The lysosomal trafficking of acid sphingomyelinase is mediated by sortilin and mannose 6-phosphate receptor

Acid sphingomyelinase (ASM), a member of the saposin-like protein (SAPLIP) family, is a lysosomal hydrolase that converts sphingomyelin to ceramide. Deficiency of ASM causes a variant form of Niemann-Pick disease. The mechanism of lysosomal targeting of ASM is poorly known. Previous studies suggest that ASM could use in part the mannose 6-phosphate receptor (M6P-Rc). Sortilin, a type I transmembrane glycoprotein that belongs to a novel family of receptor proteins, presents structural features of receptors involved in lysosomal targeting. In this study we examined the hypothesis that sortilin may be implicated in the trafficking of ASM to the lysosomes. Using a dominant-negative sortilin construct lacking the cytoplasmic tail, which is essential to recruit adaptor proteins and clathrin, we demonstrated that sortilin is also involved in the lysosomal targeting of ASM. Confocal microscopy revealed that truncated sortilin partially inhibited the lysosomal trafficking of ASM in COS-7 cells and abolished the lysosomal targeting of ASM in I-cells. Pulse-chase experiments corroborated that sortilin is involved in normal sorting of newly synthesized ASM. Furthermore, over-expression of truncated sortilin accelerated and enhanced the secretion of ASM from COS-7 cells and I-cells. Co-immunoprecipitation assays confirmed the interaction between sortilin and ASM. In conclusion, ASM uses sortilin as an alternative receptor to be targeted to the lysosomes.     

In vivo activation of pro-form Bombyx cysteine protease (BCP) in silkmoth eggs: localization of yolk proteins and BCP,and acidification of yolk granules

The present study was designed to investigate the process of acidification of yolk granules during embryogenesis. In oocytes of mature Bombyx mori silkmoth, yolk proteins and a cysteine protease (pro-form BCP) were found in yolk granules. BCP was localized in small sized yolk granules (SYG, 3-6 microm in diameter) and yolk proteins in large sized granules (LYG, 6-11 microm in diameter), which might result in a spatial separation of protease and its substrates to avoid unnecessary hydrolysis. The granules were isolated on Percoll density gradient centrifugation. Although separation of LYG and SYG was incomplete, the granules sedimented in different fractions when using unfertilized egg extract, in which LYG was recovered from heavier fractions and BCP from lighter fractions. Acid phosphatase, as well as other lysosomal marker enzymes tested, was recovered from LYG-containing fractions. When extracts were prepared from developing eggs (day 3), some BCP-containing granules co-sedimented with LYG. The inactive pro-form BCP was activated in vivo, in parallel with yolk protein degradation, and as demonstrated previously in vitro under acidic conditions (). These results suggest that acidification occurs in yolk granules during embryogenesis. This was also confirmed using acridine orange fluorescent dye. In early development, most yolk granules were neutral, but became acidic during embryonic development. SYG were progressively recovered in heavier density fractions, displaying acidic interior. In this fraction, BCP-containing granules seem to be associated with larger granules (6-11 microm in size). In addition, SYG (BCP containing granules) were likely to be acidified earlier than LYG. Our results suggest that acidification initiates yolk degradation through activation of pro-form BCP.     

Homology of lysosomal enzymes and related proteins: prediction of posttranslational modification sites including phosphorylation of mannose and potential epitopic and substrate binding sites in the alpha- and beta-subunits of hexosaminidases, alpha-glucosidase, and rabbit and human isomaltase

Recently developed computer programs, including secondary structure and epitopic site predictions, have been used to align lysosomal proteins for maximum homology, based on conservative interchanges, and the aligned sequences have been searched for potential sites for posttranslational modification, glycosylation, and binding and catalysis of substrate. The homology and prediction of the posttranslational modification of the alpha- and beta-subunits of hexosaminidase is in good agreement with previous observations, and an explanation of the differing substrate specificities of the two subunits is advanced. We show that the striking homology between alpha-glucosidase and isomaltase is reflected in the apparent conservation of the active site in both enzymes. Nonhomologous regions have been examined in detail in a search for binding sites for glycogen and maltose, and two such sites have been tentatively identified. A highly redundant consensus sequence for the phosphorylation of mannose in lysosomal proteins, YXX(Y, W, or F), is suggested.     

