Solution structure of the cytohesin-1 (B2–1) Sec7 domain and its interaction with the GTPase ADP ribosylation factor 1

Cytohesin-1 (B2–1) is a guanine nucleotide exchange factor for human ADP ribosylation factor (Arf) GTPases, which are important for vesicular protein trafficking and coatamer assembly in the cell. Cytohesin-1 also has been reported to promote cellular adhesion via binding to the β2 integrin cytoplasmic domain. The solution structure of the Sec7 domain of cytohesin-1, which is responsible for both the protein’s guanine nucleotide exchange factor function and β2 integrin binding, was determined by NMR spectroscopy. The structure consists of 10 α-helices that form a unique tertiary fold. The binding between the Sec7 domain and a soluble, truncated version of human Arf-1 was investigated by examining ¹H-¹⁵N and ¹H-¹³C chemical shift changes between the native protein and the Sec7/Arf-1 complex. We show that the binding to Arf-1 occurs through a large surface on the C-terminal subdomain that is composed of both hydrophobic and polar residues. Structure-based mutational analysis of the cytohesin-1 Sec7 domain has been used to identify residues important for binding to Arf and for mediating nucleotide exchange. Investigations into the interaction between the Sec7 domain and the β2 integrin cytoplasmic domain suggest that the two proteins do not interact in the solution phase.     

The structure of the yeast NADH dehydrogenase (Ndi1) reveals overlapping binding sites for water- and lipid-soluble substrates

Bioenergy is efficiently produced in the mitochondria by the respiratory system consisting of complexes I–V. In various organisms, complex I can be replaced by the alternative NADH-quinone oxidoreductase (NDH-2), which catalyzes the transfer of an electron from NADH via FAD to quinone, without proton pumping. The Ndi1 protein from Saccharomyces cerevisiae is a monotopic membrane protein, directed to the matrix. A number of studies have investigated the potential use of Ndi1 as a therapeutic agent against complex I disorders, and the NDH-2 enzymes have emerged as potential therapeutic targets for treatments against the causative agents of malaria and tuberculosis. Here we present the crystal structures of Ndi1 in its substrate-free, NAD⁺- and ubiquinone- (UQ2) complexed states. The structures reveal that Ndi1 is a peripheral membrane protein forming an intimate dimer, in which packing of the monomeric units within the dimer creates an amphiphilic membrane-anchor domain structure. Crucially, the structures of the Ndi1–NAD⁺ and Ndi1–UQ2 complexes show overlapping binding sites for the NAD⁺ and quinone substrates.     

ADP ribosylation factor regulates spectrin binding to the Golgi complex

Homologues of two major components of the well-characterized erythrocyte plasma-membrane-skeleton, spectrin (a not-yet-cloned isoform, βIΣ* spectrin) and ankyrin (Ank_G119 and an ≈195-kDa ankyrin), associate with the Golgi complex. ADP ribosylation factor (ARF) is a small G protein that controls the architecture and dynamics of the Golgi by mechanisms that remain incompletely understood. We find that activated ARF stimulates the in vitro association of βIΣ* spectrin with a Golgi fraction, that the Golgi-associated βIΣ* spectrin contains epitopes characteristic of the βIΣ2 spectrin pleckstrin homology (PH) domain known to bind phosphatidylinositol 4,5-bisphosphate (PtdInsP₂), and that ARF recruits βIΣ* spectrin by inducing increased PtdInsP₂ levels in the Golgi. The stimulation of spectrin binding by ARF is independent of its ability to stimulate phospholipase D or to recruit coat proteins (COP)-I and can be blocked by agents that sequester PtdInsP₂. We postulate that a PH domain within βIΣ* Golgi spectrin binds PtdInsP₂ and acts as a regulated docking site for spectrin on the Golgi. Agents that block the binding of spectrin to the Golgi, either by blocking the PH domain interaction or a constitutive Golgi binding site within spectrin’s membrane association domain I, inhibit the transport of vesicular stomatitis virus G protein from endoplasmic reticulum to the medial compartment of the Golgi complex. Collectively, these results suggest that the Golgi-spectrin skeleton plays a central role in regulating the structure and function of this organelle.     

Recognition of the antigen-presenting molecule MR1 by a Vδ3+ γδ T cell receptor

Unlike conventional αβ T cells, γδ T cells typically recognize nonpeptide ligands independently of major histocompatibility complex (MHC) restriction. Accordingly, the γδ T cell receptor (TCR) can potentially recognize a wide array of ligands; however, few ligands have been described to date. While there is a growing appreciation of the molecular bases underpinning variable (V)δ1⁺ and Vδ2⁺ γδ TCR-mediated ligand recognition, the mode of Vδ3⁺ TCR ligand engagement is unknown. MHC class I–related protein, MR1, presents vitamin B metabolites to αβ T cells known as mucosal-associated invariant T cells, diverse MR1-restricted T cells, and a subset of human γδ T cells. Here, we identify Vδ1/2⁻ γδ T cells in the blood and duodenal biopsy specimens of children that showed metabolite-independent binding of MR1 tetramers. Characterization of one Vδ3Vγ8 TCR clone showed MR1 reactivity was independent of the presented antigen. Determination of two Vδ3Vγ8 TCR-MR1-antigen complex structures revealed a recognition mechanism by the Vδ3 TCR chain that mediated specific contacts to the side of the MR1 antigen-binding groove, representing a previously uncharacterized MR1 docking topology. The binding of the Vδ3⁺ TCR to MR1 did not involve contacts with the presented antigen, providing a basis for understanding its inherent MR1 autoreactivity. We provide molecular insight into antigen-independent recognition of MR1 by a Vδ3⁺ γδ TCR that strengthens an emerging paradigm of antibody-like ligand engagement by γδ TCRs.

