Replication stress promotes cell elimination by extrusion.

Cell extrusion is a mechanism of cell elimination that is used by organisms as diverse as sponges, nematodes, insects and mammals1-3. During extrusion, a cell detaches from a layer of surrounding cells while maintaining the continuity of that layer4. Vertebrate epithelial tissues primarily eliminate cells by extrusion, and the dysregulation of cell extrusion has been linked to epithelial diseases, including cancer1,5. The mechanisms that drive cell extrusion remain incompletely understood. Here, to analyse cell extrusion by Caenorhabditis elegans embryos3, we conducted a genome-wide RNA interference screen, identified multiple cell-cycle genes with S-phase-specific function, and performed live-imaging experiments to establish how those genes control extrusion. Extruding cells experience replication stress during S phase and activate a replication-stress response via homologues of ATR and CHK1. Preventing S-phase entry, inhibiting the replication-stress response, or allowing completion of the cell cycle blocked cell extrusion. Hydroxyurea-induced replication stress6,7 triggered ATR-CHK1- and p53-dependent cell extrusion from a mammalian epithelial monolayer. We conclude that cell extrusion induced by replication stress is conserved among animals and propose that this extrusion process is a primordial mechanism of cell elimination with a tumour-suppressive function in mammals.

Analyzing human knockouts to validate GPR151 as a therapeutic target for reduction of body mass index.

Novel drug targets for sustained reduction in body mass index (BMI) are needed to curb the epidemic of obesity, which affects 650 million individuals worldwide and is a causal driver of cardiovascular and metabolic disease and mortality. Previous studies reported that the Arg95Ter nonsense variant of GPR151, an orphan G protein-coupled receptor, is associated with reduced BMI and reduced risk of Type 2 Diabetes (T2D). Here, we further investigate GPR151 with the Pakistan Genome Resource (PGR), which is one of the largest exome biobanks of human homozygous loss-of-function carriers (knockouts) in the world. Among PGR participants, we identify eleven GPR151 putative loss-of-function (plof) variants, three of which are present at homozygosity (Arg95Ter, Tyr99Ter, and Phe175LeufsTer7), with a cumulative allele frequency of 2.2%. We confirm these alleles in vitro as loss-of-function. We test if GPR151 plof is associated with BMI, T2D, or other metabolic traits and find that GPR151 deficiency in complete human knockouts is not associated with clinically significant differences in these traits. Relative to Gpr151+/+ mice, Gpr151-/- animals exhibit no difference in body weight on normal chow and higher body weight on a high-fat diet. Together, our findings indicate that GPR151 antagonism is not a compelling therapeutic approach to treatment of obesity.

Programmed elimination of cells by caspase-independent cell extrusion in C. elegans.

The elimination of unnecessary or defective cells from metazoans occurs during normal development and tissue homeostasis, as well as in response to infection or cellular damage. Although many cells are removed through caspase-mediated apoptosis followed by phagocytosis by engulfing cells, other mechanisms of cell elimination occur, including the extrusion of cells from epithelia through a poorly understood, possibly caspase-independent, process. Here we identify a mechanism of cell extrusion that is caspase independent and that can eliminate a subset of the Caenorhabditis elegans cells programmed to die during embryonic development. In wild-type animals, these cells die soon after their generation through caspase-mediated apoptosis. However, in mutants lacking all four C. elegans caspase genes, these cells are eliminated by being extruded from the developing embryo into the extra-embryonic space of the egg. The shed cells show apoptosis-like cytological and morphological characteristics, indicating that apoptosis can occur in the absence of caspases in C. elegans. We describe a kinase pathway required for cell extrusion involving PAR-4, STRD-1 and MOP-25.1/-25.2, the C. elegans homologues of the mammalian tumour-suppressor kinase LKB1 and its binding partners STRADα and MO25α. The AMPK-related kinase PIG-1, a possible target of the PAR-4­STRD-1­MOP-25 kinase complex, is also required for cell shedding. PIG-1 promotes shed-cell detachment by preventing the cell-surface expression of cell-adhesion molecules. Our findings reveal a mechanism for apoptotic cell elimination that is fundamentally distinct from that of canonical programmed cell death.

