Transition paths,diffusive processes,and preequilibria of protein folding

Fundamental relationships between the thermodynamics and kinetics of protein folding were investigated using chain models of natural proteins with diverse folding rates by extensive comparisons between the distribution of conformations in thermodynamic equilibrium and the distribution of conformations sampled along folding trajectories. Consistent with theory and single-molecule experiment, duration of the folding transition paths exhibits only a weak correlation with overall folding time. Conformational distributions of folding trajectories near the overall thermodynamic folding/unfolding barrier show significant deviations from preequilibrium. These deviations, the distribution of transition path times, and the variation of mean transition path time for different proteins can all be rationalized by a diffusive process that we modeled using simple Monte Carlo algorithms with an effective coordinate-independent diffusion coefficient. Conformations in the initial stages of transition paths tend to form more nonlocal contacts than typical conformations with the same number of native contacts. This statistical bias, which is indicative of preferred folding pathways, should be amenable to future single-molecule measurements. We found that the preexponential factor defined in the transition state theory of folding varies from protein to protein and that this variation can be rationalized by our Monte Carlo diffusion model. Thus, protein folding physics is different in certain fundamental respects from the physics envisioned by a simple transition-state picture. Nonetheless, transition state theory can be a useful approximate predictor of cooperative folding speed, because the height of the overall folding barrier is apparently a proxy for related rate-determining physical properties.Protein folding is an intriguing phenomenon at the interface of physics and biology. In the early days of folding kinetics studies, folding was formulated almost exclusively in terms of mass-action rate equations connecting the folded, unfolded, and possibly, one or a few intermediate states (1, 2). With the advent of site-directed mutagenesis, the concept of free energy barriers from transition state theory (TST) (3) was introduced to interpret mutational data (4), and subsequently, it was adopted for the Φ-value analysis (5). Since the 1990s, the availability of more detailed experimental data (6), in conjunction with computational development of coarse-grained chain models, has led to an energy landscape picture of folding (7–15). This perspective emphasizes the diversity of microscopic folding trajectories, and it conceptualizes folding as a diffusive process (16–25) akin to the theory of Kramers (26).For two-state-like folding, the transition path (TP), i.e., the sequence of kinetic events that leads directly from the unfolded state to the folded state (27, 28), constitutes only a tiny fraction of a folding trajectory that spends most of the time diffusing, seemingly unproductively, in the vicinity of the free energy minimum of the unfolded state. The development of ultrafast laser spectroscopy (29, 30) and single-molecule (27, 28, 31) techniques have made it possible to establish upper bounds on the transition path time (t_TP) ranging from <200 and <10 μs by earlier (27) and more recent (28), respectively, direct single-molecule FRET to <2 μs (30) by bulk relaxation measurements. Consistent with these observations, recent extensive atomic simulations have also provided estimated t_TP values of the order of ∼1 μs (32, 33). These advances offer exciting prospects of characterizing the productive events along folding TPs.It is timely, therefore, to further the theoretical investigation of TP-related questions (19). To this end, we used coarse-grained C_α models (14) to perform extensive simulations of the folding trajectories of small proteins with 56- to 86-aa residues. These tractable models are useful, because despite significant progress, current atomic models cannot provide the same degree of sampling coverage for proteins of comparable sizes (32, 33). In addition to structural insights, this study provides previously unexplored vantage points to compare the diffusion and TST pictures of folding. Deviations of folding behaviors from TST predictions are not unexpected, because TST is mostly applicable to simple gas reactions; however, the nature and extent of the deviations have not been much explored. Our explicit-chain simulation data conform well to the diffusion picture but not as well to TST. In particular, the preexponential factors of the simulated folding rates exhibit a small but appreciable variation that depends on native topology. These findings and others reported below underscore the importance of single-molecule measurements (13, 27, 28, 31, 34, 35) in assessing the merits of proposed scenarios and organizing principles of folding (7–25, 36, 37).     

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.     

