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Nature Communications Mar 2024Protein-protein interactions are at the heart of all cellular processes, with the ribosome emerging as a platform, orchestrating the nascent-chain interplay dynamics....
Protein-protein interactions are at the heart of all cellular processes, with the ribosome emerging as a platform, orchestrating the nascent-chain interplay dynamics. Here, to study the characteristics governing co-translational protein folding and complex assembly, we combine selective ribosome profiling, imaging, and N-terminomics with all-atoms molecular dynamics. Focusing on conserved N-terminal acetyltransferases (NATs), we uncover diverging co-translational assembly pathways, where highly homologous subunits serve opposite functions. We find that only a few residues serve as "hotspots," initiating co-translational assembly interactions upon exposure at the ribosome exit tunnel. These hotspots are characterized by high binding energy, anchoring the entire interface assembly. Alpha-helices harboring hotspots are highly thermolabile, folding and unfolding during simulations, depending on their partner subunit to avoid misfolding. In vivo hotspot mutations disrupted co-translational complexation, leading to aggregation. Accordingly, conservation analysis reveals that missense NATs variants, causing neurodevelopmental and neurodegenerative diseases, disrupt putative hotspot clusters. Expanding our study to include phosphofructokinase, anthranilate synthase, and nucleoporin subcomplex, we employ AlphaFold-Multimer to model the complexes' complete structures. Computing MD-derived interface energy profiles, we find similar trends. Here, we propose a model based on the distribution of interface energy as a strong predictor of co-translational assembly.
Topics: Protein Biosynthesis; Models, Molecular; Ribosomes; Protein Folding; Protein Processing, Post-Translational
PubMed: 38528060
DOI: 10.1038/s41467-024-46881-w -
Marine Environmental Research May 2024Marine hypoxia poses a significant challenge in the contemporary marine environment. The horseshoe crab, an ancient benthic marine organism, is confronted with the...
Marine hypoxia poses a significant challenge in the contemporary marine environment. The horseshoe crab, an ancient benthic marine organism, is confronted with the potential threat of species extinction due to hypoxia, making it an ideal candidate for studying hypoxia tolerance mechanisms. In this experiment, juvenile Tachypleus tridentatus were subjected to a 21-day trial at DO:2 mg/L (hypoxia) and DO:6 mg/L conditions. The experimental timeline included a 14-day exposure phase followed by a 7-day recovery period. Sampling occurred on days 0, 7, 14, and 21, where the period from day 14 to day 21 corresponds to seven days of recuperation. Several enzymatic activities of important proteins throughout this investigation were evaluated, such as succinate dehydrogenase (SDH), phosphofructokinase (PFK), hexokinase (HK), lactate dehydrogenase (LDH), and pyruvate kinase (PK). Concurrently, the relative expression of hexokinase-1 (HK), hypoxia-inducible factor 1-alpha inhibitor (FIH), and hypoxia-inducible factor 1-alpha (HIF-1α), pyruvate dehydrogenase phosphatase (PDH), succinate dehydrogenase assembly factor 4 (SDH), and Glucose-6-phosphatase (G6Pase) were also investigated. These analyses aimed to elucidate alterations in the hypoxia signaling pathway and respiratory energy metabolism. It is revealed that juvenile T. tridentatus initiated the HIF pathway under hypoxic conditions, resulting in an upregulation of HIF-1α and FIH-1 gene expression, which in turn, influenced a shift in metabolic patterns. Particularly, the activity of glycolysis-related enzymes was promoted significantly, including PK, HK, PKF, LDH, and the related HK gene. In contrast, enzymes linked to aerobic respiration, PDH, and SDH, as well as the related PDH and SDH genes, displayed down-regulation, signifying a transition from aerobic to anaerobic metabolism. Additionally, the activity of gluconeogenesis-related enzymes such as PK and G6Pase gene expression were significantly elevated, indicating the activation of gluconeogenesis and glycogenolysis pathways. Consequently, juvenile T. tridentatus demonstrated an adaptive response to hypoxic conditions, marked by changes in respiratory energy metabolism modes and the activation of hypoxia signaling pathways.
Topics: Animals; Horseshoe Crabs; Succinate Dehydrogenase; Hexokinase; Hypoxia; Signal Transduction; Glucose; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit
PubMed: 38520956
DOI: 10.1016/j.marenvres.2024.106467 -
International Journal of Biological... Apr 2024Uncoordinated (Unc) 51-like kinase (ulk1) and ulk2 are closely involved in autophagy activation, but little is known about their roles in regulating glucose homeostasis....
