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

The Hormel Instittute - Rhoderick Brown

RE Brown Lab – Current Scientific Research Interests

The following avenues of research are being actively pursued:

  • Regulation of Inflammation by Sphingolipid (C1P) and Phosphoglyceride (PIP2) Activators of Cytoplasmic Phospholipase A2α: Insights by Structure/Function AnalysesWe are currently investigating the molecular basis by which human cytoplasmic phospholipase A2 (cPLA2α) initially promotes inflammation but then subsequently helps reverse and resolve sepsis. Gaining new insights could lead to new avenues for treating this pathological process and inflammation associated with other pathologic conditions such as cancer, diabetes and dementia.  These recently initiated studies are being pursued in collaboration with Charles Chalfant (Univ. South Florida), Dinshaw Patel (Memorial Sloan Kettering Cancer Ctr.), and Edward (Ted) Hinchcliffe (Univ. Minnesota). They extend previous collaborative work involving ceramide-1-phosphate (C1P) transfer protein (CPTP). The previous work led to the model (right panel), that first appeared in our 2013 Nature paper, showing how CPTP depletion in cells can stimulate the action of cPLA2α to promote pro-inflammatory eicosanoid production.In recent studies, we have determined that CPTP functions an endogenous regulator of autophagy and of inflammasome assembly to help drive interleukin release (IL1B and IL18).  The report describing this work will soon be published in Autophagy.
    • Mishra SK, Gao Y-G, Deng Y, Chalfant CE, Hinchcliffe EH, Brown RE (2017) CPTP: A sphingolipid transfer protein that regulates autophagy and inflammasome activation, Autophagy (08.2017; in press)
  • Sphingolipid Trafficking by GLTP Superfamily Members: Differential Regulation and Targeting to Select Membranes via Specific Lipid Docking Sites
    In ongoing studies, we are identifying and characterizing surface sites that function as lipid interaction sites that activate and potentially guide the intracellular localization of various GLTP-fold family members.  An example is the work recently reported in The Journal of Biological Chemistry

    • Zhai X, Gao Y-G, Mishra SK, Simanshu DK, Boldyrev IA, Benson LM, Bergen III HR, Malinina L, Mundy J, Molotkovsky JG, Patel DJ, Brown RE (2017) Phosphatidylserine stimulates ceramide-1-phosphate (C1P) intermembrane transfer by C1P transfer proteins, J. Biol. Chem. 292, 2531–2541
  • GLTP-fold Structural Features That Regulate Sphingolipid Selectivity and Enable Formation of Sphingolipid Binding Compartments

    • Invited review for Annual Review of Biochemistry
      Malinina L, Patel DK, Brown RE (2017) How α-Helical Motifs Form Functionally Diverse Lipid-Binding          Compartments, Annu. Rev. Biochem. 86, 609-63

      Complimentary one-time downloading for your own personal use is possible using URL:
      http://www.annualreviews.org/eprint/veSkeNhIhwnmPHEcfdyS/full/10.1146/annurev-biochem-061516-044445
      (Any further/multiple distribution, publication, or commercial usage of this copyrighted material requires submission of a permission request addressed to the Annual Reviews Copyright Clearance Center.)

    •  Invited review in Quarterly Reviews of Biophysics
      Malinina L, Simanshu DK, Zhai X, Samygina VR, Kamlekar RK, Kenoth R, Ochoa-Lizarralde B, Malakhova ML, Molotkovsky JG, Patel DK, Brown RE (2015) Sphingolipid transfer proteins defined by the GLTP-fold. Quart. Rev. Biophys. 48, 281-322.
    • Structure/Function Analysis of FAPP2 Glycolipid Transfer Protein Homology Domain (GLTPH)
      Currently in final stages of preparation is a new paper describing the first crystal structure of the GLTPH domain of FAPP2.  The work, which reveals previously unknown regulatory elements that occur in the GLTP-fold, was pursued by collaboration with Dr. Lucy Malinina.

RE Brown Lab Scientific Research Highlights (w/ representative publications)

Complete listing of published works available at:
https://experts.umn.edu/en/persons/rhoderick-e-brown/publications/
Our research with sphingolipid transfer proteins was featured in the ‘Lipid News’ section of the March 2015 issue of ASBMB TODAY.