Both the caspase CSP-1 and a caspase-independent pathway promote programmed cell death in parallel to the canonical pathway for apoptosis in Caenorhabditis elegans.

Caspases are cysteine proteases that can drive apoptosis in metazoans and have critical functions in the elimination of cells during development, the maintenance of tissue homeostasis, and responses to cellular damage. Although a growing body of research suggests that programmed cell death can occur in the absence of caspases, mammalian studies of caspase-independent apoptosis are confounded by the existence of at least seven caspase homologs that can function redundantly to promote cell death. Caspase-independent programmed cell death is also thought to occur in the invertebrate nematode Caenorhabditis elegans. The C. elegans genome contains four caspase genes (ced-3, csp-1, csp-2, and csp-3), of which only ced-3 has been demonstrated to promote apoptosis. Here, we show that CSP-1 is a pro-apoptotic caspase that promotes programmed cell death in a subset of cells fated to die during C. elegans embryogenesis. csp-1 is expressed robustly in late pachytene nuclei of the germline and is required maternally for its role in embryonic programmed cell deaths. Unlike CED-3, CSP-1 is not regulated by the APAF-1 homolog CED-4 or the BCL-2 homolog CED-9, revealing that csp-1 functions independently of the canonical genetic pathway for apoptosis. Previously we demonstrated that embryos lacking all four caspases can eliminate cells through an extrusion mechanism and that these cells are apoptotic. Extruded cells differ from cells that normally undergo programmed cell death not only by being extruded but also by not being engulfed by neighboring cells. In this study, we identify in csp-3; csp-1; csp-2 ced-3 quadruple mutants apoptotic cell corpses that fully resemble wild-type cell corpses: these caspase-deficient cell corpses are morphologically apoptotic, are not extruded, and are internalized by engulfing cells. We conclude that both caspase-dependent and caspase-independent pathways promote apoptotic programmed cell death and the phagocytosis of cell corpses in parallel to the canonical apoptosis pathway involving CED-3 activation.

SPK-1, an SR protein kinase, inhibits programmed cell death in Caenorhabditis elegans.

To identify genes involved in protecting cells from programmed cell death in Caenorhabditis elegans, we performed a genetic screen to isolate mutations that cause an increase in the number of programmed cell deaths. We screened for suppressors of the cell-death defect caused by a partial loss-of-function mutation in ced-4, which encodes an Apaf-1 homolog that promotes programmed cell death by activating the caspase CED-3. We identified one extragenic ced-4 suppressor, which has a mutation in the gene spk-1. The spk-1 gene encodes a protein homologous to serine-arginine-rich (SR) protein kinases, which are thought to regulate splicing. Previous work suggests that ced-4 can be alternatively spliced and that the splice variants function oppositely, with the longer transcript (ced-4L) inhibiting programmed cell death. spk-1 might promote cell survival by increasing the amount of the protective ced-4L splice variant. We conclude that programmed cell death in C. elegans is regulated by an alternative splicing event controlled by the SR protein kinase SPK-1.

Identification and characterization of human GDF15 knockouts.

Growth differentiation factor 15 (GDF15) is a secreted protein that regulates food intake, body weight and stress responses in pre-clinical models1. The physiological function of GDF15 in humans remains unclear. Pharmacologically, GDF15 agonism in humans causes nausea without accompanying weight loss2, and GDF15 antagonism is being tested in clinical trials to treat cachexia and anorexia. Human genetics point to a role for GDF15 in hyperemesis gravidarum, but the safety or impact of complete GDF15 loss, particularly during pregnancy, is unknown3-7. Here we show the absence of an overt phenotype in human GDF15 loss-of-function carriers, including stop gains, frameshifts and the fully inactivating missense variant C211G3. These individuals were identified from 75,018 whole-exome/genome-sequenced participants in the Pakistan Genomic Resource8,9 and recall-by-genotype studies with family-based recruitment of variant carrier probands. We describe 8 homozygous ('knockouts') and 227 heterozygous carriers of loss-of-function alleles, including C211G. GDF15 knockouts range in age from 31 to 75 years, are fertile, have multiple children and show no consistent overt phenotypes, including metabolic dysfunction. Our data support the hypothesis that GDF15 is not required for fertility, healthy pregnancy, foetal development or survival into adulthood. These observations support the safety of therapeutics that block GDF15.

