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 interplay between E2 and E3 enzymes regulates the polyubiquitination of substrates in eukaryotes. Among the several RING-domain E3 ligases in humans, many utilize two distinct E2s for polyubiquitination. For example, the cell cycle regulatory E3, human anaphase-promoting complex/cyclosome (APC/C), relies on UBE2C to prime substrates with ubiquitin (Ub) and on UBE2S to extend polyubiquitin chains. However, the potential coordination between these steps in ubiquitin chain formation remains undefined. While numerous studies have unveiled how RING E3s stimulate individual E2s for Ub transfer, here we change perspective to describe a case where the chain-elongating E2 UBE2S feeds back and directly stimulates the E3 APC/C to promote substrate priming and subsequent multiubiquitination by UBE2C. Our work reveals an unexpected model for the mechanisms of RING E3-dependent ubiquitination and for the diverse and complex interrelationship between components of the ubiquitination cascade.

Numerous mutations that impair retrograde membrane trafficking between endosomes and the Golgi apparatus lead to neurodegenerative diseases. For example, mutations in the endosomal retromer complex are implicated in Alzheimer's and Parkinson's diseases, and mutations of the Golgi-associated retrograde protein (GARP) complex cause progressive cerebello-cerebral atrophy type 2 (PCCA2). However, how these mutations cause neurodegeneration is unknown. GARP mutations in yeast, including one causing PCCA2, result in sphingolipid abnormalities and impaired cell growth that are corrected by treatment with myriocin, a sphingolipid synthesis inhibitor, suggesting that alterations in sphingolipid metabolism contribute to cell dysfunction and death. Here we tested this hypothesis in wobbler mice, a murine model with a homozygous partial loss-of-function mutation in Vps54 (GARP protein) that causes motor neuron disease. Cytotoxic sphingoid long-chain bases accumulated in embryonic fibroblasts and spinal cords from wobbler mice. Remarkably, chronic treatment of wobbler mice with myriocin markedly improved their wellness scores, grip strength, neuropathology, and survival. Proteomic analyses of wobbler fibroblasts revealed extensive missorting of lysosomal proteins, including sphingolipid catabolism enzymes, to the Golgi compartment, which may contribute to the sphingolipid abnormalities. Our findings establish that altered sphingolipid metabolism due to GARP mutations contributes to neurodegeneration and suggest that inhibiting sphingolipid synthesis might provide a useful strategy for treating these disorders.

Haploinsufficiency of GRN causes frontotemporal dementia (FTD). The GRN locus produces progranulin (PGRN), which is cleaved to lysosomal granulin polypeptides. The function of lysosomal granulins and why their absence causes neurodegeneration are unclear. Here we discover that PGRN-deficient human cells and murine brains, as well as human frontal lobes from GRN-mutation FTD patients have increased levels of gangliosides, glycosphingolipids that contain sialic acid. In these cells and tissues, levels of lysosomal enzymes that catabolize gangliosides were normal, but levels of bis(monoacylglycero)phosphates (BMP), lipids required for ganglioside catabolism, were reduced with PGRN deficiency. Our findings indicate that granulins are required to maintain BMP levels to support ganglioside catabolism, and that PGRN deficiency in lysosomes leads to gangliosidosis. Lysosomal ganglioside accumulation may contribute to neuroinflammation and neurodegeneration susceptibility observed in FTD due to PGRN deficiency and other neurodegenerative diseases.

The multi-subunit translation initiation factor eIF2B is a control node for protein synthesis. eIF2B activity is canonically modulated through stress-responsive phosphorylation of its substrate eIF2. The eIF2B regulatory subcomplex is evolutionarily related to sugar-metabolizing enzymes, but the biological relevance of this relationship was unknown. To identify natural ligands that might regulate eIF2B, we conduct unbiased binding- and activity-based screens followed by structural studies. We find that sugar phosphates occupy the ancestral catalytic site in the eIF2Bα subunit, promote eIF2B holoenzyme formation and enhance enzymatic activity towards eIF2. A mutant in the eIF2Bα ligand pocket that causes Vanishing White Matter disease fails to engage and is not stimulated by sugar phosphates. These data underscore the importance of allosteric metabolite modulation for proper eIF2B function. We propose that eIF2B evolved to couple nutrient status via sugar phosphate sensing with the rate of protein synthesis, one of the most energetically costly cellular processes.

