A genome-wide screen identifies Yos9p as essential for ER-associated degradation of glycoproteins

We undertook a growth-based screen exploiting the degradation of CTL*, a chimeric membrane-bound ERAD substrate derived from soluble lumenal CPY*. We screened the Saccharomyces cerevisiae genomic deletion library containing approximately 5000 viable strains for mutants defective in endoplasmic reticulum (ER) protein quality control and degradation (ERAD). Among the new gene products we identified Yos9p, an ER-localized protein previously involved in the processing of GPI anchored proteins. We show that deficiency in Yos9p affects the degradation only of glycosylated ERAD substrates. Degradation of non-glycosylated substrates is not affected in cells lacking Yos9p. We propose that Yos9p is a lectin or lectin-like protein involved in the quality control of N-glycosylated proteins. It may act sequentially or in concert with the ERAD lectin Htm1p/Mnl1p (EDEM) to prevent secretion of malfolded glycosylated proteins and deliver them to the cytosolic ubiquitin-proteasome machinery for elimination.     

A genome-wide synthetic dosage lethality screen reveals multiple pathways that require the functioning of ubiquitin-binding proteins Rad23 and Dsk2

Background  

Ubiquitin regulates a myriad of important cellular processes through covalent attachment to its substrates. A classic role for ubiquitin is to flag proteins for destruction by the proteasome. Recent studies indicate that ubiquitin-binding proteins (e.g. Rad23, Dsk2, Rpn10) play a pivotal role in transferring ubiquitylated proteins to the proteasome. However, the specific role of these ubiquitin receptors remains poorly defined. A key to unraveling the functions of these ubiquitin receptors is to identify their cellular substrates and biological circuits they are involved in. Although many strategies have been developed for substrate isolation, the identification of physiological targets of proteolytic pathways has proven to be quite challenging.     

A genomic screen identifies AUT8 as a novel gene essential for autophagy in the yeast Saccharomyces cerevisiae.

Autophagy is a starvation-induced transport pathway delivering parts of the cytosol into the lysosome (vacuole) for degradation. Autophagy significantly differs from other transport pathways by using double membrane layered transport intermediates. Based on the identification of autophagy genes in Saccharomyces cerevisiae, which served as a pacemaker for higher cells, our mechanistic knowledge of autophagy notably increased over the past few years. We here identify AUT8 as a novel gene essential for autophagy by screening a collection of approximately 5000 yeast deletion strains, each containing a defined deletion in an individual gene. This collection is a result of the world-wide Saccharomyces deletion project and covers the non-essential genes of the whole yeast genome. Homozygous aut8 Delta cells are impaired in maturation of proaminopeptidase I, and they fail to undergo the cell differentiation process of sporulation. The essential function of AUT8 for autophagy is further demonstrated by the lack of accumulation of autophagic vesicles in the vacuoles of aut8 Delta cells starved of nitrogen in the presence of the proteinase B inhibitor phenylmethylsulfonyl fluoride.     

Ubiquitin chains in the Dsk2 UBL domain mediate Dsk2 stability and protein degradation in yeast

Ubiquitin-like (UBL)–ubiquitin-associated (UBA) proteins, including Dsk2 and Rad23, act as delivery factors that target polyubiquitinated substrates to the proteasome. We report here that the Dsk2 UBL domain is ubiquitinated in yeast cells and that Dsk2 ubiquitination of the UBL domain is involved in Dsk2 stability, depending on the Dsk2 UBA domain. Also, Dsk2 lacking ubiquitin chains impaired ubiquitin-dependent protein degradation and decreased the interaction of Dsk2 with polyubiquitinated proteins in cells. Moreover, Dsk2 ubiquitination affected ability to restore the temperature-sensitive growth defect of dsk2Δ. These results indicate that ubiquitination in the UBL domain of Dsk2 has in vivo functions in the ubiquitin–proteasome pathway in yeast.     

