Local generation of multineuronal spike sequences in the hippocampal CA1 region

Sequential activity of multineuronal spiking can be observed during theta and high-frequency ripple oscillations in the hippocampal CA1 region and is linked to experience, but the mechanisms underlying such sequences are unknown. We compared multineuronal spiking during theta oscillations, spontaneous ripples, and focal optically induced high-frequency oscillations (“synthetic” ripples) in freely moving mice. Firing rates and rate modulations of individual neurons, and multineuronal sequences of pyramidal cell and interneuron spiking, were correlated during theta oscillations, spontaneous ripples, and synthetic ripples. Interneuron spiking was crucial for sequence consistency. These results suggest that participation of single neurons and their sequential order in population events are not strictly determined by extrinsic inputs but also influenced by local-circuit properties, including synapses between local neurons and single-neuron biophysics.A hypothesized hallmark of cognition is self-organized sequential activation of neuronal assemblies (1). Self-organized neuronal sequences have been observed in several cortical structures (2–5) and neuronal models (6–7). In the hippocampus, sequential activity of place cells (8) may be induced by external landmarks perceived by the animal during spatial navigation (9) and conveyed to CA1 by the upstream CA3 region or layer 3 of the entorhinal cortex (10). Internally generated sequences have been also described in CA1 during theta oscillations in memory tasks (4, 11), raising the possibility that a given neuronal substrate is responsible for generating sequences at multiple time scales. The extensive recurrent excitatory collateral system of the CA3 region has been postulated to be critical in this process (4, 7, 12, 13).The sequential activity of place cells is “replayed” during sharp waves (SPW) in a temporally compressed form compared with rate modulation of place cells (14–20) and may arise from the CA3 recurrent excitatory networks during immobility and slow wave sleep. The SPW-related convergent depolarization of CA1 neurons gives rise to a local, fast oscillatory event in the CA1 region (“ripple,” 140–180 Hz; refs. 8 and 21). Selective elimination of ripples during or after learning impairs memory performance (22–24), suggesting that SPW ripple-related replay assists memory consolidation (12, 13). Although the local origin of the ripple oscillations is well demonstrated (25, 26), it has been tacitly assumed that the ripple-associated, sequentially ordered firing of CA1 neurons is synaptically driven by the upstream CA3 cell assemblies (12, 15), largely because excitatory recurrent collaterals in the CA1 region are sparse (27). However, sequential activity may also emerge by local mechanisms, patterned by the different biophysical properties of CA1 pyramidal cells and their interactions with local interneurons, which discharge at different times during a ripple (28–30). A putative function of the rich variety of interneurons is temporal organization of principal cell spiking (29–32). We tested the “local-circuit” hypothesis by comparing the probability of participation and sequential firing of CA1 neurons during theta oscillations, natural spontaneous ripple events, and “synthetic” ripples induced by local optogenetic activation of pyramidal neurons.     

From the Cover: Why the proportion of transmission during early-stage HIV infection does not predict the long-term impact of treatment on HIV incidence

Antiretroviral therapy (ART) reduces the infectiousness of HIV-infected persons, but only after testing, linkage to care, and successful viral suppression. Thus, a large proportion of HIV transmission during a period of high infectiousness in the first few months after infection (“early transmission”) is perceived as a threat to the impact of HIV “treatment-as-prevention” strategies. We created a mathematical model of a heterosexual HIV epidemic to investigate how the proportion of early transmission affects the impact of ART on reducing HIV incidence. The model includes stages of HIV infection, flexible sexual mixing, and changes in risk behavior over the epidemic. The model was calibrated to HIV prevalence data from South Africa using a Bayesian framework. Immediately after ART was introduced, more early transmission was associated with a smaller reduction in HIV incidence rate—consistent with the concern that a large amount of early transmission reduces the impact of treatment on incidence. However, the proportion of early transmission was not strongly related to the long-term reduction in incidence. This was because more early transmission resulted in a shorter generation time, in which case lower values for the basic reproductive number (R₀) are consistent with observed epidemic growth, and R₀ was negatively correlated with long-term intervention impact. The fraction of early transmission depends on biological factors, behavioral patterns, and epidemic stage and alone does not predict long-term intervention impacts. However, early transmission may be an important determinant in the outcome of short-term trials and evaluation of programs.Recent studies have confirmed that effective antiretroviral therapy (ART) reduces the transmission of HIV among stable heterosexual couples (1–3). This finding has generated interest in understanding the population-level impact of HIV treatment on reducing the rate of new HIV infections in generalized epidemic settings (4). Research, including mathematical modeling (5–10), implementation research (11), and major randomized controlled trials (12–14), are focused on how ART provision might be expanded strategically to maximize its public health benefits (15, 16).One concern is that if a large fraction of HIV transmission occurs shortly after a person becomes infected, before the person can be diagnosed and initiated on ART, this will limit the potential impact of HIV treatment on reducing HIV incidence (9, 17, 18). Data suggest that persons are more infectious during a short period of “early infection” after becoming infected with HIV (19–22), although there is debate about the extent, duration, and determinants of elevated infectiousness (18, 23). The amount of transmission that occurs also will depend on patterns of sexual behavior and sexual networks (17, 24–27). There have been estimates for the contribution of early infection to transmission from mathematical models (7, 17, 21, 24–26) and phylogenetic analyses (28–31), but these vary widely, from 5% to above 50% (23).In this study, we use a mathematical model to quantify how the proportion of transmission that comes from persons who have been infected recently affects the impact of treatment scale-up on HIV incidence. The model is calibrated to longitudinal HIV prevalence data from South Africa using a Bayesian framework. Thus, the model accounts for not only the early epidemic growth rate highlighted in previous research (5, 9, 18), but also the heterogeneity and sexual behavior change to explain the peak and decline in HIV incidence observed in sub-Saharan African HIV epidemics (32, 33).The model calibration allows uncertainty about factors that determine the amount of early transmission, including the relative infectiousness during early infection, heterogeneity in propensity for sexual risk behavior, assortativity in sexual partner selection, reduction in risk propensity over the life course, and population-wide reductions in risk behavior in response to the epidemic (32, 33). This results in multiple combinations of parameter values that are consistent with the observed epidemic and variation in the amount of early transmission. We simulated the impact of a treatment intervention and report how the proportion of early transmission correlates with the reduction in HIV incidence from the intervention over the short- and long-term.     