Interplay between insulin signaling,juvenile hormone,and vitellogenin regulates maternal effects on polyphenism in ants

Polyphenism is the phenomenon in which alternative phenotypes are produced by a single genotype in response to environmental cues. An extreme case is found in social insects, in which reproductive queens and sterile workers that greatly differ in morphology and behavior can arise from a single genotype. Experimental evidence for maternal effects on caste determination, the differential larval development toward the queen or worker caste, was recently documented in Pogonomyrmex seed harvester ants, in which only colonies with a hibernated queen produce new queens. However, the proximate mechanisms behind these intergenerational effects have remained elusive. We used a combination of artificial hibernation, hormonal treatments, gene expression analyses, hormone measurements, and vitellogenin quantification to investigate how the combined effect of environmental cues and hormonal signaling affects the process of caste determination in Pogonomyrmex rugosus. The results show that the interplay between insulin signaling, juvenile hormone, and vitellogenin regulates maternal effects on the production of alternative phenotypes and set vitellogenin as a likely key player in the intergenerational transmission of information. This study reveals how hibernation triggers the production of new queens in Pogonomyrmex ant colonies. More generally, it provides important information on maternal effects by showing how environmental cues experienced by one generation can translate into phenotypic variation in the next generation.Many plants and animals can express specific adaptive responses to their environment through phenotypic plasticity, whereby a given genotype can develop into different phenotypes depending on environmental conditions (1, 2). Maternal effects, through which the environmental conditions experienced by the mother are translated into phenotypic variation in the offspring (3, 4), contribute to many phenotypic traits in a wide variety of taxa (5, 6) and have important ecological and evolutionary consequences (7, 8). Investigating the mechanisms of cross-generational transmission of information underlying maternal effects is needed to better understand the optimization of phenotypes in changing environments (6) and, more generally, the evolution of life history strategies (9).In many insect species, maternal effects are known to affect polyphenism (3, 10), an extreme form of phenotypic plasticity characterized by the production of alternative and discrete phenotypes from a single genotype (1, 11–13). Such maternal effects allow adequate responses to environmental cues such as temperature, photoperiod, nutrition, and population density in many species (10). Examples of maternal effects on insect polyphenism include the production of sexual versus parthenogenetic morphs in aphids (14, 15), winged versus wingless morphs in firebugs (16), and dispersal versus solitary morphs in locusts (17, 18). The endocrine system was found to play a role in the regulation of some maternal effects on insect polyphenisms (19–21), but the nature of the physiological and genetic pathways interacting with the hormonal system to translate environmental cues into offspring polyphenism remains mostly unknown (22).The most striking example of polyphenism is found in insect societies (23), where a reproductive division of labor leads to the coexistence of fertile queens and sterile workers that greatly differ in morphology and behavior (24, 25). Even though recent studies revealed genetic influences on caste determination in social insects (reviewed in ref. 26), female caste fate is primarily influenced by environmental factors in most species studied (27–39). In ants, several studies suggested that maternal factors such as temperature or queen age may affect caste determination (40–44). However, it is only recently that the first example of maternal effects on female caste polyphenism was documented experimentally (45). Cross-fostering of eggs between hibernated and nonhibernated Pogonomyrmex colonies revealed strong maternal effects on caste production, as only eggs produced by a hibernated queen were able to develop into queens, irrespective of the hibernation status of the rest of the colony (45). Such maternal effects on the caste fate of the female offspring require that the hibernation triggers changes in the queen that affect polyphenism in the offspring. Hormones may be involved in this process in Pogonomyrmex ants, as Pogonomyrmex rugosus queen- and worker-destined eggs differed in their ecdysteroid content (45) and Pogonomyrmex barbatus mature queens treated with juvenile hormone (JH) were recently found to produce larger workers (46).Studies on the mechanisms regulating insect polyphenisms (reviewed in ref. 10) suggest that the insulin/insulin-like growth factor signaling (IIS), JH, and vitellogenin (Vg) pathways, known to regulate reproduction in adult insects (47–51), play predominant roles in modulating larval development in response to environmental cues. A well-known example illustrating the role of these pathways is the caste fate of the female brood (queen or worker) in the honey bee Apis mellifera (52–58). In this species, worker-triggered differences in larval diet induce changes in IIS that affect JH (57), possibly through the release of neuropeptides (e.g., allatostatin and allatotropin) that influence JH production by the corpus allatum, as found in Drosophila (59). Changes in JH in turn affect the production of Vg (60–62), which may be involved in the process of caste determination (62, 63). Such effects of JH on Vg production, also reported in flies (64), locusts (65), and cockroaches (66), have been proposed to involve the action of ecdysteroids (62, 67–70). IIS, JH, and Vg may also play a role in the regulation of caste differentiation of larvae in ants, as caste-specific expressions of genes involved in the IIS pathway were documented in Solenopsis invicta (71) and Diacamma sp. (72). Interestingly, caste-specific differences in IIS, JH, and Vg were also documented in adult ants and bees (48, 73–78), suggesting further roles of these pathways in the regulation of social life (74, 79).We propose that the interplay between IIS, JH, and Vg regulates maternal effects on caste polyphenism in ants by translating the environmental conditions experienced by the queen during hibernation into the production of alternative phenotypes in the offspring. Under this hypothesis, IIS would translate environmental cues into changes in JH, which would, in turn, affect the amount of Vg in queens and in eggs, thus possibly affecting the caste fate of the offspring (62, 63). This hypothesis makes four predictions. First, a pharmacological increase of JH in queens should mimic the effect of hibernation and stimulate the production of queens. Second, hibernation should affect IIS and the production of JH in queens. Third, both hibernation and a JH increase should stimulate the production of Vg in queens. Finally, Vg content should differ between queen- and worker-destined eggs. We tested these predictions by performing artificial hibernation, hormonal treatments, gene expression analyses, hormone measurements, and Vg quantification in Pogonomyrmex rugosus, an ant species in which temperature-triggered changes in the queen had previously been shown to affect the relative production of queens and workers. Each of the four predictions was confirmed by our experiments, thus revealing that the interplay between IIS, JH, and Vg regulates maternal effects on caste polyphenism in P. rugosus.     

