Toward unraveling membrane biogenesis in mammalian autophagy

Autophagy is a unique form of membrane trafficking, which delivers macromolecules and organelles from the cytoplasm to lysosomes for degradation. This fundamental and ubiquitous process in eukaryotic cells is mediated by the double-membrane-bound structures called autophagosomes, which transiently emerge in the cytoplasm. The recent remarkable explosion of knowledge of autophagy has revealed its multiple roles, especially in mammals; in addition to its basic role in turnover and reuse of cellular constituents, the process unexpectedly functions in elimination of invading bacteria and antigen presentation. Analysis of mammalian homologs of the autophagy-related (Atg) proteins identified in yeast has shed light on not only the common molecular mechanisms underlying autophagosome formation, but also specialized mechanisms that are related to the diverse functions and complex regulation of autophagy in higher organisms.     

Structure-based discovery of a novel non-peptidic small molecular inhibitor of caspase-3

Ac-DNLD-CHO is a novel caspase-3 specific peptide inhibitor that was rationally designed by our computational strategy. The specificity was shown to be due to the specific interaction of NLD moiety with the active site of caspase-3 on the basis of docking mode and site-directed mutagenesis analyses. Here, we computationally screened non-peptidic small molecular inhibitors of caspase-3 from our chemical library using a reliable pharmacophore derived from the specific binding mode of NLD. Through in vitro enzyme assay of the screened candidate compounds, we discovered a novel caspase-3 specific small molecular inhibitor, CS4566, which has a unique scaffold structure. The binding mode of CS4566 to caspase-3 mimics that of NLD, especially LD moiety. This represents a promising lead compound for creating non-peptidic pharmaceuticals for caspase-mediated diseases, such as neurodegenerative disorders.     

Localization of mammalian NAD(P)H steroid dehydrogenase-like protein on lipid droplets

Mammalian enzymes in late cholesterol biosynthesis have been localized uniformly over the endoplasmic reticulum by enzymatic methods. We report here the first mammalian cholesterol biosynthetic enzyme unequivocally localized at the surface of intracellular lipid storage droplets. NAD(P)H steroid dehydrogenase-like protein (Nsdhl), a mammalian C-3 sterol dehydrogenase involved in the conversion of lanosterol into cholesterol, was localized on lipid droplets by immunofluorescence microscopy and subcellular fractionation. Nsdhl was localized on lipid droplets even when cell growth exclusively depended on cholesterol biosynthesis mediated by this enzyme. Depletion of fatty acids in culture medium reduced the development of lipid droplets and caused Nsdhl redistribution to the endoplasmic reticulum. Elevating oleic acid in medium induced well developed, Nsdhl-positive lipid droplets, and simultaneously caused a reduction in cellular conversion of lanosterol into cholesterol. Manipulated human NSDHL with a missense mutation (G205S) causing a human embryonic developmental disorder, congenital hemidysplasia with ichthyosiform nevus and limb defects (CHILD) syndrome, could no longer be localized on lipid droplets. Although the expression of wild-type NSDHL could restore the defective growth of a CHO cholesterol auxotroph, LEX2 in cholesterol-deficient medium, the expression of NSDHL(G205S) failed to do so. These results point to functional significance of the localization of Nsdhl on lipid droplets. Functional significance was also suggested by the colocalization of Nsdhl on lipid droplets with TIP47, a cargo selection protein for mannose 6-phosphate receptors from late endosomes to the trans-Golgi network. These results add to the growing notion that the lipid droplet is an organelle endowed with more complex roles in various biological phenomena.     

Autophagosome formation in mammalian cells

Macroautophagy is an intracellular degradation system for the majority of proteins and some organelles. The molecular mechanism of autophagy has been extensively studied using the yeast, Saccharomyces cerevisiae, during these past 10 years. These studies suggested that the molecular machinery of autophagosome formation is well conserved from yeast to higher eukaryotes. Identification and characterization of the mammalian counterparts of the yeast autophagy proteins has facilitated our understanding of mammalian autophagy, particularly of autophagosome formation. These findings are now being applied to studies on the physiological roles of autophagy in mammals.     

SKD1 AAA ATPase-dependent endosomal transport is involved in autolysosome formation

Mouse SKD1 AAA ATPase is involved in the sorting and transport from endosomes; cells overexpressing a dominant-negative mutant, SKD1(E235Q) were defective in endosomal transport to both the plasma membranes and lysosomes (Yoshimori et al., 2000). In the present study, we demonstrated that overexpression of SKD1(E235Q) using an adenovirus delivery system caused a defect in autophagy-dependent bulk protein degradation. Morphological observations suggested that this inhibition of autophagy results from an impairment of autolysosome formation. SKD1(E235Q) overexpression also inhibited transport from endosomes to autophagosomes, an event normally occurring prior to fusion with lysosomes. These results indicate that SKD1-dependent endosomal membrane trafficking is required for formation of autolysosomes.     