A genomic screen for modifiers of tauopathy identifies puromycin-sensitive aminopeptidase as an inhibitor of tau-induced neurodegeneration

Neurofibrillary tangles (NFT) containing tau are a hallmark of neurodegenerative diseases, including Alzheimer's disease (AD). NFT burden correlates with cognitive decline and neurodegeneration in AD. However, little is known about mechanisms that protect against tau-induced neurodegeneration. We used a cross species functional genomic approach to analyze gene expression in multiple brain regions in mouse, in parallel with validation in Drosophila, to identify tau modifiers, including the highly conserved protein puromycin-sensitive aminopeptidase (PSA/Npepps). PSA protected against tau-induced neurodegeneration in vivo, whereas PSA loss of function exacerbated neurodegeneration. We further show that human PSA directly proteolyzes tau in vitro. These data highlight the utility of using both evolutionarily distant species for genetic screening and functional assessment to identify modifiers of neurodegeneration. Further investigation is warranted in defining the role of PSA and other genes identified here as potential therapeutic targets in tauopathy.     

A high-content chemical screen identifies ellipticine as a modulator of p53 nuclear localization

p53 regulates apoptosis and the cell cycle through actions in the nucleus and cytoplasm. Altering the subcellular localization of p53 can alter its biological function. Therefore, small molecules that change the localization of p53 would be useful chemical probes to understand the influence of subcellular localization on the function of p53. To identify such molecules, a high-content screen for compounds that increased the localization of p53 to the nucleus or cytoplasm was developed, automated, and conducted. With this image-based assay, we identified ellipticine that increased the nuclear localization of GFP-mutant p53 protein but not GFP alone in Saos-2 osteosarcoma cells. In addition, ellipticine increased the nuclear localization of endogenous p53 in HCT116 colon cancer cells with a resultant increase in the transactivation of the p21 promoter. Increased nuclear p53 after ellipticine treatment was not associated with an increase in DNA double stranded breaks, indicating that ellipticine shifts p53 to the nucleus through a mechanism independent of DNA damage. Thus, a chemical biology approach has identified a molecule that shifts the localization of p53 and enhances its nuclear activity. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. G. Wei Xu and Imtiaz A. Mawji have contributed equally to this work.     

Functional genomic screen identifies novel mediators of collagen uptake

Tissue fibrosis occurs when matrix production outpaces matrix degradation. Degradation of collagen, the main component of fibrotic tissue, is mediated through an extracellular proteolytic pathway and intracellular pathway of cellular uptake and lysosomal digestion. Recent studies demonstrate that disruption of the intracellular pathways can exacerbate fibrosis. These pathways are poorly characterized. Here we identify novel mediators of the intracellular pathway of collagen turnover through a genome-wide RNA interference screen in Drosophila S2 cells. Screening of 7505 Drosophila genes conserved among metazoans identified 22 genes that were required for efficient internalization of type I collagen. These included proteins involved in vesicle transport, the actin cytoskeleton, and signal transduction. We show further that the flotillin genes have a conserved and central role in collagen uptake in Drosophila and human cells. Short hairpin RNA–mediated silencing of flotillins in human monocyte and fibroblasts impaired collagen uptake by promoting lysosomal degradation of the endocytic collagen receptors uPARAP/Endo180 and mannose receptor. These data provide an initial characterization of intracellular pathways of collagen turnover and identify the flotillin genes as critical regulators of this process. A better understanding of these pathways may lead to novel therapies that reduce fibrosis by increasing collagen turnover.     

Recognition of specific ubiquitin conjugates is important for the proteolytic functions of the ubiquitin-associated domain proteins Dsk2 and Rad23.

Ubiquitin (Ub) regulates important cellular processes through covalent attachment to its substrates. The fate of a substrate depends on the number of ubiquitin moieties conjugated, as well as the lysine linkage of Ub-Ub conjugation. The major function of Ub is to regulate the in vivo half-life of its substrates. Once a multi-Ub chain is attached to a substrate, it must be shielded from deubiquitylating enzymes for the 26 S proteasome to recognize it. Molecular mechanisms of the postubiquitylation processes are poorly understood. Here, we have characterized a family of proteins that preferentially binds ubiquitylated substrates and multi-Ub chains through a motif termed the ubiquitin-associated domain (UBA). Our in vivo genetic analysis demonstrates that such interactions require specific lysine residues of Ub that are important for Ub chain formation. We show that Saccharomyces cerevisiae cells lacking two of these UBA proteins, Dsk2 and Rad23, are deficient in protein degradation mediated by the UFD pathway and that the intact UBA motif of Dsk2 is essential for its function in proteolysis. Dsk2 and Rad23 can form a complex(es), suggesting that they cooperate to recognize a subset of multi-Ub chains and deliver the Ub-tagged substrates to the proteasome. Our results suggest a molecular mechanism for differentiation of substrate fates, depending on the precise nature of the mono-Ub or multi-Ub lysine linkage, and provide a foundation to further investigate postubiquitylation events.     

