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Down syndrome (DS) is a genetic condition where the person is born with an extra chromosome 21. DS is associated with accelerated aging; people with DS are prone to age-related neurological conditions including an early-onset Alzheimer's disease. Using the Dp(17)3Yey/ + mice, which overexpresses a portion of mouse chromosome 17, which encodes for the transsulfuration enzyme cystathionine β-synthase (CBS), we investigated the functional role of the CBS/hydrogen sulfide (H2S) pathway in the pathogenesis of neurobehavioral dysfunction in DS. The data demonstrate that CBS is higher in the brain of the DS mice than in the brain of wild-type mice, with primary localization in astrocytes. DS mice exhibited impaired recognition memory and spatial learning, loss of synaptosomal function, endoplasmic reticulum stress, and autophagy. Treatment of mice with aminooxyacetate, a prototypical CBS inhibitor, improved neurobehavioral function, reduced the degree of reactive gliosis in the DS brain, increased the ability of the synaptosomes to generate ATP, and reduced endoplasmic reticulum stress. H2S levels in the brain of DS mice were higher than in wild-type mice, but, unexpectedly, protein persulfidation was decreased. Many of the above alterations were more pronounced in the female DS mice. There was a significant dysregulation of metabolism in the brain of DS mice, which affected amino acid, carbohydrate, lipid, endocannabinoid, and nucleotide metabolites; some of these alterations were reversed by treatment of the mice with the CBS inhibitor. Thus, the CBS/H2S pathway contributes to the pathogenesis of neurological dysfunction in DS in the current animal model. [ABSTRACT FROM AUTHOR]

Homocystinuria (HCU) due to cystathionine beta‐synthase (CBS) deficiency is the most common inborn error of sulfur amino acid metabolism. Recent work suggests that missense pathogenic mutations—regardless of their topology—cause instability of the C‐terminal regulatory domain, which likely translates into CBS misfolding, impaired assembly, and loss of function. However, it is unknown how instability of the regulatory domain translates into cellular CBS turnover and which degradation pathways are involved in CBS proteostasis. Here, we developed a human HEK293‐based cellular model lacking intrinsic CBS and stably overexpressing wild‐type (WT) CBS or its 10 most common missense HCU mutants. We found that HCU mutants, except the I278T variant, expressed similarly or better than CBS WT, with some of them showing impaired oligomerization, activity and response to allosteric activator S‐adenosylmethionine. Cellular stability of all HCU mutants, except P49L and A114V, was significantly lower than the stability of CBS WT, suggesting their increased degradation. Ubiquitination analysis of CBS WT and two representative CBS mutants (T191M and I278T) showed that proteasomal degradation is the major pathway for CBS disposal, with a minor involvement of lysosomal‐autophagic and endoplasmic reticulum‐associated degradation (ERAD) pathways for HCU mutants. Proteasomal inhibition significantly increased the half‐life and activity of T191M and I278T CBS mutants. Lysosomal and ERAD inhibition had only a minor impact on CBS turnover, but ERAD inhibition rescued the activity of T191M and I278T CBS mutants similarly as proteasomal inhibition. In conclusion, the present study provides new insights into proteostasis of CBS in HCU. [ABSTRACT FROM AUTHOR]

Cystathionine β-synthase (CBS), CSE (cystathionine γ-lyase) and 3-mercaptopyruvate sulfurtransferase (3-MST) have emerged as three significant sources of hydrogen sulfide (H2S) in various forms of mammalian cancer. Here, we investigated the functional role of CBS' and 3-MST's catalytic activity in the murine breast cancer cell line EO771. The CBS/CSE inhibitor aminooxyacetic acid (AOAA) and the 3-MST inhibitor 2-[(4-hydroxy-6-methylpyrimidin-2-yl)sulfanyl]-1-(naphthalen-1-yl)ethan-1-one (HMPSNE) were used to assess the role of endogenous H2S in the modulation of breast cancer cell proliferation, migration, bioenergetics and viability in vitro. Methods included measurements of cell viability (MTT and LDH assays), cell proliferation and in vitro wound healing (IncuCyte) and cellular bioenergetics (Seahorse extracellular flux analysis). CBS and 3-MST, as well as expression were detected by Western blotting; H2S production was measured by the fluorescent dye AzMC. The results show that EO771 cells express CBS, CSE and 3-MST protein, as well as several enzymes involved in H2S degradation (SQR, TST, and ETHE1). Pharmacological inhibition of CBS or 3-MST inhibited H2S production, suppressed cellular bioenergetics and attenuated cell proliferation. Cell migration was only inhibited by the 3-MST inhibitor, but not the CBS/CSE inhibitor. Inhibition of CBS/CSE of 3-MST did not significantly affect basal cell viability; inhibition of 3-MST (but not of CBS/CSE) slightly enhanced the cytotoxic effects of oxidative stress (hydrogen peroxide challenge). From these findings, we conclude that endogenous H2S, generated by 3-MST and to a lower degree by CBS/CSE, significantly contributes to the maintenance of bioenergetics, proliferation and migration in murine breast cancer cells and may also exert a minor role as a cytoprotectant. [ABSTRACT FROM AUTHOR]

