Marker for type VI secretion system effectors

Bacteria use diverse mechanisms to kill, manipulate, and compete with other cells. The recently discovered type VI secretion system (T6SS) is widespread in bacterial pathogens and used to deliver virulence effector proteins into target cells. Using comparative proteomics, we identified two previously unidentified T6SS effectors that contained a conserved motif. Bioinformatic analyses revealed that this N-terminal motif, named MIX (marker for type six effectors), is found in numerous polymorphic bacterial proteins that are primarily located in the T6SS genome neighborhood. We demonstrate that several MIX-containing proteins are T6SS effectors and that they are not required for T6SS activity. Thus, we propose that MIX-containing proteins are T6SS effectors. Our findings allow for the identification of numerous uncharacterized T6SS effectors that will undoubtedly lead to the discovery of new biological mechanisms.The type VI secretion system (T6SS), a recently discovered protein secretion machinery (1), is a tool used by Gram-negative bacteria to inject effector proteins into recipient cells (2). During the type VI secretion process, an intracellular tube complex composed of hexameric rings of haemolysin coregulated proteins (Hcp) capped with a trimer of valine-glycine repeat protein G (VgrG) and a proline-alanine-alanine-arginine (PAAR) repeat-containing protein is surrounded by a sheath made of VipA/VipB heterodimers (also known as TssB/TssC). Upon an extracellular signal, the sheath contracts, leading to secretion of the tube complex into an adjacent target cell (2–4). Multiple T6SSs can be encoded within a single bacterial genome (5), and each T6SS can have more than one cognate Hcp, VgrG, or PAAR repeat-containing protein (4).T6SS effectors are predicted to be loaded onto the tube complex by several distinct mechanisms: as toxin domains fused to VgrG or PAAR repeat-containing proteins, as proteins that bind the inner surface of the Hcp tube, or as proteins that interact with VgrG or PAAR repeat-containing proteins (2). Two T6SS effector families have been characterized: peptidoglycan hydrolases (6) and phospholipases (7). Additional effector activities, such as nucleases (8), actin cross-linking (9), ADP ribosylation (10), and pore-forming (11), have also been described. Notably, T6SS effectors with antibacterial activities are paired with a cognate immunity protein encoded downstream of the effector gene to prevent self-intoxication (6, 12).We have recently described an antibacterial activity for T6SS1 of the marine bacterium Vibrio parahaemolyticus, a leading cause of gastroenteritis (13), and identified the environmental conditions required for its activation (14). Surprisingly, no known T6SS effectors are found in the genome of the V. parahaemolyticus RIMD 2210633 isolate, suggesting this strain harbors previously unidentified T6SS effectors.Here, we set out to identify V. parahaemolyticus T6SS1 effectors that mediate its antibacterial activity. Using comparative proteomics, we identified several T6SS effectors and their cognate immunity proteins. Remarkably, we found a motif named MIX (marker for type six effectors) that was shared by two of the newly identified effectors. We hypothesized and subsequently showed that this motif is found in numerous bacterial proteins with diverse predicted or established bacteriocidal and virulence activities, among them several confirmed T6SS effectors. Thus, we propose that proteins containing the MIX motif are polymorphic T6SS effectors.     

Control of type III secretion activity and substrate specificity by the cytoplasmic regulator PcrG

Pathogenic Gram-negative bacteria use syringe-like type III secretion systems (T3SS) to inject effector proteins directly into targeted host cells. Effector secretion is triggered by host cell contact, and before contact is prevented by a set of conserved regulators. How these regulators interface with the T3SS apparatus to control secretion is unclear. We present evidence that the proton motive force (pmf) drives T3SS secretion in Pseudomonas aeruginosa, and that the cytoplasmic regulator PcrG interacts with distinct components of the T3SS apparatus to control two important aspects of effector secretion: (i) It coassembles with a second regulator (Pcr1) on the inner membrane T3SS component PcrD to prevent effectors from accessing the T3SS, and (ii) In conjunction with PscO, it controls protein secretion activity by modulating the ability of T3SS to convert pmf.Many Gram-negative bacterial pathogens rely on a type III secretion system (T3SS) to promote disease by directly injecting effector proteins into the cytoplasm of host cells. This apparatus consists of a base that spans the bacterial envelope and a needle that projects from the base and ends in a specialized tip structure. The bacterium secretes two translocator proteins via the T3SS, which insert into the host cell membrane to form a pore, through which effector proteins are then transferred (1, 2).One of the hallmarks of type III secretion is that export of effector proteins is triggered by host cell contact (3–5). The secretion apparatus is fully assembled before cell contact, but effector secretion is prevented through the concerted action of needle tip-associated proteins and regulators that control secretion from the bacterial cytoplasm.In most systems, the needle tip protein prevents premature effector secretion, most likely by allosterically constraining the T3SS in an effector secretion “off” conformation (6–10). PcrG, the needle tip protein chaperone, as well as PopN, a member of the YopN/MxiC family of proteins, control effector secretion from the bacterial cytoplasm in Pseudomonas aeruginosa. PcrG’s regulatory function is independent of its function in promoting the export of needle tip protein PcrV. Deletion of pcrG or pcrV results in partial deregulation of effector secretion, whereas removal of both genes results in high-level secretion of effectors (8). In some bacteria, the needle tip protein promotes its own export with the aid of a self-chaperoning domain, rather than with a separate export chaperone (11). Recent evidence suggests that in these systems, the needle tip protein itself also regulates effector secretion from the cytoplasm, in addition to its regulatory role at the T3SS needle tip (12). The mechanism of this regulation is unclear.YopN/MxiC family proteins, PopN in P. aeruginosa, are T3SS regulators that are exported once effector secretion is triggered (13–17). These proteins control effector secretion from the bacterial cytoplasm (18–20). P. aeruginosa PopN and the closely related YopN associate with three other proteins that are required to prevent premature effector secretion (21–23). For PopN, these three proteins are Pcr1, Pcr2, and PscB. Pcr2 and PscB form a heterodimeric export chaperone, and Pcr1 is thought to tether the PopN complex to the apparatus (23). The prevailing model for explaining how PopN and related regulators control effector secretion is that they partially insert and plug the secretion channel while being tethered to the T3SS, either directly via a C-terminal interaction or indirectly via a C-terminal–associated protein, i.e., Pcr1 in P. aeruginosa (19, 20). The apparatus component with which these regulators interact is unknown, however.Triggering of effector secretion results in the rapid injection of effector proteins into the host cell (4, 5). How this rapid burst of secretion is energized is a matter of some controversy. The flagellum, which also uses a type III secretion mechanism, uses the proton motive force (pmf) to catalyze the rapid export of flagellar subunits. In fact, secretion is possible in mutants lacking the flagellum-associated ATPase, FliI, if the associated regulatory protein, FliH, is eliminated as well (24–26). The pmf’s contribution to the rate of secretion relative to the ATPase has been questioned in the case of virulence-associated T3SS (27), where removal of the ATPase results in a complete block of secretion (28, 29) that is not alleviated by deletion of the associated FliH homolog (30).Here we present evidence that export via the P. aeruginosa T3SS is energized primarily by the pmf, thereby offering a unified model for how protein secretion is energized in all T3SSs. The cytoplasmic T3SS regulator PcrG controls both the access of effectors to the T3SS and, surprisingly, the secretion activity of the apparatus. These two functions are controlled by separate regions of PcrG. Control of secretion activity involves the central portion of PcrG as well as PscO, which regulate the pmf-dependent export of secretion substrates. Mutants that up-regulate translocator secretion without turning on effector export confirm that effector secretion is not blocked by physical obstruction of the secretion channel. Instead, access of effectors to the T3SS is controlled by the C terminus of PcrG in conjunction with the PopN complex through an interaction with the inner membrane T3SS component PcrD. This protein complex likely blocks an acceptor site for effectors. Thus, PcrG is a multifaceted protein that, along with its export chaperone function, serves as a brake and a switch to control effector secretion.     

