Calpain hydrolysis of alpha- and beta2-adaptins decreases clathrin-dependent endocytosis and may promote neurodegeneration.

Clathrin-dependent endocytosis is mediated by a tightly regulated network of molecular interactions that provides essential protein-protein and protein-lipid binding activities. Here we report the hydrolysis of the alpha- and beta2-subunits of the tetrameric adaptor protein complex 2 by calpain. Calcium-dependent alpha- and beta2-adaptin hydrolysis was observed in several rat tissues, including brain and primary neuronal cultures. Neuronal alpha- and beta2-adaptin cleavage was inducible by glutamate stimulation and was accompanied by the decreased endocytosis of transferrin. Heterologous expression of truncated forms of the beta2-adaptin subunit significantly decreased the membrane recruitment of clathrin and inhibited clathrin-mediated receptor endocytosis. Moreover, the presence of truncated beta2-adaptin sensitized neurons to glutamate receptor-mediated excitotoxicity. Proteolysis of alpha- and beta2-adaptins, as well as the accessory clathrin adaptors epsin 1, adaptor protein 180, and the clathrin assembly lymphoid myeloid leukemia protein, was detected in brain tissues after experimentally induced ischemia and in cases of human Alzheimer disease. The present study further clarifies the central role of calpain in regulating clathrin-dependent endocytosis and provides evidence for a novel mechanism through which calpain activation may promote neurodegeneration: the sensitization of cells to glutamate-mediated excitotoxicity via the decreased internalization of surface receptors.

Plasmodium yoelii: identification of a gene encoding a putative ADP-ribosylation factor-1 GTPase-activating Protein, PyAG1

The PyAG1 gene, identified by the screening of a Plasmodium yoelii genomic DNA library with a rhoptry-specific Mab, encodes a protein with a zinc finger structure immediately followed by the consensus sequence of the Arf GAP catalytic site. The serum of mice immunized with the recombinant protein recognized specifically the rhoptries of the late infected erythrocytic stages. Blast analysis using the Genbank database gave the highest scores with four proteins presenting an Arf1 GAP activity. If presenting also this activity, the PyAG1 protein could be involved in the regulation of the secreted protein vesicular transport and, consequently, in the rhoptry biogenesis.

Microautophagy of the nucleus coincides with a vacuolar diffusion barrier at nuclear-vacuolar junctions.

Nuclei bind yeast vacuoles via nucleus-vacuole (NV) junctions. Under nutrient restriction, NV junctions invaginate and release vesicles filled with nuclear material into vacuoles, resulting in piecemeal microautophagy of the nucleus (PMN). We show that the electrochemical gradient across the vacuolar membrane promotes invagination of NV junctions. Existing invaginations persist independently of the gradient, but final release of PMN vesicles requires again V-ATPase activity. We find that NV junctions form a diffusion barrier on the vacuolar membrane that excludes V-ATPase but is enriched in the VTC complex and accessible to other membrane-integral proteins. V-ATPase exclusion depends on the NV junction proteins Nvj1p,Vac8p, and the electrochemical gradient. It also depends on factors of lipid metabolism, such as the oxysterol binding protein Osh1p and the enoyl-CoA reductase Tsc13p, which are enriched in NV junctions, and on Lag1p and Fen1p. Our observations suggest that NV junctions form in two separable steps: Nvj1p and Vac8p suffice to establish contact between the two membranes. The electrochemical potential and lipid-modifying enzymes are needed to establish the vacuolar diffusion barrier, invaginate NV junctions, and form PMN vesicles.

Mechanisms of eosinophil cytokine release

Human eosinophils have been demonstrated to contain a multitude of cytokines and chemokines that exist pre-formed within these cells. This content of pre-formed cytokines, with diverse potential biologic activities, provides eosinophils with capabilities distinct from most other leukocytes. The localization of pre-formed cytokines within eosinophils is both within specific granules and associated with substantial numbers of morphologically distinct cytoplasmic vesicles. Stimulation for release of specific cytokines, such as IL-4, leads to a regulated signal transduction cascade, which is dependent on the formation of leukotriene C4 within eosinophils where it acts as an intracrine mediator. IL-4 release occurs selectively and is by means of vesicular transport. The capabilities of eosinophils not only to rapidly release pre-formed cytokines but also to differentially regulate which cytokines are released endow eosinophils with distinct abilities in innate and acquired immunity.

Expression of prox1, lymphatic endothelial nuclear transcription factor, in Kaposiform hemangioendothelioma and tufted angioma.