Differential functions of Hrs and ESCRT proteins in endocytic membrane trafficking

A ubiquitin-binding endosomal protein machinery is responsible for sorting endocytosed membrane proteins into intraluminal vesicles of multivesicular endosomes (MVEs) for subsequent degradation in lysosomes. The Hrs-STAM complex and endosomal sorting complex required for transport (ESCRT)-I, -II and -III are central components of this machinery. Here, we have performed a systematic analysis of their importance in four trafficking pathways through endosomes. Neither Hrs, Tsg101 (ESCRT-I), Vps22/EAP30 (ESCRT-II), nor Vps24/CHMP3 (ESCRT-III) was required for ligand-mediated internalization of epidermal growth factor (EGF) receptors (EGFRs) or for recycling of cation-independent mannose 6-phosphate receptors (CI-M6PRs) from endosomes to the trans-Golgi network (TGN). In contrast, both Hrs and ESCRT subunits were equally required for degradation of both endocytosed EGF and EGFR. Whereas depletion of Hrs or Tsg101 caused enhanced recycling of endocytosed EGFRs, this was not the case with depletion of Vps22 or Vps24. Depletion of Vps24 instead caused a strong increase in the levels of CI-M6PRs and a dramatic redistribution of the Golgi and the TGN. These results indicate that, although Hrs-STAM and ESCRT-I, -II and -III have a common function in degradative protein sorting, they play differential roles in other trafficking pathways, probably reflecting their functions at distinct stages of the endocytic pathway.     

Kinetics of insulin-like growth factor II (IGF-II) interaction with domain 11 of the human IGF-II/mannose 6-phosphate receptor: function of CD and AB loop solvent-exposed residues

Ligands of the IGF-II/mannose 6-phosphate receptor (IGF2R) include IGF-II and mannose 6-phosphate modified proteins. Disruption of the negative regulatory effects of IGF2R on IGF-II-induced growth can lead to embryonic lethality and cancer promotion. Of the 15 IGF2R extracellular domains, domains 1-3 and 11 are known to have a conserved beta-barrel structure similar to that of avidin and the cation-dependent mannose 6-phosphate receptor, yet only domain 11 binds IGF-II with high specificity and affinity. In order to define the functional basis of this critical biological interaction, we performed alanine mutagenesis of structurally determined solvent-exposed loop residues of the IGF-II-binding site of human domain 11, expressed these mutant forms in Pichia pastoris, and determined binding kinetics with human IGF-II using isothermal calorimetry and surface plasmon resonance with transition state thermodynamics. Two hydrophobic residues in the CD loop (F1567 and I1572) were essential for binding, with a further non-hydrophobic residue (T1570) that slows the dissociation rate. Aside from alanine mutations of AB loop residues that decrease affinity by modifying dissociation rates (e.g. Y1542), a novel mutation (E1544A) of the AB loop enhanced affinity by threefold compared to wild-type. Conversion from an acidic to a basic residue at this site (E1544K) results in a sixfold enhancement of affinity via modification principally of the association rate, with enhanced salt-dependence, decreased entropic barrier and retained specificity. These data suggest that a functional hydrophobic binding site core is formed by I1572 and F1567 located in the CD loop, which initially anchors IGF-II. Within the AB loop, residues normally act to either stabilise or function as negative regulators of the interaction. These findings have implications for the molecular architecture and evolution of the domain 11 IGF-II-binding site, and the potential interactions with other domains of IGF2R.     

