Lysosomal storage disease proteo/lipidomic profiling using nMOST links ferritinophagy with mitochondrial iron deficiencies in cells lacking NPC2.

Lysosomal storage diseases (LSDs) comprised ~50 monogenic diseases characterized by the accumulation of cellular material in lysosomes and associated defects in lysosomal function, but systematic molecular phenotyping is lacking. Here, we develop a nanoflow-based multi-omic single-shot technology (nMOST) workflow allowing simultaneously quantify HeLa cell proteomes and lipidomes from more than two dozen LSD mutants, revealing diverse molecular phenotypes. Defects in delivery of ferritin and its autophagic receptor NCOA4 to lysosomes (ferritinophagy) were pronounced in NPC2-/- cells, which correlated with increased lyso-phosphatidylcholine species and multi-lamellar membrane structures visualized by cryo-electron-tomography. Ferritinophagy defects correlated with loss of mitochondrial cristae, MICOS-complex components, and electron transport chain complexes rich in iron-sulfur cluster proteins. Strikingly, mitochondrial defects were alleviated when iron was provided through the transferrin system. This resource reveals how defects in lysosomal function can impact mitochondrial homeostasis in trans and highlights nMOST as a discovery tool for illuminating molecular phenotypes across LSDs.

Proteome census upon nutrient stress reveals Golgiphagy membrane receptors.

During nutrient stress, macroautophagy degrades cellular macromolecules, thereby providing biosynthetic building blocks while simultaneously remodelling the proteome1,2. Although the machinery responsible for initiation of macroautophagy has been well characterized3,4, our understanding of the extent to which individual proteins, protein complexes and organelles are selected for autophagic degradation, and the underlying targeting mechanisms, is limited. Here we use orthogonal proteomic strategies to provide a spatial proteome census of autophagic cargo during nutrient stress in mammalian cells. We find that macroautophagy has selectivity for recycling membrane-bound organelles (principally Golgi and endoplasmic reticulum). Through autophagic cargo prioritization, we identify a complex of membrane-embedded proteins, YIPF3 and YIPF4, as receptors for Golgiphagy. During nutrient stress, YIPF3 and YIPF4 interact with ATG8 proteins through LIR motifs and are mobilized into autophagosomes that traffic to lysosomes in a process that requires the canonical autophagic machinery. Cells lacking YIPF3 or YIPF4 are selectively defective in elimination of a specific cohort of Golgi membrane proteins during nutrient stress. Moreover, YIPF3 and YIPF4 play an analogous role in Golgi remodelling during programmed conversion of stem cells to the neuronal lineage in vitro. Collectively, the findings of this study reveal prioritization of membrane protein cargo during nutrient-stress-dependent proteome remodelling and identify a Golgi remodelling pathway that requires membrane-embedded receptors.

Spatial snapshots of amyloid precursor protein intramembrane processing via early endosome proteomics.

Degradation and recycling of plasma membrane proteins occurs via the endolysosomal system, wherein endosomes bud into the cytosol from the plasma membrane and subsequently mature into degradative lysosomal compartments. While methods have been developed for rapid selective capture of lysosomes (Lyso-IP), analogous methods for isolation of early endosome intermediates are lacking. Here, we develop an approach for rapid isolation of early/sorting endosomes through affinity capture of the early endosome-associated protein EEA1 (Endo-IP) and provide proteomic and lipidomic snapshots of EEA1-positive endosomes in action. We identify recycling, regulatory and membrane fusion complexes, as well as candidate cargo, providing a proteomic landscape of early/sorting endosomes. To demonstrate the utility of the method, we combined Endo- and Lyso-IP with multiplexed targeted proteomics to provide a spatial digital snapshot of amyloid precursor protein (APP) processing by ß and γ-Secretases, which produce amyloidogenic Aß species, and quantify small molecule modulation of Secretase action on endosomes. We anticipate that the Endo-IP approach will facilitate systematic interrogation of processes that are coordinated on EEA1-positive endosomes.

Deficiency of the frontotemporal dementia gene GRN results in gangliosidosis.

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.

Mechanisms Controlling Selective Elimination of Damaged Lysosomes.

