Lethality of mice bearing a knockout of the Ngly1-gene is partially rescued by the additional deletion of the Engase gene.

The cytoplasmic peptide:N-glycanase (Ngly1 in mammals) is a de-N-glycosylating enzyme that is highly conserved among eukaryotes. It was recently reported that subjects harboring mutations in the NGLY1 gene exhibited severe systemic symptoms (NGLY1-deficiency). While the enzyme obviously has a critical role in mammals, its precise function remains unclear. In this study, we analyzed Ngly1-deficient mice and found that they are embryonic lethal in C57BL/6 background. Surprisingly, the additional deletion of the gene encoding endo-ß-N-acetylglucosaminidase (Engase), which is another de-N-glycosylating enzyme but leaves a single GlcNAc at glycosylated Asn residues, resulted in the partial rescue of the lethality of the Ngly1-deficient mice. Additionally, we also found that a change in the genetic background of C57BL/6 mice, produced by crossing the mice with an outbred mouse strain (ICR) could partially rescue the embryonic lethality of Ngly1-deficient mice. Viable Ngly1-deficient mice in a C57BL/6 and ICR mixed background, however, showed a very severe phenotype reminiscent of the symptoms of NGLY1-deficiency subjects. Again, many of those defects were strongly suppressed by the additional deletion of Engase in the C57BL/6 and ICR mixed background. The defects observed in Ngly1/Engase-deficient mice (C57BL/6 background) and Ngly1-deficient mice (C57BL/6 and ICR mixed background) closely resembled some of the symptoms of patients with an NGLY1-deficiency. These observations strongly suggest that the Ngly1- or Ngly1/Engase-deficient mice could serve as a valuable animal model for studies related to the pathogenesis of the NGLY1-deficiency, and that cytoplasmic ENGase represents one of the potential therapeutic targets for this genetic disorder.

Endo-ß-N-acetylglucosaminidase forms N-GlcNAc protein aggregates during ER-associated degradation in Ngly1-defective cells.

The cytoplasmic peptide:N-glycanase (PNGase; Ngly1 in mice) is a deglycosylating enzyme involved in the endoplasmic reticulum (ER)-associated degradation (ERAD) process. The precise role of Ngly1 in the ERAD process, however, remains unclear in mammals. The findings reported herein, using mouse embryonic fibroblast (MEF) cells, that the ablation of Ngly1 causes dysregulation of the ERAD process. Interestingly, not only delayed degradation but also the deglycosylation of a misfolded glycoprotein was observed in Ngly1(-/-) MEF cells. The unconventional deglycosylation reaction was found to be catalyzed by the cytosolic endo-ß-N-acetylglucosaminidase (ENGase), generating aggregation-prone N-GlcNAc proteins. The ERAD dysregulation in cells lacking Ngly1 was restored by the additional knockout of ENGase gene. Thus, our study underscores the functional importance of Ngly1 in the ERAD process and provides a potential mechanism underlying the phenotypic consequences of a newly emerging genetic disorder caused by mutation of the human NGLY1 gene.

The Neurospora peptide:N-glycanase ortholog PNG1 is essential for cell polarity despite its lack of enzymatic activity.

Secretory proteins are subjected to a stringent endoplasmic reticulum-based quality control system that distinguishes aberrant from correctly folded proteins. The cytoplasmic peptide:N-glycanase cleaves oligosaccharides from misfolded glycoproteins and prepares them for degradation by the 26 S proteasome. In contrast to abundant in vitro data on its enzymatic function, the in vivo relevance of peptide:N-glycanase activity remains unclear. Here we show that the PNG1 ortholog from the filamentous ascomycete Neurospora crassa is an essential protein, and its deletion results in strong polarity defects. PNG1 and its predicted binding partner RAD23 have distinct functions in N. crassa and are involved in cell wall integrity and DNA repair, respectively. Moreover, wild type PNG1 has substitutions in essential catalytic amino acids, and its deglycosylation activity is lost. These substitutions are conserved in many PNG1 orthologs of the fungal kingdom, implying a so far unrecognized enzyme-independent function of PNG1 that may only become apparent in highly polar cells such as fungal hyphae.

Discovery of AS-0141, a Potent and Selective Inhibitor of CDC7 Kinase for the Treatment of Solid Cancers.

CDC7, a serine-threonine kinase, plays conserved and important roles in regulation of DNA replication and has been recognized as a potential anticancer target. We report here the optimization of a series of furanone analogues starting from compound 1 with a focus on ADME properties suitable for clinical development. By replacing the 2-chlorobenzene moiety in 1 with various aliphatic groups, we identified compound 24 as a potent CDC7 inhibitor with excellent kinase selectivity and favorable oral bioavailability in multiple species. Oral administration of 24 demonstrated robust in vivo antitumor efficacy in a colorectal cancer xenograft model. Compound 24 (AS-0141) is currently in phase I clinical trials for the treatment of solid cancers.

