Membrane Targeting of the Spir·Formin Actin Nucleator Complex Requires a Sequential Handshake of Polar Interactions

Spir and formin (FMN)-type actin nucleators initiate actin polymerization at vesicular membranes necessary for long range vesicular transport processes. Here we studied in detail the membrane binding properties and protein/protein interactions that govern the assembly of the membrane-associated Spir·FMN complex. Using biomimetic membrane models we show that binding of the C-terminal Spir-2 FYVE-type zinc finger involves both the presence of negatively charged lipids and hydrophobic contributions from the turret loop that intrudes the lipid bilayer. In solution, we uncovered a yet unknown intramolecular interaction between the Spir-2 FYVE-type domain and the N-terminal kinase non-catalytic C-lobe domain (KIND) that could not be detected in the membrane-bound state. Interestingly, we found that the intramolecular Spir-2 FYVE/KIND and the trans-regulatory Fmn-2-FSI/Spir-2-KIND interactions are competitive. We therefore characterized co-expressed Spir-2 and Fmn-2 fluorescent protein fusions in living cells by fluorescence cross-correlation spectroscopy. The data corroborate a model according to which Spir-2 exists in two different states, a cytosolic monomeric conformation and a membrane-bound state in which the KIND domain is released and accessible for subsequent Fmn-2 recruitment. This sequence of interactions mechanistically couples membrane binding of Spir to the recruitment of FMN, a pivotal step for initiating actin nucleation at vesicular membranes.     

Localization and functional characterization of the rat Oatp4c1 transporter in an in vitro cell system and rat tissues

The organic anion transporting polypeptide 4c1 (Oatp4c1) was previously identified as a novel uptake transporter predominantly expressed at the basolateral membrane in the rat kidney proximal tubules. Its functional role was suggested to be a vectorial transport partner of an apically-expressed efflux transporter for the efficient translocation of physiological substrates into urine, some of which were suggested to be uremic toxins. However, our in vitro studies with MDCKII cells showed that upon transfection rat Oatp4c1 polarizes to the apical membrane. In this report, we validated the trafficking and function of Oatp4c1 in polarized cell systems as well as its subcellular localization in rat kidney. Using several complementary biochemical, molecular and proteomic methods as well as antibodies amenable to immunohistochemistry, immunofluorescence, and immunobloting we investigated the expression pattern of Oatp4c1 in polarized cell systems and in the rat kidney. Collectively, these data demonstrate that rat Oatp4c1 traffics to the apical cell surface of polarized epithelium and localizes primarily in the proximal straight tubules, the S3 fraction of the nephron. Drug uptake studies in Oatp4c1-overexpressing cells demonstrated that Oatp4c1-mediated estrone-3-sulfate (E3S) uptake was pH-dependent and ATP-independent. These data definitively demonstrate the subcellular localization and histological location of Oatp4c1 and provide additional functional evidence that reconciles expression-function reports found in the literature.     

Aging and aerobic fitness affect the contribution of noradrenergic sympathetic nerves to the rapid cutaneous vasodilator response to local heating

Sedentary aging results in a diminished rapid cutaneous vasodilator response to local heating. We investigated whether this diminished response was due to altered contributions of noradrenergic sympathetic nerves by assessing 1) the age-related decline and 2) the effect of aerobic fitness. Using laser-Doppler flowmetry, we measured skin blood flow (SkBF) in young (24 ± 1 yr) and older (64 ± 1 yr) endurance-trained and sedentary men (n = 7 per group) at baseline and during 35 min of local skin heating to 42°C at 1) untreated forearm sites, 2) forearm sites treated with bretylium tosylate (BT), which prevents neurotransmitter release from noradrenergic sympathetic nerves, and 3) forearm sites treated with yohimbine + propranolol (YP), which antagonizes α- and β-adrenergic receptors. SkBF was converted to cutaneous vascular conductance (CVC = SkBF/mean arterial pressure) and normalized to maximal CVC (%CVC(max)) achieved by skin heating to 44°C. Pharmacological agents were administered using microdialysis. In the young trained group, the rapid vasodilator response was reduced at BT and YP sites (P < 0.05); by contrast, in the young sedentary and older trained groups, YP had no effect (P > 0.05), but BT did (P > 0.05). Neither BT nor YP affected the rapid vasodilator response in the older sedentary group (P > 0.05). These data suggest that the age-related reduction in the rapid vasodilator response is due to an impairment of sympathetic-dependent mechanisms, which can be partly attenuated with habitual aerobic exercise. Rapid vasodilation involves noradrenergic neurotransmitters in young trained men and nonadrenergic sympathetic cotransmitters (e.g., neuropeptide Y) in young sedentary and older trained men, possibly as a compensatory mechanism. Finally, in older sedentary men, the rapid vasodilation appears not to involve the sympathetic system.     

