Differential effect of aneuploidy on the X chromosome and genes with sex-biased expression in Drosophila

Global analysis of gene expression via RNA sequencing was conducted for trisomics for the left arm of chromosome 2 (2L) and compared with the normal genotype. The predominant response of genes on 2L was dosage compensation in that similar expression occurred in the trisomic compared with the diploid control. However, the male and female trisomic/normal expression ratio distributions for 2L genes differed in that females also showed a strong peak of genes with increased expression and males showed a peak of reduced expression relative to the opposite sex. For genes in other autosomal regions, the predominant response to trisomy was reduced expression to the inverse of the altered chromosomal dosage (2/3), but a minor peak of increased expression in females and further reduced expression in males were also found, illustrating a sexual dimorphism for the response to aneuploidy. Moreover, genes with sex-biased expression as revealed by comparing amounts in normal males and females showed responses of greater magnitude to trisomy 2L, suggesting that the genes involved in dosage-sensitive aneuploid effects also influence sex-biased expression. Each autosomal chromosome arm responded to 2L trisomy similarly, but the ratio distributions for X-linked genes were distinct in both sexes, illustrating an X chromosome-specific response to aneuploidy.Changes in chromosomal dosage have long been known to affect the phenotype or viability of an organism (1–4). Altering the dosage of individual chromosomes typically has a greater impact than varying the whole genome (5–7). This general rule led to the concept of “genomic balance” in that dosage changes of part of the genome produce a nonoptimal relationship of gene products. The interpretation afforded these observations was that genes on the aneuploid chromosome produce a dosage effect for the amount of gene product present in the cell (8).However, when gene expression studies were conducted on aneuploids, it became known that transacting modulations of gene product amounts were also more prevalent with aneuploidy than with whole-genome changes (9–14). Assays of enzyme activities, protein, and RNA levels revealed that any one chromosomal segment could modulate in trans the expression of genes throughout the genome (9–15). These modulations could be positively or negatively correlated with the changed chromosomal segment dosage, but inverse correlations were the most common (10–13). For genes on the varied segment, not only were dosage effects observed, but dosage compensation was also observed, which results from a cancelation of gene dosage effects by inverse effects operating simultaneously on the varied genes (9, 10, 14–18). This circumstance results in “autosomal” dosage compensation (14, 16–18). Studies of trisomic X chromosomes examining selected endogenous genes or global RNA sequencing (RNA-seq) studies illustrate that the inverse effect can also account for sex chromosome dosage compensation in Drosophila (15, 19–21). In concert, autosomal genes are largely inversely affected by trisomy of the X chromosome (15, 19, 21).The dosage effects of aneuploidy can be reduced to the action of single genes whose functions tend to be involved in heterogeneous aspects of gene regulation but which have in common membership in macromolecular complexes (8, 22–24). This fact led to the hypothesis that genomic imbalance effects result from the altered stoichiometry of subunits that affects the function of the whole and that occurs from partial but not whole-genome dosage change (8, 22–25). Genomic balance also affects the evolutionary trajectory of duplicate genes differently based on whether the mode of duplication is partial or whole-genome (22, 23).Here we used RNA-seq to examine global patterns of gene expression in male and female larvae trisomic for the left arm of chromosome 2 (2L). The results demonstrate the strong prevalence of aneuploidy dosage compensation and of transacting inverse effects. Furthermore, because both trisomic males and females could be examined, a sexual dimorphism of the aneuploid response was discovered. Also, the response of the X chromosome to trisomy 2L was found to be distinct from that of the autosomes, illustrating an X chromosome-specific effect. Genes with sex-biased expression, as determined by comparing normal males and females, responded more strongly to trisomy 2L. Collectively, the results illustrate the prevalence of the inverse dosage effect in trisomic Drosophila and suggest that the X chromosome has evolved a distinct response to genomic imbalance as would be expected under the hypothesis that X chromosome dosage compensation uses the inverse dosage effect as part of its mechanism (15).     

