Consideraciones anestésicas de la cirugía de la escoliosis grave en la edad pediátrica / On anesthesia for pediatric surgery to treat severe scoliosis

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Sorting of mannose 6-phosphate receptors mediated by the GGAs.

The delivery of soluble hydrolases to lysosomes is mediated by the cation-independent and cation-dependent mannose 6-phosphate receptors. The cytosolic tails of both receptors contain acidic-cluster-dileucine signals that direct sorting from the trans-Golgi network to the endosomal-lysosomal system. We found that these signals bind to the VHS domain of the Golgi-localized, gamma-ear-containing, ARF-binding proteins (GGAs). The receptors and the GGAs left the trans-Golgi network on the same tubulo-vesicular carriers. A dominant-negative GGA mutant blocked exit of the receptors from the trans-Golgi network. Thus, the GGAs appear to mediate sorting of the mannose 6-phosphate receptors at the trans-Golgi network.

An intact dilysine-like motif in the carboxyl terminus of MAL is required for normal apical transport of the influenza virus hemagglutinin cargo protein in epithelial Madin-Darby canine kidney cells.

The MAL proteolipid, a component of the integral protein sorting machinery, has been demonstrated as being necessary for normal apical transport of the influenza virus hemagglutinin (HA) and the overall apical membrane proteins in Madin-Darby canine kidney (MDCK) cells. The MAL carboxy terminus ends with the sequence Arg-Trp-Lys-Ser-Ser (RWKSS), which resembles dilysine-based motifs involved in protein sorting. To investigate whether the RWKSS pentapeptide plays a role in modulating the distribution of MAL and/or its function in apical transport, we have expressed MAL proteins with distinct carboxy terminus in MDCK cells whose apical transport was impaired by depletion of endogenous MAL. Apical transport of HA was restored to normal levels by expression of MAL with an intact but not with modified carboxyl terminal sequences bearing mutations that impair the functioning of dilysine-based sorting signals, although all the MAL proteins analyzed incorporated efficiently into lipid rafts. Ultrastructural analysis indicated that compared with MAL bearing an intact RWKSS sequence, a mutant with lysine -3 substituted by serine showed a twofold increased presence in clathrin-coated cytoplasmic structures and a reduced expression on the plasma membrane. These results indicate that the carboxyl-terminal RWKSS sequence modulates the distribution of MAL in clathrin-coated elements and is necessary for HA transport to the apical surface.

The GGAs promote ARF-dependent recruitment of clathrin to the TGN.

The GGAs constitute a family of modular adaptor-related proteins that bind ADP-ribosylation factors (ARFs) and localize to the trans-Golgi network (TGN) via their GAT domains. Here, we show that binding of the GAT domain stabilizes membrane-bound ARF1.GTP due to interference with the action of GTPase-activating proteins. We also show that the hinge and ear domains of the GGAs interact with clathrin in vitro, and that the GGAs promote recruitment of clathrin to liposomes in vitro and to TGN membranes in vivo. These observations suggest that the GGAs could function to link clathrin to membrane-bound ARF.GTP.

BENE, a novel raft-associated protein of the MAL proteolipid family, interacts with caveolin-1 in human endothelial-like ECV304 cells.

The MAL proteolipid, an integral protein present in glycolipid- and cholesterol-enriched membrane (GEM) rafts, is an element of the machinery necessary for apical sorting in polarized epithelial Madin-Darby canine kidney cells. MAL was the first member identified of an extended family of proteins that have significant overall sequence identity. In this study we have used a newly generated monoclonal antibody to investigate an unedited member of this family, named BENE, which was found to be expressed in endothelial-like ECV304 cells and normal human endothelium. Human BENE was characterized as a proteolipid protein predominantly present in GEM rafts in ECV304 cells. Coimmunoprecipitation experiments revealed that BENE interacted with caveolin-1. Confocal immunofluorescence and electron microscopic analyses indicated that BENE mainly accumulated into intracellular vesicular/tubular structures that partially colocalize with internal caveolin-1. In response to cell surface cholesterol oxidation, BENE redistributed to the dilated vesicular structures that concentrate most of the caveolin-1 originally on the cell surface. After cessation of cholesterol oxidation, a detectable fraction of the BENE molecules migrated to the plasmalemma accompanying caveolin-1 and then returned progressively to its steady state distribution. Together, these features highlight the BENE proteolipid as being an element of the machinery for raft-mediated trafficking in endothelial cells.