Farnesylation of Pex19p Is Required for Its Structural Integrity and Function in Peroxisome Biogenesis

The conserved CaaX box peroxin Pex19p is known to be modified by farnesylation. The possible involvement of this lipid modification in peroxisome biogenesis, the degree to which Pex19p is farnesylated, and its molecular function are unknown or controversial. We resolve these issues by first showing that the complete pool of Pex19p is processed by farnesyltransferase in vivo and that this modification is independent of peroxisome induction or the Pex19p membrane anchor Pex3p. Furthermore, genomic mutations of PEX19 prove that farnesylation is essential for proper matrix protein import into peroxisomes, which is supposed to be caused indirectly by a defect in peroxisomal membrane protein (PMP) targeting or stability. This assumption is corroborated by the observation that mutants defective in Pex19p farnesylation are characterized by a significantly reduced steady-state concentration of prominent PMPs (Pex11p, Ant1p) but also of essential components of the peroxisomal import machinery, especially the RING peroxins, which were almost depleted from the importomer. In vivo and in vitro, PMP recognition is only efficient when Pex19p is farnesylated with affinities differing by a factor of 10 between the non-modified and wild-type forms of Pex19p. Farnesylation is likely to induce a conformational change in Pex19p. Thus, isoprenylation of Pex19p contributes to substrate membrane protein recognition for the topogenesis of PMPs, and our results highlight the importance of lipid modifications in protein-protein interactions.A large number of eukaryotic intracellular proteins are post-translationally modified by the covalent attachment of either 15 or 20 carbon isoprenoids known as farnesyl or geranylgeranyl, respectively. This process (referred to as protein prenylation) affects lipases, kinases, inositol and protein-tyrosine phosphatases, lamins, and most of the small GTPases (1–3). Protein prenylation was shown to enable reversible association of modified proteins with lipid bilayers and to modulate protein-protein interactions (4–6).The farnesyl group is attached to the cysteine of the C-terminal motif known as the CaaX box, where “a” indicates aliphatic amino acids and X is usually serine, methionine, glutamine, alanine, or threonine (3). Farnesyltransferase (FTase)³ consists of two subunits, the α-subunit and the β-subunit (Ram2p and Ram1p in yeast). The α-subunit is shared by the geranylgeranyl transferase (GGTase I), whereas the β-subunit is unique for FTase (7).The peroxisome biogenesis protein (peroxin) Pex19p is one of a few farnesylated non-GTPases that are conserved between yeast and humans. Pex19p was initially identified as a prenylated protein (PxF) (8, 9) or housekeeping gene product (HK33) (10). A loss-of-function mutation in human PEX19 is associated with complementation group CG-J/CG-14 of Zellweger syndrome (11). In the absence of Pex19p, cells lack functional peroxisomes (11–13). Pex19p is mostly cytosolic and interacts with all peroxisomal membrane proteins (PMPs) analyzed (14–16).Different and not all exclusive models have been proposed for Pex19p function. First, Pex19p might be an import receptor for PMPs that recognizes its substrates in the cytosol and delivers them to the peroxisomal membrane (15, 17, 18). This function would be analogous to that of the peroxisomal import receptors Pex5p and Pex7p, which recognize and deliver matrix proteins with PTS1 (peroxisomal targeting signal type 1) and PTS2 to peroxisomes (19). Second, Pex19p might act as a PMP chaperone that prevents newly synthesized PMPs from aggregation and degradation in the cytosol (17, 20). Third, Pex19p might act as a PMP membrane insertion factor (14, 16). Fourth, Pex19p might be required as an association/dissociation factor of membrane protein complexes (21) and has been reported to be required for the targeting of Pex3p from the ER to the peroxisomal membrane (22). Finally, Pex19p function is dependent on Pex3p, which serves as a docking factor at the peroxisomal membrane (12, 22–24). All models agree on the importance of PMP recognition for Pex19p function (25).Pex19p shows only a moderate degree of