Sonic hedgehog is basolaterally sorted from the TGN and transcytosed to the apical domain involving Dispatched-1 at Rab11-ARE.

Hedgehog proteins (Hhs) secretion from apical and/or basolateral domains occurs in different epithelial cells impacting development and tissue homeostasis. Palmitoylation and cholesteroylation attach Hhs to membranes, and Dispatched-1 (Disp-1) promotes their release. How these lipidated proteins are handled by the complex secretory and endocytic pathways of polarized epithelial cells remains unknown. We show that polarized Madin-Darby canine kidney cells address newly synthesized sonic hedgehog (Shh) from the TGN to the basolateral cell surface and then to the apical domain through a transcytosis pathway that includes Rab11-apical recycling endosomes (Rab11-ARE). Both palmitoylation and cholesteroylation contribute to this sorting behavior, otherwise Shh lacking these lipid modifications is secreted unpolarized. Disp-1 mediates first basolateral secretion from the TGN and then transcytosis from Rab11-ARE. At the steady state, Shh predominates apically and can be basolaterally transcytosed. This Shh trafficking provides several steps for regulation and variation in different epithelia, subordinating the apical to the basolateral secretion.

Negative Modulation of Macroautophagy by Stabilized HERPUD1 is Counteracted by an Increased ER-Lysosomal Network With Impact in Drug-Induced Stress Cell Survival.

Macroautophagy and the ubiquitin proteasome system work as an interconnected network in the maintenance of cellular homeostasis. Indeed, efficient activation of macroautophagy upon nutritional deprivation is sustained by degradation of preexisting proteins by the proteasome. However, the specific substrates that are degraded by the proteasome in order to activate macroautophagy are currently unknown. By quantitative proteomic analysis we identified several proteins downregulated in response to starvation independently of ATG5 expression. Among them, the most significant was HERPUD1, an ER membrane protein with low expression and known to be degraded by the proteasome under normal conditions. Contrary, under ER stress, levels of HERPUD1 increased rapidly due to a blockage in its proteasomal degradation. Thus, we explored whether HERPUD1 stability could work as a negative regulator of autophagy. In this work, we expressed a version of HERPUD1 with its ubiquitin-like domain (UBL) deleted, which is known to be crucial for its proteasome degradation. In comparison to HERPUD1-WT, we found the UBL-deleted version caused a negative role on basal and induced macroautophagy. Unexpectedly, we found stabilized HERPUD1 promotes ER remodeling independent of unfolded protein response activation observing an increase in stacked-tubular structures resembling previously described tubular ER rearrangements. Importantly, a phosphomimetic S59D mutation within the UBL mimics the phenotype observed with the UBL-deleted version including an increase in HERPUD1 stability and ER remodeling together with a negative role on autophagy. Moreover, we found UBL-deleted version and HERPUD1-S59D trigger an increase in cellular size, whereas HERPUD1-S59D also causes an increased in nuclear size. Interestingly, ER remodeling by the deletion of the UBL and the phosphomimetic S59D version led to an increase in the number and function of lysosomes. In addition, the UBL-deleted version and phosphomimetic S59D version established a tight ER-lysosomal network with the presence of extended patches of ER-lysosomal membrane-contact sites condition that reveals an increase of cell survival under stress conditions. Altogether, we propose stabilized HERPUD1 downregulates macroautophagy favoring instead a closed interplay between the ER and lysosomes with consequences in drug-cell stress survival.

Phosphatidic acid-PKA signaling regulates p38 and ERK1/2 functions in ligand-independent EGFR endocytosis.

