Mammalian Apg12p,but not the Apg12p.Apg5p conjugate,facilitates LC3 processing

A dynamic membrane rearrangement occurs in cells during autophagy to form autophagosomes. In this dynamic process, two ubiquitin-like modifications, Apg12p-conjugation and LC3-modification, are essential for the formation of autophagosomes. Apg7p and Apg10p catalyze the conjugation of Apg12p to Apg5p. The same Apg7p and Apg3p catalyze the processing of LC3 to a membrane-bound form, LC3-II. In this paper, we investigated whether Apg12p has an influence on the second LC3-modification system. A cross-linking experiment revealed that Apg3p interacts with the endogenous Apg12p.Apg5p conjugate. However, Apg3p itself interacts with free Apg12p more preferentially than the Apg12p.Apg5p conjugate, when free Apg12p exists. When Apg12p was overexpressed, LC3 processing was significantly enhanced in the presence of Apg7p. In contrast, when the Apg12p.Apg5p conjugate itself was accumulated by the overexpression of Apg12p and Apg5p, LC3 processing was dominantly inhibited, even in the presence of Apg7p. These results indicate that both Apg12p and the Apg12p.Apg5p conjugate are regulatory factors for LC3 processing.     

The von Hippel-Lindau tumor suppressor gene product promotes, but is not essential for, NEDD8 conjugation to cullin-2

We have previously shown that human cullin-2 (Cul-2) is covalently modified at Lys-689 by NEDD8 (Wada, H., Yeh, E. T. H., and Kamitani, T. (1999) Biochem. Biophys. Res. Commun. 257, 100-105). Cul-2 has also been reported to form a multiprotein complex, Cul-2.VBC, with the von Hippel-Lindau tumor suppressor gene product (pVHL) and elongins B and C. In this study, using an in vivo coexpression system in COS cells, we show that NEDD8 conjugation to Cul-2 is promoted by coexpression with wild-type pVHL and elongins B and C. Interestingly, tumorigenic mutants and deletion mutants of pVHL, which are unable to form a Cul-2.VBC complex, do not have the activity to promote NEDD8 conjugation to Cul-2. These results suggest that the complex formation is required for NEDD8 conjugation to Cul-2. Furthermore, we used a pVHL-deficient cell line, 786-0, to show that Cul-2 is poorly but clearly conjugated by NEDD8, indicating that pVHL is not the only molecule that promotes NEDD8 conjugation to Cul-2. Taken together, the VBC complex appears to have ligase activity in the conjugation of NEDD8 to Cul-2.     

Fatty acids but not dexamethasone are essential inducers for chick adipocyte differentiation in vitro

The present study was carried out to clarify the direct effect of fatty acids (FAs) on chick (Gallus gallus) adipocyte differentiation in the absence of dexmethasone (DEX), a commonly used as strong inducer for adipocyte differentiation. Adipocyte differentiation was initiated by maintaining confluent cell in serum-free medium supplemented with FAs. Upon exposure to FAs, glycerol-3-phosphate dehydrogenase activity (GPDH) as adipocyte differentiation marker rapidly increased, and was significantly higher in chick adipocyte than in control cell. The morphology of the FAs-treated cell changed from fibroblast-like to polygon, and the cells accumulated many cytoplasmic lipid droplets as estimated by Oil red O staining. Neither insulin nor bovine serum albumin, as substitutes for serum, had an effect on chick adipocyte differentiation. The FAs-treated cell had a higher protein and mRNA expression levels for peroxisome proliferator-activated receptor-γ (PPARγ), a master regulator of differentiation, compared with untreated cell. In FAs-treated cell, the mRNA expression levels of adipocyte-specific genes, such as CCAAT/enhancer binding protein-α (C/EBP α) and adipocyte fatty acid-binding protein (aP2) were higher than in control cell. These results indicated that FAs, but not DEX, are essential inducers for chick adipocyte differentiation by elevating PPARγ expression.     

