A Case of Aggressive Lung Squamous Cell Carcinoma With Aberrant Cytoplasmic p53 Aggregation.

Background: Immunohistochemistry for p53 was a well-established method for cancer diagnosis in pathology. Aberrant cytoplasmic p53 positivity reflects the accumulation of p53 aggregates, which has been shown to be associated with chemoresistance and to be a predictive marker of a worse clinical course in ovarian cancer. Case Report: A 65-year-old Japanese man was diagnosed with lung cancer, and surgical resection was performed. Multiple metastasis were found 21 months post-surgery. The lesions were resistant to chemotherapy, and he succumbed to the disease 29 months post-surgery. The resected primary lesion was pathologically diagnosed as squamous cell carcinoma, with notable cytoplasmic p53 positivity indicated by immunohistochemistry. Conclusion: Notable aberrant cytoplasmic accumulation of p53 aggregate was observed in the cancer cells of this case. Chemotherapy was ineffective for the recurrent lesions, suggesting a role of p53 aggregates in chemoresistance. Pathological analysis of p53 via immunohistochemistry may be useful in predicting chemoresistance of lung squamous cell carcinoma.

GlcNAc6ST2/CHST4 Is Essential for the Synthesis of R-10G-Reactive Keratan Sulfate/Sulfated N-Acetyllactosamine Oligosaccharides in Mouse Pleural Mesothelium.

We recently showed that 6-sulfo sialyl N-acetyllactosamine (LacNAc) in O-linked glycans recognized by the CL40 antibody is abundant in the pleural mesothelium under physiological conditions and that these glycans undergo complementary synthesis by GlcNAc6ST2 (encoded by Chst4) and GlcNAc6ST3 (encoded by Chst5) in mice. GlcNAc6ST3 is essential for the synthesis of R-10G-positive keratan sulfate (KS) in the brain. The predicted minimum epitope of the R-10G antibody is a dimeric asialo 6-sulfo LacNAc. Whether R-10G-reactive KS/sulfated LacNAc oligosaccharides are also present in the pleural mesothelium was unknown. The question of which GlcNAc6STs are responsible for R-10G-reactive glycans was an additional issue to be clarified. Here, we show that R-10G-reactive glycans are as abundant in the pulmonary pleura as CL40-reactive glycans and that GlcNAc6ST3 is only partially involved in the synthesis of these pleural R-10G glycans, unlike in the adult brain. Unexpectedly, GlcNAc6ST2 is essential for the synthesis of R-10G-positive KS/sulfated LacNAc oligosaccharides in the lung pleura. The type of GlcNAc6ST and the magnitude of its contribution to KS glycan synthesis varied among tissues in vivo. We show that GlcNAc6ST2 is required and sufficient for R-10G-reactive KS synthesis in the lung pleura. Interestingly, R-10G immunoreactivity in KSGal6ST (encoded by Chst1) and C6ST1 (encoded by Chst3) double-deficient mouse lungs was markedly increased. MUC16, a mucin molecule, was shown to be a candidate carrier protein for pleural R-10G-reactive glycans. These results suggest that R-10G-reactive KS/sulfated LacNAc oligosaccharides may play a role in mesothelial cell proliferation and differentiation. Further elucidation of the functions of sulfated glycans synthesized by GlcNAc6ST2 and GlcNAc6ST3, such as R-10G and CL40 glycans, in pathological conditions may lead to a better understanding of the underlying mechanisms of the physiopathology of the lung mesothelium.

Structural flexibility of apolipoprotein E-derived arginine-rich peptides improves their cell penetration capability.

