Structural basis of MsbA-mediated lipopolysaccharide transport

Topics overview: Type II CRISPR–Cas and Cas1–Cas2-mediated spacer, right-handed signalling pathway, how MC1R activity is modulated by ultraviolet irradiation, how ILC2 responses are regulated by other stimuli, MsbA-mediated lipopolysaccharide transport.

1. How type II CRISPR–Cas establish immunity through Cas1–Cas2-mediated spacer integration

CRISPR (clustered regularly interspaced short palindromic repeats) and the nearby cas (CRISPR-associated) operon establish an RNA-based adaptive immunity system in prokaryotes. Molecular memory is created when a short foreign DNA-derived prespacer is integrated into the CRISPR array as a new spacer. Whereas the RNA-guided CRISPR interference mechanism varies widely among CRISPR-Cas systems, the spacer integration mechanism is essentially identical. The conserved Cas1 and Cas2 proteins form an integrase complex consisting two distal Cas1 dimers bridged by a Cas2 dimer in the middle. The prespacer is bound by Cas1-Cas2 as a dual forked DNA, and the terminal 3’-OH of each 3’-overhang serves as an attacking nucleophile during integration. Importantly, the prespacer is preferentially integrated into the leader-proximal region of the CRISPR array, guided by the leader sequence and a pair of inverted repeats (IRs) inside the CRISPR repeat. Spacer integration in the most well-studied Escherichia coli Type I-E CRISPR system further relies on the bacterial Integration Host Factor (IHF). In Type II-A CRISPR, however, Cas1-Cas2 alone integrates spacer efficiently in vitro; other Cas proteins (Cas9 and Csn2) play accessory roles in prespacer biogenesis. Focusing on the Enterococcus faecalis Type II-A system, here Yibei Xiao at Department of Molecular Biology and Genetics, Cornell University in New York, USA and his colleagues report four structure snapshots of Cas1-Cas2 during spacer integration. EfaCas1-Cas2 selectively binds to a splayed 30-bp prespacer bearing 4-nt 3’-overhangs. Three molecular events take place upon encountering a target: Cas1-Cas2/prespacer first searches for half-sites stochastically, then preferentially interacts with the leader-side CRISPR repeat and catalyzes a nucleophilic attack that connects one strand of the leader-proximal repeat to the prespacer 3’-overhang. Recognition of the spacer half-site requires DNA bending and leads to full integration. The team derive a mechanistic framework explaining the stepwise spacer integration process and the leader-proximal preference.

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2. A right-handed signalling pathway drives heart looping in vertebrates

Most animals show external bilateral symmetry, which hinders the observation of multiple internal left–right (L/R) asymmetries that are fundamental to organ packaging and function. In vertebrates, left identity is mediated by the left-specific Nodal–Pitx2 axis that is repressed on the right-hand side by the epithelial–mesenchymal transition (EMT) inducer Snail1 . Despite some existing evidence, it remains unclear whether an equivalent instructive pathway provides right-hand-specific information to the embryo. Here Oscar H. Ocaña at Instituto de Neurociencias (CSIC-UMH) in Alicante, Spain and his colleagues show that, in zebrafish, BMP mediates the L/R asymmetric activation of another EMT inducer, Prrx1a, in the lateral plate mesoderm with higher levels on the right. Prrx1a drives L/R differential cell movements towards the midline, leading to a leftward displacement of the cardiac posterior pole through an actomyosin-dependent mechanism. Downregulation of Prrx1a prevents heart looping and leads to mesocardia. Two parallel and mutually repressed pathways, respectively driven by Nodal and BMP on the left and right lateral plate mesoderm, converge on the asymmetric activation of the transcription factors Pitx2 and Prrx1, which integrate left and right information to govern heart morphogenesis. This mechanism is conserved in the chicken embryo, and in the mouse SNAIL1 acts in a similar manner to Prrx1a in zebrafish and PRRX1 in the chick. Thus, a differential L/R EMT produces asymmetric cell movements and forces, more prominent from the right, that drive heart laterality in vertebrates, the authors suggest.