Hippocampal molecular mechanisms involved in the enhancement of fear extinction caused by exposure to novelty

Exposure to a novel environment enhances the extinction of contextual fear. This has been explained by tagging of the hippocampal synapses used in extinction, followed by capture of proteins from the synapses that process novelty. The effect is blocked by the inhibition of hippocampal protein synthesis following the novelty or the extinction. Here, we show that it can also be blocked by the postextinction or postnovelty intrahippocampal infusion of the NMDA receptor antagonist 2-amino-5-phosphono pentanoic acid; the inhibitor of calcium/calmodulin-dependent protein kinase II (CaMKII), autocamtide-2–related inhibitory peptide; or the blocker of L-voltage–dependent calcium channels (L-VDCCs), nifedipine. Inhibition of proteasomal protein degradation by β-lactacystin has no effect of its own on extinction or on the influence of novelty thereon but blocks the inhibitory effects of all the other substances except that of rapamycin on extinction, suggesting that their action depends on concomitant synaptic protein turnover. Thus, the tagging-and-capture mechanism through which novelty enhances fear extinction involves more molecular processes than hitherto thought: NMDA receptors, L-VDCCs, CaMKII, and synaptic protein turnover.Frey and Morris (1, 2) and their collaborators (3–7) proposed a mechanism whereby relatively “weak” hippocampal long-term potentiation (LTP) or long-term depression (LTD) lasting only a few minutes can nevertheless “tag” the synapses involved with proteins synthesized ad hoc, so that other plasticity-related proteins (PRPs) produced at other sets of synapses by other LTPs or LTDs can be captured by the tagged synapses and strengthen their activity to “long” LTPs or LTDs lasting hours or days (8). LTDs and LTPs can “cross”-tag each other; that is, LTDs can enhance both LTDs and LTPs, and vice versa (6, 8). Because many learned behaviors rely on hippocampal LTP or LTD (7–9), among them the processing of novelty (9, 10) and the making of extinction (11–13), interactions between consecutive learnings can also be explained by the “tagging-and-capture” hypothesis (9, 10, 13), whose application to behavior became known as “behavioral tagging and capture” (5, 7, 9, 13). Typically, exposure to a novel environment [e.g., a nonanxiogenic 50 × 50 × 40-cm open field (OF) (5, 7, 9, 10, 14)] is interpolated before testing for another task, which becomes enhanced (4–10, 13). The usual reaction to novelty is orienting and exploration (14), followed by habituation of this response (14–16). Habituation is perhaps the simplest form of learning, and it consists of inhibition of the orienting/exploratory response (14, 16).We recently showed that the brief exposure of rats to a novel environment (the OF) within a limited time window enhances the extinction of contextual fear conditioning (CFC) through a mechanism of synaptic tagging and capture (13), which is a previously unidentified example of behavioral tagging of inhibitory learning. Fear extinction is most probably due to LTD in the hippocampus (11, 12), although the possibility that it may also involve LTP is not discarded (13). The enhancement of extinction by novelty probably relies on the habituation to the novel environment, which is also probably due to LTD (15, 16). The enhancement of extinction by the exposure to novelty depends on hippocampal gene expression and ribosomal protein synthesis following extinction training and on both ribosomal and nonribosomal protein synthesis caused by the novel experience (13). Nonribosomal protein synthesis that can be blocked by rapamycin is believed to be dendritic (13, 17), so it would be strategically located for tagging-and-capture processes, but it has not been studied in synaptic tagging to date (3–8) or in other forms of behavioral tagging (7–10). As occurs with the interactions between LTPs and/or LTDs (4), the enhancement of extinction by novelty relies on hippocampal but not amygdalar processes (13).Recent findings indicate that several hippocampal processes related to learning and memory, such as the reconsolidation of spatial learning, are highly dependent on NMDA glutamate receptors, calcium/calmodulin protein kinase II (CaMKII), and long-term voltage channel blockers (L-VDCCs), which, in turn, rely on the proteasomal degradation of proteins (18). Here, we study the effects of an NMDA blocker, 2-amino-5-phosphono pentanoic acid (AP5); the L-VDCC blocker nifedipine (Nife); a CaMKII inhibitor, the autocamtide-2–related inhibitory peptide (AIP); and the irreversible proteasome blocker β-lactacystin (12, 13) on the interaction between novelty and extinction (11). As will be seen, we found that both the setting up of tags by extinction and the presumable production of PRPs by the processing of novelty are dependent on NMDA receptors, CaMKII, and L-VDCCs. This endorses and expands the hypothesis that the novelty–extinction interaction relies on synaptic tagging and capture (13).     

