Emerging Role of GCN1 in Disease and Homeostasis.

GCN1 is recognized as a factor that is essential for the activation of GCN2, which is a sensor of amino acid starvation. This function is evolutionarily conserved from yeast to higher eukaryotes. However, recent studies have revealed non-canonical functions of GCN1 that are independent of GCN2, such as its participation in cell proliferation, apoptosis, and the immune response, beyond the borders of species. Although it is known that GCN1 and GCN2 interact with ribosomes to accomplish amino acid starvation sensing, recent studies have reported that GCN1 binds to disomes (i.e., ribosomes that collide each other), thereby regulating both the co-translational quality control and stress response. We propose that GCN1 regulates ribosome-mediated signaling by dynamically changing its partners among RWD domain-possessing proteins via unknown mechanisms. We recently demonstrated that GCN1 is essential for cell proliferation and whole-body energy regulation in mice. However, the manner in which ribosome-initiated signaling via GCN1 is related to various physiological functions warrants clarification. GCN1-mediated mechanisms and its interaction with other quality control and stress response signals should be important for proteostasis during aging and neurodegenerative diseases, and may be targeted for drug development.

Exploration of Malignant Characteristics in Neoadjuvant Chemotherapy-Resistant Rectal Cancer, Focusing on Extramural Lesions.

BACKGROUND: Extramural vascular invasion (EMVI) and tumor deposits (TD) are poor prognostic factors in rectal cancer (RC), especially when resistant to neoadjuvant chemotherapy (NAC). We aimed to define differential expression in NAC responders and non-responders with concomitant EMVI and TD. METHODS: From 52 RC surgical patients, post-NAC resected specimens were extracted, comprising two groups: cases with residual EMVI and TD (NAC-resistant) and cases without (NAC-effective). Proteomic analysis was conducted to define differential protein expression in the two groups. To validate the findings, immunohistochemistry was performed in another cohort that included 58 RC surgical patients. Based on the findings, chemosensitivity and prognosis were compared. RESULTS: The NAC-resistant group was associated with a lower 3-year disease-free survival rate than the NAC-effective group (p = 0.041). Discriminative proteins in the NAC-resistant group were highly associated with the sulfur metabolism pathway. Among these pathway constituents, selenium-binding protein 1 (SELENBP1) expression in the NAC-resistant group decreased to less than one-third of that of the NAC-effective group. Immunohistochemistry in another RC cohort consistently validated the relationship between decreased SELENBP1 and poorer NAC sensitivity, in both pre-NAC biopsy and post-NAC surgery specimens. Furthermore, decrease in SELENBP1 was associated with a lower 3-year disease-free survival rate (p = 0.047). CONCLUSIONS: We defined one of the differentially expressed proteins in NAC responders and non-responders, concomitant with EMVI and TD. SELENBP1 was suspected to contribute to NAC resistance and poor prognosis in RC.

Development of transgenic mice overexpressing mouse carbonyl reductase 1.

BACKGROUND: Carbonyl reductase 1 (CBR1) is a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductase with broad substrate specificity. CBR1 catalyzes the reduction of numerous carbonyl compounds, including quinones, prostaglandins, menadione, and multiple xenobiotics, while also participating in various cellular processes, such as carcinogenesis, apoptosis, signal transduction, and drug resistance. In this study, we aimed to generate transgenic mice overexpressing mouse Cbr1 (mCbr1), characterize the mCbr1 expression in different organs, and identify changes in protein expression patterns. METHODS AND RESULTS: To facilitate a deeper understanding of the functions of CBR1, we generated transgenic mice overexpressing CBR1 throughout the body. These transgenic mice overexpress 3xFLAG-tagged mCbr1 (3xFLAG-mCbr1) under the CAG promoter. Two lines of transgenic mice were generated, one with 3xFLAG-mCbr1 expression in multiple tissues, and the other, with specific expression of 3xFLAG-mCbr1 in the heart. Pathway and network analysis using transgenic mouse hearts identified 73 proteins with levels of expression correlating with mCbr1 overexpression. The expression of voltage-gated anion channels, which may be directly related to calcium ion-related myocardial contraction, was also upregulated. CONCLUSION: mCbr1 transgenic mice may be useful for further in vivo analyses of the molecular mechanisms regulated by Cbr1; such analyses will provide a better understanding of its effects on carcinogenesis and cardiotoxicity of certain cancer drugs.