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3. Palmitoylation-dependent activation of MC1R prevents melanomagenesis

The melanocortin-1 receptor (MC1R), a G-protein-coupled receptor, has a crucial role in human and mouse pigmentation. Activation of MC1R in melanocytes by α-melanocyte-stimulating hormone (α-MSH)stimulates cAMP signalling and melanin production and enhances DNA repair after ultraviolet irradiation. Individuals carrying MC1R variants, especially those associated with red hair colour, fair skin and poor tanning ability (denoted as RHC variants), are associated with higher risk of melanoma. However, how MC1R activity is modulated by ultraviolet irradiation, why individuals with red hair are more prone to developing melanoma, and whether the activity of RHC variants might be restored for therapeutic benefit are unknown. Here Shuyang Chen at Boston University School of Medicine in Massachusetts, USA and his colleagues demonstrate a potential MC1R-targeted intervention strategy in mice to rescue loss-of-function MC1R in MC1R RHC variants for therapeutic benefit by activating MC1R protein palmitoylation. MC1R palmitoylation, primarily mediated by the protein-acyl transferase ZDHHC, is essential for activating MC1R signalling, which triggers increased pigmentation, ultraviolet-B-induced G1-like cell cycle arrest and control of senescence and melanomagenesis in vitro and in vivo. Using C57BL/6J-Mc1re/eJ mice, in which endogenous MC1R is prematurely terminated, expressing Mc1r RHC variants, they show that pharmacological activation of palmitoylation rescues the defects of Mc1r RHC variants and prevents melanomagenesis. The results highlight a central role for MC1R palmitoylation in pigmentation and protection against melanoma.

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4. The neuropeptide neuromedin U stimulates innate lymphoid cells and type 2 inflammation

The type 2 cytokines interleukin (IL)-4, IL-5, IL-9 and IL-13 have important roles in stimulating innate and adaptive immune responses that are required for resistance to helminth infection, promotion of allergic inflammation, metabolic homeostasis and tissue repair. Group 2 innate lymphoid cells (ILC2s) produce type 2 cytokines, and although advances have been made in understanding the cytokine milieu that promotes ILC2 responses, how ILC2 responses are regulated by other stimuli remains poorly understood. Here Christoph S. N. Klose at Cornell University in New York , USA and his colleagues demonstrate that ILC2s in the mouse gastrointestinal tract co-localize with cholinergic neurons that express the neuropeptide neuromedin U (NMU). In contrast to other haematopoietic cells, ILC2s selectively express the NMU receptor 1 (NMUR1). In vitro stimulation of ILC2s with NMU induced rapid cell activation, proliferation, and secretion of the type 2 cytokines IL-5, IL-9 and IL-13 that was dependent on cell-intrinsic expression of NMUR1 and Gαq protein. In vivo administration of NMU triggered potent type 2 cytokine responses characterized by ILC2 activation, proliferation and eosinophil recruitment that was associated with accelerated expulsion of the gastrointestinal nematode Nippostrongylus brasiliensis or induction of lung inflammation. Conversely, worm burden was higher in Nmur1−/− mice than in control mice. Furthermore, use of gene-deficient mice and adoptive cell transfer experiments revealed that ILC2s were necessary and sufficient to mount NMU-elicited type 2 cytokine responses. Together, these data indicate that the NMU–NMUR1 neuronal signalling circuit provides a selective mechanism through which the enteric nervous system and innate immune system integrate to promote rapid type 2 cytokine responses that can induce anti-microbial, inflammatory and tissue-protective type 2 responses at mucosal sites.

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5. Structural basis of MsbA-mediated lipopolysaccharide transport

Lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria is critical for the assembly of their cell envelopes. LPS synthesized in the cytoplasmic leaflet of the inner membrane is flipped to the periplasmic leaflet by MsbA, an ATP-binding cassette transporter. Despite substantial efforts, the structural mechanisms underlying MsbA-driven LPS flipping remain elusive. Here Wei Mi at Harvard Medical School in Massachusetts, USA and his colleagues use single-particle cryo-electron microscopy to elucidate the structures of lipid-nanodisc-embedded MsbA in three functional states. The 4.2 Å-resolution structure of the transmembrane domains of nucleotide-free MsbA reveals that LPS binds deep inside MsbA at the height of the periplasmic leaflet, establishing extensive hydrophilic and hydrophobic interactions with MsbA. Two sub-nanometre-resolution structures of MsbA with ADP-vanadate and ADP reveal an unprecedented closed and an inward-facing conformation, respectively. Their study uncovers the structural basis for LPS recognition, delineates the conformational transitions of MsbA to flip LPS, and paves the way for structural characterization of other lipid flippases.

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