Intramolecular cohesion of coils mediated by phenylalanine--glycine motifs in the natively unfolded domain of a nucleoporin.

The nuclear pore complex (NPC) provides the sole aqueous conduit for macromolecular exchange between the nucleus and the cytoplasm of cells. Its diffusion conduit contains a size-selective gate formed by a family of NPC proteins that feature large, natively unfolded domains with phenylalanine-glycine repeats (FG domains). These domains of nucleoporins play key roles in establishing the NPC permeability barrier, but little is known about their dynamic structure. Here we used molecular modeling and biophysical techniques to characterize the dynamic ensemble of structures of a representative FG domain from the yeast nucleoporin Nup116. The results showed that its FG motifs function as intramolecular cohesion elements that impart order to the FG domain and compact its ensemble of structures into native premolten globular configurations. At the NPC, the FG motifs of nucleoporins may exert this cohesive effect intermolecularly as well as intramolecularly to form a malleable yet cohesive quaternary structure composed of highly flexible polypeptide chains. Dynamic shifts in the equilibrium or competition between intra- and intermolecular FG motif interactions could facilitate the rapid and reversible structural transitions at the NPC conduit needed to accommodate passing karyopherin-cargo complexes of various shapes and sizes while simultaneously maintaining a size-selective gate against protein diffusion.

Anti-tubulins DEPendably induce apoptosis.

Anti-tubulin drugs are life-saving chemotherapeutics that kill cancer cells by stabilizing or disrupting microtubules. Despite their clinical utility, the molecular mechanisms by which anti-tubulins cause apoptotic cell death remain poorly understood. It is now shown that microtubule disruption can inhibit anti-apoptotic Bcl-2 family proteins through an evolutionarily conserved signalling axis involving DEPDC1 (LET-99 in Caenorhabditis elegans) and JNK.

Xk-related protein 8 and CED-8 promote phosphatidylserine exposure in apoptotic cells.

A classic feature of apoptotic cells is the cell-surface exposure of phosphatidylserine (PtdSer) as an "eat me" signal for engulfment. We show that the Xk-family protein Xkr8 mediates PtdSer exposure in response to apoptotic stimuli. Mouse Xkr8(-/-) cells or human cancer cells in which Xkr8 expression was repressed by hypermethylation failed to expose PtdSer during apoptosis and were inefficiently engulfed by phagocytes. Xkr8 was activated directly by caspases and required a caspase-3 cleavage site for its function. CED-8, the only Caenorhabditis elegans Xk-family homolog, also promoted apoptotic PtdSer exposure and cell-corpse engulfment. Thus, Xk-family proteins have evolutionarily conserved roles in promoting the phagocytosis of dying cells by altering the phospholipid distribution in the plasma membrane.

Rapid evolution exposes the boundaries of domain structure and function in natively unfolded FG nucleoporins.

Nucleoporins with phenylalanine-glycine repeats (FG Nups) function at the nuclear pore complex (NPC) to facilitate nucleocytoplasmic transport. In Saccharomyces cerevisiae, each FG Nup contains a large natively unfolded domain that is punctuated by FG repeats. These FG repeats are surrounded by hydrophilic amino acids (AAs) common to disordered protein domains. Here we show that the FG domain of Nups from human, fly, worm, and other yeast species is also enriched in these disorder-associated AAs, indicating that structural disorder is a conserved feature of FG Nups and likely serves an important role in NPC function. Despite the conservation of AA composition, FG Nup sequences from different species show extensive divergence. A comparison of the AA substitution rates of proteins with syntenic orthologs in four Saccharomyces species revealed that FG Nups have evolved at twice the rate of average yeast proteins with most substitutions occurring in sequences between FG repeats. The rapid evolution of FG Nups is poorly explained by parameters known to influence AA substitution rate, such as protein expression level, interactivity, and essentiality; instead their rapid evolution may reflect an intrinsic permissiveness of natively unfolded structures to AA substitutions. The overall lack of AA sequence conservation in FG Nups is sharply contrasted by discrete stretches of conserved sequences. These conserved sequences highlight known karyopherin and nucleoporin binding sites as well as other uncharacterized sites that may have important structural and functional properties.