Formation of the approximately 350-kDa Apg12-Apg5.Apg16 multimeric complex, mediated by Apg16 oligomerization, is essential for autophagy in yeast

Autophagy, responsible for the delivery of cytoplasmic components to the lysosome/vacuole for degradation, is the major degradative pathway in eukaryotic cells. This process requires a ubiquitin-like protein conjugation system, in which Apg12 is covalently bound to Apg5. In the yeast Saccharomyces cerevisiae, the Apg12-Apg5 conjugate further interacts with a small coiled-coil protein, Apg16. The Apg12-Apg5 and Apg16 are localized in the cytosol and pre-autophagosomal structures and play an essential role in autophagosome formation. Here we show that the Apg12-Apg5 conjugate and Apg16 form a approximately 350-kDa complex in the cytosol. Because Apg16 was suggested to form a homo-oligomer, we generated an in vivo system that allowed us to control the oligomerization state of Apg16. With this system, we demonstrated that formation of the approximately 350-kDa complex and autophagic activity depended on the oligomerization state of Apg16. These results suggest that the Apg12-Apg5 conjugate and Apg16 form a multimeric complex mediated by the Apg16 homo-oligomer, and formation of the approximately 350-kDa complex is required for autophagy in yeast.     

Sphingolipids are essential for differentiation but not growth in Leishmania

Sphingolipids (SLs) play critical roles in eukaryotic cells in the formation of lipid rafts, membrane trafficking, and signal transduction. Here we created a SL null mutant in the protozoan parasite Leishmania major through targeted deletion of the key de novo biosynthetic enzyme serine palmitoyltransferase subunit 2 (SPT2). Although SLs are typically essential, spt2- Leishmania were viable, yet were completely deficient in de novo sphingolipid synthesis, and lacked inositol phosphorylceramides and other SLs. Remarkably, spt2- parasites maintained 'lipid rafts' as defined by Triton X-100 detergent resistant membrane formation. Upon entry to stationary phase spt2- failed to differentiate to infective metacyclic parasites and died instead. Death occurred not by apoptosis or changes in metacyclic gene expression, but from catastrophic problems leading to accumulation of small vesicles characteristic of the multivesicular body/multivesicular tubule network. Stage specificity may reflect changes in membrane structure as well as elevated demands in vesicular trafficking required for parasite remodeling during differentiation. We suggest that SL-deficient Leishmania provide a useful biological setting for tests of essential SL enzymes in other organisms where SL perturbation is lethal.     

The mouse APG10 homologue,an E2-like enzyme for Apg12p conjugation,facilitates MAP-LC3 modification

Autophagy is a process for the bulk degradation of cytosolic compartments by lysosomes/vacuoles. The formation of autophagosomes involves a dynamic rearrangement of the membrane for which two ubiquitin-like modifications (the conjugation of Apg12p and the modification of a soluble form of MAP-LC3 to a membrane-bound form) are essential. In yeast, Apg10p is an E2-like enzyme essential for Apg12p conjugation. The isolated mouse APG10 gene product interacts with mammalian Apg12p dependent on mammalian Apg7p (E1-like enzyme), and facilitates Apg12p conjugation. The interaction of Apg10p with Apg12p is dependent on the carboxyl-terminal glycine of Apg12p. Mutational analysis of the predicted active site cysteine (Cys161) within mouse Apg10p shows that mutant Apg10pC161S, which can form a stable intermediate with Apg12p, inhibits Apg12p conjugation even in the presence of Apg7p, while overexpression of Apg7p facilitates formation of an Apg12p-Apg5p conjugate. Furthermore, the coexpression of Apg10p with Apg7p facilitates the modification of a soluble form of MAP-LC3 to a membrane-bound form, a second modification essential for autophagy. Mouse Apg10p interacts with MAP-LC3 in HEK293 cells, while no mutant Apg10pC161S forms any intermediate with MAP-LC3. Direct interaction between Apg10p and MAP-LC3 is also demonstrated by yeast two-hybrid analysis. The inability of mutant Apg10pC161S to form any intermediate with MAP-LC3 has ruled out the possibility that MAP-LC3 interacts with Apg10p as a substrate.     