The endoplasmic reticulum (ER) employs a diverse proteome landscape to orchestrate many cellular functions, ranging from protein and lipid synthesis to calcium ion flux and inter-organelle communication. A case in point concerns the process of neurogenesis, where a refined tubular ER network is assembled via ER shaping proteins into the newly formed neuronal projections to create highly polarized dendrites and axons. Previous studies have suggested a role for autophagy in ER remodelling, as autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic boutons, and the membrane-embedded ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy. However, our understanding of the mechanisms underlying selective removal of the ER and the role of individual ER-phagy receptors is limited. Here we combine a genetically tractable induced neuron (iNeuron) system for monitoring ER remodelling during in vitro differentiation with proteomic and computational tools to create a quantitative landscape of ER proteome remodelling via selective autophagy. Through analysis of single and combinatorial ER-phagy receptor mutants, we delineate the extent to which each receptor contributes to both the magnitude and selectivity of ER protein clearance. We define specific subsets of ER membrane or lumenal proteins as preferred clients for distinct receptors. Using spatial sensors and flux reporters, we demonstrate receptor-specific autophagic capture of ER in axons, and directly visualize tubular ER membranes within autophagosomes in neuronal projections by cryo-electron tomography. This molecular inventory of ER proteome remodelling and versatile genetic toolkit provide a quantitative framework for understanding the contributions of individual ER-phagy receptors for reshaping ER during cell state transitions.

Cell size varies greatly between cell types, yet within a specific cell type and growth condition, cell size is narrowly distributed. Why maintenance of a cell-type specific cell size is important remains poorly understood. Here we show that growing budding yeast and primary mammalian cells beyond a certain size impairs gene induction, cell-cycle progression, and cell signaling. These defects are due to the inability of large cells to scale nucleic acid and protein biosynthesis in accordance with cell volume increase, which effectively leads to cytoplasm dilution. We further show that loss of scaling beyond a certain critical size is due to DNA becoming limiting. Based on the observation that senescent cells are large and exhibit many of the phenotypes of large cells, we propose that the range of DNA:cytoplasm ratio that supports optimal cell function is limited and that ratios outside these bounds contribute to aging.

53BP1 is a chromatin-associated protein that regulates the DNA damage response. In this study, we identify the TPX2/Aurora A heterodimer, nominally considered a mitotic kinase complex, as a novel binding partner of 53BP1. We find that TPX2/Aurora A plays a previously unrecognized role in DNA damage repair and replication fork stability by counteracting 53BP1 function. Loss of TPX2 or Aurora A compromises DNA end resection, BRCA1 and Rad51 recruitment, and homologous recombination. Furthermore, loss of TPX2 or Aurora A causes deprotection of stalled replication forks upon replication stress induction. This fork protection pathway counteracts MRE11 nuclease activity but functions in parallel to BRCA1. Strikingly, concurrent loss of 53BP1 rescues not only BRCA1/Rad51 recruitment but also the fork instability induced upon TPX2 loss. Our work suggests the presence of a feedback mechanism by which 53BP1 is regulated by a novel binding partner and uncovers a unique role for 53BP1 in replication fork stability.

Parkinson's disease (PD) is a neurodegenerative disease caused by the loss of dopaminergic neurons in the substantia nigra. PARK2 mutations cause early-onset forms of PD. PARK2 encodes an E3 ubiquitin ligase, Parkin, that can selectively translocate to dysfunctional mitochondria to promote their removal by autophagy. However, Parkin knockout (KO) mice do not display signs of neurodegeneration. To assess Parkin function in vivo, we utilized a mouse model that accumulates dysfunctional mitochondria caused by an accelerated generation of mtDNA mutations (Mutator mice). In the absence of Parkin, dopaminergic neurons in Mutator mice degenerated causing an L-DOPA reversible motor deficit. Other neuronal populations were unaffected. Phosphorylated ubiquitin was increased in the brains of Mutator mice, indicating PINK1-Parkin activation. Parkin loss caused mitochondrial dysfunction and affected the pathogenicity but not the levels of mtDNA somatic mutations. A systemic loss of Parkin synergizes with mitochondrial dysfunction causing dopaminergic neuron death modeling PD pathogenic processes.

HERC2 is a large E3 ubiquitin ligase with multiple structural domains that has been implicated in an array of cellular processes. Mutations in HERC2 are linked to developmental delays and impairment caused by nervous system dysfunction, such as Angelman Syndrome and autism-spectrum disorders. However, HERC2 cellular activity and regulation remain poorly understood. We used a broad proteomic approach to survey the landscape of cellular proteins that interact with HERC2. We identified nearly 300 potential interactors, a subset of which we validated binding to HERC2. The potential HERC2 interactors included the eukaryotic translation initiation factor 3 complex, the intracellular transport COPI coatomer complex, the glycogen regulator phosphorylase kinase, beta-catenin, PI3 kinase, and proteins involved in fatty acid transport and iron homeostasis. Through a complex bioinformatic analysis of potential interactors, we linked HERC2 to cellular processes including intracellular protein trafficking and transport, metabolism of cellular energy, and protein translation. Given its size, multidomain structure, and association with various cellular activities, HERC2 may function as a scaffold to integrate protein complexes and bridge critical cellular pathways. This work provides a significant resource with which to interrogate HERC2 function more deeply and evaluate its contributions to mechanisms governing cellular homeostasis and disease.