The haem enzyme indoleamine 2,3-dioxygenase 1 (IDO1) catalyses the rate-limiting step in the kynurenine pathway of tryptophan metabolism and plays an essential role in immunity, neuronal function, and ageing. Expression of IDO1 in cancer cells results in the suppression of an immune response, and therefore IDO1 inhibitors have been developed for use in anti-cancer immunotherapy. Here, we report an extension of our previously described highly efficient haem-binding 1,2,3-triazole and 1,2,4-triazole inhibitor series, the best compound having both enzymatic and cellular IC50 values of 34 nM. We provide enzymatic inhibition data for almost 100 new compounds and X-ray diffraction data for one compound in complex with IDO1. Structural and computational studies explain the dramatic drop in activity upon extension to pocket B, which has been observed in diverse haem-binding inhibitor scaffolds. Our data provides important insights for future IDO1 inhibitor design. [ABSTRACT FROM AUTHOR]

Recently, a CRISPR-Cas9 genome-editing system was developed with introduced sequential 'driver' mutations in the WNT, MAPK, TGF-β, TP53 and PI3K pathways into organoids derived from normal human intestinal epithelial cells. Prior studies have demonstrated that isogenic organoids harboring mutations in the tumor suppressor genes APC, SMAD4 and TP53, as well as the oncogene KRAS, assumed more proliferative and invasive properties in vitro and in vivo. A separate body of studies implicates the role of various hydrogen sulfide (H2S)-producing enzymes in the pathogenesis of colon cancer. The current study was designed to determine if the sequential mutations in the above pathway affect the expression of various H2S producing enzymes. Western blotting was used to detect the expression of the H2S-producing enzymes cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST), as well as several key enzymes involved in H2S degradation such as thiosulfate sulfurtransferase/rhodanese (TST), ethylmalonic encephalopathy 1 protein/persulfide dioxygenase (ETHE1) and sulfide-quinone oxidoreductase (SQR). H2S levels were detected by live-cell imaging using a fluorescent H2S probe. Bioenergetic parameters were assessed by Extracellular Flux Analysis; markers of epithelial-mesenchymal transition (EMT) were assessed by Western blotting. The results show that the consecutive mutations produced gradual upregulations in CBS expression—in particular in its truncated (45 kDa) form—as well as in CSE and 3-MST expression. In more advanced organoids, when the upregulation of H2S-producing enzymes coincided with the downregulation of the H2S-degrading enzyme SQR, increased H2S generation was also detected. This effect coincided with the upregulation of cellular bioenergetics (mitochondrial respiration and/or glycolysis) and an upregulation of the Wnt/β-catenin pathway, a key effector of EMT. Thus sequential mutations in colon epithelial cells according to the Vogelstein sequence are associated with a gradual upregulation of multiple H2S generating pathways, which, in turn, translates into functional changes in cellular bioenergetics and dedifferentiation, producing more aggressive and more invasive colon cancer phenotypes. [ABSTRACT FROM AUTHOR]

Neuroblastoma (NB) is one of the most deadly solid tumors of the young child, for which new efficient and targeted therapies are strongly needed. The CXCR4/CXCR7/CXCL12 chemokine axis has been involved in the progression and organ-specific dissemination of various cancers. In NB, CXCR4 expression was shown to be associated to highly aggressive undifferentiated tumors, while CXCR7 expression was detected in more differentiated and mature neuroblastic tumors. As investigated in vivo, using an orthotopic model of tumor cell implantation of chemokine receptor-overexpressing NB cells (IGR-NB8), the CXCR4/CXCR7/CXCL12 axis was shown to regulate NB primary and secondary growth, although without any apparent influence on organ selective metastasis. In the present study, we addressed the selective role of CXCR4 and CXCR7 receptors in the homing phase of metastatic dissemination using an intravenous model of tumor cell implantation. Tail vein injection into NOD-scid-gamma mice of transduced IGR-NB8 cells overexpressing CXCR4, CXCR7, or both receptors revealed that all transduced cell variants preferentially invaded the adrenal gland and typical NB metastatic target organs, such as the liver and the bone marrow. However, CXCR4 expression favored NB cell dissemination to the liver and the lungs, while CXCR7 was able to strongly promote NB cell homing to the adrenal gland and the liver. Finally, coexpression of CXCR4 and CXCR7 receptors significantly and selectively increased NB dissemination toward the bone marrow. In conclusion, CXCR4 and CXCR7 receptors may be involved in a complex and organ-dependent control of NB growth and selective homing, making these receptors and their inhibitors potential new therapeutic targets. [ABSTRACT FROM AUTHOR]