Ligand-independent epidermal growth factor receptor (EGFR) endocytosis is inducible by a variety of stress conditions converging upon p38 kinase. A less known pathway involves phosphatidic acid (PA) signaling toward the activation of type 4 phosphodiesterases (PDE4) that decrease cAMP levels and protein kinase A (PKA) activity. This PA/PDE4/PKA pathway is triggered with propranolol used to inhibit PA hydrolysis and induces clathrin-dependent and clathrin-independent endocytosis, followed by reversible accumulation of EGFR in recycling endosomes. Here we give further evidence of this signaling pathway using biosensors of PA, cAMP, and PKA in live cells and then show that it activates p38 and ERK1/2 downstream the PKA inhibition. Clathrin-silencing and IN/SUR experiments involved the activity of p38 in the clathrin-dependent route, while ERK1/2 mediates clathrin-independent EGFR endocytosis. The PA/PDE4/PKA pathway selectively increases the EGFR endocytic rate without affecting LDLR and TfR constitute endocytosis. This selectiveness is probably because of EGFR phosphorylation, as detected in Th1046/1047 and Ser669 residues. The EGFR accumulates at perinuclear recycling endosomes colocalizing with TfR, fluorescent transferrin, and Rab11, while a small proportion distributes to Alix-endosomes. A non-selective recycling arrest includes LDLR and TfR in a reversible manner. The PA/PDE4/PKA pathway involving both p38 and ERK1/2 expands the possibilities of EGFR transmodulation and interference in cancer.

D-Propranolol Impairs EGFR Trafficking and Destabilizes Mutant p53 Counteracting AKT Signaling and Tumor Malignancy.

Cancer therapy may be improved by the simultaneous interference of two or more oncogenic pathways contributing to tumor progression and aggressiveness, such as EGFR and p53. Tumor cells expressing gain-of-function (GOF) mutants of p53 (mutp53) are usually resistant to EGFR inhibitors and display invasive migration and AKT-mediated survival associated with enhanced EGFR recycling. D-Propranolol (D-Prop), the non-beta blocker enantiomer of propranolol, was previously shown to induce EGFR internalization through a PKA inhibitory pathway that blocks the recycling of the receptor. Here, we first show that D-Prop decreases the levels of EGFR at the surface of GOF mutp53 cells, relocating the receptor towards recycling endosomes, both in the absence of ligand and during stimulation with high concentrations of EGF or TGF-α. D-Prop also inactivates AKT signaling and reduces the invasive migration and viability of these mutp53 cells. Unexpectedly, mutp53 protein, which is stabilized by interaction with the chaperone HSP90 and mediates cell oncogenic addiction, becomes destabilized after D-Prop treatment. HSP90 phosphorylation by PKA and its interaction with mutp53 are decreased by D-Prop, releasing mutp53 towards proteasomal degradation. Furthermore, a single daily dose of D-Prop reproduces most of these effects in xenografts of aggressive gallbladder cancerous G-415 cells expressing GOF R282W mutp53, resulting in reduced tumor growth and extended mice survival. D-Prop then emerges as an old drug endowed with a novel therapeutic potential against EGFR- and mutp53-driven tumor traits that are common to a large variety of cancers.

Altered Glutaminase 1 Activity During Neurulation and Its Potential Implications in Neural Tube Defects.

The neurulation process is regulated by a large amount of genetic and environmental factors that determine the establishment, folding, and fusion of the neural plate to form the neural tube, which develops into the main structure of the central nervous system. A recently described factor involved in this process is glutamate. Through NMDA ionotropic receptor, glutamate modifies intracellular Ca2+ dynamics allowing the oriented cell migration and proliferation, essentials processes in neurulation. Glutamate synthesis depends on the mitochondrial enzyme known as glutaminase 1 (GLS1) that is widely expressed in brain and kidney. The participation of GLS 1 in prenatal neurogenic processes and in the adult brain has been experimentally established, however, its participation in early stages of embryonic development has not been described. The present investigation describes for the first time the presence and functionality of GLS1 in Xenopus laevis embryos during neurulation. Although protein expression levels remains constant, the catalytic activity of GLS1 increases significantly (~66%) between early (stage 12) and middle to late (stages 14-19) neurulation process. Additionally, the use of 6-diazo-5-oxo-L-norleucine (L-DON, competitive inhibitor of glutamine-depend enzymes), reduced significantly the GLS1 specific activity during neurulation (~36%) and induce the occurrence of neural tube defects involving its possible participation in the neural tube closure in Xenopus laevis embryos.