A multidrug resistance plasmid contains the molecular switch for type VI secretion in Acinetobacter baumannii

Infections with Acinetobacter baumannii, one of the most troublesome and least studied multidrug-resistant superbugs, are increasing at alarming rates. A. baumannii encodes a type VI secretion system (T6SS), an antibacterial apparatus of Gram-negative bacteria used to kill competitors. Expression of the T6SS varies among different strains of A. baumannii, for which the regulatory mechanisms are unknown. Here, we show that several multidrug-resistant strains of A. baumannii harbor a large, self-transmissible resistance plasmid that carries the negative regulators for T6SS. T6SS activity is silenced in plasmid-containing, antibiotic-resistant cells, while part of the population undergoes frequent plasmid loss and activation of the T6SS. This activation results in T6SS-mediated killing of competing bacteria but renders A. baumannii susceptible to antibiotics. Our data show that a plasmid that has evolved to harbor antibiotic resistance genes plays a role in the differentiation of cells specialized in the elimination of competing bacteria.Antibiotic-resistant bacteria that cause hospital-acquired infections are a mounting concern for health care systems globally (1). Multidrug-resistant (MDR) Acinetobacter baumannii is emerging as a frequent cause of difficult-to-treat nosocomial infections, and some isolates are resistant to all clinically relevant antibiotics (2, 3). A. baumannii is often isolated from polymicrobial infections and therefore spends at least a part of its time competing with other bacteria (4). Antagonistic interactions between bacteria manifest in a variety of different ways (5), and the type VI secretion system (T6SS) is a potent weapon used by many Gram-negative bacteria to kill competitors (6–8). The multicomponent T6SS apparatus facilitates a dynamic contact-dependent injection of toxic effector proteins into prey cells (9, 10), and expression of cognate immunity proteins prevents self-inflicted intoxication (9, 11). The T6SS is composed of several conserved proteins involved in the formation of the secretory apparatus (12, 13). One of these components, hemolysin-coregulated protein (Hcp), forms hexameric tubule structures that are robustly secreted to the culture supernatants in bacteria with an active T6SS, allowing it to be used as a molecular marker for T6SS activity (6, 14).T6SS is a dynamic apparatus (15). Its biogenesis follows energetically costly cycles of assembly/disassembly, and therefore, in most bacteria, T6SS appears to be exquisitely regulated. T6SS is silenced in most strains and only activated under specific conditions, such as an attack from another bacterium or in environments leading to membrane perturbations (16–19). Many Acinetobacter spp. encode the genes for a T6SS, including Acinetobacter noscomialis and Acinetobacter baylyi, which possess a constitutively active antibacterial T6SS (20–24). A. baumannii strains have been shown by us and others to secrete Hcp (21, 25), but to our knowledge a T6SS-dependent phenotype has not been ascribed to this species. Furthermore, our previous results showed that Hcp secretion is highly variable between A. baumannii strains, with some isolates carrying an inactive system (21). The precise regulatory mechanism(s) underlying T6SS suppression in some A. baumannii is unknown.Here, we show that a large resistance plasmid of A. baumannii functions to repress the T6SS by encoding negative regulators of its activity. Analysis of colonies from a clinical isolate showed that the plasmid is readily lost in a subset of the population. This leads to the activation of the T6SS, which imparts the ability to kill other bacteria, with the simultaneous loss of antibiotic resistance. We propose that the differentiation into T6SS+ MDR– and T6SS– MDR+ phenotypes may constitute a novel survival strategy of this organism.     