Conjugated linoleic acid (CLA) is a group of isomers of linoleic acid (LA) with several potential health benefits which justifies the efforts to obtain foods with enhanced CLA contents. A possible way to obtain CLA-enriched products is via fermentation as several micro-organisms, many of which are employed in the manufacture of foods, are known to convert free LA into CLA. However, due to several constraints, the use of CLA-producing bacteria still remains challenging and continuous efforts are required. This review summarises the current knowledge on microbial CLA production by food-grade micro-organisms, its technical issues and limitations, potential applications and bioactivities. [ABSTRACT FROM AUTHOR]

[Display omitted] Hydrogen sulfide (H 2 S) is an important endogenous gaseous transmitter mediator, which regulates a variety of cellular functions in autocrine and paracrine manner. The enzymes responsible for the biological generation of H 2 S include cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST). Increased expression of these enzymes and overproduction of H 2 S has been implicated in essential processes of various cancer cells, including the stimulation of metabolism, maintenance of cell proliferation and cytoprotection. Cancer cell identity is characterized by so-called "transition states". The progression from normal (epithelial) to transformed (mesenchymal) state is termed epithelial-to-mesenchymal transition (EMT) whereby epithelial cells lose their cell-to-cell adhesion capacity and gain mesenchymal characteristics. The transition process can also proceed in the opposite direction, and this process is termed mesenchymal-to-epithelial transition (MET). The current project was designed to determine whether inhibition of endogenous H 2 S production in colon cancer cells affects the EMT/MET balance in vitro. Inhibition of H 2 S biosynthesis in HCT116 human colon cancer cells was achieved either with aminooxyacetic acid (AOAA) or 2-[(4-hydroxy-6-methylpyrimidin-2-yl)sulfanyl]-1-(naphthalen-1-yl)ethan-1-one (HMPSNE). These inhibitors induced an upregulation of E-cadherin and Zonula occludens-1 (ZO-1) expression and downregulation of fibronectin expression, demonstrating that H 2 S biosynthesis inhibitors can produce a pharmacological induction of MET in colon cancer cells. These actions were functionally reflected in an inhibition of cell migration, as demonstrated in an in vitro "scratch wound" assay. The mechanisms involved in the action of endogenously produced H 2 S in cancer cells in promoting (or maintaining) EMT (or tonically inhibiting MET) relate, at least in part, in the induction of ATP citrate lyase (ACLY) protein expression, which occurs via upregulation of ACLY mRNA (via activation of the ACLY promoter). ACLY in turn, regulates the Wnt-β-catenin pathway, an essential regulator of the EMT/MET balance. Taken together, pharmacological inhibition of endogenous H 2 S biosynthesis in cancer cells induces MET. We hypothesize that this may contribute to anti-cancer / anti-metastatic effects of H 2 S biosynthesis inhibitors. [ABSTRACT FROM AUTHOR]

Homocystinuria (HCU) due to cystathionine beta-synthase (CBS) deficiency is the most common inborn error of sulfur amino acid metabolism. Recent work suggests that missense pathogenic mutations-regardless of their topology-cause instability of the C-terminal regulatory domain, which likely translates into CBS misfolding, impaired assembly, and loss of function. However, it is unknown how instability of the regulatory domain translates into cellular CBS turnover and which degradation pathways are involved in CBS proteostasis. Here, we developed a human HEK293-based cellular model lacking intrinsic CBS and stably overexpressing wild-type (WT) CBS or its 10 most common missense HCU mutants. We found that HCU mutants, except the I278T variant, expressed similarly or better than CBS WT, with some of them showing impaired oligomerization, activity and response to allosteric activator S-adenosylmethionine. Cellular stability of all HCU mutants, except P49L and A114V, was significantly lower than the stability of CBS WT, suggesting their increased degradation. Ubiquitination analysis of CBS WT and two representative CBS mutants (T191M and I278T) showed that proteasomal degradation is the major pathway for CBS disposal, with a minor involvement of lysosomal-autophagic and endoplasmic reticulum-associated degradation (ERAD) pathways for HCU mutants. Proteasomal inhibition significantly increased the half-life and activity of T191M and I278T CBS mutants. Lysosomal and ERAD inhibition had only a minor impact on CBS turnover, but ERAD inhibition rescued the activity of T191M and I278T CBS mutants similarly as proteasomal inhibition. In conclusion, the present study provides new insights into proteostasis of CBS in HCU., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)