A combined bioinformatic approach oriented to the analysis and design of peptides with high affinity to MHC class I molecules

We report on a new method to compute the antigenic degree of peptides from available experimental data on peptide binding affinity to class I MHC molecules. The methodology is a combination of two strategies at different levels of information. The first, at the primary structure level, consists in expressing the peptides binding activity as a profile of amino acid contributions, amino acid similarity being accounted for by their characteristic physicochemical properties and their position within the sequence. The higher level of the strategy is based on a meticulous analysis of the contact interface of the peptides with the cleft constituting the receptor region of a particular class I MHC molecule. Interaction interfaces are inferred by docking the peptide onto the receptor groove of the MHC molecule; evaluation of the affinity of the peptide to the receptor is then performed by analysis of the electrostatic and hydrophobic energies on points of the interaction interface.The result is a robust system for analysis of peptide affinity to class I MHC molecules since while the first analysis dictates the composition of active sequences at the amino acid level, the second translates this information to the atomic level, where the molecular interaction can be analyzed in terms of the intrinsic interatomic forces and energies. Evaluation results for the methodology are encouraging since high affinity peptides are reflected by high scores at both levels of information, and are proportionally lower for peptides of medium and lower affinity for which interaction surfaces show relatively lower electrostatic complementarity and hydrophobic correlation than for the former.     

Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3

During the process of autophagy, autophagosomes undergo a maturation process consisting of multiple fusions with endosomes and lysosomes, which provide an acidic environment and digestive function to the interior of the autophagosome. Here we found that a fusion protein of monomeric red-fluorescence protein and LC3, the most widely used marker for autophagosomes, exhibits a quite different localization pattern from that of GFP-LC3. GFP-LC3 loses fluorescence due to lysosomal acidic and degradative conditions but mRFP-LC3 does not, indicating that the latter can label the autophagic compartments both before and after fusion with lysosomes. Taking advantage of this property, we devised a novel method for dissecting the maturation process of autophagosomes. mRFP-GFP tandem fluorescent-tagged LC3 (tfLC3) showed a GFP and mRFP signal before the fusion with lysosomes, and exhibited only the mRFP signal subsequently. Using this method, we provided evidence that overexpression of a dominant negative form of Rab7 prevented the fusion of autophagosomes with lysosomes, suggesting that Rab7 is involved in this step. This method will be of general utility for analysis of the autophagosome maturation process.     

Retinoic acid treatment elevates matrix metalloproteinase-2 protein and mRNA levels in avian growth plate chondrocyte cultures

Matrix metalloproteinases (MMPs) play a crucial role in tissue remodeling. In growth plate (GP) cartilage, extensive remodeling occurs at the calcification front. To study the potential involvement of MMPs in retinoic acid (RA) regulation of skeletal development, we studied the effect of all-trans-RA on MMPs levels in mineralizing chicken epiphyseal chondrocyte primary cultures. When treated for 4 day periods on days 10 and 17, RA increased levels of an ∼70 kDa gelatinase activity. The N-terminal sequence of the first 20 amino acid residues of the purified enzyme was identical to that deduced from chicken MMP-2 cDNA. Time-course studies indicated that RA elevated MMP-2 activity levels in the cultures within 16 h. This increase was inhibited by cycloheximide and was enhanced by forskolin. The increase in MMP-2 activity induced by RA was accompanied by an increase in MMP-2 mRNA levels and was abolished by treatment with cycloheximide. This upregulation of MMP levels by RA in GP chondrocytes is consistent with its effects on osteoblasts and osteosarcoma cells and opposite its inhibitory effects on fibroblasts and endothelial cells. It may well be related to the breakdown of the extracellular matrix in the GP and would be governed by the availability of RA at the calcification front where extensive vascularization also occurs. J. Cell. Biochem. 68:90–99, 1998. © 1998 Wiley-Liss, Inc.     

Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi network

Autophagy is an intracellular bulk protein degradation system. Beclin is known to be involved in this process; however, its role is unclear. In this study, we showed that Beclin was co-immunoprecipitated with phosphatidylinositol (PtdIns) 3-kinase, which is also required for autophagy, suggesting that Beclin is a component of the PtdIns 3-kinase complex. Quantitative analyses using a cross-linker showed that all Beclin forms a complex with PtdIns 3-kinase, whereas ~50% of PtdIns 3-kinase remains free from Beclin. Indirect immunofluorescence microscopy demonstrated that the majority of Beclin and PtdIns 3-kinase localize to the trans-Golgi network (TGN). Some PtdIns 3-kinase is also distributed in the late endosome. These results suggest that Beclin and PtdIns 3-kinase control autophagy as a complex at the TGN.