Galectin-8 mediates fibrogenesis induced by cyclosporine in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: During cyclosporine-induced gingival overgrowth, the homeostatic balance of gingival connective tissue is disrupted leading to fibrosis. Galectins are glycan-binding proteins that can modulate a variety of cellular processes including fibrosis in several organs. Here, we study the role of galectin-8 (Gal-8) in the response of gingival connective tissue cells to cyclosporine. METHODS: We used human gingival fibroblasts and mouse NIH3T3 cells treated with recombinant Gal-8 and/or cyclosporine for analyzing specific mRNA and protein levels through immunoblot, real-time polymerase chain reaction, ELISA and immunofluorescence, pull-down with Gal-8-Sepharose for Gal-8-to-cell surface glycoprotein interactions, short hairpin RNA for Gal-8 silencing and Student's t test and ANOVA for statistical analysis. RESULTS: Galectin-8 stimulated type I collagen and fibronectin protein levels and potentiated CTGF protein levels in TGF-ß1-stimulated human gingival fibroblasts. Gal-8 interacted with α5ß1-integrin and type II TGF-ß receptor. Gal-8 stimulated fibronectin protein and mRNA levels, and this response was dependent on FAK activity but not Smad2/3 signaling. Cyclosporine and tumor necrosis factor alpha (TNF-α) increased Gal-8 protein levels. Finally, silencing of galectin-8 in NIH3T3 cells abolished cyclosporine-induced fibronectin protein levels. CONCLUSION: Taken together, these results reveal for the first time Gal-8 as a fibrogenic stimulus exerted through ß1-integrin/FAK pathways in human gingival fibroblasts, which can be triggered by cyclosporine. Further studies should explore the involvement of Gal-8 in human gingival tissues and its role in drug-induced gingival overgrowth.

Fact or Fiction, It Is Time for a Verdict on Vasculogenic Mimicry?

The term vasculogenic mimicry (VM) refers to the capacity of certain cancer cells to form fluid-conducting structures within a tumor in an endothelial cell (EC)-free manner. Ever since its first report by Maniotis in 1999, the existence of VM has been an extremely contentious issue. The overwhelming consensus of the literature suggests that VM is frequently observed in highly aggressive tumors and correlates to lower patient survival. While the presence of VM in vivo in animal and patient tumors are claimed upon the strong positive staining for glycoproteins (Periodic Acid Schiff, PAS), it is by no means universally accepted. More controversial still is the existence of an in vitro model of VM that principally divides the scientific community. Original reports demonstrated that channels or tubes occur in cancer cell monolayers in vitro when cultured in matrigel and that these structures may support fluid movement. However, several years later many papers emerged stating that connections formed between cancer cells grown on matrigel represented VM. We speculate that this became accepted by the cancer research community and now the vast majority of the scientific literature reports both presence and mechanisms of VM based on intercellular connections, not the presence of fluid conducting tubes. In this opinion paper, we call upon evidence from an exhaustive review of the literature and original data to argue that the majority of in vitro studies presented as VM do not correspond to this phenomenon. Furthermore, we raise doubts on the validity of concluding the presence of VM in patient samples and animal models based solely on the presence of PAS+ staining. We outline the requirement for new biomarkers of VM and present criteria by which VM should be defined in vitro and in vivo.

GALECTIN-8 Is a Neuroprotective Factor in the Brain that Can Be Neutralized by Human Autoantibodies.