The MAL proteolipid is necessary for the overall apical delivery of membrane proteins in the polarized epithelial Madin-Darby canine kidney and fischer rat thyroid cell lines.

The MAL proteolipid has been recently demonstrated as being necessary for correct apical sorting of the transmembrane influenza virus hemagglutinin (HA) in Madin-Darby canine kidney (MDCK) cells. The fact that, in contrast to MDCK cells, Fischer rat thyroid (FRT) cells target the majority of glycosylphosphatidylinositol (GPI)-anchored proteins to the basolateral membrane provides us with the opportunity to determine the role of MAL in apical transport of membrane proteins under conditions in which the majority of GPI-anchored proteins are (MDCK cells) or are not (FRT cells) targeted to the apical surface. Using an antisense oligonucleotide-based strategy to deplete endogenous MAL, we have observed that correct transport of apical transmembrane proteins associated (HA) or not (exogenous neurotrophin receptor and endogenous dipeptidyl peptidase IV) with lipid rafts, as well as that of the bulk of endogenous apical membrane, takes place in FRT cells by a pathway that requires normal MAL levels. Even transport of placental alkaline phosphatase, a GPI-anchored protein that is targeted apically in FRT cells, was dependent on normal MAL levels. Similarly, in addition to the reported effect of MAL on HA transport, depletion of MAL in MDCK cells caused a dramatic reduction in the apical delivery of the GPI-anchored gD1-DAF protein, neurotrophin receptor, and the bulk of membrane proteins. These results suggest that MAL is necessary for the overall apical transport of membrane proteins in polarized MDCK and FRT cells.

GGAs: a family of ADP ribosylation factor-binding proteins related to adaptors and associated with the Golgi complex.

Formation of intracellular transport intermediates and selection of cargo molecules are mediated by protein coats associated with the cytosolic face of membranes. Here, we describe a novel family of ubiquitous coat proteins termed GGAs, which includes three members in humans and two in yeast. GGAs have a modular structure consisting of a VHS domain, a region of homology termed GAT, a linker segment, and a region with homology to the ear domain of gamma-adaptins. Immunofluorescence microscopy showed colocalization of GGAs with Golgi markers, whereas immunoelectron microscopy of GGA3 revealed its presence on coated vesicles and buds in the area of the TGN. Treatment with brefeldin A or overexpression of dominant-negative ADP ribosylation factor 1 (ARF1) caused dissociation of GGAs from membranes. The GAT region of GGA3 was found to: target a reporter protein to the Golgi complex; induce dissociation from membranes of ARF-regulated coats such as AP-1, AP-3, AP-4, and COPI upon overexpression; and interact with activated ARF1. Disruption of both GGA genes in yeast resulted in impaired trafficking of carboxypeptidase Y to the vacuole. These observations suggest that GGAs are components of ARF-regulated coats that mediate protein trafficking at the TGN.

Incorporation of MAL, an integral protein element of the machinery for the glycolipid and cholesterol-mediated apical pathway of transport, into artificial membranes requires neither of these lipid species.

The MAL proteolipid, an integral membrane protein with selective residence in glycolipid- and cholesterol-enriched membrane (GEM) microdomains, has recently been identified as being an element of the integral protein machinery necessary for apical transport in MDCK cells. With the use of a recombinant baculovirus, we have expressed and purified polyhistidine-tagged MAL to determine whether MAL has special lipid requirements for becoming incorporated into membranes. In contrast with caveolin-1, a component of GEMs that requires cholesterol for its integration into artificial membranes, MAL incorporation took place with dimyristoylphosphatidylcholine as the only lipid component. The presence of cholesterol, sphingomyelin, or galactocerebrosides did not affect the efficiency of this process. These results indicated that MAL is compatible with membranes containing either only phospholipids or also glycolipids and cholesterol and are consistent with the reported requirement of a sorting event for the specific targeting of MAL to GEM microdomains.

MAL, an integral element of the apical sorting machinery, is an itinerant protein that cycles between the trans-Golgi network and the plasma membrane.