Characterized by both innate and adaptive immune cell functions, γδ T cells are an unconventional T cell subset. While the functional role of γδ T cells is yet to be fully established, they can play a central role in antimicrobial immunity (1), antitumor immunity (2), tissue homeostasis, and mucosal immunity (3). Owing to a lack of clarity on activating ligands and phenotypic markers, γδ T cells are often delineated into subsets based on the expression of T cell receptor (TCR) variable (V) δ gene usage, grouped as Vδ2⁺ or Vδ2⁻.The most abundant peripheral blood γδ T cell subset is an innate-like Vδ2⁺subset that comprises ∼1 to 10% of circulating T cells (4). These cells generally express a Vγ9 chain with a focused repertoire in fetal peripheral blood (5) that diversifies through neonatal and adult life following microbial challenge (6, 7). Indeed, these Vγ9/Vδ2⁺ T cells play a central role in antimicrobial immune response to Mycobacterium tuberculosis (8) and Plasmodium falciparum (9). Vγ9/Vδ2⁺ T cells are reactive to prenyl pyrophosphates that include isopentenyl pyrophosphate and (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (8) in a butyrophilin 3A1- and BTN2A1-dependent manner (10–13). Alongside the innate-like protection of Vγ9/Vδ2⁺ cells, a Vγ9⁻ population provides adaptive-like immunobiology with clonal expansions that exhibit effector function (14).The Vδ2⁻ population encompasses the remaining γδ T cells but most notably the Vδ1⁺ and Vδ3⁺ populations. Vδ1⁺ γδ T cells are an abundant neonatal lineage that persists as the predominating subset in adult peripheral tissue including the gut and skin (15–18). Vδ1⁺ γδ T cells display potent cytokine production and respond to virally infected and cancerous cells (19). Vδ1⁺ T cells were recently shown to compose a private repertoire that diversifies, from being unfocused to a selected clonal TCR pool upon antigen exposure (20–23). Here, the identification of both Vδ1⁺ T_naive and Vδ1⁺ T_effector subsets and the Vδ1⁺ T_naive to T_effector differentiation following in vivo infection point toward an adaptive phenotype (22).The role of Vδ3⁺ γδ T cells has remained unclear, with a poor understanding of their lineage and functional role. Early insights into Vδ3⁺ γδ T cell immunobiology found infiltration of Vδ3⁺ intraepithelial lymphocytes (IEL) within the gut mucosa of celiac patients (24). More recently it was shown that although Vδ3⁺ γδ T cells represent a prominent γδ T cell component of the gut epithelia and lamina propria in control donors, notwithstanding pediatric epithelium, the expanding population of T cells in celiac disease were Vδ1⁺ (25). Although Vδ3⁺ IELs compose a notable population of gut epithelia and lamina propria T cells (∼3 to 7%), they also formed a discrete population (∼0.2%) of CD4⁻CD8⁻ T cells in peripheral blood (26). These Vδ3⁺ DN γδ T cells are postulated to be innate-like due to the expression of NKG2D, CD56, and CD161 (26). When expanded in vitro, these cells degranulated and killed cells expressing CD1d and displayed a T helper (Th) 1, Th2, and Th17 response in addition to promoting dendritic cell maturation (26). Peripheral Vδ3⁺ γδ T cells frequencies are known to increase in systemic lupus erythematosus patients (27, 28), and upon cytomegalovirus (29) and HIV infection (30), although, our knowledge of their exact role and ligands they recognize remains incomplete.The governing paradigms of antigen reactivity, activation principles, and functional roles of γδ T cells remain unresolved. This is owing partly due to a lack of knowledge of bona fide γδ T cell ligands. Presently, Vδ1⁺ γδ T cells remain the best characterized subset with antigens including Major Histocompatibility Complex (MHC)-I (31), monomorphic MHC-I–like molecules such as CD1b (32), CD1c (33), CD1d (34), and MR1 (35), as well as more diverse antigens such as endothelial protein coupled receptor (EPCR) and phycoerythrin (PE) (36, 37). The molecular determinants of this reactivity were first established for Vδ1⁺ TCRs in complex with CD1d presenting sulfatide (38) and α-galactosylceramide (α-GalCer) (34), which showed an antigen-dependent central focus on the presented lipids and docked over the antigen-binding cleft.In humans, mucosal-associated invariant T (MAIT) cells are an abundant innate-like αβ T cell subset typically characterized by a restricted TCR repertoire (39–43) and reactivity to the monomorphic molecule MR1 presenting vitamin B precursors and drug-like molecules of bacterial origin (41, 44–46). Recently, populations of atypical MR1-restricted T cells have been identified in mice and humans that utilize a more diverse TCR repertoire for MR1-recognition (42, 47, 48). Furthermore, MR1-restricted γδ T cells were identified in blood and tissues including Vδ1⁺, Vδ3⁺, and Vδ5⁺ clones (35). As seen with TRAV 1-2⁻, unconventional MAITs cells the isolated γδ T cells exhibited MR1-autoreactivity with some capacity for antigen discrimination within the responding compartment (35, 48). Structural insight into one such MR1-reactive Vδ1⁺ γδ TCR showed a down-under TCR engagement of MR1 in a manner that is thought to represent a subpopulation of MR1-reactive Vδ1⁺ T cells (35). However, biochemical evidence suggested other MR1-reactive γδ T cell clones would likely employ further unusual docking topologies for MR1 recognition (35).Here, we expanded our understanding of a discrete population of human Vδ3⁺ γδ T cells that display reactivity to MR1. We provide a molecular basis for this Vδ3⁺ γδ T cell reactivity and reveal a side-on docking for MR1 that is distinct from the previously determined Vδ1⁺ γδ TCR-MR1-Ag complex. A Vδ3⁺ γδ TCR does not form contacts with the bound MR1 antigen, and we highlight the importance of non–germ-line Vδ3 residues in driving this MR1 restriction. Accordingly, we have provided key insights into the ability of human γδ TCRs to recognize MR1 in an antigen-independent manner by contrasting mechanisms.     

