Biology subjects

Guaitoli, G.

Publications and source records attributed to Guaitoli, G..

6 recordsLinked to original sources

bioRxiv2024

The Cellular and Extra-Cellular Proteomic Signature of Human Dopaminergic Neurons Carrying the LRRK2 G2019S Mutation

Extracellular vesicles are easily accessible in various biofluids and allow the assessment of disease-related changes of the proteome. This has made them a promising target for biomarker studies, especially in the field of neurodegeneration where access to diseased tissue is very limited. Genetic variants in the LRRK2 gene have been linked to both familial and sporadic forms of Parkinsons disease. With LRRK2 inhibitors entering clinical trials, there is an unmet need for biomarkers that reflect LRRK2-specific pathology and target engagement. In this study, we used induced pluripotent stem cells derived from a patient with Parkinsons disease carrying the LRRK2 G2019S mutation and an isogenic gene corrected control to generate human dopaminergic neurons. We isolated extracellular vesicles and neuronal cell lysates and characterized their proteomic signature using data-independent acquisition proteomics. We performed differential expression analysis and identified 595 significantly differentially regulated proteins in extracellular vesicles and 3205 in cell lysates. Next, we performed gene ontology enrichment analyses on the dysregulated proteins and found close association to biological processes relevant in neurodegeneration and Parkinsons disease. Finally, we focused on proteins that were dysregulated in both the extracellular and cellular proteomes and provide a list of ten promising biomarker candidates that are functionally relevant in neurodegeneration and linked to LRRK2 associated pathology. Among those was the sonic hedgehog signaling molecule, a protein that has tightly been linked to LRRK2-related disruption of cilia function. In conclusion, we characterized the cellular and extracellular proteome of dopaminergic neurons carrying the LRRK2 G2019S mutation and propose an experimentally based list of promising biomarker candidates for future studies.

neuroscience↗

bioRxiv2023

Structural insights in the GTP-driven monomerization and activation of a bacterial LRRK2 homologue using allosteric nanobodies

The Roco proteins are a family of GTPases, characterized by the conserved presence of a Roc-COR tandem domain. These proteins entered the limelight after mutations in human LRRK2 were identified as a major cause of familial Parkinsons disease. LRRK2 is a large and complex protein combining a GTPase and protein kinase activity, and disease mutations increase the kinase activity, while presumably decreasing the GTPase activity. Although a cross-communication between both catalytic activities has been suggested, the underlying mechanisms and the regulatory role of the GTPase domain remain unknown. Recently, several structures of LRRK2 have been reported, but so far structures of Roco proteins in their activated GTP-bound state are lacking. Here, we use single particle cryo-EM to solve the structure of a simpler bacterial Roco protein (CtRoco) in its GTP-bound state, aided by the use of two conformation-specific nanobodies: NbRoco1 and NbRoco2. This structure presents CtRoco in an active monomeric state, featuring very significant conformational changes compared to the previously solved nucleotide-free dimer structure. In particular, the structure shows a very large GTP-induced conformational change of the LRR domain, unleashing it from the Roc-COR domains, using the LRR-Roc linker as a hinge. Furthermore, this structure shows how NbRoco1 and NbRoco2 collaborate to activate CtRoco in an allosteric way. Altogether, our data provide important new insights in the activation mechanism of Roco proteins, with relevance to LRRK2 regulation, and suggest new routes for the allosteric modulation of their GTPase activity.

biochemistry↗

bioRxiv2023

Biophysical analysis reveals autophosphorylation as an important negative regulator of LRRK2 dimerization

Leucine-rich repeat kinase 2 (LRRK2) is a large, multi-domain protein which is associated with Parkinsons disease. Although high-resolution structures of LRRK2 are available, little is known about the complex dynamics behind the inter-domain regulation of LRRK2 and its perturbation by pathogenic variants. Previous studies have demonstrated that LRRK2 goes through an oligomerization cycle at the membrane, however it remains unclear in which form it exerts its kinase activity. Moreover, the LRRK2 monomer-dimer equilibrium and associated functional implications at a molecular level also need further investigation. In the present work, we used a multi-faceted approach to better understand LRRK2 oligomerization and suggest a functional model of how LRRK2 interacts with its substrates. To this end, we combined nano differential scanning calorimetry and mass photometry with molecular modelling. The thermal analysis resulted in a multistep denaturation profile, elucidating novel insights into the composite structural organization of the multi-domain protein LRRK2. Furthermore, LRRK2 shows a remarkable thermal stability, confirming its oligomeric nature. By using mass photometry, we could observe a monomer-dimer equilibrium which is altered by R1441G, a pathogenic variant within the Roc-COR interface. Most importantly, we could demonstrate that autophosphorylation induces LRRK2 monomerization, indicating a novel intramolecular feedback mechanism. Finally, we investigated the interaction of LRRK2 with its substrate, RAB10 by integrative computational modelling. The resulting models suggest that the monomeric form of LRRK2 is the favored protein conformation for the interaction with its substrate, leading to an increasing interest in the monomer-dimer equilibrium as a possible intervention point for the pathology.