Sulforaphane Increase Mitochondrial Biogenesis-Related Gene Expression in the Hippocampus and Suppresses Age-Related Cognitive Decline in Mice.

Sulforaphane (SFN) is a potent activator of the transcriptional factor, Nuclear Factor Erythroid 2 (NF-E2)-Related factor 2 (NRF2). SFN and its precursor, glucoraphanin (sulforaphane glucosinolate, SGS), have been shown to ameliorate cognitive function in clinical trials and in vivo studies. However, the effects of SGS on age-related cognitive decline in Senescence-Accelerated Mouse Prone 8 (SAMP8) is unknown. In this study, we determined the preventive potential of SGS on age-related cognitive decline. One-month old SAMP8 mice or control SAM resistance 1 (SAMR1) mice were fed an ad libitum diet with or without SGS-containing broccoli sprout powder (0.3% w/w SGS in diet) until 13 months of age. SGS significantly improved long-term memory in SAMP8 at 12 months of age. Interestingly, SGS increased hippocampal mRNA and protein levels of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC1α) and mitochondrial transcription factor A (TFAM), which are master regulators of mitochondrial biogenesis, both in SAMR1 and SAMP8 at 13 months of age. Furthermore, mRNAs for nuclear respiratory factor-1 (NRF-1) and mitochondrial DNA-encoded respiratory complex enzymes, but not mitochondrial DNA itself, were increased by SGS in SAMP8 mice. These results suggest that SGS prevents age-related cognitive decline by maintaining mitochondrial function in senescence-accelerated mice.

Inducible Systemic Gcn1 Deletion in Mice Leads to Transient Body Weight Loss upon Tamoxifen Treatment Associated with Decrease of Fat and Liver Glycogen Storage.

GCN1 is an evolutionarily-conserved ribosome-binding protein that mediates the amino acid starvation response as well as the ribotoxic stress response. We previously demonstrated that Gcn1 mutant mice lacking the GCN2-binding domain suffer from growth retardation and postnatal lethality via GCN2-independent mechanisms, while Gcn1-null mice die early in embryonic development. In this study, we explored the role of GCN1 in adult mice by generating tamoxifen-inducible conditional knockout (CKO) mice. Unexpectedly, the Gcn1 CKO mice showed body weight loss during tamoxifen treatment, which gradually recovered following its cessation. They also showed decreases in liver weight, hepatic glycogen and lipid contents, blood glucose and non-esterified fatty acids, and visceral white adipose tissue weight with no changes in food intake and viability. A decrease of serum VLDL suggested that hepatic lipid supply to the peripheral tissues was primarily impaired. Liver proteomic analysis revealed the downregulation of mitochondrial ß-oxidation that accompanied increases of peroxisomal ß-oxidation and aerobic glucose catabolism that maintain ATP levels. These findings show the involvement of GCN1 in hepatic lipid metabolism during tamoxifen treatment in adult mice.

Novel roles of glycosaminoglycans in the degradation of type I collagen by cathepsin K.

