Exploring the potential of asparagine restriction in solid cancer treatment: recent discoveries, therapeutic implications, and challenges.

Asparagine is a non-essential amino acid crucial for protein biosynthesis and function, and therefore cell maintenance and growth. Furthermore, this amino acid has an important role in regulating several metabolic pathways, such as tricarboxylic acid cycle and the urea cycle. When compared to normal cells, tumor cells typically present a higher demand for asparagine, making it a compelling target for therapy. In this review article, we investigate different facets of asparagine bioavailability intricate role in malignant tumors raised from solid organs. We take a comprehensive look at asparagine synthetase expression and regulation in cancer, including the impact on tumor growth and metastasis. Moreover, we explore asparagine depletion through L-asparaginase as a potential therapeutic method for aggressive solid tumors, approaching different formulations of the enzyme and combinatory therapies. In summary, here we delve into studies about endogenous and exogenous asparagine availability in solid cancers, analyzing therapeutic implications and future challenges.

In-solution behavior and protective potential of asparagine synthetase A from Trypanosoma cruzi.

l-Asparagine synthetase (AS) acts in asparagine formation and can be classified into two families: AS-A or AS-B. AS-A is mainly found in prokaryotes and can synthetize asparagine from ammonia. Distinct from other eukaryotes, Trypanosoma cruzi produces an AS-A. AS-A from Trypanosoma cruzi (Tc-AS-A) differs from prokaryotic AS-A due to its ability to catalyze asparagine synthesis using both glutamine and ammonia as nitrogen sources. Regarding these peculiarities, this work uses several biophysical techniques to provide data concerning the Tc-AS-A in-solution behavior. Tc-AS-A was produced as a recombinant and purified by three chromatography steps. Circular dichroism, dynamic light scattering, and analytical size exclusion chromatography showed that Tc-AS-A has the same fold and quaternary arrangement of prokaryotic AS-A. Despite the tendency of protein to aggregate, stable dimers were obtained when solubilization occurred at pH ≤ 7.0. We also demonstrate the protective efficacy against T. cruzi infection in mice immunized with Tc-AS-A. Our results indicate that immunization with Tc-AS-A might confer partial protection to infective forms of T. cruzi in this particular model.

Natural variation in non-coding regions underlying phenotypic diversity in budding yeast.

Linkage mapping studies in model organisms have typically focused their efforts in polymorphisms within coding regions, ignoring those within regulatory regions that may contribute to gene expression variation. In this context, differences in transcript abundance are frequently proposed as a source of phenotypic diversity between individuals, however, until now, little molecular evidence has been provided. Here, we examined Allele Specific Expression (ASE) in six F1 hybrids from Saccharomyces cerevisiae derived from crosses between representative strains of the four main lineages described in yeast. ASE varied between crosses with levels ranging between 28% and 60%. Part of the variation in expression levels could be explained by differences in transcription factors binding to polymorphic cis-regulations and to differences in trans-activation depending on the allelic form of the TF. Analysis on highly expressed alleles on each background suggested ASN1 as a candidate transcript underlying nitrogen consumption differences between two strains. Further promoter allele swap analysis under fermentation conditions confirmed that coding and non-coding regions explained aspartic and glutamic acid consumption differences, likely due to a polymorphism affecting Uga3 binding. Together, we provide a new catalogue of variants to bridge the gap between genotype and phenotype.

Flooding of the root system in soybean: biochemical and molecular aspects of N metabolism in the nodule during stress and recovery.

Nitrogen fixation of the nodule of soybean is highly sensitive to oxygen deficiency such as provoked by waterlogging of the root system. This study aimed to evaluate the effects of flooding on N metabolism in nodules of soybean. Flooding resulted in a marked decrease of asparagine (the most abundant amino acid) and a concomitant accumulation of γ-aminobutyric acid (GABA). Flooding also resulted in a strong reduction of the incorporation of (15)N2 in amino acids. Nodule amino acids labelled before flooding rapidly lost (15)N during flooding, except for GABA, which initially increased and declined slowly thereafter. Both nitrogenase activity and the expression of nifH and nifD genes were strongly decreased on flooding. Expression of the asparagine synthetase genes SAS1 and SAS2 was reduced, especially the former. Expression of genes encoding the enzyme glutamic acid decarboxylase (GAD1, GAD4, GAD5) was also strongly suppressed except for GAD2 which increased. Almost all changes observed during flooding were reversible after draining. Possible changes in asparagine and GABA metabolism that may explain the marked fluctuations of these amino acids during flooding are discussed. It is suggested that the accumulation of GABA has a storage role during flooding stress.

Hap2-3-5-Gln3 determine transcriptional activation of GDH1 and ASN1 under repressive nitrogen conditions in the yeast Saccharomyces cerevisiae.

The transcriptional activation response relies on a repertoire of transcriptional activators, which decipher regulatory information through their specific binding to cognate sequences, and their capacity to selectively recruit the components that constitute a given transcriptional complex. We have addressed the possibility of achieving novel transcriptional responses by the construction of a new transcriptional regulator--the Hap2-3-5-Gln3 hybrid modulator--harbouring the HAP complex polypeptides that constitute the DNA-binding domain (Hap2-3-5) and the Gln3 activation domain, which usually act in an uncombined fashion. The results presented in this paper show that transcriptional activation of GDH1 and ASN1 under repressive nitrogen conditions is achieved through the action of the novel Hap2-3-5-Gln3 transcriptional regulator. We propose that the combination of the Hap DNA-binding and Gln3 activation domains results in a hybrid modulator that elicits a novel transcriptional response not evoked when these modulators act independently.