Crystal structure of the high-affinity Na+,K+-ATPase–ouabain complex with Mg2+ bound in the cation binding site

The Na⁺,K⁺-ATPase maintains electrochemical gradients for Na⁺ and K⁺ that are critical for animal cells. Cardiotonic steroids (CTSs), widely used in the clinic and recently assigned a role as endogenous regulators of intracellular processes, are highly specific inhibitors of the Na⁺,K⁺-ATPase. Here we describe a crystal structure of the phosphorylated pig kidney Na⁺,K⁺-ATPase in complex with the CTS representative ouabain, extending to 3.4 Å resolution. The structure provides key details on CTS binding, revealing an extensive hydrogen bonding network formed by the β-surface of the steroid core of ouabain and the side chains of αM1, αM2, and αM6. Furthermore, the structure reveals that cation transport site II is occupied by Mg²⁺, and crystallographic studies indicate that Rb⁺ and Mn²⁺, but not Na⁺, bind to this site. Comparison with the low-affinity [K₂]E2–MgF_x–ouabain structure [Ogawa et al. (2009) Proc Natl Acad Sci USA 106(33):13742–13747) shows that the CTS binding pocket of [Mg]E2P allows deep ouabain binding with possible long-range interactions between its polarized five-membered lactone ring and the Mg²⁺. K⁺ binding at the same site unwinds a turn of αM4, dragging residues Ile318–Val325 toward the cation site and thereby hindering deep ouabain binding. Thus, the structural data establish a basis for the interpretation of the biochemical evidence pointing at direct K⁺–Mg²⁺ competition and explain the well-known antagonistic effect of K⁺ on CTS binding.     

γ-Actin is required for cytoskeletal maintenance but not development

β_cyto-Actin and γ_cyto-actin are ubiquitous proteins thought to be essential building blocks of the cytoskeleton in all non-muscle cells. Despite this widely held supposition, we show that γ_cyto-actin null mice (Actg1^−/−) are viable. However, they suffer increased mortality and show progressive hearing loss during adulthood despite compensatory up-regulation of β_cyto-actin. The surprising viability and normal hearing of young Actg1^−/− mice means that β_cyto-actin can likely build all essential non-muscle actin-based cytoskeletal structures including mechanosensory stereocilia of hair cells that are necessary for hearing. Although γ_cyto-actin–deficient stereocilia form normally, we found that they cannot maintain the integrity of the stereocilia actin core. In the wild-type, γ_cyto-actin localizes along the length of stereocilia but re-distributes to sites of F-actin core disruptions resulting from animal exposure to damaging noise. In Actg1^−/− stereocilia similar disruptions are observed even without noise exposure. We conclude that γ_cyto-actin is required for reinforcement and long-term stability of F-actin–based structures but is not an essential building block of the developing cytoskeleton.     

Restoration of electron transport without proton pumping in mammalian mitochondria

We have restored the CoQ oxidative capacity of mouse mtDNA-less cells (ρ° cells) by transforming them with the alternative oxidase Aox of Emericella nidulans. Cotransforming ρ° cells with the NADH dehydrogenase of Saccharomyces cerevisiae, Ndi1 and Aox recovered the NADH DH/CoQ reductase and the CoQ oxidase activities. CoQ oxidation by AOX reduces the dependence of ρ° cells on pyruvate and uridine. Coexpression of AOX and NDI1 further improves the recycling of NAD⁺. Therefore, 2 single-protein enzymes restore the electron transport in mammalian mitochondria substituting >80 nuclear DNA-encoded and 11 mtDNA-encoded proteins. Because those enzymes do not pump protons, we were able to split electron transport and proton pumping (ATP synthesis) and inquire which of the metabolic deficiencies associated with the loss of oxidative phosphorylation should be attributed to each of the 2 processes.     