    1. Support: The research projects in the REB lab have been supported for ~25 years by NIGMS GM45228 and the Hormel Foundation with additional support provided by NHLBI-HL08214 and NCI-CA121493 and recently, by NHLBI-HL125353.
  1. Structure and Function of Glycolipid Transfer Proteins (GLTP). The molecular cloning and crystalliza­tion of human GLTP in our lab sparked major advances by enabling X-ray determination of GLTP molecular structure in collaboration with the DJ Patel lab (Sloan Kettering). The novel GLTP-fold (designated by the Protein Data Bank in Cambridge) is highly conserved among eukaryotes but sometimes displays localized conformational features that alter the glycolipid selectivity such as in a fungal GLTP ortholog. The structure/function insights have aided the successful development of ‘designer human GLTPs’ engineered by point mutations to achieve more focused glycolipid selectivity. The findings have also guided current ideas regarding subtle structural features in the GLTP-fold that control the glycolipid selectivity differences of human FAPP2 and GLTP.
    • Malinina L, Simanshu DK, Zhai X, Samygina VR, Kamlekar R-K, Kenoth R, Ochoa-Lizarralde B, Malakhova ML, Molotkovsky JG, Patel DJ*, Brown RE* (2015) Sphingolipid transfer proteins defined by the GLTP-fold.
      Quarterly Rev. Biophysics 48, 281-322 (*cspd. authors)
    • Kamlekar R-K, Simanshu DK, Gao Y-G, Kenoth R, Pike HM, Prendergast FG, Malinina L, Molotkovsky JG, Venyaminov SY, Patel DJ, Brown RE (2013) The glycolipid transfer protein (GLTP) domain of phosphoinositol 4‑phosphate adaptor protein-2 (FAPP2): Structure drives preference for simple neutral glycosphingolipids. Biochim. Biophys. Acta 1831, 417–427.
    • Malinina L, Malakhova M, Teplov A, Brown RE*, Patel DJ* (2004) Structural basis for glycosphingolipid transfer specificity, Nature 430, 1048-1053 (*cspd. authors).
    • Lin X, Mattjus P, Pike HM, Windebank AJ, Brown RE. (2000) Cloning and expression of glycolipid transfer protein from bovine and porcine brain, Biol. Chem. 275, 5104-5110.
    • Samygina VR, Popov AN, Cabo-Bilbao A, Ochoa-Lizarralde B, Goni-De-Derio F, Zhai X, Molotkovsky JG, Patel DJ, Brown RE*, Malinina L* (2011) Enhanced selectivity for sulfatide by engineered human glycolipid transfer protein, Structure 19: 1644-1654 (*cspd. authors)
    • Kamlekar R-K, Gao Y, Kenoth R, Pike HM, Molotkovsky JG, Prendergast FG, Malinina L, Patel DJ, Wessels W, Venyaminov, SY, Brown RE. (2010) Human GLTP: Three distinct functions for the three tryptophans in a novel peripheral amphitropic fold. Biophys. J. 90: 2626-2635.
    • Kenoth R, Simanshu DK, Kamlekar R-K, Pike HM, Molotkovsky JG, Benson LM, Bergen III HR, Prendergast FG, Malinina L, Venyaminov SY, Patel DJ, Brown RE (2010) Structural determination and tryptophan fluorescence of Heterokaryon Incompatibility C2 Protein (HET-C2), a fungal glycolipid transfer protein (GLTP), provide novel insights into glycolipid specificity and membrane interaction by the GLTP-fold. J. Biol. Chem. 285:13066–13078.
    • Zhai X,  Malakhova ML, Pike HM, Benson LM, Bergen III HR, Sugár IP, Malinina L, Patel DJ, Brown RE. (2009) Glycolipid acquisition by human glycolipid transfer protein dramatically alters intrinsic tryptophan fluorescence: insights into glycolipid binding affinity. J. Biol. Chem. 284:13620-13628.
    • Malinina L, Malakhova M, Kanack AT, Lu M, Abagyan R,  Brown RE*, Patel DJ* (2006) The liganding mode of glycolipid transfer protein is controlled by glycosphingolipid structure. PLoS Biol. 4: e362 (*cspd. authors)