Glycobiology in the cytosol: the bitter side of a sweet world.

Progress in glycobiology has undergone explosive growth over the past decade with more of the researchers now realizing the importance of glycan chains in various inter- and intracellular processes. However, there is still an area of glycobiology awaiting exploration. This is especially the case for the field of "glycobiology in the cytosol" which remains rather poorly understood. Yet evidence is accumulating to demonstrate that the glycoconjugates and their recognition molecules (i.e. lectins) are often present in this subcellular compartment.

Accumulation of free complex-type N-glycans in MKN7 and MKN45 stomach cancer cells.

During the N-glycosylation reaction, it has been shown that 'free' N-glycans are generated either from lipid-linked oligosaccharides or from misfolded glycoproteins. In both cases, occurrence of high mannose-type free glycans is well-documented, and the molecular mechanism for their catabolism in the cytosol has been studied. On the other hand, little, if anything, is known with regard to the accumulation of more processed, complex-type free oligosaccharides in the cytosol of mammalian cells. During the course of comprehensive analysis of N-glycans in cancer cell membrane fractions [Naka et al. (2006) J. Proteome Res. 5, 88-97], we found that a significant amount of unusual, complex-type free N-glycans were accumulated in the stomach cancer-derived cell lines, MKN7 and MKN45. The most abundant and characteristic glycan found in these cells was determined to be NeuAcalpha2-6Galbeta1-4GlcNAcbeta1-2Manalpha1-3Manbeta1-4GlcNAc. Biochemical analyses indicated that those glycans found were cytosolic glycans derived from lysosomes due to low integrity of the lysosomal membrane. Since the accumulation of these free N-glycans was specific to only two cell lines among the various cancer cell lines examined, these cytosolic N-glycans may serve as a specific biomarker for diagnosis of specific tumours. A cytosolic sialidase, Neu2, was shown to be involved in the degradation of these sialoglycans, indicating that the cytosol of mammalian cells might be equipped for metabolism of complex-type glycans.

Man2C1, an alpha-mannosidase, is involved in the trimming of free oligosaccharides in the cytosol.

The endoplasmic-reticulum-associated degradation of misfolded (glyco)proteins ensures that only functional, correctly folded proteins exit from the endoplasmic reticulum and that misfolded ones are degraded by the ubiquitin-proteasome system. During the degradation of misfolded glycoproteins, they are deglycosylated by the PNGase (peptide:N-glycanase). The free oligosaccharides released by PNGase are known to be further catabolized by a cytosolic alpha-mannosidase, although the gene encoding this enzyme has not been identified unequivocally. The findings in the present study demonstrate that an alpha-mannosidase, Man2C1, is involved in the processing of free oligosaccharides that are formed in the cytosol. When the human Man2C1 orthologue was expressed in HEK-293 cells, most of the enzyme was localized in the cytosol. Its activity was enhanced by Co2+, typical of other known cytosolic alpha-mannosidases so far characterized from animal cells. The down-regulation of Man2C1 activity by a small interfering RNA drastically changed the amount and structure of oligosaccharides accumulating in the cytosol, demonstrating that Man2C1 indeed is involved in free oligosaccharide processing in the cytosol. The oligosaccharide processing in the cytosol by PNGase, endo-beta-N-acetylglucosaminidase and alpha-mannosidase may represent the common 'non-lysosomal' catabolic pathway for N-glycans in animal cells, although the molecular mechanism as well as the functional importance of such processes remains to be determined.

Discovery of novel furanone derivatives as potent Cdc7 kinase inhibitors.

Cdc7 is a serine-threonine kinase and plays a conserved and important role in DNA replication, and it has been recognized as a potential anticancer target. Herein, we report the design, synthesis and structure-activity relationship of novel furanone derivatives as Cdc7 kinase inhibitors. Compound 13 was identified as a strong inhibitor of Cdc7 with an IC50 value of 0.6 nM in the presence of 1 mM ATP and showed excellent kinase selectivity. In addition, it exhibited slow off-rate characteristics, which may offer advantages over known Cdc7 inhibitors in its potential to yield prolonged inhibitory effects in vivo. Compound 13 potently inhibited Cdc7 activity in cancer cells, and effectively induced cell death.

Co-Expression of NEU2 and GBA3 Causes a Drastic Reduction in Cytosolic Sialyl Free N-glycans in Human MKN45 Stomach Cancer Cells-Evidence for the Physical Interaction of NEU2 and GBA3.

