The Role of the ��DELSEED-loop of ATP Synthase

ATP synthase uses a unique rotational mechanism to convert chemical energy into mechanical energy and back into chemical energy. The helix-turn-helix motif, termed “DELSEED-loop,” in the C-terminal domain of the β subunit was suggested to be involved in coupling between catalysis and rotation. Here, the role of the DELSEED-loop was investigated by functional analysis of mutants of Bacillus PS3 ATP synthase that had 3–7 amino acids within the loop deleted. All mutants were able to catalyze ATP hydrolysis, some at rates several times higher than the wild-type enzyme. In most cases ATP hydrolysis in membrane vesicles generated a transmembrane proton gradient, indicating that hydrolysis occurred via the normal rotational mechanism. Except for two mutants that showed low activity and low abundance in the membrane preparations, the deletion mutants were able to catalyze ATP synthesis. In general, the mutants seemed less well coupled than the wild-type enzyme, to a varying degree. Arrhenius analysis demonstrated that in the mutants fewer bonds had to be rearranged during the rate-limiting catalytic step; the extent of this effect was dependent on the size of the deletion. The results support the idea of a significant involvement of the DELSEED-loop in mechanochemical coupling in ATP synthase. In addition, for two deletion mutants it was possible to prepare an α₃β₃γ subcomplex and measure nucleotide binding to the catalytic sites. Interestingly, both mutants showed a severely reduced affinity for MgATP at the high affinity site.F₁F₀-ATP synthase catalyzes the final step of oxidative phosphorylation and photophosphorylation, the synthesis of ATP from ADP and inorganic phosphate. F₁F₀-ATP synthase consists of the membrane-embedded F₀ subcomplex, with, in most bacteria, a subunit composition of ab₂c₁₀, and the peripheral F₁ subcomplex, with a subunit composition of α₃β₃γδε. The energy necessary for ATP synthesis is derived from an electrochemical transmembrane proton (or, in some organisms, a sodium ion) gradient. Proton flow down the gradient through F₀ is coupled to ATP synthesis on F₁ by a unique rotary mechanism. The protons flow through (half) channels at the interface of the a and c subunits, which drives rotation of the ring of c subunits. The c₁₀ ring, together with F₁ subunits γ and ε, forms the rotor. Rotation of γ leads to conformational changes in the catalytic nucleotide binding sites on the β subunits, where ADP and P_i are bound. The conformational changes result in the formation and release of ATP. Thus, ATP synthase converts electrochemical energy, the proton gradient, into mechanical energy in the form of subunit rotation and back into chemical energy as ATP. In bacteria, under certain physiological conditions, the process runs in reverse. ATP is hydrolyzed to generate a transmembrane proton gradient, which the bacterium requires for such functions as nutrient import and locomotion (for reviews, see Refs. 1–6).F₁ (or F₁-ATPase) has three catalytic nucleotide binding sites located on the β subunits at the interface to the adjacent α subunit. The catalytic sites have pronounced differences in their nucleotide binding affinity. During rotational catalysis, the sites switch their affinities in a synchronized manner; the position of γ determines which catalytic site is the high affinity site (K_d1 in the nanomolar range), which site is the medium affinity site (K_d2 ≈ 1 μm), and which site is the low affinity site (K_d3 ≈ 30–100 μm; see Refs. 7 and 8). In the original crystal structure of bovine