Toxin Kid uncouples DNA replication and cell division to enforce retention of plasmid R1 in Escherichia coli cells

Worldwide dissemination of antibiotic resistance in bacteria is facilitated by plasmids that encode postsegregational killing (PSK) systems. These produce a stable toxin (T) and a labile antitoxin (A) conditioning cell survival to plasmid maintenance, because only this ensures neutralization of toxicity. Shortage of antibiotic alternatives and the link of TA pairs to PSK have stimulated the opinion that premature toxin activation could be used to kill these recalcitrant organisms in the clinic. However, validation of TA pairs as therapeutic targets requires unambiguous understanding of their mode of action, consequences for cell viability, and function in plasmids. Conflicting with widespread notions concerning these issues, we had proposed that the TA pair kis-kid (killing suppressor-killing determinant) might function as a plasmid rescue system and not as a PSK system, but this remained to be validated. Here, we aimed to clarify unsettled mechanistic aspects of Kid activation, and of the effects of this for kis-kid–bearing plasmids and their host cells. We confirm that activation of Kid occurs in cells that are about to lose the toxin-encoding plasmid, and we show that this provokes highly selective restriction of protein outputs that inhibits cell division temporarily, avoiding plasmid loss, and stimulates DNA replication, promoting plasmid rescue. Kis and Kid are conserved in plasmids encoding multiple antibiotic resistance genes, including extended spectrum β-lactamases, for which therapeutic options are scarce, and our findings advise against the activation of this TA pair to fight pathogens carrying these extrachromosomal DNAs.Plasmids serve as extrachromosomal DNA platforms for the reassortment, mobilization, and maintenance of antibiotic resistance genes in bacteria, enabling host cells to colonize environments flooded with antimicrobials and to take advantage of resources freed by the extinction of nonresistant competitors. Fueled by these selective forces and aided by their itinerant nature, plasmids disseminate resistance genes worldwide shortly after new antibiotics are developed, which is a major clinical concern (1–3). However, in antibiotic-free environments, such genes are dispensable. There, the cost that plasmid carriage imposes on cells constitutes a disadvantage in the face of competition from other cells and, because plasmids depend on their hosts to survive, also a threat to their own existence.Many plasmids keep low copy numbers (CNs) to minimize the problem above, because it reduces burdens to host cells. However, this also decreases their chances to fix in descendant cells, a new survival challenge (4). To counteract this, plasmids have evolved stability functions. Partition systems pull replicated plasmid copies to opposite poles in host cells, facilitating their inheritance by daughter cells (5). Plasmids also bear postsegregational killing (PSK) systems, which encode a stable toxin and a labile antitoxin (TA) pair that eliminates plasmid-free cells produced by occasional replication or partition failures. Regular production of the labile antitoxin protects plasmid-containing cells from the toxin. However, antitoxin replenishment is not possible in cells losing the plasmid, and this triggers their elimination (5).TA pairs are common in plasmids disseminating antibiotic resistance in bacterial pathogens worldwide (2, 6–10). The link of these systems to PSK and the exiguous list of alternatives in the pipeline have led some to propose that chemicals activating these TA pairs may constitute a powerful antibiotic approach against these organisms (5, 11–13). However, the appropriateness of these TA pairs as therapeutic targets requires unequivocal understanding of their function in plasmids. Although PSK systems encode TA pairs, not all TA pairs might function as PSK systems, as suggested by their abundance in bacterial chromosomes, where PSK seems unnecessary (14–16). Moreover, the observation that many plasmids bear several TA pairs (6–10) raises the intriguing question of why they would need more than one PSK system, particularly when they increase the metabolic burden that plasmids impose on host cells (17). Because PSK functions are not infallible, their gathering may provide a mechanism for reciprocal failure compensation, minimizing the number of cells that escape killing upon plasmid loss (5). Alternatively, some TA pairs may stabilize plasmids by mechanisms different from PSK, and their grouping might not necessarily reflect functional redundancy (18).This may be the case in plasmid R1, which encodes TA pairs hok-sok (host killing-suppressor of killing) and kis(pemI)-kid(pemK) (19–23). Inconsistent with PSK, we had noticed that activation of toxin Kid occurred in cells that still contained R1, and that this happened when CNs were insufficient to ensure plasmid transmission to descendant cells. We also found that Kid cleaved mRNA at UUACU sites, which appeared well suited to trigger a response that prevented plasmid loss and increased R1 CNs without killing cells, as suggested by our results. In view of all this, we argued that Kid and Kis functioned as a rescue system for plasmid R1, and not as a PSK system (24). This proposal cannot be supported by results elsewhere, suggesting that Kid may cleave mRNA at simpler UAH sites (with H being A, C, or U) (25, 26), a view that has prevailed in the literature (14, 16, 27–29). Moreover, other observations indicate that our past experiments may have been inappropriate to conclude that Kid does not kill Escherichia coli cells (30, 31). Importantly, Kid, Kis, and other elements that we found essential for R1 rescue are conserved in plasmids conferring resistance to extended-spectrum β-lactamases, a worrying threat to human health (1, 6–10, 32). Therapeutic options to fight pathogens carrying these plasmids are limited, and activation of Kid may be perceived as a good antibiotic alternative. Because the potential involvement of this toxin in plasmid rescue advises against such approach, we aimed to ascertain here the mode of action; the effects on cells; and, ultimately, the function of Kid (and Kis) in R1.     

