5. RELEVANCIA ECONÓMICA Y FINANCIERA DE UN SISTEMA DE
5.7 La regulación de las inversiones de las pensiones
All members of the Rab32 subfamily are involved in the organisation of the trans-‐Golgi network. Rab32/38 are best characterised for their role in melanogenesis and the regulation of lysosome-‐related organelles, while the related Rab29 is implicated in retromer-‐mediated trafficking [176, 179]. Rab32/38 bind to the ankyrin domain of their shared effector VARP, which regulates endosomal dynamics [182]. However, in the case of LRRK2, Rab32/38 was shown in interact with the N-‐terminal armadillo repeats as opposed to the adjacent ankyrin
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distinguishing between similar domains within different proteins. Through binding with the ARM domain, Rab32/38 were shown to localise LRRK2 to transport vesicles and recycling endosomes [300]. The biological significance of this has not yet been established.
The related Rab29 GTPase recruits LRRK2 to the trans-‐Golgi network, and was reported to interact via the ankyrin domain. This was inferred from the introduction of mutations within the ankyrin repeats that prevented Rab29-‐mediated kinase activation, but no direct interaction with the LRRK2 ankyrin domain had been demonstrated [306]. In this work, we have clarified that all three members of the subfamily bind within the first 552 residues of the LRRK2 armadillo domain, and no interaction with the ankyrin repeats is observed. The ankyrin domain is found in close proximity to the kinase in the full length LRRK2 structure [330], and so the disruption of intra-‐domain interactions from the introduction of ankyrin mutations could potentially explain the elimination of kinase activity.
From competition assays it appears the Rab32 family share the same or an overlapping binding site within the LRRK2 armadillo repeats. Structural studies were then undertaken to identify molecular determinants of the interaction. Human LRRK2 has proven notoriously difficult to crystallise, with extensive efforts being carried out to overcome this hurdle [464]. Attempts by other groups have focused primarily on LRRK2 residues 1327 – 2527, comprising the Roc-‐COR domains, the kinase domain and the WD40 repeats. In our hands, crystallisation of the LRRK2 N-‐terminus was also unsuccessful. In place of an experimental structure, a homology model of LRRK21-‐552 was generated and a negatively charged surface towards the C-‐terminus of this construct was identified as the putative Rab binding domain. Mutational studies demonstrated a reduction in Rab binding at this site following the removal of surface charge, and suggests these negatively-‐charged residues contribute to complex formation.
The structures of Rab32(GTP) in complex with VARP, and Rab32(GDP) bound to the Salmonella protease GtgE are published and deposited in the Protein Data Bank [182, 207]. The uncomplexed structure of Rab32(GTP) is also available in our lab (unpublished). Here, two novel X-‐ray structures of active Rab38 and inactive Rab29 are reported at high resolution. Thus, structural information is now available for all members of the Rab32 subfamily. Examination of the active Rab38 and Rab32 proteins identified positively charged residues in
the nucleotide-‐dependent switches that may be important for mediating the interaction. The structure of Rab29 GTPase was solved in its inactive conformation, and reveals an unusual open Switch 1 conformation. Additionally, the position of the glutamate 68 residue pointing into the nucleotide-‐binding cavity is also found in the structure of Rab32(GDP) and may play a role in conferring GtgE specificity to the Rab32 subfamily.
Biophysical analyses give a mechanistic insight into the LRRK2:Rab interaction, and it was determined that all three members of the Rab32 subfamily bind with equal affinities to LRRK2. Dissociation constants were measured in the low micromolar range, and to the best of our knowledge, this is the first time binding affinities have been reported for the interaction. We also provide clear evidence of dependency on GTP for binding. This data hints that LRRK2 can be considered a classical effector of the Rab32 subfamily. However, evidence for a direct biological function arising as a consequence of the interaction must be identified in vivo for the definition to strictly apply.
Despite high sequence identity, a shared binding site and equivalent affinities documented in
vitro, an important biological distinction exists between Rab29 and Rab32/38. Rab29 is
capable of activating LRRK2 kinase while Rab32/38 do not possess this ability. Mutational studies of Rab32/38 revealed an arginine residue in Switch 1 that is critical for mediating the interaction with LRRK2. A surprising result emerged when it was found that mutation of the equivalent Switch 1 lysine in Rab29 did not elicit the same effect. In this case, removal of the positive charge had no observable impact on binding LRRK2. While Rab specificity is multi-‐ factorial, preliminary results from the substitution of this single residue implies a unique mode of Rab29 binding. While proteins interact with one another through a combination of hydrogen bonding, electrostatic interactions, hydrophobic and Van der Waals forces, the identification of charged, solvent-‐exposed residues is a convenient starting point for experimentally mapping a binding site in the absence of an available complex structure. It will be fascinating to further dissect the subtle differences that distinguish Rab29 from Rab32/38 going forward.
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site, and are not downstream substrates of LRRK2. Unlike other Rab substrates, Rab29 possesses two adjacent phosphorylation sites, Thr71 and Ser72, and both residues have been shown to be phosphorylated [149]. It was previously determined that double phosphorylation within Switch 2 prevented Rab29-‐mediated activation of LRRK2 kinase, while phosphorylation of either individual site had no effect [306]. Following the generation of phosphomimetic Rab29 constructs, we also observe a similar trend in this study. Here, introduction of the double phosphorylation mutant reduced Rab29 interaction with the LRRK2 ARM domain, while the single site mimetics remained comparable to wild-‐type. It will be important to confirm these observations hold true for the intrinsic phosphorylation of Rab29 Switch 2, as phosphomimetic substitutions have rendered Rab GTPases non-‐functional in some circumstances [408]. Nonetheless, these initial findings may link phosphorylation-‐mediated kinase inhibition directly to a disruption in Rab29 binding the LRRK2 ARM domain. This in turn may elude to a robust intrinsic regulatory mechanism whereby a high or sustained level of kinase activity resulting in the dual phosphorylation of Rab29 dampens further any activation via the ARM domain interactions.
Finally, a fascinating observation was made by Kalogeropulou et al. (2018) when they showed a LRRK2 truncation missing the N-‐terminus (LRRK2970-‐2527) could no longer phosphorylate Rab29, yet the phosphorylation of Rab8 and was Rab10 was unaffected [297]. This suggests a distinction between substrates and lends further credence to the importance of the LRRK2 N-‐ terminus is the regulation of kinase activity. One could speculate from this data that LRRK2 might phosphorylate active Rab29 when associated with the ARM domain at the membrane, and the resulting Switch 2 modifications result in a weakened affinity and dissociation of the complex. It will be exciting to see how the precise mechanisms of this interplay are unravelled.