sequence conservation, with less than 20% amino acid identity between yeast and human Pex19p. Its CaaX box, however, has been retained throughout evolution (see Fig. 1). Information on the status and the requirement of Pex19p farnesylation has so far been available only through often conflicting side observations. Mammalian PEX19 was described to be partially farnesylated in CHO-K1 cells (11), but other studies with human fibroblasts challenged the relevance of Pex19p farnesylation (15, 26). It was speculated that in Saccharomyces cerevisiae, farnesylation is required for an essential aspect of Pex19p function (12). This notion was recently contradicted (27). Work on other yeasts similarly suggested that farnesylation would be dispensable for Pex19p function (13, 28, 29).Open in a separate windowFIGURE 1.Pex19p is completely farnesylated in vivo, independent of peroxisome induction and Pex3p. A and B, Pex19p is fully modified by yeast FTase in vivo. Whole cell lysates from non-induced cells of the indicated strains were analyzed by immunoblotting. Blots were probed with anti-Pex19p antibodies. The non-farnesylated form of Pex19p of a Δram1 mutant (arrowhead) cannot be detected in extracts from wild-type yeast (arrow) (A), whereas it reappears after reintroduction of Ram1p (B). C, the yeast farnesylation machinery can be saturated by overexpression of GST-Pex19p. A Coomassie-stained gel of purified farnesylated and non-farnesylated Pex19p is shown. GST-Pex19p was expressed under control of a copper-inducible promoter in Δpex19 and Δram1 strains and isolated by affinity chromatography. In Δram1 (right), only the non-farnesylated GST-Pex19p can be detected. In Δpex19 (left) two bands appear, corresponding to non-farnesylated GST-Pex19p (upper band) and farnesylated GST-Pex19p (lower band). D, Pex19p farnesylation levels are independent of peroxisome induction and are not affected by the absence of the Pex19p membrane anchor Pex3p. Cells were grown on YPD medium and, where indicated, washed and grown on 0.1% oleate medium for 17 h for peroxisome induction. Lysates were fractionated by centrifugation (20,000 × g, 1 h, 4 °C) and analyzed as in A. Blots were probed with antibodies against Pex19p. E, evolutionary conservation of the Pex19p farnesylation site in fungi, plant, and metazoa.In this study, we determined the in vivo farnesylation status of Pex19p and its dependence on peroxisome induction and on Pex3p. We discovered that Pex19p is fully modified by FTase and investigated whether Pex19p farnesylation is required for PMP recognition and stability. By peptide blots, two-hybrid analysis, and fluorescence polarization titration, we showed that farnesylation increases the affinity for PMPs by a factor of about 10. Last, we provide evidence that the interaction between farnesylated Pex19p and PMPs is achieved through a farnesylation-induced structural change in Pex19p rather than through direct farnesyl-PMP interaction. Our results exemplify the biological relevance of isoprenylation-dependent protein-protein interactions.     

Interactions between hepatic Mrp4 and Sult2a as revealed by the constitutive androstane receptor and Mrp4 knockout mice

The ABC transporter, Mrp4, transports the sulfated steroid DHEA-s, and sulfated bile acids interact with Mrp4 with high affinity. Hepatic Mrp4 levels are low, but increase under cholestatic conditions. We therefore inferred that up-regulation of Mrp4 during cholestasis is a compensatory mechanism to protect the liver from accumulation of hydrophobic bile acids. We determined that the nuclear receptor CAR is required to coordinately up-regulate hepatic expression of Mrp4 and an enzyme known to sulfate hydroxy-bile acids and steroids, Sult2a1. CAR activators increased Mrp4 and Sult2a1 expression in primary human hepatocytes and HepG2, a human liver cell line. Sult2a1 was down-regulated in Mrp4-null mice, further indicating an inter-relation between Mrp4 and Sult2a1 gene expression. Based on the hydrophilic nature of sulfated bile acids and the Mrp4 capability to transport sulfated steroids, our findings suggest that Mrp4 and Sult2a1 participate in an integrated pathway mediating elimination of sulfated steroid and bile acid metabolites from the liver.     