Predictive Value of Brain Natriuretic Peptides in Patients on Peritoneal Dialysis: Results from the ADEMEX Trial

Background and objectives: Natriuretic peptides have been suggested to be of value in risk stratification in dialysis patients. Data in patients on peritoneal dialysis remain limited.Design, setting, participants, & measurements: Patients of the ADEMEX trial (ADEquacy of peritoneal dialysis in MEXico) were randomized to a control group [standard 4 × 2L continuous ambulatory peritoneal dialysis (CAPD); n = 484] and an intervention group (CAPD with a target creatinine clearance ≥60L/wk/1.73 m²; n = 481). Natriuretic peptides were measured at baseline and correlated with other parameters as well as evaluated for effects on patient outcomes.Results: Control group and intervention group were comparable at baseline with respect to all measured parameters. Baseline values of natriuretic peptides were elevated and correlated significantly with levels of residual renal function but not with body size or diabetes. Baseline values of N-terminal fragment of B-type natriuretic peptide (NT-proBNP) but not proANP(1–30), proANP(31–67), or proANP(1–98) were independently highly predictive of overall survival and cardiovascular mortality. Volume removal was also significantly correlated with patient survival.Conclusions. NT-proBNP have a significant predictive value for survival of CAPD patients and may be of value in guiding risk stratification and potentially targeted therapeutic interventions.Plasma levels of cardiac natriuretic peptides are elevated in patients with chronic kidney disease, owing to impairment of renal function, hypertension, hypervolemia, and/or concomitant heart disease (1–7). Atrial natriuretic peptide (ANP) and particularly brain natriuretic peptide (BNP) levels are linked independently to left ventricular mass (3–5,8–16) and function (3,6–17) and predict total and cardiovascular mortality (1,3,8,10,12,18) as well as cardiac events (12,19). ANP and BNP decrease significantly during hemodialysis treatment but increase again during the interdialytic interval (1,2,4,6,7,14,17,20–23). Levels in patients on peritoneal dialysis (PD) have been found to be lower than in patients on hemodialysis (11,24–26), but the correlations with left ventricular function and structure are maintained in both types of dialysis modalities (11,15,27,28).The high mortality of patients on peritoneal dialysis and the failure of dialytic interventions to alter this mortality (29,30) necessitate renewed attention into novel methods of stratification and identification of patients at highest risk to be targeted for specific interventions. Cardiac natriuretic peptides are increasingly considered to fulfill this role in nonrenal patients. Evaluations of cardiac natriuretic peptides in patients on PD have been limited by small numbers (3,9,11,12,15,24–26) and only one study examined correlations between natriuretic peptide levels and outcomes (12). The PD population enrolled in the ADEMEX trial offered us the opportunity to evaluate cardiac natriuretic peptides and their value in predicting outcomes in the largest clinical trial ever performed on PD (29,30). It is hoped that such an evaluation would identify patients at risk even in the absence of overt clinical disease and hence facilitate or encourage interventions with salutary outcomes.     