Kaposiform hemangioendothelioma (KHE) and tufted angioma (TA) are rare tumors mainly occurring in early childhood. Our recent results showed that ectopic overexpression of human Prox1 gene, a lymphatic endothelial nuclear transcription factor, promoted an aggressive behavior in 2 murine models of KHE. This dramatic Prox1-induced phenotype prompted us to investigate immunohistochemical staining pattern of Prox1, podoplanin (D2-40), LYVE-1, and Prox1/CD34 as well as double immunofluorescent staining pattern of LYVE-1/CD31 in KHE and TA, compared with other pediatric vascular tumors. For this purpose, we examined 75 vascular lesions: KHE (n=18), TA (n=13), infantile hemangioma (n=13), pyogenic granuloma (n=18), and granulation tissue (n=13). Overall, KHE and TA shared an identical endothelial immunophenotype: the neoplastic spindle cells were Prox1, podoplanin, LYVE-1, CD31, and CD34, whereas endothelial cells within glomeruloid foci were Prox1, podoplanin, LYVE-1, CD31, and CD34. The lesional cells of all infantile hemangiomas and pyogenic granulomas were negative for Prox1 in the presence of positive internal control. These findings provide immunophenotypic evidence to support a preexisting notion that KHE and TA are closely related, if not identical. Overall, our results show, for the first time, that Prox1 is an immunohistochemical biomarker helpful in confirming the diagnosis of KHE/TA and in distinguishing it from infantile hemangioma and pyogenic granuloma.

Autophagy controls IL-1beta secretion by targeting pro-IL-1beta for degradation.

Autophagy is a key regulator of cellular homeostasis that can be activated by pathogen-associated molecules and recently has been shown to influence IL-1β secretion by macrophages. However, the mechanisms behind this are unclear. Here, we describe a novel role for autophagy in regulating the production of IL-1β in antigen-presenting cells. After treatment of macrophages with Toll-like receptor ligands, pro-IL-1β was specifically sequestered into autophagosomes, whereas further activation of autophagy with rapamycin induced the degradation of pro-IL-1β and blocked secretion of the mature cytokine. Inhibition of autophagy promoted the processing and secretion of IL-1β by antigen-presenting cells in an NLRP3- and TRIF-dependent manner. This effect was reduced by inhibition of reactive oxygen species but was independent of NOX2. Induction of autophagy in mice in vivo with rapamycin reduced serum levels of IL-1β in response to challenge with LPS. These data demonstrate that autophagy controls the production of IL-1β through at least two separate mechanisms: by targeting pro-IL-1β for lysosomal degradation and by regulating activation of the NLRP3 inflammasome.

The high road and the low road: trafficking choices in plants.

Polar transport of the signaling molecule auxin is critical for plant development and depends on both the polar distribution of auxin efflux carriers, which pump auxin out of the cell and the alignment of these polarized cells. Two papers in this issue of Cell (Michniewicz et al., 2007; Jaillais et al., 2007) address how polar transport of these carriers occurs and describe the endosomal pathways involved.

Regulation of the intracellular distribution, cell surface expression, and protein levels of AMPA receptor GluR2 subunits by the monocarboxylate transporter MCT2 in neuronal cells.

The neuronal monocarboxylate transporter, MCT2, is not only an energy substrate carrier but it is also purported to be a binding partner for the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR2 subunit. To unravel a putative role of MCT2 in the regulation of GluR2 subcellular distribution, Neuro2A cells and primary cultures of mouse cortical neurons were co-transfected with plasmids containing sequences to express the fluorescent proteins mStrawberry (mStb)-fused MCT2 and Venus-fused GluR2. Subsequently, their subcellular distribution was visualized by fluorescence microscopy. GluR2 was led to form perinuclear and dendritic clusters together with MCT2 when co-transfected in Neuro2A cells or in neurons, following the original distribution of MCT2. MCT2 co-transfection had no effect on the intracellular distribution of several other post-synaptic proteins, although it partially affected the intracellular distribution of GluR1 similarly to GluR2. Both cell surface and total protein expression levels of GluR2 were significantly reduced by co-expression with MCT2. Finally, partial perinuclear and dendritic co-localization between MCT2 and Rab8, a member of the small GTPase family involved in membrane trafficking of AMPA receptors, was also observed in co-transfected neurons. These results suggest that MCT2 could influence AMPA receptor trafficking within neurons by modulating GluR2 sorting between different subcellular compartments.