Crystal structure and versatile functional roles of the COP9 signalosome subunit 1

The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) plays key roles in many biological processes, such as repression of photomorphogenesis in plants and protein subcellular localization, DNA-damage response, and NF-κB activation in mammals. It is an evolutionarily conserved eight-protein complex with subunits CSN1 to CSN8 named following the descending order of molecular weights. Here, we report the crystal structure of the largest CSN subunit, CSN1 from Arabidopsis thaliana (atCSN1), which belongs to the Proteasome, COP9 signalosome, Initiation factor 3 (PCI) domain containing CSN subunit family, at 2.7 Å resolution. In contrast to previous predictions and distinct from the PCI-containing 26S proteasome regulatory particle subunit Rpn6 structure, the atCSN1 structure reveals an overall globular fold, with four domains consisting of helical repeat-I, linker helix, helical repeat-II, and the C-terminal PCI domain. Our small-angle X-ray scattering envelope of the CSN1–CSN7 complex agrees with the EM structure of the CSN alone (apo-CSN) and suggests that the PCI end of each molecule may mediate the interaction. Fitting of the CSN1 structure into the CSN–Skp1-Cul1-Fbox (SCF) EM structure shows that the PCI domain of CSN1 situates at the hub of the CSN for interaction with several other subunits whereas the linker helix and helical repeat-II of CSN1 contacts SCF using a conserved surface patch. Furthermore, we show that, in human, the C-terminal tail of CSN1, a segment not included in our crystal structure, interacts with IκBα in the NF-κB pathway. Therefore, the CSN complex uses multiple mechanisms to hinder NF-κB activation, a principle likely to hold true for its regulation of many other targets and pathways.The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) is a more than 300-kDa complex that was first identified as a negative regulator of Constitutive Photomorphogenesis (COP) in plants (1, 2). In the subsequent years, the highly conserved protein complex was also found in fungi (3, 4), Caenorhabditis elegans (5), Drosophila melanogaster (6), and mammals (7, 8). The most studied function of the CSN complex in eukaryotes is the regulation of protein degradation through two pathways, deneddylation (9–11) and deubiquitination (12, 13). In the deneddylation pathway, the CSN complex can influence the cullin-RING ligase activity by removing Nedd8, a ubiquitin-like protein, from a cullin (9, 14). On the other hand, the CSN complex can also suppress cullin activity through recruitment of the deubiquitination enzyme USP15 (12) or Ubp12p, the Schizosaccharomyces pombe ortholog of human USP15 (13). Other functions of the CSN complex identified in mammalian cells include regulating the phosphorylation of ubiquitin–proteasome pathway substrates through CSN-associated kinases (7, 15–18). Overall, the CSN complex appears to be a key player in protein subcellular localization (19, 20), DNA-damage response (21), NF-κB activation (22), development, and cell cycle control (23, 24). Thus, the functions of the CSN complex are beyond the regulation of light-dependent reaction in plants.The CSN complex in most of the species contains eight subunits named CSN1 to CSN8, in order of decreasing size. All eight subunits share homologous sequences with “lid” components of the 26S proteasome regulatory particle and the eukaryotic translation initiation factor 3 (eIF3) (7, 25). Among these eight subunits, CSN6 and catalytic CSN5 contain a conserved MPN-domain (MOV34, Pad1N-terminal) (26), and the rest of the CSN subunits bear a PCI-domain (Proteasome, COP9 signalosome, Initiation factor eIF3). The MPN-domain within CSN5 contains a metal chelating site and forms the catalytic region of the isopeptidase for deneddylation (27). Recently, the crystal structures of the CSN6–MPN domain and the CSN5 subunit have been revealed (28, 29). Interestingly, amino acids 97–131, a flexible segment within the CSN5–MPN domain, were proven to be essential in regulating the isopeptidase states of CSN5 (29). PCI is an ∼200-amino acid domain, beginning with an N-terminal helical bundle arrangement and ending with a globular winged-helix subdomain (30, 31). A number of interactions between PCI domains of CSN subunits have been identified by the yeast two-hybrid system (32, 33). Dessau et al. reported the crystallographic data of the PCI domain of Arabidopsis thaliana subunit 7, and their in vitro studies also suggested that the PCI domain mediates and stabilizes protein–protein interactions within the complex (34).Although many speculated on how the CSN subunits interact with each other and come into a functional unit, the architecture of the CSN complex was accessed by electron microscopy (EM) (35, 36) and native mass spectrometry approaches (37). These studies confirmed structural similarities among CSN, the proteasome lid, and eIF3. Furthermore, the CSN appears to contain two dominant subcomplexes, CSN1/2/3/8 and CSN 4/5/6/7 (37), which correspond to the large and the small segments, respectively, in an EM study of the CSN alone (apo-CSN) (36). An EM study of the CSN in complex with an Skp1-Cul1-Fbox (SCF) E3 ligase was also reported, showing reciprocal regulation between CSN and SCF (38). To date, unfortunately, there is no high-resolution mapping on these subunit interactions.To further define the CSN structure and to study its functional significance, we feel the need to obtain structures of CSN subunits at an atomic level. In our study, we used Arabidopsis thaliana CSN1 (atCSN1) as a guide to facilitate our understandings of the PCI-containing CSN subunits. The atCSN1, encoded in the chromosome 3, has 441 amino acids that are 45% identical in sequence to Homo sapiens CSN1. Among all of the subunits of the complex, CSN1 is known to be the longest and to play a crucial role in complex integrity (39–41). Here, we report the crystal structure of atCSN1 and describe its integration within the complex as well as its interaction with IκBα in the NF-κB signaling pathway.     