It is well known that the "free" form of glycans that are structurally related to asparagine (N)-linked glycans ("free N-glycans") are found in a wide variety of organisms. The mechanisms responsible for the formation/degradation of high mannose-type free N-glycans have been extensively studied in mammalian cells. Recent evidence, however, also suggests that sialylated, complex-type free N-glycans are also present in the cytosol of various mammalian-derived cultured cells/tissues. We report herein on an investigation of the mechanism responsible for the degradation of such sialyl free N-glycans. The findings show that the amount of glycans is dramatically reduced upon the co-expression of cytosolic sialidase NEU2 with cytosolic ß-glycosidase GBA3 in human stomach cancer-derived MKN45 cells. The physical interaction between NEU2 and GBA3 was confirmed by co-precipitation analyses as well as gel filtration assays. The NEU2 protein was found to be stabilized in the presence of GBA3 both in cellulo and in vitro. Our results thus indicate that cytosolic GBA3 is likely involved in the catabolism of cytosolic sialyl free N-glycans, possibly by stabilizing the activity of the NEU2 protein.

Genome-wide microarray analysis of TGFbeta signaling in the Drosophila brain.

BACKGROUND: Members of TGFbeta superfamily are found to play important roles in many cellular processes, such as proliferation, differentiation, development, apoptosis, and cancer. In Drosophila, there are seven ligands that function through combinations of three type I receptors and two type II receptors. These signals can be roughly grouped into two major TGFbeta pathways, the dpp/BMP and activin pathways, which signal primarily through thick veins (tkv) and baboon (babo). Few downstream targets are known for either pathway, especially targets expressed in the Drosophila brain. RESULTS: tkv and babo both affect the growth of tissues, but have varying effects on patterning. We have identified targets for the tkv and babo pathways by employing microarray techniques using activated forms of the receptors expressed in the brain. In these experiments, we compare the similarities of target genes of these two pathways in the brain. About 500 of 13,500 examined genes changed expression at 95% confidence level (P < 0.05). Twenty-seven genes are co-regulated 1.5 fold by both the tkv and babo pathways. These regulated genes cluster into various functional groups such as DNA/RNA binding, signal transducers, enzymes, transcription regulators, and neuronal regulators. RNAi knockdown experiments of homologs of several of these genes show abnormal growth regulation, suggesting these genes may execute the growth properties of TGFbeta. CONCLUSIONS: Our genomic-wide microarray analysis has revealed common targets for the tkv and babo pathways and provided new insights into downstream effectors of two distinct TGFbeta like pathways. Many of these genes are novel and several genes are implicated in growth control. Among the genes regulated by both pathways is ultraspiracle, which further connects TGFbeta with neuronal remodeling.

Evidence for an essential deglycosylation-independent activity of PNGase in Drosophila melanogaster.

BACKGROUND: Peptide:N-glycanase (PNGase) is an enzyme which releases N-linked glycans from glycopeptides/glycoproteins. This enzyme plays a role in the ER-associated degradation (ERAD) pathway in yeast and mice, but the biological importance of this activity remains unknown. PRINCIPAL FINDINGS: In this study, we characterized the ortholog of cytoplasmic PNGases, PNGase-like (Pngl), in Drosophila melanogaster. Pngl was found to have a molecular weight of approximately 74K and was mainly localized in the cytosol. Pngl lacks a CXXC motif that is critical for enzymatic activity in other species and accordingly did not appear to possess PNGase activity, though it still retains carbohydrate-binding activity. We generated microdeletions in the Pngl locus in order to investigate the functional importance of this protein in vivo. Elimination of Pngl led to a serious developmental delay or arrest during the larval and pupal stages, and surviving mutant adult males and females were frequently sterile. Most importantly, these phenotypes were rescued by ubiquitous expression of Pngl, clearly indicating that those phenotypic consequences were indeed due to the lack of functional Pngl. Interestingly, a putative "catalytic-inactive" mutant could not rescue the growth-delay phenotype, indicating that a biochemical activity of this protein is important for its biological function. CONCLUSION: Pngl was shown to be inevitable for the proper developmental transition and the biochemical properties other than deglycosylation activity is important for its biological function.

Free N-linked oligosaccharide chains: formation and degradation.

There is growing evidence that N-linked glycans play pivotal roles in protein folding and intra- and/or intercellular trafficking of N-glycosylated proteins. It has been shown that during the N-glycosylation of proteins, significant amounts of free oligosaccharides (free OSs) are generated in the lumen of the endoplasmic reticulum (ER) by a mechanism which remains to be clarified. Free OSs are also formed in the cytosol by enzymatic deglycosylation of misfolded glycoproteins, which are subjected to destruction by a cellular system called "ER-associated degradation (ERAD)." While the precise functions of free OSs remain obscure, biochemical studies have revealed that a novel cellular process enables them to be catabolized in a specialized manner, that involves pumping free OSs in the lumen of the ER into the cytosol where further processing occurs. This process is followed by entry into the lysosomes. In this review we summarize current knowledge about the formation, processing and degradation of free OSs in eukaryotes and also discuss the potential biological significance of this pathway. Other evidence for the occurrence of free OSs in various cellular processes is also presented.