The ability of the cellular immune system to discriminate self from foreign antigens depends on the appropriate calibration of the T cell receptor (TCR) signalling threshold. The lymphocyte homeostatic cytokine interleukin 7 (IL-7) is known to affect TCR thresholding, but the molecular mechanism is not fully elucidated. A better understanding of this process is highly relevant in the context of autoimmune disease therapy and cancer immunotherapy. We sought to characterise the early signalling events attributable to IL-7 priming; in particular, the altered phosphorylation of signal transduction proteins and their molecular localisation to the TCR. By integrating high-resolution proximity- phospho-proteomic and imaging approaches using primary T cells, rather than engineered cell lines or an in vitro expanded T cell population, we uncovered transduction events previously not linked to IL-7. We show that IL-7 leads to dephosphorylation of cytohesin interacting protein (CYTIP) at a hitherto undescribed phosphorylation site (pThr280) and alters the co-localisation of cytohesin-1 with the TCR and LFA-1 integrin. These results show that IL-7, acting via CYTIP and cytohesin-1, may impact TCR activation thresholds by enhancing the co-clustering of TCR and LFA-1 integrin.

Inhibitor of kappa B (IκB) kinase β (IKKβ) has long been viewed as the dominant IKK in the canonical nuclear factor-κB (NF-κB) signalling pathway, with IKKα being more important in non-canonical NF-κB activation. Here we have investigated the role of IKKα and IKKβ in canonical NF-κB activation in colorectal cells using CRISPR–Cas9 knock-out cell lines, siRNA and selective IKKβ inhibitors. IKKα and IKKβ were redundant for IκBα phosphorylation and turnover since loss of IKKα or IKKβ alone had little (SW620 cells) or no (HCT116 cells) effect. However, in HCT116 cells IKKα was the dominant IKK required for basal phosphorylation of p65 at S536, stimulated phosphorylation of p65 at S468, nuclear translocation of p65 and the NF-κB-dependent transcriptional response to both TNFα and IL-1α. In these cells, IKKβ was far less efficient at compensating for the loss of IKKα than IKKα was able to compensate for the loss of IKKβ. This was confirmed when siRNA was used to knock-down the non-targeted kinase in single KO cells. Critically, the selective IKKβ inhibitor BIX02514 confirmed these observations in WT cells and similar results were seen in SW620 cells. Notably, whilst IKKα loss strongly inhibited TNFα-dependent p65 nuclear translocation, IKKα and IKKβ contributed equally to c-Rel nuclear translocation indicating that different NF-κB subunits exhibit different dependencies on these IKKs. These results demonstrate a major role for IKKα in canonical NF-κB signalling in colorectal cells and may be relevant to efforts to design IKK inhibitors, which have focused largely on IKKβ to date.

Bacteriophage endolysins degrade peptidoglycan and have been identified as antibacterial candidates to combat antimicrobial resistance. Considering the catalytic and structural diversity of endolysins, there is a paucity of structural data to inform how these enzymes work at the molecular level - key data that is needed to realize the potential of endolysin-based antibacterial agents. Here, we determine the atomic structure and define the enzymatic function of Escherichia coli O157:H7 phage FTEBc1 endolysin, LysT84. Bioinformatic analysis reveals that LysT84 is a modular endolysin, which is unusual for Gram-negative endolysins, comprising a peptidoglycan binding domain and an enzymatic domain. The crystal structure of LysT84 (2.99 Å) revealed a mostly α-helical protein with two domains connected by a linker region but packed together. LysT84 was determined to be a monomer in solution using analytical ultracentrifugation. Small-angle X-ray scattering data revealed that LysT84 is a flexible protein but does not have the expected bimodal P(r) function of a multidomain protein, suggesting that the domains of LysT84 pack closely creating a globular protein as seen in the crystal structure. Structural analysis reveals two key glutamate residues positioned on either side of the active site cavity; mutagenesis demonstrating these residues are critical for peptidoglycan degradation. Molecular dynamic simulations suggest that the enzymatically active domain is dynamic, allowing the appropriate positioning of these catalytic residues for hydrolysis of the β(1-4) bond. Overall, our study defines the structural basis for peptidoglycan degradation by LysT84 which supports rational engineering of related endolysins into effective antibacterial agents.