Uncoordinated (Unc) 51-like kinase (ulk1) and ulk2 are closely involved in autophagy activation, but little is known about their roles in regulating glucose homeostasis. In this study, the genes of ulk1a, ulk1b and ulk2 were cloned and characterized in fish Megalobrama amblycephala. All the three genes shared the approximate N-terminal kinase domain and the C-terminal Atg1-like_tMIT domain structure, while the amino acid sequence identity of them are different between M. amblycephala and other vertebrates. Their transcripts were widely observed in various tissues (brain, muscle, gill, heart, spleen, eye, liver, intestine, abdominal adipose and kidney), but differed in tissue expression patterns. During the glucose tolerance test and the insulin tolerance test, the up-regulated transcriptions of ulk1a, ulk1b and ulk2 were all found despite some differences in the temporal patterns. At the same time, the activities of glycolytic enzymes like hexokinase and phosphofructokinase both showed parallel increases. Furthermore, the feeding of a high-carbohydrate diet decreased the transcriptions of ulk1a, ulk1b and ulk2. Collectively, this study demonstrated that ulk1a, ulk1b and ulk2 in M. amblycephala had similar molecular characterizations, but with different conservation and tissue expression patterns. In addition, ulk1/2 might play important roles in maintaining the glucose homeostasis in fish through regulating the glycolytic pathway.
Topics: Animals; Cypriniformes; Amino Acid Sequence; Cloning, Molecular; Glucose; Cyprinidae; Fish Proteins; Phylogeny
PubMed: 38518944
DOI: 10.1016/j.ijbiomac.2024.130985 -
Enzyme and Microbial Technology Jun 2024d-mannose has been widely used in food, medicine, cosmetic, and food-additive industries. To date, chemical synthesis or enzymatic conversion approaches based on...
d-mannose has been widely used in food, medicine, cosmetic, and food-additive industries. To date, chemical synthesis or enzymatic conversion approaches based on iso/epimerization reactions for d-mannose production suffered from low conversion rate due to the reaction equilibrium, necessitating intricate separation processes for obtaining pure products on an industrial scale. To circumvent this challenge, this study showcased a new approach for d-mannose synthesis from glucose through constructing a phosphorylation-dephosphorylation pathway in an engineered strain. Specifically, the gene encoding phosphofructokinase (PfkA) in glycolytic pathway was deleted in Escherichia coli to accumulate fructose-6-phosphate (F6P). Additionally, one endogenous phosphatase, YniC, with high specificity to mannose-6-phosphate, was identified. In ΔpfkA strain, a recombinant synthetic pathway based on mannose-6-phosphate isomerase and YniC was developed to direct F6P to mannose. The resulting strain successfully produced 25.2 g/L mannose from glucose with a high conversion rate of 63% after transformation for 48 h. This performance surpassed the 15% conversion rate observed with 2-epimerases. In conclusion, this study presents an efficient method for achieving high-yield mannose synthesis from cost-effective glucose.
Topics: Mannose; Escherichia coli; Phosphorylation; Glucose; Escherichia coli Proteins; Mannosephosphates; Metabolic Engineering; Fructosephosphates; Mannose-6-Phosphate Isomerase; Phosphoric Monoester Hydrolases; Glycolysis
PubMed: 38518553
DOI: 10.1016/j.enzmictec.2024.110427 -
Proceedings of the National Academy of... Mar 2024During metazoan development, how cell division and metabolic programs are coordinated with nutrient availability remains unclear. Here, we show that nutrient...
During metazoan development, how cell division and metabolic programs are coordinated with nutrient availability remains unclear. Here, we show that nutrient availability signaled by the neuronal cytokine, ILC-17.1, switches development between reproductive growth and dormancy by controlling the activity of the tumor suppressor p53 ortholog, CEP-1. Specifically, upon food availability, ILC-17.1 signaling by amphid neurons promotes glucose utilization and suppresses CEP-1/p53 to allow growth. In the absence of ILC-17.1, CEP-1/p53 is activated, up-regulates cell-cycle inhibitors, decreases phosphofructokinase and cytochrome C expression, and causes larvae to arrest as stress-resistant, quiescent dauers. We propose a model whereby ILC-17.1 signaling links nutrient availability and energy metabolism to cell cycle progression through CEP-1/p53. These studies describe ancestral functions of IL-17 s and the p53 family of proteins and are relevant to our understanding of neuroimmune mechanisms in cancer. They also reveal a DNA damage-independent function of CEP-1/p53 in invertebrate development and support the existence of a previously undescribed dauer pathway.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Tumor Suppressor Protein p53; Interleukin-17; DNA Damage
PubMed: 38483995
DOI: 10.1073/pnas.2315248121 -
BMC Urology Mar 2024Bladder cancer (BC) is one of the most common malignancies of the genitourinary system. Phosphofructokinase 1 (PFK-1) is one of member of PFK, which plays an important...