Glycosaminoglycans (GAGs) and collagen are the major organic components of bone matrix. However, their roles and functional relationships remain elusive. To investigate the role of GAGs in bone matrix degradation, the effects of GAGs on collagen were examined under acidic conditions that recapitulate the microenvironment of osteoclast resorption pits. We found that sulfated GAGs protect collagen fibrils against acid denaturation. Scanning electron microscopy demonstrated that collagen fibrils retain the fibril structure at pH 4.0 in the presence of chondroitin 6-sulfate. By surface plasmon resonance analysis, we found that sulfated GAGs, but not non-sulfated GAGs, bind to triple-helix type I collagen below pH 4.5. The binding of collagen in an acidic solution was dependent upon the GAG sugar chain length. Functionally, the acid-resistant collagen fibrils generated in the presence of sulfated GAGs were resistant to cathepsin K degradation in vitro below pH 4.0. As the pH increased from 4.0 to 5.0, the acid-resistant collagen fibrils were degraded by cathepsin K. Our results highlight the possibility that the interaction between GAGs and collagen under acidic conditions has a regulatory impact on cathepsin K-mediated bone degradation.

Chondroitin sulfate cluster of epiphycan from salmon nasal cartilage defines binding specificity to collagens.

Epiphycan (EPY) from salmon nasal cartilage has a glycosaminoglycan (GAG) domain that is heavily modified by chondroitin 4-sulfate and chondroitin 6-sulfate. The functional role of the GAG domain has not been investigated. The interaction of EPY with collagen was examined in vitro using surface plasmon resonance analysis. EPY was found to bind to type I collagen via clustered chondroitin sulfate (CS), while a single chain of CS was unable to bind. Types I, III, VII, VIII and X collagen showed high binding affinity with EPY, whereas types II, IV, V, VI and IX showed low binding affinities. Chemical modification of lysine residues in collagen decreased the affinity with the clustered CS. These results suggest that lysine residues of collagen are involved in the interaction with the clustered CS, and the difference in lysine modification defines the binding affinity to EPY. The clustered CS was also involved in an inter-saccharide interaction, and formed self-associated EPY. CS of EPY promoted fibril formation of type I collagen.

Preparation of proteoglycan from salmon nasal cartilage under nondenaturing conditions.

Salmon nasal cartilage was micronized in ethanol using a rotor-stator homogenizer for the high yield of proteoglycan extraction. This procedure also brought about depressing the degradation of proteoglycan and the contamination of collagens. Proteoglycan was extracted by 4 M magnesium chloride and isolated by anion-exchange chromatography. The gel filtration HPLC and the antibody reactivity showed that the core protein was intact.

Purification and Characterization of α-Mannosidase from Onion, Allium cepa.

α-Mannosidase (ALMAN) extracted from onion (Allium cepa) was purified by column chromatography such as hydrophobic and gel filtration. ALMAN is an acidic α-mannosidase that exhibits maximum activity against pNP-α-Man at pH 4.0-5.0 at 50°C. Amino acid sequence analysis of ALMAN was consistent with α-mannosidase deduced from Allium cepa transcriptome analysis. The gene alman was amplified by PCR using mRNA extracted from onions, and a full-length gene of 3,054 bp encoding a protein of 1,018 amino acid residues was revealed. ALMAN is classified as Glycoside Hydrolase Family (GH) 38 and showed homology with other plant-derived α-mannosidases such as tomato and hot pepper.

Overexpression of carbonyl reductase 1 in ovarian cancer cells suppresses proliferation and activates the eIF2 signaling pathway.

High expression of carbonyl reductase 1 (CBR1) protein in ovarian cancer cells inhibits tumor growth and metastasis. However, the underlying mechanism is unknown. To investigate the mechanism by which CBR1 suppresses tumor growth, the present study generated ovarian cancer cells that constitutively overexpress human CBR1 (hCBR1) protein. Ovarian cancer cell lines (OVCAR-3 and SK-OV-3) were transfected with a plasmid encoding hCBR1, followed by selection with G418 to isolate hCBR1-overexpressing lines. The proliferation rates of hCBR1-overexpressing cells were then compared with those of negative control and wild-type cells. Overexpression of hCBR1 led to significant inhibition of proliferation (P<0.05). Subsequently, to investigate changes in intracellular signaling pathways, cellular proteins were extracted and subjected to proteome analysis using liquid chromatography followed by mass spectrometry. There was an inverse correlation between CBR1 protein expression and cell proliferation. In addition, Ingenuity Pathway Analysis of hCBR1-overexpressing cell lines was performed, which revealed changes in the expression of proteins involved in signaling pathways related to growth regulation. Of these, the eukaryotic translation initiation factor 2 (eIF2) signaling pathway was upregulated most prominently. Thus, alterations in multiple tumor-related signaling pathways, including eIF2 signaling, may lead to growth suppression. Taken together, the present data may lead to the development of new drugs that target CBR1 and related signaling pathways, thereby improving outcomes for patients with ovarian cancer.