Galectin-8 (Gal-8) is a glycan-binding protein that modulates a variety of cellular processes interacting with cell surface glycoproteins. Neutralizing anti-Gal-8 antibodies that block Gal-8 functions have been described in autoimmune and inflammatory disorders, likely playing pathogenic roles. In the brain, Gal-8 is highly expressed in the choroid plexus and accordingly has been detected in human cerebrospinal fluid. It protects against central nervous system autoimmune damage through its immune-suppressive potential. Whether Gal-8 plays a direct role upon neurons remains unknown. Here, we show that Gal-8 protects hippocampal neurons in primary culture against damaging conditions such as nutrient deprivation, glutamate-induced excitotoxicity, hydrogen peroxide (H2O2)-induced oxidative stress, and ß-amyloid oligomers (Aßo). This protective action is manifested even after 2 h of exposure to the harmful condition. Pull-down assays demonstrate binding of Gal-8 to selected ß1-integrins, including α3 and α5ß1. Furthermore, Gal-8 activates ß1-integrins, ERK1/2, and PI3K/AKT signaling pathways that mediate neuroprotection. Hippocampal neurons in primary culture produce and secrete Gal-8, and their survival decreases upon incubation with human function-blocking Gal-8 autoantibodies obtained from lupus patients. Despite the low levels of Gal-8 expression detected by real-time PCR in hippocampus, compared with other brain regions, the complete lack of Gal-8 in Gal-8 KO mice determines higher levels of apoptosis upon H2O2 stereotaxic injection in this region. Therefore, endogenous Gal-8 likely contributes to generate a neuroprotective environment in the brain, which might be eventually counteracted by human function-blocking autoantibodies.

Galectin-8 induces partial epithelial-mesenchymal transition with invasive tumorigenic capabilities involving a FAK/EGFR/proteasome pathway in Madin-Darby canine kidney cells.

Epithelial cells can acquire invasive and tumorigenic capabilities through epithelial-mesenchymal-transition (EMT). The glycan-binding protein galectin-8 (Gal-8) activates selective ß1-integrins involved in EMT and is overexpressed by certain carcinomas. Here we show that Gal-8 overexpression or exogenous addition promotes proliferation, migration, and invasion in nontumoral Madin-Darby canine kidney (MDCK) cells, involving focal-adhesion kinase (FAK)-mediated transactivation of the epidermal growth factor receptor (EGFR), likely triggered by α5ß1integrin binding. Under subconfluent conditions, Gal-8-overexpressing MDCK cells (MDCK-Gal-8H) display hallmarks of EMT, including decreased E-cadherin and up-regulated expression of vimentin, fibronectin, and Snail, as well as increased ß-catenin activity. Changes related to migration/invasion included higher expression of α5ß1 integrin, extracellular matrix-degrading MMP13 and urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) protease systems. Gal-8-stimulated FAK/EGFR pathway leads to proteasome overactivity characteristic of cancer cells. Yet MDCK-Gal-8H cells still develop apical/basolateral polarity reverting EMT markers and proteasome activity under confluence. This is due to the opposite segregation of Gal-8 secretion (apical) and ß1-integrins distribution (basolateral). Strikingly, MDCK-Gal-8H cells acquired tumorigenic potential, as reflected in anchorage-independent growth in soft agar and tumor generation in immunodeficient NSG mice. Therefore, Gal-8 can promote oncogenic-like transformation of epithelial cells through partial and reversible EMT, accompanied by higher proliferation, migration/invasion, and tumorigenic properties.

Structural and functional identification of vasculogenic mimicry in vitro.

Vasculogenic mimicry (VM) describes a process by which cancer cells establish an alternative perfusion pathway in an endothelial cell-free manner. Despite its strong correlation with reduced patient survival, controversy still surrounds the existence of an in vitro model of VM. Furthermore, many studies that claim to demonstrate VM fail to provide solid evidence of true hollow channels, raising concerns as to whether actual VM is actually being examined. Herein, we provide a standardized in vitro assay that recreates the formation of functional hollow channels using ovarian cancer cell lines, cancer spheres and primary cultures derived from ovarian cancer ascites. X-ray microtomography 3D-reconstruction, fluorescence confocal microscopy and dye microinjection conclusively confirm the existence of functional glycoprotein-rich lined tubular structures in vitro and demonstrate that many of structures reported in the literature may not represent VM. This assay may be useful to design and test future VM-blocking anticancer therapies.

Serine proteases activity is important for the interaction of nematophagous fungus Duddingtonia flagrans with infective larvae of trichostrongylides and free-living nematodes Panagrellus spp.