C-terminal amino acids are essential for human heat shock protein 70 dimerization

The human inducible heat shock protein 70 (hHsp70), which is involved in several major pathologies, including neurodegenerative disorders and cancer, is a key molecular chaperone and contributes to the proper protein folding and maintenance of a large number of protein structures. Despite its role in disease, the current structural knowledge of hHsp70 is almost exclusively based on its Escherichia coli homolog, DnaK, even though these two proteins only share ~50 % amino acid identity. For the first time, we describe a complete heterologous production and purification strategy that allowed us to obtain a large amount of soluble, full-length, and non-tagged hHsp70. The protein displayed both an ATPase and a refolding activity when combined to the human Hsp40. Multi-angle light scattering and bio-layer interferometry analyses demonstrated the ability of hHsp70 to homodimerize. The role of the C-terminal part of hHsp70 was identified and confirmed by a study of a truncated version of hHsp70 that could neither dimerize nor present refolding activity.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-014-0526-3) contains supplementary material, which is available to authorized users.     

VirB3 to VirB6 and VirB8 to VirB11, but not VirB7, are essential for mediating persistence of Brucella in the reticuloendothelial system

The Brucella abortus virB locus contains 12 open reading frames, termed virB1 through virB12, which encode a type IV secretion system. Polar mutations in the virB locus markedly reduce the ability of B. abortus to survive in cultured macrophages or to persist in organs of mice. While a nonpolar deletion of the virB2 gene reduces survival in cultured macrophages and in organs of mice, a nonpolar deletion of virB1 only reduces survival in macrophages, whereas virB12 is dispensable for either virulence trait. Here we investigated the role of the remaining genes in the virB locus during survival in macrophages and virulence in mice. Mutants carrying nonpolar deletions of the virB3, virB4, virB5, virB6, virB7, virB8, virB9, virB10, or virB11 gene were constructed and characterized. All mutations reduced the ability of B. abortus to survive in J774A.1 mouse macrophage-like cells to a degree similar to that caused by a deletion of the entire virB locus. Deletion of virB3, virB4, virB5, virB6, virB8, virB9, virB10, or virB11 markedly reduced the ability of B. abortus to persist in the spleens of mice at 8 weeks after infection. Interestingly, deletion of virB7 did not reduce the ability of B. abortus to persist in spleens of mice. We conclude that virB2, virB3, virB4, virB5, virB6, virB8, virB9, virB10, and virB11 are essential for virulence of B. abortus in mice, while functions encoded by the virB1, virB7, and virB12 genes are not required for persistence in organs with this animal model.     

In vitro reconstitution of plant Atg8 and Atg12 conjugation systems essential for autophagy

Genetic and biochemical analyses using yeast Saccharomyces cerevisiae showed that two ubiquitin-like conjugation systems, the Atg8 and Atg12 systems, exist and play essential roles in autophagy, the bulk degradation system conserved in yeast and mammals. These conjugation systems are also conserved in Arabidopsis thaliana; however, further detailed study of plant ATG (autophagy-related) conjugation systems in relation to those in yeast and mammals is needed. Here, we describe the in vitro reconstitution of Arabidopsis thaliana ATG8 and ATG12 (AtATG8 and AtATG12) conjugation systems using purified recombinant proteins. AtATG12b was conjugated to AtATG5 in a manner dependent on AtATG7, AtATG10, and ATP, whereas AtATG8a was conjugated to phosphatidylethanolamine (PE) in a manner dependent on AtATG7, AtATG3, and ATP. Other AtATG8 homologs (AtATG8b-8i) were similarly conjugated to PE. The AtATG8 conjugates were deconjugated by AtATG4a and AtATG4b. These results support the hypothesis that the ATG conjugation systems in Arabidopsis are very similar to those in yeast and mammals. Intriguingly, in vitro analyses showed that AtATG12-AtATG5 conjugates accelerated the formation of AtATG8-PE, whereas AtATG3 inhibited the formation of AtATG12-AtATG5 conjugates. The in vitro conjugation systems reported here will afford a tool with which to investigate the cross-talk mechanism between two conjugation systems.     