Generation of reactive oxygen species by lethal attacks from competing microbes

Whether antibiotics induce the production of reactive oxygen species (ROS) that contribute to cell death is an important yet controversial topic. Here, we report that lethal attacks from bacterial and viral species also result in ROS production in target cells. Using soxS as an ROS reporter, we found soxS was highly induced in Escherichia coli exposed to various forms of attacks mediated by the type VI secretion system (T6SS), P1vir phage, and polymyxin B. Using a fluorescence ROS probe, we found enhanced ROS levels correlate with induced soxS in E. coli expressing a toxic T6SS antibacterial effector and in E. coli treated with P1vir phage or polymyxin B. We conclude that both contact-dependent and contact-independent interactions with aggressive competing bacterial species and viruses can induce production of ROS in E. coli target cells.Microbial species exist predominantly in complex communities in the natural environment and animal hosts. To survive in a multispecies environment, bacteria have developed various strategies to compete with other species. For example, some bacteria can exert long-range inhibitory effects by secreting diffusible molecules, such as antibiotics, bacteriocins, and H₂O₂ (1), whereas others require direct cell-to-cell contact to kill nearby organisms (2, 3). One such contact-dependent inhibitory system is the type VI secretion system (T6SS), a protein translocating nanomachine expressed by many Gram-negative bacterial pathogens that can kill both bacterial and eukaryotic cells (3–5). Structurally analogous to an inverted bacteriophage tail, the T6SS delivers effectors into target cells by using a contractile sheath to propel an inner tube out of the producer cell and into nearby target cells. The inner tube (composed of Hcp protein) is thought to carry toxic effector proteins within its lumen or on its tip, which is decorated with VgrG and PAAR proteins (4, 6, 7). Given that some cells can detect T6SS attack but not suffer any measurable loss in viability (8, 9), it would seem that cell killing is likely due to the toxicity of effectors rather than membrane disruptions caused by insertion of the spear-like VgrG/PAAR/Hcp tube complex. T6SS-dependent effectors can attack a number of essential cellular targets, including the cell wall (10, 11), membranes (11, 12), and nucleic acids (13), and thus can mimic the actions of antibiotics and bacteriocins. As a model prey or target organism, Escherichia coli can be killed by the T6SS activities of a number of bacteria including Vibrio cholerae (14), Pseudomonas aeruginosa (10, 15), and Acinetobacter baylyi ADP1 (7).Collins and coworkers (16–18) have reported that antibiotic treatment of E. coli elicits the production of reactive oxygen species (ROS) resulting from a series of events involving perturbation of the central metabolic pathway, NADPH depletion, and the Fenton reaction. ROS can cause lethal damage to DNA, lipid, and proteins (19, 20) and thus can contribute to cell death in combination with the deleterious effects of antibiotics on their primary targets. The idea that antibiotics kill bacterial cells, in part, through the action of ROS has been supported by a number of follow-up studies (18, 21–23) but has also been challenged by others as a result of observations contradictory to a model where ROS is the sole mediator of antibiotic lethality (24–26). These observations include the fact that antibiotics kill under anaerobic conditions, oxidation of the hydroxyphenyl fluorescein fluorescence dye used to measure ROS levels is nonspecific, and the extracellular level of H₂O₂ is not elevated by antibiotic treatment (24, 26). To address these concerns, Dwyer et al. (27) used a panel of ROS-detection fluorescence dyes, a defined growth medium under stringent anaerobic conditions, and an in vivo H₂O₂ enzymatic assay to study the effects of antibiotics on cells. The results further support that antibiotics induce ROS generation, which contributes to the efficacy of antibiotics in addition to their primary lethal actions (18, 27, 28).     

Decreased abundance of type III secretion system-inducing signals in Arabidopsis mkp1 enhances resistance against Pseudomonas syringae

Genes encoding the virulence-promoting type III secretion system (T3SS) in phytopathogenic bacteria are induced at the start of infection, indicating that recognition of signals from the host plant initiates this response. However, the precise nature of these signals and whether their concentrations can be altered to affect the biological outcome of host–pathogen interactions remain speculative. Here we use a metabolomic comparison of resistant and susceptible genotypes to identify plant-derived metabolites that induce T3SS genes in Pseudomonas syringae pv tomato DC3000 and report that mapk phosphatase 1 (mkp1), an Arabidopsis mutant that is more resistant to bacterial infection, produces decreased levels of these bioactive compounds. Consistent with these observations, T3SS effector expression and delivery by DC3000 was impaired when infecting the mkp1 mutant. The addition of bioactive metabolites fully restored T3SS effector delivery and suppressed the enhanced resistance in the mkp1 mutant. Pretreatment of plants with pathogen-associated molecular patterns (PAMPs) to induce PAMP-triggered immunity (PTI) also restricts T3SS effector delivery and enhances resistance by unknown mechanisms, and the addition of the bioactive metabolites similarly suppressed both aspects of PTI. Together, these results demonstrate that DC3000 perceives multiple signals derived from plants to initiate its T3SS and that the level of these host-derived signals impacts bacterial pathogenesis.Plants evoke resistance against invading bacteria using plasma membrane-localized pattern recognition receptors (PRRs) to detect the presence of pathogen-associated molecular patterns (PAMPs) in the extracellular space (1). Activation of PRRs by PAMPs results in numerous defense responses that limit bacterial growth (1). However, the actual mechanisms by which plants suppress virulence and restrict bacterial growth remain unclear. Pseudomonas syringae is a model bacterial pathogen that infects a wide range of economically important crops as well as the laboratory model plant Arabidopsis (2). P. syringae uses several different virulence strategies to suppress host defenses, including a type III secretion system (T3SS) that secretes up to 30 effector proteins into plant cells (3, 4). Many effectors function to suppress PRR-induced signaling, thereby allowing the bacteria to avoid detection and proliferate (4). Mutants of P. syringae lacking a functional T3SS are not fully virulent, demonstrating that this system is essential for a successful infection (5, 6). Moreover, recent studies have revealed that PAMP-triggered immunity (PTI) leads to a restriction in the delivery of type III effectors into host cells, suggesting that plants possess an unknown mechanism(s) to block type III secretion (7, 8).Despite the critical role of the T3SS in P. syringae virulence, T3SS structural components and effectors are not constitutively present but are produced at the onset of infection (9, 10). Early attempts to identify plant signals perceived by P. syringae revealed that synthetic medium mimicking the plant apoplast, namely a minimal nutrient medium with acidic pH and including a sugar such as fructose, is capable of inducing T3SS-associated genes (9–12). However, in some instances expression of the T3SS was higher in planta than in synthetic medium, indicating that additional plant-derived factors likely were required for full induction (10, 12). These results imply the presence of plant-derived signal(s) that induce the T3SS, and various signals have been proposed to be capable of inducing the T3SS in different plant pathogenic bacteria based largely on in vitro experiments (10, 12–17). However, whether any of these signals affect the biological outcome of the host–pathogen interaction remains speculative because of the lack of genetic mutants altering the abundance of these chemical signals in the host.In the present work, we identify host chemical signals that DC3000 uses to switch to its virulence program and demonstrate that this recognition event plays an important role in a successful infection. The identification of an Arabidopsis mutant, MAPK phosphatase 1 (mkp1), in which the delivery of the P. syringae pv tomato DC3000 effector is suppressed, provided an important genetic model for investigating the basis for T3SS induction. Using a metabolomics comparison of mutant and WT plant exudates, we identified several plant-derived metabolites that are present at lower levels in mkp1 and induce the T3SS in DC3000. The biological significance of these compounds was demonstrated by showing that reintroducing these T3SS-inducing metabolites can overcome both the suppression of effector delivery and the enhanced resistance in mkp1 plants. Furthermore, the addition of these metabolites also can overcome enhanced resistance induced in plants pretreated with PAMPs. Together, these results demonstrate that DC3000 perceives multiple signals derived from plants to initiate its T3SS and that the levels of these host-derived signals contribute to susceptibility or resistance.     