The nematode-trapping fungus Duddingtonia flagrans has been studied as a possible control method for gastrointestinal nematodes of livestock animals. These fungi capture and infect the nematode by cuticle penetration, immobilization, and digestion of the internal contents. It has been suggested that this sequence of events occurs by a combination of physical and enzymatical activities. The aim of this study was to investigate the participation of proteolytic enzymatic activity during the interaction of the nematophagous fungus D. flagrans with infective larvae of trichostrongylides and the free-living nematode Panagrellus spp. Protease inhibitors used interfered in the predatory activity of D. flagrans. However, only PMSF significantly reduced the mean number of Panagrellus spp. captured by D. flagrans in comparison with the control. The experiment with fluorogenic substrate showed that maximum urokinase activity during the interaction of the fungus with the infective larvae of trichostrongylides or Panagrellus spp. occurred within 7 or 1 h of incubation, respectively. The protease activity, especially of the serine class, may be important during the interaction between the fungus and nematodes.

Morphological evidence for a permeability barrier in the testis and spermatic duct of Gymnotus carapo (Teleostei: Gymnotidae).

Cell-cell interactions play essential roles in the regulation of gametogenesis. The involvement of junctional complexes in permeability barriers, for example, provides structural and physiological support for male germ-cell development. This study describes morphological characteristics of the reproductive system of Gymnotus carapo, a neo-tropical freshwater fish widely distributed in South and Central America, focusing on the detection of permeability barriers using morphological and biochemical approaches. Ultrastructural analysis of testes treated with the lanthanum nitrate exclusion technique showed that the tracer penetrated the interstitial compartment of the testis, surrounding and appearing within cysts containing spermatogonia and spermatocytes in early stages of meiosis, but was not detected in the spermatid cysts or inside the lumen of spermatogenic tubules. These results suggest the presence of a permeability barrier that is stabilized after meiosis is completed and serves to protect the haploid cells from the vascular system. In the spermatic-duct region, the tracer was obstructed near the lumen of the duct. Junctional complexes and focal tight junctions between adjacent cells were observed in the testis and spermatic duct. Freeze-fracture methods indeed confirmed the presence of tight junctions, which were visualized as parallel rows of individual particles between adjacent cells. More evidence supporting the existence of a permeability barrier was gathered from differences observed in the electrophoretic protein profiles of testis and spermatic-duct fluids compared to blood plasma. Together, these observations demonstrate the existence of a permeability barrier formed by tight junctions in the testis and spermatic duct of G. carapo.

Pathogenicity of lupus anti-ribosomal P antibodies: role of cross-reacting neuronal surface P antigen in glutamatergic transmission and plasticity in a mouse model.

OBJECTIVE: To assess whether autoantibodies against ribosomal P (anti-P), which are possibly pathogenic in neuropsychiatric systemic lupus erythematosus (NPSLE), alter glutamatergic synaptic transmission and to what extent the cross-reacting neuronal surface P antigen (NSPA) is involved. METHODS: We analyzed glutamatergic transmission and long-term potentiation (LTP) mediated by AMPA receptor (AMPAR) and N-methyl-d-aspartate receptor (NMDAR) by field excitatory postsynaptic potential (EPSP) at the CA3-CA1 synapse. AMPAR activation by patch-clamp recordings in primary ventral spinal cord neurons was analyzed. In primary hippocampal neurons, NSPA distribution was assessed by double immunofluorescence, and intracellular calcium changes were evaluated using Fura-2 AM. NSPA-LacZ reporter-knockin mice expressing a truncated NSPA were used to assess NSPA expression pattern and function in the brain using ß-galactosidase staining and comparative electrophysiology, calcium responses, and water maze memory tests. RESULTS: NSPA was expressed in the brain in hippocampal CA1, dentate gyrus and ventral, but not dorsal, CA3 regions, encompassing postsynaptic regions and partial colocalization with NMDAR. Notably, NSPA-LacZ reporter-knockin mice showed impaired memory, and decreased NMDAR activity and LTP, with neurons insensitive to anti-P autoantibodies. Anti-P autoantibodies enhanced CA1 postsynaptic transmission, increasing AMPAR and NMDAR activity and leading to LTP abrogation after prolonged (20-minute) incubation. CONCLUSION: Our findings indicate that the neuronal cell surface target of anti-P, NSPA, is involved in glutamatergic synaptic transmission and plasticity related to memory in the hippocampus, and mediates the deleterious effects of anti-P on these processes. Cognitive impairment, as well as other diffuse NPSLE manifestations, may develop when anti-P autoantibodies have access to brain regions coexpressing NSPA, AMPAR, and NMDAR.