Yos9p detects and targets misfolded glycoproteins for ER-associated degradation

Endoplasmic reticulum (ER) quality control mechanisms monitor the folding of nascent secretory and membrane polypeptides. Immature molecules are actively retained in the folding compartment whereas proteins that fail to fold are diverted to proteasome-dependent degradation pathways. We report that a key pathway of ER quality control consists of a two-lectin receptor system consisting of Yos9p and Htm1/Mnl1p that recognizes N-linked glycan signals embedded in substrates. This pathway recognizes lumenally oriented determinants of soluble and membrane proteins. Yos9p binds directly to substrates to discriminate misfolded from folded proteins. Substrates displaying cytosolic determinants can be degraded independently of this system. Our studies show that mechanistically divergent systems collaborate to guard against passage and accumulation of misfolded proteins in the secretory pathway.     

The UBA2 domain functions as an intrinsic stabilization signal that protects Rad23 from proteasomal degradation

The proteasome-interacting protein Rad23 is a long-lived protein. Interaction between Rad23 and the proteasome is required for Rad23's functions in nucleotide excision repair and ubiquitin-dependent degradation. Here, we show that the ubiquitin-associated (UBA)-2 domain of yeast Rad23 is a cis-acting, transferable stabilization signal that protects Rad23 from proteasomal degradation. Disruption of the UBA2 domain converts Rad23 into a short-lived protein that is targeted for degradation through its N-terminal ubiquitin-like domain. UBA2-dependent stabilization is required for Rad23 function because a yeast strain expressing a mutant Rad23 that lacks a functional UBA2 domain shows increased sensitivity to UV light and, in the absence of Rpn10, severe growth defects. The C-terminal UBA domains of Dsk2, Ddi1, Ede1, and the human Rad23 homolog hHR23A have similar protective activities. Thus, the UBA2 domain of Rad23 is an evolutionarily conserved stabilization signal that allows Rad23 to interact with the proteasome without facing destruction.     

A genetic screen identifies BRCA2 and PALB2 as key regulators of G2 checkpoint maintenance

To identify key connections between DNA-damage repair and checkpoint pathways, we performed RNA interference screens for regulators of the ionizing radiation-induced G2 checkpoint, and we identified the breast cancer gene BRCA2. The checkpoint was also abrogated following depletion of PALB2, an interaction partner of BRCA2. BRCA2 and PALB2 depletion led to premature checkpoint abrogation and earlier activation of the AURORA A-PLK1 checkpoint-recovery pathway. These results indicate that the breast cancer tumour suppressors and homologous recombination repair proteins BRCA2 and PALB2 are main regulators of G2 checkpoint maintenance following DNA-damage.     

A genetic screen identifies genes essential for development of myelinated axons in zebrafish

The myelin sheath insulates axons in the vertebrate nervous system, allowing rapid propagation of action potentials via saltatory conduction. Specialized glial cells, termed Schwann cells in the PNS and oligodendrocytes in the CNS, wrap axons to form myelin, a compacted, multilayered sheath comprising specific proteins and lipids. Disruption of myelinated axons causes human diseases, including multiple sclerosis and Charcot-Marie-Tooth peripheral neuropathies. Despite the progress in identifying human disease genes and other mutations disrupting glial development and myelination, many important unanswered questions remain about the mechanisms that coordinate the development of myelinated axons. To address these questions, we began a genetic dissection of myelination in zebrafish. Here we report a genetic screen that identified 13 mutations, which define 10 genes, disrupting the development of myelinated axons. We present the initial characterization of seven of these mutations, defining six different genes, along with additional characterization of mutations that we have described previously. The different mutations affect the PNS, the CNS, or both, and phenotypic analyses indicate that the genes affect a wide range of steps in glial development, from fate specification through terminal differentiation. The analysis of these mutations will advance our understanding of myelination, and the mutants will serve as models of human diseases of myelin.     