  2. Structure and Function of Ceramide-1-Phosphate Transfer Proteins (CPTPs). This research provided the first insights into human ceramide-1-phosphate transfer protein (CPTP), which uses a GLTP-fold with an evolutionarily-modified lipid recognition center and is coded on chromosome 1 (locus 1p36.33). After verifying the existence of CPTP mRNA predicted by computer annotation of the human genome, we cloned CPTP, solved its molecular structure complexed with various species of C1P in collaboration with DJ Patel lab (Sloan Kettering), and discovered that CPTP can modulate C1P levels in the trans-Golgi and regulate cPLA2α activity that drives pro-inflammatory eicosanoid production in collaboration with the EH Hinchcliffe (UMN) and CE Chalfant (VCU) labs. In parallel, we studied the structure and function of Arabidopsis ACD11, a plant ortholog of human CPTP, and showed its essential role in regulating both C1P and ceramide levels in Arabidopsis in collabora­tion with the J Mundy (U Copenhagen) and DJ Patel labs. These findings provide the first evidence for the existence of a new CPTP protein family, with a modified GLTP-fold, within the GLTP superfamily.
    • Zhai X, Gao Y-G, Mishra SK, Simanshu DK, Boldyrev IA, Benson LM, Bergen III HR, Malinina L, Mundy J, Molotkovsky JG, Patel DJ, Brown RE* (2017) Phosphatidylserine stimulates ceramide-1-phosphate (C1P) intermembrane transfer by C1P transfer proteins, J. Biol. Chem. 292, 2531–2541 (Epub12/23/2016)
    • Malinina L, Simanshu DK, Zhai X, Samygina VR, Kamlekar R-K, Kenoth R, Ochoa-Lizarralde B, Malakhova ML, Molotkovsky JG, Patel DJ*, Brown RE* (2015) Sphingolipid transfer proteins defined by the GLTP-fold. Quarterly Rev. Biophysics 48, 281-322 (*cspd. authors)
    • Simanshu DK, Zhai X, Munch D, Hofius D, Markham JE, Bielawski J, Bielawska A, Malinina L, Molotkovsky JG, Mundy J*, Patel DS*, Brown RE*. (2014) Arabidopsis accelerated-cell-death11, ACD11, is a ceramide-1-phosphate transfer protein and intermediary regulator of phytoceramide levels. Cell Reports 6:388-399.
    • Simanshu DK, Kamlekar R-K, Wijesinghe DS Zou X, Zhai X, Mishra SK, Molotkovsky JG Malinina L, Hinchcliffe EH*, Chalfant CE*, Brown RE*, Patel DS*. (2013) Non-vesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids. Nature 500:463-467 (*cspd. authors).
    • Brodersen P, Petersen M, Pike HM, Olszak B, Skov S, Odum N, Jorgensen LB, Brown RE, Mundy J. (2002) Knockout of Arabidopsis ACCELERATED-CELL-DEATH11 encoding a sphingosine transfer protein causes activation of programmed cell death and defense, Genes Devel. 16, 490-502.
  3. Molecular and Cell Biological Aspects of GLTPs & CPTPs. GLTP gene organiza­tion, transcriptional status, phylogenetic, and evolutionary relationships has been studied in our lab. The single-copy GLTP gene on chromosome 12 (locus 12q24.11) is the source of transcribed human GLTP. Phylogenetic and evolutionary analyses show a 5-exon/4-intron gene organizational pattern that is highly conserved in therian mammals and other vertebrates. A second intronless GLTP gene on chromosome 11 (locus 11p15.1) is a nontranscribed pseudogene present only in primates, consistent with recent evolution­ary development.  We also have identified and characterized the constitutive and basal human GLTP gene promoters. Four GC-boxes were shown to be functional Sp1/Sp3 transcription factor binding sites. Mutation of one GC-box was particularly detrimental to GLTP transcriptional activity. Sp1/Sp3 RNA silencing and mithramycin-A treat­ment significantly affected GLTP promoter activity.  Among various sphingolipids, only ceramide induced GLTP promoter activity and partially blocked activity decreases induced by Sp1/Sp3 RNAi, thereby linking human GLTP expression to sphingolipid homeostasis through ceramide. GLTP over­expression in epithelial cells (HeLa and HEK-293) was found to induce cell rounding. Cell shape was unaffected by overexpression of W96A-GLTP, a ligand-site point mutant with abrogated glyco­lipid transfer activity. The round adherent cells exhibit diminished motility in wound healing assays and an inability to endocytose cholera toxin but remain viable and non-apoptotic. Interaction of GLTP with δ-catenin accelerates the transition to the rounded phenotype while δ‑catenin overexpression alone induces dendritic outgrowths. Regarding CPTP, our studies showed that CPTP depletion, but not GLTP depletion, induces pro-inflammatory eicosanoid generation. Currently underway are studies of CPTP involvement in triggering autophagy by upregulation of autophagosome formation that can drive interleukin release.
    • Simanshu DK, Kamlekar R-K, Wijesinghe DS Zou X, Zhai X, Mishra SK, Molotkovsky JG Malinina L, Hinchcliffe EH*, Chalfant CE*, Brown RE*, Patel DS*. (2013) Non-vesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids. Nature 500:463-467 (*cspd. authors).
    • Gao Y, Chung T, Zou X, Pike HM, Brown RE (2011) Human glycolipid transfer protein (GLTP) modulates cell shape. PLoS ONE 6:e19990.
    • Zou X, Gao Y, Ruvolo VR, Gardner, TL, Ruvolo PP, Brown RE (2011) Human glycolipid transfer protein gene (GLTP) expression is regulated by Sp1 and Sp3: Involvement of the bioactive sphingolipid J. Biol. Chem. 286:1301-1311(Epub-Nov.2010).
    • Zou X, Chung T, Lin X, Malakhova ML, Pike HM, Brown RE (2008) Human glycolipid transfer protein (GLTP) genes: organization, transcriptional status and evolution. BMC Genomics 9:72