Anti-ribosomal P protein autoantibodies from patients with neuropsychiatric lupus impair memory in mice.

OBJECTIVE: To define whether anti-ribosomal P (anti-P) autoantibodies from patients with neuropsychiatric systemic lupus erythematosus (NPSLE) impair the function of hippocampal neurons that express the neuronal surface P antigen (NSPA) when accessing the brain via circulating blood. METHODS: We used anti-P antibodies from patients with NPSLE and rabbit-generated anti-P and anti-NSPA antibodies. Primary hippocampal neurons from mice were analyzed to determine antibody cell surface binding (double immunofluorescence), intracellular calcium variations (Fura 2 AM), and apoptosis (caspase 3 activation). Hippocampal-dependent spatial flexible memory was assessed in mice subjected to a water maze test 24 hours after an intravenous injection of anti-P or anti-NSPA, using lipopolysaccharide (LPS) to permeate the blood-brain barrier. Presence of antibodies and apoptosis in the hippocampus was studied using immunohistochemistry and TUNEL assays. RESULTS: Hippocampal neurons expressed NSPA on the cell surface, as revealed by anti-P and anti-NSPA staining colocalization, and responded to both anti-P and anti-NSPA by exhibiting increased intracellular calcium levels. Neuronal apoptosis was induced when anti-P was directly injected by stereotaxis into the hippocampus or added to primary cultures. Upon LPS treatment, intravenously injected anti-P impaired memory but did not elicit neuronal apoptosis in the hippocampus, where it was detectable in low amounts. Anti-NSPA antibodies also impaired memory. CONCLUSION: Anti-P antibodies interact with NSPA on the surface of hippocampal neurons leading to apoptotic death or to functional perturbations, results that are likely dependent on the concentration of these antibodies. Circulating anti-P can access the hippocampus and impair memory without requiring neuronal death when the blood-brain barrier is disrupted. NSPA can mediate antibody-driven diffuse brain dysfunction, and anti-P might contribute to the cognitive impairment that is frequently observed in SLE.

Epidermal growth factor receptor endocytic traffic perturbation by phosphatidate phosphohydrolase inhibition: new strategy against cancer.

Epidermal growth factor receptor (EGFR) exaggerated (oncogenic) function is currently targeted in cancer treatment with drugs that block receptor ligand binding or tyrosine kinase activity. Because endocytic trafficking is a crucial regulator of EGFR function, its pharmacological perturbation might provide a new anti-tumoral strategy. Inhibition of phosphatidic acid (PA) phosphohydrolase (PAP) activity has been shown to trigger PA signaling towards type 4 phosphodiesterase (PDE4) activation and protein kinase A inhibition, leading to internalization of empty/inactive EGFR. Here, we used propranolol, its l- and d- isomers and desipramine as PAP inhibitors to further explore the effects of PAP inhibition on EGFR endocytic trafficking and its consequences on EGFR-dependent cancer cell line models. PAP inhibition not only made EGFR inaccessible to stimuli but also prolonged the signaling lifetime of ligand-activated EGFR in recycling endosomes. Strikingly, such endocytic perturbations applied in acute/intermittent PAP inhibitor treatments selectively impaired cell proliferation/viability sustained by an exaggerated EGFR function. Phospholipase D inhibition with FIPI (5-fluoro-2-indolyl des-chlorohalopemide) and PDE4 inhibition with rolipram abrogated both the anti-tumoral and endocytic effects of PAP inhibition. Prolonged treatments with a low concentration of PAP inhibitors, although without detectable endocytic effects, still counteracted cell proliferation, induced apoptosis and decreased anchorage-independent growth of cells bearing EGFR oncogenic influences. Overall, our results show that PAP inhibitors can counteract EGFR oncogenic traits, including receptor overexpression or activating mutations resistant to current tyrosine kinase inhibitors, perturbing EGFR endocytic trafficking and perhaps other as yet unknown processes, depending on treatment conditions. This puts PAP activity forward as a new suitable target against EGFR-driven malignancy.