An outer membrane channel protein of Mycobacterium tuberculosis with exotoxin activity

The ability to control the timing and mode of host cell death plays a pivotal role in microbial infections. Many bacteria use toxins to kill host cells and evade immune responses. Such toxins are unknown in Mycobacterium tuberculosis. Virulent M. tuberculosis strains induce necrotic cell death in macrophages by an obscure molecular mechanism. Here we show that the M. tuberculosis protein Rv3903c (channel protein with necrosis-inducing toxin, CpnT) consists of an N-terminal channel domain that is used for uptake of nutrients across the outer membrane and a secreted toxic C-terminal domain. Infection experiments revealed that CpnT is required for survival and cytotoxicity of M. tuberculosis in macrophages. Furthermore, we demonstrate that the C-terminal domain of CpnT causes necrotic cell death in eukaryotic cells. Thus, CpnT has a dual function in uptake of nutrients and induction of host cell death by M. tuberculosis.Toxins were recognized more than a century ago to play a major role in bacterial infectious diseases (1). Subsequently, hundreds of toxins from pathogenic bacteria have been characterized. Based on bioinformatic analysis of its genome, toxins appear to be absent from Mycobacterium tuberculosis (2–4), the causative agent of tuberculosis, a devastating disease with nine million new cases every year (5). Survival within host macrophages is a key trait enabling M. tuberculosis to persist in the human body (6), where it can reactivate after decades of quiescence (7). This so-called latent infection is poorly understood and is one of the reasons why tuberculosis remains a global public health problem. Alveolar macrophages engulf inhaled M. tuberculosis, contribute to killing of the bacteria, reduce inflammation of lung tissue, and limit uptake of M. tuberculosis by migratory dendritic cells to prevent bacterial dissemination (8). However, M. tuberculosis has evolved effective strategies to subvert this bactericidal response (6, 9). Death of M. tuberculosis-infected macrophages is caused by two processes: necrosis and apoptosis. Necrosis is characterized by metabolic collapse and loss of membrane integrity and is used by M. tuberculosis to exit destroyed cells, evade host defenses, and disseminate to other tissues and eventually to new hosts (10). By contrast, apoptosis of the infected macrophages helps the host to control the bacterial infection (11). Virulent M. tuberculosis strains induce a necrosis-like cell death and concomitantly suppress apoptosis of macrophages (12). Although M. tuberculosis is known to secrete virulence factors that interfere with phagosome maturation (9), it is unknown how M. tuberculosis kills macrophages.Gram-negative bacterial pathogens use complex nanomachines such as type I–VI secretion systems to secrete effector proteins mediating host cell death and subverting immune responses (13). Similarly, proteins secreted by M. tuberculosis need to cross both an inner and an outer membrane (14), a barrier of notoriously low permeability in M. tuberculosis (15). However, the only known secretion systems capable of translocating proteins across both M. tuberculosis membranes are the type VII secretion systems encoded by esx operons. Although inner membrane proteins of ESX secretion systems have been characterized (16), channel proteins that are required for protein translocation across the outer membrane are currently unknown in M. tuberculosis. We hypothesized that deletion or inactivation of outer membrane channel proteins in M. tuberculosis may result in increased antibiotic resistance, as has been described for Gram-negative bacteria and Mycobacterium smegmatis (17, 18). Here we show that this approach identified a protein that enables uptake of small, hydrophilic molecules via its N-terminal pore domain and induces host cell necrosis by its secreted toxic C-terminal domain (CTD).     