  4. Development of new BODIPY-lipid probes for membrane research. In collaboration with JG Molotkovsky and Ivan Boldyrev (Russian Acad. Sciences), we characterized a lipid fluorophores with BODIPY omega-linked to a fatty acid of phospho­glycer­ides or sphingolipids for useful­ness in membrane studies. We showed how these BODIPY probes (marketed as TopFluorTM by Avanti Polar Lipids) can provide nanoscale insights into lipid organiza­tion and mixing in monolayers. This work relied on a modified Langmuir film balance (designed by the HL Brockman lab, UMN) that acquires fluorescence spectra, i.e. >200 emission spectra of BODIPY-labeled-PC, ‑SM, or -GalCer during a single monolayer compression scan. The concen­tra­tion-dependent emission changes of BODIPY-lipids that occur simultaneously with acquisition of the surface pressure versus molecular area isotherms provide nano­scale insights into the lipid packing and lateral mixing.
    • Zhai X, Boldyrev IA, Mizuno NK, Momsen MM, Molotkovsky JG, Brockman HL*, Brown RE* (2014) Nanoscale packing differences in sphingomyelin and phosphatidylcholine revealed by BODIPY fluorescence in monolayers: physiological implications. Langmuir 30, 3154-3164 (*cspd. authors)
    • Zhai X, Momsen WE, Malakhov DA, Boldyrev IA, Momsen MM, Molotkovsky JG*, Brockman HL*, Brown RE* (2013) GLTP-fold interaction with planar phosphatidylcholine surfaces is synergistically stimulated by phosphatidic acid and phosphatidylethanol­amine, Lipid Res. 54, 1103-1113 (*cspd. authors).
    • Brown RE* & Brockman HL* (2007) Using monomolecular films to characterize lipid lateral interactions. Mol. Biology 398:41-58, (Lipid Rafts, McIntosh TJ, ed.) Humana Press, Totowa NJ (*cspd. authors).
    • Boldyrev IA, Zhai X, Momsen MM, Brockman HL, Brown RE*, Molotkovsky JG* (2007) New BODIPY lipid probes for fluorescence studies of membranes. Lipid Res. 48: 1518-1532 (*cspd. authors).
  5. Sphingolipid-Cholesterol Rafts: Physical characterization of their liquid-ordered (LO) packing state. We investigated the mixing behavior of glyco­sphingolipids and sphingomyelins with phosphatidylcholines and cholesterol to gain insights into ‘raft’ microdomain physical properties. We showed that SL aliphatic chain saturation and head­group chemistry both are key factors that regulate packing with other membrane lipids. The lack of commercial availability in the 1990s prompted us to synthesize chain-pure SLs. Using a high-precision, automated, Langmuir film balance [collaboration w/ HL Brockman lab (UMN)], we found that lipid lateral packing elasticity (surface compressional modulus; Cs-1) provides a quantifiable measure of the ordered but nonrigid environ­ment in SL-cholesterol mixtures, i.e. liquid-ordered phase, that characterizes lipid rafts. Cs-1 reflects a macroscopic pro­p­erty of the lipid phase state. To gain sights at the mole­cular level, we used the custom-modified Langmuir film balance that acquires >200 fluorescence spectra during a single monolayer compression scan. The concentra­tion-dependent emission changes of BODIPY-PC during monolayer compression revealed nano­scale differences in the lipid packing/phase states of sphingomyelins versus saturated-chain phophatidylcholines.