Phenylketonuria (PKU, phenylalanine hydroxylase deficiency), an inborn error of metabolism, can be detected through newborn screening for hyperphenylalaninemia (HPA). Most individuals with HPA harbor mutations in the gene encoding phenylalanine hydroxylase (PAH), and a small proportion (2%) exhibit tetrahydrobiopterin (BH4) deficiency with additional neurotransmitter (dopamine and serotonin) deficiency. Here we report six individuals from four unrelated families with HPA who exhibited progressive neurodevelopmental delay, dystonia, and a unique profile of neurotransmitter deficiencies without mutations in PAH or BH4 metabolism disorder-related genes. In these six affected individuals, whole-exome sequencing (WES) identified biallelic mutations in DNAJC12, which encodes a heat shock co-chaperone family member that interacts with phenylalanine, tyrosine, and tryptophan hydroxylases catalyzing the BH4-activated conversion of phenylalanine into tyrosine, tyrosine into L-dopa (the precursor of dopamine), and tryptophan into 5-hydroxytryptophan (the precursor of serotonin), respectively. DNAJC12 was undetectable in fibroblasts from the individuals with null mutations. PAH enzyme activity was reduced in the presence of DNAJC12 mutations. Early treatment with BH4 and/or neurotransmitter precursors had dramatic beneficial effects and resulted in the prevention of neurodevelopmental delay in the one individual treated before symptom onset. Thus, DNAJC12 deficiency is a preventable and treatable cause of intellectual disability that should be considered in the early differential diagnosis when screening results are positive for HPA. Sequencing of DNAJC12 may resolve any uncertainty and should be considered in all children with unresolved HPA.

Asymmetric disassembly of the synaptonemal complex (SC) is crucial for proper meiotic chromosome segregation. However, the signaling mechanisms that directly regulate this process are poorly understood. Here we show that the mammalian Rho GEF homolog, ECT-2, functions through the conserved RAS/ERK MAP kinase signaling pathway in the C. elegans germline to regulate the disassembly of SC proteins. We find that SYP-2, a SC central region component, is a potential target for MPK-1-mediated phosphorylation and that constitutively phosphorylated SYP-2 impairs the disassembly of SC proteins from chromosomal domains referred to as the long arms of the bivalents. Inactivation of MAP kinase at late pachytene is critical for timely disassembly of the SC proteins from the long arms, and is dependent on the crossover (CO) promoting factors ZHP-3/RNF212/Zip3 and COSA-1/CNTD1. We propose that the conserved MAP kinase pathway coordinates CO designation with the disassembly of SC proteins to ensure accurate chromosome segregation.

Mitochondrial dysfunction is associated with the development of many age-related human diseases. Therefore recognizing and correcting the early signs of malfunctioning mitochondria is of critical importance for cellular welfare and survival. We previously demonstrated that VCP/Cdc48-associated mitochondrial stress responsive 1 (Vms1) is a component of a mitochondrial surveillance system that mediates the stress-responsive degradation of mitochondrial proteins by the proteasome. Here we propose novel mechanisms through which Vms1 monitors the status of mitochondria and is recruited to damaged or stressed mitochondria. We find that Vms1 contains a highly conserved region that is necessary and sufficient for mitochondrial targeting (the mitochondrial targeting domain [MTD]). Of interest, MTD-mediated mitochondrial targeting of Vms1 is negatively regulated by a direct interaction with the Vms1 N-terminus. Using laser-induced generation of mitochondrial reactive oxygen species, we also show that Vms1 is preferentially recruited to mitochondria subjected to oxidative stress. These studies define cellular and biochemical mechanisms by which Vms1 locali-zation to mitochondria is controlled to enable an efficient protein quality control system.

N-terminal acetylation is an abundant modification influencing protein functions. Because ∼80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation-dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors are highly selective with respect to other protein acetyl-amide-binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets and provide insights into targeting multiprotein E2-E3 ligases.

The PINK1 protein kinase activates the PARK2 ubiquitin ligase to promote mitochondrial ubiquitylation and recruitment of ubiquitin-binding mitophagy receptors typified by OPTN and TAX1BP1. Here, we combine proximity biotinylation of OPTN and TAX1BP1 with CRISPR-Cas9-based screens for mitophagic flux to develop a spatial proteogenetic map of PARK2-dependent mitophagy. Proximity labeling of OPTN allowed visualization of a "mitochondrial-autophagosome synapse" upon mitochondrial depolarization. Proximity proteomics of OPTN and TAX1BP1 revealed numerous proteins at the synapse, including both PARK2 substrates and autophagy components. Parallel mitophagic flux screens identified proteins with roles in autophagy, vesicle formation and fusion, as well as PARK2 targets, many of which were also identified via proximity proteomics. One protein identified in both approaches, HK2, promotes assembly of a high-molecular weight complex of PINK1 and phosphorylation of ubiquitin in response to mitochondrial damage. This work provides a resource for understanding the spatial and molecular landscape of PARK2-dependent mitophagy.