The MAL proteolipid is a nonglycosylated integral membrane protein found in glycolipid-enriched membrane microdomains. In polarized epithelial Madin-Darby canine kidney cells, MAL is necessary for normal apical transport and accurate sorting of the influenza virus hemagglutinin. MAL is thus part of the integral machinery for glycolipid-enriched membrane-mediated apical transport. At steady state, MAL is predominantly located in perinuclear vesicles that probably arise from the trans-Golgi network (TGN). To act on membrane traffic and to prevent their accumulation in the target compartment, integral membrane elements of the protein-sorting machinery should be itinerant proteins that cycle between the donor and target compartments. To establish whether MAL is an itinerant protein, we engineered the last extracellular loop of MAL by insertion of sequences containing the FLAG epitope or with sequences containing residues that became O-glycosylated within the cells or that displayed biotinylatable groups. The ectopic expression of these modified MAL proteins allowed us to investigate the surface expression of MAL and its movement through different compartments after internalization with the use of a combination of assays, including surface biotinylation, surface binding of anti-FLAG antibodies, neuraminidase sensitivity, and drug treatments. Immunofluorescence and flow cytometric analyses indicated that, in addition to its Golgi localization, MAL was also expressed on the cell surface, from which it was rapidly internalized. This retrieval implies transport through the endosomal pathway and requires endosomal acidification, because it can be inhibited by drugs such as chloroquine, monensin, and NH(4)Cl. Resialylation experiments of surface MAL treated with neuraminidase indicated that approximately 30% of the internalized MAL molecules were delivered to the TGN, probably to start a new cycle of cargo transport. Together, these observations suggest that, as predicted for integral membrane members of the late protein transport machinery, MAL is an itinerant protein cycling between the TGN and the plasma membrane.

Substitution of the two carboxyl-terminal serines by alanine causes retention of MAL, a component of the apical sorting machinery, in the endoplasmic reticulum.

MAL, a selective resident of glycolipid-enriched membranes (GEMs), is an integral membrane protein necessary for apical transport and accurate sorting of the influenza virus hemagglutinin in MDCK cells. The carboxyl-terminal end of MAL has the sequence Phe-Ser-Leu-Ile-Arg-Trp-Lys-Ser-Ser (FSLIRWKSS), which includes the LIRW motif necessary for sorting MAL to GEMs, and whose last five amino acids resemble dilysine-based signals involved in endoplasmic reticulum (ER) retention. We have addressed the influence of the carboxyl-terminal serines in both MAL distribution and incorporation into GEMs. Substitution of the serines by alanine impeded the access of MAL to GEMs and changed its distribution from a perinuclear distribution to an ER pattern. The RWKSS sequence appended to the carboxyl-terminus of CD4 caused retention of the chimera in the ER. Thus, although this pentapeptide can function producing ER retention in other protein context, the presence of the carboxyl-terminal serines in the intact MAL molecule prevents its use as an ER-retention signal.

The MAL proteolipid is necessary for normal apical transport and accurate sorting of the influenza virus hemagglutinin in Madin-Darby canine kidney cells.

The MAL (MAL/VIP17) proteolipid is a nonglycosylated integral membrane protein expressed in a restricted pattern of cell types, including T lymphocytes, myelin-forming cells, and polarized epithelial cells. Transport of the influenza virus hemagglutinin (HA) to the apical surface of epithelial Madin-Darby canine kidney (MDCK) cells appears to be mediated by a pathway involving glycolipid- and cholesterol- enriched membranes (GEMs). In MDCK cells, MAL has been proposed previously as being an element of the protein machinery for the GEM-dependent apical transport pathway. Using an antisense oligonucleotide-based strategy and a newly generated monoclonal antibody to canine MAL, herein we have approached the effect of MAL depletion on HA transport in MDCK cells. We have found that MAL depletion diminishes the presence of HA in GEMs, reduces the rate of HA transport to the cell surface, inhibits the delivery of HA to the apical surface, and produces partial missorting of HA to the basolateral membrane. These effects were corrected by ectopic expression of MAL in MDCK cells whose endogenous MAL protein was depleted. Our results indicate that MAL is necessary for both normal apical transport and accurate sorting of HA.