An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis

Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required to cause lethal infections in animals. The only pathway known to target proteins for proteasomal degradation in bacteria is pupylation, which is functionally analogous to eukaryotic ubiquitylation. However, evidence suggests that the M. tuberculosis proteasome contributes to pupylation-independent pathways as well. To identify new proteasome cofactors that might contribute to such pathways, we isolated proteins that bound to proteasomes overproduced in M. tuberculosis and found a previously uncharacterized protein, Rv3780, which formed rings and capped M. tuberculosis proteasome core particles. Rv3780 enhanced peptide and protein degradation by proteasomes in an adenosine triphosphate (ATP)-independent manner. We identified putative Rv3780-dependent proteasome substrates and found that Rv3780 promoted robust degradation of the heat shock protein repressor, HspR. Importantly, an M. tuberculosis Rv3780 mutant had a general growth defect, was sensitive to heat stress, and was attenuated for growth in mice. Collectively, these data demonstrate that ATP-independent proteasome activators are not confined to eukaryotes and can contribute to the virulence of one the world''s most devastating pathogens.Proteasomes are compartmentalized proteases essential for the viability of all eukaryotes and archaea. The proteolytically active component of the proteasome is the 20S core particle (CP), a ∼700-kDa complex composed of four heptameric rings stacked axially to form a barrel (reviewed in refs. 1 and 2). The active site of the 20S CP is buried deep within its core, rendering it inaccessible to folded proteins (3, 4). Further protection of the active site is afforded by “gating” amino acids that block the entrance to the proteasome core. Thus, proteasomal degradation is critically dependent on proteasome activators, which bind to either end of the 20S CP to reposition its gating residues and allow access to doomed substrates (reviewed in ref. 5).The best-characterized proteasome activator is the eukaryotic 19S regulatory particle (RP). The 19S RP is a ∼700-kDa heteromeric complex that binds either or both ends of the 20S CP to form the 26S proteasome, which is responsible for degrading proteins in an adenosine triphosphate (ATP)-dependent manner (6–8). The unfolding activities of the 19S RP come from its base, a heterohexameric ring of ATPases associated with diverse cellular activities (AAA ATPases) that directly bind to the 20S CP (reviewed in ref. 2). Ubiquitylation, which is the posttranslational modification of doomed proteins with the small protein ubiquitin, is the major signal that targets proteins for degradation by the 26S proteasome. Because ubiquitylation is an essential regulator of virtually every cellular pathway in eukaryotes, a considerable amount of work has focused on the biochemistry and biology of the 26S proteasome, leading to the identification of many substrates and the reconstitution of robust in vitro degradation (reviewed in refs. 2 and 9).In addition to the 19S RP, there are several cofactors that activate proteasome activity without ATP. These include Blm10/PA200 (10, 11), archaeal PbaB (12), and the 11S activators. The 11S activators are a family of small proteins that form heptameric rings and enhance peptide degradation upon binding to 20S CPs. Simple eukaryotes encode a single 11S activator, PA26 (13), whereas higher eukaryotes encode three 11S activator isoforms: PA28-α, -β, and -γ (14–16). An abundance of work has detailed the biochemical characteristics of the 11S activators, which has been crucial in understanding the structural basis of proteasomal gate opening (17–21). However, their precise biological roles remain controversial. A multitude of studies have demonstrated that PA28-α/β alter the peptide products of proteasomal degradation to influence major histocompatibility complex class I antigen presentation (22, 23). However, PA28-α/β–deficient mice are not remarkably defective in antigen presentation or immunity (24). Similarly, only a handful of PA28-γ–dependent protein substrates have been described, and few studies have demonstrated a significant physiologic role for PA28-γ in mammals (25–29). As a result, our understanding of the biological relevance of eukaryotic ATP-independent activators has lagged behind that of the 19S RP.Although the bulk of research into proteasomes has been in eukaryotes, they are also present in bacteria of the orders Nitrospirales and Actinomycetales, which includes the human pathogen Mycobacterium tuberculosis. M. tuberculosis infects one-third of the world’s population, killing >1.5 million people each year (www.who.int/mediacentre/factsheets/fs104/en/). There is an urgent need for the identification of novel antitubercular drug targets because resistance to currently available drugs is on the rise. One potential target is the prokaryotic ubiquitin-like protein (Pup)-proteasome system (PPS), a pathway used by M. tuberculosis and other proteasome-bearing bacteria to target proteins for degradation. The PPS centers on the small protein modifier Pup (30). The C terminus of Pup is covalently attached to a lysine of a target protein by the Pup ligase proteasome accessory factor A (PafA) (31) and is then bound by the homohexameric AAA ATPase mycobacterial proteasome ATPase (Mpa), which resembles the Rpt ATPases found in the bases of eukaryotic 19S RPs (32–35). Mpa unfolds pupylated proteins to be degraded by the M. tuberculosis 20S CP, which is structurally similar to the eukaryotic 20S CP but is composed of just two unique subunits, PrcA and PrcB (36, 37). Because the PPS is chemically distinct from the eukaryotic ubiquitin–proteasome pathway, and because proteasomal degradation of pupylated proteins is required for full M. tuberculosis virulence (32, 38–40), the PPS represents an attractive therapeutic target for tuberculosis.To date, pupylation is the only pathway known to target proteins for proteasomal degradation in bacteria. However, several studies suggest that the 20S CP may have pupylation-independent functions, because chemical inhibition of the 20S CP or genetic deletion of the 20S CP genes, but not mpa or genes involved in pupylation, produces a severe growth defect in liquid culture and on solid medium (38–41). Furthermore, although mpa and pupylation mutants are severely attenuated for growth in mice, they are able to persist for the natural lifespan of infected animals; in contrast, 20S CP mutants fail to persist in mice (40, 41). It is therefore likely that the M. tuberculosis proteasome has Pup-independent functions.In an effort to identify proteasomal cofactors in M. tuberculosis that might contribute to new degradation pathways, we used a catalytically inactive proteasome trap to stabilize normally transient interactions of the M. tuberculosis 20S CP. The proteasome trap interacted with Rv3780, a previously uncharacterized protein. Rv3780 formed oligomeric rings and bound to M. tuberculosis 20S CPs. This interaction required a conserved C-terminal motif in Rv3780, reminiscent of what has been described for eukaryotic proteasome activators. We show that Rv3780 promoted the robust ATP-independent proteasomal degradation of short peptides, an unfolded protein, and a native M. tuberculosis protein in vitro. Importantly, Rv3780 contributed to M. tuberculosis resistance to heat stress and virulence in a mouse infection model.     