Prenylation of Ras facilitates hSOS1-promoted nucleotide exchange, upon Ras binding to the regulatory site

The oncoprotein Ras is anchored in lipid membranes due to its C-terminal lipid modification. The ubiquitously expressed Ras nucleotide exchange-factor hSOS1 promotes nucleotide exchange and thus Ras activation. This reaction is enhanced by a positive feedback loop whereby activated Ras binds to an allosteric site of SOS to enhance GEF activity. Here we present biochemical data showing that prenylation of both active site bound and allosterically bound N-Ras is required for efficient hSOS1-promoted nucleotide exchange. Our results indicate that prenyl sensitivity of the allosteric feedback-activation is mediated by the PH domain of hSOS1. Farnesylation of Ras thereby allows hSOS1 to bind even GDP-loaded allosteric regulator to maintain basal hSOS1-activity.     

Ex vivo cytokine mRNA levels correlate with changing clinical status of ethiopian TB patients and their contacts over time

There is an increasing body of evidence which suggests that IL-4 plays a role in the pathogenesis of TB, but a general consensus on its role remains elusive. We have previously published data from a cohort of Ethiopian TB patients, their contacts, and community controls suggesting that enhanced IL-4 production is associated with infection with M. tuberculosis, rather than overt disease and that long-term protection in infected community controls is associated with co-production of the IL-4 antagonist IL-4d2, alongside elevated IL-4. Here, for the first time, we compare data on expression of IFN-gamma, IL-4 and IL-4delta2 over time in TB patients and their household contacts. During the follow-up period, the TB patients completed therapy and ceased to display TB-like symptoms. This correlated with a decrease in the relative amount of IL-4 expressed. Over the same period, the clinical status of some of their contacts also changed, with a number developing TB-like symptoms or clinically apparent TB. IL-4 expression was disproportionately increased in this group. The findings support the hypothesis that elevated IL-4 production is generally associated with infection, but that TB disease is associated with a relatively increased expression of IL-4 compared to IFN-gamma and IL-4delta2. However, the data also suggest that there are no clear-cut differences between groups: the immune response over time appears to include changes in the expression of IFN-gamma, IL-4 and IL-4delta2, and it is the relative, not absolute levels of cytokine expression that are characteristic of clinical status.     

Immunity against HIV/AIDS, malaria, and tuberculosis during co-infections with neglected infectious diseases: recommendations for the European Union research priorities

Infectious diseases remain a major health and socioeconomic problem in many low-income countries, particularly in sub-Saharan Africa. For many years, the three most devastating diseases, HIV/AIDS, malaria, and tuberculosis (TB) have received most of the world's attention. However, in rural and impoverished urban areas, a number of infectious diseases remain neglected and cause massive suffering. It has been calculated that a group of 13 neglected infectious diseases affects over one billion people, corresponding to a sixth of the world's population. These diseases include infections with different types of worms and parasites, cholera, and sleeping sickness, and can cause significant mortality and severe disabilities in low-income countries. For most of these diseases, vaccines are either not available, poorly effective, or too expensive. Moreover, these neglected diseases often occur in individuals who are also affected by HIV/AIDS, malaria, or TB, making the problem even more serious and indicating that co-infections are the rule rather than the exception in many geographical areas. To address the importance of combating co-infections, scientists from 14 different countries in Africa and Europe met in Addis Ababa, Ethiopia, on September 9-11, 2007. The message coming from these scientists is that the only possibility for winning the fight against infections in low-income countries is by studying, in the most global way possible, the complex interaction between different infections and conditions of malnourishment. The new scientific and technical tools of the post-genomic era can allow us to reach this goal. However, a concomitant effort in improving education and social conditions will be needed to make the scientific findings effective.