Structure-based mechanism of lipoteichoic acid synthesis by Staphylococcus aureus LtaS

Staphylococcus aureus synthesizes polyglycerol-phosphate lipoteichoic acid (LTA) from phosphatidylglycerol. LtaS, a predicted membrane protein with 5 N-terminal transmembrane helices followed by a large extracellular part (eLtaS), is required for staphylococcal growth and LTA synthesis. Here, we report the first crystal structure of the eLtaS domain at 1.2-Å resolution and show that it assumes a sulfatase-like fold with an α/β core and a C-terminal part composed of 4 anti-parallel β-strands and a long α-helix. Overlaying eLtaS with sulfatase structures identified active site residues, which were confirmed by alanine substitution mutagenesis and in vivo enzyme function assays. The cocrystal structure with glycerol-phosphate and the coordination of a Mn²⁺ cation allowed us to propose a reaction mechanism, whereby the active site threonine of LtaS functions as nucleophile for phosphatidylglycerol hydrolysis and formation of a covalent threonine–glycerolphosphate intermediate. These results will aid in the development of LtaS-specific inhibitors for S. aureus and many other Gram-positive pathogens.     

Modulation of actin structure and function by phosphorylation of Tyr-53 and profilin binding

On starvation, Dictyostelium cells aggregate to form multicellular fruiting bodies containing spores that germinate when transferred to nutrient-rich medium. This developmental cycle correlates with the extent of actin phosphorylation at Tyr-53 (pY53-actin), which is low in vegetative cells but high in viable mature spores. Here we describe high-resolution crystal structures of pY53-actin and unphosphorylated actin in complexes with gelsolin segment 1 and profilin. In the structure of pY53-actin, the phosphate group on Tyr-53 makes hydrogen-bonding interactions with residues of the DNase I-binding loop (D-loop) of actin, resulting in a more stable conformation of the D-loop than in the unphosphorylated structures. A more rigidly folded D-loop may explain some of the previously described properties of pY53-actin, including its increased critical concentration for polymerization, reduced rates of nucleation and pointed end elongation, and weak affinity for DNase I. We show here that phosphorylation of Tyr-53 inhibits subtilisin cleavage of the D-loop and reduces the rate of nucleotide exchange on actin. The structure of profilin–Dictyostelium-actin is strikingly similar to previously determined structures of profilin–β-actin and profilin–α-actin. By comparing this representative set of profilin–actin structures with other structures of actin, we highlight the effects of profilin on the actin conformation. In the profilin–actin complexes, subdomains 1 and 3 of actin close around profilin, producing a 4.7° rotation of the two major domains of actin relative to each other. As a result, the nucleotide cleft becomes moderately more open in the profilin–actin complex, probably explaining the stimulation of nucleotide exchange on actin by profilin.     

Interferon-gamma and interleukin-4 reciprocally regulate CD8 expression in CD8+ T cells

The CD8 co-receptor can modulate CD8⁺ T cell function through its contributions to T cell receptor (TCR) binding and signaling. Here we show that IFN-γ and IL-4 exert opposing effects on the expression of CD8α mRNA and surface CD8 protein during CD8⁺ T cell activation. IL-4 caused down-regulation of surface CD8 on ovalbumin (OVA)_257–264-specific TCR-transgenic OT-I CD8⁺ T cells activated with OVA_257–264-coated antigen presenting cells or polyclonal stimuli, and on wild type CD8⁺ T cells activated with polyclonal stimuli. This effect was enhanced in each case when the cells lacked a functional IFN-γ or IFN-γR gene. When WT or IFN-γ-deficient OT-I CD8⁺ T cells were analyzed 9 days after co-injection with control or IL-4-expressing OVA⁺ tumor cells into RAG-2^−/−γc^−/− mice, CD8 levels were highest on WT donor cells from mice that received the control tumor and lowest on IFN-γ-deficient donor cells from mice that received the IL-4-expressing tumor. The latter CD8^low cells displayed markedly impaired binding of OVA_257–264/MHC tetramers and peptide/MHC-dependent degranulation. The data reveal an unexpected role for IFN-γ in tuning the CD8 co-receptor during primary CD8⁺ T cell activation both in vitro and in vivo.     

TRIM5α Restriction of HIV-1-N74D Viruses in Lymphocytes Is Caused by a Loss of Cyclophilin A Protection

The core of HIV-1 viruses bearing the capsid change N74D (HIV-1-N74D) do not bind the human protein CPSF6. In primary human CD4⁺ T cells, HIV-1-N74D viruses exhibit an infectivity defect when compared to wild-type. We first investigated whether loss of CPSF6 binding accounts for the loss of infectivity. Depletion of CPSF6 in human CD4⁺ T cells did not affect the early stages of wild-type HIV-1 replication, suggesting that defective infectivity in the case of HIV-1-N74D viruses is not due to the loss of CPSF6 binding. Based on our previous result that cyclophilin A (Cyp A) protected HIV-1 from human tripartite motif-containing protein 5α (TRIM5α_hu) restriction in CD4⁺ T cells, we found that depletion of TRIM5α_hu in CD4⁺ T cells rescued the infectivity of HIV-1-N74D, suggesting that HIV-1-N74D cores interacted with TRIM5α_hu. Accordingly, TRIM5α_hu binding to HIV-1-N74D cores was increased compared with that of wild-type cores, and consistently, HIV-1-N74D cores lost their ability to bind Cyp A. In agreement with the notion that N74D capsids are defective in their ability to bind Cyp A, we found that HIV-1-N74D viruses were 20-fold less sensitive to TRIMCyp restriction when compared to wild-type viruses in OMK cells. Structural analysis revealed that N74D hexameric capsid protein in complex with PF74 is different from wild-type hexameric capsid protein in complex with PF74, which explains the defect of N74D capsids to interact with Cyp A. In conclusion, we showed that the decreased infectivity of HIV-1-N74D in CD4⁺ T cells is due to a loss of Cyp A protection from TRIM5α_hu restriction activity.     