Targeting of MAL, a putative element of the apical sorting machinery, to glycolipid-enriched membranes requires a pre-golgi sorting event.

The MAL proteolipid is a nonglycosylated polytopic membrane protein with specific residence in glycolipid-enriched membrane (GEM) microdomains. MAL has been proposed as an element of the machinery for apical transport in polarized epithelial cells. Previous work demonstrated that MAL requires its four carboxyl-terminal amino acids to be targeted to GEMs. In the present work, we have engineered MAL with N-glycosylation consensus sequences to delimit the site at which commitment of MAL to access into GEMs takes place. Comparison of engineered MAL proteins bearing either an intact or a truncated carboxyl terminus revealed that whereas the former acquired endo H-sensitive and endo H-resistant mature glycosylation, the protein with a deleted carboxyl terminus did not. These results indicate that although MAL incorporation into GEMs takes place mainly in the Golgi, commitment of MAL to enter GEMs is a pre-Golgi event.

A short peptide motif at the carboxyl terminus is required for incorporation of the integral membrane MAL protein to glycolipid-enriched membranes.

The MAL (VIP17, MVP17) proteolipid, an integral membrane protein with specific residence in glycolipid-enriched membrane (GEM) microdomains, has been recently proposed as a component of the protein machinery for GEM vesiculation. In this work, we have searched the COOH terminus of MAL for sorting determinants responsible for targeting to GEMs. This has allowed the identification of the sequence Leu-Ile-Arg-Trp (LIRW) as necessary for the access of MAL to GEMs. This motif requires at least one additional amino acid at its COOH end for full effectiveness. The arginine within the LIRW motif is the most crucial residue for targeting to GEMs, tryptophan replacement affects targeting to a lesser extent, and the leucine-isoleucine pair tolerates substitution by valine, but not by alanine, without effect on targeting. Pulse-chase experiments indicate that the LIRW tetrapeptide is required for access to GEMs early after MAL biosynthesis. Interestingly, the loss of the capacity of the MAL protein to be incorporated into GEMs correlated with the loss of its response to brefeldin A treatment. This is the first identification of a juxtamembrane peptide motif required for incorporation of an integral membrane protein into GEMs.

Recombinant expression of the MAL proteolipid, a component of glycolipid-enriched membrane microdomains, induces the formation of vesicular structures in insect cells.

The MAL proteolipid has been identified as a component of glycolipid-enriched membrane microdomains resistant to detergent solubilization in epithelial Madin-Darby canine cells, as well as in T lymphocytes and in myelin-forming cells. To study the function of the MAL proteolipid we have ectopically expressed a tagged form of MAL in both mammalian and insect cellular backgrounds. Immunofluorescence analysis in transiently transfected COS-7 cells showed the presence of MAL in large vesicular structures, and biochemical analysis identified MAL in the fraction of membranes resistant to Triton X-100 solubilization. Electron microscopic analysis showed that the expression of MAL in Sf21 cells morphologically resulted in the intracellular accumulation of large vesicles with a diameter from 200 to greater than 700 nm that were absent in uninfected or control infected cultures. Thus, ectopic expression of MAL in this heterologous expression system was sufficient to drive the formation of vesicles with a size similar to that of the vesicles detected in mammalian cells. These vesicles were clearly different from the caveolae-like vesicles induced by caveolin expression, as evidenced by co-infection experiments using a recombinant caveolin baculovirus. Taken together, these results suggest that the MAL proteolipid might play a role as a component of the machinery of vesiculation of glycolipid-enriched membranes.

A study of genetic association between manic-depressive illness and a highly polymorphic marker from the GABRbeta-1 gene.

We report on an association study between a tetranucleotide repeat polymorphism in the GABR beta1 gene and manic-depressive illness in a Spanish population. This gene may be an important candidate for bipolar affective disorders since severe GABergic alterations have been described in patients. Although our results do not reveal a clear evidence for association between manic-depressive illness and GABR beta1, we have found significant differences between patients and controls in the female subpopulation.

Caveolin and MAL, two protein components of internal detergent-insoluble membranes, are in distinct lipid microenvironments in MDCK cells.

