Membrane Biochemistry
Section Leader
Rhoderick E. Brown, Ph.D.
Professor

The Hormel Instittute - Rhoderick Brown

When people read or hear the word “lipid”, the picture that often comes to mind is the fat or grease associated with meat or body tissue. Generally unappreciated and misunderstood is the fact that many lipids play other key roles that are fundamental for healthy cell function and survival. For instance, formation of the thin, flexible barriers that surround cells and divide the cell interior into specialized compartments depends on lipids. These lipids are specially modified at the molecular level so that they are polar at one end and nonpolar at the opposite end. As the polar ends prefer to be in contact with water and the nonpolar ends do not, these special lipids readily form layers only two molecules thick, i.e. bilayers, commonly known as cell membranes. The thin, flexible nature of cell membranes enables them to act as selective permeability barriers to control what gets in and out of cells. Interestingly, there are many more varieties of lipids found in membranes than are needed to form bilayers. Over the past two decades or so, it has become clear that some membrane lipids can function as intracellular messenger signals that regulate cell growth, proliferation, and programmed cell death and survival processes, while other membrane lipids can cluster together to form membrane microdomains that control the spatial distribution and lateral interactions of certain membrane proteins. The discovery of these new functions for membrane lipids underscores why biomembranes so often come under direct attack during cancer and infectious disease.

 

Our research focuses on membrane lipids known as sphingolipids. Certain sphingolipids serve as key components needed for formation of .raft. microdomains in membranes. Rafts appear to function as organizing regions for some signaling kinases as well as target sites for certain viruses and bacteria. In earlier investigations, our research focused on rigorously defining the physical basis for raft microdomain functionality. To do so, we developed ways to quantitatively measure the lateral elasticity within model membranes as well as accurately assess and quantify physical changes that occur within the raft microdomain environment when the content and structure of sphingolipids and sterols becomes altered. This research helped identify structural features of sphingolipids that regulate their interactions with other membrane lipids and provided fundamental insights into the unique physical features of membrane microdomains at the heart of their lateral organizing functionality. The findings have been proven to be important for understanding how changes in membrane lipid composition can regulate interaction with proteins that need to translocate onto membranes to function.

Formation and maintenance of sphingolipid-enriched microdomains in cells are likely to involve 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 have found that GLTP functionality is regulated by lipid composition and packing within membranes. To gain insights into the 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 balances, NMR, microcalorimetry). We have applied this basic knowledge to decipher the functional regulation of GLTP, i.e. exactly how GLTPs accomplish the intermembrane transfer of glycolipids. To do so, we carried out the first molecular cloning of human GLTP and showed the existence of related homologs in mammals, plants, and fungi. Molecular biological approaches involving polyermase chain reaction (PCR) enabled amplification of mRNA transcript open reading frames and production/purification of human GLTP and related homologs using bacterial expression systems. The successes enabled application of X-ray crystallographic approaches that led to molecular structure determination of GLTP and related homologs in glycolipid-free form and complexed with different glycolipids in collaboration with structural biologists in the D.J. Patel lab at Memorial Sloan Kettering Cancer Center in New York and in the L. Malinina lab at CIC bioGUNE in Derio/Bilbao, Spain. Our work showed that human GLTP forms a novel structural fold among known proteins. The Worldwide Protein Data Bank has designated human GLTP as the founding member and prototype of the GLTP superfamily, enabling our research findings to be published in Nature, PLoS Biology, Structure, The Journal of Biological Chemistry, Biophysical Journal, Biochemistry, and Journal of Lipid Research. The studies have shed light on the: i) structural adaptation used by GLTP to accommodate different glycolipids within its binding site; ii) functional role played by intrinsic tryptophan residues in glycolipid binding and membrane interaction; and iii) structural basis for the more focused glycolipid selectivity of a fungal GLTP ortholog as well as the GLTPH domain of human FAPP2.

In studies of the model plant, Arabidopsis thaliana, carried out in collaboration with Dr. 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 showed 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 recently was published in Cell Reports. In other recent investigations, we reported the discovery of a new GLTP structural homolog in human cells that we named ceramide-1- phosphate transfer protein (CPTP). Remarkably, the lipid specificity of CPTP has evolved for binding/transfer of ceramide-1-phosphate rather than glycolipids even though CPTP still forms a GLTP-fold encoded by a completely different gene than GLTP. In collaboration with Dr. Ted Hinchcliffe at The Hormel Institute, University of Minnesota, we have tracked the location of CPTP in mammalian cells using state-of-the-art fluorescence microscopy approaches.

The Hormel Institute - Rhoderick Brown Lab

(Left to right) Liudmila (Lucy) Malinina, Xiuhong Zhai, Rick Brown, Shrawan Kimar Mishra, Helen Pike, Tommy Dvergsten

In collaboration with the Charles Chalfant lab at Virginia Commonwealth University, we showed that depletion of CPTP levels in human cells by RNA interference leads to over-accumulaion 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. CPTP previously was unknown and unstudied prior to our investigations. The recent successful CPTP research efforts, published in Nature, have stimulated a new research project on sepsis with our collaborators at Virginia Commonwealth University; Memorial Sloan Kettering Cancer Center in New York; and The Hormel Institute, University of Minnesota focused. Investigations now are underway to decipher the molecular means to control and reverse inflammation, which is critically important for successful resolution and recovery from sepsis.

We anticipate that elucidation of the fundamental structure-function relationships governing GTLP and CPTP action will facilitate the development of pharmacological ways to modulate GLTP and CPTP while enhancing 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.

“The discovery of these new functions for membrane lipids
under-scores the reasons why biomembranes come under
direct attack during cancer and infectious disease.”
Dr. Rhoderick E. Brown

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; Virginia Commonwealth University in Richmond; The Russian Academy of Sciences in Moscow; The University of Copenhagen in Denmark; CIC bioGUNE in Derio/Bilbao, Spain; and the Mayo Clinic in Rochester, MN. Our research continues because of financial support received from the National Institute of General Medical Sciences; the National Cancer Institute of NIH; the National Heart, Lung, and Blood 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:
My NCBI Collections (REB):
http://www.ncbi.nlm.nih.gov/myncbi/browse/
collection/42052948/?sort=date&direction=descending

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-a17c-
.202e43bc0884%29/publications.html