Dairy consumption, bone turnover biomarkers, and osteo sono assessment index in Japanese adults: A cross-sectional analysis of data from the Iwaki Health Promotion Project.

Purpose: Dairy foods are nutritional sources of calcium, phosphorus, protein, and other nutrients that improve bone health. However, the effects of dairy consumption on bone biomarkers in the Japanese population remain unclear. This study explored the association between dairy consumption and bone biomarkers in Japanese adults. Methods: This cross-sectional study was conducted as part of the Iwaki Health Promotion Project in Aomori, Japan. In total, 1063 adults were included in the analysis. Bone turnover marker levels were measured in local citizens during their annual medical checkups. The calcaneus osteo sono assessment index (OSI) was calculated using a quantitative ultrasound technique. The dietary intake of foods and nutrients was estimated using a food frequency questionnaire. Linear regression models were established using dairy consumption and bone biomarkers with adjustments. Statistic significance was considered by P < 0.05. Results: In multivariate models, the tartrate-resistant acid phosphatase 5b and parathyroid hormone concentrations were inversely associated with dietary dairy consumption after adjusting for age and sex. The undercarboxylated osteocalcin, a procollagen type I N-terminal peptide to bone alkaline phosphatase ratio, and OSI were the directly associated with dairy consumption in multivariate models with adjustment. Conclusions: Dairy consumption is partially associated with bone turnover biomarkers and OSI in adult Japanese participants. Habitual consumption of dairy foods may contribute to the nutritional supplementation for maintaining bone health, including turnover and structure. Clinical trial registry number and website where it was obtained: The Japanese Clinical Trials Registry (UMIN000040459), https://center6.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000046175.

Novel insight into nicotinamide adenine dinucleotide and related metabolites in cancer patients undergoing surgery.

Nicotinamide adenine dinucleotide (NAD +) plays a pivotal role in numerous cellular functions. Reduced NAD + levels are postulated to be associated with cancer. As interest in understanding NAD + dynamics in cancer patients with therapeutic applications in mind grows, there remains a shortage of comprehensive data. This study delves into NAD + dynamics in patients undergoing surgery for different digestive system cancers. This prospective study enrolled 99 patients with eight different cancers. Fasting blood samples were obtained during the perioperative period. The concentrations of NAD + , nicotinamide mononucleotide (NMN), and nicotinamide riboside were analyzed using tandem mass spectrometry. After erythrocyte volume adjustment, NAD + remained relatively stable after surgery. Meanwhile, NMN decreased the day after surgery and displayed a recovery trend. Interestingly, liver and pancreatic cancer patients exhibited poor postoperative NMN recovery, suggesting a potential cancer type-specific influence on NAD + metabolism. This study illuminated the behavior of NAD + in surgically treated cancer patients. We identified which cancer types have particularly low levels and at what point depletion occurs during the perioperative period. These insights suggest the need for personalized NAD + supplementation strategies, calibrated to individual patient needs and treatment timelines. Clinical trial registration jRCT1020210066.

Epiphycan from salmon nasal cartilage is a novel type of large leucine-rich proteoglycan.

