Identification of hepatic protein-protein interaction targets for betaine homocysteine S-methyltransferase.

Protein-protein interactions are an important mechanism for the regulation of enzyme function allowing metabolite channeling, crosstalk between pathways or the introduction of post-translational modifications. Therefore, a number of high-throughput studies have been carried out to shed light on the protein networks established under different pathophysiological settings. Surprisingly, this type of information is quite limited for enzymes of intermediary metabolism such as betaine homocysteine S-methyltransferase, despite its high hepatic abundancy and its role in homocysteine metabolism. Here, we have taken advantage of two approaches, affinity purification combined with mass spectrometry and yeast two-hybrid, to further uncover the array of interactions of betaine homocysteine S-methyltransferase in normal liver of Rattus norvegicus. A total of 131 non-redundant putative interaction targets were identified, out of which 20 were selected for further validation by coimmunoprecipitation. Interaction targets validated by two different methods include: S-methylmethionine homocysteine methyltransferase or betaine homocysteine methyltransferase 2, methionine adenosyltransferases α1 and α2, cAMP-dependent protein kinase catalytic subunit alpha, 4-hydroxyphenylpyruvic acid dioxygenase and aldolase b. Network analysis identified 122 nodes and 165 edges, as well as a limited number of KEGG pathways that comprise: the biosynthesis of amino acids, cysteine and methionine metabolism, the spliceosome and metabolic pathways. These results further expand the connections within the hepatic methionine cycle and suggest putative cross-talks with additional metabolic pathways that deserve additional research.

Cloning, overexpression, and purification of glucose-6-phosphate dehydrogenase of Pseudomonas aeruginosa.

Glucose-6-phosphate dehydrogenase (G6PDH) (EC 1.1.1.363) plays an important role in the human pathogen Pseudomonas aeruginosa because it generates NADPH, an essential cofactor for several biosynthetic pathways and antioxidant enzymes. P. aeruginosa G6PDH is also a key enzyme in the metabolism of various carbon sources, such as glucose, glycerol, fructose, and mannitol. Understanding the kinetic characteristics and mechanisms that control the activity of this enzyme is crucial for future studies in this context. However, one of the impediments to achieving this goal is the limited amount of protein obtained when current purification protocols are implemented, a factor curtailing its biochemical characterization. In this study, we report a fast, efficient and reproducible procedure for the purification of P. aeruginosa G6PDH that can be implemented in a short period (2 days). In order to establish this protocol, the zwf gene, which encodes for this enzyme, was cloned and overexpressed in Escherichia coli cells. In contrast to other procedures, our method is based on protein precipitation with CaCl2 and further purification by ion exchange chromatography. Using this protocol, we were able to obtain 31 mg/L of pure protein that manifested specific activity of 145.7 U/mg. The recombinant enzyme obtained in this study manifested similar physicochemical and kinetic properties to those reported in previous works for this molecule. The large quantities of active enzyme obtained using this procedure will facilitate its structural characterization and identify differences between P. aeruginosa- and human G6PDH, thus contributing to the search for selective inhibitors against the bacterial enzyme.

The study of protein-protein interactions in bacteria.

Many of the functions fulfilled by proteins in the cell require specific protein-protein interactions (PPI). During the last decade, the use of high-throughput experimental technologies, primarily based on the yeast 2-hybrid system, generated extensive data currently located in public databases. This information has been used to build interaction networks for different species. Unfortunately, due to the nature of the yeast 2-hybrid system, these databases contain many false positives and negatives, thus they require purging. A method for confirming these PPI is to test them using a technique that operates in vivo and detects binary PPI. This article comprises an overview of the study of PPI and describes the main techniques that have been used to identify bacterial PPI, prioritizing those that can be used for their verification, and it also mentions a number of PPI that have been identified or confirmed using these methods.