Necroptosis, a cell death pathway mediated by the RIPK1-RIPK3-MLKL signaling cascade downstream of tumor necrosis factor α (TNF-α), has been implicated in many inflammatory diseases. Members of the TAM (Tyro3, Axl, and Mer) family of receptor tyrosine kinases are known for their anti-apoptotic, oncogenic, and anti-inflammatory roles. Here, we identify an unexpected role of TAM kinases as promoters of necroptosis, a pro-inflammatory necrotic cell death. Pharmacologic or genetic targeting of TAM kinases results in a potent inhibition of necroptotic death in various cellular models. We identify phosphorylation of MLKL Tyr376 as a direct point of input from TAM kinases into the necroptosis signaling. The oligomerization of MLKL, but not its membranal translocation or phosphorylation by RIPK3, is controlled by TAM kinases. Importantly, both knockout and inhibition of TAM kinases protect mice from systemic inflammatory response syndrome. In conclusion, this study discovers that immunosuppressant TAM kinases are promoters of pro-inflammatory necroptosis, shedding light on the biological complexity of the regulation of inflammation.

Cells respond to nutrient stress by trafficking cytosolic contents to lysosomes for degradation via macroautophagy. The endoplasmic reticulum (ER) serves as an initiation site for autophagosomes and is also remodeled in response to nutrient stress through ER-phagy, a form of selective autophagy. Quantitative proteome analysis during nutrient stress identified an unstudied single-pass transmembrane ER protein, TEX264, as an ER-phagy receptor. TEX264 uses an LC3-interacting region (LIR) to traffic into ATG8-positive puncta that often initiate from three-way ER tubule junctions and subsequently fuse with lysosomes. Interaction and proximity biotinylation proteomics identified a cohort of autophagy regulatory proteins and cargo adaptors located near TEX264 in an LIR-dependent manner. Global proteomics and ER-phagy flux analysis revealed the stabilization of a cohort of ER proteins in TEX264-/- cells during nutrient stress. This work reveals TEX264 as an unrecognized ER-phagy receptor that acts independently of other candidate ER-phagy receptors to remodel the ER during nutrient stress.

Flux through kinase and ubiquitin-driven signaling systems depends on the modification kinetics, stoichiometry, primary site specificity, and target abundance within the pathway, yet we rarely understand these parameters and their spatial organization within cells. Here we develop temporal digital snapshots of ubiquitin signaling on the mitochondrial outer membrane in embryonic stem cell-derived neurons, and we model HeLa cell systems upon activation of the PINK1 kinase and PARKIN ubiquitin ligase by proteomic counting of ubiquitylation and phosphorylation events. We define the kinetics and site specificity of PARKIN-dependent target ubiquitylation, and we demonstrate the power of this approach to quantify pathway modulators and to mechanistically define the role of PARKIN UBL phosphorylation in pathway activation in induced neurons. Finally, through modulation of pS65-Ub on mitochondria, we demonstrate that Ub hyper-phosphorylation is inhibitory to mitophagy receptor recruitment, indicating that pS65-Ub stoichiometry in vivo is optimized to coordinate PARKIN recruitment via pS65-Ub and mitophagy receptors via unphosphorylated chains.

The mechanisms of cellular energy sensing and AMPK-mediated mTORC1 inhibition are not fully delineated. Here, we discover that RIPK1 promotes mTORC1 inhibition during energetic stress. RIPK1 is involved in mediating the interaction between AMPK and TSC2 and facilitate TSC2 phosphorylation at Ser1387. RIPK1 loss results in a high basal mTORC1 activity that drives defective lysosomes in cells and mice, leading to accumulation of RIPK3 and CASP8 and sensitization to cell death. RIPK1-deficient cells are unable to cope with energetic stress and are vulnerable to low glucose levels and metformin. Inhibition of mTORC1 rescues the lysosomal defects and vulnerability to energetic stress and prolongs the survival of RIPK1-deficient neonatal mice. Thus, RIPK1 plays an important role in the cellular response to low energy levels and mediates AMPK-mTORC1 signaling. These findings shed light on the regulation of mTORC1 during energetic stress and unveil a point of crosstalk between pro-survival and pro-death pathways.

Cell state changes are associated with proteome remodeling to serve newly emergent cell functions. Here, we show that NGN2-driven conversion of human embryonic stem cells to induced neurons (iNeurons) is associated with increased PINK1-independent mitophagic flux that is temporally correlated with metabolic reprogramming to support oxidative phosphorylation. Global multiplex proteomics during neurogenesis revealed large-scale remodeling of functional modules linked with pluripotency, mitochondrial metabolism, and proteostasis. Differentiation-dependent mitophagic flux required BNIP3L and its LC3-interacting region (LIR) motif, and BNIP3L also promoted mitophagy in dopaminergic neurons. Proteomic analysis of ATG12-/- iNeurons revealed accumulation of endoplasmic reticulum, Golgi, and mitochondria during differentiation, indicative of widespread organelle remodeling during neurogenesis. This work reveals broad organelle remodeling of membrane-bound organelles during NGN2-driven neurogenesis via autophagy, identifies BNIP3L's central role in programmed mitophagic flux, and provides a proteomic resource for elucidating how organelle remodeling and autophagy alter the proteome during changes in cell state.

The ubiquitin ligase Parkin, protein kinase PINK1, USP30 deubiquitylase, and p97 segregase function together to regulate turnover of damaged mitochondria via mitophagy, but our mechanistic understanding in neurons is limited. Here, we combine induced neurons (iNeurons) derived from embryonic stem cells with quantitative proteomics to reveal the dynamics and specificity of Parkin-dependent ubiquitylation under endogenous expression conditions. Targets showing elevated ubiquitylation in USP30-/- iNeurons are concentrated in components of the mitochondrial translocon, and the ubiquitylation kinetics of the vast majority of Parkin targets are unaffected, correlating with a modest kinetic acceleration in accumulation of pS65-Ub and mitophagic flux upon mitochondrial depolarization without USP30. Basally, ubiquitylated translocon import substrates accumulate, suggesting a quality control function for USP30. p97 was dispensable for Parkin ligase activity in iNeurons. This work provides an unprecedented quantitative landscape of the Parkin-modified ubiquitylome in iNeurons and reveals the underlying specificity of central regulatory elements in the pathway.