Chum salmon (Oncorhynchus keta) nasal cartilage was examined by next-generation DNA sequencing and mass spectrometric analyses, and 14 types of proteoglycans including epiphycan (EPY) were found. A cDNA encoding EPY was cloned and sequenced. The cDNA encoded 589 amino acids comprised a glycosaminoglycan (GAG) domain containing 55 potential GAG-modified sites (Ser-Gly and/or Gly-Ser), a cysteine cluster and 6 leucine-rich repeats. EPY was purified from salmon nasal cartilage and the structure of the GAG was characterized. As a result of unsaturated disaccharide analysis, GAG was found to be composed of chondroitin 6-sulfate (58.0%), chondroitin 4-sulfate (26.5%) and non-sulfated chondroitin (15.3%). The average molecular weight of GAG was estimated to be 3.0 × 10(4). Ser-100 and Ser-103 were identified as serine residues substituted by GAG chains by chemical modification and mass spectrometric analysis. More than 50 serine residues were assumed to be substituted by GAG chains. EPY is heavily substituted by chondroitin sulfate, giving an overall molecular weight of just under 2 × 10(6). EPY from salmon nasal cartilage is a novel type of large leucine-rich proteoglycan.

Proteomics and secreted lipidomics of mouse-derived bone marrow cells exposed to a lethal level of ionizing radiation.

High doses of ionizing radiation (IR) exposure can lead to the development of severe acute radiation syndrome with bone marrow failure. Defining risk factors that predict adverse events is a critical mission to guide patient selection for personalized treatment protocols. Since non-hematopoietic stem cells act as feeder cells in the niche and their secreted lipids may regulate hematopoietic stem cells, we focused on non-hematopoietic stem cells and aimed to discover biomarkers that can assess radiation exposure from their secreted lipids. Bone marrow stromal cells (BMSCs) and osteoblast differentiation-inducing cells (ODICs) isolated from mouse femurs were exposed to lethal doses of IR and the proteomic differences between BMSC and ODIC cell layers were compared. We observed an increased Nrf2-mediated oxidative stress response and IL6 expression in ODICs and decreased expression of mitochondrial proteins in BMSCs. To elucidate secreted factors, lipidomics of the cultures were profiled; the relevant lipids distinguishing IR-exposed and control groups of BMSC were acyl-acyl phosphatidylcholine (PC aa C34:1 and PC aa C34:4), lysophosphatidylcholine (lyso-PC a C18:0 and lyso PC a C17:0) and sphingomyelin (SM C20:2). These analyses suggest that certain lipids are candidate markers for the toxic effects of IR.

Novel dicarbonyl metabolic pathway via mitochondrial ES1 possessing glyoxalase III activity.

Glycation, caused by reactive dicarbonyls, plays a role in various diseases by forming advanced glycation end products. In live cells, reactive dicarbonyls such as glyoxal (GO) and methylglyoxal (MGO) are produced during cell metabolism, and these should be removed consistently. However, the dicarbonyl metabolic system in the mitochondria remains unclear. It has been speculated that the mammalian mitochondrial protein ES1 is a homolog of bacterial elbB possessing glyoxalase III (GLO3) activity. Therefore, in this study, to investigate ES1 functions and GLO3 activity, we generated ES1-knockout (KO) mice and recombinant mouse ES1 protein and investigated the biochemical and histological analyses. In the mitochondrial fraction obtained from ES1-KO mouse brains, the GO metabolism and cytochrome c oxidase activity were significantly lower than those in the mitochondrial fraction obtained from wildtype (WT) mouse brains. However, the morphological features of the mitochondria did not change noticeably in the ES1-KO mouse brains compared with those in the WT mouse brains. The mitochondrial proteome analysis showed that the MGO degradation III pathway and oxidative phosphorylation-related proteins were increased. These should be the response to the reduced GO metabolism caused by ES1 deletion to compensate for the dicarbonyl metabolism and damaged cytochrome c oxidase by elevated GO. Recombinant mouse ES1 protein exhibited catalytic activity of converting GO to glycolic acid. These results indicate that ES1 possesses GLO3 activity and modulates the metabolism of GO in the mitochondria. To our knowledge, this is the first study to show a novel metabolic pathway for reactive dicarbonyls in mitochondria.