Regulation of the expression of components of the exocytotic machinery of insulin-secreting cells by microRNAs

Fine-tuning of insulin secretion from pancreatic beta-cells participates in blood glucose homeostasis. Defects in this process can lead to chronic hyperglycemia and diabetes mellitus. Several proteins controlling insulin exocytosis have been identified, but the mechanisms regulating their expression remain poorly understood. Here, we show that two non-coding microRNAs, miR124a and miR96, modulate the expression of proteins involved in insulin exocytosis and affect secretion of the beta-cell line MIN6B1. miR124a increases the levels of SNAP25, Rab3A and synapsin-1A and decreases those of Rab27A and Noc2. Inhibition of Rab27A expression is mediated by direct binding to the 3'-untranslated region of Rab27A mRNA. The effect on the other genes is indirect and linked to changes in mRNA levels. Over-expression of miR124a leads to exaggerated hormone release under basal conditions and a reduction in glucose-induced secretion. miR96 increases mRNA and protein levels of granuphilin, a negative modulator of insulin exocytosis, and decreases the expression of Noc2, resulting in lower capacity of MIN6B1 cells to respond to secretagogues. Our data identify miR124a and miR96 as novel regulators of the expression of proteins playing a critical role in insulin exocytosis and in the release of other hormones and neurotransmitters

Genetic evidence that the higher plant Rab-D1 and Rab-D2 GTPases exhibit distinct but overlapping interactions in the early secretory pathway.

GTPases of the Rab1 subclass are essential for membrane traffic between the endoplasmic reticulum (ER) and Golgi complex in animals, fungi and plants. Rab1-related proteins in higher plants are unusual because sequence comparisons divide them into two putative subclasses, Rab-D1 and Rab-D2, that are conserved in monocots and dicots. We tested the hypothesis that the Rab-D1 and Rab-D2 proteins of Arabidopsis represent functionally distinct groups. RAB-D1 and RAB-D2a each targeted fluorescent proteins to the same punctate structures associated with the Golgi stacks and trans-Golgi-network. Dominant-inhibitory N121I mutants of each protein inhibited traffic of diverse cargo proteins at the ER but they appeared to act via distinct biochemical pathways as biosynthetic traffic in cells expressing either of the N121I mutants could be restored by coexpressing the wild-type form of the same subclass but not the other subclass. The same interaction was observed in transgenic seedlings expressing RAB-D1 [N121I]. Insertional mutants confirmed that the three Arabidopsis Rab-D2 genes were extensively redundant and collectively performed an essential function that could not be provided by RAB-D1, which was non-essential. However, plants lacking RAB-D1, RAB-D2b and RAB-D2c were short and bushy with low fertility, indicating that the Rab-D1 and Rab-D2 subclasses have overlapping functions.

Characterization of a protein of the plastid inner envelope having homology to animal inorganic phosphate, chloride and organic-anion transporters.

A protein from Arabidopsis thaliana (L.) Heynh. showing homology to animal proteins of the NaPi-1 family, involved in the transport of inorganic phosphate, chloride, glutamate and sialic acid, has been characterized. This protein, named ANTR2 (for anion transporters) was shown by chloroplast subfractionation to be localized to the plastid inner envelope in both A. thaliana and Spinacia oleracea (L.). Immunolocalization revealed that ANTR2 was expressed in the leaf mesophyll cells as well as in the developing embryo at the upturned-U stage. Five additional homologues of ANTR2 are found in the Arabidopsis genome, of which one was shown by green fluorescent protein (GFP) fusion to be also located in the chloroplast. All ANTR proteins share homology to the animal NaPi-1 family, as well as to other organic-anion transporters that are members of the Anion:Cation Symporter (ACS) family, and share the main features of transporters from this family, including the presence of 12 putative transmembrane domains and of a 7-amino acid motif in the fourth putative transmembrane domain. ANTR2 thus represent a novel protein of the plastid inner envelope that is likely to be involved in anion transport.

Munc18c in adipose tissue is downregulated in obesity and is associated with insulin.

OBJECTIVE Munc18c is associated with glucose metabolism and could play a relevant role in obesity. However, little is known about the regulation of Munc18c expression. We analyzed Munc18c gene expression in human visceral (VAT) and subcutaneous (SAT) adipose tissue and its relationship with obesity and insulin. MATERIALS AND METHODS We evaluated 70 subjects distributed in 12 non-obese lean subjects, 23 overweight subjects, 12 obese subjects and 23 nondiabetic morbidly obese patients (11 with low insulin resistance and 12 with high insulin resistance). RESULTS The lean, overweight and obese persons had a greater Munc18c gene expression in adipose tissue than the morbidly obese patients (p<0.001). VAT Munc18c gene expression was predicted by the body mass index (B = -0.001, p = 0.009). In SAT, no associations were found by different multiple regression analysis models. SAT Munc18c gene expression was the main determinant of the improvement in the HOMA-IR index 15 days after bariatric surgery (B = -2148.4, p = 0.038). SAT explant cultures showed that insulin produced a significant down-regulation of Munc18c gene expression (p = 0.048). This decrease was also obtained when explants were incubated with liver X receptor alpha (LXRα) agonist, either without (p = 0.038) or with insulin (p = 0.050). However, Munc18c gene expression was not affected when explants were incubated with insulin plus a sterol regulatory element-binding protein-1c (SREBP-1c) inhibitor (p = 0.504). CONCLUSIONS Munc18c gene expression in human adipose tissue is down-regulated in morbid obesity. Insulin may have an effect on the Munc18c expression, probably through LXRα and SREBP-1c.