Lysosomes are subjected to physiological and patho-physiological insults over the course of their life cycle and are accordingly repaired or recycled. Lysophagy, the selective degradation of lysosomes via autophagy, occurs upon unrepairable lysosomal membrane rupture; galectins bind to glycosylated macromolecules in the lysosome lumen, orchestrating a series of cellular responses to promote autophagic recycling of damaged lysosomes and transcriptional upregulation of lysosomal genes. Damaged lysosomes are ubiquitylated, resulting in the recruitment of ubiquitin-binding autophagy receptors, which promote assembly of an autophagosome around damaged lysosomes for delivery to healthy lysosomes for degradation. Here, we review the current state of our understanding of mechanisms used to mark and eliminate damaged lysosomes, and discuss the complexities of galectin function and ubiquitin-chain linkage types. Finally, we discuss the limitations of available data and challenges with the goal of understanding the mechanistic basis of key steps in lysophagic flux.

Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy.

Removal of damaged organelles via the process of selective autophagy constitutes a major form of cellular quality control. Damaged organelles are recognized by a dedicated surveillance machinery, leading to the assembly of an autophagosome around the damaged organelle, prior to fusion with the degradative lysosomal compartment. Lysosomes themselves are also prone to damage and are degraded through the process of lysophagy. While early steps involve recognition of ruptured lysosomal membranes by glycan-binding galectins and ubiquitylation of transmembrane lysosomal proteins, many steps in the process, and their interrelationships, remain poorly understood, including the role and identity of cargo receptors required for completion of lysophagy. Here, we employ quantitative organelle capture and proximity biotinylation proteomics of autophagy adaptors, cargo receptors, and galectins in response to acute lysosomal damage, thereby revealing the landscape of lysosome-associated proteome remodeling during lysophagy. Among the proteins dynamically recruited to damaged lysosomes were ubiquitin-binding autophagic cargo receptors. Using newly developed lysophagic flux reporters including Lyso-Keima, we demonstrate that TAX1BP1, together with its associated kinase TBK1, are both necessary and sufficient to promote lysophagic flux in both HeLa cells and induced neurons (iNeurons). While the related receptor Optineurin (OPTN) can drive damage-dependent lysophagy when overexpressed, cells lacking either OPTN or CALCOCO2 still maintain significant lysophagic flux in HeLa cells. Mechanistically, TAX1BP1-driven lysophagy requires its N-terminal SKICH domain, which binds both TBK1 and the autophagy regulatory factor RB1CC1, and requires upstream ubiquitylation events for efficient recruitment and lysophagic flux. These results identify TAX1BP1 as a central component in the lysophagy pathway and provide a proteomic resource for future studies of the lysophagy process.

Inhibition of sphingolipid synthesis improves outcomes and survival in GARP mutant wobbler mice, a model of motor neuron degeneration.

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.

Architecture of the human interactome defines protein communities and disease networks.

The physiology of a cell can be viewed as the product of thousands of proteins acting in concert to shape the cellular response. Coordination is achieved in part through networks of protein-protein interactions that assemble functionally related proteins into complexes, organelles, and signal transduction pathways. Understanding the architecture of the human proteome has the potential to inform cellular, structural, and evolutionary mechanisms and is critical to elucidating how genome variation contributes to disease. Here we present BioPlex 2.0 (Biophysical Interactions of ORFeome-derived complexes), which uses robust affinity purification-mass spectrometry methodology to elucidate protein interaction networks and co-complexes nucleated by more than 25% of protein-coding genes from the human genome, and constitutes, to our knowledge, the largest such network so far. With more than 56,000 candidate interactions, BioPlex 2.0 contains more than 29,000 previously unknown co-associations and provides functional insights into hundreds of poorly characterized proteins while enhancing network-based analyses of domain associations, subcellular localization, and co-complex formation. Unsupervised Markov clustering of interacting proteins identified more than 1,300 protein communities representing diverse cellular activities. Genes essential for cell fitness are enriched within 53 communities representing central cellular functions. Moreover, we identified 442 communities associated with more than 2,000 disease annotations, placing numerous candidate disease genes into a cellular framework. BioPlex 2.0 exceeds previous experimentally derived interaction networks in depth and breadth, and will be a valuable resource for exploring the biology of incompletely characterized proteins and for elucidating larger-scale patterns of proteome organization.