WWOX,the common fragile site FRA16D gene product,regulates ATM activation and the DNA damage response

Genomic instability is a hallmark of cancer. The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor spanning the common chromosomal fragile site FRA16D. Here, we report a direct role of WWOX in DNA damage response (DDR) and DNA repair. We show that Wwox deficiency results in reduced activation of the ataxia telangiectasia-mutated (ATM) checkpoint kinase, inefficient induction and maintenance of γ-H2AX foci, and impaired DNA repair. Mechanistically, we show that, upon DNA damage, WWOX accumulates in the cell nucleus, where it interacts with ATM and enhances its activation. Nuclear accumulation of WWOX is regulated by its K63-linked ubiquitination at lysine residue 274, which is mediated by the E3 ubiquitin ligase ITCH. These findings identify a novel role for the tumor suppressor WWOX and show that loss of WWOX expression may drive genomic instability and provide an advantage for clonal expansion of neoplastic cells.Genomic instability is a common characteristic of human cancers. The DNA damage response (DDR) maintains the integrity of the genome in response to DNA damage. DDR is a complex signaling process that results in cell cycle arrest followed by either DNA repair or apoptosis if the DNA damage is too extensive to be repaired (1–3). Key mammalian damage response sensors are ataxia telangiectasia-mutated (ATM), ATM and Rad3-related, and DNA-dependent PKs (4, 5). Disruption of the DDR machinery in human cells leads to genomic instability and an increased risk of cancer progression (6, 7).The WW domain-containing oxidoreductase (WWOX) gene spans the common fragile site (CFS) FRA16D (8, 9). Genomic alterations affecting the WWOX locus have been reported in several types of cancer and include homozygous and hemizygous deletions (10–13). Ectopic expression of WWOX in WWOX-negative cancer cells attenuates cell growth and suppresses tumor growth in immunocompromised mice (10, 11, 14). Importantly, targeted ablation of Wwox in mice results in higher incidence of spontaneous lesions resembling osteosarcomas and lung and mammary tumors (14–16). These findings suggest WWOX as a tumor suppressor. The WWOX protein contains two N-terminal WW domains mediating WWOX interaction with PP(proline)x(amino acid)Y(tyrosine)-containing proteins (11, 17) and a central short-chain deyhdrogenase/reductase domain that has been proposed to function in steroidogenesis (18). Recent characterization of WWOX domains revealed that they interact, mainly through the WW1 domain, with multiprotein networks (3). The mechanism by which WWOX suppresses tumorigenicity is, however, not well-known.In vitro, CFSs are defined as gaps or breaks on metaphase chromosomes that occur in cells treated with inhibitors of DNA replication (19, 20). In vivo, CFSs are preferential targets of replication stress in preneoplastic lesions (21), and emerging evidence suggests that they represent early warning sensors for DNA damage (22–24). Both genetic and epigenetic factors are thought to regulate the fragility of CFS (25, 26). Recent profiling studies of CFS provide evidence that the functional fragility of CFS is tissue-specific (27–29). High-throughput genomic analyses of 3,131 cancer specimens (12) and 746 cancer cell lines (13) have recently identified large deletions in CFSs, including the FRA16D/WWOX locus. Although these deletions have been linked to the presence of DNA replication stress (30), the molecular function of gene products of CFSs, including the WWOX protein, is poorly understood. Here, we identify a direct role of WWOX in the DDR and show that the WWOX gene product functions as a modulator of the DNA damage checkpoint kinase ATM.     

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.     

