Projecto nº:016613

Referência do Projecto:PTDC/BEX-BCM/2311/2014 (POCI-01-0125-FEDER-016613)

Título:The molecular mechanisms of peroxisome biogenesis

Montante envolvidos:

Investimento total: 187.716,00 €

Apoio FEDER: 159.558,60€

Apoio OE: 25.157,40€

Localização do projecto: Porto, Portugal

Sintese do projecto:

Peroxisomes are single membrane-bound organelles involved in several metabolic pathways such as beta-oxidation of very long chain fatty acids, synthesis of ether glycerophospholipids and bile acids and reactive oxygen species detoxification. The vital importance of these organelles in human health and development is underscored by a group of genetic diseases, the peroxisomal biogenesis disorders, in which peroxisomes are partially or even completely dysfunctional. These devastating disorders are caused by mutations in genes encoding proteins specifically involved in the biogenesis of peroxisomes, the so-called peroxins or PEX proteins. From the 16 peroxins presently known in humans, 10 are mechanistically involved in sorting peroxisomal enzymes to the matrix of the organelle. 

Sorting of newly synthesized proteins to the peroxisomal matrix is a complex process involving the shuttling receptors PEX5 and PEX7, the ubiquitin-conjugating cascade and 2 peroxisomal membrane protein complexes - the docking/ translocation machinery (DTM) and the receptor-export module (REM). Collectively, these components ensure that the 50 or so different peroxisomal enzymes arrive at their final destination thus yielding a functional peroxisome.

Our group has been studying the mammalian peroxisomal protein import machinery (PIM) for some years now. During this time we have developed and refined several biochemical tools to study this machinery. Undoubtedly the most powerful one is a cell-free in vitro import/export system that recapitulates all the steps of the peroxisomal protein import pathway. The information we gathered with this system, together with biochemical data on the properties of some central peroxins (e.g., their protein interaction networks and their membrane topologies) are at the basis of our current model. In essence, we have proposed that the PIM operates through a syringe-like mechanism, in which a soluble shuttling receptor (PEX5 or a PEX5-PEX7 complex) binds a newly synthesized peroxisomal matrix protein in the cytosol and, acting as a plunge, pushes this protein all the way through the peroxisomal transmembrane DTM (the barrel) into the matrix of the organelle. Insertion of cargo-loaded receptor into the barrel  is an ATPindependent process, whereas its extraction involves the ubiquitin conjugating cascade and the action of the ATPdependent mechanoenzymes that comprise the REM.

Although our knowledge on the general mechanism of this protein import pathway seems fairly detailed, there are still many fundamental questions that remain unanswered. In this project we will address the following ones:

1) What is the mechanism ensuring that only cargo-loaded PEX5 enters the DTM? All the available data suggest that PEX5 is an autoregulated protein, exposing its DTM-interacting domain only when bound to a cargo. However, direct evidence supporting this idea is still lacking.

2) Which DTM components interact with PEX5 at the docking and translocation steps? Although the mammalian DTM comprises only five peroxins (PEX2, PEX10, PEX12, PEX13 and PEX14) it is still unknown which peroxins serve a docking sites and which peroxins are in contact with PEX5 when this receptor inserts into the DTM. Determining the identities of the DTM peroxins that bind PEX5 at the docking and insertion steps will provide invaluable data also on the architecture of the PIM and thus on the structure of the hydrophilic channel through which cargoes are translocated.

3) How does the REM recognize and extract PEX5 from the DTM? After delivering a cargo protein to the peroxisome matrix PEX5 has to be extracted from the DTM in order to catalyze further protein transport cycles. We do know that DTM-embedded PEX5 has to be mono-ubiquitinated at a conserved cysteine residue in order to be extracted back into the cytosol in an ATP-dependent manner by the REM. However, nothing else is known regarding the mechanism of this process.

Finally, in an independent task we will try to apply all the experimental strategies we developed in the last years to characterize a set of mutant PEX7 proteins responsible for Rhizomelic Chondrodysplasia Punctata type 1. One of the aims of this task is to gather structural/functional data that may be useful in any future attempt to treat this disorder using pharmacologic therapies.

Our team comprises researchers with a vast experience in mammalian peroxisome biogenesis. The two laboratories involved use different but complementary approaches to successfully address the topics of this project.

 

Galeria de fotos do projeto


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