β-Catenin in dendritic cells exerts opposite functions in cross-priming and maintenance of CD8+ T cells through regulation of IL-10

Recent studies have demonstrated that β-catenin in DCs serves as a key mediator in promoting both CD4⁺ and CD8⁺ T-cell tolerance, although how β-catenin exerts its functions remains incompletely understood. Here we report that activation of β-catenin in DCs inhibits cross-priming of CD8⁺ T cells by up-regulating mTOR-dependent IL-10, suggesting blocking β-catenin/mTOR/IL-10 signaling as a viable approach to augment CD8⁺ T-cell immunity. However, vaccination of DC–β-catenin^−/− (CD11c-specific deletion of β-catenin) mice surprisingly failed to protect them against tumor challenge. Further studies revealed that DC–β-catenin^−/− mice were deficient in generating CD8⁺ T-cell immunity despite normal clonal expansion, likely due to impaired IL-10 production by β-catenin^−/− DCs. Deletion of β-catenin in DCs or blocking IL-10 after clonal expansion similarly led to reduced CD8⁺ T cells, suggesting that β-catenin in DCs plays a positive role in CD8⁺ T-cell maintenance postclonal expansion through IL-10. Thus, our study has not only identified mTOR/IL-10 as a previously unidentified mechanism for β-catenin–dependent inhibition of cross-priming, but also uncovered an unexpected positive role that β-catenin plays in maintenance of CD8⁺ T cells. Despite β-catenin’s opposite functions in regulating CD8⁺ T-cell responses, selectively blocking β-catenin with a pharmacological inhibitor during priming phase augmented DC vaccine-induced CD8⁺ T-cell immunity and improved antitumor efficacy, suggesting manipulating β-catenin signaling as a feasible therapeutic strategy to improve DC vaccine efficacy.As the initiators of antigen-specific immune responses, dendritic cells (DCs) play a central role in regulating both T-cell immunity and tolerance (1). β-Catenin, a major component in Wnt signaling pathway, has emerged as a key factor in DC differentiation and function (2). Previous studies have shown that β-catenin regulates DC-mediated CD4⁺ T-cell responses and promotes CD4⁺ T-cell tolerance in murine models of autoimmune diseases (3, 4). Consistently, activation of β-catenin in DCs has recently been shown to suppress CD8⁺ T-cell immunity in a DC-targeted vaccine model (5), suggesting that β-catenin in DCs might similarly serve as a tolerizing signal that shifts the balance between CD8⁺ T-cell immunity and tolerance. Although the underlying mechanisms of how β-catenin mediates CD8⁺ T-cell tolerance remain largely unclear, we have shown that activation of β-catenin in DCs genetically or induced by tumors suppresses CD8⁺ T-cell immunity by inhibiting cross-priming (5). Exploiting their ability to potentiate host effector and memory CD8⁺ T-cell responses, DC vaccines have emerged as a leading strategy for cancer immunotherapy (6). However, one major obstacle for their success is host DC-mediated immunosuppression (7–9). Given that cross-priming plays a major role in generating antitumor CD8⁺ T-cell immunity (7, 10), activation of β-catenin in DCs might be a key mechanism for tumors to achieve immunosuppression. Thus, manipulating β-catenin function in cross-priming might be a viable approach to overcome DC-mediated immunosuppression and improve DC vaccine efficacy. However, The underlying mechanisms of how β-catenin in DCs achieves immunosuppression, in particular how β-catenin negatively regulates cross-priming to suppress CD8⁺ T-cell immunity, remain poorly understood.Although the mechanisms for DC-mediated priming of antitumor CD8⁺ T cells through cross-presentation remain incompletely understood, DC subsets, DC maturation status and cytokines have been shown to possibly affect their capacity in cross-priming (7, 10, 11). Although the role of cytokines in cross-priming has not been directly tested, cytokines as “signal 3” have been shown in principal to play a critical role in priming and effector differentiation of antitumor CD8⁺ T cells (12). β-Catenin in DCs has been shown to play a critical role in regulating cytokine induction (3, 4), thus suggesting that β-catenin might regulate DC cytokine production to achieve its effects on cross-priming.In this report we have identified mTOR/IL-10 signaling as a mechanism for β-catenin–dependent inhibition of cross-priming. Activation of β-catenin in DCs inhibited cross-priming of CD8⁺ T cells by up-regulating mTOR-dependent IL-10, and blocking mTOR or IL-10 led to restored cross-priming by β-catenin^active DCs. Surprisingly, mice with DC-specific deletion of β-catenin (DC–β-catenin^−/− mice) exhibited reduced antitumor immunity upon vaccination, despite the fact that deletion of β-catenin in DCs abrogated tumor-induced inhibition of cross-priming. Further studies showed that DC–β-catenin^−/− mice were deficient in generating CD8⁺ T-cell immunity despite normal clonal expansion, and β-catenin in DCs was required to maintain primed CD8⁺ T cells postclonal expansion. Thus, β-catenin in DCs exerts negative and positive functions in cross-priming and maintenance of CD8⁺ T cells, respectively. Importantly, we have demonstrated blocking β-catenin selectively at priming phase as a feasible strategy to improve DC vaccine efficacy.     

