Protein pores play key roles in fundamental biological processes 1 and biotechnological applications such as DNA nanopore sequencing 2 – 4 , and hence the design of pore-containing proteins is of considerable scientific and biotechnological interest. Synthetic amphiphilic peptides have been found to form ion channels 5 , 6 , and there have been recent advances in de novo membrane protein design 7 , 8 and in redesigning naturally occurring channel-containing proteins 9 , 10 . However, the de novo design of stable, well-defined transmembrane protein pores capable of conducting ions selectively or large enough to allow passage of small-molecule fluorophores remains an outstanding challenge 11 , 12 . Here, we report the computational design of protein pores formed by two concentric rings of ɑ-helices that are stable and mono-disperse in both water-soluble and transmembrane forms. Crystal structures of the water-soluble forms of a 12 helical and a 16 helical pore are close to the computational design models. Patch-clamp electrophysiology experiments show that the transmembrane form of the 12-helix pore expressed in insect cells allows passage of ions across the membrane with high selectivity for potassium over sodium, which is blocked by specific chemical modification at the pore entrance. The transmembrane form of the 16-helix pore, but not the 12-helix pore, allows passage of biotinylated Alexa Fluor 488 when incorporated into liposomes using in vitro protein synthesis. A cryo-EM structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer pores for a wide variety of applications.
ObjectiveOur goals were to evaluate the antitumour efficacy of Lactobacillus rhamnosus GG (LGG) in combination with immune checkpoint blockade (ICB) immunotherapies on tumour growth and to investigate the underlying mechanisms.DesignWe used murine models of colorectal cancer and melanoma to evaluate whether oral administration of LGG improves the efficacy of ICB therapies. We performed the whole genome shotgun metagenome sequencing of intestinal contents and RNA sequencing of dendritic cells (DCs). In a series of in vitro and in vivo experiments, we further defined the immunological and molecular mechanisms of LGG-mediated antitumour immunity.ResultsWe demonstrate that oral administration of live LGG augmented the antitumour activity of anti-programmed cell death 1 (PD-1) immunotherapy by increasing tumour-infiltrating DCs and T cells. Moreover, the combination treatment shifted the gut microbial community towards enrichment in Lactobacillus murinus and Bacteroides uniformis, that are known to increase DC activation and CD8+tumour recruitment. Mechanistically, treatment with live LGG alone or in combination with anti-PD-1 antibody triggered type I interferon (IFN) production in DCs, enhancing the cross-priming of antitumour CD8+ T cells. In DCs, cyclic GMP-AMP synthase (cGAS)/stimulator of IFN genes (STING) was required for IFN-β induction in response to LGG, as evidenced by the significant decrease in IFN-β levels in cGAS or STING-deficient DCs. LGG induces IFN-β production via the cGAS/STING/TANK binding kinase 1/interferon regulatory factor 7 axis in DCs.ConclusionOur findings have offered valuable insight into the molecular mechanisms of live LGG-mediated antitumour immunity and establish an empirical basis for developing oral administration of live LGG as a combination agent with ICB for cancer therapies.
