Structural basis for recognition of phosphodiester-containing lysosomal enzymes by the cation-independent mannose 6-phosphate receptor

Mannose 6-phosphate (Man-6-P)-dependent trafficking is vital for normal development. The biogenesis of lysosomes, a major cellular site of protein, carbohydrate, and lipid catabolism, depends on the 300-kDa cation-independent Man-6-P receptor (CI-MPR) that transports newly synthesized acid hydrolases from the Golgi. The CI-MPR recognizes lysosomal enzymes bearing the Man-6-P modification, which arises by the addition of GlcNAc-1-phosphate to mannose residues and subsequent removal of GlcNAc by the uncovering enzyme (UCE). The CI-MPR also recognizes lysosomal enzymes that elude UCE maturation and instead display the Man-P-GlcNAc phosphodiester. This ability of the CI-MPR to target phosphodiester-containing enzymes ensures lysosomal delivery when UCE activity is deficient. The extracellular region of the CI-MPR is comprised of 15 repetitive domains and contains three distinct Man-6-P binding sites located in domains 3, 5, and 9, with only domain 5 exhibiting a marked preference for phosphodiester-containing lysosomal enzymes. To determine how the CI-MPR recognizes phosphodiesters, the structure of domain 5 was determined by NMR spectroscopy. Although domain 5 contains only three of the four disulfide bonds found in the other seven domains whose structures have been determined to date, it adopts the same fold consisting of a flattened β-barrel. Structure determination of domain 5 bound to N-acetylglucosaminyl 6-phosphomethylmannoside, along with mutagenesis studies, revealed the residues involved in diester recognition, including Y679. These results show the mechanism by which the CI-MPR recognizes Man-P-GlcNAc-containing ligands and provides new avenues to investigate the role of phosphodiester-containing lysosomal enzymes in the biogenesis of lysosomes.     

Transgenic analysis of the BmBLOS2 gene that governs the translucency of the larval integument of the silkworm,Bombyx mori

The larval integument of the silkworm, Bombyx mori, is opaque because urate granules accumulate in the epidermis. Although the biosynthetic pathway of uric acid is well studied, little is known about how uric acid accumulates as urate granules in epidermal cells. In the distinct oily (od) mutant silkworm, the larval integument is translucent because of the inability to construct urate granules. Recently, we have found that the od mutant has a genomic deletion in the B. mori homologue of the human biogenesis of lysosome‐related organelles complex1, subunit 2 (BLOS2) gene (BmBLOS2). Here, we performed a molecular and functional characterization of BmBLOS2. Northern blot analysis showed that BmBLOS2 was ubiquitously expressed in various tissues. We analysed the structure of a newly isolated mutant (od^B) allelic to od and found a premature stop codon in the coding sequence of BmBLOS2 in this new mutation. Moreover, the translucent phenotype was rescued by the germ‐line transformation of the wild‐type BmBLOS2 allele into the od mutant. Our results suggest that BmBLOS2 is responsible for the od mutant phenotype and plays a crucial role in biogenesis of urate granules in the larval epidermis of the silkworm. The relationships amongst Hermansky–Pudlak syndrome (HPS) genes in mammals, granule group genes in Drosophila and translucent mutant genes in B. mori are discussed.     

溶酶体在新城疫病毒诱导肿瘤细胞凋亡和自噬中的作用

溶瘤病毒(oncolytic virus, OV)具有对肿瘤细胞选择性杀伤而对正常细胞无影响的特性,在针对肿瘤治疗方面有着巨大的潜力。凋亡与自噬在溶瘤病毒杀伤肿瘤细胞的过程中扮演者重要的角色,而溶酶体与二者有着密切的联系。新城疫病毒(newcastle disease virus, NDV)在感染肿瘤细胞后,溶酶体不仅会触发凋亡和溶酶体细胞死亡途径,而且会抑制细胞发生保护性自噬。因此,了解溶酶体在NDV诱导凋亡和自噬的确切作用可以提高NDV治疗各种癌症的治疗潜力,为今后攻克肿瘤提供理论基础。     

Targeting autophagy enhances the anticancer effect of artemisinin and its derivatives

Artemisinin and its derivatives, with their outstanding clinical efficacy and safety, represent the most effective and impactful antimalarial drugs. Apart from its antimalarial effect, artemisinin has also been shown to exhibit selective anticancer properties against multiple cancer types both in vitro and in vivo. Specifically, our previous studies highlighted the therapeutic effects of artemisinin on autophagy regulation. Autophagy is a well-conserved degradative process that recycles cytoplasmic contents and organelles in lysosomes to maintain cellular homeostasis. The deregulation of autophagy is often observed in cancer cells, where it contributes to tumor adaptation to nutrient-deficient tumor microenvironments. This review discusses recent advances in the anticancer properties of artemisinin and its derivatives via their regulation of autophagy, mitophagy, and ferritinophagy. In particular, we will discuss the mechanisms of artemisinin activation in cancer and novel findings regarding the role of artemisinin in regulating autophagy, which involves changes in multiple signaling pathways. More importantly, with increasing failure rates and the high cost of the development of novel anticancer drugs, the strategy of repurposing traditional therapeutic Chinese medicinal agents such as artemisinin to treat cancer provides a more attractive alternative. We believe that the topics covered here will be important in demonstrating the potential of artemisinin and its derivatives as safe and potent anticancer agents.     