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.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

Differential gradients of interaction affinities drive efficient targeting and recycling in the GET pathway

Efficient and accurate localization of membrane proteins requires a complex cascade of interactions between protein machineries. This requirement is exemplified in the guided entry of tail-anchored (TA) protein (GET) pathway, where the central targeting factor Get3 must sequentially interact with three distinct binding partners to ensure the delivery of TA proteins to the endoplasmic reticulum (ER) membrane. To understand the molecular principles that provide the vectorial driving force of these interactions, we developed quantitative fluorescence assays to monitor Get3–effector interactions at each stage of targeting. We show that nucleotide and substrate generate differential gradients of interaction energies that drive the ordered interaction of Get3 with successive effectors. These data also provide more molecular details on how the targeting complex is captured and disassembled by the ER receptor and reveal a previously unidentified role for Get4/5 in recycling Get3 from the ER membrane at the end of the targeting reaction. These results provide general insights into how complex protein interaction cascades are coupled to energy inputs in biological systems.Membrane proteins comprise ∼30% of the proteome; their efficient and accurate localization is crucial for the structure and function of all cells. Although the well-studied cotranslational signal recognition particle pathway delivers numerous endoplasmic reticulum (ER) -destined proteins (1), many membrane proteins use posttranslational targeting pathways with mechanisms that are far less well understood. A salient example is tail-anchored (TA) proteins, which comprise 3–5% of the eukaryotic membrane proteome and play essential roles in numerous processes, including protein translocation, vesicular trafficking, quality control, and apoptosis (2–5). Because their sole transmembrane domain is at the extreme C terminus, TA proteins cannot engage the cotranslational signal recognition particle machinery and instead, must use posttranslational pathways for localization (6).In the guided entry of TA protein (GET) pathway, TA proteins are initially captured by the yeast cochaperone Sgt2 (or mammalian SGTA) (2, 7). The Get4/5 complex then enables loading of the TA substrate from Sgt2 onto Get3 (or mammalian TRC40), the central targeting factor (7–9). The Get3/TA complex binds a receptor complex on the ER membrane comprised of Get1 and Get2, through which the TA protein is released from Get3 and inserted into the membrane (10–12). Dissociation from Get1/2 is then needed to recycle Get3 for additional rounds of targeting (11–13). Knockout of Get3 (or TRC40) confers stress sensitivity in yeast and embryonic lethality in mammals, underscoring its essential role in the proper functioning of the cell (10, 14, 15).TA protein targeting is driven by the ATPase cycle of Get3, a member of the signal recognition particle, MinD and BioD class of nucleotide hydrolases (8, 16). Crystallographic studies revealed that Get3 is an obligate homodimer, in which the ATPase domains bridge the dimer interface and are connected to helical domains (17, 18). Notably, the conformation of Get3 can be tuned by its nucleotide state, the TA substrate, and its binding partners (11, 12, 17, 19). Apo-Get3 is in an open conformation, in which the helical domains are