Reptilian MPR 300 is also the IGF-IIR: Cloning, sequencing and functional characterization of the IGF-II binding domain

The mammalian cation-independent mannose 6-phosphate/insulin-like growth factor (IGF)-II receptor binds IGF-II with high affinity. Ligands transported by the MPR 300/IGF-IIR include IGF-II and mannose 6-phosphate-modified proteins. By targeting IGF-II to lysosomal degradation, it plays a key role in the maintenance of correct IGF-II levels in the circulation and in target tissues. Although, from our studies we found homologous receptor in calotes but its functional significance was not known. We present here the first report on the calotes MPR 300/IGF-IIR binds IGF-II with K_d of 12.02 nM; these findings provide new and strong evidence that MPR 300/IGF-IIR in Calotes versicolor binds IGFII with high affinity.     

Intracellular distribution and late endosomal effects of the ocular albinism type 1 gene product: consequences of disease-causing mutations and implications for melanosome biogenesis

To investigate the function of ocular albinism type 1 ( OA1 ), the gene responsible for X-linked ocular albinism, we employed a construct containing murine Oa1 fused to green fluorescent protein (GFP) in a heterologous COS cell expression system. The cellular distribution of wild-type (WT) Oa1 protein and Oa1 proteins reflecting mutations causing X-linked ocular albinism were examined. Comparison with different organelle markers revealed that Oa1-GFP localized to the late endolysosomal compartments. Some Oa1 mutant proteins failed to exit the endoplasmic reticulum (ER) (Class I mutants), while other mutants partially (Class II mutants) or fully (Class III mutants) exited the ER and trafficked to endolysosomal compartments. We observed that expression of WT Oa1-GFP in COS cells caused an apparent enlargement of late endosomes and a redistribution of the mannose-6-phosphate receptor (M6PR). None of the mutants displayed the full range of effects on the redistribution of M6PR exhibited by WT Oa1. The effects of Oa1 on late endosome structure and content are thus likely to reflect an important biological property of Oa1. We propose that OA1 is involved in reorganizing the endolysosomal compartment as a necessary step in ocular melanosome biogenesis.     

Dominant-negative mutant of BIG2, an ARF-guanine nucleotide exchange factor,specifically affects membrane trafficking from the trans-Golgi network through inhibiting membrane association of AP-1 and GGA coat proteins

BIG2 is one of the guanine nucleotide exchange factors (GEFs) for the ADP-ribosylation factor (ARF) family of small GTPases, which regulate membrane association of COPI and AP-1 coat protein complexes and GGA proteins. Brefeldin A (BFA), an ARF-GEF inhibitor, causes redistribution of the coat proteins from membranes to the cytoplasm and membrane tubulation of the Golgi complex and the trans-Golgi network (TGN). We have recently shown that BIG2 overexpression blocks BFA-induced redistribution of the AP-1 complex but not TGN membrane tubulation. In the present study, we constructed a dominant-negative BIG2 mutant and found that when expressed in cells it induced redistribution of AP-1 and GGA1 and membrane tubulation of the TGN. By contrast, the mutant did not induce COPI redistribution or Golgi membrane tubulation. These observations indicate that BIG2 is involved in trafficking from the TGN by regulating membrane association of AP-1 and GGA through activating ARF.     

Mannose 6-Phosphate Receptor Proteins from Reptiles and Amphibians: Evidence for the Presence of MPR 300 and MPR 46

Two mannose 6-phosphate receptors (MPR 300 and MPR 46) are involved in transport of lysosomal enzymes. Both receptors are expressed in all mammalian species studied so far and in chicken. Here we present the first report on affinity purification of both MPRs from the liver tissues of reptiles and amphibians using Sepharose divinyl sulfone phosphomannan at pH 7.0. MPR 300 from both species show similar electrophoretic mobility as mammalian MPR 300 and cross-react with an antibody directed against MPR 300 from goat liver. Furthermore, MPR 46 from reptilian liver and amphibian oocytes cross-react with peptide-specific antibodies against the cytoplasmic domain of human MPR 46 (anti-MSC1).     

