Anno: 
2017
Nome e qualifica del proponente del progetto: 
sb_p_501500
Abstract: 

Carnitine, a crucial metabolite in eukariotes energy metabolism, is responsible for the transport of acyl and acetyl activated groups into the mitochondrial matrix, where their oxidation takes place. Mammals obtain carnitine primarily from diet, although they are able to synthesise it. The carnitine biosinthesis pathway has been well-characterized in invertebrates, yeasts and mammals. In mammals, the genes encoding three out of the four enzymes involved in the pathway have been identified and their enzyme products characterized. The identity of the remaining enzyme, which has been dubbed 3-hydroxy-N6-trimethyllysine aldolase (HTMLA) and catalyses the retroaldol cleavage of HTML (a ß-hydroxyamino acid) into glycyne and 4-N-trimethylaminobutyraldehyde (TMABA), remains a mystery.

It has been proposed that HTMLA coincides with cytosolic serine hydroxymethyltransferase (SHMT1), a pyridoxal 5'-phosphate (PLP)-dependent enzyme that also exists as a mitochondrial isoform (SHMT2). However, this hypothesis has never been verified. Other PLP-dependent enzymes may have the capability to catalyse HTML cleavage. Threonine aldolases, for instance, are good candidates. Notably, although both mouse and human genomes possess a threonine aldolase gene, in humans this gene is not expressed.

The goal of this project is to identify the enzyme that plays the role of HTMLA in humans. A computational screening of all human PLP-dependent enzymes, including SHMT1 and SHMT2, will evaluate their potential capability to bind HTML and catalyse its retroaldol cleavage into glycine and TMABA. Human SHMT1, SHMT2, and any other PLP-dependent enzyme selected in the computational screening, will be characterized in vitro with respect to their actual capability to cleave HTML into glycine and TMABA. In vivo studies on cancer cell lines, in which SHMT1 and SHMT2 expression will be knockdown, will establish whether these enzymes are actually involved in the carnitine biosynthesis pathway.

Componenti gruppo di ricerca: 
sb_cp_is_748811
sb_cp_es_107236
sb_cp_es_107237
sb_cp_es_107235
sb_cp_es_107238
sb_cp_es_107239
sb_cp_es_107240
Innovatività: 

INNOVATIVE ASPECTS OF RESEARCH.
Although a lot of work has been done on carnitine biosynthesis, the identity of the PLP-dependent enzyme that has been dubbed HTMLA and catalyses the retroaldol cleavage of HTML into glycine and TMABA is still a mystery. The goal of our research project is to shed light on this puzzle. Although SHMT1 is the best candidate for the role of HTMLA in humans, this hypothesis has never been verified. The experimental evidence in favour of SHMT1 derives from experiments carried out on rabbit cytosolic SHMT, which was able to catalyse the HMTLA reaction with a Km of 3.3 mM (kcat was not determined [1]). This result does not preclude the possibility that a threonine aldolase may be responsible for the HTMLA activity in rabbit. In humans, that lack threonine aldolase, SHMT1 may have developed a more efficient HTMLA activity. However, the actual capability of human SHMT1 to catalyse the HTMLA reaction has never been tested and neither has that of SHMT2. If SHMT1 coincides with HTMLA in humans, SHMT2 is expected to show a lower activity in the retroaldol cleavage of HTML. We are going to clarify all these aspects, also thanks to in vivo experiments that will test the actual involvement of SHMT1 and SHMT2 in carnitine biosynthesis. SHMT is a very versatile enzyme that apparently plays many cellular functions. In particular, it is a pivotal enzyme in cancer metabolic reprogramming. In the last years, attention of the international community has focused on SHMT especially because of this role of the enzyme. The possible identification of SHMT1 as HTMLA in carnitine biosynthesis will represent a further step forward in the knowledge of the multifaceted cellular role of this enzyme and will surely have a large resonance.
This research project, if funded, has a very high probability to solve the mystery that surrounds HTMLA. The longstanding experience of our research group on SHMT, the mastery of all techniques planned in the project and the collaboration with external experienced researchers will make our goal an actually achievable one. Our group and our collaborators have already successfully tested most methods described in the project. Other methods, such as the organic synthesis of HTML, are fully described in the literature. That is the reason why, although this research program may seem too ambitious, we feel like to guarantee its success, within the frame time of the project and with the requested budget.

FUTURE RESEARCH ACTIVITIES.
In the event that a PLP-dependent enzyme different from SHMT1 and SHMT2 will be identified by the computational and in vitro studies as a better candidate for HTMLA activity (this information will also have to match the results obtained in the in vivo studies on the role of SHMT1 and SHMT2 in carnitine biosynthesis), other in vivo experiments will be required to demonstrate the actual involvement of this enzyme in carnitine biosynthesis. These studies will be the focus of a future research program.

1. Henderson et al., 1982 Fed Proc 41, 2843-7.

Codice Bando: 
501500
Keywords: 

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