disconnected (18). ATP biases Get3 to more closed structures, in which the helical domains form a contiguous hydrophobic surface implicated in TA protein binding (17, 18, 20). The Get4/5 complex further locks Get3 into an occluded conformation, in which ATP is tightly bound, but its hydrolysis is delayed, priming Get3 into the optimal state to capture the TA substrate (19, 21). TA proteins induce additional association of Get3 dimers to form a closed tetramer, which stimulates rapid ATP hydrolysis and delays ADP release (19, 22). Finally, Get1 strongly binds apo-Get3 in the open conformation (see below), likely at the end of the targeting reaction (11, 12, 23).The GET pathway demands a sequential cascade of interactions of Get3 with three distinct binding partners: the Get4 subunit in the Get4/5 complex and the Get1 and Get2 subunits in the Get1/2 receptor complex. All three partners share overlapping binding sites on Get3 (Fig. S1) (21), raising intriguing questions as to the mechanisms that ensure the high spatial and temporal accuracy of these protein interactions. For example, Get3 must first interact with Get4/5 in the cytosol to facilitate the loading of TA substrate (7, 9). It is unclear what then drives the release of Get3 from Get4/5 and enables its transit to the ER membrane, where it interacts with the Get1/2 receptor instead.Similarly, how Get3 and the Get3/TA complex transit between different subunits of the Get1/2 receptor at the ER membrane remains unclear. Get1/2 (WRB/CAML in mammals) is necessary and sufficient for TA protein insertion at the ER membrane (12, 13, 24, 25). Crystallographic analyses revealed that Get1 binds strongly to apo- or ADP-bound Get3 in the open conformation (11, 12, 23), whereas Get2 can bind Get3 in semiclosed or closed states (11, 12). In vitro reconstitution experiments showed that high concentrations of Get1 but not Get2 can trigger substrate release from Get3 (12). These observations led to the model that Get2 first captures Get3, whereas Get1 is responsible for disassembling the targeting complex (2, 13). Nevertheless, the subunit that is responsible for capturing the Get3/TA targeting complex has not been experimentally addressed, and whether Get1 or Get2 can discriminate different substrate-bound states of Get3 also has not been addressed.At the end of targeting, Get1 is tightly bound to apo-Get3 (11–13). Experiments with the cytosolic domain (CD) of Get1 show that its interaction with Get3 is strongly antagonized by ATP, leading to the current model that ATP drives the recycling of Get3 from the ER membrane (11, 12). However, two observations raise difficulties with this minimal model. In experiments with intact ER membranes or Get1/2 proteoliposomes (PLs), ATP is insufficient to completely release Get3 from the membrane (12, 13). Furthermore, the tight interaction of Get1 with Get3 raises the possibility that their dissociation is slow (11), which could pose potential barriers for subsequent rounds of TA protein targeting.To address these issues, we developed fluorescence assays to report on the interaction of Get3 with its effectors. Quantitative measurements show that both substrate and nucleotide regulate the interaction of Get3 with Get4/5 and Get1/2, generating differential gradients of interaction energies that drive the ordered transit of Get3 from one binding partner to the next. These results also reveal an active role of ATP in displacing Get3 from Get1, which together with Get4/5, ensures the effective recycling of Get3 back to the cytosol.     