Effect of the extracts from Glycyrrhiza uralensis Fisch on the growth characteristics of human cell lines: Anti-tumor and immune activation activities

Immune modulating activity of ethanol extracts from Glycyrrhiza uralensis Fisch was investigated by conserving growth characteristics of several human cell lines. All of the samples did not show severe cytotoxicity on normal human liver cell line, WRL-68, showing less than 25% inhibition of cell growth. The crude extract and its fractionized samples (F1 and F3) inhibited the growth of human hepatoma, Hep3B, down to ca. 70% of normal cell growth in adding 1.0 g l^-1 of fraction F3. The result of anticancer experiments was well matched to the results of antimutagenicity using Chinese Hamster Lung cell lines(CHL V79). In adding 1.0 g l^-1 of fraction F1, the growth of human B cell was enhanced, up to 60% of control growth. The secretion of two kinds of cytokines, Interleukin-6 and Tumor Necrosis Factor-α from human B cells was also enhanced in adding the crude extract, but not the standards such as Glycyrrhizin (GL) or 18,β-glycyrrhetinic acid (GM). It was found that both of the apoptosis and differentiation were more accelerated in supplementing the crude extract and fraction F1 than in adding the standards. A spot was found only in the crude extract and fractions, not standards by Thin Layer Chromatography(TLC) analysis. It tells that there must be another unknown component in crude and/or fraction F1 as a possible candidate of immune modulators. This component seems to be a derivative of a monomer, GM since its R_f was close to the monomer. It was also interesting that glycyrrhizin, a major component in G. uralensis Fisch was biologically activated by first being hydrolyzed by an enzyme. This revised version was published online in August 2006 with corrections to the Cover Date.     

Transfer of a lipophilic drug (temoporfin) between small unilamellar liposomes and human plasma proteins: influence of membrane composition on vesicle integrity and release characteristics

Abstract

The introduction of PEG lipid conjugates into lipid bilayers leads to long circulating liposomes with improved pharmacokinetics and pharmacodynamics characteristics. The concentration range of PEG-lipids is limited by their micelle forming properties. We investigated two phosphatidyl oligoglycerols as potential alternatives to PEG-lipid conjugates and compared their micelle forming properties after incorporation of increasing amounts of oligoglycerols into gel-phase liposomes via cryo-transmission electron microscopy. The incorporation of highly hydrophobic drugs into liposomes makes water soluble formulations possible and improves the therapeutic properties of the drug. We incorporated the hydrophobic photosensitizer temoporfin into liposomes varying in membrane fluidity and nature of surface modifying agents. The main purpose of this study was the investigation of liposome integrity and temoporfin incorporation stability in the presence of plasma. After incubation of temoporfin-loaded liposomes with human plasma for different time intervals, liposomes and the single lipoprotein fractions were separated via size-exclusion chromatography. Liposome stability and temoporfin distribution profile over the lipoprotein fractions were determined with the help of a non-exchangeable ³H-lipid label and ¹⁴C-labeled temoporfin. The results demonstrate that both oligoglycerols are suitable alternatives to PEG-lipid conjugates because of the lack of micelle forming properties, comparable liposome stability, and a reduced temoporfin transfer rate compared to PEG-lipids. Furthermore, the incorporation stability of temoporfin is – at least to some extent – influenced by membrane fluidity, indicating that fluid membranes may be better suited for retention of lipophilic drugs.     

Successful amphiphiles as the key to crystallization of membrane proteins: Bridging theory and practice

Background

Membrane proteins constitute a major group of proteins and are of great significance as pharmaceutical targets, but underrepresented in the Protein Data Bank. Particular reasons are their low expression yields and the constant need for cautious and diligent handling in a sufficiently stable hydrophobic environment substituting for the native membrane. When it comes to protein crystallization, such an environment is often established by detergents.

