Ravikanth Kamlekar | VIT - Academia.edu (original) (raw)

Papers by Ravikanth Kamlekar

Biophysical Journal, 2014

Nature, 2013

Phosphorylated sphingolipids ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) have em... more Phosphorylated sphingolipids ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) have emerged as key regulators of cell growth, survival, migration and inflammation. C1P produced by ceramide kinase is an activator of group IVA cytosolic phospholipase A2α (cPLA2α), the rate-limiting releaser of arachidonic acid used for pro-inflammatory eicosanoid production, which contributes to disease pathogenesis in asthma or airway hyper-responsiveness, cancer, atherosclerosis and thrombosis. To modulate eicosanoid action and avoid the damaging effects of chronic inflammation, cells require efficient targeting, trafficking and presentation of C1P to specific cellular sites. Vesicular trafficking is likely but non-vesicular mechanisms for C1P sensing, transfer and presentation remain unexplored. Moreover, the molecular basis for selective recognition and binding among signalling lipids with phosphate headgroups, namely C1P, phosphatidic acid or their lyso-derivatives, remains unclear. Here, a ubiquitously expressed lipid transfer protein, human GLTPD1, named here CPTP, is shown to specifically transfer C1P between membranes. Crystal structures establish C1P binding through a novel surface-localized, phosphate headgroup recognition centre connected to an interior hydrophobic pocket that adaptively expands to ensheath differing-length lipid chains using a cleft-like gating mechanism. The two-layer, α-helically-dominated 'sandwich' topology identifies CPTP as the prototype for a new glycolipid transfer protein fold subfamily. CPTP resides in the cell cytosol but associates with the trans-Golgi network, nucleus and plasma membrane. RNA interference-induced CPTP depletion elevates C1P steady-state levels and alters Golgi cisternae stack morphology. The resulting C1P decrease in plasma membranes and increase in the Golgi complex stimulates cPLA2α release of arachidonic acid, triggering pro-inflammatory eicosanoid generation.

Biophysical Journal, 2009

The orientation of membrane-associated alpha-helical peptides was investigated using novel method... more The orientation of membrane-associated alpha-helical peptides was investigated using novel methodologies of oriented Synchrotron Radiation Circular Dichroism (SRCD) and linear dichroism (SRLD) spectroscopies. Because of its enhanced signal-to-noise and a detector geometry that minimised optical artefacts associated with conventional CD studies of membrane suspensions, SRCD enabled the measurement of oriented CD spectra. To accomplish this a specially-designed sample cell holder was produced which would maintain constant humidity in hydrated film samples. Distinct spectra were obtained for peptides oriented parallel or normal to the direction of the beam, corresponding to the parallel and perpendicular pi to pi* and n to pi* electronic transitions.

Biophysical Journal, 2009

Biophysical Journal, 2009

Title: Folding/Unfolding of Glycolipid Transfer Protein: Molten Globule-Like Intermediates? Autho... more Title: Folding/Unfolding of Glycolipid Transfer Protein: Molten Globule-Like Intermediates? Authors:Kamlekar, Ravi Kanth; Kenoth, Roopa; Pike, Helen M.; Prendergast, Franklyn G.; Venyaminov, Sergei Yu; Brown, Rhoderick E. Publication: Biophysical Journal, vol. ...

Biochemistry, 2011

The glycolipid transfer protein (GLTP) superfamily is defined by the human GLTP fold that represe... more The glycolipid transfer protein (GLTP) superfamily is defined by the human GLTP fold that represents a novel motif for lipid binding and transfer and for reversible interaction with membranes, i.e., peripheral amphitropic proteins. Despite limited sequence homology with human GLTP, we recently showed that HET-C2 GLTP of Podospora anserina is organized conformationally as a GLTP fold. Currently, insights into the folding stability and conformational states that regulate GLTP fold activity are almost nonexistent. To gain such insights into the disulfide-less GLTP fold, we investigated the effect of a change in pH on the fungal HET-C2 GLTP fold by taking advantage of its two tryptophans and four tyrosines (compared to three tryptophans and 10 tyrosines in human GLTP). pH-induced conformational alterations were determined by changes in (i) intrinsic tryptophan fluorescence (intensity, emission wavelength maximum, and anisotropy), (ii) circular dichroism over the near-UV and far-UV ranges, including thermal stability profiles of the derivatized molar ellipticity at 222 nm, (iii) fluorescence properties of 1-anilinonaphthalene-8-sulfonic acid, and (iv) glycolipid intermembrane transfer activity monitored by Förster resonance energy transfer. Analyses of our recently determined crystallographic structure of HET-C2 (1.9 Å) allowed identification of side chain electrostatic interactions that contribute to HET-C2 GLTP fold stability and can be altered by a change in pH. Side chain interactions include numerous salt bridges and interchain cation-π interactions, but not intramolecular disulfide bridges. Histidine residues are especially important for stabilizing the local positioning of the two tryptophan residues and the conformation of adjacent chains. Induction of a low-pH-induced, molten globule-like state inhibited glycolipid intermembrane transfer by the HET-C2 GLTP fold.