Centromere proteins CENP-C and CAL1 functionally interact in meiosis for centromere clustering,pairing, and chromosome segregation

Meiotic chromosome segregation involves pairing and segregation of homologous chromosomes in the first division and segregation of sister chromatids in the second division. Although it is known that the centromere and kinetochore are responsible for chromosome movement in meiosis as in mitosis, potential specialized meiotic functions are being uncovered. Centromere pairing early in meiosis I, even between nonhomologous chromosomes, and clustering of centromeres can promote proper homolog associations in meiosis I in yeast, plants, and Drosophila. It was not known, however, whether centromere proteins are required for this clustering. We exploited Drosophila mutants for the centromere proteins centromere protein-C (CENP-C) and chromosome alignment 1 (CAL1) to demonstrate that a functional centromere is needed for centromere clustering and pairing. The cenp-C and cal1 mutations result in C-terminal truncations, removing the domains through which these two proteins interact. The mutants show striking genetic interactions, failing to complement as double heterozygotes, resulting in disrupted centromere clustering and meiotic nondisjunction. The cluster of meiotic centromeres localizes to the nucleolus, and this association requires centromere function. In Drosophila, synaptonemal complex (SC) formation can initiate from the centromere, and the SC is retained at the centromere after it disassembles from the chromosome arms. Although functional CENP-C and CAL1 are dispensable for assembly of the SC, they are required for subsequent retention of the SC at the centromere. These results show that integral centromere proteins are required for nuclear position and intercentromere associations in meiosis.Centromeres are the control centers for chromosomes, and thus are essential for accurate segregation in cell division. Centromeres are the DNA regions with a specialized chromatin structure upon which the kinetochore is built. The kinetochore is a complex of at least 100 proteins that contains the proteins to bind microtubules, motors to move on or destabilize microtubules, as well as checkpoint proteins monitoring kinetochore–microtubule attachment (1). The ability of the kinetochore to control microtubule binding and chromosome movement is essential for proper segregation in both mitosis and meiosis. In meiosis, additional constraints are placed on kinetochore function to ensure that homologs segregate in the first division and that segregation of sister chromatids is deferred until the second division (2). Recent studies indicate that in addition, the centromere itself may influence homolog segregation by controlling homolog pairing and formation of the synaptonemal complex (SC) (3).In prophase of meiosis I, the homologs must pair and ultimately become attached, usually by recombination and crossing-over. By quantifying centromere number through prophase I, it has been observed that centromeres pair in yeast, plants, and Drosophila (3). Perhaps unexpectedly, this pairing can be between nonhomologous centromeres; in yeast, this has been proposed as a mechanism to prevent recombination around the centromere, as centromere pairing resolves from initially being nonhomologous to being homologous (4, 5). Homologous centromere pairing may play a critical role in ensuring segregation of chromosomes that do not undergo crossing-over, possibly by affecting orientation of the kinetochores (3, 6–8).The centromere also regulates synapsis via the formation of the SC. SC formation initiates at the centromere and sites of cross-over formation in yeast, and the centromere is the first site for SC formation in Drosophila prophase I (9, 10). In addition, the SC persists at the centromere in yeast and Drosophila after the SC present along the chromosome arms has disassembled late in prophase I (7, 9, 11). Although SC assembly does not begin at centromeres in mouse meiosis, it persists at the centromeres and appears to promote proper segregation (12, 13).Another centromere property has been observed in Drosophila oocytes. In most organisms, the centromeres are clustered together at one site at the onset of meiosis, likely a remnant of their configuration in mitosis, but this clustering breaks down as centromeres arrange in pairs (3, 4). In Drosophila, however, the centromeres remain clustered until exit from prophase I at oocyte maturation (9, 10, 14). Although an essential role for centromere clustering has not been demonstrated, it may facilitate homolog pairing, synapsis, or accurate segregation, particularly given that the homologous telomeres do not pair into a bouquet formation in Drosophila meiosis (15, 16). Components of the SC are necessary for centromere clustering, as is the cohesion protein ORD (9, 14).The studies on centromere pairing and clustering define centromere geography within the meiotic nucleus, but they did not test whether centromere structure or function was involved. Centromeres have specialized nucleosomes with a histone H3 variant, centromere protein-A (CENP-A) (17). Incorporation of CENP-A into centromere chromatin is regulated precisely, although it occurs at distinct cell cycle times in different cell types, varying between late mitosis and G1 (17). In vertebrates, a complex of 15 proteins, the constitutive centromere-associated network (CCAN), is present on the CENP-A chromatin throughout the cell cycle and is crucial for assembling kinetochore proteins (1). In Drosophila, the entire CCAN complex has not been identified, although the CENP-C protein is present (18). Another Drosophila protein, CAL1, binds to CENP-A (called CID in Drosophila) in a prenucleosomal complex, and CAL1 is required for loading CID (19–22). CAL1 interacts with both CID and CENP-C, and all three proteins show interdependency for centromere localization (21, 23).Little is known about the activities of these centromere proteins in meiosis. In fission yeast, CENP-C has been demonstrated to be critical for kinetochore–microtubule binding in meiosis and also to control kinetochore orientation in meiosis I (24). The timing of assembly of kinetochore and centromere proteins onto meiotic chromosomes has been examined in mouse spermatocytes (25) and in Drosophila spermatocytes and sperm (26, 27). RNAi studies have shown that CAL1 and CENP-C (the latter to a lesser extent) are needed for CID localization in Drosophila male meiosis, with reduction in the levels of any of these three proteins being associated with meiotic segregation errors (26). Drosophila males differ from most organisms in not undergoing recombination or forming an SC, and centromere clustering does not occur (28). A question of particular interest that has yet to be addressed is whether centromere architecture and function are required for centromere clustering and pairing in meiosis.     