Endosome-ER Contacts Control Actin Nucleation and Retromer Function through VAP-Dependent Regulation of PI4P.

VAP (VAPA and VAPB) is an evolutionarily conserved endoplasmic reticulum (ER)-anchored protein that helps generate tethers between the ER and other membranes through which lipids are exchanged across adjacent bilayers. Here, we report that by regulating PI4P levels on endosomes, VAP affects WASH-dependent actin nucleation on these organelles and the function of the retromer, a protein coat responsible for endosome-to-Golgi traffic. VAP is recruited to retromer budding sites on endosomes via an interaction with the retromer SNX2 subunit. Cells lacking VAP accumulate high levels of PI4P, actin comets, and trans-Golgi proteins on endosomes. Such defects are mimicked by downregulation of OSBP, a VAP interactor and PI4P transporter that participates in VAP-dependent ER-endosomes tethers. These results reveal a role of PI4P in retromer-/WASH-dependent budding from endosomes. Collectively, our data show how the ER can control budding dynamics and association with the cytoskeleton of another membrane by direct contacts leading to bilayer lipid modifications.

Genome-wide identification of phospho-regulators of Wnt signaling in Drosophila.

Evolutionarily conserved intercellular signaling pathways regulate embryonic development and adult tissue homeostasis in metazoans. The precise control of the state and amplitude of signaling pathways is achieved in part through the kinase- and phosphatase-mediated reversible phosphorylation of proteins. In this study, we performed a genome-wide in vivo RNAi screen for kinases and phosphatases that regulate the Wnt pathway under physiological conditions in the Drosophila wing disc. Our analyses have identified 54 high-confidence kinases and phosphatases capable of modulating the Wnt pathway, including 22 novel regulators. These candidates were also assayed for a role in the Notch pathway, and numerous phospho-regulators were identified. Additionally, each regulator of the Wnt pathway was evaluated in the wing disc for its ability to affect the mechanistically similar Hedgehog pathway. We identified 29 dual regulators that have the same effect on the Wnt and Hedgehog pathways. As proof of principle, we established that Cdc37 and Gilgamesh/CK1γ inhibit and promote signaling, respectively, by functioning at analogous levels of these pathways in both Drosophila and mammalian cells. The Wnt and Hedgehog pathways function in tandem in multiple developmental contexts, and the identification of several shared phospho-regulators serve as potential nodes of control under conditions of aberrant signaling and disease.

Hipk proteins dually regulate Wnt/Wingless signal transduction.

The Wnt/Wingless (Wg) pathway is an evolutionarily conserved signaling system that is used reiteratively, both spatially and temporally, to control the development of multicellular animals. The stability of cytoplasmic ß-catenin/Armadillo, the transcriptional effector of the pathway, is controlled by sequential N-terminal phosphorylation and ubiquitination that targets it for proteasome-mediated degradation. Orthologous members of the Homeodomain-interacting protein kinase family from Drosophila to vertebrates have been implicated in the regulation of Wnt/Wingless signaling. In Drosophila, as a consequence of Hipk activity, cells accumulate stabilized Armadillo that directs the expression of Wg-specific target genes. Hipk promotes the stabilization of Armadillo by inhibiting its ubiquitination (and hence subsequent degradation) by the SCF(Slimb) E3 ubiquitin ligase complex. Vertebrate Hipk2 impedes ß-catenin ubiquitination to promote its stability and the Wnt signal in a mechanism that is functionally conserved. Moreover, we describe here that Hipk proteins have a role independent of their effect on ß-catenin/Armadillo stability to enhance Wnt/Wingless signaling.

Wnt/Wingless signaling in Drosophila.

The Wingless (Wg) pathway represents one of the best-characterized intercellular signaling networks. Studies performed in Drosophila over the last 30 years have contributed to our understanding of the role of Wg signaling in the regulation of tissue growth, polarity, and patterning. These studies have revealed mechanisms conserved in the vertebrate Wnt pathways and illustrate the elegance of using the Drosophila model to understand evolutionarily conserved modes of gene regulation. In this article, we describe the function of Wg signaling in patterning the Drosophila embryonic epidermis and wing imaginal disc. As well, we present an overview of the establishment of the Wg morphogen gradient and discuss the differential modes of Wg-regulated gene expression.