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1997

N-acylethanolamines (NAEs) have attracted the attention of researchers in the last two decades du... more N-acylethanolamines (NAEs) have attracted the attention of researchers in the last two decades due to their occurrence in biological membranes under conditions of stress as well as under normal conditions. Differential scanning calorimetric studies have been carried out on dry and hydrated samples of a homologous series of N-acylethanolamines containing saturated acyl chains of even number of carbon atoms (n=8–20).

Glycolipid transfer proteins (GLTPs) originally were identified as small (~24 kDa), soluble, amph... more Glycolipid transfer proteins (GLTPs) originally were identified as small (~24 kDa), soluble, amphitropic proteins that specifically accelerate the intermembrane transfer of glycolipids. GLTPs and related homologs now are known to adopt a unique, helically dominated, two-layer ‘sandwich’ architecture defined as the GLTP-fold that provides the structural underpinning for the eukaryotic GLTP superfamily. Recent advances now provide exquisite insights into structural features responsible for lipid headgroup selectivity as well as the adaptability of the hydrophobic compartment for accommodating hydrocarbon chains of differing length and unsaturation. A new understanding of the structural versatility and evolutionary premium placed on the GLTP motif has emerged. Human GLTP-motifs have evolved to function not only as glucosylceramide binding/transferring domains for phosphoinositol 4-phosphate adaptor protein-2 during glycosphingolipid biosynthesis but also as selective binding/transfer proteins for ceramide-1-phosphate. The latter, known as ceramide-1-phosphate transfer protein, recently has been shown to form GLTP-fold while critically regulating Group-IV cytoplasmic phospholipase A2 activity and pro-inflammatory eicosanoid production.

Nature, Aug 22, 2013

"Phosphorylated sphingolipids ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) have ... more "Phosphorylated sphingolipids ceramide-1-phosphate (C1P) and
sphingosine-1-phosphate (S1P) have emerged as key regulators of
cell growth, survival, migration and inflammation1–5. C1P produced
by ceramide kinase is an activator of group IVA cytosolic phospholipase
A2a (cPLA2a), the rate-limiting releaser of arachidonic acid
used for pro-inflammatory eicosanoid production3,6–9, which contributes
todisease pathogenesis in asthmaor airwayhyper-responsiveness,
cancer, atherosclerosis and thrombosis.Tomodulate eicosanoid action
and avoid the damaging effects of chronic inflammation, cells require
efficient targeting, trafficking and presentation of C1P to specific
cellular sites.Vesicular trafficking is likely10but non-vesicularmechanisms
forC1Psensing, transfer andpresentation remain unexplored11,12.
Moreover, the molecular basis for selective recognition and binding
among signalling lipids with phosphate headgroups, namely C1P,
phosphatidic acid or their lyso-derivatives, remains unclear. Here,
a ubiquitously expressed lipid transfer protein, human GLTPD1,
named here CPTP, is shown to specifically transfer C1P between
membranes. Crystal structures establish C1P binding through a novel
surface-localized, phosphate headgroup recognition centre connected
to an interior hydrophobic pocket that adaptively expands to ensheath
differing-length lipid chains using a cleft-like gating mechanism.
The two-layer, a-helically-dominated ‘sandwich’ topology identifies
CPTP as the prototype for a new glycolipid transfer protein fold13
subfamily.CPTPresides in the cell cytosol but associates with the trans-
Golgi network, nucleus and plasma membrane. RNA interferenceinduced
CPTP depletion elevates C1P steady-state levels and alters
Golgi cisternae stack morphology. The resulting C1P decrease in
plasma membranes and increase in the Golgi complex stimulates
cPLA2a release of arachidonic acid, triggering pro-inflammatory
eicosanoid generation."