Kinesin processivity is gated by phosphate release

Kinesin-1 is a dimeric motor protein, central to intracellular transport, that steps hand-over-hand toward the microtubule (MT) plus-end, hydrolyzing one ATP molecule per step. Its remarkable processivity is critical for ferrying cargo within the cell: over 100 successive steps are taken, on average, before dissociation from the MT. Despite considerable work, it is not understood which features coordinate, or “gate,” the mechanochemical cycles of the two motor heads. Here, we show that kinesin dissociation occurs subsequent to, or concomitant with, phosphate (P_i) release following ATP hydrolysis. In optical trapping experiments, we found that increasing the steady-state population of the posthydrolysis ADP·P_i state (by adding free P_i) nearly doubled the kinesin run length, whereas reducing either the ATP binding rate or hydrolysis rate had no effect. The data suggest that, during processive movement, tethered-head binding occurs subsequent to hydrolysis, rather than immediately after ATP binding, as commonly suggested. The structural change driving motility, thought to be neck linker docking, is therefore completed only upon hydrolysis, and not ATP binding. Our results offer additional insights into gating mechanisms and suggest revisions to prevailing models of the kinesin reaction cycle.Since its discovery nearly 30 years ago (1), kinesin-1—the founding member of the kinesin protein superfamily—has emerged as an important model system for studying biological motors (2, 3). During “hand-over-hand” stepping, kinesin dimers alternate between a two–heads-bound (2-HB) state, with both heads attached to the microtubule (MT), and a one–head-bound (1-HB) state, where a single head, termed the tethered head, remains free of the MT (4, 5). The catalytic cycles of the two heads are maintained out of phase by a series of gating mechanisms, thereby enabling the dimer to complete, on average, over 100 steps before dissociating from the MT (6–8). A key structural element for this coordination is the neck linker (NL), a ∼14-aa segment that connects each catalytic head to a common stalk (9). In the 1-HB state, nucleotide binding is thought to induce a structural reconfiguration of the NL, immobilizing it against the MT-bound catalytic domain (2, 3, 10–17). This transition, called “NL docking,” is believed to promote unidirectional motility by biasing the position of the tethered head toward the next MT binding site (2, 3, 10–17). The completion of an 8.2-nm step (18) entails the binding of this tethered head to the MT, ATP hydrolysis, and detachment of the trailing head, thereby returning the motor to the ATP-waiting state (2, 3, 10–17). Prevailing models of the kinesin mechanochemical cycle (2, 3, 10, 14, 15, 17), which invoke NL docking upon ATP binding, explain the highly directional nature of kinesin motility and offer a compelling outline of the sequence of events following ATP binding. Nevertheless, these abstractions do not speak directly to the branching transitions that determine whether kinesin dissociates from the MT (off-pathway) or continues its processive reaction cycle (on-pathway). The distance moved by an individual motor before dissociating—the run length—is limited by unbinding from the MT. The propensity for a dimer to unbind involves a competition among multiple, force-dependent transitions in the two heads, which are not readily characterized by traditional structural or bulk biochemical approaches. Here, we implemented high-resolution single-molecule optical trapping techniques to determine transitions in the kinesin cycle that govern processivity.     