Amphipathic arginine-rich peptide, A2-17, exhibits moderate perturbation of lipid membranes and the highest cell penetration among its structural isomers. We investigated the direct cell-membrane penetration mechanism of the A2-17 peptide while focusing on structural flexibility. We designed conformationally constrained versions of A2-17, stapled (StpA2-17) and stitched (StchA2-17), whose α-helical conformations were stabilized by chemical crosslinking. Circular dichroism confirmed that StpA2-17 and StchA2-17 had higher α-helix content than A2-17 in aqueous solution. Upon liposome binding, only A2-17 exhibited a coil-to-helix transition. Confocal microscopy revealed that A2-17 had higher cell penetration efficiency than StpA2-17, whereas StchA2-17 remained on the cell membrane without cell penetration. Although the tryptophan fluorescence analysis suggested that A2-17 and its analogs had similar membrane-insertion positions between the interface and hydrophobic core, StchA2-17 exhibited a higher membrane affinity than A2-17 or StpA2-17. Atomic force microscopy demonstrated that A2-17 reduced the mechanical rigidity of liposomes to a greater extent than StpA2-17 and StchA2-17. Finally, electrophysiological analysis showed that A2-17 induced a higher charge influx through transient pores in a planer lipid bilayer than StpA2-17 and StchA2-17. These findings indicate that structural flexibility, which enables diverse conformations of A2-17, leads to a membrane perturbation mode that contributes to cell membrane penetration.

Amyloidogenic 60-71 deletion/ValThr insertion mutation of apolipoprotein A-I generates a new aggregation-prone segment that promotes nucleation through entropic effects.

The N-terminal fragment of apolipoprotein A-I (apoA-I), comprising residues 1-83, contains three segments prone to aggregation: residues 14-22, 53-58, and 67-72. We previously demonstrated that residues 14-22 are critical in apoA-I fibril formation while residues 53-58 entropically drove the nucleation process. Here, we investigated the impact of amyloidogenic mutations (Δ60-71/VT, Δ70-72, and F71Y) located around residues 67-72 on fibril formation by the apoA-I 1-83 fragment. Thioflavin T fluorescence assay demonstrated that the Δ60-71/VT mutation significantly enhances both nucleation and fibril elongation rates, whereas the Δ70-72 and F71Y mutations had minimal effects. Circular dichroism measurements and microscopic observations revealed that all variant fragments formed straight fibrils, transitioning from random coils to ß-sheet structures. Kinetic analysis demonstrated that primary nucleation is the dominant step in fibril formation, with fibril elongation reaching saturation at high protein concentrations. Thermodynamically, both nucleation and fibril elongation were enthalpically and entropically unfavorable in all apoA-I 1-83 variants, in which the entropic barrier of nucleation was almost eliminated for the Δ60-71/VT variant. Taken together, our results suggest the presence of new aggregation-prone segment in the Δ60-71/VT variant that promotes nucleation through entropic effects.

Calnexin Is Involved in Forskolin-Induced Syncytialization in Cytotrophoblast Model BeWo Cells.

Calnexin (CNX), a membrane-bound molecular chaperone, is involved in protein folding and quality control of nascent glycoproteins in the endoplasmic reticulum. We previously suggested critical roles of calreticulin, a functional paralogue of CNX, in placentation, including invasion of extravillous trophoblasts and syncytialization of cytotrophoblasts. However, the roles of CNX in placentation are unclear. In human choriocarcinoma BeWo cells, which serve as an experimental model of syncytialization, CNX knockdown suppressed forskolin-induced cell fusion and ß-human chorionic gonadotropin (ß-hCG) induction. Cell-surface luteinizing hormone/chorionic gonadotropin receptor, a ß-hCG receptor, was significantly down-regulated in CNX-knockdown cells, which suggested the presence of a dysfunctional autocrine loop of ß-hCG up-regulation. In this study, we also found abundant CNX expression in normal human placentas. Collectively, our results revealed the critical role of CNX in the syncytialization-related signaling in a villous trophoblast model and suggest a link between CNX expression and placenta development.

Complementary Role of GlcNAc6ST2 and GlcNAc6ST3 in Synthesis of CL40-Reactive Sialylated and Sulfated Glycans in the Mouse Pleural Mesothelium.

