. Here we describe the properties of the soluble and membrane-bound monoubiquitinated Pex5p species (Ub-Pex5p). Our data suggest that 1) Ub-Pex5p is deubiquitinated by a combination of context-dependent enzymatic and nonenzymatic mechanisms; 2) soluble Ub-Pex5p retains the capacity to interact with the peroxisomal import machinery in a cargo-dependent manner; and 3) substitution of the conserved cysteine residue of Pex5p by a lysine results in a quite functional protein both in vitro and in vivo. Additionally, we show that MG132, a proteasome inhibitor, blocks the import of a peroxisomal reporter protein in vivo.

PEX5 Protein Binds Monomeric Catalase Blocking Its Tetramerization and Releases It upon Binding the N-terminal Domain of PEX14

Background: PEX5 binds newly synthesized peroxisomal proteins in the cytosol and releases them in the organelle matrix. Results: PEX5 binds monomeric catalase and releases it in the presence of PEX14. Conclusion: PEX14 participates in the cargo release step. Significance: Knowing how PEX5 interacts with cargo proteins and which factors disrupt this interaction are crucial for understanding this protein sorting pathway. Newly synthesized peroxisomal matrix proteins are targeted to the organelle by PEX5. PEX5 has a dual role in this process. First, it acts as a soluble receptor recognizing these proteins in the cytosol. Subsequently, at the peroxisomal docking/translocation machinery, PEX5 promotes their translocation across the organelle membrane. Despite significant advances made in recent years, several aspects of this pathway remain unclear. Two important ones regard the formation and disruption of the PEX5-cargo protein interaction in the cytosol and at the docking/translocation machinery, respectively. Here, we provide data on the interaction of PEX5 with catalase, a homotetrameric enzyme in its native state. We found that PEX5 interacts with monomeric catalase yielding a stable protein complex; no such complex was detected with tetrameric catalase. Binding of PEX5 to monomeric catalase potently inhibits its tetramerization, a property that depends on domains present in both the N- and C-terminal halves of PEX5. Interestingly, the PEX5-catalase interaction is disrupted by the N-terminal domain of PEX14, a component of the docking/translocation machinery. One or two of the seven PEX14-binding diaromatic motifs present in the N-terminal half of PEX5 are probably involved in this phenomenon. These results suggest the following: 1) catalase domain(s) involved in the interaction with PEX5 are no longer accessible upon tetramerization of the enzyme; 2) the catalase-binding interface in PEX5 is not restricted to its C-terminal peroxisomal targeting sequence type 1-binding domain and also involves PEX5 N-terminal domain(s); and 3) PEX14 participates in the cargo protein release step.