Rhoderick E. Brown, Ph.D.
Membrane Biochemistry

Essential for the existence of cells are barriers to envelope their contents. The barriers must be selectively permeable for nutrient entry and toxic by-product export. To meet this need, cells produce specialized lipids that are polar at one end and nonpolar at the opposite end. The polar ends are well-suited for contact with water but the nonpolar ends are not. As a result, they readily form thin, flexible layers only two molecules thick, i.e. bilayer, which forms the basic structural platform for cell membranes. In addition to corralling the cell contents, membranes serve as internal partitions that enable formation of functionally-specialized compartments within cells. Interestingly, there are many more varieties of lipids found in membranes than are needed to form bilayers. What is clear is that certain membrane lipids can function as messenger signals that regulate cell growth, proliferation, inflammation, and programmed cell death processes, while other membrane lipids appear to cluster together in bilayers to form microdomains that regulate the spatial distribution and lateral interactions of membrane proteins. The discovery of these membrane lipid functions under scores why biomembranes so often come under direct attack during cancer and infectious disease.

Our research focuses on membrane lipids known as sphingolipids. Certain sphingo¬lipids along with cholesterol can form ‘raft’ microdomains in membranes. Rafts appear to function as organizing regions for certain signaling kinases as well as target sites for certain viruses and bacteria. In earlier investigations, we focused on rigorously defining the physical basis for raft micro¬domain functionality. To gain insights into lipid structural features that control both the lateral and transmembrane distributions of sphingolipids, we have used a combination of biophysical approaches (fluorescence spectroscopy, Langmuir surface balance approaches, calorimetry, NMR). We developed ways to quantitatively measure the lateral elasticity within model membranes, to accurately assess the physical changes that occur within the ‘raft environment’ when the content and structure of sphingolipids and sterols become altered, as well as assess changes in sphingolipid lateral and transbilayer distributions. Our research has elucidated structural features of sphingolipids that regulate their interactions with other membrane lipids and provided insights into the unique physical features at the heart of the lateral organizing functionality of sphingolipid-enriched microdomains. The findings have proved to be important for understanding how the spatial organization of lipids in membranes can regulate proteins that translocate onto membranes to function.

Formation and maintenance of sphingolipid-enriched microdomains in cells likely involves specific proteins that can bind and transfer sphingolipids between membrane surfaces. Hence, much recent effort in our lab has been directed toward a protein family known as glycolipid transfer proteins (GLTPs) that can specifically bind and transfer glycosphingolipids between membranes. We found that GLTP functionality is regulated by lipid composition and packing within membranes. We applied this basic Membrane Biochemistry Rhoderick E. Brown, Ph.D. Section Leader Professor knowledge to begin elucidating exactly how GLTPs accomplish glycolipid intermembrane transfer. To do so, we cloned human GLTP and showed the existence of closely related homologs in mammals, plants, and fungi. Polymerase chain reaction (PCR) approaches enabled amplification of mRNA transcript open reading frames encoding human GLTP and related homologs followed by expression in bacterial expression systems, and purification of sufficient quantities to crystallize the proteins. Our efforts led to the molecular structural determination of GLTP and related homologs both in glycolipid-free form and complexed with different glycolipids, in collaboration with the D.J. Patel lab at Memorial Sloan Kettering Cancer Center in New York and the L. Malinina lab at CIC bioGUNE in Derio/Bilbao, Spain. Our studies shed light on: i) how GLTP adapts to accommodate different glycolipids within its binding site; ii) the functional role played by intrinsic tryptophan residues in glycolipid binding and membrane interaction; iii) the structural basis for the more focused glycolipid selectivity of a fungal GLTP ortholog as well as the GLTPH domain of human FAPP2 . More importantly, our work revealed that human GLTP forms a novel structural fold among known proteins. As a result, the Protein Data Bank has designated human GLTP as the founding member and prototype of the new GLTP superfamily, enabling our findings to be published in Nature, PLoS Biology, Structure, The Journal of Biological Chemistry, Biophysical Journal, Biochemistry, Journal of Lipid Research, and Quarterly Reviews of Biophysics.

In very recent investigations also published in Nature, we reported the discovery of a new GLTP structural homolog in human cells. Remarkably, the lipid specificity of the new protein has evolved for binding/transfer of ceramide-1-phosphate rather than glycolipids even though the new protein still forms a GLTP-fold encoded by a completely different gene than GLTP. For this reason, the protein is named ceramide-1-phosphate transfer protein (CPTP). In collaboration with Ted Hinchcliffe here at the UMN-Hormel Institute, we have tracked the location of CPTP in mammalian cells using state-of-the-art fluorescence microscopy approaches. With collaborating investigators in the Charles Chalfant lab at Virginia Common¬wealth University, we have shown that depletion of CPTP levels in human cells by RNA interference leads to over-accumulation of newly synthesized ceramide-1-phosphate in the trans-Golgi. The over-accumulation triggers cytoplasmic phospholipase A2 action, generating arachidonic acid that then is further metabolized into pro inflammatory eicosanoids. In studies of the model plant, Arabidopsis thaliana, carried out in collaboration with the John Mundy at the University of Copenhagen, we showed that a gene originally identified by its ability to induce accelerated cell death, known as acd11, actually encodes a plant GLTP ortholog. X-ray structural determinations revealed that ACD11 is a GLTP-fold that has evolved to bind and transfer ceramide-1-phosphate. Disruption of the acd11 gene results in impaired development and dwarfed plants in which the ceramide-1-phosphate and ceramide levels are severely altered. This research study was published in Cell Reports.

We anticipate that elucidation of the fundamental structure-function relationships governing GTLP and CPTP action will facilitate development of the means to pharmacologically modulate GLTP and enhance their potential use as biotechnological resources, i.e. nanotools, for targeted manipulation of cellular sphingolipid composition. Such strategies could provide new ways to introduce specific sphingolipid antigens to help achieve the targeted destruction of cancer cells via immunotherapeutic means, and lead to new therapeutic approaches to treat disease processes involving sphingolipids. Our exciting progress to date emphasizes the need for continuing studies into the workings of GLTP, CPTP, and other proteins containing GLTP-like motifs using comprehensive strategies involving biophysical, cell, and molecular biological approaches. Our recent investigations of the gene organization and transcriptional status in humans and other mammals now provide a firm foundation for identification and characterization of inherited diseases involving GLTP and CPTP. Our ongoing efforts benefit from collaborations with researchers at Memorial Sloan Kettering Cancer Center in New York, The University of Copenhagen in Denmark, Virginia Commonwealth University in Richmond, The Russian Academy of Sciences in Moscow, CIC bioGUNE in Derio/Bilbao, Spain and the Mayo Clinic. Our research continues because of financial support received from the National Institute of General Medical Sciences, the National Cancer Institute of NIH and The Hormel Foundation.

For more details regarding research expertise and scientific publications of our lab, please visit the following web sites:

Experts-UMN (REB): http://experts.umn.edu/en/persons/rhoderick-e-brown%28b67653a3-667a-4e50-a17c-202e43bc0884%29.html

Experts-UMN (REB publications): http://experts.umn.edu/en/persons/rhoderick-e-brown%28b67653a3-667a-4e50-a17c202e43bc0884%29/publications.html