Brain system for mental orientation in space,time, and person

Orientation is a fundamental mental function that processes the relations between the behaving self to space (places), time (events), and person (people). Behavioral and neuroimaging studies have hinted at interrelations between processing of these three domains. To unravel the neurocognitive basis of orientation, we used high-resolution 7T functional MRI as 16 subjects compared their subjective distance to different places, events, or people. Analysis at the individual-subject level revealed cortical activation related to orientation in space, time, and person in a precisely localized set of structures in the precuneus, inferior parietal, and medial frontal cortex. Comparison of orientation domains revealed a consistent order of cortical activity inside the precuneus and inferior parietal lobes, with space orientation activating posterior regions, followed anteriorly by person and then time. Core regions at the precuneus and inferior parietal lobe were activated for multiple orientation domains, suggesting also common processing for orientation across domains. The medial prefrontal cortex showed a posterior activation for time and anterior for person. Finally, the default-mode network, identified in a separate resting-state scan, was active for all orientation domains and overlapped mostly with person-orientation regions. These findings suggest that mental orientation in space, time, and person is managed by a specific brain system with a highly ordered internal organization, closely related to the default-mode network.Orientation in space, time, and person is a fundamental cognitive function and the bedrock of neurological and psychiatric mental status examination (1, 2). Orientation is defined as the “tuning between the subject and the internal representation he forms of the corresponding public reference system”: that is, the external world (1). Although the representation of the external world by means of a cognitive map has been widely investigated (3–5), the way in which the self refers to this map has yet to be understood. Moreover, the behaving self refers not only to spatial landmarks but also to remembered or imagined events, or to people around, yielding a “cognitive mapping” of the time and person domains of the mental world (2, 6–9). However, it is still unknown whether mental orientation in space, time, and person relies on similar or distinct neurocognitive systems.Several lines of research support the idea that similar neurocognitive systems underlie orientation in these three domains. Behavioral studies indicate a common psychological metric for proximity estimations (“cognitive distance”) in space, time, and person (7); for example, manipulation of stimuli’s distance in one orientation domain affects the perceived distance in the other two domains (10, 11). Accordingly, a recent neuroimaging study mapped cognitive distance estimations in the three domains to a single region in the inferior parietal lobe (IPL) (12). However, other neuroimaging studies that investigated processing of places, events, and people separately have found activation in brain regions besides the IPL, including the precuneus and posterior cingulate cortices, medial prefrontal cortex (mPFC), and lateral frontal and temporal lobes (6, 13–29). Notably, these regions constitute a part of the default-mode network (DMN), a system involved in self-referential processes (24, 30–35). These findings suggest a common brain system for orientation across domains, possibly related to the DMN.Clinical observations in patients with disorientation in space, time, and person are less clear: on the one hand, clinical syndromes may involve disorientation in several domains simultaneously, and disorientation disorders in the three domains involve lesions in similar brain regions, usually overlapping with the DMN (1, 2). On the other hand, disorientation may be limited to one specific domain – space, time, or person (2, 36–40). In addition, patients with traumatic brain injury or after electro-convulsive therapy regain their orientation gradually, from personal to spatial and temporal orientation (41, 42) whereas patients with Alzheimer’s disease typically lose orientation in time first, then in place, and then in person, suggesting that partially separate systems underlie orientation in each domain.Here, we investigated the neurocognitive system underlying orientation in space, time, and person and its relation to the DMN. To this aim, we used a mental-orientation task, with individually tailored stimuli in the space (places), time (events), and person (people) domains. To gain high anatomical specificity, we used high-resolution 7-Tesla functional MRI (fMRI). To capitalize on the spatial acuity of the high-resolution fMRI, we applied a strategy of analyzing each subject individually in native space and combined the results to compare activations for the three domains. Finally, we compared our results to the DMN as identified in each individual subject by analysis of resting-state fMRI. We hypothesized that orientation across different domains relies on a shared “core” brain system, in close relation to the DMN, yet orientation in specific domains may involve additional specialized subsystems.     

Site-specific cation release drives actin filament severing by vertebrate cofilin

Actin polymerization powers the directed motility of eukaryotic cells. Sustained motility requires rapid filament turnover and subunit recycling. The essential regulatory protein cofilin accelerates network remodeling by severing actin filaments and increasing the concentration of ends available for elongation and subunit exchange. Although cofilin effects on actin filament assembly dynamics have been extensively studied, the molecular mechanism of cofilin-induced filament severing is not understood. Here we demonstrate that actin filament severing by vertebrate cofilin is driven by the linked dissociation of a single cation that controls filament structure and mechanical properties. Vertebrate cofilin only weakly severs Saccharomyces cerevisiae actin filaments lacking this “stiffness cation” unless a stiffness cation-binding site is engineered into the actin molecule. Moreover, vertebrate cofilin rescues the viability of a S. cerevisiae cofilin deletion mutant only when the stiffness cation site is simultaneously introduced into actin, demonstrating that filament severing is the essential function of cofilin in cells. This work reveals that site-specific interactions with cations serve a key regulatory function in actin filament fragmentation and dynamics.Actin polymerization powers the directed motility of eukaryotic cells and some pathogenic bacteria (1–3). Actin assembly also plays critical roles in endocytosis, cytokinesis, and establishment of cell polarity. Sustained motility requires filament disassembly and subunit recycling. The essential regulatory protein cofilin severs actin filaments (4–6), which accelerates actin network reorganization by increasing the concentration of filament ends available for subunit exchange (7).Cofilin binding alters the structure and mechanical properties of filaments, which effectively introduces local “defects” that compromise filament integrity and promote severing (5). Filaments with bound cofilin have altered twist (8, 9) and are more compliant in both bending and twisting than bare filaments (10–13). It has been suggested that deformations in filament shape promote fragmentation at or near regions of topological and mechanical discontinuities, such as boundaries between bare and cofilin-decorated segments along partially decorated filaments (5, 12, 14–18).Cations modulate actin filament structure and mechanical properties (19) and cofilin dissociates filament-associated cations (20), leading us to hypothesize that cation-binding interactions regulate filament severing by cofilin. Cations bind filaments at two discrete and specific sites positioned between adjacent subunits along the long-pitch helix of the filament (19, 21). These cation binding sites are referred to as “polymerization” and “stiffness” sites based on their roles in filament assembly and mechanics, respectively. These discrete sites bind both monovalent and divalent cations with a range of affinities (low millimolar for divalent and tens of millimolar for monovalent cations) (19, 21) but are predominantly occupied by Mg²⁺ and K⁺ under physiological conditions. Here we demonstrate that cation release from the stiffness site plays a central role in filament severing by vertebrate cofilin, both in vitro and in cells.     