Atg8L/Apg8L is the fourth mammalian modifier of mammalian Atg8 conjugation mediated by human Atg4B, Atg7 and Atg3

Murine Atg8L/Apg8L has significant homology with the other known mammalian Atg8 homologs, LC3, GABARAP and GATE-16. However, it is unclear whether murine Atg8L modification is mediated by human Atg4B, Atg7 and Atg3. Expression of Atg8L in HEK293 cells led to cleavage of its C-terminus. In vitro, the C-terminus of Atg8L was cleaved by human Atg4B, but not human Atg4A or Atg4C. Atg8L-I formed an E1-substrate intermediate with Atg7(C572S), and an E2-substrate intermediate with Atg3(C264S). A modified form of Atg8L was detected in the pelletable fraction in the presence of lysosomal protease inhibitors under nutrient-rich conditions. Cyan fluorescent protein (CFP)-Atg8L colocalized with yellow fluorescent protein (YFP)-LC3 in HeLa cells in the presence of the inhibitors. However, little accumulation of the modified form of Atg8L was observed under conditions of starvation. These results indicate that Atg8L is the fourth modifier of mammalian Atg8 conjugation.     

Systemic, but not intestinal, IL-7 is essential for the persistence of chronic colitis

We previously demonstrated that IL-7 is produced by intestinal goblet cells and is essential for the persistence of colitis. It is well known, however, that goblet cells are decreased or depleted in the chronically inflamed mucosa of animal colitis models or human inflammatory bowel diseases. Thus, in this study, we assess whether intestinal IL-7 is surely required for the persistence of colitis using a RAG-1/2-/- colitis model induced by the adoptive transfer of CD4+CD45RBhigh T cells in combination with parabiosis system. Surprisingly, both IL-7-/-xRAG-1-/- and IL-7+/+xRAG-1-/- host mice developed colitis 4 wk after parabiosis to a similar extent of colitic IL-7+/+xRAG-1-/- donor mice that were previously transferred with CD4+CD45RBhigh T cells. Of note, although the number of CD4+ T cells recovered from the spleen or the bone marrow of IL-7-/-xRAG-1-/- host mice was significantly decreased compared with that of IL-7+/+xRAG-1-/- host mice, an equivalent number of CD4+ T cells was recovered from the lamina propria of both mice, indicating that the expansion of CD4+ T cells in the spleen or in the bone marrow is dependent on IL-7, but not in the lamina propria. Development of colitis was never observed in parabionts between IL-7+/+xRAG-1-/- host and noncolitic IL-7-/-xRAG-1-/- donor mice that were transferred with CD4+CD45RBhigh T cells. Collectively, systemic, but not intestinal, IL-7 is essential for the persistence of colitis, suggesting that therapeutic approaches targeting the systemic IL-7/IL-7R signaling pathway may be feasible in the treatment of inflammatory bowel diseases.     

The reversible modification regulates the membrane-binding state of Apg8/Aut7 essential for autophagy and the cytoplasm to vacuole targeting pathway

Autophagy and the Cvt pathway are examples of nonclassical vesicular transport from the cytoplasm to the vacuole via double-membrane vesicles. Apg8/Aut7, which plays an important role in the formation of such vesicles, tends to bind to membranes in spite of its hydrophilic nature. We show here that the nature of the association of Apg8 with membranes changes depending on a series of modifications of the protein itself. First, the carboxy-terminal Arg residue of newly synthesized Apg8 is removed by Apg4/Aut2, a novel cysteine protease, and a Gly residue becomes the carboxy-terminal residue of the protein that is now designated Apg8FG. Subsequently, Apg8FG forms a conjugate with an unidentified molecule "X" and thereby binds tightly to membranes. This modification requires the carboxy-terminal Gly residue of Apg8FG and Apg7, a ubiquitin E1-like enzyme. Finally, the adduct Apg8FG-X is reversed to soluble or loosely membrane-bound Apg8FG by cleavage by Apg4. The mode of action of Apg4, which cleaves both newly synthesized Apg8 and modified Apg8FG, resembles that of deubiquitinating enzymes. A reaction similar to ubiquitination is probably involved in the second modification. The reversible modification of Apg8 appears to be coupled to the membrane dynamics of autophagy and the Cvt pathway.     