The evolution of self-control

Cognition presents evolutionary research with one of its greatest challenges. Cognitive evolution has been explained at the proximate level by shifts in absolute and relative brain volume and at the ultimate level by differences in social and dietary complexity. However, no study has integrated the experimental and phylogenetic approach at the scale required to rigorously test these explanations. Instead, previous research has largely relied on various measures of brain size as proxies for cognitive abilities. We experimentally evaluated these major evolutionary explanations by quantitatively comparing the cognitive performance of 567 individuals representing 36 species on two problem-solving tasks measuring self-control. Phylogenetic analysis revealed that absolute brain volume best predicted performance across species and accounted for considerably more variance than brain volume controlling for body mass. This result corroborates recent advances in evolutionary neurobiology and illustrates the cognitive consequences of cortical reorganization through increases in brain volume. Within primates, dietary breadth but not social group size was a strong predictor of species differences in self-control. Our results implicate robust evolutionary relationships between dietary breadth, absolute brain volume, and self-control. These findings provide a significant first step toward quantifying the primate cognitive phenome and explaining the process of cognitive evolution.Since Darwin, understanding the evolution of cognition has been widely regarded as one of the greatest challenges for evolutionary research (1). Although researchers have identified surprising cognitive flexibility in a range of species (2–40) and potentially derived features of human psychology (41–61), we know much less about the major forces shaping cognitive evolution (62–71). With the notable exception of Bitterman’s landmark studies conducted several decades ago (63, 72–74), most research comparing cognition across species has been limited to small taxonomic samples (70, 75). With limited comparable experimental data on how cognition varies across species, previous research has largely relied on proxies for cognition (e.g., brain size) or metaanalyses when testing hypotheses about cognitive evolution (76–92). The lack of cognitive data collected with similar methods across large samples of species precludes meaningful species comparisons that can reveal the major forces shaping cognitive evolution across species, including humans (48, 70, 89, 93–98).To address these challenges we measured cognitive skills for self-control in 36 species of mammals and birds (Fig. 1 and Tables S1–S4) tested using the same experimental procedures, and evaluated the leading hypotheses for the neuroanatomical underpinnings and ecological drivers of variance in animal cognition. At the proximate level, both absolute (77, 99–107) and relative brain size (108–112) have been proposed as mechanisms supporting cognitive evolution. Evolutionary increases in brain size (both absolute and relative) and cortical reorganization are hallmarks of the human lineage and are believed to index commensurate changes in cognitive abilities (52, 105, 113–115). Further, given the high metabolic costs of brain tissue (116–121) and remarkable variance in brain size across species (108, 122), it is expected that the energetic costs of large brains are offset by the advantages of improved cognition. The cortical reorganization hypothesis suggests that selection for absolutely larger brains—and concomitant cortical reorganization—was the predominant mechanism supporting cognitive evolution (77, 91, 100–106, 120). In contrast, the encephalization hypothesis argues that an increase in brain volume relative to body size was of primary importance (108, 110, 111, 123). Both of these hypotheses have received support through analyses aggregating data from published studies of primate cognition and reports of “intelligent” behavior in nature—both of which correlate with measures of brain size (76, 77, 84, 92, 110, 124).Open in a separate windowFig. 