Sialyl 6-sulfo Lewis X (6-sulfo sLeX) and its derivative sialyl 6-sulfo N-acetyllactosamine (LacNAc) are sialylated and sulfated glycans of sialomucins found in the high endothelial venules (HEVs) of secondary lymphoid organs. A component of 6-sulfo sLeX present in the core 1-extended O-linked glycans detected by the MECA-79 antibody was previously shown to exist in the lymphoid aggregate vasculature and bronchial mucosa of allergic and asthmatic lungs. The components of 6-sulfo sLeX in pulmonary tissues under physiological conditions remain to be analyzed. The CL40 antibody recognizes 6-sulfo sLeX and sialyl 6-sulfo LacNAc in O-linked and N-linked glycans, with absolute requirements for both GlcNAc-6-sulfation and sialylation. Immunostaining of normal mouse lungs with CL40 was performed and analyzed. The contribution of GlcNAc-6-O-sulfotransferases (GlcNAc6STs) to the synthesis of the CL40 epitope in the lungs was also elucidated. Here, we show that the expression of the CL40 epitope was specifically detected in the mesothelin-positive mesothelium of the pulmonary pleura. Moreover, GlcNAc6ST2 (encoded by Chst4) and GlcNAc6ST3 (encoded by Chst5), but not GlcNAc6ST1 (encoded by Chst2) or GlcNAc6ST4 (encoded by Chst7), are required for the synthesis of CL40-positive glycans in the lung mesothelium. Furthermore, neither GlcNAc6ST2 nor GlcNAc6ST3 is sufficient for in vivo expression of the CL40 epitope in the lung mesothelium, as demonstrated by GlcNAc6ST1/3/4 triple-knock-out and GlcNAc6ST1/2/4 triple-knock-out mice. These results indicate that CL40-positive sialylated and sulfated glycans are abundant in the pleural mesothelium and are synthesized complementarily by GlcNAc6ST2 and GlcNAc6ST3, under physiological conditions in mice.

Design and Synthesis of 6-O-Phosphorylated Heparan Sulfate Oligosaccharides to Inhibit Amyloid ß Aggregation.

Dysregulation of amyloidogenic proteins and their abnormal processing and deposition in tissues cause systemic and localized amyloidosis. Formation of amyloid ß (Aß) fibrils that deposit as amyloid plaques in Alzheimer's disease (AD) brains is an earliest pathological hallmark. The polysulfated heparan sulfate (HS)/heparin (HP) is one of the non-protein components of Aß deposits that not only modulates Aß aggregation, but also acts as a receptor for Aß fibrils to mediate their cytotoxicity. Interfering with the interaction between HS/HP and Aß could be a therapeutic strategy to arrest amyloidosis. Here we have synthesized the 6-O-phosphorylated HS/HP oligosaccharides and reported their competitive effects on the inhibition of HP-mediated Aß fibril formation in vitro using a thioflavin T fluorescence assay and a tapping mode atomic force microscopy.

Effect of hydrophobic moment on membrane interaction and cell penetration of apolipoprotein E-derived arginine-rich amphipathic α-helical peptides.

We previously developed an amphipathic arginine-rich peptide, A2-17, which has high ability to directly penetrate across cell membranes. To understand the mechanism of the efficient cell-penetrating ability of the A2-17 peptide, we designed three structural isomers of A2-17 having different values of the hydrophobic moment and compared their membrane interaction and direct cell penetration. Confocal fluorescence microscopy revealed that cell penetration efficiency of peptides tends to increase with their hydrophobic moment, in which A2-17 L14R/R15L, an A2-17 isomer with the highest hydrophobic moment, predominantly remains on plasma cell membranes. Consistently, Trp fluorescence analysis indicated the deepest insertion of A2-17 L14R/R15L into lipid membranes among all A2-17 isomers. Electrophysiological analysis showed that the duration and charge flux of peptide-induced pores in lipid membranes were prominent for A2-17 L14R/R15L, indicating the formation of stable membrane pores. Indeed, the A2-17 L14R/R15L peptide exhibited the strongest membrane damage to CHO-K1 cells. Atomic force microscopy quantitatively defined the peptide-induced membrane perturbation as the decrease in the stiffness of lipid vesicles, which was correlated with the hydrophobic moment of all A2-17 isomers. These results indicate that optimal membrane perturbation by amphipathic A2-17 peptide is critical for its efficient penetration into cells without inducing stabilized membrane pores.

