As described above, we are now beginning to understand the molecular mechanisms by which sphingolipid production is regulated according to metabolic need and in conjunction with other basic cellular processes. Despite these advances, there are many areas in which our understanding of sphingolipid homeostasis remains rudimentary. We discuss below these directions for future research and emerging technologies that will enable these questions to be addressed.
One immediate goal is to define the full spectrum of mechanisms by which sphingolipid metabolism is regulated. Indeed it is quite likely that our current inventory of regulators is highly incomplete, particularly given the large number of sphingolipid metabolites that have potent biological activities that must be tightly controlled. The need for multiple feedback controls is further underscored by the existence of distinct sub-cellular pools of sphingolipids that may each need to be precisely regulated.
A specific area in which our understanding remains poor is transcriptional control of sphingolipid metabolism. Given that master transcriptional regulators have been identified for glycerolipids and sterols in both yeast and mammals (Nohturfft and Zhang, 2009
), it is surprising that this aspect of sphingolipid biosynthesis is not yet understood. Although it is possible that sphingolipid regulation is primarily post-transcriptional, there is preliminary evidence for coordinated transcription of sterol and sphingolipid genes in yeast. For example, the transcription factors Ecm22 and Upc2 that control ergosterol production also have consensus binding sequences in the promoters of the serine palmitoyltransferase genes LCB1
(Vik and Rine, 2001
). Similarly, in the course of re-examining published yeast microarray studies (Ihmels et al., 2002
), we have found evidence for co-regulation of ergosterol and sphingolipid biosynthesis across a range of experimental conditions (our unpublished observations). It is also interesting to note that RPD3
, which encode the yeast homologs of the S1P-regulated histone deacetylases characterized by Hait et al. (2006)
, were previously identified as TSC
genes (Beeler et al., 1998
) and are known to participate in transcriptional control of glycerolipid metabolism (Wagner et al., 2001
). Whether LCB-Ps also regulate Rpd3/Sin3 in yeast and how this deacetylase controls sphingolipid metabolism will be exciting questions for future investigation.
Beyond obtaining a comprehensive list of sphingolipid regulators, a second major area for future research is understanding how cells modulate the activity of these regulators in response to sphingolipid levels. Particularly, what is the physical basis for sensing sphingolipids and what cellular machinery performs this function? Given that sphingolipid metabolites are membrane-embedded and vary widely in their physical properties and cellular concentrations, sensing sphingolipids is hardly a trivial task. Furthermore, sensors likely need to distinguish subtle chemical features because small differences in acyl chain length or unsaturation can be functionally important (as in Alvarez et al., 2010
and Menuz et al., 2009
Broadly speaking, two potential mechanisms for detecting sphingolipids can be imagined. In the first case, sphingolipid sensors may bind specifically to lipid metabolites in a fashion similar to canonical ligand-receptor interactions. Alternatively, sphingolipid sensors could instead detect sphingolipid-dependent changes in membrane properties. To date, evidence for both mechanisms exists. For example, specific binding of sphingolipid metabolites is carried out by S1P-specific GPCRs (Rosen et al., 2009
), the ubiquitin ligase TRAF2 (Alvarez et al., 2010
), and the histone deacetylases HDAC1 and HDAC2 (Hait et al., 2009
), and crystallographic studies are now providing structural insights into such mechanisms of protein-sphingolipid recognition (Kudo et al., 2008
; Malinina et al., 2006
). There is also precedent for sensors that detect the physical properties of membranes. For example, sphingolipids impact membrane thickness (Lingwood and Simons, 2010
), and a mechanism for responding to changes in membrane thickness has recently been described in bacteria (Cybulski et al., 2010
). Similarly, dynamic association of a sensor protein with sphingolipid-dependent membrane microdomains could be used to monitor sphingolipid levels. Intriguingly, the yeast trans-membrane protein Nce102 was recently shown to utilize this type of membrane domain-dependent regulation to control Pkh kinase activity (Frohlich et al., 2009
). Given that sphingolipids interact with functional effectors by a variety of mechanisms, it seems likely that multiple modes of molecular recognition are utilized to ensure sphingolipid homeostasis.
Efforts to identify and characterize sensors and regulators of sphingolipid metabolism will likely depend on a number of newly emerging technologies. Indeed, the substantial progress made in the past several years has been enabled by new methodologies such as lipidome analysis by mass spectrometry (Shevchenko and Simons, 2010
), identification of sphingolipid-binding proteins with affinity resins (Hait et al., 2009
), and the use of functional genomic approaches to characterize new regulators of sphingolipid production (Aguilar et al., 2010
; Breslow et al., 2010
; Denic and Weissman, 2007
). Moving forward, technologies that allow researchers to modulate and monitor sphingolipid metabolism with high temporal and spatial resolution would be of particularly value. In this regard, in vivo
reporters based on fusions of fluorescent proteins to lipid-binding domains have been key tools to study phosphoinositides and are now being developed for sphingolipids (Bakrac et al., 2010
). Similarly, previous work on phosphoinositides suggests that inducible targeting of sphingolipid-generating and sphingolipid-degrading enzymes to specific sub-cellular locations will be a powerful method to reveal local functions of sphingolipid metabolites (Birbes et al., 2001
Enabled by new technologies, future studies will be poised to meet the key challenge of understanding how different regulators of sphingolipid metabolism act together to ensure sphingolipid homeostasis. For example, it is unlikely that Orm proteins, SMSr, and CERT regulate serine palmitoyltransferase, ceramide levels, and ceramide transport by three isolated, independent mechanisms. Rather, multiple sphingolipid regulatory pathways likely act in concert, and characterizing the nature of these connections will be critical to understanding the systems-level properties of sphingolipid metabolism. More broadly, a comprehensive and integrated view of sphingolipid homeostasis promises to provide fundamental insights into lipid signaling and membrane function in cell biology and disease.