SUMOylation at K340 inhibits tau degradation through deregulating its phosphorylation and ubiquitination

Intracellular accumulation of the abnormally modified tau is hallmark pathology of Alzheimer’s disease (AD), but the mechanism leading to tau aggregation is not fully characterized. Here, we studied the effects of tau SUMOylation on its phosphorylation, ubiquitination, and degradation. We show that tau SUMOylation induces tau hyperphosphorylation at multiple AD-associated sites, whereas site-specific mutagenesis of tau at K340R (the SUMOylation site) or simultaneous inhibition of tau SUMOylation by ginkgolic acid abolishes the effect of small ubiquitin-like modifier protein 1 (SUMO-1). Conversely, tau hyperphosphorylation promotes its SUMOylation; the latter in turn inhibits tau degradation with reduction of solubility and ubiquitination of tau proteins. Furthermore, the enhanced SUMO-immunoreactivity, costained with the hyperphosphorylated tau, is detected in cerebral cortex of the AD brains, and β-amyloid exposure of rat primary hippocampal neurons induces a dose-dependent SUMOylation of the hyperphosphorylated tau. Our findings suggest that tau SUMOylation reciprocally stimulates its phosphorylation and inhibits the ubiquitination-mediated tau degradation, which provides a new insight into the AD-like tau accumulation.Alzheimer’s disease (AD) is the most common neurodegenerative disorder in the elderly. Intracellular accumulation of neurofibrillary tangles (NFTs) and extracellular precipitation of senile plaques are the most prominent pathological hallmarks of AD (1–3). The clinical-to-pathological correlation studies have demonstrated that the number of NFTs consisting of hyperphosphorylated tau correlates with the degree of dementia in AD (4–6). Tau is the major microtubule-associated protein that normally contains 2–3 mol of phosphate per mole of tau protein. In AD brains, tau is abnormally hyperphosphorylated (namely AD-P-tau) and the phosphate level increases to 5–9 mol phosphate per mole tau (4). AD-P-tau does not bind to tubulin and become incompetent in promoting microtubule assembly and maintaining the stability of the microtubules. The AD-P-tau also sequesters normal tau from microtubules (7), and serves as a template for the conversion of normal tau into misfolded protein in a prion-like manner (8). In addition to hyperphosphorylation, tau is also contains other posttranslational modifications, such as ubiquitination and SUMOylation (5, 9–11). The abnormal modification of tau also decreases its solubility, and ∼40% of the hyperphosphorylated tau in AD brains has been isolated as sedimentable nonfibril cytosolic protein (1, 12). Although the mechanisms underlying the formation of the NFTs remain unclear, the altered tau modifications and impaired degradation are believed to play a role. Therefore, clarifying the mechanism that may cause tau accumulation is of great significance for understanding the pathogenesis of AD and for developing new therapeutics.Like other proteins, tau can be degraded by autophagy-lysosomal and ubiqutin-proteasomal systems under physiological conditions. In mouse cortical neurons, a C-terminal–truncated form of tau that mimics tau cleaved at Asp421 (tauΔC) is removed by macroautophagic and lysosomal mechanisms (13). Lysosomal perturbation inhibits the clearance of tau with accumulation and aggregation of tau in M1C cells (14). Cathepsin D released from lysosome can degrade tau in cultured hippocampal slices (15). Inhibition of the autophagic vacuole formation leads to a noticeable accumulation of tau (14). Studies also suggest that tau protein is degraded in an ubiquitin-, ATP-, and 26S proteasome-, but not a 20S proteasome-dependent manner under normal conditions (16). When the cells are exposed to the stresses, CHIP, a ubiquitin ligase that interacts directly with Hsp70/90, can induce tau ubiquitination and thus selectively reduce the level of detergent insoluble tau (17). The compensatory activation of autophagy-lysosomal or ubiqutin-proteasomal system can antagonize tau aggregation; therefore, tau accumulation does not show in the early stage of AD. During the evolution of AD, a gradual impairment of autophagy-lysosomal system and ubiqutin-proteasomal system has been detected at later stage of the disease (18–20). Studies suggest that the ubiquitin-mediated degradation pathway seems ineffective in removing the tau-positive fibrillar structures in the AD brains (21–23); however, the mechanisms underlying the impairment of the ubiqutin-proteasomal system are elusive.Ubiquitin is an important component of the cellular defense system that tags abnormal proteins for their degradation by ATP-dependent nonlysosomal proteases (24). Monoclonal antibodies 3-39 and 5-25 raised against paired helical filaments of NFTs have been shown to recognize ubiquitin (25). Meanwhile, tau can be sumoylated at K340 in vitro by SUMO-1 (small ubiquitin-like modifier protein-1) and to a lesser extent by SUMO2 and SUMO3 (9–11). Moreover, SUMO-1 immunoreactivity was colocalized with tau aggregates in neuritic plaques of APP transgenic mice (11). It is well known that SUMO share similarities with ubiquitin in both the structure and the biochemistry of their conjugation (26). Therefore, tau SUMOylation may compete against its ubiquitination and thus suppress tau degradation. In the present study, we found that tau SUMOylation reciprocally stimulates its phosphorylation and thus inhibits the ubiquitination and degradation of tau proteins.     

