Abstract
The pioneering work of Eugene Kennedy in the 1950s established the choline pathway for phosphatidylcholine (PC) biosynthesis. However, the regulation of PC biosynthesis was poorly understood at that time. When I started my lab at the University of British Columbia in the 1970s, this was the focus of my research. This article provides my reflections on these studies that began with enzymology and the use of cultured mammalian cells, and progressed to utilize the techniques of molecular biology and gene-targeted mice. The research in my lab and others demonstrated that the regulated and rate-limiting step in the choline pathway for PC biosynthesis was catalyzed by CTP:phosphocholine cytidylyltransferase. This enzyme is regulated by its movement from a soluble form (largely in the nucleus) to a membrane-associated form where the enzyme becomes activated. Gene targeting in mice subsequently demonstrated that this gene is essential for development of mouse embryos. The other mammalian pathway for PC biosynthesis is catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT) that converts phosphatidylethanolamine to PC. Understanding of the regulation and function of the integral membrane protein PEMT was improved when the enzyme was purified (a masochistic endeavor) in 1987, leading to the cloning of the Pemt cDNA. Generation of knock-out mice that lacked PEMT showed that they were protected from atherosclerosis, diet-induced obesity, and insulin resistance. The protection from atherosclerosis appears to be due to decreased secretion of lipoproteins from the liver. We continue to investigate the mechanism(s) by which Pemt-/- mice are protected from weight gain and insulin resistance.
Highlights
The pioneering work of Eugene Kennedy in the 1950s established the choline pathway for phosphatidylcholine (PC) biosynthesis
The mechanism for the latter effect appeared to be that the concentration of cytoplasmic CTP increased and accelerated the rate-limiting step in PC biosynthesis catalyzed by CTP:phosphocholine cytidylyltransferase, which converts phosphocholine to CDP-choline [9] (Fig. 3)
The choline pathway for PC biosynthesis is present in all nucleated mammalian cells, an alternative pathway in which phosphatidylethanolamine (PE) is converted to PC by phosphatidylethanolamine N-methyltransferase (PEMT) is present in the liver, but only at very low levels (Ͻ1% of the activity in liver) in a few other tissues
Summary
The pioneering work of Eugene Kennedy in the 1950s established the choline pathway for phosphatidylcholine (PC) biosynthesis. One evening Dr Naff organized a talk on cholesterol biosynthesis by Dr Charles (Chuck) Sweeley (Fig. 1) from the University of Pittsburgh At this time (1963), biochemistry was not taught at the undergraduate level in most colleges and universities in the United States (biochemistry was taught in medical schools). I was very fortunate to be able to join the laboratory of David Feingold in the Department of Microbiology where I learned enzymology In his lab, I developed a technique for N-terminal sequencing of proteins and peptides using Edman degradation [5]. We were able to obtain the N-terminal sequence for up to 4 amino acids of a peptide without difficulty During this time, I took the opportunity to broaden my horizons by attending the medical student lectures in microbiology and virology
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