PLA2G15 is a Lysosomal BMP Hydrolase with Ester Position Specificity and its Targeting Ameliorates Lysosomal Disease.

Lysosomes catabolize lipids and other biological molecules, a function essential for cellular and organismal homeostasis. Key to lipid catabolism in the lysosome is bis(monoacylglycero)phosphate (BMP), a major lipid constituent of intralysosomal vesicles (ILVs) and a stimulator of lipid-degrading enzymes. BMP levels are altered in a broad spectrum of human conditions, including neurodegenerative diseases. Although BMP synthase was recently discovered, it has long been thought that BMP's unique stereochemistry confers resistance to acid phospholipases, a requirement for its role in the lysosome. Here, we demonstrate that PLA2G15, a major lysosomal phospholipase, efficiently hydrolyzes BMP with primary esters regardless of stereochemistry. Interestingly, we discover that BMP's unique esterification position is what confers resistance to hydrolysis. Purified PLA2G15 catabolizes most BMP species derived from cell and tissue lysosomes under acidic conditions. Furthermore, PLA2G15 catalytic activity against synthesized BMP stereoisomers with primary esters was comparable to its canonical substrates. Conversely, BMP with secondary esters is intrinsically stable in vitro and requires acyl migration for hydrolysis in lysosomes. Consistent with our biochemical data, PLA2G15-deficient tissues and cells accumulate multiple BMP species, a phenotype reversible by supplementing wildtype PLA2G15 but not its catalytically dead mutant. Increasing BMP levels by targeting PLA2G15 reverses the cholesterol accumulation phenotype in Niemann Pick Disease Type C (NPC1) patient fibroblasts and significantly ameliorate disease pathologies in NPC1-deficient mice leading to extended lifespan. Our findings establish the rules that govern the stability of BMP in the lysosome and identify PLA2G15 as a lysosomal BMP hydrolase and as a potential target for modulating BMP levels for therapeutic intervention.

MND1 enables homologous recombination in somatic cells primarily outside the context of replication.

Faithful and timely repair of DNA double-strand breaks (DSBs) is fundamental for the maintenance of genomic integrity. Here, we demonstrate that the meiotic recombination co-factor MND1 facilitates the repair of DSBs in somatic cells. We show that MND1 localizes to DSBs, where it stimulates DNA repair through homologous recombination (HR). Importantly, MND1 is not involved in the response to replication-associated DSBs, implying that it is dispensable for HR-mediated repair of one-ended DSBs. Instead, we find that MND1 specifically plays a role in the response to two-ended DSBs that are induced by irradiation (IR) or various chemotherapeutic drugs. Surprisingly, we find that MND1 is specifically active in G2 phase, whereas it only marginally affects repair during S phase. MND1 localization to DSBs is dependent on resection of the DNA ends and seemingly occurs through direct binding of MND1 to RAD51-coated ssDNA. Importantly, the lack of MND1-driven HR repair directly potentiates the toxicity of IR-induced damage, which could open new possibilities for therapeutic intervention, specifically in HR-proficient tumors.

A Mediator-cohesin axis controls heterochromatin domain formation.

The genome consists of regions of transcriptionally active euchromatin and more silent heterochromatin. We reveal that the formation of heterochromatin domains requires cohesin turnover on DNA. Stabilization of cohesin on DNA through depletion of its release factor WAPL leads to a near-complete loss of heterochromatin domains. We observe the opposite phenotype in cells deficient for subunits of the Mediator-CDK module, with an almost binary partition of the genome into dense H3K9me3 domains, and regions devoid of H3K9me3 spanning the rest of the genome. We suggest that the Mediator-CDK module might contribute to gene expression by limiting the formation of dense heterochromatin domains. WAPL deficiency prevents the formation of heterochromatin domains, and allows for gene expression even in the absence of the Mediator-CDK subunit MED12. We propose that cohesin and Mediator affect heterochromatin in different ways to enable the correct distribution of epigenetic marks, and thus to ensure proper gene expression.