Extracellular endosulfatase Sulf-2 harbors a chondroitin/dermatan sulfate chain that modulates its enzyme activity.

Sulfs represent a class of unconventional sulfatases which provide an original post-synthetic regulatory mechanism for heparan sulfate polysaccharides and are involved in multiple physiopathological processes, including cancer. However, Sulfs remain poorly characterized enzymes, with major discrepancies regarding their in vivo functions. Here we show that human Sulf-2 (HSulf-2) harbors a chondroitin/dermatan sulfate glycosaminoglycan (GAG) chain, attached to the enzyme substrate-binding domain. We demonstrate that this GAG chain affects enzyme/substrate recognition and tunes HSulf-2 activity in vitro and in vivo. In addition, we show that mammalian hyaluronidase acts as a promoter of HSulf-2 activity by digesting its GAG chain. In conclusion, our results highlight HSulf-2 as a proteoglycan-related enzyme and its GAG chain as a critical non-catalytic modulator of the enzyme activity. These findings contribute to clarifying the conflicting data on the activities of the Sulfs.

Beta3Gn-T7 Is a Keratan Sulfate ß1,3 N-Acetylglucosaminyltransferase in the Adult Brain.

Keratan sulfate (KS) glycan is covalently attached to a core protein of proteoglycans. KS is abundant in neuropils and presents densely in close proximity to the perineuronal region of the perineuronal net-positive neurons in the adult brain under physiological conditions. We previously showed that the synthesis of KS positive for the R-10G antibody in the adult brain is mediated by GlcNAc-6-sulfotransferase 3 (GlcNAc6ST3; encoded by Chst5). Deficiency in both GlcNAc6ST3 and GlcNAc6ST1, encoded by Chst2, completely abolished KS. Protein-tyrosine phosphatase receptor type z1 (Ptprz1)/phosphacan was identified as a KS scaffold. KS requires the extension of GlcNAc by ß1,3 N-acetylglucosaminyltransferase (Beta3Gn-T). Members of the Beta3Gn-T family involved in the synthesis of adult brain KS have not been identified. In this study, we show by a method of gene targeting that Beta3Gn-T7, encoded by B3gnt7, is a major Beta3Gn-T for the synthesis of KS in neuropils and the perineuronal region in the adult brain. Intriguingly, the B3gnt7 gene is selectively expressed in oligodendrocyte precursor cells (OPCs) and oligodendrocytes similar to that of GlcNAc6ST3. These results indicate that Beta3Gn-T7 in oligodendrocyte lineage cells may play a role in the formation of neuropils and perineuronal nets in the adult brain through the synthesis of R-10G-positive KS-modified proteoglycan.

C-Mannosylated tryptophan-containing WSPW peptide binds to actinin-4 and alters E-cadherin subcellular localization in lung epithelial-like A549 cells.