1.A phylogeny of the species included in this study. Branch lengths are proportional to time except where long branches have been truncated by parallel diagonal lines (split between mammals and birds ∼292 Mya).With respect to selective pressures, both social and dietary complexities have been proposed as ultimate causes of cognitive evolution. The social intelligence hypothesis proposes that increased social complexity (frequently indexed by social group size) was the major selective pressure in primate cognitive evolution (6, 44, 48, 50, 87, 115, 120, 125–141). This hypothesis is supported by studies showing a positive correlation between a species’ typical group size and the neocortex ratio (80, 81, 85–87, 129, 142–145), cognitive differences between closely related species with different group sizes (130, 137, 146, 147), and evidence for cognitive convergence between highly social species (26, 31, 148–150). The foraging hypothesis posits that dietary complexity, indexed by field reports of dietary breadth and reliance on fruit (a spatiotemporally distributed resource), was the primary driver of primate cognitive evolution (151–154). This hypothesis is supported by studies linking diet quality and brain size in primates (79, 81, 86, 142, 155), and experimental studies documenting species differences in cognition that relate to feeding ecology (94, 156–166).Although each of these hypotheses has received empirical support, a comparison of the relative contributions of the different proximate and ultimate explanations requires (i) a cognitive dataset covering a large number of species tested using comparable experimental procedures; (ii) cognitive tasks that allow valid measurement across a range of species with differing morphology, perception, and temperament; (iii) a representative sample within each species to obtain accurate estimates of species-typical cognition; (iv) phylogenetic comparative methods appropriate for testing evolutionary hypotheses; and (v) unprecedented collaboration to collect these data from populations of animals around the world (70).Here, we present, to our knowledge, the first large-scale collaborative dataset and comparative analysis of this kind, focusing on the evolution of self-control. We chose to measure self-control—the ability to inhibit a prepotent but ultimately counterproductive behavior—because it is a crucial and well-studied component of executive function and is involved in diverse decision-making processes (167–169). For example, animals require self-control when avoiding feeding or mating in view of a higher-ranking individual, sharing food with kin, or searching for food in a new area rather than a previously rewarding foraging site. In humans, self-control has been linked to health, economic, social, and academic achievement, and is known to be heritable (170–172). In song sparrows, a study using one of the tasks reported here found a correlation between self-control and song repertoire size, a predictor of fitness in this species (173). In primates, performance on a series of nonsocial self-control control tasks was related to variability in social systems (174), illustrating the potential link between these skills and socioecology. Thus, tasks that quantify self-control are ideal for comparison across taxa given its robust behavioral correlates, heritable basis, and potential impact on reproductive success.In this study we tested subjects on two previously implemented self-control tasks. In the A-not-B task (27 species, n = 344), subjects were first familiarized with finding food in one location (container A) for three consecutive trials. In the test trial, subjects initially saw the food hidden in the same location (container A), but then moved to a new location (container B) before they were allowed to search (Movie S1). In the cylinder task (32 species, n = 439), subjects were first familiarized with finding a piece of food hidden inside an opaque cylinder. In the following 10 test trials, a transparent cylinder was substituted for the opaque cylinder. To successfully retrieve the food, subjects needed to inhibit the impulse to reach for the food directly (bumping into the cylinder) in favor of the detour response they had used during the familiarization phase (Movie S2).Thus, the test trials in both tasks required subjects to inhibit a prepotent motor response (searching in the previously rewarded location or reaching directly for the visible food), but the nature of the correct response varied between tasks. Specifically, in the A-not-B task subjects were required to inhibit the response that was previously successful (searching in location A) whereas in the cylinder task subjects were required to perform the same response as in familiarization trials (detour response), but in the context of novel task demands (visible food directly in front of the subject).     

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.