E3 ubiquitin ligase Mindbomb 1 facilitates nuclear delivery of adenovirus genomes.

The journey from plasma membrane to nuclear pore is a critical step in the lifecycle of DNA viruses, many of which must successfully deposit their genomes into the nucleus for replication. Viral capsids navigate this vast distance through the coordinated hijacking of a number of cellular host factors, many of which remain unknown. We performed a gene-trap screen in haploid cells to identify host factors for adenovirus (AdV), a DNA virus that can cause severe respiratory illness in immune-compromised individuals. This work identified Mindbomb 1 (MIB1), an E3 ubiquitin ligase involved in neurodevelopment, as critical for AdV infectivity. In the absence of MIB1, we observed that viral capsids successfully traffic to the proximity of the nucleus but ultimately fail to deposit their genomes within. The capacity of MIB1 to promote AdV infection was dependent on its ubiquitination activity, suggesting that MIB1 may mediate proteasomal degradation of one or more negative regulators of AdV infection. Employing complementary proteomic approaches to characterize proteins proximal to MIB1 upon AdV infection and differentially ubiquitinated in the presence or absence of MIB1, we observed an intersection between MIB1 and ribonucleoproteins (RNPs) largely unexplored in mammalian cells. This work uncovers yet another way that viruses utilize host cell machinery for their own replication, highlighting a potential target for therapeutic interventions that counter AdV infection.

The SPPL3-Defined Glycosphingolipid Repertoire Orchestrates HLA Class I-Mediated Immune Responses.

HLA class I (HLA-I) glycoproteins drive immune responses by presenting antigens to cognate CD8+ T cells. This process is often hijacked by tumors and pathogens for immune evasion. Because options for restoring HLA-I antigen presentation are limited, we aimed to identify druggable HLA-I pathway targets. Using iterative genome-wide screens, we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augmented B3GNT5 enzyme activity, resulting in upregulation of surface neolacto-series GSLs. These GSLs sterically impeded antibody and receptor interactions with HLA-I and diminished CD8+ T cell activation. Furthermore, a disturbed SPPL3-B3GNT5 pathway in glioma correlated with decreased patient survival. We show that the immunomodulatory effect could be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that inhibits immune recognition and represents a potential therapeutic target in cancer, infection, and autoimmunity.

Quantitative genetic screening reveals a Ragulator-FLCN feedback loop that regulates the mTORC1 pathway.

Forward genetic screens in mammalian cell lines, such as RNAi and CRISPR-Cas9 screens, have made major contributions to the elucidation of diverse signaling pathways. Here, we exploited human haploid cells as a robust comparative screening platform and report a set of quantitative forward genetic screens for identifying regulatory mechanisms of mTORC1 signaling, a key growth control pathway that senses diverse metabolic states. Selected chemical and genetic perturbations in this screening platform, including rapamycin treatment and genetic ablation of the Ragulator subunit LAMTOR4, revealed the known core mTORC1 regulatory signaling complexes and the intimate interplay of the mTORC1 pathway with lysosomal function, validating the approach. In addition, we identified a differential requirement for LAMTOR4 and LAMTOR5 in regulating the mTORC1 pathway under fed and starved conditions. Furthermore, we uncovered a previously unknown "synthetic-sick" interaction between the tumor suppressor folliculin and LAMTOR4, which may have therapeutic implications in cancer treatment. Together, our study demonstrates the use of iterative "perturb and observe" genetic screens to uncover regulatory mechanisms driving complex mammalian signaling networks.

Functional Radiogenetic Profiling Implicates ERCC6L2 in Non-homologous End Joining.