mitochondrial F₁ (9), one of the three catalytic sites, was filled with the ATP analog AMP-PNP,² a second was filled with ADP (plus azide) (see Ref. 10), and the third site was empty. Hence, the β subunits are referred to as β_TP, β_DP, and β_E. The occupied β subunits, β_TP and β_DP, were in a closed conformation, and the empty β_E subunit was in an open conformation. The main difference between these two conformations is found in the C-terminal domain. Here, the “DELSEED-loop,” a helix-turn-helix structure containing the conserved DELSEED motif, is in an “up” position when the catalytic site on the respective β subunit is filled with nucleotide and in a “down” position when the site is empty (Fig. 1A). When all three catalytic sites are occupied by nucleotide, the previously open β_E subunit assumes an intermediate, half-closed (β_HC) conformation. It cannot close completely because of steric clashes with γ (11).Open in a separate windowFIGURE 1.The βDELSEED-loop. A, interaction of the β_TP and β_E subunits with theγ subunit.β subunits are shown in yellow andγ in blue. The DELSEED-loop (shown in orange, with the DELSEED motif itself in green)of β_TP interacts with the C-terminal helixγ and the short helix that runs nearly perpendicular to the rotation axis. The DELSEED-loop of β_E makes contact with the convex portion of γ, formed mainly by the N-terminal helix. A nucleotide molecule (shown in stick representation) occupies the catalytic site of β_TP, and the subunit is in the closed conformation. The catalytic site on β_E is empty, and the subunit is in the open conformation. This figure is based on Protein Data Bank file 1e79 (32). B, deletions in the βDELSEED-loop. The loop was “mutated” in silico to represent the PS3 ATP synthase. The 3–4-residue segments that are removed in the deletion mutants are color-coded as follows: ³⁸⁰LQDI³⁸³, pink; ³⁸⁴IAIL³⁸⁷, green; ³⁸⁸GMDE³⁹¹, yellow; ³⁹²LSD³⁹⁴, cyan; ³⁹⁵EDKL³⁹⁸, orange; ³⁹⁹VVHR⁴⁰², blue. Residues that are the most involved in contacts with γ are labeled. All figures were generated using the program PyMOL (DeLano Scientific, San Carlos, CA).The DELSEED-loop of each of the three β subunits makes contact with the γ subunit. In some cases, these contacts consist of hydrogen bonds or salt bridges between the negatively charged residues of the DELSEED motif and positively charged residues on γ. The interactions of the DELSEED-loop with γ, its movement during catalysis, the conservation of the DELSEED motif (see 12–14). Thus, the finding that an AALSAAA mutant in the α₃β₃γ complex of ATP synthase from the thermophilic Bacillus PS3, where several hydrogen bonds/salt bridges to γ are removed simultaneously, could drive rotation of γ with the same torque as the wild-type enzyme (14) came as a surprise. On the other hand, it seems possible that it is the bulk of the DELSEED-loop, more so than individual interactions, that drives rotation of γ. According to a model favored by several authors (6, 15, 16) (see also Refs. 17–19), binding of ATP (or, more precisely, MgATP) to the low affinity catalytic site on β_E and the subsequent closure of this site, accompanied by its conversion into the high affinity site, are responsible for driving the large (80–90°) rotation substep during ATP hydrolysis, with the DELSEED-loop acting as a “pushrod.” A recent molecular dynamics (20) study supports this model and implicates mainly the region around several hydrophobic residues upstream of the DELSEED motif (specifically βI386 and βL387)³ as being responsible for making contact with γ during the large rotation substep.