Visualization of the type III secretion sorting platform of Shigella flexneri

Bacterial type III secretion machines are widely used to inject virulence proteins into eukaryotic host cells. These secretion machines are evolutionarily related to bacterial flagella and consist of a large cytoplasmic complex, a transmembrane basal body, and an extracellular needle. The cytoplasmic complex forms a sorting platform essential for effector selection and needle assembly, but it remains largely uncharacterized. Here we use high-throughput cryoelectron tomography (cryo-ET) to visualize intact machines in a virulent Shigella flexneri strain genetically modified to produce minicells capable of interaction with host cells. A high-resolution in situ structure of the intact machine determined by subtomogram averaging reveals the cytoplasmic sorting platform, which consists of a central hub and six spokes, with a pod-like structure at the terminus of each spoke. Molecular modeling of wild-type and mutant machines allowed us to propose a model of the sorting platform in which the hub consists mainly of a hexamer of the Spa47 ATPase, whereas the MxiN protein comprises the spokes and the Spa33 protein forms the pods. Multiple contacts among those components are essential to align the Spa47 ATPase with the central channel of the MxiA protein export gate to form a unique nanomachine. The molecular architecture of the Shigella type III secretion machine and its sorting platform provide the structural foundation for further dissecting the mechanisms underlying type III secretion and pathogenesis and also highlight the major structural distinctions from bacterial flagella.Type III secretion systems (T3SSs) are essential virulence determinants for many Gram-negative pathogens. The injectisome, also known as the needle complex, is the central T3SS machine required to inject effector proteins from the bacterium into eukaryotic host cells (1, 2). The injectisome has three major components: an extracellular needle, a basal body, and a cytoplasmic complex (3). Contact with a host cell membrane triggers activation of the injectisome and the insertion of a translocon pore into the target cell membrane. The entire complex then serves as a conduit for direct translocation of effectors (1, 2). Assembly of a functional T3SS requires recognition and sorting of specific secretion substrates in a well-defined order by the cytoplasmic complex (4, 5). Furthermore, genes encoding the cytoplasmic complex are regulated by physical and environmental signals (6), providing temporal control of the injection of effector proteins and thereby optimizing invasion and virulence.Significant progress has been made in elucidating T3SS structures from many different bacteria (7, 8). 3D reconstructions of purified injectisomes from Salmonella and Shigella, together with the atomic structures of major basal body proteins, have provided a detailed view of basal body architecture (9, 10). Recent in situ structures of injectisomes from Shigella flexneri, Salmonella enterica, and Yersinia enterocolitica revealed an export gate and the structural flexibility of the basal body (11, 12). Unfortunately, these in situ structures from intact bacteria (11, 12) did not reveal any evident densities related to the proposed model of the cytoplasmic complex (8, 13).The flagellar C ring is the cytoplasmic complex in evolutionarily related flagellar systems. It is composed of flagellar proteins FliG, FliM, and FliN and plays an essential role in flagellar assembly, rotation, and switching (14). Large drum-shaped structures of the flagellar C ring have been determined in both purified basal bodies (15, 16) and in situ motors (17–19). Similarly, electron microscopy analysis in Shigella indicated that the Spa33 protein (a homolog of the flagellar proteins FliN and FliM) is localized beneath the basal body via interactions with MxiG and MxiJ and is an essential component of the putative C ring (20). Recent experimental evidence suggests that the putative C ring provides a sorting platform for the recognition and secretion of the substrates in S. enterica (5). This sorting platform consists of three proteins, SpaO, OrgA, and OrgB, which are highly conserved among other T3SSs (21) (SI Appendix, Table S1). Despite its critical roles, little is still known about the structure and assembly of the cytoplasmic sorting platform in T3SS. In this study, we choose S. flexneri as a model system to study the intact T3SS machine and its cytoplasmic complex, mainly because a wealth of structural, biochemical, and functional information is available for the S. flexneri T3SS (22).     

Bacteroides fragilis type VI secretion systems use novel effector and immunity proteins to antagonize human gut Bacteroidales species