Male-specific lethal complex in Drosophila counteracts histone acetylation and does not mediate dosage compensation

Dosage compensation is achieved in male Drosophila by a twofold up-regulation of the single X chromosome to reach the level of the two X chromosomes in females. A popular hypothesis to explain this phenomenon is that the male-specific lethal (MSL) complex, which is present at high levels on the male X, mediates this modulation of gene expression. One member of the complex, MOF, a histone acetyltransferase, acetylates lysine 16 of histone H4 and another, MSL2, which is only expressed in males, triggers its assembly. Here, we find that when a GAL4-MOF fusion protein is targeted to an upstream-activating sequence linked to a miniwhite reporter, up-regulation occurs in females but down-regulation in males, even though in the latter the whole MSL complex is recruited to the reporter genes and produces an increased histone acetylation. The expression of a GAL4-MSL2 fusion protein does not cause dosage compensation of X and autosomal reporters in females, although its expression causes the organization of the MSL complex on the reporter genes, leading to increased histone acetylation. RNAseq analysis of global endogenous gene expression in females with ectopic expression of MSL2 to coat the X chromosomes shows no evidence of increased expression compared with normal females. These data from multiple approaches indicate that the MSL complex does not mediate dosage compensation directly, but rather its activity overrides the high level of histone acetylation and counteracts the potential overexpression of X-linked genes to achieve the proper twofold up-regulation in males.Dosage compensation is achieved in male Drosophila by an approximate twofold up-regulation of the X chromosome to equal the two X chromosomes in females (1, 2). For several decades, it has been proposed that the components of the male-specific lethal (MSL) complex are present on the male X chromosome and produce the twofold modulation (3, 4). One member of the complex, MOF, a histone acetyltransferase, acetylates lysine 16 of histone H4 (5) and another, MSL2, which is male-specific, triggers its X chromosome assembly (6). The MSL2 protein is not expressed in females because it is blocked at translation by the female-specific sex-lethal (SXL) protein (7). Gene-expression data taken in support of the MSL hypothesis have normalized X expression to autosomal expression and found a reduced X/A ratio that was interpreted as a loss of compensation when the MSL complex was dissociated (3, 8–10). However, when gene expression is assayed phenotypically or in absolute terms, rather than as a ratio of X to autosomal expression, data from ectopic assembly of the complex in females failed to demonstrate up-regulation of the X chromosomes (11, 12) and the dissolution of the complex does not eliminate compensation (11–15). Many assayed autosomal genes were increased in expression in the maleless (mle) mutant males (11, 13, 14), providing an explanation for the reduced ratios when X values were normalized to the autosomes.The retention of dosage compensation and elevated autosomal expression conforms to the prediction that the X monosomic state triggers an inverse dosage effect that is commonly found in aneuploids (16–23). We hypothesize that this general reaction to genomic imbalance appears to have been modified and selected for the process of dosage compensation, which could account for the twofold up-regulation in males. However, because in normal males the autosomes are similarly expressed to those of females, it was hypothesized that the MSL complex present only in males sequesters MOF from the autosomes to mute any autosomal inverse effect, and counteracts the action of the resulting very high levels of histone acetylation on the X to allow the proper twofold modulation for compensation (11, 14). To examine further the function of the MSL complex, MOF and MSL2 were targeted to reporter genes to determine their impact on gene expression and dosage compensation and a global study of gene expression was conducted in females with ectopic MSL complex on their X chromosomes.     

