The serine/threonine protein phosphatase 1 (PP1) dephosphorylates hundreds of key biological targets by associating with nearly 200 regulatory proteins to form highly specific holoenzymes. However, how these proteins direct PP1 specificity and the ability to predict how these PP1 interacting proteins bind PP1 from sequence alone is still missing. PP1 nuclear targeting subunit (PNUTS) is a PP1 targeting protein that, with PP1, plays a central role in the nucleus, where it regulates chromatin decondensation, RNA processing, and the phosphorylation state of fundamental cell cycle proteins, including the retinoblastoma protein (Rb), p53, and MDM2. The molecular function of PNUTS in these processes is completely unknown. Here, we show that PNUTS, which is intrinsically disordered in its free form, interacts strongly with PP1 in a highly extended manner. Unexpectedly, PNUTS blocks one of PP1's substrate binding grooves while leaving the active site accessible. This interaction site, which we have named the arginine site, allowed us to define unique PP1 binding motifs, which advances our ability to predict how more than a quarter of the known PP1 regulators bind PP1. Additionally, the structure shows how PNUTS inhibits the PP1-mediated dephosphorylation of critical substrates, especially Rb, by blocking their binding sites on PP1, insights that are providing strategies for selectively enhancing Rb activity.nuclear phosphatases | enzyme regulation | enzyme specificity | X-ray crystal structure | nuclear magnetic resonance P rotein phosphatase 1 (PP1; ∼38.5 kDa), a single-domain protein, is the most widely expressed and abundant serine/threonine phosphatase (1). By dephosphorylating a variety of protein substrates, PP1 regulates diverse biological processes, including protein synthesis, muscle contraction, carbohydrate metabolism, neuronal signaling and, of specific interest for this work, cell-cycle progression. Although the intrinsic substrate specificity of PP1 is very low, by interacting with regulatory proteins (∼200 biochemically confirmed PP1 interactors), PP1 achieves high specificity (2-5). The majority of PP1 regulators and some substrates bind PP1 via a primary PP1-binding motif, the RVxF motif, which binds to a hydrophobic groove on PP1 ∼20 Å distal from its catalytic center (6). Outside of the RVxF motif, PP1 regulatory proteins mostly lack any apparent sequence similarity. Thus, additional interaction sites, such as the SILK (7), the MyPhoNE (8), and the recently identified ΦΦ motif (9) can only be identified by structural analysis, a major challenge for a comprehensive understanding of PP1 regulation. Only when the primary sequences of PP1 regulators are correlated with specific PP1 binding modes and activities will the PP1 interactome, and the biological processes it regulates, become a viable drug target for the multitude of PP1-associated diseases, such as multiple cancers.One of the key PP1 regulatory targeting proteins in the nucleus, in addition to nuclear inhibitor of PP1 (NIPP1) and Repoman, is the...
Summary The existence of innate predator aversion evoked by predator-derived chemostimuli called kairomones offers a strong selective advantage for potential prey animals. However, it is unclear how chemically-diverse kairomones can elicit similar avoidance behaviors. Using a combination of behavioral analyses and single-cell Ca2+ imaging in wild-type and gene-targeted mice, we show that innate predator-evoked avoidance is driven by parallel, non-redundant processing of volatile and nonvolatile kairomones through the activation of multiple olfactory subsystems including the Grueneberg ganglion, the vomeronasal organ, and chemosensory neurons within the main olfactory epithelium. Perturbation of chemosensory responses in specific subsystems through disruption of genes encoding key sensory transduction proteins (Cnga3, Gnao1) or by surgical axotomy abolished avoidance behaviors and/or cellular Ca2+ responses to different predator odors. Stimulation of these different subsystems resulted in the activation of widely distributed target regions in the olfactory bulb, as assessed by c-Fos expression. However, in each case this c-Fos increase was observed within the same subnuclei of the medial amygdala and ventromedial hypothalamus, regions implicated in fear, anxiety and defensive behaviors. Thus, the mammalian olfactory system has evolved multiple, parallel mechanisms for kairomone detection that converge in the brain to facilitate a common behavioral response. Our findings provide significant insights into the genetic substrates and circuit logic of predator-driven, innate aversion and may serve as a valuable model for studying instinctive fear [1] and human emotional and panic disorders [2, 3].
AAA+ proteases and remodeling machines couple hydrolysis of ATP to mechanical unfolding and translocation of proteins following recognition of sequence tags called degrons. Here, we use singlemolecule optical trapping to determine the mechanochemistry of two AAA+ proteases, Escherichia coli ClpXP and ClpAP, as they unfold and translocate substrates containing multiple copies of the titin I27 domain during degradation initiated from the N terminus. Previous studies characterized degradation of related substrates with C-terminal degrons. We find that ClpXP and ClpAP unfold the wild-type titin I27 domain and a destabilized variant far more rapidly when pulling from the N terminus, whereas translocation speed is reduced only modestly in the N-to-C direction. These measurements establish the role of directionality in mechanical protein degradation, show that degron placement can change whether unfolding or translocation is rate limiting, and establish that one or a few power strokes are sufficient to unfold some protein domains.protein degradation | AAA+ proteases | directional unfolding | AAA+ motors
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