The p75 neurotrophin receptor evades the endolysosomal route in neuronal cells, favouring multivesicular bodies specialised for exosomal release.

The p75 neurotrophin receptor (p75, also known as NGFR) is a multifaceted signalling receptor that regulates neuronal physiology, including neurite outgrowth, and survival and death decisions. A key cellular aspect regulating neurotrophin signalling is the intracellular trafficking of their receptors; however, the post-endocytic trafficking of p75 is poorly defined. We used sympathetic neurons and rat PC12 cells to study the mechanism of internalisation and post-endocytic trafficking of p75. We found that p75 internalisation depended on the clathrin adaptor protein AP2 and on dynamin. More surprisingly, p75 evaded the lysosomal route at the level of the early endosome, instead accumulating in two different types of endosomes, Rab11-positive endosomes and multivesicular bodies (MVBs) positive for CD63, a marker of the exosomal pathway. Consistently, depolarisation by KCl induced the liberation of previously endocytosed full-length p75 into the extracellular medium in exosomes. Thus, p75 defines a subpopulation of MVBs that does not mature to lysosomes and is available for exosomal release by neuronal cells.

The adaptor protein-1 µ1B subunit expands the repertoire of basolateral sorting signal recognition in epithelial cells.

An outstanding question in protein sorting is why polarized epithelial cells express two isoforms of the µ1 subunit of the AP-1 clathrin adaptor complex: the ubiquitous µ1A and the epithelial-specific µ1B. Previous studies led to the notion that µ1A and µ1B mediate basolateral sorting predominantly from the trans-Golgi network (TGN) and recycling endosomes, respectively. Using improved analytical tools, however, we find that µ1A and µ1B largely colocalize with each other. They also colocalize to similar extents with TGN and recycling endosome markers, as well as with basolateral cargoes transiting biosynthetic and endocytic-recycling routes. Instead, the two isoforms differ in their signal-recognition specificity. In particular, µ1B preferentially binds a subset of signals from cargoes that are sorted basolaterally in a µ1B-dependent manner. We conclude that expression of distinct µ1 isoforms in epithelial cells expands the repertoire of signals recognized by AP-1 for sorting of a broader range of cargoes to the basolateral surface.

Activity of recombinant and natural defensins from Vigna unguiculata seeds against Leishmania amazonensis.

Antimicrobial peptides (AMPs), which are differentiated from other antibiotic peptides, such as gramicidins and polymyxins, because they are synthesized by large enzymatic complex and bear modified amino acids including d-amino acids, are short polymers of l-amino acids synthesized by ribosomes upon which all living organisms rely to defend themselves from invaders or competitor microorganisms. AMPs have received a great deal of attention from the scientific community as potential new drugs for neglected diseases such as Leishmaniasis. In plants, they include several families of compounds, including the plant defensins. The aim of the present study was to improve the expression of recombinant defensin from Vigna unguiculata seeds (Vu-Defr) and to test its activity against Leishmania amazonensis promatigotes. Recombinant expression was performed in LB and TB media and under different conditions. The purification of Vu-Defr was achieved by immobilized metal ion affinity and reversed-phase chromatography. The purified Vu-Defr was analyzed by circular dichroism (CD), and its biological activity was tested against L. amazonenis promastigotes. To demonstrate that the recombinant production of Vu-Defr did not interfere with its fold and biological activity, the results of all experiments were compared with the results from the natural defensin (Vu-Def). The CD spectra of both peptides presented good superimposition indicating that both peptides present very similar secondary structure and that the Vu-Defr was correctly folded. L. amazonensis treated with Vu-Defr led to the elimination of 54.3% and 46.9% of the parasites at 24 and 48h of incubation time, respectively. Vu-Def eliminated 50% and 54.8% of the parasites at 24 and 48 h, respectively. Both were used at a concentration of 100 µg/mL. These results suggested the potential for plant defensins to be used as new antiparasitic substances.