BACKGROUND
Bladder cancer (BC) is one of the most common malignancies of the genitourinary system. Phosphofructokinase 1 (PFK-1) is one of member of PFK, which plays an important role in reprogramming cancer metabolism, such as lactylation modification. Zinc finger E-box-binding homeobox 1 (ZEB1) has been demonstrated to be a oncogene in many cancers. Therefore, this study was performed to explore the effects of PFK-1 on the lactylation of ZEB1 in BC development.
METHODS
Cell viability was measured using the CCK-8 kit. The glucose assay kit and lactate assay kit were used to detect glucose utilization and lactate production. The DNA was purified and quantified by qRT-PCR.
RESULTS
In the present study, we found that ZEB1 expression levels were significantly elevated in bladder cancer cells. Impaired PFK-1 expression inhibits proliferation, migration, and invasion of BC cells and suppresses tumour growth in vivo. We subsequently found that knockdown of PFK-1 decreases glycolysis, including reduced glucose consumption, lactate production and total extracellular acidification rate (ECAR). Mechanistically, PFK-1 inhibits histone lactylation of bladder cancer cells, and thus inhibits the transcription activity of ZEB1.
CONCLUSION
Our results suggest that PFK-1 can inhibit the malignant phenotype of bladder cancer cells by mediating the lactylation of ZEB1. These findings suggested PFK-1 to be a new potential target for bladder cancer therapy.
Topics: Humans; Cell Line, Tumor; Cell Movement; Urinary Bladder Neoplasms; Phosphofructokinase-1; Lactates; Glucose; Cell Proliferation; Gene Expression Regulation, Neoplastic; Zinc Finger E-box-Binding Homeobox 1
PubMed: 38481182
DOI: 10.1186/s12894-024-01444-5 -
Chinese Medicine Mar 2024Cutaneous melanoma is a kind of skin malignancy with low morbidity but high mortality. Cryptotanshinone (CPT), an important component of salvia miltiorrhiza has potent...
BACKGROUND
Cutaneous melanoma is a kind of skin malignancy with low morbidity but high mortality. Cryptotanshinone (CPT), an important component of salvia miltiorrhiza has potent anti-tumor activity and also indicates therapeutic effect on dermatosis. So we thought that CPT maybe a potential agent for therapy of cutaneous melanoma.
METHODS
B16F10 and A375 melanoma cells were used for in vitro assay. Tumor graft models were made in C57BL/6N and BALB/c nude mice for in vivo assay. Seahorse XF Glycolysis Stress Test Kit was used to detect extracellular acidification rate and oxygen consumption rate. Si-RNAs were used for knocking down adenosine monophosphate-activated protein kinase (AMPK) expression in melanoma cells.
RESULTS
CPT could inhibit the proliferation of melanoma cells. Meanwhile, CPT changed the glucose metabolism and inhibited phosphofructokinase (PFK)-mediated glycolysis in melanoma cells to a certain extent. Importantly, CPT activated AMPK and inhibited the expression of hypoxia inducible factor 1α (HIF-1α). Both AMPK inhibitor and silencing AMPK could partially reverse CPT's effect on cell proliferation, cell apoptosis and glycolysis. Finally, in vivo experimental data demonstrated that CPT blocked the growth of melanoma, in which was dependent on the glycolysis-mediated cell proliferation.
CONCLUSIONS
CPT activated AMPK and then inhibited PFK-mediated aerobic glycolysis leading to inhibition of growth of cutaneous melanoma. CPT should be a promising anti-melanoma agent for clinical melanoma therapy.
PubMed: 38454519
DOI: 10.1186/s13020-024-00913-1 -
FASEB Journal : Official Publication of... Mar 2024Although elevated glycolysis has been widely recognized as a hallmark for highly proliferating cells like stem cells and cancer, its regulatory mechanisms are still...