CaMKIIbeta binding to stable F-actin in vivo regulates F-actin filament stability

Ca2⁺/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine kinase that is best known for its role in synaptic plasticity and memory. Multiple roles of CaMKII have been identified in the hippocampus, yet its role in developing neurons is less well understood. We show here that endogenous CaMKIIβ, but not CaMKIIα, localized to prominent F-actin-rich structures at the soma in embryonic cortical neurons. Fluorescence recovery after photobleaching analyses of GFP-CaMKIIβ binding interactions with F-actin in this CaMKIIα-free system indicated CaMKIIβ binding depended upon a putative F-actin binding domain in the variable region of CaMKIIβ. Furthermore, CaMKIIα decreased CaMKIIβ binding to F-actin. We examined the interaction of CaMKIIβ with stable and dynamic actin and show that CaMKIIβ binding to F-actin was dramatically prolonged when F-actin was stabilized. CaMKIIβ binding to stable F-actin was disrupted when it was bound by Ca2⁺/calmodulin or when it was highly phosphorylated, but not by kinase inactivity. Whereas CaMKIIβ over-expression increased the prevalence of the F-actin-rich structures, disruption of CaMKIIβ binding to F-actin reduced them. Taken together, these data suggest that CaMKIIβ binding to stable F-actin is important for the in vivo maintenance of polymerized F-actin.     

Structures and characterization of digoxin- and bufalin-bound Na+,K+-ATPase compared with the ouabain-bound complex

Cardiotonic steroids (CTSs) are specific and potent inhibitors of the Na⁺,K⁺-ATPase, with highest affinity to the phosphoenzyme (E2P) forms. CTSs are comprised of a steroid core, which can be glycosylated, and a varying number of substituents, including a five- or six-membered lactone. These functionalities have specific influence on the binding properties. We report crystal structures of the Na⁺,K⁺-ATPase in the E2P form in complex with bufalin (a nonglycosylated CTS with a six-membered lactone) and digoxin (a trisaccharide-conjugated CTS with a five-membered lactone) and compare their characteristics and binding kinetics with the previously described E2P–ouabain complex to derive specific details and the general mechanism of CTS binding and inhibition. CTSs block the extracellular cation exchange pathway, and cation-binding sites I and II are differently occupied: A single Mg²⁺ is bound in site II of the digoxin and ouabain complexes, whereas both sites are occupied by K⁺ in the E2P–bufalin complex. In all complexes, αM4 adopts a wound form, characteristic for the E2P state and favorable for high-affinity CTS binding. We conclude that the occupants of the cation-binding site and the type of the lactone substituent determine the arrangement of αM4 and hypothesize that winding/unwinding of αM4 represents a trigger for high-affinity CTS binding. We find that the level of glycosylation affects the depth of CTS binding and that the steroid core substituents fine tune the configuration of transmembrane helices αM1–2.Cardiotonic steroids (CTSs) induce diverse physiological effects on, for example, heart muscle and blood pressure regulation, but the underlying mechanisms remain unknown, despite a long history of therapeutic applications and model studies. It is widely recognized that they target Na⁺,K⁺-ATPase, and a direct consequence of their binding is an inhibition of the enzyme. Their positive inotropic effect in cardiomyocytes has been related to coupling between Na⁺,K⁺-ATPase and Na⁺/Ca²⁺-exchanger through the intracellular Na⁺ concentration, whereas numerous other outcomes observed on the cellular level have led to hypotheses of the existence of signaling cascade mechanisms with Na⁺,K⁺-ATPase acting as a receptor. The minimal functional unit of the enzyme is an αβ-complex, and because there exist four α- and three β-isoforms of the Na⁺,K⁺-ATPase, the variations in the heterodimer composition and a vast number of CTSs differing in apparent isoform specificities (1) add to the complexity and multiplicity of reported physiological responses.The conserved structural core shared by all CTSs includes a cis-trans-cis ring-fused steroid core with two methyl substituents at steroid positions C10_β and C13_β, two hydroxyl groups (OH3_β and OH14_β), and an unsaturated lactone ring at the C17_β position, among which the lactone and OH14_β are critical for binding to Na⁺,K⁺-ATPase (2, 3). The type of lactone at the C17_β position divides natural CTSs into cardenolides (five-membered lactone rings) and bufadienolides (six-membered lactone rings). Finally, many CTSs are glycosylated by one to four carbohydrate residues at OH3_β (Fig. S1). It has been shown that glycosylation improves CTS affinity toward the Na⁺,K⁺-ATPase and contributes (at least in the case of digoxin and digitoxin) to their Na⁺,K⁺-ATPase isoform selectivity, with up to fourfold preference for α2/α3 over α1 (1).The recently published crystal structure of the Na⁺,K⁺-ATPase phosphoenzyme (E2P) in complex with the widely studied CTS ouabain (4, 5) showed that the high-affinity CTS-binding site is constituted by the transmembrane helices αM1–6 of the catalytic α-subunit, forming a pocket exposed to the extracellular side and overlapping with the extracellular ion exchange pathway (6). The E2P–ouabain structure also revealed details on protein–ligand interactions facilitating high-affinity CTS binding compared with a low-affinity ouabain complex (7). Among the important features brought to view by the high-affinity complex structure were (i) a Mg²⁺ ion occupying cation-binding site II, (ii) the rearrangement of αM4, forming the structural basis for the well-known antagonistic effect of K⁺ on ouabain binding, and (iii) an E2P-specific configuration of αM1–2 on the cytoplasmic side, whereas the extracellular end of this helix pair closes in on the CTS-binding site (4). Biochemical experiments showing competitive interactions between K⁺ and Mg²⁺ suggested that the nature of the cation in site II is a determinant for ouabain affinity. In addition, long-range interactions between the unsaturated, polarized five-membered lactone ring of ouabain and the Mg²⁺ ion were suggested as a factor for CTS recognition and differentiation. Despite previous reports showing that glycosylated CTSs have higher Na⁺,K⁺-ATPase affinity than their aglycones, no specific interactions were observed between the sugar moiety of ouabain and the protein to explain that effect.To gain a better understanding of the structure–activity relationship of the CTSs, we have crystallized the E2P form of the pig kidney Na⁺,K⁺-ATPase (α₁β₁γ) in complex with two CTSs: bufalin (a nonglycosylated bufadienolide) and digoxin (a trisaccharide-conjugated cardenolide) (Fig. 1A), which also are pharmacological agents. We further performed experiments on CTS binding to Na⁺,K⁺-ATPase, including the aglycones digitoxigenin and ouabagenin (Fig. S1). The data revealed notable qualitative differences in kinetics of the enzyme interactions with the glycosylated vs. nonglycosylated CTSs as well as a remarkable insensitivity of bufalin binding to K⁺. The time course of Na⁺,K⁺-ATPase inhibition under steady-state conditions, mimicking the interactions with CTSs in vivo, revealed that binding occurs in two steps. The impact of separate structural components, such as sugar and lactone moieties, on the individual steps of CTS binding is discussed on the basis of our structural and biochemical data.Open in a separate windowFig. 1.Structural comparison of the crystal structures of the high-affinity Na⁺,K⁺-ATPase α₁β₁γ E2P–CTS complexes. The phosphoenzyme stabilized by bufalin, digoxin, and ouabain (5) is depicted in blue, green, and gray cartoons, respectively, and the bufalin, digoxin, and ouabain molecules are represented by magenta, orange, and dark gray sticks, respectively. The K⁺ and Mg²⁺ ions are represented by purple and yellow spheres, respectively. (A) Structural representation of the CTSs digoxin, bufalin, and ouabain. (B and C) The final 2F_o-F_c electron density maps of the E2P–bufalin and E2P–digoxin, respectively, complexes (contoured at 1.0σ level). The maps are represented by gray mesh. (D) Structural alignment of the E2P–bufalin, E2P–digoxin, and E2P–ouabain complexes performed on the segments αM7–10 showing a high degree of overall structural similarity. (E) The CTS-binding site visualized from the extracellular site based on the same alignment as above. The alignment reveals similar hydrophobic interactions between the α-surface of the CTS core and αM4–6. In contrast, different interactions are formed between the substituents at the β-surface of the CTS core and αM1–2, leading to minor CTS-induced rearrangements. (F) The CTS-binding site visualized from αM1–2. αM4 overlays well for Mg²⁺-bound complexes of E2P–digoxin and E2P–ouabain as well as the E2P–bufalin complex, despite potassium bound in the cation-binding sites.     