Degenerate target sites mediate rapid primed CRISPR adaptation

Prokaryotes encode adaptive immune systems, called CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR associated), to provide resistance against mobile invaders, such as viruses and plasmids. Host immunity is based on incorporation of invader DNA sequences in a memory locus (CRISPR), the formation of guide RNAs from this locus, and the degradation of cognate invader DNA (protospacer). Invaders can escape type I-E CRISPR-Cas immunity in Escherichia coli K12 by making point mutations in the seed region of the protospacer or its adjacent motif (PAM), but hosts quickly restore immunity by integrating new spacers in a positive-feedback process termed “priming.” Here, by using a randomized protospacer and PAM library and high-throughput plasmid loss assays, we provide a systematic analysis of the constraints of both direct interference and subsequent priming in E. coli. We have defined a high-resolution genetic map of direct interference by Cascade and Cas3, which includes five positions of the protospacer at 6-nt intervals that readily tolerate mutations. Importantly, we show that priming is an extremely robust process capable of using degenerate target regions, with up to 13 mutations throughout the PAM and protospacer region. Priming is influenced by the number of mismatches, their position, and is nucleotide dependent. Our findings imply that even outdated spacers containing many mismatches can induce a rapid primed CRISPR response against diversified or related invaders, giving microbes an advantage in the coevolutionary arms race with their invaders.Bacteria and Archaea are regularly exposed to bacteriophages and other mobile genetic elements, such as plasmids. To control the competing effects of horizontal gene transfer, a spectrum of resistance strategies have evolved in prokaryotes (1). One of the most widespread and well-characterized are the CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated) systems, which provide bacterial “adaptive immunity” (1–8). Simply, CRISPR-Cas functions in three major steps. First, in a process termed “adaptation,” short sequences are derived from the invading element and incorporated into a CRISPR array (9). CRISPR arrays are composed of short repeats that are separated by the foreign-derived sequences, termed “spacers.” Second, CRISPRs are transcribed into a pre-CRISPR RNA (pre-crRNA), which is then processed into short crRNAs, which encompass portions of the repeats and most—or all—of the spacer. Finally, as part of a Cas ribonucleoprotein complex, the crRNAs guide a sequence-specific targeting of complementary nucleic acids (for recent reviews, see refs. 1–7).CRISPR-Cas systems are divided into three major types (I–III) and further categorized into subtypes (e.g., I-A to I-F) (10). The mechanisms of both crRNA generation and interference differ between the types and there are even significant differences between closely related subtypes. However, Cas1 and Cas2 are the only two Cas proteins completely conserved across all CRISPR-Cas systems and they are crucial for adaptation in Escherichia coli (10–12). The acquisition of new spacers is the most poorly understood stage in CRISPR-Cas immunity, mainly hindered by the paucity of robust laboratory assays to monitor this process (reviewed in ref. 9). Streptococcus thermophilus is highly proficient at spacer acquisition and provided much of the early insight into adaptation, showing that new spacers are typically acquired at one end of the CRISPR array from either phages (13–15) or plasmids (16). Recently, spacer acquisition has been detected in a variety of other systems (11, 12, 17–20). Adjacent to the expanding end of the array is the leader region, which harbors the promoter for pre-crRNA expression and sequences important for spacer acquisition (12, 21). Recent studies in E. coli in the type I-E system have shown that spacer acquisition can occur from phages and plasmids either when the Cas1 and Cas2 proteins are overexpressed or if the native cas genes are up-regulated, because of deletion of hns (11, 12, 20–22). The DNA targets (termed “protospacers”) of newly acquired spacers are consistently flanked by protospacer-adjacent motifs (PAMs), with the E. coli type I-E consensus 5′-protospacer-CTT-3′. PAMs were originally identified computationally (23) and were shown to play a role in interference in an early study (14). The importance of PAMs in the recognition and selection of precursor-spacers (prespacers) during adaptation was demonstrated unequivocally using assays that were independent of interference (12, 21). The simple overexpression of Cas1 and Cas2, in the absence of other cas genes, demonstrated these are the only Cas proteins essential for adaptation and are likely to recognize PAMs (12).Adaptation consists of two related stages, termed “naïve” and “primed” (9). Naïve adaptation occurs when a bacterium harboring a CRISPR-Cas system is infected by a new foreign element that it has not previously encountered. Although the acquisition of a new spacer can result in effective protection from the element, point mutations within the protospacer or PAM allow the element to escape CRISPR-Cas targeting (14, 24, 25). This aspect had been viewed as a weakness of CRISPR-Cas interference, but recent studies show that a positive feedback loop—called priming—occurs, which enables one or more new spacers to be acquired (11, 20, 22). Specifically, single mutations within either the PAM or the seed region of the protospacer, although inactive for interference, promote the rapid acquisition of new spacers from the same target (11). Priming is proposed to allow an effective response against viral or plasmid escapees through the incorporation of new spacers. Unlike naïve adaptation, priming is more complex, and in type I-E systems requires Cas1, Cas2, crRNA, the targeting complex termed Cascade [CRISPR-associated complex for antiviral defence, composed of Cse1, Cse2, Cas7, Cas5, and Cas6e (26–28)] and the Cas3 nuclease/helicase (11). Interestingly, the vast majority of spacers acquired through priming are derived from the same DNA strand as the original priming spacer (11, 20, 22). In addition, priming in E. coli was abolished by two mutations in the protospacer and PAM regions (11).In this study, we generated a mutagenic variant library of a protospacer and PAM region and used both individual high-throughput plasmid-loss assays and next-generation sequencing to determine the limits of both direct interference and indirect interference through priming. Our results demonstrate that direct interference tolerates mutations mostly at very specific positions in the protospacer, whereas priming tolerates extensive mutation of the PAM and protospacer regions. The results have wide evolutionary consequences for primed acquisition and could explain the retention of multiple “older” spacers in CRISPR arrays.     