Although elevated glycolysis has been widely recognized as a hallmark for highly proliferating cells like stem cells and cancer, its regulatory mechanisms are still being updated. Here, we found a previously unappreciated mechanism of mammalian target of rapamycin complex 2 (mTORC2) in regulating glycolysis in intestinal stem cell maintenance and cancer progression. mTORC2 key subunits expression levels and its kinase activity were specifically upregulated in intestinal stem cells, mouse intestinal tumors, and human colorectal cancer (CRC) tissues. Genetic ablation of its key scaffolding protein Rictor in both mouse models and cell lines revealed that mTORC2 played an important role in promoting intestinal stem cell proliferation and self-renewal. Moreover, utilizing mouse models and organoid culture, mTORC2 loss of function was shown to impair growth of gut adenoma and tumor organoids. Based on these findings, we performed RNA-seq and noticed significant metabolic reprogramming in Rictor conditional knockout mice. Among all the pathways, carbohydrate metabolism was most profoundly altered, and further studies demonstrated that mTORC2 promoted glycolysis in intestinal epithelial cells. Most importantly, we showed that a rate-limiting enzyme in regulating glycolysis, 6-phosphofructo-2-kinase (PFKFB2), was a direct target for the mTORC2-AKT signaling. PFKFB2 was phosphorylated upon mTORC2 activation, but not mTORC1, and this process was AKT-dependent. Together, this study has identified a novel mechanism underlying mTORC2 activated glycolysis, offering potential therapeutic targets for treating CRC.
Topics: Animals; Humans; Mice; Disease Models, Animal; Epithelial Cells; Glycolysis; Mammals; Mechanistic Target of Rapamycin Complex 2; Mice, Knockout; Neoplasms; Phosphofructokinase-2; Proto-Oncogene Proteins c-akt; Sirolimus
PubMed: 38451470
DOI: 10.1096/fj.202301833RR -
The European Journal of Neuroscience May 2024Globally, the incidence of diabetes mellitus (DM) and Alzheimer's disease (AD) is increasing year by year, causing a huge economic and social burden, and their... (Review)
Review
Globally, the incidence of diabetes mellitus (DM) and Alzheimer's disease (AD) is increasing year by year, causing a huge economic and social burden, and their pathogenesis and aetiology have been proven to have a certain correlation. In recent years, more and more studies have shown that vacuolar adenosine triphosphatases (v-ATPases) in eukaryotes, which are biomolecules regulating lysosomal acidification and glycolipid metabolism, play a key role in DM and AD. This article describes the role of v-ATPase in DM and AD, including its role in glycolysis, insulin secretion and insulin resistance (IR), as well as its relationship with lysosomal acidification, autophagy and β-amyloid (Aβ). In DM, v-ATPase is involved in the regulation of glucose metabolism and IR. v-ATPase is closely related to glycolysis. On the one hand, v-ATPase affects the rate of glycolysis by affecting the secretion of insulin and changing the activities of key glycolytic enzymes hexokinase (HK) and phosphofructokinase 1 (PFK-1). On the other hand, glucose is the main regulator of this enzyme, and the assembly and activity of v-ATPase depend on glucose, and glucose depletion will lead to its decomposition and inactivation. In addition, v-ATPase can also regulate free fatty acids, thereby improving IR. In AD, v-ATPase can not only improve the abnormal brain energy metabolism by affecting lysosomal acidification and autophagy but also change the deposition of Aβ by affecting the production and degradation of Aβ. Therefore, v-ATPase may be the bridge between DM and AD.
Topics: Alzheimer Disease; Humans; Vacuolar Proton-Translocating ATPases; Animals; Diabetes Mellitus; Glycolysis; Insulin Resistance; Lysosomes; Autophagy
PubMed: 38419188
DOI: 10.1111/ejn.16286 -
Zhongguo Xue Xi Chong Bing Fang Zhi Za... Feb 2024To investigate the effects of on the phenotypic transformations of glucose metabolism, polarization types and inflammatory responses in macrophages, so as to provide...
OBJECTIVE
To investigate the effects of on the phenotypic transformations of glucose metabolism, polarization types and inflammatory responses in macrophages, so as to provide insights into elucidation of echinococcosis pathogenesis.