Spectrins: A structural platform for stabilization and activation of membrane channels,receptors and transporters

This review focuses on structure and functions of spectrin as a major component of the membrane skeleton. Recent advances on spectrin function as an interface for signal transduction mediation and a number of data concerning interaction of spectrin with membrane channels, adhesion molecules, receptors and transporters draw a picture of multifaceted protein. Here, we attempted to show the current depiction of multitask role of spectrin in cell physiology. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.     

Expression,stabilization and purification of membrane proteins via diverse protein synthesis systems and detergents involving cell-free associated with self-assembly peptide surfactants

G-protein coupled receptors (GPCRs) are involved in regulating most of physiological actions and metabolism in the bodies, which have become most frequently addressed therapeutic targets for various disorders and diseases. Purified GPCR-based drug discoveries have become routine that approaches to structural study, novel biophysical and biochemical function analyses. However, several bottlenecks that GPCR-directed drugs need to conquer the problems including overexpression, solubilization, and purification as well as stabilization. The breakthroughs are to obtain efficient protein yield and stabilize their functional conformation which are both urgently requiring of effective protein synthesis system methods and optimal surfactants. Cell-free protein synthesis system is superior to the high yields and post-translation modifications, and early signs of self-assembly peptide detergents also emerged to superiority in purification of membrane proteins. We herein focus several predominant protein synthesis systems and surfactants involving the novel peptide detergents, and uncover the advantages of cell-free protein synthesis system with self-assembling peptide detergents in purification of functional GPCRs. This review is useful to further study in membrane proteins as well as the new drug exploration.     

Reconstitution into liposomes of the glutamine/amino acid transporter from renal cell plasma membrane: functional characterization, kinetics and activation by nucleotides

The glutamine/amino acid transporter was solubilized from rat renal apical plasma membrane (brush-border membrane) with C₁₂E₈ and reconstituted into liposomes by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite XAD-4. The reconstitution was optimised with respect to the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. The reconstituted glutamine/amino acid transporter catalysed a first-order antiport reaction stimulated by external, not internal, Na⁺. Optimal activity was found at pH 7.0. The sulfhydryl reagents HgCl₂, mersalyl and p-hydroxymercuribenzoate and the amino acids alanine, serine, threonine, cysteine, asparagine, methionine and valine strongly inhibited the transport, whereas the amino acid analogue methylaminoisobutyrate had no effect. Glutamine, alanine, serine, asparagine, threonine were efficiently translocated from outside to inside and from inside to outside the proteoliposomes as well. Cysteine and valine were translocated preferentially from outside to inside. The K_m for glutamine on the external and internal side of the transporter was 0.47 and 11 mM, respectively; the values were not influenced by the type of the counter substrate. The transporter is functionally asymmetrical and it is unidirectionally inserted into the proteoliposomal membrane with an orientation corresponding to that of the native membrane. By a bisubstrate kinetic analysis of the glutamine antiport, a random simultaneous mechanism was found. The glutamine antiport was strongly stimulated by internal nucleoside triphosphates and, to a lower extent, by pyrophoshate. The reconstituted glutamine/amino acid transporter functionally corresponds to the ASCT2 protein.     

Lysophosphatidylethanolamine stimulates chemotactic migration and cellular invasion in SK-OV3 human ovarian cancer cells: involvement of pertussis toxin-sensitive G-protein coupled receptor

We investigated whether lysophosphatidylethanolamine (LPE) modulates cellular signaling in different cell types. SK-OV3 ovarian cancer cells and OVCAR-3 ovarian cancer cells were responsive to LPE. LPE-stimulated intracellular calcium concentration ([Ca(2+)](i)) increase was inhibited by U-73122, suggesting that LPE stimulates calcium signaling via phospholipase C activation. Moreover, pertussis toxin (PTX) almost completely inhibited [Ca(2+)](i) increase by LPE, indicating the involvement of PTX-sensitive G-proteins. Furthermore, we found that LPE stimulated chemotactic migration and cellular invasion in SK-OV3 ovarian cancer cells. We examined the role of lysophosphatidic acid receptors on LPE-stimulated cellular responses using HepG2 cells transfected with different LPA receptors, and found that LPE failed to stimulate nuclear factor kappa B-driven luciferase. We suggest that LPE stimulates a membrane bound receptor, different from well known LPA receptors, resulting in chemotactic migration and cellular invasion in SK-OV3 ovarian cancer cells.     

