Structurally targeted mutagenesis identifies key residues supporting α -synuclein misfolding in multiple system atrophy.

Multiple system atrophy (MSA) and Parkinson's disease (PD) are caused by misfolded α -synuclein spreading throughout the central nervous system. While familial PD is linked to several point mutations in α -synuclein, there are no known mutations associated with MSA. Our previous work investigating differences in α -synuclein misfolding between the two disorders showed that the familial PD mutation E46K inhibits replication of MSA prions both in vitro and in vivo, providing key evidence to support the hypothesis that α -synuclein adopts unique strains in patients. Here, to further interrogate α -synuclein misfolding, we engineered a panel of cell lines harboring both PD-linked and novel mutations designed to identify key residues that facilitate α -synuclein misfolding in MSA. These data were paired with in silico analyses using Maestro software to predict the effect of each mutation on the ability of α -synuclein to misfold into one of the reported MSA cryo-electron microscopy conformations. In many cases, our modeling accurately identified mutations that facilitated or inhibited MSA replication. However, Maestro was occasionally unable to predict the effect of a mutation on MSA propagation in vitro, demonstrating the challenge of using computational tools to investigate intrinsically disordered proteins. Finally, we used our cellular models to determine the mechanism underlying the E46K-driven inhibition of MSA replication, finding that the E46/K80 salt bridge is necessary to support α -synuclein misfolding. Overall, our studies use a structure-based approach to investigate α -synuclein misfolding, resulting in the creation of a powerful panel of cell lines that can be used to interrogate MSA strain biology.

The ER membrane protein complex restricts mitophagy by controlling BNIP3 turnover.

Lysosomal degradation of autophagy receptors is a common proxy for selective autophagy. However, we find that two established mitophagy receptors, BNIP3 and BNIP3L/NIX, are constitutively delivered to lysosomes in an autophagy-independent manner. This alternative lysosomal delivery of BNIP3 accounts for nearly all its lysosome-mediated degradation, even upon mitophagy induction. To identify how BNIP3, a tail-anchored protein in the outer mitochondrial membrane, is delivered to lysosomes, we performed a genome-wide CRISPR screen for factors influencing BNIP3 flux. This screen revealed both known modifiers of BNIP3 stability as well as a pronounced reliance on endolysosomal components, including the ER membrane protein complex (EMC). Importantly, the endolysosomal system and the ubiquitin-proteosome system regulated BNIP3 independently. Perturbation of either mechanism is sufficient to modulate BNIP3-associated mitophagy and affect underlying cellular physiology. More broadly, these findings extend recent models for tail-anchored protein quality control and install endosomal trafficking and lysosomal degradation in the canon of pathways that tightly regulate endogenous tail-anchored protein localization.

The ER membrane protein complex governs lysosomal turnover of a mitochondrial tail-anchored protein, BNIP3, to restrict mitophagy.

Lysosomal degradation of autophagy receptors is a common proxy for selective autophagy. However, we find that two established mitophagy receptors, BNIP3 and BNIP3L/NIX, violate this assumption. Rather, BNIP3 and NIX are constitutively delivered to lysosomes in an autophagy-independent manner. This alternative lysosomal delivery of BNIP3 accounts for nearly all of its lysosome-mediated degradation, even upon mitophagy induction. To identify how BNIP3, a tail-anchored protein in the outer mitochondrial membrane, is delivered to lysosomes, we performed a genome-wide CRISPR screen for factors influencing BNIP3 flux. By this approach, we revealed both known modifiers of BNIP3 stability as well as a pronounced reliance on endolysosomal components, including the ER membrane protein complex (EMC). Importantly, the endolysosomal system regulates BNIP3 alongside, but independent of, the ubiquitin-proteosome system (UPS). Perturbation of either mechanism is sufficient to modulate BNIP3-associated mitophagy and affect underlying cellular physiology. In short, while BNIP3 can be cleared by parallel and partially compensatory quality control pathways, non-autophagic lysosomal degradation of BNIP3 is a strong post-translational modifier of BNIP3 function. More broadly, these data reveal an unanticipated connection between mitophagy and TA protein quality control, wherein the endolysosomal system provides a critical axis for regulating cellular metabolism. Moreover, these findings extend recent models for tail-anchored protein quality control and install endosomal trafficking and lysosomal degradation in the canon of pathways that ensure tight regulation of endogenous TA protein localization.

Consequences of variability in α-synuclein fibril structure on strain biology.

Synucleinopathies are a group of clinically and neuropathologically distinct protein misfolding diseases caused by unique α-synuclein conformations, or strains. While multiple atomic resolution cryo-electron microscopy structures of α-synuclein fibrils are now deposited in Protein Data Bank, significant gaps in the biological consequences arising from each conformation have yet to be unraveled. Mutations in the α-synuclein gene (SNCA), cofactors, and the solvation environment contribute to the formation and maintenance of each disease-causing strain. This review highlights the impact of each of these factors on α-synuclein misfolding and discusses the implications of the resulting structural variability on therapeutic development.