The Trp-x-x-Trp (W-x-x-W) peptide motif, a consensus site for C-mannosylation, is the functional motif in cytokine type I receptors or thrombospondin type I repeat (TSR) superfamily proteins. W-x-x-W motifs are important for physiological and pathological functions of their parental proteins, but effects of C-mannosylation on protein functions remain to be elucidated. By using chemically synthesized WSPW peptides and C-mannosylated WSPW peptides (C-Man-WSPW), we herein investigated whether C-mannosylation of WSPW peptides confer additional biological functions to WSPW peptides. C-Man-WSPW peptide, but not non-mannosylated WSPW, reduced E-cadherin levels in A549 cells. Via peptide mass fingerprinting analysis, we identified actinin-4 as a C-Man-WSPW-binding protein in A549 cells. Actinin-4 partly co-localized with E-cadherin or ß-catenin, despite no direct interaction between actinin-4 and E-cadherin. C-Man-WSPW reduced co-localization of E-cadherin and actinin-4; non-mannosylated WSPW had no effect on localization. In actinin-4-knockdown cells, E-cadherin was upregulated and demonstrated a punctate staining pattern in the cytoplasm, which suggests that actinin-4 regulated cell-surface E-cadherin localization. Thus, C-mannosylation of WSPW peptides is required for interaction with actinin-4 that subsequently alters expression and subcellular localization of E-cadherin and morphology of epithelial-like cells. Our results therefore suggest a regulatory role of C-mannosylation of the W-x-x-W motif in interactions between the motif and its binding partner and will thereby enhance understanding of protein C-mannosylation.

Thrombospondin type 1 repeat-derived C-mannosylated peptide attenuates synaptogenesis of cortical neurons induced by primary astrocytes via TGF-ß.

C-Mannosylation is a rare type of protein glycosylation and is reportedly critical for the proper folding and secretion of parental proteins. Still, the effects of C-mannosylation on the biological functions of these modified proteins remain to be elucidated. The Trp-x-x-Trp (WxxW) sequences, whose first tryptophan (Trp) can be C-mannosylated, constitute the consensus motifs for this glycosylation modification and are commonly found in thrombospondin type 1 repeats that regulate molecular functions of thrombospondin 1 in binding and activation of transforming growth factor ß (TGF-ß). TGF-ß plays critical roles in the control of the central nervous system including synaptogenesis. Here, we investigated whether C-mannosylation of the synthetic Trp-Ser-Pro-Trp (WSPW) peptide may confer certain functions to this peptide in TGF-ß-mediated synaptogenesis. By using primary cultured rat astrocytes and cortical neurons, we found that the C-mannosylated WSPW (C-Man-WSPW) peptide, but not non-mannosylated WSPW peptide, suppressed astrocyte-conditioned medium (ACM)-stimulated synaptogenesis. C-Man-WSPW peptide inhibited both ACM- and recombinant mature TGF-ß1-induced activations of Smad 2, an important mediator in TGF-ß signaling. Interactions of recombinant mature TGF-ß with the C-Man-WSPW peptide were similar to those with non-C-mannosylated WSPW peptide. Taken together, our results reveal a novel function of C-mannosylation of the WxxW motif in signaling and synaptogenesis mediated by TGF-ß. Molecular details of how C-mannosylation affects the biological functions of WxxW motifs deserve future study for clarification.

Cytoplasmic p53 aggregates accumulated in p53-mutated cancer correlate with poor prognosis.

Recent studies suggested that aggregates of mutant p53 proteins may propagate and impair normal p53 functioning in recipient cells. Our previous study showed that cancer cell-derived p53 aggregates that cells internalized interfered with p53-dependent apoptosis in recipient cells. However, involvement of p53 aggregate propagation in cancer pathology has not been fully elucidated. Here, we screened patients with high-grade serous ovarian carcinoma, which is characterized by an extremely high frequency of TP53 gene mutations, to show that patients with cytoplasmic p53 deposits have a poor prognosis compared with patients with complete p53 absence or strong nuclear p53 positivity. Cytoplasmic p53 in the patients with poor prognosis consisted of protein aggregates, which suggests that p53 aggregates are oncogenic drivers. Indeed, an inhibitor of p53 aggregation restored cellular apoptosis, a proper p53 function, in p53 aggregate-bearing patient-derived tumor organoids. In cell-based assays, endogenous and exogenous mutant p53 aggregates hindered chemotherapeutic activity of cisplatin, which depends on normal p53 functions. This inhibition was reduced by blocking p53 aggregation or internalization of p53 aggregates. Our study, thus indicates the involvement of p53 aggregate transmission in poor prognosis and in chemotherapy resistance in cancers.