Using genome-wide radiogenetic profiling, we functionally dissect vulnerabilities of cancer cells to ionizing radiation (IR). We identify ERCC6L2 as a major determinant of IR response, together with classical DNA damage response genes and members of the recently identified shieldin and CTC1-STN1-TEN1 (CST) complexes. We show that ERCC6L2 contributes to non-homologous end joining (NHEJ), and it may exert this function through interactions with SFPQ. In addition to causing radiosensitivity, ERCC6L2 loss restores DNA end resection and partially rescues homologous recombination (HR) in BRCA1-deficient cells. As a consequence, ERCC6L2 deficiency confers resistance to poly (ADP-ribose) polymerase (PARP) inhibition in tumors deficient for both BRCA1 and p53. Moreover, we show that ERCC6L2 mutations are found in human tumors and correlate with a better overall survival in patients treated with radiotherapy (RT); this finding suggests that ERCC6L2 is a predictive biomarker of RT response.

A Genome-Wide Haploid Genetic Screen Identifies Heparan Sulfate-Associated Genes and the Macropinocytosis Modulator TMED10 as Factors Supporting Vaccinia Virus Infection.

Vaccinia virus is a promising viral vaccine and gene delivery candidate and has historically been used as a model to study poxvirus-host cell interactions. We employed a genome-wide insertional mutagenesis approach in human haploid cells to identify host factors crucial for vaccinia virus infection. A library of mutagenized HAP1 cells was exposed to modified vaccinia virus Ankara (MVA). Deep-sequencing analysis of virus-resistant cells identified host factors involved in heparan sulfate synthesis, Golgi organization, and vesicular protein trafficking. We validated EXT1, TM9SF2, and TMED10 (TMP21/p23/p24δ) as important host factors for vaccinia virus infection. The critical roles of EXT1 in heparan sulfate synthesis and vaccinia virus infection were confirmed. TM9SF2 was validated as a player mediating heparan sulfate expression, explaining its contribution to vaccinia virus infection. In addition, TMED10 was found to be crucial for virus-induced plasma membrane blebbing and phosphatidylserine-induced macropinocytosis, presumably by regulating the cell surface expression of the TAM receptor Axl.IMPORTANCE Poxviruses are large DNA viruses that can infect a wide range of host species. A number of these viruses are clinically important to humans, including variola virus (smallpox) and vaccinia virus. Since the eradication of smallpox, zoonotic infections with monkeypox virus and cowpox virus are emerging. Additionally, poxviruses can be engineered to specifically target cancer cells and are used as a vaccine vector against tuberculosis, influenza, and coronaviruses. Poxviruses rely on host factors for most stages of their life cycle, including attachment to the cell and entry. These host factors are crucial for virus infectivity and host cell tropism. We used a genome-wide knockout library of host cells to identify host factors necessary for vaccinia virus infection. We confirm a dominant role for heparin sulfate in mediating virus attachment. Additionally, we show that TMED10, previously not implicated in virus infections, facilitates virus uptake by modulating the cellular response to phosphatidylserine.

LZTR1 is a regulator of RAS ubiquitination and signaling.

In genetic screens aimed at understanding drug resistance mechanisms in chronic myeloid leukemia cells, inactivation of the cullin 3 adapter protein-encoding leucine zipper-like transcription regulator 1 (LZTR1) gene led to enhanced mitogen-activated protein kinase (MAPK) pathway activity and reduced sensitivity to tyrosine kinase inhibitors. Knockdown of the Drosophila LZTR1 ortholog CG3711 resulted in a Ras-dependent gain-of-function phenotype. Endogenous human LZTR1 associates with the main RAS isoforms. Inactivation of LZTR1 led to decreased ubiquitination and enhanced plasma membrane localization of endogenous KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog). We propose that LZTR1 acts as a conserved regulator of RAS ubiquitination and MAPK pathway activation. Because LZTR1 disease mutations failed to revert loss-of-function phenotypes, our findings provide a molecular rationale for LZTR1 involvement in a variety of inherited and acquired human disorders.

Loss of Kif18A Results in Spindle Assembly Checkpoint Activation at Microtubule-Attached Kinetochores.