Crystal structure of LpxK, the 4'-kinase of lipid A biosynthesis and atypical P-loop kinase functioning at the membrane interface

In Gram-negative bacteria, the hydrophobic anchor of the outer membrane lipopolysaccharide is lipid A, a saccharolipid that plays key roles in both viability and pathogenicity of these organisms. The tetraacyldisaccharide 4′-kinase (LpxK) of the diverse P-loop–containing nucleoside triphosphate hydrolase superfamily catalyzes the sixth step in the biosynthetic pathway of lipid A, and is the only known P-loop kinase to act upon a lipid substrate at the membrane. Here, we report the crystal structures of apo- and ADP/Mg²⁺-bound forms of Aquifex aeolicus LpxK to a resolution of 1.9 Å and 2.2 Å, respectively. LpxK consists of two α/β/α sandwich domains connected by a two-stranded β-sheet linker. The N-terminal domain, which has most structural homology to other family members, is responsible for catalysis at the P-loop and positioning of the disaccharide-1-phosphate substrate for phosphoryl transfer on the inner membrane. The smaller C-terminal domain, a substructure unique to LpxK, helps bind the nucleotide substrate and Mg²⁺ cation using a 25° hinge motion about its base. Activity was severely reduced in alanine point mutants of conserved residues D138 and D139, which are not directly involved in ADP or Mg²⁺ binding in our structures, indicating possible roles in phosphoryl acceptor positioning or catalysis. Combined structural and kinetic studies have led to an increased understanding of the enzymatic mechanism of LpxK and provided the framework for structure-based antimicrobial design.     