Type VI secretion systems (T6SSs) are multiprotein complexes best studied in Gram-negative pathogens where they have been shown to inhibit or kill prokaryotic or eukaryotic cells and are often important for virulence. We recently showed that T6SS loci are also widespread in symbiotic human gut bacteria of the order Bacteroidales, and that these T6SS loci segregate into three distinct genetic architectures (GA). GA1 and GA2 loci are present on conserved integrative conjugative elements (ICE) and are transferred and shared among diverse human gut Bacteroidales species. GA3 loci are not contained on conserved ICE and are confined to Bacteroides fragilis. Unlike GA1 and GA2 T6SS loci, most GA3 loci do not encode identifiable effector and immunity proteins. Here, we studied GA3 T6SSs and show that they antagonize most human gut Bacteroidales strains analyzed, except for B. fragilis strains with the same T6SS locus. A combination of mutation analyses, trans-protection analyses, and in vitro competition assays, allowed us to identify novel effector and immunity proteins of GA3 loci. These proteins are not orthologous to known proteins, do not contain identified motifs, and most have numerous predicted transmembrane domains. Because the genes encoding effector and immunity proteins are contained in two variable regions of GA3 loci, GA3 T6SSs of the species B. fragilis are likely the source of numerous novel effector and immunity proteins. Importantly, we show that the GA3 T6SS of strain 638R is functional in the mammalian gut and provides a competitive advantage to this organism.Bacteria that live in communities have numerous mechanisms to compete with other strains and species. The ability to acquire nutrients is a major factor dictating the success of a species in a community. In addition, the production of secreted factors, such as bacteriocins, that competitively interfere or antagonize other strains/species, also contributes to a member’s fitness in a community. In the microbe-dense human gut ecosystem, such factors and mechanisms of antagonism by predominant members are just beginning to be described, as are models predicting the relevance of these competitive interactions to the microbial community (1). Bacteroidales is the most abundant order of bacteria in the human colonic microbiota, and also the most temporally stable (2). The fact that numerous gut Bacteroidales species stably cocolonize the human gut at high density raises the question of how these related species and strains interact with each other to promote or limit each other’s growth. We previously showed that coresident Bacteroidales strains intimately interact with each other and exchange large amounts of DNA (3) and also cooperate in the utilization of dietary polysaccharides (4). To date, two types of antagonistic factors/systems have been shown to be produced by human gut Bacteroidales species: secreted antimicrobial proteins (5) and T6SSs (3, 6, 7). However, neither of these antagonistic processes has been analyzed to determine if they provide a competitive advantage in the mammalian intestine.Type VI secretion systems (T6SSs) are contact-dependent antagonistic systems used by some Gram-negative bacteria to intoxicate other bacteria or eukaryotic cells. The T6 apparatus is a multiprotein, cell envelope spanning complex comprised of core Tss proteins. A key component of the machinery is a needle-like structure, similar to the T4 contractile bacteriophage tail, which is assembled in the cytoplasm where it is loaded with toxic effectors (8–10). Contraction of the sheath surrounding the needle apparatus drives expulsion of the needle from the cell, delivering the needle and associated effectors either into the supernatant of in vitro grown bacteria, or across the membrane of prey cells. Identified T6SS effectors include cell wall degrading enzymes (11), proteins that affect cell membranes such as phospholipases (12) and pore-forming toxins (13, 14), proteins that degrade NAD(P)+ (15), and nucleases (16). The effector protein is produced with a cognate immunity protein, typically encoded by the adjacent downstream gene (17), which protects the producing cell from the toxicity of the effector. Although both eukaryotic and bacterial cells are targeted by T6SS effectors (18), most described T6SSs target Gram-negative bacteria.We previously performed a comprehensive analysis of all sequenced human gut Bacteroidales stains and found that more than half contain T6SS loci (7). These T6SSs are similar to the well-described T6SSs of Proteobacteria in that remote orthologs of many Proteobacterial Tss proteins are encoded by Bacteroidales T6SS regions, with the exception of proteins that likely comprise the transmembrane complex, which are distinct. The T6SS loci of human gut Bacteroidales species segregate into three distinct genetic architectures (GA), designated GA1, GA2, and GA3, each with highly identical segments within a GA comprising the core tss genes (7). GA1 and GA2 T6SS loci are present on large ∼80- to 120-kb integrative conjugative elements (ICE) that are extremely similar at the DNA level within a GA. Due to the ability of these T6SS regions to be transferred between strains via ICE, GA1 and GA2 T6SS loci are present in diverse human gut Bacteroidales species. GA3 T6SS loci are confined to Bacteroides fragilis and are not contained on conserved ICE (7).Although T6SS loci of a particular GA are highly identical to each other, each GA has internal regions of variability where the genes differ between strains (7). The variable regions of GA1 and GA2 T6SS loci contain genes encoding the identifiable toxic effector and cognate immunity proteins found in these regions. Unlike the GA1 and GA2 T6SS loci, there are no identifiable genes encoding toxin or immunity proteins in the two variable regions or other areas of GA3 T6SS loci. The present study was designed to answer three fundamental questions regarding GA3 T6SS loci: (i) Because no known effectors/immunity proteins are encoded by these regions, are they involved in bacterial antagonism? And if so, what prey cells do they target? (ii) Do the variable regions contain genes encoding effector and immunity proteins? and (iii) If GA3 T6SSs mediate bacterial antagonism, do they provide a competitive advantage in the mammalian gut?     

Mitigation of acute kidney injury by cell-cycle inhibitors that suppress both CDK4/6 and OCT2 functions

Acute kidney injury (AKI) is a potentially fatal syndrome characterized by a rapid decline in kidney function caused by ischemic or toxic injury to renal tubular cells. The widely used chemotherapy drug cisplatin accumulates preferentially in the renal tubular cells and is a frequent cause of drug-induced AKI. During the development of AKI the quiescent tubular cells reenter the cell cycle. Strategies that block cell-cycle progression ameliorate kidney injury, possibly by averting cell division in the presence of extensive DNA damage. However, the early signaling events that lead to cell-cycle activation during AKI are not known. In the current study, using mouse models of cisplatin nephrotoxicity, we show that the G1/S-regulating cyclin-dependent kinase 4/6 (CDK4/6) pathway is activated in parallel with renal cell-cycle entry but before the development of AKI. Targeted inhibition of CDK4/6 pathway by small-molecule inhibitors palbociclib (PD-0332991) and ribociclib (LEE011) resulted in inhibition of cell-cycle progression, amelioration of kidney injury, and improved overall survival. Of additional significance, these compounds were found to be potent inhibitors of organic cation transporter 2 (OCT2), which contributes to the cellular accumulation of cisplatin and subsequent kidney injury. The unique cell-cycle and OCT2-targeting activities of palbociclib and LEE011, combined with their potential for clinical translation, support their further exploration as therapeutic candidates for prevention of AKI.Cell division is a fundamental biological process that is tightly regulated by evolutionarily conserved signaling pathways (1, 2). The initial decision to start cell division, the fidelity of subsequent DNA replication, and the final formation of daughter cells is monitored and regulated by these essential pathways (2–6). The cyclin-dependent kinases (CDKs) are the central players that orchestrate this orderly progression through the cell cycle (1, 2, 6, 7). The enzymatic activity of CDKs is regulated by complex mechanisms that include posttranslational modifications and expression of activating and inhibitory proteins (1, 2, 6, 7). The spatial and temporal changes in the activity of these CDK complexes are thought to generate the distinct substrate specificities that lead to sequential and unidirectional progression of the cell cycle (1, 8, 9).Cell-cycle deregulation is a universal feature of human cancer and a long-sought-after target for anticancer therapy (1, 10–13). Frequent genetic or epigenetic changes in mitogenic pathways, CDKs, cyclins, or CDK inhibitors are observed in various human cancers (1, 4, 11). In particular, the G1/S-regulating CDK4/6–cyclin D–inhibitors of CDK4 (INK4)–retinoblastoma (Rb) protein pathway frequently is disrupted in cancer cells (11, 14). These observations provided an impetus to develop CDK inhibitors as anticancer drugs. However, the earlier class of CDK inhibitors had limited specificity, inadequate clinical activity, poor pharmacokinetic properties, and unacceptable toxicity profiles (10, 11, 14, 15). These disappointing initial efforts now have been followed by the development of the specific CDK4/6 inhibitors palbociclib (PD0332991), ribociclib (LEE011), and abemaciclib (LY2835219), which have demonstrated manageable toxicities, improved pharmacokinetic properties, and impressive antitumor activity, especially in certain forms of breast cancer (14, 16). Successful early clinical trials with these three CDK4/6 inhibitors have generated cautious enthusiasm that these drugs may emerge as a new class of anticancer agents (14, 17). Palbociclib recently was approved by Food and Drug Administration for the treatment of metastatic breast cancer and became the first CDK4/6 inhibitor approved for anticancer therapy (18).In addition to its potential as an anticancer strategy, CDK4/6 inhibition in normal tissues could be exploited therapeutically for wide-ranging clinical conditions. For example, radiation-induced myelosuppression, caused by cell death of proliferating hematopoietic stem/progenitor cells, can be rescued by palbociclib (19, 20). Furthermore, cytotoxic anticancer agents cause significant toxicities to normal proliferating cells, which possibly could be mitigated by the concomitant use of CDK4/6 inhibitors (20, 21). More broadly, cell-cycle inhibition could have beneficial effects in disorders in which maladaptive proliferation of normal cells contributes to the disease pathology, as observed in vascular proliferative diseases, hyperproliferative skin diseases, and autoimmune disorders (22, 23). In support of this possibility, palbociclib treatment recently was reported to ameliorate disease progression in animal models of rheumatoid arthritis through cell-cycle inhibition of synovial fibroblasts (24).Abnormal cellular proliferation also is a hallmark of various kidney diseases (25), and cell-cycle inhibition has been shown to ameliorate significantly the pathogenesis of polycystic kidney disease (26), nephritis (27), and acute kidney injury (AKI) (28). Remarkably, during AKI, the normally quiescent renal tubular cells reenter the cell cycle (29–34), and blocking cell-cycle progression can reduce renal injury (28). Here, we provide evidence that the CDK4/6 pathway is activated early during AKI and demonstrate significant protective effects of CDK4/6 inhibitors in animal models of cisplatin-induced AKI. In addition, we found that the CDK4/6 inhibitors palbociclib and LEE011 are potent inhibitors of organic cation transporter 2 (OCT2), a cisplatin uptake transporter highly expressed in renal tubular cells (35–37). Our findings provide a rationale for the clinical development of palbociclib and LEE011 for the prevention and treatment of AKI.     