Lipid Droplet Accumulation Independently Predicts Poor Clinical Prognosis in High-Grade Serous Ovarian Carcinoma.

High-grade serous ovarian carcinoma (HGSOC) is an epithelial cancer that accounts for most ovarian cancer deaths. Metabolic abnormalities such as extensive aerobic glycolysis and aberrant lipid metabolism are well-known characteristics of cancer cells. Indeed, accumulation of lipid droplets (LDs) in certain types of malignant tumors has been known for more than 50 years. Here, we investigated the correlation between LD accumulation and clinical prognosis. In 96 HGSOC patients, we found that high expression of the LD marker adipophilin was associated with poor progression-free and overall survival (p = 0.0022 and p = 0.014, respectively). OVCAR-3 ovarian carcinoma cells accumulated LDs in a glucose-dependent manner, which suggested the involvement of aerobic glycolysis and subsequently enhanced lipogenesis, with a result being LD accumulation. The acyl-CoA: cholesterol acyltransferase 1 inhibitor K604 and the hydroxymethylglutaryl-CoA reductase inhibitor pitavastatin blocked LD accumulation in OVCAR-3 cells and reduced phosphorylation of the survival-related kinases Akt and ERK1/2, both of which have been implicated in malignancy. Our cell-based assays thus suggested that enhanced aerobic glycolysis resulted in LD accumulation and activation of survival-related kinases. Overall, our results support the idea that cancers with lipogenic phenotypes are associated with poor clinical prognosis, and we suggest that adipophilin may serve as an independent indicator of a poor prognosis in HGSOC.

Protein C-Mannosylation and C-Mannosyl Tryptophan in Chemical Biology and Medicine.

C-Mannosylation is a post-translational modification of proteins in the endoplasmic reticulum. Monomeric α-mannose is attached to specific Trp residues at the first Trp in the Trp-x-x-Trp/Cys (W-x-x-W/C) motif of substrate proteins, by the action of C-mannosyltransferases, DPY19-related gene products. The acceptor substrate proteins are included in the thrombospondin type I repeat (TSR) superfamily, cytokine receptor type I family, and others. Previous studies demonstrated that C-mannosylation plays critical roles in the folding, sorting, and/or secretion of substrate proteins. A C-mannosylation-defective gene mutation was identified in humans as the disease-associated variant affecting a C-mannosylation motif of W-x-x-W of ADAMTSL1, which suggests the involvement of defects in protein C-mannosylation in human diseases such as developmental glaucoma, myopia, and/or retinal defects. On the other hand, monomeric C-mannosyl Trp (C-Man-Trp), a deduced degradation product of C-mannosylated proteins, occurs in cells and extracellular fluids. Several studies showed that the level of C-Man-Trp is upregulated in blood of patients with renal dysfunction, suggesting that the metabolism of C-Man-Trp may be involved in human kidney diseases. Together, protein C-mannosylation is considered to play important roles in the biosynthesis and functions of substrate proteins, and the altered regulation of protein C-manosylation may be involved in the pathophysiology of human diseases. In this review, we consider the biochemical and biomedical knowledge of protein C-mannosylation and C-Man-Trp, and introduce recent studies concerning their significance in biology and medicine.

Extracellularly Released Calreticulin Induced by Endoplasmic Reticulum Stress Impairs Syncytialization of Cytotrophoblast Model BeWo Cells.