The E3 ubiquitin ligase Itch regulates sorting nexin 9 through an unconventional substrate recognition domain.

The level of intracellular proteins is mainly regulated through modifications by ubiquitin ligases that target them for degradation. Members of the NEDD4 family of E3 ubiquitin ligases, such as Itch (atrophin-1 interacting protein 4), possess up to four WW domains for specific association with PY motif-containing substrates. We have identified sorting nexin 9 (SNX9), a protein involved in endocytic processes, as a new substrate of Itch. Itch ubiquitylates SNX9 and regulates intracellular SNX9 levels. Using truncated proteins, we found that the interaction with SNX9 is mediated by the proline-rich domain (PRD) of Itch, a domain distinct from the conventional WW recognition domain, and the SH3 domain of SNX9. Interaction with the PRD of Itch is essential for SNX9 ubiquitylation and degradation. Furthermore, this effect is specific for Itch, as NEDD4, a related PRD-containing E3 ligase, does not bind SNX9. SNX18, a second member of the SNX family containing an SH3 domain, was also found to bind to Itch. Our results indicate that the pool of substrates of NEDD4 family E3 ubiquitin ligases extends beyond proteins containing PY motifs.

Cell-free reconstitution of vacuole membrane fragmentation reveals regulation of vacuole size and number by TORC1.

Size and copy number of organelles are influenced by an equilibrium of membrane fusion and fission. We studied this equilibrium on vacuoles-the lysosomes of yeast. Vacuole fusion can readily be reconstituted and quantified in vitro, but it had not been possible to study fission of the organelle in a similar way. Here we present a cell-free system that reconstitutes fragmentation of purified yeast vacuoles (lysosomes) into smaller vesicles. Fragmentation in vitro reproduces physiological aspects. It requires the dynamin-like GTPase Vps1p, V-ATPase pump activity, cytosolic proteins, and ATP and GTP hydrolysis. We used the in vitro system to show that the vacuole-associated TOR complex 1 (TORC1) stimulates vacuole fragmentation but not the opposing reaction of vacuole fusion. Under nutrient restriction, TORC1 is inactivated, and the continuing fusion activity then dominates the fusion/fission equilibrium, decreasing the copy number and increasing the volume of the vacuolar compartment. This result can explain why nutrient restriction not only induces autophagy and a massive buildup of vacuolar/lysosomal hydrolases, but also leads to a concomitant increase in volume of the vacuolar compartment by coalescence of the organelles into a single large compartment.

Changes in microRNA expression contribute to pancreatic β-cell dysfunction in prediabetic NOD mice.

During the initial phases of type 1 diabetes, pancreatic islets are invaded by immune cells, exposing β-cells to proinflammatory cytokines. This unfavorable environment results in gene expression modifications leading to loss of β-cell functions. To study the contribution of microRNAs (miRNAs) in this process, we used microarray analysis to search for changes in miRNA expression in prediabetic NOD mice islets. We found that the levels of miR-29a/b/c increased in islets of NOD mice during the phases preceding diabetes manifestation and in isolated mouse and human islets exposed to proinflammatory cytokines. Overexpression of miR-29a/b/c in MIN6 and dissociated islet cells led to impairment in glucose-induced insulin secretion. Defective insulin release was associated with diminished expression of the transcription factor Onecut2, and a consequent rise of granuphilin, an inhibitor of β-cell exocytosis. Overexpression of miR-29a/b/c also promoted apoptosis by decreasing the level of the antiapoptotic protein Mcl1. Indeed, a decoy molecule selectively masking the miR-29 binding site on Mcl1 mRNA protected insulin-secreting cells from apoptosis triggered by miR-29 or cytokines. Taken together, our findings suggest that changes in the level of miR-29 family members contribute to cytokine-mediated β-cell dysfunction occurring during the initial phases of type 1 diabetes.