The spindle assembly checkpoint (SAC) halts anaphase progression until all kinetochores have obtained bipolar, stable attachments to the mitotic spindle. Upon initial attachment, chromosomes undergo oscillatory movements to reach metaphase. Once a chromosome is correctly attached and positioned, these oscillatory movements are reduced by the motor protein Kif18A, and loss of Kif18A results in chromosome hyper-oscillations. By using a haploid genetic approach, we found that loss of Kif18A is lethal in wild-type human HAP1 cells, but not in SAC-deficient HAP1 cells. Unexpectedly, we found that the hyper-oscillations after Kif18A loss are not associated with chromosome missegregations. Rather, we found that loss of Kif18A results in a loss of tension across a subset of kinetochores accompanying SAC activation. Strikingly, the SAC-active kinetochores appear to have established fully functional kinetochore-microtubule (k-Mt) attachments, allowing proper chromosome segregation. These findings shed new light on the role of Kif18A in chromosome segregation and demonstrate that the SAC can be activated at kinetochores that are occupied by fully functional k-Mts that lack tension.

KREMEN1 Is a Host Entry Receptor for a Major Group of Enteroviruses.

Human type A Enteroviruses (EV-As) cause diseases ranging from hand-foot-and-mouth disease to poliomyelitis-like disease. Although cellular receptors are identified for some EV-As, they remain elusive for the majority of EV-As. We identify the cell surface molecule KREMEN1 as an entry receptor for coxsackievirus A10 (CV-A10). Whereas loss of KREMEN1 renders cells resistant to CV-A10 infection, KREMEN1 overexpression enhances CV-A10 binding to the cell surface and increases susceptibility to infection, indicating that KREMEN1 is a rate-limiting factor for CV-A10 infection. Furthermore, the extracellular domain of KREMEN1 binds CV-A10 and functions as a neutralizing agent during infection. Kremen-deficient mice are resistant to CV-A10-induced lethal paralysis, emphasizing the relevance of Kremen for infection in vivo. KREMEN1 is also essential for infection by a phylogenetic and pathogenic related group of EV-As. Collectively these findings highlight the importance of KREMEN1 for these emerging pathogens and its potential as an antiviral therapeutic target.

Haploid genetic screens identify genetic vulnerabilities to microtubule-targeting agents.

The absence of biomarkers to accurately predict anticancer therapy response remains a major obstacle in clinical oncology. We applied a genome-wide loss-of-function screening approach in human haploid cells to characterize genetic vulnerabilities to classical microtubule-targeting agents. Using docetaxel and vinorelbine, two well-established chemotherapeutic agents, we sought to identify genetic alterations sensitizing human HAP1 cells to these drugs. Despite the fact that both drugs act on microtubules, a set of distinct genes were identified whose disruption affects drug sensitivity. For docetaxel, this included a number of genes with a function in mitosis, while for vinorelbine we identified inactivation of FBXW7, RB1, and NF2, three frequently mutated tumor suppressor genes, as sensitizing factors. We validated these genes using independent knockout clones and confirmed FBXW7 as an important regulator of the mitotic spindle assembly. Upon FBXW7 depletion, vinorelbine treatment led to decreased survival of cells due to defective mitotic progression and subsequent mitotic catastrophe. We show that haploid insertional mutagenesis screens are a useful tool to study genetic vulnerabilities to classical chemotherapeutic drugs by identifying thus far unknown sensitivity factors. These results provide a rationale for investigating patient response to vinca alkaloid-based anticancer treatment in relation to the mutational status of these three tumor suppressor genes, and could in the future lead to the establishment of novel predictive biomarkers or suggest new drug combinations based on molecular mechanisms of drug sensitivity.

BUB1 Is Essential for the Viability of Human Cells in which the Spindle Assembly Checkpoint Is Compromised.