Trichomonas vaginalis homolog of macrophage migration inhibitory factor induces prostate cell growth,invasiveness, and inflammatory responses

The human-infective parasite Trichomonas vaginalis causes the most prevalent nonviral sexually transmitted infection worldwide. Infections in men may result in colonization of the prostate and are correlated with increased risk of aggressive prostate cancer. We have found that T. vaginalis secretes a protein, T. vaginalis macrophage migration inhibitory factor (TvMIF), that is 47% similar to human macrophage migration inhibitory factor (HuMIF), a proinflammatory cytokine. Because HuMIF is reported to be elevated in prostate cancer and inflammation plays an important role in the initiation and progression of cancers, we have explored a role for TvMIF in prostate cancer. Here, we show that TvMIF has tautomerase activity, inhibits macrophage migration, and is proinflammatory. We also demonstrate that TvMIF binds the human CD74 MIF receptor with high affinity, comparable to that of HuMIF, which triggers activation of ERK, Akt, and Bcl-2–associated death promoter phosphorylation at a physiologically relevant concentration (1 ng/mL, 80 pM). TvMIF increases the in vitro growth and invasion through Matrigel of benign and prostate cancer cells. Sera from patients infected with T. vaginalis are reactive to TvMIF, especially in males. The presence of anti-TvMIF antibodies indicates that TvMIF is released by the parasite and elicits host immune responses during infection. Together, these data indicate that chronic T. vaginalis infections may result in TvMIF-driven inflammation and cell proliferation, thus triggering pathways that contribute to the promotion and progression of prostate cancer.Prostate cancer is the most common noncutaneous cancer of men in the United States, affecting one in six men (1). Although the causes of prostate cancer are poorly understood, inflammation has been implicated in both initiation and progression of the disease (2, 3). The origin of inflammation in prostate cancer is unclear, although chronic infections are believed to promote and establish a tumor-enhancing proinflammatory environment.Trichomonas vaginalis is the causative agent of the most common nonviral sexually transmitted infection, infecting ∼275 million people worldwide (4). T. vaginalis is a flagellated, protozoan parasite that infects the prostate epithelium (5, 6). Over 75% of men harboring T. vaginalis are asymptomatic and may not seek treatment, resulting in chronic inflammation (5). Several studies have positively associated T. vaginalis infection with increased incidence and severity of prostate cancer, as well as benign prostate hyperplasia (2, 6–9). The magnitude of the association between T. vaginalis seropositivity and overall prostate cancer risk is between 1.23 and 1.43 based on two large, nested case–control studies (7, 8). Additionally there is a statistically significant increase in risk of extraprostatic cancer [odds ratio (OR) = 2.17] or cancer-specific death (OR = 2.69) with T. vaginalis seropositive status (7).Our research focuses on the potential contribution of a proinflammatory protein, T. vaginalis macrophage migration inhibitory factor (TvMIF), to prostate cancer, because the human homolog has a role in the growth and invasion of prostate cancer (10, 11). Human macrophage migration inhibitory factor (HuMIF) has been implicated in a broad array of conditions associated with inflammation, including autoimmunity, cell proliferation, angiogenesis, and tumorigenesis (12–15). Increased expression of HuMIF has been reported in several cancers, including prostate cancer (16–18). Studies show high expressers of HuMIF have a heightened risk of prostate cancer, as well as a significant increase in prostate cancer progression and drug resistance (17–23). Several clinical studies have shown HuMIF production correlates with both tumor aggressiveness and metastatic potential (23, 24). Additionally, the expression of the HuMIF receptor CD74 is increased in prostate cancer (10, 25).HuMIF induces ERK1/2, MAPK, and Akt activation via binding with the extracellular domain of the MIF receptor CD74 (26). HuMIF has multiple functions with regard to regulating the immune system, including protecting monocytes and macrophages from activation-induced apoptosis, which results in sustained inflammation (27, 28). Consequently, HuMIF is implicated in the pathogenesis of several inflammatory and autoimmune diseases in addition to cancer (29, 30).Recent studies have shown that several parasitic eukaryotes encode MIF-like proteins with considerable structural and biological similarity to their mammalian hosts (31–34). These parasite MIF proteins have been shown to modulate host immune responses and regulate pathways to promote parasite survival. Here, we characterize TvMIF and show that it can act as a molecular mimic of HuMIF. We find that TvMIF binding to the human CD74 receptor activates extracellular signal-regulated kinases (ERK)1/2 and Akt protein kinase/proapoptotic Bcl-2–associated death promoter (BAD) pathways as well as secretion of proinflammatory IL-8 from monocytes, reduces monocyte migration, and increases growth and invasiveness of benign prostate hyperplasia (BPH-1) and prostate cancer (PC3) cells. This research is, to our knowledge, the first to identify a human inflammatory cytokine mimic in T. vaginalis and to begin to explore the link between this sexually transmitted infection and prostate cancer.     

