Felix Boos

Engulfment by macrophages is critical for waste clearance in the vertebrate brain. Understanding clearance mechanisms may open new therapeutic possibilities to counter brain aging and neurodegenerative diseases. However, few in vivo models exist to study engulfment in the brain and characterize this process during aging and across species. Here we present a genetic model for secretion of a fluorescent protein by neurons in the brain of the African turquoise killifish, the shortest-lived vertebrate that can be bred in captivity. We use this model to identify a population of brain macrophages in the killifish responsible for engulfment of material from the brain extracellular space. Intriguingly, many of these cells bear similarities to mammalian border-associated and monocyte-derived macrophages, rare subsets of macrophages in mouse and human brains noted for their engulfment capabilities. We also find that in our model, killifish brain macrophages decline in engulfment capacity with age. This work highlights how vertebrate brain macrophages, particularly those at brain border regions, can play a critical role in clearance and provides an opportunity to test interventions that can boost engulfment by these macrophages to promote brain resilience in old age and disease.

Upon inhibition of mitochondrial protein import, yeast cells activate the unfolded protein response of the endoplasmic reticulum (UPR^ER). Mitochondrial proteins are targeted to the ER under these conditions. The UPR-ER is important for cellular fitness upon mitochondrial dysfunction, as well as when metabolic stimuli increase the synthesis of mitochondrial precursor proteins.

Cellular functionality relies on a well-balanced, but highly dynamic proteome. Dysfunction of mitochondrial protein import leads to the cytosolic accumulation of mitochondrial precursor proteins which compromise cellular proteostasis and trigger a mitoprotein-induced stress response. To dissect the effects of mitochondrial dysfunction on the cellular proteome as a whole, we developed pre-post thermal proteome profiling. This multiplexed time-resolved proteome-wide thermal stability profiling approach with isobaric peptide tags in combination with a pulsed SILAC labelling elucidated dynamic proteostasis changes in several dimensions: In addition to adaptations in protein abundance, we observed rapid modulations of the thermal stability of individual cellular proteins. Different functional groups of proteins showed characteristic response patterns and reacted with group-specific kinetics, allowing the identification of functional modules that are relevant for mitoprotein-induced stress. Thus, our new pre-post thermal proteome profiling approach uncovered a complex response network that orchestrates proteome homeostasis in eukaryotic cells by time-controlled adaptations of the abundance and the conformation of proteins.

Cellular functionality relies on a well-balanced, but highly dynamic proteome. Dysfunction of mitochondrial protein import leads to the cytosolic accumulation of mitochondrial precursor proteins which compromise cellular proteostasis and trigger the mitoprotein-induced stress response. To dissect the effects of mitochondrial dysfunction on the cellular proteome as a whole, we developed pre-post thermal proteome profiling (ppTPP). This multiplexed time-resolved proteome-wide thermal stability profiling approach with isobaric peptide tags in combination with a pulsed SILAC labeling elucidated dynamic proteostasis changes in several dimensions: In addition to adaptations in protein abundance, we observed rapid modulations of the thermal stability of individual cellular proteins. Strikingly, different functional groups of proteins showed characteristic response patterns and reacted with group-specific kinetics, allowing the identification of the functional modules that are relevant for mitoprotein-induced stress. Thus, our new ppTPP approach uncovered a complex response network that orchestrates proteome homeostasis in eukaryotic cells by time-controlled adaptations of protein abundance and protein stability.

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Almost all mitochondrial proteins are synthesized in the cytosol and subsequently targeted to mitochondria. The accumulation of non-imported precursor proteins occurring upon mitochondrial dysfunction can challenge cellular protein homeostasis. Here we show that blocking protein translocation into mitochondria results in the accumulation of mitochondrial membrane proteins at the endoplasmic reticulum, thereby triggering the unfolded protein response (UPR ^ER ). Moreover, we find that mitochondrial membrane proteins are also routed to the ER under physiological conditions. The levels of ER-resident mitochondrial precursors is enhanced by import defects as well as metabolic stimuli that increase the expression of mitochondrial proteins. Under such conditions, the UPR ^ER is crucial to maintain protein homeostasis and cellular fitness. We propose the ER serves as a physiological buffer zone for those mitochondrial precursors that can’t be immediately imported into mitochondria while engaging the UPR ^ER to adjust the ER proteostasis capacity to the extent of precursor accumulation.

Most mitochondrial proteins are synthesized in the cytosol as precursors that carry N-terminal presequences. After import into mitochondria, these targeting signals are cleaved off by the mitochondrial processing peptidase MPP, giving rise to shorter mature proteins. Using the mitochondrial tandem protein Arg5,6 as a model substrate, we demonstrate that MPP has an additional role in preprotein maturation, beyond the removal of presequences. Arg5,6 is synthesized as a polyprotein precursor that is imported into the mitochondrial matrix and subsequently separated into two distinct enzymes that function in arginine biogenesis. This internal processing is performed by MPP, which cleaves the Arg5,6 precursor both at its N-terminus and at an internal site between the Arg5 and Arg6 parts. The peculiar organization and biogenesis of Arg5,6 is conserved across fungi and might preserve the mode of co-translational subunit association of the arginine biosynthesis complex of the polycistronic arginine operon in prokaryotic mitochondrial ancestors. Putative MPP cleavage sites are also present at the junctions in other mitochondrial fusion proteins from fungi, plants and animals. Our data suggest that, in addition to its role as “ticket canceller” for the removal of presequences, MPP exhibits a second, widely conserved activity as internal processing peptidase for complex mitochondrial precursor proteins.