A transgene-assisted genetic screen identifies essential regulators of vascular development in vertebrate embryos

Formation of a functional vasculature during mammalian development is essential for embryonic survival. In addition, imbalance in blood vessel growth contributes to the pathogenesis of numerous disorders. Most of our understanding of vascular development and blood vessel growth comes from investigating the Vegf signaling pathway as well as the recent observation that molecules involved in axon guidance also regulate vascular patterning. In order to take an unbiased, yet focused, approach to identify novel genes regulating vascular development, we performed a three-step ENU mutagenesis screen in zebrafish. We first screened live embryos visually, evaluating blood flow in the main trunk vessels, which form by vasculogenesis, and the intersomitic vessels, which form by angiogenesis. Embryos that displayed reduced or absent circulation were fixed and stained for endogenous alkaline phosphatase activity to reveal blood vessel morphology. All putative mutants were then crossed into the Tg(flk1:EGFP)(s843) transgenic background to facilitate detailed examination of endothelial cells in live and fixed embryos. We screened 4015 genomes and identified 30 mutations affecting various aspects of vascular development. Specifically, we identified 3 genes (or loci) that regulate the specification and/or differentiation of endothelial cells, 8 genes that regulate vascular tube and lumen formation, 8 genes that regulate vascular patterning, and 11 genes that regulate vascular remodeling, integrity and maintenance. Only 4 of these genes had previously been associated with vascular development in zebrafish illustrating the value of this focused screen. The analysis of the newly defined loci should lead to a greater understanding of vascular development and possibly provide new drug targets to treat the numerous pathologies associated with dysregulated blood vessel growth.     

A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis

Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-β-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death.Programmed cell death has a crucial role in a variety of biological processes ranging from normal tissue development to diverse pathological conditions.^{1, 2} Necroptosis is a form of regulated cell death that has been shown to occur during pathogen infection or sterile injury-induced inflammation in conditions where apoptosis signaling is compromised.^{3, 4, 5, 6} Given that many viruses have developed strategies to circumvent apoptotic cell death, necroptosis constitutes an important, pro-inflammatory back-up mechanism that limits viral spread in vivo.^{7, 8, 9} In contrast, in the context of sterile inflammation, necroptotic cell death contributes to disease pathology, outlining potential benefits of therapeutic intervention.¹⁰ Necroptosis can be initiated by death receptors of the tumor necrosis factor (TNF) superfamily,¹¹ Toll-like receptor 3 (TLR3),¹² TLR4,¹³ DNA-dependent activator of IFN-regulatory factors¹⁴ or interferon receptors.¹⁵ Downstream signaling is subsequently conveyed via RIPK1¹⁶ or TIR-domain-containing adapter-inducing interferon-β,^{8, 17} and converges on RIPK3-mediated^{13, 18, 19, 20} activation of MLKL.²¹ Phosphorylated MLKL triggers membrane rupture,^{22, 23, 24, 25, 26} releasing pro-inflammatory cellular contents to the extracellular space.²⁷ Studies using the RIPK1 inhibitor necrostatin-1 (Nec-1) ²⁸ or RIPK3-deficient mice have established a role for necroptosis in the pathophysiology of pancreatitis,¹⁹ artherosclerosis,²⁹ retinal cell death,³⁰ ischemic organ damage and ischemia-reperfusion injury in both the kidney³¹ and the heart.³² Moreover, allografts from RIPK3-deficient mice are better protected from rejection, suggesting necroptosis inhibition as a therapeutic option to improve transplant outcome.³³ Besides Nec-1, several tool compounds inhibiting different pathway members have been described,^{12, 16, 21, 34, 35} however, no inhibitors of necroptosis are available for clinical use so far.^{2, 10} In this study we screened a library of FDA approved drugs for the precise purpose of identifying already existing and generally safe chemical agents that could be used as necroptosis inhibitors. We identified the two structurally distinct kinase inhibitors pazopanib and ponatinib as potent blockers of necroptosis targeting the key enzymes RIPK1/3.     