TABLE 1

Conservation of residues in the DELSEED-loop Amino acids found in selected species in the turn region of the DELSEED-loop. Listed are all positions subjected to deletions in the present study. Residue numbers refer to the PS3 enzyme. Consensus annotation: p, polar residue; s, small residue; h, hydrophobic residue; –, negatively charged residue; +, positively charged residue.Open in a separate windowIn the present study, we investigated the function of the DELSEED-loop using an approach less focused on individual residues, by deleting stretches of 3–7 amino acids between positions β380 and β402 of ATP synthase from the thermophilic Bacillus PS3. We analyzed the functional properties of the deletion mutants after expression in Escherichia coli. The mutants showed ATPase activities, which were in some cases surprisingly high, severalfold higher than the activity of the wild-type control. On the other hand, in all cases where ATP synthesis could be measured, the rates where below or equal to those of the wild-type enzyme. In Arrhenius plots, the hydrolysis rates of the mutants were less temperature-dependent than those of wild-type ATP synthase. In those cases where nucleotide binding to the catalytic sites could be tested, the deletion mutants had a much reduced affinity for MgATP at high affinity site 1. The functional role of the DELSEED-loop will be discussed in light of the new information.     

Deficiency of p110δ Isoform of the Phosphoinositide 3 Kinase Leads to Enhanced Resistance to Leishmania donovani

Background

Visceral leishmaniasis is the most clinically relevant and dangerous form of human leishmaniasis. Most traditional drugs for treatment of leishmaniasis are toxic, possess many adverse reactions and drug resistance is emerging. Therefore, there is urgent need for identification of new therapeutic targets. Recently, we found that mice with an inactivating knock-in mutation in the p110δ isoform of pi3k, (p110δ^d910a) are hyper resistant to L. major, develop minimal cutaneous lesion and rapidly clear their parasite. Here, we investigated whether pi3k signaling also regulates resistance to L. donovani, one of the causative agents of visceral leishmaniasis.

Methodology/Principal Findings

WT and p110δ^D910A mice (on a BALB/c background) were infected with L. donovani. At different time points, parasite burden and granuloma formation were assessed. T and B cell responses in the liver and spleen were determined. In addition, Tregs were expanded in vivo and its impact on resistance was assessed. We found that p110δ^D910A mice had significantly reduced splenomegaly and hepatomegaly and these organs harbored significantly fewer parasites than those of WT mice. Interestingly, infected p110δ^D910A mice liver contains fewer and less organized granulomas than their infected WT counterparts. Cells from p110δ^D910A mice were significantly impaired in their ability to produce cytokines compared to WT mice. The percentage and absolute numbers of Tregs in infected p110δ^D910A mice were lower than those in WT mice throughout the course of infection. In vivo expansion of Tregs in infected p110δ^D910A mice abolished their enhanced resistance to L. donovani infection.

Conclusions/Significance

Our results indicate that the enhanced resistance of p110δ^D910A mice to L. donovani infection is due to impaired activities of Tregs. They further show that resistance to Leishmania in the absence of p110δ signaling is independent of parasite species, suggesting that targeting the PI3K signaling pathway may be useful for treatment of both visceral and cutaneous leishmaniasis.     

γ-Sarcoglycan Deficiency Leads to Muscle Membrane Defects and Apoptosis Independent of Dystrophin

γ-Sarcoglycan is a transmembrane, dystrophin-associated protein expressed in skeletal and cardiac muscle. The murine γ-sarcoglycan gene was disrupted using homologous recombination. Mice lacking γ-sarcoglycan showed pronounced dystrophic muscle changes in early life. By 20 wk of age, these mice developed cardiomyopathy and died prematurely. The loss of γ-sarcoglycan produced secondary reduction of β- and δ-sarcoglycan with partial retention of α- and ε-sarcoglycan, suggesting that β-, γ-, and δ-sarcoglycan function as a unit. Importantly, mice lacking γ-sarco- glycan showed normal dystrophin content and local- ization, demonstrating that myofiber degeneration occurred independently of dystrophin alteration. Furthermore, β-dystroglycan and laminin were left intact, implying that the dystrophin–dystroglycan–laminin mechanical link was unaffected by sarcoglycan deficiency. Apoptotic myonuclei were abundant in skeletal muscle lacking γ-sarcoglycan, suggesting that programmed cell death contributes to myofiber degeneration. Vital staining with Evans blue dye revealed that muscle lacking γ-sarcoglycan developed membrane disruptions like those seen in dystrophin-deficient muscle. Our data demonstrate that sarcoglycan loss was sufficient, and that dystrophin loss was not necessary to cause membrane defects and apoptosis. As a common molecular feature in a variety of muscular dystrophies, sarcoglycan loss is a likely mediator of pathology.     

The Carboxyl-terminal Domain of Atypical Protein Kinase C�� Binds to Ceramide and Regulates Junction Formation in Epithelial Cells