Structural basis for membrane recruitment and allosteric activation of cytohesin family Arf GTPase exchange factors

Membrane recruitment of cytohesin family Arf guanine nucleotide exchange factors depends on interactions with phosphoinositides and active Arf GTPases that, in turn, relieve autoinhibition of the catalytic Sec7 domain through an unknown structural mechanism. Here, we show that Arf6-GTP relieves autoinhibition by binding to an allosteric site that includes the autoinhibitory elements in addition to the PH domain. The crystal structure of a cytohesin-3 construct encompassing the allosteric site in complex with the head group of phosphatidyl inositol 3,4,5-trisphosphate and N-terminally truncated Arf6-GTP reveals a large conformational rearrangement, whereby autoinhibition can be relieved by competitive sequestration of the autoinhibitory elements in grooves at the Arf6/PH domain interface. Disposition of the known membrane targeting determinants on a common surface is compatible with multivalent membrane docking and subsequent activation of Arf substrates, suggesting a plausible model through which membrane recruitment and allosteric activation could be structurally integrated.Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing exchange of GDP for GTP (1). Because many GEFs are recruited to membranes through interactions with phospholipids, active GTPases, or other membrane-associated proteins (1–5), GTPase activation can be restricted or amplified by spatial–temporal overlap of GEFs with binding partners. GEF activity can also be controlled by autoregulatory mechanisms, which may depend on membrane recruitment (6–11). Structural relationships between these mechanisms are poorly understood.Arf GTPases function in trafficking and cytoskeletal dynamics (5, 12, 13). Membrane partitioning of a myristoylated (myr) N-terminal amphipathic helix primes Arfs for activation by Sec7 domain GEFs (14–17). Cytohesins comprise a metazoan Arf GEF family that includes the mammalian proteins cytohesin-1 (Cyth1), ARNO (Cyth2), and Grp1 (Cyth3). The Drosophila homolog steppke functions in insulin-like growth factor signaling, whereas Cyth1 and Grp1 have been implicated in insulin signaling and Glut4 trafficking, respectively (18–20). Cytohesins share a modular architecture consisting of heptad repeats, a Sec7 domain with exchange activity for Arf1 and Arf6, a PH domain that binds phosphatidyl inositol (PI) polyphosphates, and a C-terminal helix (CtH) that overlaps with a polybasic region (PBR) (21–28). The overlapping CtH and PBR will be referred to as the CtH/PBR. The phosphoinositide specificity of the PH domain is influenced by alternative splicing, which generates diglycine (2G) and triglycine (3G) variants differing by insertion of a glycine residue in the β1/β2 loop (29). Despite similar PI(4,5)P₂ (PIP₂) affinities, the 2G variant has 30-fold higher affinity for PI(3,4,5)P₃ (PIP₃) (30). In both cases, PIP₃ is required for plasma membrane (PM) recruitment (23, 26, 31–33), which is promoted by expression of constitutively active Arf6 or Arl4d and impaired by PH domain mutations that disrupt PIP₃ or Arf6 binding, or by CtH/PBR mutations (8, 34–36).Cytohesins are autoinhibited by the Sec7-PH linker and CtH/PBR, which obstruct substrate binding (8). Autoinhibition can be relieved by Arf6-GTP binding in the presence of the PIP₃ head group (8). Active myr-Arf1 and myr-Arf6 also stimulate exchange activity on PIP₂-containing liposomes (37). Whether this effect is due to relief of autoinhibition per se or enhanced membrane recruitment is not yet clear. Phosphoinositide recognition by PH domains, catalysis of nucleotide exchange by Sec7 domains, and autoinhibition in cytohesins are well characterized (8, 16, 17, 30, 38–43). How Arf-GTP binding relieves autoinhibition and promotes membrane recruitment is unknown. Here, we determine the structural basis for relief of autoinhibition and investigate potential mechanistic relationships between allosteric regulation, phosphoinositide binding, and membrane targeting.     

Dissecting neural pathways for forgetting in Drosophila olfactory aversive memory