J. Phys. Chem B, Jun 24, 2013

N-Acyldopamines (NADAs), which are present in mammalian nervous tissues, exhibit interesting bio... more N-Acyldopamines (NADAs), which are present in mammalian
nervous tissues, exhibit interesting biological and pharmacological properties.
In the present study, a homologous series of NADAs with varying acyl chains
(n = 12−20) have been synthesized and characterized. Differential scanning
calorimetric studies show that in the dry state the transition temperatures,
enthalpies, and entropies of NADAs exhibit odd−even alternation with the
values corresponding to the even chain length series being slightly higher.
Both even and odd chain length NADAs display a linear dependence of the
transition enthalpies and entropies on the chain length. However, odd−even
alternation was not observed in the calorimetric properties upon hydration,
although the transition enthalpies and entropies exhibit linear dependence.
Linear least-squares analyses yielded incremental values contributed by each
methylene group to the transition enthalpy and entropy and the
corresponding end contributions. N-Lauroyldopamine (NLDA) crystallized in the monoclinic space group C2/c with eight
symmetry-related molecules in the unit cell. Single-crystal X-ray diffraction studies show that NLDA molecules are organized in
the bilayer form, with a head-to-head (and tail-to-tail) arrangement of the molecules. Water-mediated hydrogen bonds between
the hydroxyl groups of the dopamine moieties of opposing layers and N−H···O hydrogen bonds between the amide groups of
adjacent molecules in the same layer stabilize the crystal packing. These results provide a thermodynamic and structural basis for
investigating the interaction of NADAs with other membrane lipids, which are expected to provide clues to understand how they
function in vivo, e.g., as signaling molecules in the modulation of pain.

Phosphoinositol 4-phosphate adaptor protein-2 (FAPP2) plays a key role in glycosphingolipid (GSL)... more Phosphoinositol 4-phosphate adaptor protein-2 (FAPP2) plays a key role in glycosphingolipid (GSL) production using its C-terminal domain to transport newly synthesized glucosylceramide away from the cytosol-facing glucosylceramide synthase in the cis-Golgi for further anabolic processing. Structural homology modeling against human glycolipid transfer protein (GLTP) predicts a GLTP-fold for FAPP2 C-terminal domain, but no experimental support exists to warrant inclusion in the GLTP superfamily. Here, the biophysical properties and glycolipid transfer specificity of FAPP2-C-terminal domain have been characterized and compared with other established GLTP-folds. Experimental evidence for a GLTP-fold includes: i) far-UV circular dichroism (CD) showing secondary structure with high alpha-helix content and a low thermally-induced unfolding transition (~ 41 °C); ii) near-UV-CD indicating only subtle tertiary conformational change before/after interaction with membranes containing/lacking glycolipid; iii) Red-shifted tryptophan (Trp) emission wavelength maximum (λmax ~ 352 nm) for apo-FAPP2-C-terminal domain consistent with surface exposed intrinsic Trp residues; iv) ‘signature’ GLTP-fold Trp fluorescence response, i.e., intensity decrease (~ 30%) accompanied by strongly blue-shifted λmax (~ 14 nm) upon interaction with membranes containing glycolipid, supporting direct involvement of Trp in glycolipid binding and enabling estimation of partitioning affinities. A structurally-based preference for other simple uncharged GSLs, in addition to glucosylceramide, makes human FAPP2-GLTP more similar to fungal HET-C2 than to plant AtGLTP1 (glucosylceramide-specific) or to broadly GSL-selective human GLTP. These findings along with the distinct mRNA exon/intron organizations originating from single-copy genes on separate human chromosomes suggest adaptive evolutionary divergence by these two GLTP-folds.► FAPP2 enables glycosphingolipid synthesis in the Golgi by transfer of glucosylceramide. ► FAPP2 contains a modified GLTP-fold that can also transfer other neutral glycosphingolipids. ► Glycolipid selectivity of FAPP2-GLTP is more focused than human GLTP. ► Glycolipid compartmentation appears to mute evolutionary selection pressure and divergence.