Neurons depend on autophagy to maintain cellular homeostasis, and defects in autophagy are pathological hallmarks of neurodegenerative disease. To probe the role of basal autophagy in the maintenance of neuronal health, we isolated autophagic vesicles from mouse brain tissue and used proteomics to identify the major cargos engulfed within autophagosomes, validating our findings in rodent primary and human iPSC-derived neurons. Mitochondrial proteins were identified as a major cargo in the absence of mitophagy adaptors such as OPTN. We found that nucleoid-associated proteins are enriched compared with other mitochondrial components. In the axon, autophagic engulfment of nucleoid-enriched mitochondrial fragments requires the mitochondrial fission machinery Drp1. We proposed that localized Drp1-dependent fission of nucleoid-enriched fragments in proximity to the sites of autophagosome biogenesis enhances their capture. The resulting efficient autophagic turnover of nucleoids may prevent accumulation of mitochondrial DNA in the neuron, thus mitigating activation of proinflammatory pathways that contribute to neurodegeneration.

How activation of PINK1 and Parkin leads to elimination of damaged mitochondria by mitophagy is largely based on cell lines with few studies in neurons. Here, we have undertaken proteomic analysis of mitochondria from mouse neurons to identify ubiquitylated substrates of endogenous Parkin. Comparative analysis with human iNeuron datasets revealed a subset of 49 PINK1 activation–dependent diGLY sites in 22 proteins conserved across mouse and human systems. We use reconstitution assays to demonstrate direct ubiquitylation by Parkin in vitro. We also identified a subset of cytoplasmic proteins recruited to mitochondria that undergo PINK1 and Parkin independent ubiquitylation, indicating the presence of alternate ubiquitin E3 ligase pathways that are activated by mitochondrial depolarization in neurons. Last, we have developed an online resource to search for ubiquitin sites and enzymes in mitochondria of neurons, MitoNUb. These findings will aid future studies to understand Parkin activation in neuronal subtypes.

Parkinson's disease (PD) is a complex and highly variable neurodegenerative disease. Familial PD is caused by mutations in several genes with diverse and mostly unknown functions. It is unclear how dysregulation of these genes results in the relatively selective death of nigral dopaminergic neurons (DNs). To address this question, we modeled PD by knocking out the PD genes PARKIN (PRKN), DJ-1 (PARK7), and ATP13A2 (PARK9) in independent isogenic human pluripotent stem cell (hPSC) lines. We found increased levels of oxidative stress in all PD lines. Increased death of DNs upon differentiation was found only in the PARKIN knockout line. Using quantitative proteomics, we observed dysregulation of mitochondrial and lysosomal function in all of the lines, as well as common and distinct molecular defects caused by the different PD genes. Our results suggest that precise delineation of PD subtypes will require evaluation of molecular and clinical data.

Aneuploidy, a condition characterized by chromosome gains and losses, causes reduced fitness and numerous cellular stresses, including increased protein aggregation. Here, we identify protein complex stoichiometry imbalances as a major cause of protein aggregation in aneuploid cells. Subunits of protein complexes encoded on excess chromosomes aggregate in aneuploid cells, which is suppressed when expression of other subunits is coordinately altered. We further show that excess subunits are either degraded or aggregate and that protein aggregation is nearly as effective as protein degradation at lowering levels of excess proteins. Our study explains why proteotoxic stress is a universal feature of the aneuploid state and reveals protein aggregation as a form of dosage compensation to cope with disproportionate expression of protein complex subunits.

Organelle identity depends on protein composition. How mistargeted proteins are selectively recognized and removed from organelles is incompletely understood. Here, we found that the orphan P5A-adenosine triphosphatase (ATPase) transporter ATP13A1 (Spf1 in yeast) directly interacted with the transmembrane segment (TM) of mitochondrial tail-anchored proteins. P5A-ATPase activity mediated the extraction of mistargeted proteins from the endoplasmic reticulum (ER). Cryo-electron microscopy structures of Saccharomyces cerevisiae Spf1 revealed a large, membrane-accessible substrate-binding pocket that alternately faced the ER lumen and cytosol and an endogenous substrate resembling an α-helical TM. Our results indicate that the P5A-ATPase could dislocate misinserted hydrophobic helices flanked by short basic segments from the ER. TM dislocation by the P5A-ATPase establishes an additional class of P-type ATPase substrates and may correct mistakes in protein targeting or topogenesis.