Atypical protein kinase Cs (PKCs) (aPKCζ and λ/ι) have emerged as important binding partners for ceramide, a membrane-resident cell signaling lipid that is involved in the regulation of apoptosis as well as cell polarity. Using ceramide overlay assays with proteolytic fragments of PKCζ and vesicle binding assays with ectopically expressed protein, we show that a protein fragment comprising the carboxyl-terminal 20-kDa sequence of PKCζ (C20ζ, amino acids 405–592) bound to C16:0 ceramide. This sequence is not identical to the C1 domain (amino acids 131–180), which has been suggested to serve as a potential ceramide binding domain. Using immunocytochemistry, we found that a C20ζ protein fragment ectopically expressed in two epithelial cell types (neural progenitors and Madin-Darby canine kidney cells) co-distributed with ceramide. Stable expression of C20ζ-EGFP in Madin-Darby canine kidney cells disrupted the formation of adherens and tight junctions and impaired the epithelium integrity by reducing transepithelial electrical resistance. Disruption of cell adhesion and loss of transepithelial electrical resistance was prevented by incubation with C16:0 ceramide. Our results show, for the first time, that there is a novel ceramide binding domain (C20ζ) in the carboxyl terminus of aPKC. Our results also show that the interaction of ceramide with this binding domain is essential for cell-to-cell contacts in epithelia. Therefore, ceramide interaction with the C20ζ binding domain is a potential mechanism by which ceramide and aPKC regulate the formation of junctional complexes in epithelial cells.Epithelial cells play essential roles in multicellular organisms by forming physiological and mechanical barriers and controlling tissue architecture, because they acquire apicobasal and cell-to-cell (planar) polarity (1, 2). Adherens junctions (AJs)² and tight junctions (TJs) are major structures responsible for cell-to-cell adhesion in epithelial cells (3). The regulation of junction formation requires endocytosis, redistribution, and recycling of junctional proteins, such as E-cadherin (4), and ZO-1. Many factors, including EGF, EGFR, Src kinase, Rho family GTPases Cdc42 and Rac1, and atypical PKC (aPKC), have been found to regulate junction formation (5–9). In Madin-Darby canine kidney (MDCK) cells, Cdc42 modulates AJs by regulating E-cadherin ubiquitination and degradation (9), whereas aPKC directly localized at TJs is required for the asymmetric differentiation of the premature junction complex during epithelial cell polarization (1, 10).The protein kinase C (PKC) family comprises serine/threonine kinases, which consist of a carboxyl-terminal catalytic domain and an amino-terminal regulatory domain (Fig. 1A). The regulatory domain includes an inhibitory pseudosubstrate domain and allosteric sites for activation by phosphatidylserine and, depending on the isoform, calcium (C2 domain) and/or diacylglycerol (C1 domain). aPKC is a subfamily of PKC, which consists of the isoforms ζ and λ/ι. The aPKC isoforms contain only half of the C1 domain, and hence, their activity is not affected by calcium or diacylglycerol/phorbol esters (see Fig. 1A and Refs. 11–13).Open in a separate windowFIGURE 1.Binding of ceramide to the COOH terminus of PKCζ. A, primary structure of aPKC, the caspase 3 proteolytic fragment ζCasp II, and the NH₂-terminal deletion mutant C20ζ-EGFP. B, 2 μg of recombinant His-tagged PKCζ was proteolytically digested by 20 ng of recombinant caspase 3. Proteolysis by caspase 3 occurred first after amino acid 239 (4-h incubation) and then after amino acid 459 (10-h incubation, ζCasp II). C, binding to ceramide spotted on nitrocellulose (overlay assay). FL PKCζ and the COOH-terminal proteolytic fragment ζCasp II bound to C16 ceramide. D, C16 ceramide vesicle binding assay (LIMAC). Ectopically expressed C20ζ-EGFP prepared from a cell lysate was bound to ceramide vesicles; EGFP was not. Protein was detected using anti-aPKC and anti-GFP antibodies. Lanes 1–3, loading input for ceramide vesicles; lanes 4–6, eluate of vesicle binding columns (output). Lanes 7 (input) and 8 (output) show that PKCζ-EGFP did not bind to vesicles prepared with sphingomyelin (SM) instead of ceramide. E, subcellular fractionation of cells expressing FL PKCζ-EGFP or C20ζ-EGFP.Apart from its function in apoptosis (13–15) and cell growth (16), aPKC has been found to play a pivotal role in cell polarity, both in neuroepithelial cells (17–20) or other epithelial cell types (1, 10). Consistently, the gene knock-out of aPKC shows loss of cell junction formation and detachment of neural progenitor cells from the neuroepithelium (8, 21–23). We and others have found that the sphingolipid ceramide activates aPKC, recruits it to structured microdomains, and regulates cell polarity and motility (24–28). Using lipid vesicle-mediated affinity chromatography (LIMAC) assays, we showed for the first time that ceramide directly binds to aPKC (25). Yet which domain of aPKC binds to ceramide is not known.Using lipid overlay and LIMAC assays, we show here that a COOH-terminal 20-kDa domain of PKCζ (C20ζ) binds to ceramide. Similar to its full-length counterpart, the C20ζ protein fragment resides in cellular membranes, where it co-distributes with ceramide in both C17.2 (neural progenitor) and MDCK cells. To study the function of this ceramide binding domain, we established a stably transfected MDCK cell line expressing C20ζ-EGFP. In these cells, the protein level of E-cadherin is reduced, and the cellular distribution of E-cadherin, ZO-1, and β-catenin is disrupted when compared with EGFP-transfected cell lines. Further, transepithelial electrical resistance (TER) assays show that the C20ζ-EGFP cell line has reduced impedance when compared with the control cell line expressing EGFP. This finding suggests that the C20ζ protein fragment is a dominant negative mutant of PKCζ. The effects of this dominant negative mutant can be, at least partially, rescued by incubation with C16:0 ceramide, suggesting that ceramide regulates aPKC and aPKC-dependent cell junction formation by interaction with the COOH-terminal domain.     