Exchange protein directly activated by cAMP plays a critical role in bacterial invasion during fatal rickettsioses

Rickettsiae are responsible for some of the most devastating human infections. A high infectivity and severe illness after inhalation make some rickettsiae bioterrorism threats. We report that deletion of the exchange protein directly activated by cAMP (Epac) gene, Epac1, in mice protects them from an ordinarily lethal dose of rickettsiae. Inhibition of Epac1 suppresses bacterial adhesion and invasion. Most importantly, pharmacological inhibition of Epac1 in vivo using an Epac-specific small-molecule inhibitor, ESI-09, completely recapitulates the Epac1 knockout phenotype. ESI-09 treatment dramatically decreases the morbidity and mortality associated with fatal spotted fever rickettsiosis. Our results demonstrate that Epac1-mediated signaling represents a mechanism for host–pathogen interactions and that Epac1 is a potential target for the prevention and treatment of fatal rickettsioses.Rickettsiae are responsible for some of the most devastating human infections (1–4). It has been forecasted that temperature increases attributable to global climate change will lead to more widespread distribution of rickettsioses (5). These tick-borne diseases are caused by obligately intracellular bacteria of the genus Rickettsia, including Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever (RMSF) in the United States and Latin America (2, 3), and Rickettsia conorii, the causative agent of Mediterranean spotted fever endemic to southern Europe, North Africa, and India (6). A high infectivity and severe illness after inhalation make some rickettsiae (including Rickettsia prowazekii, R. rickettsii, Rickettsia typhi, and R. conorii) bioterrorism threats (7). Although the majority of rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated (1, 3) and 5% of treated RMSF cases (8) result in a fatal outcome caused by acute disseminated vascular endothelial infection and damage (9). Fatality rates as high as 32% have been reported in hospitalized patients diagnosed with Mediterranean spotted fever (10). In addition, strains of R. prowazekii resistant to tetracycline and chloramphenicol have been developed in laboratories (11). Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central features of SFG rickettsioses (1, 2, 9). The molecular mechanisms involved in rickettsial infection remain incompletely elucidated (9, 12). A comprehensive understanding of rickettsial pathogenesis and the development of novel mechanism-based treatment are urgently needed.Living organisms use intricate signaling networks for sensing and responding to changes in the external environment. cAMP, a ubiquitous second messenger, is an important molecular switch that translates environmental signals into regulatory effects in cells (13). As such, a number of microbial pathogens have evolved a set of diverse virulence-enhancing strategies that exploit the cAMP-signaling pathways of their hosts (14). The intracellular functions of cAMP are predominantly mediated by the classic cAMP receptor, protein kinase A (PKA), and the more recently discovered exchange protein directly activated by cAMP (Epac) (15). Thus, far, two isoforms, Epac1 and Epac2, have been identified in humans (16, 17). Epac proteins function by responding to increased intracellular cAMP levels and activating the Ras superfamily small GTPases Ras-proximate 1 and 2 (Rap1 and Rap2). Accumulating evidence demonstrates that the cAMP/Epac1 signaling axis plays key regulatory roles in controlling various cellular functions in endothelial cells in vitro, including cell adhesion (18–21), exocytosis (22), tissue plasminogen activator expression (23), suppressor of cytokine signaling 3 (SOCS-3) induction (24–27), microtubule dynamics (28, 29), cell–cell junctions, and permeability and barrier functions (30–37). Considering the critical importance of endothelial cells in rickettsioses, we examined the functional roles of Epac1 in rickettsial pathogenesis in vivo, taking advantage of the recently generated Epac1 knockout mouse (38) and Epac-specific inhibitors (39, 40) generated from our laboratory. Our studies demonstrate that Epac1 plays a key role in rickettsial infection and represents a therapeutic target for fatal rickettsioses.     