The FAP motif within human ATG7, an autophagy-related E1-like enzyme, is essential for the E2-substrate reaction of LC3 lipidation

ATG7 is an autophagy-related E1-like enzyme that is essential for two ubiquitination-like reactions, ATG12-conjugation and LC3-lipidation. The existence of functional sequences at the amino-terminal region of human ATG7 remains uncertain. Mutational analyses of ATG7 revealed that both mutant ATG7ΔFAP lacking the FAP motif and ATG7FAPtoDDD, in which the Phe15-Ala16-Pro17 sequence was changed to Asp-Asp-Asp, could not complement defects in endogenous ATG12-conjugation and LC3-lipidation when expressed in Atg7-deficient mouse embryonic fibroblasts (MEFs). However, wild-type ATG7 complemented the defects in these cells. Overexpression of GFP-ATG10 and GFP-ATG12 rescued a defect in ATG12-conjugation in Atg7-deficient MEFs expressing mutant ATG7ΔFAP and ATG7FAPtoDDD, whereas overexpression of all ATG proteins related to ATG12-conjugation and LC3-lipidation could not rescue a defect in LC3-lipidation in Atg7-deficient MEFs expressing these ATG7 mutants. Both ATG7ΔFAP and ATG7FAPtoDDD mutants showed severe defects in the formation of an E2-substrate intermediate of ATG3 with LC3 in LC3-lipidation, but were able to form an E1-substrate intermediate of ATG7 with LC3 and the E1- and E2-substrate intermediates in ATG12-conjugation with reduced efficiency. These ATG7 mutants could also form the ATG12-ATG3 conjugate. Co-immunoprecipitation experiments revealed that the FAP motif of ATG7 is essential for the interaction of ATG7 with ATG3, but not for ATG7-homodimerization. These results indicated that the FAP motif of ATG7 is indispensable for formation of the ATG3-LC3 E2-substrate intermediate through the interaction of ATG7 with ATG3.     

Primary cultures of human hepatocytes but not HepG2 hepatoblastoma cells are suitable for the study of glycosidic conjugation of bile acids

To define the role of glycosidic conjugation of bile acids in humans, an in vitro model system is desirable. We studied the formation of glycosidic conjugates of bile acids in primary cultures of human hepatocytes, isolated from organ donor liver, and the human hepatoblastoma cell line, HepG2. Cells were incubated with 100 microM bile acids (chenodeoxycholic, CDCA; hyodeoxycholic, HDCA; and isoursodeoxycholic acids, isoUDCA) and 1-2 mM uridine diphosphoglycosides (UDP-glucose, UDP-Glc; UDP-glucuronic acid, UDP-GlcA, and UDP-N-acetylglucosamine, UDP-GlcNAc), and octyl glucoside. Media were analysed by electrospray-/gas chromatography-mass spectrometry and electrospray with collision induced dissociation. Primary cultures of human hepatocytes formed glycosidic bile acid conjugates with UDP-sugars (6alpha-Glc-HDCA, 6alpha-GlcA-HDCA, and 7beta-GlcNAc-isoUDCA) and octyl glucoside as sugar donors (3alpha-Glc-CDCA). HDCA was completely metabolised to either Glc-HDCA, a compound yet not found in vivo, or GlcA-HDCA. No glycosidic bile acid conjugate was found in media from experiments with HepG2. Thus, primary cultures of human hepatocytes, but not HepG2, are suitable in vitro systems for the study of glycosidic bile acid conjugation reactions.     