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.     

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.     

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.     

Crystal structure of Mycobacterium tuberculosis ClpP1P2 suggests a model for peptidase activation by AAA+ partner binding and substrate delivery

Caseinolytic peptidase P (ClpP), a double-ring peptidase with 14 subunits, collaborates with ATPases associated with diverse activities (AAA+) partners to execute ATP-dependent protein degradation. Although many ClpP enzymes self-assemble into catalytically active homo-tetradecamers able to cleave small peptides, the Mycobacterium tuberculosis enzyme consists of discrete ClpP1 and ClpP2 heptamers that require a AAA+ partner and protein–substrate delivery or a peptide agonist to stabilize assembly of the active tetradecamer. Here, we show that cyclic acyldepsipeptides (ADEPs) and agonist peptides synergistically activate ClpP1P2 by mimicking AAA+ partners and substrates, respectively, and determine the structure of the activated complex. Our studies establish the basis of heteromeric ClpP1P2 assembly and function, reveal tight coupling between the conformations of each ring, show that ADEPs bind only to one ring but appear to open the axial pores of both rings, provide a foundation for rational drug development, and suggest strategies for studying the roles of individual ClpP1 and ClpP2 rings in Clp-family proteolysis.The self-compartmentalized caseinolytic peptidase P (ClpP) functions in collaboration with the ATPases associated with diverse activities (AAA+) ClpX, ClpA, or ClpC enzymes to carry out ATP-dependent proteolysis in bacteria and eukaryotic organelles (1). The physiological importance of these proteolytic complexes is reflected in their requirement for the viability and/or virulence of some bacteria and the observation that loss-of-function mutations in mammals are linked to developmental defects and disease (2–8). Most well-characterized ClpP enzymes come from organisms that have a single clpP gene and consist of identical heptameric rings, which stack face-to-face to enclose a degradation chamber in which 14 active sites mediate peptide-bond hydrolysis (1, 9, 10). Importantly, the proteolytic chamber is accessible only via narrow axial pores that allow entry of small peptides, greatly slow entry of larger peptides or unfolded proteins, and block access of native proteins (11, 12). Degradation of proteins is mediated by the ClpXP, ClpAP, or ClpCP proteolytic complexes. In these enzymes, the AAA+ partner forms a ring hexamer that binds peptide degrons in target proteins, unfolds native structure if necessary, and translocates the unfolded polypeptide through a central channel and into the lumen of ClpP for degradation (13).When AAA+ partner proteins bind to ClpP, one consequence is opening of the narrow axial pores (12, 14, 15). Binding is mediated in part by tripeptide motifs [typically Ile-Gly-Phe or Leu-Gly-Phe (LGF)] in flexible loops in the AAA+ hexamer, which dock into hydrophobic pockets at subunit interfaces on each ClpP heptamer (16–19). In a remarkable example of protein mimicry by a natural product, cyclic acyldepsipeptide (ADEP) antibiotics bind in the same hydrophobic pockets on ClpP and also open the axial pores, potentially leading to unregulated protein degradation and cell death (14, 15, 20, 21).In contrast to organisms with one ClpP, two or more ClpP isoforms are characteristic of two large bacterial phyla (Actinobacteria and Cyanobacteria) and also occur in individual species from other phyla (22, 23). For example, Mycobacterium tuberculosis, a pathogenic actinobacterium, encodes cotranscribed clpP1 and clpP2 genes (24, 25). The importance of Clp-family proteolysis in M. tuberculosis is highlighted by the facts that the clpP1, clpP2, clpX, and clpC1 genes are all essential and that mechanism-based ClpP inhibitors suppress growth (24, 26–28). Recent studies indicate that M. tuberculosis ClpP1 and ClpP2 form discrete heptameric rings that assemble into an active ClpP1P2 tetradecamer only in the presence of a ClpX or ClpC1 AAA+ partner and one additional factor, either protein substrates being actively translocated into the degradation chamber or N-blocked peptide agonists (23, 29). Because M. tuberculosis resistance to conventional antibacterial drugs is a major health hazard, there is substantial interest in developing drugs that target ClpP1P2. At the outset of this work, however, there was no structure of M. tuberculosis ClpP1P2 or any heteromeric ClpP enzyme to guide design efforts.Here, we show that a catalytically active ClpP1P2 tetradecamer can be stabilized by the combination of a novel ADEP and an agonist peptide, which allowed crystallization and determination of the 3D structure. Together, our structural and biochemical results reveal the basis for ClpP1P2 assembly and activation, establish that the conformations of the ClpP1 and ClpP2 rings are tightly coupled, show that ADEPs bind exclusively to one ring, and suggest strategies for the design of active ClpP1 or ClpP2 tetradecamers for studies of AAA+ partner specificity and biological function.     

Southern East Asian origin and coexpansion of Mycobacterium tuberculosis Beijing family with Han Chinese