The Saccharomyces cerevisiae nucleoporin Nup2p is a natively unfolded protein.

Little is known about the structure of the individual nucleoporins that form eukaryotic nuclear pore complexes (NPCs). We report here in vitro physical and structural characterizations of a full-length nucleoporin, the Saccharomyces cerevisiae protein Nup2p. Analyses of the Nup2p structure by far-UV circular dichroism (CD) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, protease sensitivity, gel filtration, and sedimentation velocity experiments indicate that Nup2p is a "natively unfolded protein," belonging to a class of proteins that exhibit little secondary structure, high flexibility, and low compactness. Nup2p possesses a very large Stokes radius (79 A) in gel filtration columns, sediments slowly in sucrose gradients as a 2.9 S particle, and is highly sensitive to proteolytic digestion by proteinase K; these characteristics suggest a structure of low compactness and high flexibility. Spectral analyses (CD and FTIR spectroscopy) provide additional evidence that Nup2p contains extensive regions of structural disorder with comparatively small contributions of ordered secondary structure. We address the possible significance of natively unfolded nucleoporins in the mechanics of nucleocytoplasmic trafficking across NPCs.

Disorder in the nuclear pore complex: the FG repeat regions of nucleoporins are natively unfolded.

Nuclear transport proceeds through nuclear pore complexes (NPCs) that are embedded in the nuclear envelope of eukaryotic cells. The Saccharomyces cerevisiae NPC is comprised of 30 nucleoporins (Nups), 13 of which contain phenylalanine-glycine repeats (FG Nups) that bind karyopherins and facilitate the transport of karyopherin-cargo complexes. Here, we characterize the structural properties of S. cerevisiae FG Nups by using biophysical methods and predictive amino acid sequence analyses. We find that FG Nups, particularly the large FG repeat regions, exhibit structural characteristics typical of "natively unfolded" proteins (highly flexible proteins that lack ordered secondary structure). Furthermore, we use protease sensitivity assays to demonstrate that most FG Nups are disordered in situ within the NPCs of purified yeast nuclei. The conclusion that FG Nups constitute a family of natively unfolded proteins supports the hypothesis that the FG repeat regions of Nups form a meshwork of random coils at the NPC through which nuclear transport proceeds.

Sleep deprivation effects on growth factor expression in neonatal rats: a potential role for BDNF in the mediation of delta power.

The sleeping brain differs from the waking brain in its electrophysiological and molecular properties, including the expression of growth factors and immediate early genes (IEG). Sleep architecture and homeostatic regulation of sleep in neonates is distinct from that of adults. Hence, the present study addressed the question whether the unique homeostatic response to sleep deprivation in neonates is reflected in mRNA expression of the IEG cFos, brain-derived nerve growth factor (BDNF), and basic fibroblast growth factor (FGF2) in the cortex. As sleep deprivation is stressful to developing rats, we also investigated whether the increased levels of corticosterone would affect the expression of growth factors in the hippocampus, known to be sensitive to glucocorticoid levels. At postnatal days 16, 20, and 24, rats were subjected to sleep deprivation, maternal separation without sleep deprivation, sleep deprivation with 2 h recovery sleep, or no intervention. mRNA expression was quantified in the cortex and hippocampus. cFos was increased after sleep deprivation and was similar to control level after 2 h recovery sleep irrespective of age or brain region. BDNF was increased by sleep deprivation in the cortex at P20 and P24 and only at P24 in the hippocampus. FGF2 increased during recovery sleep at all ages in both brain regions. We conclude that cortical BDNF expression reflects the onset of adult sleep-homeostatic response, whereas the profile of expression of both growth factors suggests a trophic effect of mild sleep deprivation.