The spindle assembly checkpoint (SAC) ensures faithful segregation of chromosomes. Although most mammalian cell types depend on the SAC for viability, we found that human HAP1 cells can grow SAC independently. We generated MAD1- and MAD2-deficient cells and mutagenized them to identify synthetic lethal interactions, revealing that chromosome congression factors become essential upon SAC deficiency. Besides expected hits, we also found that BUB1 becomes essential in SAC-deficient cells. We found that the BUB1 C terminus regulates alignment as well as recruitment of CENPF. Second, we found that BUBR1 was not essential in SAC-deficient HAP1 cells. We confirmed that BUBR1 does not regulate chromosome alignment in HAP1 cells and that BUB1 does not regulate chromosome alignment through BUBR1. Taken together, our data resolve some long-standing questions about the interplay between BUB1 and BUBR1 and their respective roles in the SAC and chromosome alignment.

NRP2 and CD63 Are Host Factors for Lujo Virus Cell Entry.

Arenaviruses cause fatal hemorrhagic disease in humans. Old World arenavirus glycoproteins (GPs) mainly engage α-dystroglycan as a cell-surface receptor, while New World arenaviruses hijack transferrin receptor. However, the Lujo virus (LUJV) GP does not cluster with New or Old World arenaviruses. Using a recombinant vesicular stomatitis virus containing LUJV GP as its sole attachment and fusion protein (VSV-LUJV), we demonstrate that infection is independent of known arenavirus receptor genes. A genome-wide haploid genetic screen identified the transmembrane protein neuropilin 2 (NRP2) and tetraspanin CD63 as factors for LUJV GP-mediated infection. LUJV GP binds the N-terminal domain of NRP2, while CD63 stimulates pH-activated LUJV GP-mediated membrane fusion. Overexpression of NRP2 or its N-terminal domain enhances VSV-LUJV infection, and cells lacking NRP2 are deficient in wild-type LUJV infection. These findings uncover this distinct set of host cell entry factors in LUJV infection and are attractive focus points for therapeutic intervention.

Vasohibins encode tubulin detyrosinating activity.

Tubulin is subjected to a number of posttranslational modifications to generate heterogeneous microtubules. The modifications include removal and ligation of the C-terminal tyrosine of ⍺-tubulin. The enzymes responsible for detyrosination, an activity first observed 40 years ago, have remained elusive. We applied a genetic screen in haploid human cells to find regulators of tubulin detyrosination. We identified SVBP, a peptide that regulates the abundance of vasohibins (VASH1 and VASH2). Vasohibins, but not SVBP alone, increased detyrosination of ⍺-tubulin, and purified vasohibins removed the C-terminal tyrosine of ⍺-tubulin. We found that vasohibins play a cell type-dependent role in detyrosination, although cells also contain an additional detyrosinating activity. Thus, vasohibins, hitherto studied as secreted angiogenesis regulators, constitute a long-sought missing link in the tubulin tyrosination cycle.

Diverse Viruses Require the Calcium Transporter SPCA1 for Maturation and Spread.

Respiratory and arthropod-borne viral infections are a global threat due to the lack of effective antivirals and vaccines. A potential strategy is to target host proteins required for viruses but non-essential for the host. To identify such proteins, we performed a genome-wide knockout screen in human haploid cells and identified the calcium pump SPCA1. SPCA1 is required by viruses from the Paramyxoviridae, Flaviviridae, and Togaviridae families, including measles, dengue, West Nile, Zika, and chikungunya viruses. Calcium transport activity is required for SPCA1 to promote virus spread. SPCA1 regulates proteases within the trans-Golgi network that require calcium for their activity and are critical for virus glycoprotein maturation. Consistent with these findings, viral glycoproteins fail to mature in SPCA1-deficient cells preventing viral spread, which is evident even in cells with partial loss of SPCA1. Thus, SPCA1 is an attractive antiviral host target for a broad spectrum of established and emerging viral infections.

Genetic wiring maps of single-cell protein states reveal an off-switch for GPCR signalling.