BtuB is a TonB-dependent transport protein that binds and carries vitamin B 12 across the outer membrane of Gram negative bacteria such as Escherichia coli. Previous work has demonstrated that the Ton box, a highly conserved segment near the N-terminus of the protein, undergoes an order-todisorder transition upon the binding of substrate. Here we incorporate pairs of nitroxide spin labels into membrane reconstituted BtuB and utilize a four-pulse double electron-electron resonance (DEER) experiment to measure distances between the Ton box and the periplasmic surface of the transporter with and without substrate. During the reconstitution, the labeled membrane protein was diluted with wild-type protein, which significantly reduced the intermolecular electron spin-spin relaxation rate and increased the DEER signal-to-noise ratio. In the absence of substrate, each spin pair gives rise to a single distribution of distances that is consistent with the crystal structure obtained for BtuB; however, distances that are much longer are found in the presence of substrate, and the data are consistent with the existence of an equilibrium between folded and unfolded states of the Ton box. From these distances, a model for the position of the Ton box was constructed, and indicates that the N-terminal end of the Ton box extends approximately 20 to 30 Å into the periplasm upon the addition of substrate. We propose that this substrate-induced extension provides the signal that initiates interactions between BtuB and the inner membrane protein TonB.TonB-dependent transporters are a unique class of high-affinity transport proteins found in the outer membrane (OM) of Escherichia Coli and other Gram-negative bacteria. In these systems, energy for transport is extracted from the proton potential across the inner membrane by coupling to the inner membrane protein TonB. Crystal structures have been obtained for a number of TonB-dependent transporters, including the iron transporters FhuA (1,2), FepA (3), FecA (4) and the vitamin B 12 (cyanocobalamin (CNCbl)) transporter BtuB (5). These membrane transporters have homologous structures formed from a 22-stranded β-barrel where the N-terminal region of the protein forms a core (or hatch) that occludes the interior of the barrel (see Figure 1).At the present time, the mechanism of transport in TonB-dependent systems is unclear. Coupling between the transporter and TonB is thought to take place through a conserved sequence near the N-terminus termed the Ton box, and there is evidence for an interaction between the transporter and TonB, that is substrate-dependent (6-8). the Ton box is resolved, but is not resolved when substrate is bound. In BtuB, the crystal structures show a minor change in the Ton box configuration with substrate addition, but it is resolved and remains folded within the barrel of the protein. The results of site-directed spin labeling (SDSL) present a different picture for the Ton box of BtuB, where the Ton box is seen to undergo an order-to-disorder transition upo...
Elevated cholesterol decreases agonist-induced contractility and enhances stone formation in the gallbladder. The current study was conducted to determine if and how the electrical properties and ionic conductances of gallbladder smooth muscle are altered by elevated cholesterol. Cholesterol was delivered as a complex with cyclodextrin, and effects were evaluated with intracellular recordings from intact gallbladder and whole cell patch-clamp recordings from isolated cells. Cholesterol significantly attenuated the spontaneous action potentials of intact tissue. Furthermore, calcium-dependent action potentials and calcium currents were reduced in the intact tissue and in isolated cells, respectively. However, neither membrane potential hyperpolarizations induced by the ATP-sensitive potassium channel opener, pinacidil, nor voltage-activated outward potassium currents were affected by cholesterol. Hyperpolarizations elicited by calcitonin gene-related peptide were reduced by cholesterol enrichment, indicating potential changes in receptor ligand binding and/or second messenger interactions. These data indicate that excess cholesterol can contribute to gallbladder stasis by affecting calcium channel activity, whereas potassium channels remained unaffected. In addition, cholesterol enrichment may also modulate receptor ligand behavior and/or second messenger interactions.
The binding and recognition of ligands by bacterial outer membrane transport proteins is mediated in part by interactions made through their extracellular loops. Here, site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy were used to examine the effect of stabilizing solutes on the extracellular loops in BtuB, the vitamin B12 transporter, and FecA, the ferric citrate transporter. EPR spectra from the extracellular loops of FecA and BtuB arise from dynamic backbone segments, and distance measurements made by double electron-electron resonance indicate that the second extracellular loop in BtuB samples a wide range of conformations. These conformations are dramatically restricted upon substrate binding. In addition, the EPR spectra from nitroxide labels attached to the extracellular loops in BtuB and FecA are highly sensitive to solutes, and at every site examined the motion of the label is significantly reduced in the presence of stabilizing osmolytes, such as polyethylene glycols. For the second extracellular loop in BtuB, the solute-induced structural changes are small, but they are sufficient to bring spin-labeled side chains into tertiary contact with other portions of the protein. The spectroscopic changes seen by SDSL suggest that high concentrations of stabilizing solutes, such as those used to generate membrane protein crystals, result in a more compact and ordered state of the protein than is seen under more physiological conditions.
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