Mapping of SUMO sites and analysis of SUMOylation changes induced by external stimuli

SUMOylation is an essential ubiquitin-like modification involved in important biological processes in eukaryotic cells. Identification of small ubiquitin-related modifier (SUMO)-conjugated residues in proteins is critical for understanding the role of SUMOylation but remains experimentally challenging. We have set up a powerful and high-throughput method combining quantitative proteomics and peptide immunocapture to map SUMOylation sites and have analyzed changes in SUMOylation in response to stimuli. With this technique we identified 295 SUMO1 and 167 SUMO2 sites on endogenous substrates of human cells. We further used this strategy to characterize changes in SUMOylation induced by listeriolysin O, a bacterial toxin that impairs the host cell SUMOylation machinery, and identified several classes of host proteins specifically deSUMOylated in response to this toxin. Our approach constitutes an unprecedented tool, broadly applicable to various SUMO-regulated cellular processes in health and disease.Posttranslational modifications (PTMs) are key mechanisms used by both prokaryotes and eukaryotes to regulate protein activity specifically, locally, and temporally. Ubiquitin and ubiquitin-like proteins (UBLs) constitute a specific class of small protein modifiers that can be covalently attached to a target protein via the formation of an isopeptide bond in a reversible manner. Small ubiquitin-related modifier (SUMO), one of these UBLs, is an essential PTM in eukaryotic cells that is involved in various cellular functions including gene expression regulation, DNA repair, intracellular transport, and response to viral and bacterial infections (1–5). The human genome encodes three different functional SUMO isoforms (SUMO1, SUMO2, and SUMO3) that are conjugated to distinct but overlapping sets of target proteins (1, 2, 6). Conjugation of SUMO to its targets in humans requires an E1-activating enzyme (the SAE1/SAE2 heterodimer), an E2-conjugating enzyme (Ubc9), and several E3 SUMO enzymes. Once conjugated to its target, SUMO can be deconjugated by several different SUMO isopeptidases that tightly regulate the SUMOylation levels of proteins (7).Since the discovery of SUMO two decades ago, much effort has been dedicated to the identification of SUMO-conjugated proteins in different organisms including yeast, plants, and mammals (8). However, isolation of SUMOylated proteins has proven to be challenging. Indeed, for most SUMO substrates, only a small proportion of the total amount of protein is SUMO-modified. In addition, the high activity of SUMO isopeptidases in cell lysates results in the rapid loss of SUMO conjugation in the absence of appropriate inhibitors. Thus, the most common approach used to isolate SUMOylated proteins is based on the expression of His-tagged versions of SUMO allowing the purification of SUMO-conjugated proteins by nickel chromatography under denaturing conditions (8, 9). Denaturing conditions inactivate SUMO isopeptidases and also prevent contamination by proteins interacting noncovalently with SUMO via specific domains such as SUMO-interacting motifs (SIMs) (2). Once SUMOylated proteins have been isolated, their analysis by mass spectrometry (MS) has been widely used to identify SUMO-modified proteins and, albeit less successfully, SUMO-conjugation sites.Mapping the exact lysine residue to which SUMO is attached in modified proteins is a critical step to get further insight into the function of SUMOylation. Indeed, the identification of SUMO sites allows the generation of non-SUMOylatable mutants and the study of associated phenotypes. Identification of SUMO sites by MS is not straightforward (8). Unlike ubiquitin, which leaves a small diglycine (GG) signature tag on the modified lysine residue after trypsin digestion, SUMO leaves a larger signature that severely hampers the identification of modified peptides.In addition to the identification of the SUMO site per se, a comparison of the SUMOylation status of sites in different cell-growth conditions is critical for better characterizing the biological implications of SUMOylation. For example, analysis of SUMOylation changes induced after heat shock, arsenic treatment, inhibition of the proteasome, or during the cell cycle has led to numerous insights into the role of SUMOylation in cell physiology (refs. 10–14 and reviewed in ref. 2). Here, we devised a performant approach which combines the use of SUMO variants, peptide immunocapture, and quantitative proteomics for high-throughput identification of SUMO sites. We then show that our approach is able to characterize global changes in the cell SUMOylome in response to a given stimulus, such as exposure to a bacterial toxin, listeriolysin O (LLO).     