Multiomics and artificial intelligence enabled peripheral blood-based prediction of amnestic mild cognitive impairment.

BACKGROUND: Since dementia is preventable with early interventions, biomarkers that assist in diagnosing early stages of dementia, such as mild cognitive impairment (MCI), are urgently needed. METHODS: Multiomics analysis of amnestic MCI (aMCI) peripheral blood (n = 25) was performed covering the transcriptome, microRNA, proteome, and metabolome. Validation analysis for microRNAs was conducted in an independent cohort (n = 12). Artificial intelligence was used to identify the most important features for predicting aMCI. FINDINGS: We found that hsa-miR-4455 is the best biomarker in all omics analyses. The diagnostic index taking a ratio of hsa-miR-4455 to hsa-let-7b-3p predicted aMCI patients against healthy subjects with 97% overall accuracy. An integrated review of multiomics data suggested that a subset of T cells and the GCN (general control nonderepressible) pathway are associated with aMCI. INTERPRETATION: The multiomics approach has enabled aMCI biomarkers with high specificity and illuminated the accompanying changes in peripheral blood. Future large-scale studies are necessary to validate candidate biomarkers for clinical use.

Hyaluronan-chondroitin hybrid oligosaccharides as new life science research tools.

Hyaluronan and chondroitin are glycosaminoglycans well-known as components of pharmaceutical agents and health foods. From these attractive molecules, using transglycosylation reaction of testicular hyaluronidase, we synthesized hybrid neo-oligosaccharides not found in nature. We also found a new site between the chondroitin disaccharide unit and hyaluronan disaccharide unit recognized by a hyaluronan lyase specific to hyaluronan using these hybrid oligosaccharides as substrates. We hope that these hybrid oligosaccharides will help to elucidate the involvement of hyaluronan, chondroitin, and chondroitin sulfates in the mechanisms of cell functions and diseases, based on the structures of these glycosaminoglycans.

Heterogeneity of metabolic adaptive capacity affects the prognosis among pancreatic ductal adenocarcinomas.

BACKGROUND: Evolutionary cancer has a supply mechanism to satisfy higher energy demands even in poor-nutrient conditions. Metabolic reprogramming is essential to supply sufficient energy. The relationship between metabolic reprogramming and the clinical course of pancreatic ductal adenocarcinoma (PDAC) remains unclear. We aimed to clarify the differences in metabolic status among PDAC patients. METHODS: We collected clinical data from 128 cases of resectable PDAC patients undergoing surgery. Sixty-three resected tissues, 15 tissues from the low carbohydrate antigen 19-9 (CA19-9), 38-100 U/mL, and high CA19-9, > 500 U/mL groups, and 33 non-tumor control parts, were subjected to tandem mass spectrometry workflow to systematically explore metabolic status. Clinical and proteomic data were compared on the most used PDAC biomarker, preoperative CA19-9 value. RESULTS: Higher CA19-9 levels were clearly associated with higher early recurrence (p < 0.001), decreased RFS (p < 0.001), and decreased DSS (p = 0.025). From proteomic analysis, we discovered that cancer evolution-related as well as various metabolism-related pathways were more notable in the high group. Using resected tissue immunohistochemical staining, we learned that high CA19-9 PDAC demonstrated aerobic glycolysis enhancement, yet no decrease in protein synthesis. We found a heterogeneity of various metabolic processes, including carbohydrates, proteins, amino acids, lipids, and nucleic acids, between the low and the high groups, suggesting differences in metabolic adaptive capacity. CONCLUSIONS: Our study found metabolic adaptation differences among PDAC cases, pertaining to both cancer evolution and the prognosis. CA19-9 can help estimate the metabolic adaptive capacity of energy supply for PDAC evolution.