Recent studies have identified molecular pathways driving forgetting and supported the notion that forgetting is a biologically active process. The circuit mechanisms of forgetting, however, remain largely unknown. Here we report two sets of Drosophila neurons that account for the rapid forgetting of early olfactory aversive memory. We show that inactivating these neurons inhibits memory decay without altering learning, whereas activating them promotes forgetting. These neurons, including a cluster of dopaminergic neurons (PAM-β′1) and a pair of glutamatergic neurons (MBON-γ4>γ1γ2), terminate in distinct subdomains in the mushroom body and represent parallel neural pathways for regulating forgetting. Interestingly, although activity of these neurons is required for memory decay over time, they are not required for acute forgetting during reversal learning. Our results thus not only establish the presence of multiple neural pathways for forgetting in Drosophila but also suggest the existence of diverse circuit mechanisms of forgetting in different contexts.Although forgetting commonly has a negative connotation, it is a functional process that shapes memory and cognition (1–4). Recent studies, including work in relatively simple invertebrate models, have started to reveal basic biological mechanisms underlying forgetting (5–15). In Drosophila, single-session Pavlovian conditioning by pairing an odor (conditioned stimulus, CS) with electric shock (unconditioned stimulus, US) induces aversive memories that are short-lasting (16). The memory performance of fruit flies is observed to drop to a negligible level within 24 h, decaying rapidly early after training and slowing down thereafter (17). Memory decay or forgetting requires the activation of the small G protein Rac, a signaling protein involved in actin remodeling, in the mushroom body (MB) intrinsic neurons (6). These so-called Kenyon cells (KCs) are the neurons that integrate CS–US information (18, 19) and support aversive memory formation and retrieval (20–22). In addition to Rac, forgetting also requires the DAMB dopamine receptor (7), which has highly enriched expression in the MB (23). Evidence suggests that the dopamine-mediated forgetting signal is conveyed to the MB by dopamine neurons (DANs) in the protocerebral posterior lateral 1 (PPL1) cluster (7, 24). Therefore, forgetting of olfactory aversive memory in Drosophila depends on a particular set of intracellular molecular pathways within KCs, involving Rac, DAMB, and possibly others (25), and also receives modulation from extrinsic neurons. Although important cellular evidence supporting the hypothesis that memory traces are erased under these circumstances is still lacking, these findings lend support to the notion that forgetting is an active, biologically regulated process (17, 26).Although existing studies point to the MB circuit as essential for forgetting, several questions remain to be answered. First, whereas the molecular pathways for learning and forgetting of olfactory aversive memory are distinct and separable (6, 7), the neural circuits seem to overlap. Rac-mediated forgetting has been localized to a large population of KCs (6), including the γ-subset, which is also critical for initial memory formation (21, 27). The site of action of DAMB for forgetting has yet to be established; however, the subgroups of PPL1-DANs implicated in forgetting are the same as those that signal aversive reinforcement and are required for learning (28–30). It leaves open the question of whether the brain circuitry underlying forgetting and learning is dissociable, or whether forgetting and learning share the same circuit but are driven by distinct activity patterns and molecular machinery (26). Second, shock reinforcement elicits multiple memory traces through at least three dopamine pathways to different subdomains in the MB lobes (28, 29). Functional imaging studies have also revealed Ca²⁺-based memory traces in different KC populations (31). It is poorly understood how forgetting of these memory traces differs, and it remains unknown whether there are multiple regulatory neural pathways. Notably, when PPL1-DANs are inactivated, forgetting still occurs, albeit at a lower rate (7). This incomplete block suggests the existence of an additional pathway(s) that conveys forgetting signals to the MB. Third, other than memory decay over time, forgetting is also observed through interference (32, 33), when new learning or reversal learning is introduced after training (6, 34, 35). Time-based and interference-based forgetting shares a similar dependence on Rac and DAMB (6, 7). However, it is not known whether distinct circuits underlie forgetting in these different contexts.In the current study, we focus on the diverse set of MB extrinsic neurons (MBENs) that interconnect the MB lobes with other brain regions, which include 34 MB output neurons (MBONs) of 21 types and ∼130 dopaminergic neurons of 20 types in the PPL1 and protocerebral anterior medial (PAM) clusters (36, 37). These neurons have been intensively studied in olfactory memory formation, consolidation, and retrieval in recent years (e.g., 24, 28–30, 38–48); however, their roles in forgetting have not been characterized except for the aforementioned PPL1-DANs. In a functional screen, we unexpectedly found that several Gal4 driver lines of MBENs showed significantly better 3-h memory retention when the Gal4-expressing cells were inactivated. The screen has thus led us to identify two types of MBENs that are not involved in initial learning but play important and additive roles in mediating memory decay. Furthermore, neither of these MBEN types is required for reversal learning, supporting the notion that there is a diversity of neural circuits that drive different forms of forgetting.     

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.     

N terminus of ASPP2 binds to Ras and enhances Ras/Raf/MEK/ERK activation to promote oncogene-induced senescence

The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras–GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras–GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras–GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12–induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras–GTP to stimulate Ras-induced senescence in nontransformed human cells.ASPP2, also known as 53BP2L, is a tumor suppressor whose expression is altered in human cancers (1). Importantly, targeting of the ASPP2 allele in two different mouse models reveals that ASPP2 heterozygous mice are prone to spontaneous and γ-irradiation–induced tumors, which rigorously demonstrates the role of ASPP2 as a tumor suppressor (2, 3). ASPP2 binds p53 via the C-terminal ankyrin-repeat and SH3 domain (4–6), is damage-inducible, and can enhance damage-induced apoptosis in part through a p53-mediated pathway (1, 2, 7–10). However, it remains unclear what biologic pathways and mechanisms mediate ASPP2 tumor suppressor function (1). Indeed, accumulating evidence demonstrates that ASPP2 also mediates nonapoptotic p53-independent pathways (1, 3, 11–15).The induction of cellular senescence forms an important barrier to tumorigenesis in vivo (16–21). It is well known that oncogenic Ras signaling induces senescence in normal nontransformed cells to prevent tumor initiation and maintain complex growth arrest pathways (16, 18, 21–24). The level of oncogenic Ras activation influences its capacity to activate senescence; high levels of oncogenic H-RasV12 signaling leads to low grade tumors with senescence markers, which progress to invasive cancers upon senescence inactivation (25). Thus, tight control of Ras signaling is critical to ensure the proper biologic outcome in the correct cellular context (26–28).The ASPP2 C terminus is important for promoting p53-dependent apoptosis (7). The ASPP2 N terminus may also suppress cell growth (1, 7, 29–33). Alternative splicing can generate the ASPP2 N-terminal truncated protein BBP (also known as 53BP2S) that is less potent in suppressing cell growth (7, 34, 35). Although the ASPP2 C terminus mediates nuclear localization, full-length ASPP2 also localizes to the cytoplasm and plasma membrane to mediate extranuclear functions (7, 11, 12, 36). Structural studies of the ASPP2 N terminus reveal a β–Grasp ubiquitin-like fold as well as a potential Ras-binding (RB)/Ras-association (RA) domain (32). Moreover, ASPP2 can promote H-RasV12–induced senescence (13, 15). However, the molecular mechanism(s) of how ASPP2 directly promotes Ras signaling are complex and remain to be completely elucidated.Here, we explore the molecular mechanisms of how Ras-signaling is enhanced by ASPP2. We demonstrate that ASPP2: (i) binds Ras-GTP and stimulates Ras-induced ERK signaling via its N-terminal domain at the plasma membrane; (ii) enhances Ras-GTP loading and B-Raf/C-Raf dimerization and forms a ASPP2/Raf complex; (iii) stimulates Ras-induced C-Raf phosphorylation and activation; and (iv) potentiates H-RasV12–induced senescence in both primary human fibroblasts and neonatal human epidermal keratinocytes. These data provide mechanistic insight into ASPP2 function(s) and opens important avenues for investigation into its role as a tumor suppressor in human cancer.