Subtype-specific control of P2X receptor channel signaling by ATP and Mg2+

The identity and forms of activating ligands for ion channels are fundamental to their physiological roles in rapid electrical signaling. P2X receptor channels are ATP-activated cation channels that serve important roles in sensory signaling and inflammation, yet the active forms of the nucleotide are unknown. In physiological solutions, ATP is ionized and primarily found in complex with Mg²⁺. Here we investigated the active forms of ATP and found that the action of MgATP²⁻ and ATP⁴⁻ differs between subtypes of P2X receptors. The slowly desensitizing P2X2 receptor can be activated by free ATP, but MgATP²⁻ promotes opening with very low efficacy. In contrast, both free ATP and MgATP²⁻ robustly open the rapidly desensitizing P2X3 subtype. A further distinction between these two subtypes is the ability of Mg²⁺ to regulate P2X3 through a distinct allosteric mechanism. Importantly, heteromeric P2X2/3 channels present in sensory neurons exhibit a hybrid phenotype, characterized by robust activation by MgATP²⁻ and weak regulation by Mg²⁺. These results reveal the existence of two classes of homomeric P2X receptors with differential sensitivity to MgATP²⁻ and regulation by Mg²⁺, and demonstrate that both restraining mechanisms can be disengaged in heteromeric channels to form fast and sensitive ATP signaling pathways in sensory neurons.Seven subtypes of P2X receptors have been identified in mammals that can form either homomeric (P2X1, P2X2, P2X3, P2X4, P2X5, P2X7) or heteromeric (P2X1/2, P2X1/4, P2X1/5, P2X2/3, P2X2/5, P2X2/6, P2X4/6, and possibly, P2X4/7) channels (1–8). These subtypes of P2X receptors have distinct gating properties, pharmacology, and cellular distributions. P2X1 and P2X3 receptors desensitize within a few hundred milliseconds when opened by ATP, and their distributions are restricted to either smooth muscle cells and platelets (P2X1) or a subset of sensory neurons (P2X3) (1, 9–14). P2X2 and P2X4 receptors exhibit slow desensitization during prolonged ATP application, and these receptors are the most abundant subtypes in the central nervous system (15). P2X2 subunits also express in a subset of sensory neurons; however, in these cells they only form heteromeric channels with P2X3 subunits (12, 16, 17). In sensory neurons, P2X3 homomeric channels together with P2X2/3 heteromeric channels play important roles in mediating the primary sensory effects of ATP, and knock-out animals with either P2X3 deletion or P2X2 and P2X3 double-deletions have revealed critical roles in taste, pain, oxygen sensing, and bladder filling (17–20).A long-standing conundrum in P2X receptor-mediated signaling concerns the forms of ATP that activate these channels. In neutral solutions, ATP is ionized and exists mostly as free ATP (ATP⁴⁻), an efficient chelator of divalent cations such as Mg²⁺, and to a lesser extent Ca²⁺ (21). In extracellular biological compartments, such as the synaptic cleft, Ca²⁺ and Mg²⁺ are present in the millimolar range, and therefore only a relatively small fraction of ATP released from vesicles is present in the free form. Although a range of important studies have explored the regulatory effects of Ca²⁺ and Mg²⁺ on P2X receptor channels (22–30), the essential question of which forms of ATP serve as agonists remains unresolved. Several previous studies have reported that P2X2, P2X7, and the native P2X receptors in cilia are activated by ATP in solutions containing low concentrations of divalent cations, and that the addition of divalent cations shifts the concentration dependence for activation of the channels to higher ATP concentrations, suggesting that either ATP⁴⁻ is the most active form of ATP or that divalent cations regulate those subtypes through allosteric mechanisms (27, 30–36). In the present study, we investigated the form(s) of ATP that serve as agonists for a range of subtypes of P2X receptor channels. Our primary focus was to determine whether ATP⁴⁻ or MgATP²⁻ are the principal agonists and to explore whether Mg²⁺ might serve specific regulatory roles. Our results demonstrate that the action of MgATP²⁻ and ATP⁴⁻ differ between subtypes of P2X receptors, and reveal that heteromeric channels can have unique hybrid phenotypes, findings that will be crucial for understanding the physiological functions of these channels in both the peripheral and central nervous systems.     