Ctr2 regulates biogenesis of a cleaved form of mammalian Ctr1 metal transporter lacking the copper- and cisplatin-binding ecto-domain

Copper is an essential catalytic cofactor for enzymatic activities that drive a range of metabolic biochemistry including mitochondrial electron transport, iron mobilization, and peptide hormone maturation. Copper dysregulation is associated with fatal infantile disease, liver, and cardiac dysfunction, neuropathy, and anemia. Here we report that mammals regulate systemic copper acquisition and intracellular mobilization via cleavage of the copper-binding ecto-domain of the copper transporter 1 (Ctr1). Although full-length Ctr1 is critical to drive efficient copper import across the plasma membrane, cleavage of the ecto-domain is required for Ctr1 to mobilize endosomal copper stores. The biogenesis of the truncated form of Ctr1 requires the structurally related, previously enigmatic copper transporter 2 (Ctr2). Ctr2^−/− mice are defective in accumulation of truncated Ctr1 and exhibit increased tissue copper levels, and X-ray fluorescence microscopy demonstrates that copper accumulates as intracellular foci. These studies identify a key regulatory mechanism for mammalian copper transport through Ctr2-dependent accumulation of a Ctr1 variant lacking the copper- and cisplatin-binding ecto-domain.Due to its unique chemistry, the redox-active metal ion copper (Cu) is an essential element for human growth and development (1–3). Defects in Cu metabolism are associated with pathologies that include Alzheimer’s disease, peripheral neuropathy, anemia, neutropenia, cardiomyopathy, Menkes disease, and Wilson’s disease (4–9). Although many of the components responsible for Cu uptake, intracellular distribution, detoxification, and efflux have been identified, the mechanisms by which these proteins are regulated are not well understood.The copper transporter 1 (Ctr1) protein is a high-affinity Cu⁺ transporter that functions in copper accumulation in organisms ranging from yeast to mammals (10–16). In mammals Ctr1 localizes to both the plasma membrane and to intracellular vesicles (17–19). Mice bearing a systemic Ctr1 deletion fail to survive gestation, whereas tissue-specific ablation of Ctr1 in the intestinal epithelium, liver, or heart cause a range of phenotypes that include peripheral Cu deficiency, hepatic iron accumulation, and lethal cardiac hypertrophy, respectively (20–24). Moreover, both yeast and mammalian Ctr1 function in acquisition of the chemotherapeutic agent cisplatin (25–29) and Ctr1 expression levels have been correlated to the efficacy of chemotherapy and patient survival (30). The regulation of Ctr1 function and abundance is of great significance to both normal growth and development as well as to the efficacy of platinum-based chemotherapy.The general structure and function of Ctr1 is conserved from yeast to humans, with three membrane-spanning domains and a Met-X₃-Met motif in the second transmembrane domain that is essential for Cu⁺ import (16). The human and mouse protein contains a short ecto-domain with clusters of Met and His. Mutagenic and truncation studies in the context of intact yeast or human Ctr1 indicate that the ecto-domain in general, and the Met residues in particular, play an important role in high-affinity cellular Cu⁺ import, yet all but one key Met near the first transmembrane domain appear to be dispensable for function in cellular Cu⁺ import (31). Studies using model peptides suggest that the Ctr1 Met residues are direct ligands for both Cu⁺ and cisplatin (32–34). In contrast to Cu⁺ uptake, the Met-rich ecto-domain of yeast Ctr1 is required for cisplatin import (27). Moreover, as Ctr1 M-X₃-M mutants are competent for cisplatin uptake, but not Cu⁺ (35), studies suggest that Ctr1-mediated cisplatin uptake may occur via an ecto-domain–dependent receptor-mediated endocytosis mechanism, rather than as an ion channel as for Cu⁺ (27). Ctr1 has been observed in both cell lines and mouse tissues as a full-length glycosylated form and a lower-molecular-weight form, which has been reported to lack a portion of the Cu⁺ and cisplatin-binding ecto-domain (17, 36). However, neither the physiological significance of this truncated form of Ctr1, nor its mode of biogenesis, have been elucidated.The Ctr2 protein is structurally related to Ctr1 and is encoded by a linked gene in both the mouse and the human genome. Recent studies suggest that Ctr2 functions as a low-affinity Cu⁺ importer, a lysosomal Cu⁺ exporter, or as a regulator of cellular macropinocytosis (37–39). However, these studies have been performed in cultured cells, and the physiological role of Ctr2 in animals has not been reported. Here we demonstrate that Ctr2 interacts with Ctr1 in vivo and that Ctr2 knockout mice show increased levels of total copper in several tissues. Mice and mouse embryonic fibroblasts lacking Ctr2 accumulate copper in endosomal compartments and have lower levels of the truncated form of Ctr1 lacking the metal-binding ecto-domain. Whereas truncation of the Ctr1 ecto-domain reduces Cu⁺ import at the plasma membrane, truncated Ctr1 stimulates the mobilization of Cu⁺ from endosomal compartments. These studies demonstrate a critical role for Ctr2 in modulating the accumulation of Ctr1 lacking the Cu⁺ and cisplatin-binding ecto-domain of Ctr1 and, as a consequence, in the regulation of cellular copper uptake and intracellular mobilization. Given the fundamental role for Ctr1 in Cu⁺ import and cisplatin acquisition, the action of Ctr2 represents an important mechanism for the regulation of Ctr1 function.     