The MAL proteolipid and caveolin have been identified as components of internal detergent-insoluble membrane microdomains enriched in glycolipids and cholesterol. We have addressed the study of the glycolipid-enriched membranes in cells expressing endogenously only either MAL (Jurkat T cells) or caveolin (epithelial A498 cells) and in polarized MDCK cells which express both proteins simultaneously. Subcellular fractionation by centrifugation to equilibrium in sucrose gradients of Triton X-100 cell extracts from Jurkat and A498 cells revealed that MAL and caveolin are incorporated in detergent-insoluble buoyant membranes independently of the expression of each other and indicated the existence in these cells of insoluble membrane microdomains with either MAL or caveolin. Immunofluorescence analysis in MDCK cells indicated that both MAL and caveolin were located in the Golgi region, whereas caveolin was found in addition at the cell surface. Biochemical analysis in these cells revealed the existence of distinct membrane microenvironments differentially susceptible to detergent solubilization containing either internal MAL or internal plus surface caveolin. The observed heterogeneity within the internal glycolipid-enriched membrane fraction suggests the existence of distinct specialized lipid microenvironments in MDCK cells.

Structural and biochemical similarities reveal a family of proteins related to the MAL proteolipid, a component of detergent-insoluble membrane microdomains.

The MAL gene encodes a proteolipid protein displaying a cell type-specific pattern of expression, including T lymphocytes, myelin-forming cells, and epithelial renal MDCK cells, which has been recently identified as a component of detergent-insoluble membranes known to be enriched in glycolipids and cholesterol. Sequence alignment revealed a high degree of conservation of the MAL protein across species and evidenced the existence of a significant level of overall identity between MAL and two other proteins, BENE and the plasmolipin proteolipid. Moreover, using subcellular fractionation of transiently transfected COS-7 cells, both MAL and BENE were identified in detergent-resistant membranes. These results suggest the existence of a novel family of MAL-related proteins (including MAL, BENE, and plasmolipin) with primary structure homologies and with the distinctive features of having unusual biochemical properties such as lipid-like behaviour and/or the ability to reside in glycolipid-enriched membrane microdomains.

The MAL proteolipid is a component of the detergent-insoluble membrane subdomains of human T-lymphocytes.

The human mal gene, identified during a search for cDNAs selectively expressed during T-cell development, encodes a highly hydrophobic protein belonging to a group of proteins, termed proteolipids, characterized by their unusual property of being soluble in organic solvents used to extract cell lipids. To study the localization of the MAL protein we have prepared stable transfectants expressing the MAL protein tagged with a c-myc epitope (MAL/c-myc) using human epithelial A-498 cells. Immunofluorescence analysis suggested that MAL/c-myc is localized mainly to cholesterol-enriched structures with a post-Golgi location and, at low levels, in early endosomes. Moreover, extraction of A-498 cell membranes with Triton X-100 (TX100) and fractionation by centrifugation to equilibrium in sucrose gradients demonstrated the presence of MAL/c-myc in the detergent-insoluble buoyant fraction, known to be enriched in glycolipids and cholesterol. To compare the behaviour of MAL in T-cells with that in epithelial A-498 cells, we prepared stably transfected cells expressing MAL/c-myc using human Jurkat T-cells. When TX100 extracts from Jurkat cells were subjected to centrifugation to equilibrium in sucrose gradients we found MAL exclusively in the floating fractions, together with molecules characteristic of the T-cell insoluble complexes, such as the tyrosine kinase p56lck, the glycosylphosphatidylinositol-anchored protein CD59 and the ganglioside GM1. These results, taken together, indicate that the MAL proteolipid is a component of the detergent-resistant membrane microdomains present in T-lymphocytes, and suggest that MAL might play a role in modulating the function of these microdomains during T-cell differentiation.

Lack of association between manic-depressive illness and a highly polymorphic marker from GABRA3 gene.

We have carried out an association study between a dinucleotide repeat polymorphism in GABRA3 gene and manic-depressive illness in a Spanish population. This may be an important candidate gene for bipolar affective disorders since it is located in the Xq28 region, previously implicated in linkage studies. In addition, severe GABergic alterations have been reported in patients. We have not found significant differences between controls and patients in allele frequencies or genotypes.