Intercellular communication via the endo-lysosomal system: Translocation of granzymes through membrane barriers

Cytotoxic lymphocytes (CLs) are responsible for the clearance of virally infected or neoplastic cells. CLs possess specialised lysosome-related organelles called granules which contain the granzyme family of serine proteases and perforin. Granzymes may induce apoptosis in the target cell when delivered by the pore forming protein, perforin. Here we follow the perforin-granzyme pathway from synthesis and storage in the granule, to exocytosis and finally delivery into the target cell. This review focuses on the controversial subject of perforin-mediated translocation of granzymes into the target cell cytoplasm. It remains unclear whether this occurs at the cell surface with granzymes moving through a perforin pore in the plasma membrane, or if it involves internalisation of perforin and granzymes and subsequent release from an endocytic compartment. The latter mechanism would represent an example of cross talk between the endo-lysosomal pathways of individual cells. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Mammalian OS-9 Is Upregulated in Response to Endoplasmic Reticulum Stress and Facilitates Ubiquitination of Misfolded Glycoproteins

Proteins that fail to fold or assemble with partner subunits are selectively removed from the endoplasmic reticulum (ER) via the ER-associated degradation (ERAD) pathway. Proteins selected for ERAD are polyubiquitinated and retrotranslocated into the cytosol for degradation by the proteasome. Although it is unclear how proteins are initially identified by the ERAD system in mammalian cells, OS-9 was recently proposed to play a key role in this process. Here we show that OS-9 is upregulated in response to ER stress and is associated both with components of the ERAD machinery and with ERAD substrates. Using RNA interference, we show that OS-9 is required for efficient ubquitination of glycosylated ERAD substrates, suggesting that it helps transfer misfolded proteins to the ubiquitination machinery. We also find that OS-9 binds to a misfolded nonglycosylated protein destined for ERAD, but not to the properly folded wild-type protein. Surprisingly, however, OS-9 is not required for ubiquitination or degradation of this nonglycosylated ERAD substrate. We propose a model in which OS-9 recognises terminally misfolded proteins via polypeptide-based rather than glycan-based signals, but is only required for transferring those bearing N-glycans to the ubiquitination machinery.     

Neurogenic differentiation of human adipose-derived stem cells: Relevance of different signaling molecules,transcription factors,and key marker genes

Since numerous diseases affect the central nervous system and it has limited self-repair capability, a great interest in using stem cells as an alternative cell source is generated. Previous reports have shown the differentiation of adipose-derived stem cells in neuron-like cells and it has also been proved that the expression pattern of patterning, proneural, and neural factors, such as Pax6, Mash1, Ngn2, NeuroD1, Tbr2 and Tbr1, regulates and defines adult neurogenesis. Regarding this, we hypothesize that a functional parallelism between adult neurogenesis and neuronal differentiation of human adipose-derived stem cells exists. In this study we differentiate human adipose-derived stem cells into neuron-like cells and analyze the expression pattern of different patterning, proneural, neural and neurotransmitter genes, before and after neuronal differentiation. The neuron-like cells expressed neuronal markers, patterning and proneural factors characteristics of intermediate stages of neuronal differentiation. Thus we demonstrated that it is possible to differentiate adipose-derived stem cells in vitro into immature neuron-like cells and that this process is regulated in a similar way to adult neurogenesis. This may contribute to elucidate molecular mechanisms involved in neuronal differentiation of adult human non-neural cells, in aid of the development of potential therapeutic tools for diseases of the nervous system.