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

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

The structural kinetics of switch-1 and the neck linker explain the functions of kinesin-1 and Eg5

Kinesins perform mechanical work to power a variety of cellular functions, from mitosis to organelle transport. Distinct functions shape distinct enzymologies, and this is illustrated by comparing kinesin-1, a highly processive transport motor that can work alone, to Eg5, a minimally processive mitotic motor that works in large ensembles. Although crystallographic models for both motors reveal similar structures for the domains involved in mechanochemical transduction—including switch-1 and the neck linker—how movement of these two domains is coordinated through the ATPase cycle remains unknown. We have addressed this issue by using a novel combination of transient kinetics and time-resolved fluorescence, which we refer to as “structural kinetics,” to map the timing of structural changes in the switch-1 loop and neck linker. We find that differences between the structural kinetics of Eg5 and kinesin-1 yield insights into how these two motors adapt their enzymologies for their distinct functions.There are more than 42 kinesin genes in the human genome, representing 14 distinct classes (1). All are members of the P-loop NTPase superfamily of nucleotide triphosphate hydrolases (2–4). Like other NTPases, kinesins share a conserved Walker motif nucleotide-binding fold (2, 4) that consists of a central twisted β-sheet and three nucleotide-binding loops, which are termed switch-1, switch-2, and the P-loop. Kinesins also share a common microtubule (MT) binding interface, which isomerizes between states that either bind MTs weakly or strongly, and a mechanical element, termed the neck linker (NL). The NL has been proposed to isomerize between two conformations: one that is flexible and termed undocked, and the other that is ordered and termed docked, where it interacts with a cleft in the motor domain formed by the twisted β-sheet and is oriented along the MT axis (5–7). NL isomerization (5, 8) is hypothesized to be the force-generating transition in kinesin motors (6, 7, 9–11), and its position has also been proposed to coordinate the ATPase cycles of processive kinesin dimers by regulating nucleotide binding and hydrolysis (11).Spectroscopic and structural studies have led to a model to explain how kinesins generate force (5–7, 9, 10, 12–15) (summarized in SI Appendix, Fig. S1), which proposes that the conformations of the nucleotide binding site, the MT-binding interface, and the NL are all determined by the state of the catalytic site. It predicts that when unbound to the MT, the motor contains ADP in its catalytic site and its NL is undocked. MT binding accelerates ADP dissociation, thereby allowing ATP to bind, the NL to dock, and mechanical work to be performed. ATP hydrolysis and phosphate release are then followed by dissociation from the MT to complete the cycle (5, 7–10, 14). This model also argues that: (i) NL docking of the MT-attached motor domain moves the tethered, trailing head into a forward position, where it undergoes a biased diffusional search to attach to the next MT-binding site (11, 14); (ii) switch-1, which coordinates the γ-phosphate of ATP, alternates between two conformations, referred to as “open” and “closed,” and the NL alternates between docked and undocked (5, 6, 10, 13–15); and (iii) coordination between the conformations of switch-1 and the NL regulates the timing of the ATPase cycles of the two motor domains in processive kinesin dimers (11). However, the model fails to explain several features of kinesins. For example, it predicts that ATP does not bind to kinesin when the NL is docked. This prediction is inconsistent with studies of both Eg5 and kinesin-1, which suggest ATP binds more readily when the NL is docked (11, 16, 17). The model also predicts that the NL should be docked after ATP binding. However, electron paramagnetic resonance (EPR) probes attached to the NL show a significant population of both mobile and immobile NL states in the presence of both pre- and posthydrolytic ATP analogs (5). Furthermore, the model cannot explain the load dependence of stall, detachment, and back stepping, all of which require a branched pathway (11).To resolve these uncertainties, we have measured the kinetics of the structural changes that occur in switch-1 and the NL with nucleotide binding while the motor is bound to the MT in an experimental design that we refer to as “structural kinetics.” We carried out these experiments using an novel spectroscopic approach, termed transient time-resolved fluorescence resonance energy transfer, (TR)²FRET, that allows us to monitor the kinetics and thermodynamics of both the undocked/docked transition in the NL and the open/closed transition in switch-1 that accompany the process of nucleotide binding. These experiments explain differences in the enzymologies of kinesin-1 and Eg5 and suggest an interesting role for the L5 loop in controlling the timing of conformational changes in the Eg5 switch-1 and NL.     