METHODS
Bone marrow cells were isolated from C57BL/6J mice at ages of 6 to 8 weeks, and induced into bone marrow-derived macrophages (BMDMs) with mouse macrophage colony-stimulating factor (M-CSF), which served as controls (BMDMs-M0). BMDMs-M0 induced M2 macrophages by interleukin-4 for 24 hours served as the IL-4 induction group, and BMDMs-M0 co-cultured with 2.4 ng/mL cystic fluid (CF) served as the BMDM-CF co-culture group, while BMDMs-M0 co-cultured with protoscolex (PSC) at a ratio of 500:1 served as the BMDM-PSC co-culture group. The types of polarization of BMDMs co-cultured with CF and PSC were analyzed using flow cytometry, and the expression of macrophage markers, inflammatory factors, and glucose metabolism-related enzymes was quantified using fluorescent quantitative real-time PCR (qPCR) and Western blotting assays.
RESULTS
There were significant differences among the four groups in terms of Arginase-1 () ( = 1 457.00, < 0.000 1), macrophages-derived C-C motif chemokine 22 () ( = 22 203.00, < 0.000 1), resistin-like α () ( = 151.90, < 0.000 1), inducible nitric oxide synthase () ( = 107.80, < 0.001), hexokinase () ( = 9 389.00, < 0.000 1), pyruvate kinase () ( = 641.40, < 0.001), phosphofructokinase 1 () ( = 43.97, < 0.01), glucokinase () ( = 432.50, < 0.000 1), pyruvate dehydrogenase kinases1 () ( = 737.30, < 0.000 1), lactic dehydrogenase () ( = 3 632.00, < 0.000 1), glucose transporter 1 () ( = 532.40, < 0.000 1), glyceraldehyde-3-phosphate dehydrogenase () ( = 460.00, < 0.000 1), citrate synthase () ( = 5 642.00, < 0.01), glycogen synthase1 () ( = 273.30, < 0.000 1), - ( = 1 823.00, < 0.000 1), - ( = 291.70, < 0.000 1), - ( = 986.60, < 0.000 1), and tumor necrosis factor ()-α ( = 334.80, < 0.000 1) and transforming growth factor ()-β mRNA expression ( = 163.30, < 0.001). The proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM-PSC co-culture group [(22.87% ±1.48%) vs. (1.70% ±0.17%); = 24.61, < 0.001], and the proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM-CF co-culture group [(20.07% ±0.64%) vs. (1.93% ±0.25%); = 45.73, < 0.001]. The mRNA expression of M2 macrophages markers , and was significantly higher in the BMDM-CF and BMDM-PSC co-culture groups than in the control group (all values < 0.01), and no significant difference was seen in the mRNA expression of the M1 macrophage marker iNOS among the three groups ( > 0.05), while qPCR assay quantified higher mRNA expression of key glycolytic enzymes , and , as well as inflammatory factors , β, -α and -β in the BMDM-CF and BMDM-PSC co-culture groups than in the control group (all values < 0.01). Western blotting assay determined higher HK, PK and PFK protein expression in the BMDM-PSC co-culture group than in the control group (all values < 0.05), and qPCR quantified higher , and mRNA expression in the BMDM-CF co-culture group than in the control group (all values < 0.05), while higher HK, PK and PFK protein and mRNA expression (all values < 0.01), as well as lower and -α and higher -β mRNA expression (both values < 0.05) was detected in the IL-4 induction group than in the control group. Glycolytic stress test showed no significant difference in the extracellular acidification rate (ECAR) of mouse BMDM among the control group, IL-4 induction group and BMDM-PSC co-culture group ( = 124.4, < 0.05), and a higher ECAR was seen in the BMDM-PSC co-culture group and a lower ECAR was found in the IL-4 induction group than in the control group (both values < 0.05).
CONCLUSIONS
Treatment of CF or PSC mainly causes polarization of BMDM into M2 macrophages, and phenotypic transformation of glucose metabolism into high-energy and high-glycolytic metabolism, and affects inflammatory responses in BMDM.
Topics: Animals; Mice; Interleukin-10; Interleukin-4; Tumor Necrosis Factor-alpha; Echinococcus; Glucose Transporter Type 1; Interleukin-6; Mice, Inbred C57BL; Macrophages; Transforming Growth Factor beta; Oxidoreductases; Glucose; RNA, Messenger
PubMed: 38413020
DOI: 10.16250/j.32.1374.2023118