Drosophila homeodomain-interacting protein kinase inhibits the Skp1-Cul1-F-box E3 ligase complex to dually promote Wingless and Hedgehog signaling.

Drosophila Homeodomain-interacting protein kinase (Hipk) has been shown to regulate in vivo, the stability of Armadillo, the transcriptional effector of Wingless signaling. The Wingless pathway culminates in the stabilization of Armadillo that, in the absence of signaling, is sequentially phosphorylated, polyubiquitinated and degraded. Loss-of-function clones for hipk result in reduced stabilized Armadillo, whereas overexpression of hipk elevates Armadillo levels to promote Wingless-responsive target gene expression. Here, we show that overexpression of hipk can suppress the effects of negative regulators of Armadillo to prevent its degradation in the wing imaginal disc. Hipk acts to stabilize Armadillo by impeding the function of the E3 ubiquitin ligase Skp1-Cul1-F-box (SCF)(Slimb), thereby inhibiting Armadillo ubiquitination and subsequent degradation. Vertebrate Hipk2 displays a similar ability to prevent ß-catenin ubiquitination in a functionally conserved mechanism. We find that Hipk's ability to inhibit SCF(Slimb)-mediated ubiquitination is not restricted to Armadillo and extends to other substrates of SCF(Slimb), including the Hedgehog signaling effector Ci. Thus, similar to casein kinase 1 and glycogen synthase kinase 3, Hipk dually regulates both Wingless and Hedgehog signaling by controlling the stability of their respective signaling effectors, but it is the first kinase to our knowledge identified that promotes the stability of both Armadillo and Ci.

Nemo phosphorylates Even-skipped and promotes Eve-mediated repression of odd-skipped in even parasegments during Drosophila embryogenesis.

Drosophila nemo (nmo) and other Nemo-like kinase family members (Nlks) are well-established key regulators of numerous conserved signaling pathways, such as Wg and BMP. nmo mutants display pleiotropic defects at different developmental stages, including the embryo. In this study we describe a detailed characterization of embryonic cuticle patterning defects associated with maternal loss of nmo. nmo mutant embryos consistently show segmentation defects, most frequently fusions of pairs of denticle belts in alternating segments. These phenotypes are reminiscent of those associated with defects in pair-rule patterning. Genetic interaction studies demonstrate that Nmo promotes Even-skipped (Eve) activity and is required to promote the expression of the Eve target, engrailed (en), in even numbered parasegments. We find that Nmo regulates a subset of Eve activities by stimulating Eve-mediated suppression of the odd-skipped (odd) repressor. Furthermore, we isolate Nmo in a protein complex with Eve and show that Nmo phosphorylates Eve in in vitro kinase assays. These studies reveal a novel role for the Nmo kinase in embryonic pattern formation through its regulation of the homeodomain-containing transcription factor Eve.

Homeodomain-interacting protein kinases (Hipks) promote Wnt/Wg signaling through stabilization of beta-catenin/Arm and stimulation of target gene expression.

The Wnt/Wingless (Wg) pathway represents a conserved signaling cascade involved in diverse biological processes. Misregulation of Wnt/Wg signal transduction has profound effects on development. Homeodomain-interacting protein kinases (Hipks) represent a novel family of serine/threonine kinases. Members of this group (in particular Hipk2) are implicated as important factors in transcriptional regulation to control cell growth, apoptosis and development. Here, we provide genetic and phenotypic evidence that the sole Drosophila member of this family, Hipk, functions as a positive regulator in the Wg pathway. Expression of hipk in the wing rescues loss of the Wg signal, whereas loss of hipk can enhance decreased wg signaling phenotypes. Furthermore, loss of hipk leads to diminished Arm protein levels, whereas overexpression of hipk promotes the Wg signal by stabilizing Arm, resulting in activation of Wg responsive targets. In Wg transcriptional assays, Hipk enhanced Tcf/Arm-mediated gene expression in a kinase-dependent manner. In addition, Hipk can bind to Arm and Drosophila Tcf, and phosphorylate Arm. Using both in vitro and in vivo assays, Hipk was found to promote the stabilization of Arm. We observe similar molecular interactions between Lef1/beta-catenin and vertebrate Hipk2, suggesting a direct and conserved role for Hipk proteins in promoting Wnt signaling.