The Yeast E4 Ubiquitin Ligase Ufd2 Interacts with the Ubiquitin-like Domains of Rad23 and Dsk2 via a Novel and Distinct Ubiquitin-like Binding Domain

Proteins containing ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains interact with various binding partners and function as hubs during ubiquitin-mediated protein degradation. A common interaction of the budding yeast UBL-UBA proteins Rad23 and Dsk2 with the E4 ubiquitin ligase Ufd2 has been described in endoplasmic reticulum-associated degradation among other pathways. The UBL domains of Rad23 and Dsk2 play a prominent role in this process by interacting with Ufd2 and different subunits of the 26 S proteasome. Here, we report crystal structures of Ufd2 in complex with the UBL domains of Rad23 and Dsk2. The N-terminal UBL-interacting region of Ufd2 exhibits a unique sequence pattern, which is distinct from any known ubiquitin- or UBL-binding domain identified so far. Residue-specific differences exist in the interactions of these UBL domains with Ufd2, which are coupled to subtle differences in their binding affinities. The molecular details of their differential interactions point to a role for adaptive evolution in shaping these interfaces.     

p97 functions as an auxiliary factor to facilitate TM domain extraction during CFTR ER-associated degradation

The AAA-ATPase (ATPase associated with various cellular activities) p97 has been implicated in the degradation of misfolded and unassembled proteins in the endoplasmic reticulum (ERAD). To better understand its role in this process, we used a reconstituted cell-free system to define the precise contribution of p97 in degrading immature forms of the polytopic, multi-domain protein CFTR (cystic fibrosis transmembrane conductance regulator). Although p97 augmented both the rate and the extent of CFTR degradation, it was not obligatorily required for ERAD. Only a 50% decrease in degradation was observed in the complete absence of p97. Moreover, p97 specifically stimulated the degradation of CFTR transmembrane (TM) domains but had no effect on isolated cytosolic domains. Consistent with this, p97-mediated extraction of intact TM domains was independent of proteolytic cleavage and influenced by TM segment hydrophobicity, indicating that the relative contribution of p97 is partially determined by substrate stability. Thus, we propose that p97 functions in ERAD as a nonessential but important ancillary component to the proteasome where it facilitates substrate presentation and increases the degradation rate and efficiency of stable (TM) domains.     

Yos9p and Hrd1p mediate ER retention of misfolded proteins for ER-associated degradation

The endoplasmic reticulum (ER) has an elaborate quality control system, which retains misfolded proteins and targets them to ER-associated protein degradation (ERAD). To analyze sorting between ER retention and ER exit to the secretory pathway, we constructed fusion proteins containing both folded carboxypeptidase Y (CPY) and misfolded mutant CPY (CPY*) units. Although the luminal Hsp70 chaperone BiP interacts with the fusion proteins containing CPY* with similar efficiency, a lectin-like ERAD factor Yos9p binds to them with different efficiency. Correlation between efficiency of Yos9p interactions and ERAD of these fusion proteins indicates that Yos9p but not BiP functions in the retention of misfolded proteins for ERAD. Yos9p targets a CPY*-containing ERAD substrate to Hrd1p E3 ligase, thereby causing ER retention of the misfolded protein. This ER retention is independent of the glycan degradation signal on the misfolded protein and operates even when proteasomal degradation is inhibited. These results collectively indicate that Yos9p and Hrd1p mediate ER retention of misfolded proteins in the early stage of ERAD, which constitutes a process separable from the later degradation step.     

A functional genomic screen identifies a role for TAO1 kinase in spindle-checkpoint signalling

Defects in chromosome-microtubule attachment trigger spindle-checkpoint activation and delay mitotic progression. How microtubule attachment is sensed and integrated into the steps of checkpoint-signal amplification is poorly understood. In a functional genomic screen targeting human kinases and phosphatases, we identified a microtubule affinity-regulating kinase kinase, TAO1 (also known as MARKK) as an important regulator of mitotic progression, required for both chromosome congression and checkpoint-induced anaphase delay. TAO1 interacts with the checkpoint kinase BubR1 and promotes enrichment of the checkpoint protein Mad2 at sites of defective attachment, providing evidence for a regulatory step that precedes the proposed Mad2-Mad1 dependent checkpoint-signal amplification step. We propose that the dual functions of TAO1 in regulating microtubule dynamics and checkpoint signalling may help to coordinate the establishment and monitoring of correct congression of chromosomes, thereby protecting genomic stability in human cells.