ATP-binding cassette (ABC) proteins play important roles in cells as importers and exporters but as membrane proteins they are subject to well-known challenges of isolating pure and stable samples for study. One solution to this problem is to use styrene-maleic acid lipid particles (SMALPs). Styrene-maleic acid (SMA) can be added directly to membranes, forming stable nanoparticles incorporating membrane proteins and lipids. Here we use Sav1866, a well-characterised bacterial protein, as a proxy for ABC proteins in general. We show that stable and monodispersed Sav1866 can be purified at high yield using SMA. This protein can be used for biophysical characterisations showing that its overall structure is consistent with existing evidence. However, like other ABC proteins in SMALPs it does not hydrolyse ATP. The lack of ATPase activity in ABC–SMALPs may result from conformational trapping of the proteins in SMALPs. Undertaken in a controlled manner, conformational trapping is a useful tool to stabilise protein samples into a single conformation for structural studies. Due to their inability to hydrolyse ATP, the conformation of Sav1866–SMALPs cannot be altered using ATP and vanadate after purification. To achieve controlled trapping of Sav1866–SMALPs we show that Sav1866 in crude membranes can be incubated with ATP, magnesium and sodium orthovanadate. Subsequent solubilisation and purification with SMA produces a sample of Sav1866–SMALPs with enhanced stability, and in a single conformational state. This method may be generally applicable to vanadate-sensitive ABC proteins and overcomes a limitation of the SMALP system for the study of this protein family.

LASP-1 was identified as a protein following mass spectrometric analysis of phosphoproteins consequent to signaling by ErbB2 in SKOV-3 cells. It has been previously identified as an oncogene and is located on chromosomal arm 17q 0.76 Mb centromeric to ErbB2. It is expressed in serous ovarian cancer cell lines as a 40 kDa protein. In SKOV-3 cells, it was phosphorylated and was inhibited by Lapatinib and CP7274714. LASP-1 co-immunoprecipitated with ErbB2 in SKOV-3 cells, suggesting a direct interaction. This interaction and phosphorylation were independent of the kinase activity of ErbB2. Moreover, the binding of LASP-1 to ErbB2 was independent of the tyrosine phosphorylation of LASP-1. LASP-1 was neither expressed on the surface epithelium of the normal ovary nor in the fallopian tube. It was expressed in 28% of ovarian tumours (n = 101) that did not significantly correlate with other clinical factors. In tumours from patients with invasive ductal carcinoma of the breast who had ErbB2 amplification (3+), LASP-1 was expressed in 3/20 (P < 0.001). Analysis of the expression of an independent dataset of ovarian and breast tumours from TCGA showed the significant co-occurrence of ErbB2 and LASP-1 (P < 0.01). These results suggest that LASP-1 and ErbB2 interaction could be important in the pathogenesis of ovarian cancer.

Every living cell needs to get rid of leftover electrons when metabolism extracts energy through the oxidation of nutrients. Common soil microbes such as Geobacter sulfurreducens live in harsh environments that do not afford the luxury of soluble, ingestible electron acceptors like oxygen. Instead of resorting to fermentation, which requires the export of reduced compounds (e.g. ethanol or lactate derived from pyruvate) from the cell, these organisms have evolved a means to anaerobically respire by using nanowires to export electrons to extracellular acceptors in a process called extracellular electron transfer (EET) [ 1]. Since 2005, these nanowires were thought to be pili filaments [ 2]. But recent studies have revealed that nanowires are composed of multiheme cytochromes OmcS [ 3, 4] and OmcZ [ 5] whereas pili remain inside the cell during EET and are required for the secretion of nanowires [ 6]. However, how electrons are passed to these nanowires remains a mystery ( Figure 1A). Periplasmic cytochromes (Ppc) called PpcA-E could be doing the job, but only two of them (PpcA and PpcD) can couple electron/proton transfer - a necessary condition for energy generation. In a recent study, Salgueiro and co-workers selectively replaced an aromatic with an aliphatic residue to couple electron/proton transfer in PpcB and PpcE (Biochem. J. 2021, 478 (14): 2871-2887). This significant in vitro success of their protein engineering strategy may enable the optimization of bioenergetic machinery for bioenergy, biofuels, and bioelectronics applications.

Tunneling nanotubes (TNTs) are F-actin-based, membrane-enclosed tubular connections between animal cells that transport a variety of cellular cargo. Over the last 15 years since their discovery, TNTs have come to be recognized as key players in normal cell communication and organism development, and are also exploited for the spread of various microbial pathogens and major diseases like cancer and neurodegenerative disorders. TNTs have also been proposed as modalities for disseminating therapeutic drugs between cells. Despite the rapidly expanding and wide-ranging relevance of these structures in both health and disease, there is a glaring dearth of molecular mechanistic knowledge regarding the formation and function of these important but enigmatic structures. A series of fundamental steps are essential for the formation of functional nanotubes. The spatiotemporally controlled and directed modulation of cortical actin dynamics would be required to ensure outward F-actin polymerization. Local plasma membrane deformation to impart negative curvature and membrane addition at a rate commensurate with F-actin polymerization would enable outward TNT elongation. Extrinsic tactic cues, along with cognate intrinsic signaling, would be required to guide and stabilize the elongating TNT towards its intended target, followed by membrane fusion to create a functional TNT. Selected cargoes must be transported between connected cells through the action of molecular motors, before the TNT is retracted or destroyed. This review summarizes the current understanding of the molecular mechanisms regulating these steps, also highlighting areas that deserve future attention.