Carnitine Deficiency in OCTN2?/? Newborn Mice Leads to a Severe Gut and Immune Phenotype with Widespread Atrophy,Apoptosis and a Pro-Inflammatory Response

We have investigated the gross, microscopic and molecular effects of carnitine deficiency in the neonatal gut using a mouse model with a loss-of-function mutation in the OCTN2 (SLC22A5) carnitine transporter. The tissue carnitine content of neonatal homozygous (OCTN2^−/−) mouse small intestine was markedly reduced; the intestine displayed signs of stunted villous growth, early signs of inflammation, lymphocytic and macrophage infiltration and villous structure breakdown. Mitochondrial β-oxidation was active throughout the GI tract in wild type newborn mice as seen by expression of 6 key enzymes involved in β-oxidation of fatty acids and genes for these 6 enzymes were up-regulated in OCTN2^−/− mice. There was increased apoptosis in gut samples from OCTN2^−/− mice. OCTN2^−/− mice developed a severe immune phenotype, where the thymus, spleen and lymph nodes became atrophied secondary to increased apoptosis. Carnitine deficiency led to increased expression of CD45-B220⁺ lymphocytes with increased production of basal and anti-CD3-stimulated pro-inflammatory cytokines in immune cells. Real-time PCR array analysis in OCTN2^−/− mouse gut epithelium demonstrated down-regulation of TGF-β/BMP pathway genes. We conclude that carnitine plays a major role in neonatal OCTN2^−/− mouse gut development and differentiation, and that severe carnitine deficiency leads to increased apoptosis of enterocytes, villous atrophy, inflammation and gut injury.     

Rab2 Utilizes Glyceraldehyde-3-phosphate Dehydrogenase and Protein Kinase C�� to Associate with Microtubules and to Recruit Dynein