The miscibility and phase behavior of hydrated binary mixtures of two N-acylethanolamines (NAEs),... more The miscibility and phase behavior of hydrated binary mixtures of two N-acylethanolamines (NAEs), N-myristoylethanolamine (NMEA), and N-palmitoylethanolamine (NPEA), with the corresponding diacyl phosphatidylethanolamines (PEs), dimyristoylphosphatidylethanolamine (DMPE), and dipalmitoylphosphatidylethanolamine (DPPE), respectively, have been investigated by differential scanning calorimetry (DSC), spin-label electron spin resonance (ESR), and 31P-NMR spectroscopy. Temperature-composition phase diagrams for both NMEA/DMPE and NPEA/DPPE binary systems were established from high sensitivity DSC. The structures of the phases involved were determined by 31P-NMR spectroscopy. For both systems, complete miscibility in the fluid and gel phases is indicated by DSC and ESR, up to 35 mol % of NMEA in DMPE and 40 mol % of NPEA in DPPE. At higher contents of the NAEs, extensive solid-fluid phase separation and solid-solid immiscibility occur depending on the temperature. Characterization of the structures of the mixtures formed with 31P-NMR spectroscopy shows that up to 75 mol % of NAE, both DMPE and DPPE form lamellar structures in the gel phase as well as up to at least 65°C in the fluid phase. ESR spectra of phosphatidylcholine spin labeled at the C-5 position in the sn-2 acyl chain present at a probe concentration of 1 mol % exhibit strong spin-spin broadening in the low-temperature region for both systems, suggesting that the acyl chains pack very tightly and exclude the spin label. However, spectra recorded in the fluid phase do not exhibit any spin-spin broadening and indicate complete miscibility of the two components. The miscibility of NAE and diacyl PE of matched chainlengths is significantly less than that found earlier for NPEA and dipalmitoylphosphatidylcholine, an observation that is consistent with the notion that the NAEs are most likely stored as their precursor lipids (N-acyl PEs) and are generated only when the system is subjected to membrane stress.

Biophysical Journal, 2014

Nature, 2013

Phosphorylated sphingolipids ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) have em... more Phosphorylated sphingolipids ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) have emerged as key regulators of cell growth, survival, migration and inflammation. C1P produced by ceramide kinase is an activator of group IVA cytosolic phospholipase A2α (cPLA2α), the rate-limiting releaser of arachidonic acid used for pro-inflammatory eicosanoid production, which contributes to disease pathogenesis in asthma or airway hyper-responsiveness, cancer, atherosclerosis and thrombosis. To modulate eicosanoid action and avoid the damaging effects of chronic inflammation, cells require efficient targeting, trafficking and presentation of C1P to specific cellular sites. Vesicular trafficking is likely but non-vesicular mechanisms for C1P sensing, transfer and presentation remain unexplored. Moreover, the molecular basis for selective recognition and binding among signalling lipids with phosphate headgroups, namely C1P, phosphatidic acid or their lyso-derivatives, remains unclear. Here, a ubiquitously expressed lipid transfer protein, human GLTPD1, named here CPTP, is shown to specifically transfer C1P between membranes. Crystal structures establish C1P binding through a novel surface-localized, phosphate headgroup recognition centre connected to an interior hydrophobic pocket that adaptively expands to ensheath differing-length lipid chains using a cleft-like gating mechanism. The two-layer, α-helically-dominated 'sandwich' topology identifies CPTP as the prototype for a new glycolipid transfer protein fold subfamily. CPTP resides in the cell cytosol but associates with the trans-Golgi network, nucleus and plasma membrane. RNA interference-induced CPTP depletion elevates C1P steady-state levels and alters Golgi cisternae stack morphology. The resulting C1P decrease in plasma membranes and increase in the Golgi complex stimulates cPLA2α release of arachidonic acid, triggering pro-inflammatory eicosanoid generation.

Biophysical Journal, 2009

The orientation of membrane-associated alpha-helical peptides was investigated using novel method... more The orientation of membrane-associated alpha-helical peptides was investigated using novel methodologies of oriented Synchrotron Radiation Circular Dichroism (SRCD) and linear dichroism (SRLD) spectroscopies. Because of its enhanced signal-to-noise and a detector geometry that minimised optical artefacts associated with conventional CD studies of membrane suspensions, SRCD enabled the measurement of oriented CD spectra. To accomplish this a specially-designed sample cell holder was produced which would maintain constant humidity in hydrated film samples. Distinct spectra were obtained for peptides oriented parallel or normal to the direction of the beam, corresponding to the parallel and perpendicular pi to pi* and n to pi* electronic transitions.

Biophysical Journal, 2009

Biophysical Journal, 2009

Title: Folding/Unfolding of Glycolipid Transfer Protein: Molten Globule-Like Intermediates? Autho... more Title: Folding/Unfolding of Glycolipid Transfer Protein: Molten Globule-Like Intermediates? Authors:Kamlekar, Ravi Kanth; Kenoth, Roopa; Pike, Helen M.; Prendergast, Franklyn G.; Venyaminov, Sergei Yu; Brown, Rhoderick E. Publication: Biophysical Journal, vol. ...