Autophagic-lysosomal perturbation enhances tau aggregation in transfectants with induced wild-type tau expression

The intracellular assembly of tau aggregates is a pathological hallmark shared by Alzheimer's disease and other neurodegenerative disorders known collectively as tauopathies. To model how tau fibrillogenesis evolves in tauopathies, we previously established transfectant M1C cultures from human neuroblastoma BE(2)-M17D cells that inducibly express human tau. In the present study, these cells were used to determine the role of the autophagic-lysosomal system in the degradation and aggregation of wild-type tau. Tau induction for 5 days led to the accumulation of tau with nominal assembly of tau aggregates within cells. When the lysosomotropic agent, chloroquine (CQ), was added following the termination of tau induction, tau clearance was delayed. Decreased tau truncation and increased levels of intact tau were observed. When present during tau induction, CQ led to tau accumulation and promoted the formation of sarkosyl-insoluble aggregates containing both truncated and full-length tau. CQ treatment significantly decreased the activities of cathepsins D, B and L, and the inhibition of cathepsins B and L mimicked the effect of CQ and increased tau levels in cells. Additionally, exposure of cells to the autophagy inhibitor, 3-methyladenine, led to tau accumulation and aggregation. These results suggest that the autophagic-lysosomal system plays a role in the clearance of tau, and that dysfunction of this system results in the formation of tau oligomers and insoluble aggregates.     

Abnormal neuronal metabolism and storage in mucopolysaccharidosis type VI (Maroteaux-Lamy) disease

Mucopolysaccharidosis (MPS) type VI, also known as Maroteaux-Lamy disease, is an inherited disorder of glycosaminoglycan catabolism caused by deficient activity of the lysosomal hydrolase, N-acetylgalactosamine 4-sulphatase (4S). A variety of prominent visceral and skeletal defects are characteristic, but primary neurological involvement has generally been considered absent. We report here that the feline model of MPS VI exhibits abnormal lysosomal storage in occasional neurones and glia distributed throughout the cerebral cortex. Abnormal lysosomal inclusions were pleiomorphic with some resembling zebra bodies and dense core inclusions typical of other MPS diseases or the membranous storage bodies characteristic of the gangliosidoses. Pyramidal neurones were shown to contain abnormal amounts of GM2 and GM3 gangliosides by immunocytochemical staining and unesterified cholesterol by histochemical (filipin) staining. Further, Golgi staining of pyramidal neurones revealed that some possessed ectopic axon hillock neurites and meganeurites similar to those described in Tay-Sachs and other neuronal storage diseases with ganglioside storage. Some animals evaluated in this study also received allogeneic bone marrow transplants, but no significant differences in neuronal storage were noted between treated and untreated individuals. These studies demonstrate that deficiency of 4S activity can lead to metabolic abnormalities in the neurones of central nervous system in cats, and that these changes may not be readily amenable to correction by bone marrow transplantation. Given the close pathological and biochemical similarities between feline and human MPS VI, it is conceivable that children with this disease have similar neuronal involvement.     

Skeletal muscle expression of clathrin and mannose 6-phosphate receptor in experimental chloroquine-induced myopathy

Previous studies suggest that the muscle fiber lysosome system plays a central role in the increased formation of autophagosomes and autolysosomes that occurs in the context of chloroquine-induced myopathy. The goal of this study was to characterize the contribution of receptor-mediated intracellular transport, particularly the endosomal pathway, to the abnormal accumulation of vacuoles in experimental chloroquine myopathy. Expression of the mannose 6-phosphate receptor (M6PR) and clathrin were analyzed in innervated and denervated rat soleus muscles after treatment with either saline or chloroquine. Accumulation of vacuoles was observed only in chloroquine-treated denervated muscles. Further, clathrin immunostaining and M6PR messenger ribonucleic acid (mRNA) were significantly increased in denervated soleus muscle from saline- and chloroquine-treated rats compared to contralateral, innervated muscles. However, there was no difference in clathrin levels when comparing saline- and chloroquine-treated denervated muscles. These data suggest that chloroquine activates the transport of newly synthesized lysosomal enzymes from the secretory pathway via the trans-Golgi network of the Golgi apparatus (an endosomal pathway) as well as autophagosome formation (an autophagic process) in skeletal muscles. Vacuoles may subsequently accumulate secondary to abnormal formation or turnover of autolysosomes at or after fusion of autophagosomes with early endosomes.