biochemistry↗

bioRxiv2023

Intramolecular feedback regulation of the LRRK2 Roc G domain by a LRRK2 kinase dependent mechanism

The Parkinsons Disease (PD)-linked protein Leucine Rich Repeat Kinase 2 (LRRK2) consists of seven domains, including a kinase and a Roc G domain. Despite the availability of several high-resolution structures, the dynamic regulation of its unique intramolecular domain stack is nevertheless still not well understood. By in-depth biochemical analysis, assessing the Michaelis-Menten kinetics of the Roc G domain, we have confirmed that LRRK2 has, similar to other Roco protein family members, a KM value of LRRK2 that lays within the range of the physiological GTP concentrations within the cell. Furthermore, the R1441G PD variant located within a mutational hotspot in the Roc domain showed an increased catalytic efficiency. In contrast, the most common PD variant G2019S, located in the kinase domain, showed an increased KM and reduced catalytic efficiency, suggesting a negative feedback mechanism from the kinase domain to the G domain. Autophosphorylation of the G1+2 residue (T1343) in the Roc P-loop motif is critical for this phosphoregulation of both the KM as well as the kcat values of the Roc-catalyzed GTP hydrolysis, most likely by changing the monomer-dimer equilibrium. The LRRK2 T1343A variant has a similar increased kinase activity in cells compared to G2019S and the double mutant T1343A/G2019S has no further increased activity suggesting that T1343 is crucial for the negative feedback in the LRRK2 signaling cascade. Together our data reveal a novel intramolecular feedback regulation of the LRRK2 Roc G domain by a LRRK2 kinase dependent mechanism. Interestingly, PD mutants differently change the kinetics of the GTPase cycle, which might in part explain the difference in penetrance of these mutations in PD patients.

biochemistry↗

bioRxiv2021

Nanobodies as allosteric modulators of Parkinson's disease-associated LRRK2

Mutations in the gene coding for Leucine-Rich Repeat Kinase 2 (LRRK2) are a leading cause of the inherited form of Parkinsons disease (PD), while LRRK2 overactivation is also associated with the more common idiopathic form of PD. LRRK2 is a large multi-domain protein, including a GTPase as well as a Ser/Thr protein kinase domain. Common disease-causing mutations increase LRRK2 kinase activity, presenting LRRK2 as an attractive target for inhibitory drug design. Currently, drug development has mainly focused on ATP-competitive kinase inhibitors. Here, we report the identification and characterization of a variety of Nanobodies that bind to different LRRK2 domains and inhibit or activate LRRK2 activity in cells and in vitro. Importantly, diverse groups of Nanobodies were identified that inhibit LRRK2 kinase activity through a mechanism that does not involve binding to the ATP pocket or even to the kinase domain. Moreover, while certain Nanobodies completely inhibit the LRRK2 kinase activity, we also identified Nanobodies that specifically inhibit the phosphorylation of Rab protein substrates. Finally, in contrast to current type-I kinase inhibitors, the studied kinase-inhibitory Nanobodies did not induce LRRK2 microtubule association. These comprehensively characterized Nanobodies represent versatile tools to study the LRRK2 function and mechanism, and can pave the way toward novel diagnostic and therapeutic strategies for PD.

biochemistry↗

bioRxiv2019

Allosteric structural alterations and auto-regulation of Rab5 GEF activity in Rabex5

Intracellular trafficking depends on the function of Rab GTPases, whose activation is regulated by guanine exchange factors (GEFs). The Rab5 GEF, Rabex5, was previously proposed to be autoinhibited by its C-terminus. Here, we studied full-length Rabex5 and Rabaptin5 proteins as well as domain deletion Rabex5 mutants using hydrogen deuterium exchange mass spectrometry. We generated a structural model of Rabex5, using chemical crosslinking mass spectrometry and integrative modeling techniques. Our results are inconsistent with the previous model of autoinhibition. By correlating structural changes with nucleotide exchange activity for each construct, we uncovered new auto-regulatory roles for the Ubiquitin binding domains and the Linker connecting those domains to the catalytic core of Rabex5. Our results suggest a more complex auto-regulation mechanism than previously thought and imply that Ubiquitin binding serves not only to position Rabex5 but to also control its Rab5 GEF activity through allosteric structural alterations.

biophysics↗