The pathogenic protist Trypanosoma cruzi uses kissing bugs as invertebrate hosts that vectorize the infection among mammals. This parasite oxidizes proline to glutamate through two enzymatic steps and one nonenzymatic step. In insect vectors, T. cruzi differentiates from a noninfective replicating form to nonproliferative infective forms. Proline sustains this differentiation, but to date, a link between proline metabolism and differentiation has not been established. In T. cruzi, the enzymatic steps of the proline-glutamate oxidation pathway are catalyzed exclusively by the mitochondrial enzymes proline dehydrogenase [TcPRODH, EC: 1.5.5.2] and Δ1-pyrroline-5-carboxylate dehydrogenase [TcP5CDH, EC: 1.2.1.88]. Both enzymatic steps produce reducing equivalents that are able to directly feed the mitochondrial electron transport chain (ETC) and thus produce ATP. In this study, we demonstrate the contribution of each enzyme of the proline-glutamate pathway to ATP production. In addition, we show that parasites overexpressing these enzymes produce increased levels of H2O2, but only those overexpressing TcP5CDH produce increased levels of superoxide anion. We show that parasites overexpressing TcPRODH, but not parasites overexpressing TcP5CDH, exhibit a higher rate of differentiation into metacyclic trypomastigotes in vitro. Finally, insect hosts infected with parasites overexpressing TcPRODH showed a diminished parasitic load but a higher percent of metacyclic trypomastigotes, when compared with controls. Our data show that parasites overexpressing both, PRODH and P5CDH had increased mitochondrial functions that orchestrated different oxygen signaling, resulting in different outcomes in relation to the efficiency of parasitic differentiation in the invertebrate host.

The effects of small-molecule AMP-activated protein kinase (AMPK) activators in rat epididymal adipocytes were compared. SC4 was the most effective and submaximal doses of SC4 and 5-amino-4-imidazolecarboxamide (AICA) riboside were combined to study the effects of AMPK activation in white adipose tissue (WAT). Incubation of rat adipocytes with SC4 + AICA riboside inhibited noradrenaline-induced lipolysis and decreased hormone-sensitive lipase (HSL) Ser563 phosphorylation, without affecting HSL Ser565 phosphorylation. Preincubation of fat pads from wild-type (WT) mice with SC4 + AICA riboside inhibited insulin-stimulated lipogenesis from glucose or acetate and these effects were lost in AMPKα1 knockout (KO) mice, indicating AMPKα1 dependency. Moreover, in fat pads from acetyl-CoA carboxylase (ACC)1/2 S79A/S212A double knockin versus WT mice, the effect of SC4 + AICA riboside to inhibit insulin-stimulated lipogenesis from acetate was lost, pinpointing ACC as the main AMPK target. Treatment with SC4 + AICA riboside decreased insulin-stimulated glucose uptake, an effect that was still observed in fat pads from AMPKα1 KO versus WT mice, suggesting the effect was partly AMPKα1-independent. SC4 + AICA riboside treatment had no effect on the insulin-induced increase in palmitate esterification nor on sn-glycerol-3-phosphate-O-acyltransferase activity. Therefore in WAT, AMPK activation inhibits noradrenaline-induced lipolysis and suppresses insulin-stimulated lipogenesis primarily by inactivating ACC and by inhibiting glucose uptake.

PEPT1 is a proton-coupled peptide transporter that is up-regulated in PDAC cell lines and PDXs, with little expression in the normal pancreas. However, the relevance of this up-regulation to cancer progression and the mechanism of up-regulation have not been investigated. Herein, we show that PEPT1 is not just up-regulated in a large panel of PDAC cell lines and PDXs but is also functional and transport-competent. PEPT2, another proton-coupled peptide transporter, is also overexpressed in PDAC cell lines and PDXs, but is not functional due to its intracellular localization. Using glibenclamide as a pharmacological inhibitor of PEPT1, we demonstrate in cell lines in vitro and mouse xenografts in vivo that inhibition of PEPT1 reduces the proliferation of the cancer cells. These findings are supported by genetic knockdown of PEPT1 with shRNA, wherein the absence of the transporter significantly attenuates the growth of cancer cells, both in vitro and in vivo, suggesting that PEPT1 is critical for the survival of cancer cells. We also establish that the tumor-derived lactic acid (Warburg effect) in the tumor microenvironment supports the transport function of PEPT1 in the maintenance of amino acid nutrition in cancer cells by inducing MMPs and DPPIV to generate peptide substrates for PEPT1 and by generating a H+ gradient across the plasma membrane to energize PEPT1. Taken collectively, these studies demonstrate a functional link between PEPT1 and extracellular protein breakdown in the tumor microenvironment as a key determinant of pancreatic cancer growth, thus identifying PEPT1 as a potential therapeutic target for PDAC.