Rab2 requires glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and atypical protein kinase Cι (aPKCι) for retrograde vesicle formation from vesicular tubular clusters that sort secretory cargo from recycling proteins returned to the endoplasmic reticulum. However, the precise role of GAPDH and aPKCι in the early secretory pathway is unclear. GAPDH was the first glycolytic enzyme reported to co-purify with microtubules (MTs). Similarly, aPKC associates directly with MTs. To learn whether Rab2 also binds directly to MTs, a MT binding assay was performed. Purified Rab2 was found in a MT-enriched pellet only when both GAPDH and aPKCι were present, and Rab2-MT binding could be prevented by a recombinant fragment made to the Rab2 amino terminus (residues 2-70), which directly interacts with GAPDH and aPKCι. Because GAPDH binds to the carboxyl terminus of α-tubulin, we characterized the distribution of tyrosinated/detyrosinated α-tubulin that is recruited by Rab2 in a quantitative membrane binding assay. Rab2-treated membranes contained predominantly tyrosinated α-tubulin; however, aPKCι was the limiting and essential factor. Tyrosination/detyrosination influences MT motor protein binding; therefore, we determined whether Rab2 stimulated kinesin or dynein membrane binding. Although kinesin was not detected on membranes incubated with Rab2, dynein was recruited in a dose-dependent manner, and binding was aPKCι-dependent. These combined results suggest a mechanism by which Rab2 controls MT and motor recruitment to vesicular tubular clusters.The small GTPase Rab2 is essential for membrane trafficking in the early secretory pathway and associates with vesicular tubular clusters (VTCs)² located between the endoplasmic reticulum (ER) and the cis-Golgi compartment (1, 2). VTCs are pleomorphic structures that sort anterograde-directed cargo from recycling proteins and trafficking machinery retrieved to the ER (3-6). Rab2 bound to a VTC microdomain stimulates recruitment of soluble factors that results in the release of vesicles containing the recycling protein p53/p58 (7). In that regard, we have previously reported that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and atypical PKC ι (aPKCι) are Rab2 effectors that interact directly with the Rab2 amino terminus and with each other (8, 9). Their interaction requires Src-dependent tyrosine phosphorylation of GAPDH and aPKCι (10). Moreover, GAPDH is a substrate for aPKCι (11). GAPDH catalytic activity is not required for ER to Golgi transport indicating that GAPDH provides a specific function essential for membrane trafficking from VTCs independent of glycolytic function (9). Indeed, phospho-GAPDH influences MT dynamics in the early secretory pathway (11).GAPDH was the first glycolytic enzyme reported to co-purify with microtubules (MTs) (12) and subsequently was shown to interact with the carboxyl terminus of α-tubulin (13). The binding of GAPDH to MTs promotes formation of cross-linked parallel MT arrays or bundles (14, 15). GAPDH has also been reported to possess membrane fusogenic activity, which is inhibited by tubulin (16). Similarly, aPKC associates directly with tubulin and promotes MT stability and MT remodeling at specific intracellular sites (17-21). It may not be coincidental that these two Rab2 effectors influence MT dynamics because recent studies indicate that the cytoskeleton plays a central role in the organization and operation of the secretory pathway (22).MTs are dynamic structures that grow or shrink by the addition or loss of α- and β-tubulin heterodimers from the ends of protofilaments (23). Their assembly and stability is regulated by a variety of proteins traditionally referred to as microtubule-associated proteins (MAPs). In addition to the multiple α/β isoforms that are present in eukaryotes, MTs undergo an assortment of post-translational modifications, including acetylation, glycylation, glutamylation, phosphorylation, palmitoylation, and detyrosination, which further contribute to their biochemical heterogeneity (24, 25). It has been proposed that these tubulin modifications regulate intracellular events by facilitating interaction with MAPs and with other specific effector proteins (24). For example, the reversible addition of tyrosine to the carboxyl terminus of α-tubulin regulates MT interaction with plus-end tracking proteins (+TIPs) containing the cytoskeleton-associated protein glycine-rich (CAP-Gly) motif and with dynein-dynactin (27-29). Additionally, MT motility and cargo transport rely on the cooperation of the motor proteins kinesin and dynein (30). Kinesin is a plus-end directed MT motor, whereas cytoplasmic dynein is a minus-end MT-based motor, and therefore the motors transport vesicular cargo toward the opposite end of a MT track (31).Although MT assembly does not appear to be directly regulated by small GTPases, Rab proteins provide a molecular link for vesicle movement along MTs to the appropriate target (22, 32-34). In this study, the potential interaction of Rab2 with MTs and motor proteins was characterized. We found that Rab2 does not bind directly to preassembled MTs but does associate when both GAPDH and aPKCι are present and bound to MTs. Moreover, the MTs predominantly contained tyrosinated α-tubulin (Tyr-tubulin) suggesting that a dynamic pool of MTs that differentially binds MAPs/effector proteins/motors associates with VTCs in response to Rab2. To that end, we determined that Rab2-promoted dynein/dynactin binding to membranes and that the recruitment required aPKCι.