Chemoproteomic-enabled characterization of small GTPase Rab1a as a target of an N-arylbenzimidazole ligand's rescue of Parkinson's-associated cell toxicity.

The development of phenotypic models of Parkinson's disease (PD) has enabled screening and identification of phenotypically active small molecules that restore complex biological pathways affected by PD toxicity. While these phenotypic screening platforms are powerful, they do not inherently enable direct identification of the cellular targets of promising lead compounds. To overcome this, chemoproteomic platforms like Thermal Proteome Profiling (TPP) and Stability of Proteins from Rates of Oxidation (SPROX) can be implemented to reveal protein targets of biologically active small molecules. Here we utilize both of these chemoproteomic strategies to identify targets of an N-arylbenzimidazole compound, NAB2, which was previously identified for its ability to restore viability in cellular models of PD-associated α-synuclein toxicity. The combined results from our TPP and SPROX analyses of NAB2 and the proteins in a neuroblastoma-derived SHSY5Y cell lysate reveal a previously unrecognized protein target of NAB2. This newly recognized target, Rab1a, is a small GTPase that acts as a molecular switch to regulate ER-to-Golgi trafficking, a process that is disrupted by α-synuclein toxicity and restored by NAB2 treatment. Further validation reveals that NAB2 binds to Rab1a with selectivity for its GDP-bound form and that NAB2 treatment phenocopies Rab1a overexpression in alleviation of α-synuclein toxicity. Finally, we conduct a preliminary investigation into the relationship between Rab1a and the E3 ubiquitin ligase, Nedd4, a previously identified NAB2 target. Together, these efforts expand our understanding of the mechanism of NAB2 in the alleviation of α-synuclein toxicity and reinforce the utility of chemoproteomic identification of the targets of phenotypically active small molecules that regulate complex biological pathways.

Predicting PY motif-mediated protein-protein interactions in the Nedd4 family of ubiquitin ligases.

The Nedd4 family contains several structurally related but functionally distinct HECT-type ubiquitin ligases. The members of the Nedd4 family are known to recognize substrates through their multiple WW domains, which recognize PY motifs (PPxY, LPxY) or phospho-threonine or phospho-serine residues. To better understand protein interactor recognition mechanisms across the Nedd4 family, we report the development and implementation of a python-based tool, PxYFinder, to identify PY motifs in the primary sequences of previously identified interactors of Nedd4 and related ligases. Using PxYFinder, we find that, on average, half of Nedd4 family interactions are likely PY-motif mediated. Further, we find that PPxY motifs are more prevalent than LPxY motifs and are more likely to occur in proline-rich regions and that PPxY regions are more disordered on average relative to LPxY-containing regions. Informed by consensus sequences for PY motifs across the Nedd4 interactome, we rationally designed a focused peptide library and employed a computational screen, revealing sequence- and biomolecular interaction-dependent determinants of WW-domain/PY-motif interactions. Cumulatively, our efforts provide a new bioinformatic tool and expand our understanding of sequence and structural factors that contribute to PY-motif mediated interactor recognition across the Nedd4 family.

Small Molecule Improvement of Trafficking Defects in Models of Neurodegeneration.

Disrupted cellular trafficking and transport processes are hallmarks of many neurodegenerative disorders (NDs). Recently, efforts have been made toward developing and implementing experimental platforms to identify small molecules that may help restore normative trafficking functions. There have been a number of successes in targeting endomembrane trafficking with the identification of compounds that restore cell viability through rescue of protein transport and trafficking. Here, we describe some of the experimental platforms implemented for small molecule screening efforts for rescue of trafficking defects in neurodegeneration. A survey of phenotypically active small molecules identified to date is provided, including a summary of medicinal chemistry efforts and insights into putative targets and mechanisms of action. In particular, emphasis is put on ligands that demonstrate activity in more than one model of neurodegeneration as retention of phenotypic activity across ND models suggests conservation of biological targets across NDs.

Characterization of Small-Molecule-Induced Changes in Parkinson's-Related Trafficking via the Nedd4 Ubiquitin Signaling Cascade.

The benzdiimidazole NAB2 rescues α-synuclein-associated trafficking defects associated with early onset Parkinson's disease in a Nedd4-dependent manner. Despite identification of E3 ubiquitin ligase Nedd4 as a putative target of NAB2, its molecular mechanism of action has not been elucidated. As such, the effect of NAB2 on Nedd4 activity and specificity was interrogated through biochemical, biophysical, and proteomic analyses. NAB2 was found to bind Nedd4 (KDapp = 42 nM), but this binding is side chain mediated and does not alter its conformation or ubiquitination kinetics in vitro. Nedd4 co-localizes with trafficking organelles, and NAB2 exposure did not alter its co-localization. Ubiquitin enrichment coupled proteomics revealed that NAB2 stimulates ubiquitination of trafficking-associated proteins, most likely through modulating the substrate specificity of Nedd4, providing a putative protein network involved in the NAB2 mechanism and revealing trafficking scaffold protein TFG as a Nedd4 substrate.

Robust and facile purification of full-length, untagged human Nedd4 as a recombinant protein from Escherichia coli.

Nedd4 is an E3 ubiquitin ligase that has received increased attention due to its role in the maintenance of proteostasis and in cellular stress responses. Investigation of Nedd4 enzymology has revealed a complex enzymatic mechanism that involves intermolecular interactions with upstream E2 conjugating enzymes and with substrates and intramolecular interactions that serve to regulate Nedd4 function. Thus, it is imperative that investigations of Nedd4 enzymology that employ recombinant enzyme be conducted with Nedd4 in its native, untagged form. We report herein an optimized, facile method for purification of recombinant human Nedd4 in its full-length form as a stable and active recombinant enzyme. Specifically, Nedd4 can be purified through a two-step purification which employs glutathione-S-transferase and hexahistidine sequences as orthogonal affinity tags. Proteolytic cleavage of Nedd4 was optimized to enable removal of the affinity tags with TEV protease, providing access to the untagged enzyme in yields of 2-3 mg/L. Additionally, investigation of Nedd4 storage conditions reveal that the enzyme is not stable through freeze-thaw cycles, and storage conditions should be carefully considered for preservation of enzyme stability. Finally, Nedd4 activity was validated through three activity assays which measure ubiquitin chain formation, Nedd4 autoubiquitination, and monoubiquitin consumption, respectively. Comparison of the method described herein with previously reported purification methods reveal that our optimized purification strategy enables access to Nedd4 in fewer chromatographic steps and eliminates reagents and materials that are potentially cost-prohibitive. This method, therefore, is more efficient and provides a more accessible route for purifying recombinant full-length Nedd4.