NAD+ and SIRT3 control microtubule dynamics and reduce susceptibility to antimicrotubule agents

Nicotinamide adenine dinucleotide (NAD⁺) is an endogenous enzyme cofactor and cosubstrate that has effects on diverse cellular and physiologic processes, including reactive oxygen species generation, mitochondrial function, apoptosis, and axonal degeneration. A major goal is to identify the NAD⁺-regulated cellular pathways that may mediate these effects. Here we show that the dynamic assembly and disassembly of microtubules is markedly altered by NAD⁺. Furthermore, we show that the disassembly of microtubule polymers elicited by microtubule depolymerizing agents is blocked by increasing intracellular NAD⁺ levels. We find that these effects of NAD⁺ are mediated by the activation of the mitochondrial sirtuin sirtuin-3 (SIRT3). Overexpression of SIRT3 prevents microtubule disassembly and apoptosis elicited by antimicrotubule agents and knockdown of SIRT3 prevents the protective effects of NAD⁺ on microtubule polymers. Taken together, these data demonstrate that NAD⁺ and SIRT3 regulate microtubule polymerization and the efficacy of antimicrotubule agents.Nicotinamide adenine dinucleotide (NAD⁺) is an endogenous dinucleotide that is present in the cytosol, nucleus, and mitochondria. Athough it serves an important role as a redox cofactor in metabolism, NAD⁺ is also a substrate for several families of enzymes, including the poly(ADP ribose) polymerases and the sirtuin deacetylase enzymes (reviewed in refs. 1 and 2). The level of intracellular NAD⁺ is regulated by many factors, including diet and energy status (3), axonal injury (4), DNA damage (5), and certain disease states (6), suggesting that NAD⁺-dependent signaling is dynamically modulated in diverse contexts.NAD⁺-dependent signaling can be induced by treatment of cells with exogenous NAD⁺, which increases intracellular NAD⁺ levels and results in diverse effects in cells and animals. These effects include enhanced oxygen consumption and ATP production (7), as well as protection from genotoxic stress and apoptosis (3). Mice treated with nicotinamide riboside, a NAD⁺ precursor that is metabolized into NAD⁺, have enhanced oxidative metabolism, increased insulin sensitivity, and protection from high-fat diet-induced obesity (8). These results demonstrate that NAD⁺-dependent pathways can enhance metabolic function and improve a variety of disease phenotypes.An NAD⁺-regulated pathway also inhibits axonal degeneration elicited by axonal transection (4). Treatment of axons with 5–20 mM NAD⁺ markedly delays the axon degenerative process (9). Additionally, animals that express the Wallerian degeneration slow (Wld^S) protein, a fusion of the NAD⁺ biosynthetic enzyme Nicotinamide mononucleotide adenylyl transferase 1 and Ube4a, exhibit markedly delayed degeneration of the distal axonal fragment after axonal transection (10), and expression of Wld^S mitigates disease phenotypes in several neurodegenerative disease models (11–14). Thus, understanding the intracellular pathways regulated by NAD⁺ may be important for understanding the pathogenesis of numerous disorders.Despite the diverse beneficial effects of genetically and pharmacologically augmenting NAD⁺ levels, the cellular processes that are affected by NAD⁺ treatment are incompletely understood. In this study, we show that microtubule dynamics and polymer stability are markedly influenced by NAD⁺ levels. We show that elevation of intracellular NAD⁺ levels markedly alters the stability of microtubule polymers in cells, and renders these polymers resistant to depolymerization elicited by antimicrotubule agents, such as vinblastine, nocodazole, and colchicine. We find that these effects are mediated by sirtuin-3 (SIRT3), a mitochondrial NAD⁺-dependent deacetylase, and that elevated SIRT3 levels also blocks the effects of antimicrotubule agents on the cytoskeleton. Furthermore, we find that both NAD⁺ and SIRT3 reduce the sensitivity of cells to the cytotoxic effects of vinblastine. Taken together, these data identify a new role for NAD⁺ and SIRT3 in regulating the effects of antimicrotubule agents, and link the actions of NAD⁺ to microtubule stabilization in cells.     

Inhibitors of cholera toxin-induced adenosine diphosphate ribosylation of membrane-associated proteins block stem cell differentiation

Two potent inhibitors of mono-adenosine diphosphate (ADP) ribosylation have recently been described and characterized, named p- methoxylbenzylaminodecamethylene guanidine sulfate (MBAMG) and benzylaminododecylguanine hydrochloride (BADGH). We have used these agents to investigate the role of ADP ribosylation in hematopoiesis using long-term marrow cultures. The addition of MBAMG (10(-6) mol/L) or BADGH (5 X 10(-4) mol/L) led to both an inhibition of mature cell production and the development of colony-stimulating factor (CSF-1)- responsive GM-CFC, but had no effect upon spleen colony-forming units (CFU-S) or on progenitor cells which respond to the multilineage stimulating factor present in WEHI-3B cell-conditioned medium. These data indicate that these inhibitors of mono-ADP ribosylation can block the commitment and/or differentiation of stem cells and infers that ADP ribosylation may be of some importance in the hematopoietic process.     

Reversible dissociation of coatomer: Functional characterization of a β/δ-coat protein subcomplex

COPI-coated vesicles mediate protein transport within the early secretory pathway. Their coat consists of ADP ribosylation factor (ARF1, a small guanosine nucleotide binding protein), and coatomer, a cytosolic complex composed of seven subunits, α- to ζ-coat proteins (COPs). For coat formation that initiates budding of a vesicle, ARF1 is recruited to the Golgi membrane from the cytosol in its GTP-bound form, and subsequently, coatomer can bind to the membrane. To identify a minimal structure of coatomer capable to bind to Golgi membranes in an ARF1-dependent manner, we have established a procedure to dissociate coatomer under conditions that allow reassociation of the subunits to a complete and functional complex. After dissociation, subunits or subcomplexes can be isolated and may be expected to be functional. Herein we describe isolation of a subcomplex of coatomer consisting of β- and δ-COPs that is able to bind to Golgi membranes in an ARF1- and GTP-dependent manner.