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.     

Dynamic ubiquitination drives herpesvirus neuroinvasion

Neuroinvasive herpesviruses display a remarkable propensity to enter the nervous system of healthy individuals in the absence of obvious trauma at the site of inoculation. We document a repurposing of cellular ubiquitin during infection to switch the virus between two invasive states. The states act sequentially to defeat consecutive host barriers of the peripheral nervous system and together promote the potent neuroinvasive phenotype. The first state directs virus access to nerve endings in peripheral tissue, whereas the second delivers virus particles within nerve fibers to the neural ganglia. Mutant viruses locked in either state remain competent to overcome the corresponding barrier but fail to invade the nervous system. The herpesvirus “ubiquitin switch” may explain the unusual ability of these viruses to routinely enter the nervous system and, as a consequence, their prevalence in human and veterinary hosts.Herpesviruses such as herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) routinely enter the peripheral nervous system following casual contact between infected and uninfected individuals. Recent findings have provided insights into this complex phenotype, but the molecular mechanisms directing nervous system invasion are poorly understood (1–5). A critical effector of this process is a conserved herpesvirus deubiquitinase (DUB) (a cysteine protease that removes ubiquitin from target posttranslationally modified proteins) (6–8). The DUB is housed in the amino terminus of the pUL36 protein (protein encoded by the 36th open reading frame in the unique-long segment of the viral genome), amounting to less than 10% of the total mass of this large (>250 kDa) structural component of the virion (9). pUL36 is attached directly to the icosahedral capsid shell of these enveloped viruses (10–12) and, due to its location between the capsid and envelope, is referred to as a herpesvirus tegument protein. Upon virus entry into a cell, pUL36 becomes exposed to the cytosol (4, 13, 14) and directs delivery of the capsid and its DNA content to the nucleus (2, 15–19). Following replication, pUL36 is also essential for viral assembly and egress (20–22).The presence of the pUL36 DUB on the exposed surface of the intracellular viral particle suggests that removal of ubiquitin from cellular cytosolic proteins, or from the virus particle itself, is instrumental for these viruses to transmit between peripheral tissues and the neurons that innervate them (8). This necessarily implicates the prior addition of ubiquitin as a factor in disease progression, either as part of the virus neuroinvasive program or as a cellular defense mechanism that is overcome by the DUB. Although several substrates of the DUB have been identified, how this enzyme promotes the potent neuroinvasive property of these viruses is unknown (23–30). To elucidate the role of the DUB, we set out to examine the role of ubiquitination during neuroinvasive herpesvirus infection. We report that ubiquitin addition and removal from a conserved lysine residue in pUL36 switches PRV between two invasive states that act successively to promote virus dissemination into the peripheral nervous system. Supporting evidence that the “ubiquitin switch” is conserved in HSV-1, and possibly all neuroinvasive alphaherpesviruses, is provided.     

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.     

