Alzheimer's disease (AD)-linked mutations in Presenilins (PSEN) and the amyloid precursor protein (APP) lead to production of longer amyloidogenic Aβ peptides. The shift in Aβ length is fundamental to the disease; however, the underlying mechanism remains elusive. Here, we show that substrate shortening progressively destabilizes the consecutive enzyme-substrate (E-S) complexes that characterize the sequential γ-secretase processing of APP. Remarkably, pathogenic PSEN or APP mutations further destabilize labile E-S complexes and thereby promote generation of longer Aβ peptides. Similarly, destabilization of wild-type E-S complexes by temperature, compounds, or detergent promotes release of amyloidogenic Aβ. In contrast, E-Aβ stabilizers increase γ-secretase processivity. Our work presents a unifying model for how PSEN or APP mutations enhance amyloidogenic Aβ production, suggests that environmental factors may increase AD risk, and provides the theoretical basis for the development of γ-secretase/substrate stabilizing compounds for the prevention of AD.
The type II secretion system (T2SS), a protein complex spanning the bacterial envelope, is pivotal to bacterial pathogenicity. Central to T2SS function is the extrusion of protein cargos from the periplasm into the extracellular environment mediated by a pseudopilus and motorized by a cytosolic ATPase. GspF, an inner-membrane component of T2SS has long been considered to be a key player in this process, yet the structural basis of its role had remained elusive. Here, we employed single-particle electron microscopy based on XcpS (GspF) from the T2SS of pathogenic P. aeruginosa stabilized by a nanobody, to show that XcpS adopts a dimeric structure mediated by its transmembrane helices. This assembly matches in terms of overall organization and dimensions the basal inner-membrane cassette of a T2SS machinery. Thus, GspF is poised to serve as an adaptor involved in the mediation of propeller-like torque generated by the motor ATPase to the secretion pseudopilus. Non-technical author summaryAntibiotic resistance by bacteria imposes a worldwide threat that can only be overcome through a multifront approach: preventive actions and the parallel development of novel molecular strategies to combat antibiotic resistance mechanisms. One such strategy might focus on antivirulence drugs that prevent host invasion and spreading by pathogenic bacteria, without shutting down essential functions related to bacterial survival. The rationale behind such an approach is that it might limit selective pressure leading to slower evolutionary rates of resistant bacterial strains. Bacterial secretion systems are an appropriate target for such therapeutic approaches as their impairment will inhibit the secretion of a multitude of virulence factors. This study focuses on the structural characterization of one of the proteins residing in the inner-membrane cassette of the type II secretion system (T2SS), a multi-protein complex in multiple opportunistic pathogens that secretes virulence factors. The targeted protein is essential for the assembly of the pseudopilus, a rod-like supramolecular structure that propels the secretion of virulence factors by pathogenic Gram-negative bacteria. Our study crucially complements growing evidence supporting a rotational assembly model of the pseudopilus and contributes to a better understanding of the functioning of the T2SS and the related secretion systems. We envisage that such knowledge will facilitate targeting of these systems for therapeutic purposes.
Interleukin-9 (IL-9) is the hallmark cytokine in Th9 immunity and is also central to Innate Lymphocyte 2 (ILC2) biology. Furthermore, receptor signaling mediated by IL-9 has been linked to inflammatory and autoimmune diseases, and cancer. Despite its functional pleiotropy, the structure-function landscape of IL-9 had remained enigmatic. Here, we show via a combination of X-ray crystallography and NMR that human IL-9 adopts a helical bundle fold with unprecedented structural features among helical cytokines, including five disulfide bridges. Binding of IL-9 to the interdomain junction of IL-9Rα results in marked structural changes on the opposite face of IL-9 that prime the binary complex for recruiting the common gamma chain (γc) for signaling. Surprisingly, this tripartite cytokine-receptor assembly displays a markedly lower affinity than the IL-9: IL-9Rα complex, which we trace to distinct features of IL-9Rα that might destabilize the ternary complex. Furthermore, we developed monoclonal antibodies that antagonize IL-9 activity by sterically competing for the binding footprint of IL-9Rα. Collectively, we here provide a structural and mechanistic blueprint to facilitate interrogation and modulation of pleiotropic signaling outputs of IL-9 in physiology and disease.
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