Drosophila seminal protein ovulin mediates ovulation through female octopamine neuronal signaling

Across animal taxa, seminal proteins are important regulators of female reproductive physiology and behavior. However, little is understood about the physiological or molecular mechanisms by which seminal proteins effect these changes. To investigate this topic, we studied the increase in Drosophila melanogaster ovulation behavior induced by mating. Ovulation requires octopamine (OA) signaling from the central nervous system to coordinate an egg’s release from the ovary and its passage into the oviduct. The seminal protein ovulin increases ovulation rates after mating. We tested whether ovulin acts through OA to increase ovulation behavior. Increasing OA neuronal excitability compensated for a lack of ovulin received during mating. Moreover, we identified a mating-dependent relaxation of oviduct musculature, for which ovulin is a necessary and sufficient male contribution. We report further that oviduct muscle relaxation can be induced by activating OA neurons, requires normal metabolic production of OA, and reflects ovulin’s increasing of OA neuronal signaling. Finally, we showed that as a result of ovulin exposure, there is subsequent growth of OA synaptic sites at the oviduct, demonstrating that seminal proteins can contribute to synaptic plasticity. Together, these results demonstrate that ovulin increases ovulation through OA neuronal signaling and, by extension, that seminal proteins can alter reproductive physiology by modulating known female pathways regulating reproduction.Throughout internally fertilizing animals, seminal proteins play important roles in regulating female fertility by altering female physiology and, in some cases, behavior after mating (reviewed in refs. 1–3). Despite this, little is understood about the physiological mechanisms by which seminal proteins induce postmating changes and how their actions are linked with known networks regulating female reproductive physiology.In Drosophila melanogaster, the suite of seminal proteins has been identified, as have many seminal protein-dependent postmating responses, including changes in egg production and laying, remating behavior, locomotion, feeding, and in ovulation rate (reviewed in refs. 2 and 3). For example, the Drosophila seminal protein ovulin elevates ovulation rate to maximal levels during the 24 h following mating (4, 5), and the seminal protein sex peptide (SP) suppresses female mating receptivity and increases egg-laying behavior for several days after mating (6–10). However, although a receptor for SP has been identified (11), along with elements of the neural circuit in which it is required (12–14), SP’s mechanism of action has not yet been linked to regulatory networks known to control postmating behaviors. Thus, a crucial question remains: how do male-derived seminal proteins interact with regulatory networks in females to trigger postmating responses?We addressed this question by examining the stimulation of Drosophila ovulation by the seminal protein ovulin. In insects, ovulation, defined here as the release of an egg from the ovary to the uterus, is among the best understood reproductive processes in terms of its physiology and neurogenetics (15–27). In D. melanogaster, ovulation requires input from neurons in the abdominal ganglia that release the catecholaminergic neuromodulators octopamine (OA) and tyramine (17, 18, 28). Drosophila ovulation also requires an OA receptor, OA receptor in mushroom bodies (OAMB) (19, 20). Moreover, it has been proposed that OA may integrate extrinsic factors to regulate ovulation rates (17). Noradrenaline, the vertebrate structural and functional equivalent to OA (29, 30), is important for mammalian ovulation, and its dysregulation has been associated with ovulation disorders (31–38). In this paper we investigate the role of neurons that release OA and tyramine in ovulin’s action. For simplicity, we refer to these neurons as “OA neurons” to reflect the well-established role of OA in ovulation behavior (16–20, 22).We investigated how action of the seminal protein ovulin relates to the conserved canonical neuromodulatory pathway that regulates ovulation physiology (39–41). We found that ovulin increases ovulation and egg laying through OA neuronal signaling. We also found that ovulin relaxes oviduct muscle tonus, a postmating process that is also mediated by OA neuronal signaling. Finally, subsequent to these effects we detected an ovulin-dependent increase in synaptic sites between OA motor neurons and oviduct muscle, suggesting that ovulin’s stimulation of OA neurons could have increased their synaptic activity. These results suggest that ovulin affects ovulation by manipulating the gain of a neuromodulatory pathway regulating ovulation physiology.