Structural basis for the sheddase function of human meprin β metalloproteinase at the plasma membrane

Ectodomain shedding at the cell surface is a major mechanism to regulate the extracellular and circulatory concentration or the activities of signaling proteins at the plasma membrane. Human meprin β is a 145-kDa disulfide-linked homodimeric multidomain type-I membrane metallopeptidase that sheds membrane-bound cytokines and growth factors, thereby contributing to inflammatory diseases, angiogenesis, and tumor progression. In addition, it cleaves amyloid precursor protein (APP) at the β-secretase site, giving rise to amyloidogenic peptides. We have solved the X-ray crystal structure of a major fragment of the meprin β ectoprotein, the first of a multidomain oligomeric transmembrane sheddase, and of its zymogen. The meprin β dimer displays a compact shape, whose catalytic domain undergoes major rearrangement upon activation, and reveals an exosite and a sugar-rich channel, both of which possibly engage in substrate binding. A plausible structure-derived working mechanism suggests that substrates such as APP are shed close to the plasma membrane surface following an “N-like” chain trace.Physiological processes in the extracellular milieu and the circulation require finely tuned concentrations of signal molecules such as cytokines, growth factors, receptors, adhesion molecules, and peptidases. Many of these proteins are synthesized as type-I membrane protein variants or precursors consisting of a glycosylated N-terminal ectoprotein, a transmembrane helix, and a C-terminal cytosolic tail. Their localization at the cell surface restricts their field of action to autocrine or juxtacrine processes. However, to act at a distance in paracrine, synaptic, or endocrine events, they have to be released from the plasma membrane into the extracellular space as soluble factors through “protein ectodomain shedding” (1, 2). This entails limited proteolysis and is a major posttranslational regulation mechanism that affects 2–4% of the proteins on the cell surface, occurs at or near the plasma membrane (3), and apparently follows a common release mechanism (2). It may also proteolytically inactivate proteins to terminate their function on the cell surface (4). Peptidases engaged in such processing are “sheddases” and the most studied transmembrane sheddases are members of the adamalysin/ADAMs (4, 5) and matrix-metalloproteinase (MMP) (6) families within the metzincin clan of metallopeptidases (MPs) (7–9). These include ADAM-8, -9, -10, -12, -15, -17, -19, -28, and -33 (1, 4) and membrane-type 1 (MT1)-MMP, MT3-MMP, and MT5-MMP (2, 6, 10). Other confirmed transmembrane sheddases are the aspartic proteinases BACE-1 and -2 (ref. 11 and references therein) and the malarian parasite serine proteinases, PfSUB2, PfROM1, and PfROM4 (12). Distinct sheddases may participate in intercalating processes, with disparate physiological consequences: ADAM-9, -10 (α-secretases), and -17 contribute to the nonamyloidogenic pathway of human amyloid precursor protein (APP) processing, whereas BACE-1 (β-secretase) participates in the amyloidogenic pathway. Whereas the former generates innocuous peptides, the latter gives rise to the toxic β-amyloid peptides believed to be responsible for Alzheimer’s disease (11). In several instances, shedding at the membrane surface is followed by a “regulated intramembrane proteolysis” step within the membrane (1). This is the case for the processing of Notch ligand Delta1 and of APP, both carried out by γ-secretase after action of an α/β-secretase (11), and for signal-peptide peptidase, which removes remnants of the secretory protein translocation from the endoplasmic membrane (13).Recently, human meprin β (Mβ) was found to specifically process APP in vivo, which may contribute to Alzheimer’s disease (14, 15). It was also reported to activate cell-anchored α-secretase ADAM-10 and to be widely expressed in brain, intestine, kidney, and skin (14, 16–18). Disruption of Mβ in mice affects embryonic viability, birth weight, and renal gene expression profiles (19). The enzyme was further identified as a sheddase or proteolytic regulator at the plasma membrane of interleukin-1β (20), interleukin-18 (21), tumor growth factor α (22), procollagen III (23), epithelial sodium channel (24), E-cadherin (25), tenascin-C (26), and vascular endothelial growth factor A (27). Further substrates include fibroblast growth factor 19 and connective tissue growth factor. Altered expression and activity of the enzyme are associated with pathological conditions such as inflammatory bowel disease (28), tumor progression (29), nephritis (30), and fibrosis (23).Mβ is a 679-residue secreted multidomain type-I membrane MP that belongs to the astacin family within the metzincins (7, 9, 16, 31, 32). The enzyme is glycosylated and assembles into either disulfide-linked homodimers or heterodimers with the closely related meprin α-subunit (33). Mβ homodimers are essentially membrane bound but may also be shed from the surface by ADAM-10 and -17 (34, 35). To assess function, working mechanism, and activation of Mβ, we analyzed the structure of the major ectoprotein of mature Mβ (MβΔC) and of its zymogen, promeprin β (pMβΔC). With regard to transmembrane sheddases, to date only the structures of the isolated monomeric catalytic domains of ADAM-17 [Protein Data Bank (PDB) access code 1BKC], ADAM-33 (PDB 1R55), MT1-MMP (PDB 1BUV), MT3-MMP (PDB 1RM8), BACE-1 (PDB 1FKN), and BACE-2 (PDB 2EWY) have been described. Accordingly, this is a unique structural report of a multidomain oligomeric transmembrane sheddase. This report has allowed us a better understanding of the structural basis for latency and activation of this MP and to derive a plausible working mechanism for shedding of glycosylated type-I membrane substrates such as APP at the extracellular membrane surface.