    • Brown RE* & Brockman HL* (2007) Using monomolecular films to characterize lipid lateral interactions. Methods Mol. Biology 398:41-58, (Lipid Rafts, McIntosh TJ, ed.) Humana Press, Totowa NJ.
    • Li X-M, Momsen MM, Brockman HL, Brown RE (2003) Sterol structure and sphingomyelin acyl chain length modulate lateral packing elasticity and detergent solubility in model membranes, J. 85, 3788-3801.
    • Li X-M, Momsen MM, Smaby JM, Brockman HL, Brown RE (2001) Cholesterol decreases the interfacial elasticity and detergent solubility of sphingomyelins, Biochemistry 40:5954-63.
    • Brown RE (1998) Sphingolipid organization in biomembranes: what physical studies of model membranes reveal. Cell Sci. 111, 1-9.
    • Smaby JM, Momsen MM, Brockman HL, Brown RE (1997) Phosphatidylcholine acyl unsaturation modulates the decrease in interfacial elasticity induced by cholesterol. J. 73:1492-1505.
  6. Regulation of sphingolipid transmembrane distribution by bilayer curvature and lipid composition.
    Using NMR approaches, we studied how membrane curvature and lipid composition of membrane bilayers regulate the transbilayer distribution of glycosphingolipids. This work showed that sphingomyelin lateral interactions with simple glycosphingolipids play a major role in regulating glycolipid transbilayer distributions and pool size in bilayer vesicle outer and inner leaflets.

    • Malewicz B, Valiyaveettil JT, Jacob K, Byun H-S, Mattjus P, Baumann WJ, Bittman R, Brown RE (2005) The 3‑OH group and 4,5-trans double bond of sphingomyelin are essential for modulation of galactosylceramide transmembrane asymmetry, J. 88, 2670-2680.
    • Mattjus P, Malewicz B, Valiyaveettil JT, Baumann WJ, Bittman R, Brown RE (2002) Sphingomyelin modulates the transbilayer distribution of galactosylceramide in phospholipid membranes, Biol. Chem. 277, 19476-481.
  7. Cryo-EM studies of lipid nanotubes and ribbons formed via chirally-regulated lateral interactions of sphingolipids. Glycosphingolipids (e.g. galactosyl­ceramide) synthetically modified to have various homogenously monounsaturated acyl chains were biophysically characterized. Using freeze-fracture electron microscopy and differential scanning calorimetry, we discovered that galactosylceramides acylated with nervonoyl chains (24:1) form bilayer helical ribbons that self-seal into bilayer nanotubes. The chiral interactions that generate the helical packing within the bilayer nanotubes were found to support and enhance 2D-helical crystallization of proteins in collaborative cryo-EM studies with the RA Milligan lab (Scripps Res. Inst.).

    • Kulkarni VS, Boggs JM, Brown RE (1999) Modulation of nanotube formation by structural modifications of sphingolipids. J. 77, 319-330.
    • Wilson-Kubalek EM, Brown RE, Celia H, Milligan RA. (1998) Lipid nanotubes as substrates for helical crystallization of macromolecules. Natl. Acad. Sci. USA 95, 8040-8045.
    • Kulkarni VS, Anderson WH, Brown RE (1995) Bilayer nanotubes and helical ribbons formed by hydrated galactosylceramides: acyl chain and headgroup effects. J. 69, 1976–1986.
    • Brown RE, Anderson WH, Kulkarni VS. (1995) Macro-Ripple phase formation in bilayers composed of galactosylceramide and phosphatidylcholine, J. 68, 1396-1405.
  8. Spontaneous Lipid Transfer Processes. Our interest in lipid intermembrane transfer began with spontaneous processes that can occur in the absence of proteins and extended post-doc studies initiated in the T.E. Thompson lab. This kind of lipid transfer involves lipid monomer through the aqueous phase from one membrane to another or is mediated by transient collisional contacts between membrane vesicles or hemi-fusion between membrane vesicles.

    • Brown RE, Hyland KJ (1992) Spontaneous transfer of ganglioside GM1 from its micelles to lipid vesicles of differing size. Biochemistry 31: 10602-10609.
    • Brown RE (1992) Spontaneous lipid transfer between organized lipid assemblies. Biophys. Acta 1113: 375-389.
    • Brown RE (1990) Spontaneous transfer of lipids between membranes. Subcellular Biochem. 16: 333-363
    • Brown RE, Thompson TE (1987) Spontaneous transfer of ganglioside GM1 between phospholipid vesicles. Biochemistry 26, 5454-5460.
    • Brown RE, Sugar IP, Thompson TE (1985) Spontaneous transfer of gangliotetraosylceramide between phospholipid vesicles. Biochemistry 24, 4082-4091.