The Beijing family is the most successful genotype of Mycobacterium tuberculosis and responsible for more than a quarter of the global tuberculosis epidemic. As the predominant genotype in East Asia, the Beijing family has been emerging in various areas of the world and is often associated with disease outbreaks and antibiotic resistance. Revealing the origin and historical dissemination of this strain family is important for understanding its current global success. Here we characterized the global diversity of this family based on whole-genome sequences of 358 Beijing strains. We show that the Beijing strains endemic in East Asia are genetically diverse, whereas the globally emerging strains mostly belong to a more homogenous subtype known as “modern” Beijing. Phylogeographic and coalescent analyses indicate that the Beijing family most likely emerged around 30,000 y ago in southern East Asia, and accompanied the early colonization by modern humans in this area. By combining the genomic data and genotyping result of 1,793 strains from across China, we found the “modern” Beijing sublineage experienced massive expansions in northern China during the Neolithic era and subsequently spread to other regions following the migration of Han Chinese. Our results support a parallel evolution of the Beijing family and modern humans in East Asia. The dominance of the “modern” Beijing sublineage in East Asia and its recent global emergence are most likely driven by its hypervirulence, which might reflect adaption to increased human population densities linked to the agricultural transition in northern China.Tuberculosis has plagued human beings since ancient times and remains a leading cause of global morbidity and mortality. The causative agent of human tuberculosis is the Mycobacterium tuberculosis complex (MTBC), a group of organisms that harbor little genetic diversity compared with other bacteria (1). MTBC most likely originated in Africa, although its age is being debated (2–4). The human-adapted MTBC is highly clonal and is classified into seven main phylogenetic groups, designated lineage 1 through lineage 7 (2). These seven lineages show strong biogeographic associations that have been proposed to result from codiversification with different human populations (2, 5). Lineage 2 that dominates in East Asia is one of the most successful MTBC variants; more than a quarter of the global tuberculosis epidemic is caused by this lineage (6, 7). Lineage 2 contains strains that mostly belong to the so-called Beijing family (8, 9). This strain family has attracted great attentions due to its global emergence in recent decades (6, 7, 10–12), its tendency to cause disease outbreak (13–17), and its association with antibiotic resistance (12, 18). Experimental and clinical evidences suggest a hypervirulent phenotype of Beijing strains (12, 19), and a higher mutation rate compared with other strains (20).According to genotyping data from previous molecular-epidemiology studies, most Beijing strains from widespread geographic areas showed a remarkable degree of genetic similarity (6, 21), suggesting this strain family might have emerged from recent expansions. It was hypothesized that vaccination with Bacille Calmette Guerin (bacillus Calmette–Guérin) that has been widely implemented in East Asian countries might be the force driving the dominance of this strain family in this area (21). Moreover, the global emergence of the Beijing family may have been due to its hypervirulence and association with drug resistance (7, 18). However, there were discrepant results regarding the relative protective effect of bacillus Calmette–Guérin vaccination against Beijing strains from animal infection experiments (19), and many epidemiological studies failed to find any association between bacillus Calmette–Guérin vaccination and Beijing strains (22–25). The link between drug resistance and the Beijing family has primarily been observed in regions where this family has emerged recently (e.g., Cuba, South Africa, countries of the former Soviet Union) but not in East Asian, where the Beijing family has been endemic for a long time (18, 26). Furthermore, more recent studies indicate that the expansion of the Beijing family may have started long before the introduction of vaccination and antibiotic treatment (2, 3, 27).With the increased availability of genotyping data, the Beijing strains were proved more heterogeneous than initially estimated, and several Beijing sublineages have been identified (28–31). However, a full understanding of the genetic diversity of Beijing family is constrained by the low amount of nucleotide variation (8, 32). Whole-genome sequencing provides an ideal tool to study the genetic diversity of MTBC, and new insights into the origin and evolution of MTBC have been gained (2, 4, 20, 33–35). The genomic diversity of Beijing family was initially studied in a most recent study, in which a general East Asian origin and recent expansions of this strain family were suggested (36). However, the details about the origin and primary dissemination of Beijing family remain unclear. Answering of these questions is important to better understand the virulence of this lineage and its global success. Here, we combined whole-genome sequencing of key strains with detailed single nucleotide polymorphism (SNP) typing of a large collection of clinical MTBC strains isolated from across China. Our results strongly support a southern East Asian origin of the MTBC Beijing family and suggest a parallel evolution of this family with modern humans in East Asia during the last 30,000 y.