The pregnancy-specific syndrome preeclampsia is a major cause of maternal mortality throughout the world. The initial insult resulting in the development of preeclampsia is inadequate trophoblast invasion, which may lead to reduced maternal perfusion of the placenta and placental dysfunction, such as insufficient trophoblast syncytialization. Endoplasmic reticulum (ER) stress has been implicated in the pathology of preeclampsia and serves as the major risk factor. Our previous studies suggested critical roles of calreticulin (CRT), which is an ER-resident stress response protein, in extravillous trophoblast invasion and cytotrophoblast syncytialization. Here, we studied the mechanism by which ER stress exposes the placenta to the risk of preeclampsia. We found that CRT was upregulated in the serum samples, but not in the placental specimens, from preeclamptic women. By using BeWo cells, an established model of cytotrophoblasts that syncytialize in the presence of forskolin, we demonstrated that thapsigargin-induced ER stress caused extracellular release of CRT from BeWo cells and that the extracellular CRT suppressed forskolin-induced release of ß-human chorionic gonadotropin and altered subcellular localization of E-cadherin, which is a key adhesion molecule associated with syncytialization. Our results together provide evidence that induction of ER stress leads to extracellular CRT release, which may contribute to placental dysfunction by suppressing cytotrophoblast syncytialization.

Cell-to-cell transmission of p53 aggregates: a novel player in oncology?

The mutants of the tumor suppressor protein p53 form protein aggregates. It has been proposed that these aggregates propagate like prions, albeit the detailed mechanism of the propagation is unclear. Our recent study revealed that sulfated glycosaminoglycans, especially highly sulfated domains of heparan sulfate (heparan sulfate S-domains), participate in cancer pathology by mediating transcellular propagation of p53 aggregates.

Sulfated glycosaminoglycans mediate prion-like behavior of p53 aggregates.

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) are heteropolysaccharides implicated in the pathology of protein aggregation diseases including localized and systemic forms of amyloidosis. Among subdomains of sulfated GAGs, highly sulfated domains of HS, called HS S-domains, have been highlighted as being critical for HS function in amyloidoses. Recent studies suggest that the tumor suppressor p53 aggregates to form amyloid fibrils and propagates in a prion-like manner; however, molecules and mechanisms that are involved in the prion-like behavior of p53 aggregates have not been addressed. Here, we identified sulfated GAGs as molecules that mediate prion-like behavior of p53 aggregates. Sulfated GAGs at the cell surface were required for cellular uptake of recombinant and cancer cell-derived p53 aggregates and extracellular release of p53 from cancer cells. We further showed that HS S-domains accumulated within p53 deposits in human ovarian cancer tissues, and enzymatic remodeling of HS S-domains by Sulf-2 extracellular sulfatase down-regulated cellular uptake of p53 aggregates. Finally, sulfated GAG-dependent cellular uptake of p53 aggregates was critical for subsequent extracellular release of the aggregates and gain of oncogenic function in recipient cells. Our work provides a mechanism of prion-like behavior of p53 aggregates and will shed light on sulfated GAGs as a common mediator of prions.

Monomeric C-mannosyl tryptophan is a degradation product of autophagy in cultured cells.

C-Mannosyl tryptophan (C-Man-Trp) is a unique glycosylated amino acid present in various eukaryotes. The C-Man-Trp structure can be found as a monomeric form or a part of post-translational modifications within polypeptide chains in living organisms. However, the mechanism of how monomeric C-Man-Trp is produced has not been fully investigated. In this study, we assessed levels of cellular C-Man-Trp by ultra performance liquid chromatography with a mass spectrometry assay system, and investigated whether the cellular C-Man-Trp is affected by autophagy induction. The intracellular C-Man-Trp level was significantly increased under serum and/or amino acid starvation in A549, HaCaT, HepG2, NIH3T3, and NRK49F cells. The increase in C-Man-Trp was also observed in NIH3T3 cells treated with rapamycin, an autophagy inducer. The up-regulation of C-Man-Trp caused by starvation was reversed by the inhibition of lysosomal enzymes. We further showed that C-Man-Trp is produced by incubating a synthetic C-mannosylated peptide (C-Man-Trp-Ser-Pro-Trp) or thrombospondin (TSP) in a lysosomal fraction that was prepared from a mouse liver, which provides supporting evidence that C-Man-Trp is a degradation product of the C-mannosylated peptide or protein following lysosome-related proteolysis. Taken together, we propose that the autophagic pathway is a novel pathway that at least partly contributes to intracellular C-Man-Trp production under certain conditions, such as nutrient starvation.