Extracellular glucose increases the coupling capacity of the yeast V H+-ATPase and the resistance of its H+ transport activity to nitrate inhibition.

V H(+)-ATPase has an important role in a variety of key physiological processes. This enzyme is reversibly activated/partly inactivated by the addition/exhaustion of extracellular glucose. The current model of its regulation assumes the reversible disassembly/reassembly of ∼60-70% of the V1 and V0 membrane complexes, which are responsible for ATP hydrolysis and H(+) conductance, respectively. The number of assembled complexes determines the pump activity because disassembled complexes are inactive. The model predicts the identical catalytic properties for the activated and semi-active enzymes molecules. To verify the model predictions we have isolated total membranes from yeast spheroplasts that were pre-incubated either with or without glucose. Nitrate treatment of membranes revealed the similar ATPase inhibition for two enzyme states, suggesting that they have identical structures that are essential for ATP hydrolysis. However, H(+) transport was inhibited more than the ATPase activities, indicating a nitrate uncoupling action, which was significantly higher for the nonactivated enzyme. This finding suggests that the structure of the non-activated enzyme, which is essential for H(+) transport, is less stable than that of the activated enzyme. Moreover, the glucose activation of the pump increases i) its coupling capacity; ii) its K(M) for ATP hydrolysis and ATP affinity for H(+) transport; iii) the Vmax for H(+) transport in comparison with the Vmax for ATP hydrolysis and iv) the immune reactivity of catalytic subunit A and regulatory subunit B by 9.3 and 2.4 times, respectively. The protein content of subunits A and B was not changed by extracellular glucose. We propose that instead of the dissociation/reassociation of complexes V1 and V0, changes in the extracellular glucose concentration cause reversible and asymmetrical modulations in the immune reactivity of subunits A and B by their putative biochemical modifications. This response asymmetrically modulates H(+)-transport and ATP hydrolysis, exhibiting distinct properties for the activated versus non-activated enzymes.

Mobilization of reserve proteins and activities of cysteine peptidases during germinative and post-germinative events of cowpea seeds.

Cysteine peptidases are the best characterized peptidases among those involved with storage protein mobilization during seed germination. In the present work we show two major groups of cysteine peptidase activities, one of higher (55 to 97 kDa) and other with lower (15 to 20 kDa) molecular masses which are temporally activated after 24 and 48 HAI, respectively, in germinating cowpeas. The former group is found both in protein bodies and in cytoplasmic fraction, while the latter is mostly present outside protein bodies. A third cysteine peptidase activity of ~37 kDa was specifically active at quiescent cotyledons and at 12 and 60 hours after imbibition (HAI). Main peptidase activities of albumin fractions were synchronizedly detected with radicle emergence at 36 HAI. Major vicilin mobilization was more pronounced from 60 HAI onwards and steadily increased until 144 HAI, when low levels of the smallest vicilin subunit were present. Cysteine peptidases were susceptible to iodoacetamide, E-64, iodoacetic acid, pCMB and ß-mercaptoethanol, except for the ~37 kDa peptidase, which was not affected by any of the inhibitors. By a two-dimensional native/SDS-PAGE combination it was observed an apparent linear arrangement of protein breakdown products as well as of peptidase activity spots. The finding may indicate a complex set of sequential proteolytic events where peptidases induce or activate new peptidases, which may act upon different aggregates or zymogens, and these hydrolysis products appear in a line of constant decreasing Rf x Mr ratio.