The streptococcal hyaluronan synthases are inhibited by sulfhydryl-modifying reagents, but conserved cysteine residues are not essential for enzyme function.

Hyaluronan (HA) synthase (HAS) is a membrane-bound enzyme that utilizes UDP-glucuronic acid (GlcUA) and UDP-GlcNAc to synthesize HA. The HAS from Streptococcus pyogenes (spHAS, 419 amino acids) contains six Cys residues, whereas the enzyme from Streptococcus equisimilis (seHAS, 417 amino acids) contains four Cys residues. These Cys residues of seHAS are highly conserved in all Class I HAS family members. Here we investigated the structural and functional roles of these conserved cysteines in seHAS by using site-directed mutagenesis and sensitivity to sulfhydryl modifying reagents. Both seHAS and spHAS were inhibited by sulfhydryl reagents such as N-ethylmaleimide (NEM) and iodoacetamide in a dose-dependent and time-dependent manner. These inhibition curves were biphasic, indicating the presence of sensitive and insensitive components. After treatment of seHAS with NEM, the V(max) value was decreased approximately 50%, and the K(m) values changed only slightly. All the Cys-to-Ala mutants of seHAS were partially active. The least active single (C226A), double (C226A,C262A), or triple (C226A,C262A,C367A) Cys mutants retained 24, 3.2, and 1.4% activity, respectively, compared with wild-type enzyme. Surprisingly, the V(max) value of the seHAS(cys-null) mutant was approximately 17% of wild-type, although the K(m) values for both substrates were increased 3-6-fold. Cys residues, therefore, are not involved in a critical interaction necessary for either substrate binding or catalysis. However, the distribution of HA products was shifted to a smaller size in approximately 25% of the seHAS Cys mutants, particularly the triple mutants. Mass spectroscopic analysis of wild-type and Cys-null seHAS as well as the labeling of all double Cys-to-Ala mutants with [(14)C]NEM demonstrated that seHAS contains no disulfide bonds. We conclude that the four Cys residues in seHAS are not directly involved in catalysis, but that one or more of these Cys residues are located in or near substrate binding or glycosyltransferase active sites, so that their modification hinders the functions of HAS.     

Galectin-8 tandem-repeat structure is essential for T-cell proliferation but not for co-stimulation

Gal (galectin)-8 is a tandem-repeat Gal containing N-CRDs (Nterminal carbohydrate-recognition domains) and C-CRDs (C-terminal carbohydrate-recognition domains) with differential glycan-binding specificity fused by a linker peptide. Gal-8 has two distinct effects on CD4 T-cells: at high concentrations it induces antigen-independent proliferation, whereas at low concentrations it co-stimulates antigen-specific responses. Associated Gal-8 structural requirements were dissected in the present study. Recombinant homodimers N-N (two N-terminal CRD chimaera) and C-C (two C-terminal CRD chimaera), but not single C-CRDs or N-CRDs, induced proliferation; however, single domains induced co-stimulation. These results indicate that the tandem-repeat structure was essential only for the proliferative effect, suggesting the involvement of lattice formation, whereas co-stimulation could be mediated by agonistic interactions. In both cases, C-C chimaeras displayed higher activity than Gal-8, indicating that the C-CRD was mainly involved, as was further supported by the strong inhibition of proliferation and co-stimulation in the presence of blood group B antigen, specifically recognized by this domain. Classic Gal inhibitors (lactose and thiodigalactoside) prevented proliferation but not co-stimulatory activity, which was inhibited by 3-O-β-D-galactopyranosyl-D-arabinose. Interestingly, Gal-8 induced proliferation of na?ve human CD4 T-cells, varying from non- to high-responder individuals, whereas it promoted cell death of phytohaemagglutinin or CD3/CD28 pre-activated cells. The findings of the present study delineate the differential molecular requirements for Gal-8 activities on T-cells, and suggest a dual activity relying on activation state.