creating a
healthier future

Organelle Biogenesis & Function


Our group studies the molecular mechanisms of peroxisomal biogenesis, a process affected in several human genetic disorders. Our main focus is on a group of proteins which comprise the peroxisomal matrix protein Import machinery (PIM). These proteins include the RING finger peroxins (PEX2, PEX10 and PEX12) and other components of the ubiquitin-conjugating cascade (e.g., E2D1/2/3), ATPases of the AAA family (PEX1 and PEX6) as well as a group of proteins that comprise the peroxisomal protein translocon (e.g., PEX13 and PEX14). Thus, the general goal is not only to understand how newly synthesized (cytosolic) peroxisomal proteins are recognized, and translocated across the peroxisomal membrane in an already folded conformation, but also to contribute to a better understanding of the mechanisms used by AAA ATPases to remodel protein complexes and how protein activity can be modulated by transient ubiquitination. More recently, we are also interested in understanding the mechanisms of redox homeostasis in peroxisomes. We mainly use biochemical approaches to address these topics and we have developed a powerful cell-free in vitro system that recapitulates all the steps of peroxisomal protein import pathway.



In the last years, our group has made several important findings. One regards the conclusion that PEX5, the shuttle receptor for newly synthesized peroxisomal matrix proteins, is also a chaperone/holdase that binds its cargoes in the cytosol inhibiting their oligomerization. This finding, together with the observation that the monomeric versions of some peroxisomal proteins are far better substrates for the PIM than the corresponding oligomeric versions, argues against the long-proposed concept that peroxisomal proteins are imported into the organelle matrix already in their oligomeric native state.



In another study, the nature of the peroxisomal protein translocon was addressed. In contrast to current models claiming that PEX5 is a pore-forming toxin-like protein that becomes inserted into the peroxisomal membrane during the protein transport process, we found evidence suggesting that the peroxisomal protein translocon is actually the so-called docking/translocation module (a complex comprising PEX2, PEX10, PEX12, PEX13 and PEX14) which forms a large hydrophilic cavity into which PEX5 enters to release its cargoes.



Finally, new mechanistic data on PEX1 and PEX6 were also obtained. We demonstrated that DTM-embedded monoubiquitinated PEX5 is the bona fide substrate for these AAA ATPases and that PEX5 is globably unfolded during the ATP-dependent dislocation event, re-enforcing the idea that these mechanoenzymes unfold substrates using a processive threading mechanism.

Ubiquitination of human PEX5 at peroxisomes and its ATP-dependent export back into the cytosol as detected using an in vitro import/export system.


Selected Publications

Rodrigues T.A., Francisco T., Azevedo J.E.,
PEX13 Enters the RING, Lives Fast, Dies Young. Journal of Molecular Biology430(11):1559-1561, 2018. [Journal: Note] [IF: 5,1]
DOI: 10.1016/j.jmb.2018.04.005 SCOPUS: 85046171013

Barros-Barbosa A., Rodrigues T.A., Ferreira M.J., Pedrosa A.G., Teixeira N.R., Francisco T., Azevedo J.E.,
The intrinsically disordered nature of the peroxisomal protein translocation machinery. FEBS Journal286(1):24-38, 2019. [Journal: Article] [CI: 18] [IF: 4,4]
DOI: 10.1111/febs.14704 SCOPUS: 85057835122

Pedrosa A.G., Francisco T., Bicho D., Dias A.F., Barros-Barbosa A., Hagmann V., Dodt G., Rodrigues T.A., Azevedo J.E.,
Peroxisomal monoubiquitinated PEX5 interacts with the AAA ATPases PEX1 and PEX6 and is unfolded during its dislocation into the cytosol. Journal of Biological Chemistry293(29):11553-11563, 2018. [Journal: Article] [CI: 31] [IF: 4,1]
DOI: 10.1074/jbc.RA118.003669 SCOPUS: 85050390152

Francisco T., Rodrigues T.A., Dias A.F., Barros-Barbosa A., Bicho D., Azevedo J.E.,
Protein transport into peroxisomes: Knowns and unknowns. BioEssays39(10):, 2017. [Journal: Review] [CI: 54] [IF: 4,4]
DOI: 10.1002/bies.201700047 SCOPUS: 85029926379

Dias A.F., Rodrigues T.A., Pedrosa A.G., Barros-Barbosa A., Francisco T., Azevedo J.E.,
The peroxisomal matrix protein translocon is a large cavity-forming protein assembly into which PEX5 protein enters to release its cargo. Journal of Biological Chemistry292(37):15287-15300, 2017. [Journal: Article] [CI: 23] [IF: 4]
DOI: 10.1074/jbc.M117.805044 SCOPUS: 85029471726

Rodrigues T.A., Francisco T., Dias A.F., Pedrosa A.G., Grou C.P., Azevedo J.E.,
A cell-free organelle-based in vitro system for studying the peroxisomal protein import machinery. Nature Protocols11(12):2454-2469, 2016. [Journal: Article] [CI: 10] [IF: 10]
DOI: 10.1038/nprot.2016.147 SCOPUS: 84996598602

Freitas M.O., Francisco T., Rodrigues T.A., Lismont C., Domingues P., Pinto M.P., Grou C.P., Fransen M., Azevedo J.E.,
The peroxisomal protein import machinery displays a preference for monomeric substrates. Open Biology5(4):, 2015. [Journal: Article] [CI: 29] [IF: 4,8]
DOI: 10.1098/rsob.140236 SCOPUS: 84934927612

Ongoing Projects

A new component of the mammalian peroxisomal protein import machinery
Reference: 2022.08378.PTDC
Proponent: Instituto de Investigação e Inovação em Saúde - Universidade do Porto
Sponsor: FCT - Fundação para a Ciência e a Tecnologia
From 01-FEB-23 to 31-JUL-24
Unveiling peroxisomal homeostasis mechanisms – the role of LonP2
Reference: 2023.02428.RESTART
Proponent: Instituto de Investigação e Inovação em Saúde - Universidade do Porto
Sponsor: FCT - Fundação para a Ciência e a Tecnologia
From 01-DEC-23 to 31-MAY-25