As key executers of biological functions, the activity and abundance of proteins are subjected to extensive regulation. Deciphering the genetic architecture underlying this regulation is critical for understanding cellular signalling events and responses to environmental cues. Using random mutagenesis in haploid human cells, we apply a sensitive approach to directly couple genomic mutations to protein measurements in individual cells. Here we use this to examine a suite of cellular processes, such as transcriptional induction, regulation of protein abundance and splicing, signalling cascades (mitogen-activated protein kinase (MAPK), G-protein-coupled receptor (GPCR), protein kinase B (AKT), interferon, and Wingless and Int-related protein (WNT) pathways) and epigenetic modifications (histone crotonylation and methylation). This scalable, sequencing-based procedure elucidates the genetic landscapes that control protein states, identifying genes that cause very narrow phenotypic effects and genes that lead to broad phenotypic consequences. The resulting genetic wiring map identifies the E3-ligase substrate adaptor KCTD5 (ref. 1) as a negative regulator of the AKT pathway, a key signalling cascade frequently deregulated in cancer. KCTD5-deficient cells show elevated levels of phospho-AKT at S473 that could not be attributed to effects on canonical pathway components. To reveal the genetic requirements for this phenotype, we iteratively analysed the regulatory network linked to AKT activity in the knockout background. This genetic modifier screen exposes suppressors of the KCTD5 phenotype and mechanistically demonstrates that KCTD5 acts as an off-switch for GPCR signalling by triggering proteolysis of Gßγ heterodimers dissociated from the Gα subunit. Although biological networks have previously been constructed on the basis of gene expression, protein-protein associations, or genetic interaction profiles, we foresee that the approach described here will enable the generation of a comprehensive genetic wiring map for human cells on the basis of quantitative protein states.

The Cohesin Release Factor WAPL Restricts Chromatin Loop Extension.

The spatial organization of chromosomes influences many nuclear processes including gene expression. The cohesin complex shapes the 3D genome by looping together CTCF sites along chromosomes. We show here that chromatin loop size can be increased and that the duration with which cohesin embraces DNA determines the degree to which loops are enlarged. Cohesin's DNA release factor WAPL restricts this loop extension and also prevents looping between incorrectly oriented CTCF sites. We reveal that the SCC2/SCC4 complex promotes the extension of chromatin loops and the formation of topologically associated domains (TADs). Our data support the model that cohesin structures chromosomes through the processive enlargement of loops and that TADs reflect polyclonal collections of loops in the making. Finally, we find that whereas cohesin promotes chromosomal looping, it rather limits nuclear compartmentalization. We conclude that the balanced activity of SCC2/SCC4 and WAPL enables cohesin to correctly structure chromosomes.

PLA2G16 represents a switch between entry and clearance of Picornaviridae.

Picornaviruses are a leading cause of human and veterinary infections that result in various diseases, including polio and the common cold. As archetypical non-enveloped viruses, their biology has been extensively studied. Although a range of different cell-surface receptors are bound by different picornaviruses, it is unclear whether common host factors are needed for them to reach the cytoplasm. Using genome-wide haploid genetic screens, here we identify the lipid-modifying enzyme PLA2G16 (refs 8, 9, 10, 11) as a picornavirus host factor that is required for a previously unknown event in the viral life cycle. We find that PLA2G16 functions early during infection, enabling virion-mediated genome delivery into the cytoplasm, but not in any virion-assigned step, such as cell binding, endosomal trafficking or pore formation. To resolve this paradox, we screened for suppressors of the ΔPLA2G16 phenotype and identified a mechanism previously implicated in the clearance of intracellular bacteria. The sensor of this mechanism, galectin-8 (encoded by LGALS8), detects permeated endosomes and marks them for autophagic degradation, whereas PLA2G16 facilitates viral genome translocation and prevents clearance. This study uncovers two competing processes triggered by virus entry: activation of a pore-activated clearance pathway and recruitment of a phospholipase to enable genome release.