Nitrogen stress and the expression of asparagine synthetase in roots and nodules of soybean (Glycine max).

The difficulty of assaying asparagine synthetase (AS) (EC 6.3.5.4) activity in roots of soybean has been circumvented by measuring expression of the AS genes. Expression of three soybean asparagine synthetase (SAS) genes (SAS1, SAS2 and SAS3) was observed in roots of non-nodulated soybean plants cultivated on nitrate. Expression of these genes was reduced to very low levels within days after submitting the plants to a N-free medium. The subsequent return to a complete medium (containing nitrate) restored expression of all three AS genes. Roots of nodulated plants, where symbiotic nitrogen fixation was the exclusive source of N (no nitrate present), showed very weak expression of all three AS genes, but on transfer to a nitrate-containing medium, strong expression of these genes was observed within 24 h. In nodules, all three genes were expressed in the absence of nitrate. Under conditions that impair nitrogen fixation (nodules submerged in aerated hydroponics), only SAS1 expression was reduced. However, in the presence of nitrate, an inhibitor of N(2) fixation, SAS1 expression was maintained. High and low expressions of AS genes in the roots were associated with high and low ratios of Asn/Asp transported to the shoot through xylem. It is concluded that nitrate (or one of its assimilatory products) leads to the induction of AS in roots of soybean and that this underlies the variations found in xylem sap Asn/Asp ratios. Regulation of nodule AS expression is quite different from that of the root, but nodule SAS1, at least, appears to involve a product of N assimilation rather than nitrate itself.

Aspartate transport and metabolism in the protozoan parasite Trypanosoma cruzi.

Aspartate is one of the compounds that induce the differentiation process of the non-infective epimastigote stage to the infective trypomastigote stage of the protozoan parasite Trypanosoma cruzi. l-aspartate is transported by both epimastigote and trypomastigote cells at the same rate, about 3.4 pmolmin(-1) per 10(7) cells. Aspartate transport is only competed by glutamate suggesting that this transport system is specific for anionic amino acids. Aspartate uptake rates increase along the parasite growth curve, by amino acids starvation or pH decrease. The metabolic fate of the transported aspartate was predicted in silico by identification of seven putative genes coding for enzymes involved in aspartate metabolism that could be related to the differentiation process.

Metabolite and light regulation of metabolism in plants: lessons from the study of a single biochemical pathway

We are using molecular, biochemical, and genetic approaches to study the structural and regulatory genes controlling the assimilation of inorganic nitrogen into the amino acids glutamine, glutamate, aspartate and asparagine. These amino acids serve as the principal nitrogen-transport amino acids in most crop and higher plants including Arabidopsis thaliana. We have begun to investigate the regulatory mechanisms controlling nitrogen assimilation into these amino acids in plants using molecular and genetic approaches in Arabidopsis. The synthesis of the amide amino acids glutamine and asparagine is subject to tight regulation in response to environmental factors such as light and to metabolic factors such as sucrose and amino acids. For instance, light induces the expression of glutamine synthetase (GLN2) and represses expression of asparagine synthetase (ASN1) genes. This reciprocal regulation of GLN2 and ASN1 genes by light is reflected at the level of transcription and at the level of glutamine and asparagine biosynthesis. Moreover, we have shown that the regulation of these genes is also reciprocally controlled by both organic nitrogen and carbon metabolites. We have recently used a reverse genetic approach to study putative components of such metabolic sensing mechanisms in plants that may be conserved in evolution. These components include an Arabidopsis homolog for a glutamate receptor gene originally found in animal systems and a plant PII gene, which is a homolog of a component of the bacterial Ntr system. Based on our observations on the biology of both structural and regulatory genes of the nitrogen assimilatory pathway, we have developed a model for metabolic control of the genes involved in the nitrogen assimilatory pathway in plants

Metabolite and light regulation of metabolism in plants: lessons from the study of a single biochemical pathway.

We are using molecular, biochemical, and genetic approaches to study the structural and regulatory genes controlling the assimilation of inorganic nitrogen into the amino acids glutamine, glutamate, aspartate and asparagine. These amino acids serve as the principal nitrogen-transport amino acids in most crop and higher plants including Arabidopsis thaliana. We have begun to investigate the regulatory mechanisms controlling nitrogen assimilation into these amino acids in plants using molecular and genetic approaches in Arabidopsis. The synthesis of the amide amino acids glutamine and asparagine is subject to tight regulation in response to environmental factors such as light and to metabolic factors such as sucrose and amino acids. For instance, light induces the expression of glutamine synthetase (GLN2) and represses expression of asparagine synthetase (ASN1) genes. This reciprocal regulation of GLN2 and ASN1 genes by light is reflected at the level of transcription and at the level of glutamine and asparagine biosynthesis. Moreover, we have shown that the regulation of these genes is also reciprocally controlled by both organic nitrogen and carbon metabolites. We have recently used a reverse genetic approach to study putative components of such metabolic sensing mechanisms in plants that may be conserved in evolution. These components include an Arabidopsis homolog for a glutamate receptor gene originally found in animal systems and a plant PII gene, which is a homolog of a component of the bacterial Ntr system. Based on our observations on the biology of both structural and regulatory genes of the nitrogen assimilatory pathway, we have developed a model for metabolic control of the genes involved in the nitrogen assimilatory pathway in plants.