Biochemistry, 2011

The glycolipid transfer protein (GLTP) superfamily is defined by the human GLTP fold that represe... more The glycolipid transfer protein (GLTP) superfamily is defined by the human GLTP fold that represents a novel motif for lipid binding and transfer and for reversible interaction with membranes, i.e., peripheral amphitropic proteins. Despite limited sequence homology with human GLTP, we recently showed that HET-C2 GLTP of Podospora anserina is organized conformationally as a GLTP fold. Currently, insights into the folding stability and conformational states that regulate GLTP fold activity are almost nonexistent. To gain such insights into the disulfide-less GLTP fold, we investigated the effect of a change in pH on the fungal HET-C2 GLTP fold by taking advantage of its two tryptophans and four tyrosines (compared to three tryptophans and 10 tyrosines in human GLTP). pH-induced conformational alterations were determined by changes in (i) intrinsic tryptophan fluorescence (intensity, emission wavelength maximum, and anisotropy), (ii) circular dichroism over the near-UV and far-UV ranges, including thermal stability profiles of the derivatized molar ellipticity at 222 nm, (iii) fluorescence properties of 1-anilinonaphthalene-8-sulfonic acid, and (iv) glycolipid intermembrane transfer activity monitored by Förster resonance energy transfer. Analyses of our recently determined crystallographic structure of HET-C2 (1.9 Å) allowed identification of side chain electrostatic interactions that contribute to HET-C2 GLTP fold stability and can be altered by a change in pH. Side chain interactions include numerous salt bridges and interchain cation-π interactions, but not intramolecular disulfide bridges. Histidine residues are especially important for stabilizing the local positioning of the two tryptophan residues and the conformation of adjacent chains. Induction of a low-pH-induced, molten globule-like state inhibited glycolipid intermembrane transfer by the HET-C2 GLTP fold.

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1997

N-acylethanolamines (NAEs) have attracted the attention of researchers in the last two decades du... more N-acylethanolamines (NAEs) have attracted the attention of researchers in the last two decades due to their occurrence in biological membranes under conditions of stress as well as under normal conditions. Differential scanning calorimetric studies have been carried out on dry and hydrated samples of a homologous series of N-acylethanolamines containing saturated acyl chains of even number of carbon atoms (n=8–20).

Glycolipid transfer proteins (GLTPs) originally were identified as small (~24 kDa), soluble, amph... more Glycolipid transfer proteins (GLTPs) originally were identified as small (~24 kDa), soluble, amphitropic proteins that specifically accelerate the intermembrane transfer of glycolipids. GLTPs and related homologs now are known to adopt a unique, helically dominated, two-layer ‘sandwich’ architecture defined as the GLTP-fold that provides the structural underpinning for the eukaryotic GLTP superfamily. Recent advances now provide exquisite insights into structural features responsible for lipid headgroup selectivity as well as the adaptability of the hydrophobic compartment for accommodating hydrocarbon chains of differing length and unsaturation. A new understanding of the structural versatility and evolutionary premium placed on the GLTP motif has emerged. Human GLTP-motifs have evolved to function not only as glucosylceramide binding/transferring domains for phosphoinositol 4-phosphate adaptor protein-2 during glycosphingolipid biosynthesis but also as selective binding/transfer proteins for ceramide-1-phosphate. The latter, known as ceramide-1-phosphate transfer protein, recently has been shown to form GLTP-fold while critically regulating Group-IV cytoplasmic phospholipase A2 activity and pro-inflammatory eicosanoid production.

Nature, Aug 22, 2013

"Phosphorylated sphingolipids ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) have ... more "Phosphorylated sphingolipids ceramide-1-phosphate (C1P) and
sphingosine-1-phosphate (S1P) have emerged as key regulators of
cell growth, survival, migration and inflammation1–5. C1P produced
by ceramide kinase is an activator of group IVA cytosolic phospholipase
A2a (cPLA2a), the rate-limiting releaser of arachidonic acid
used for pro-inflammatory eicosanoid production3,6–9, which contributes
todisease pathogenesis in asthmaor airwayhyper-responsiveness,
cancer, atherosclerosis and thrombosis.Tomodulate eicosanoid action
and avoid the damaging effects of chronic inflammation, cells require
efficient targeting, trafficking and presentation of C1P to specific
cellular sites.Vesicular trafficking is likely10but non-vesicularmechanisms
forC1Psensing, transfer andpresentation remain unexplored11,12.
Moreover, the molecular basis for selective recognition and binding
among signalling lipids with phosphate headgroups, namely C1P,
phosphatidic acid or their lyso-derivatives, remains unclear. Here,
a ubiquitously expressed lipid transfer protein, human GLTPD1,
named here CPTP, is shown to specifically transfer C1P between
membranes. Crystal structures establish C1P binding through a novel
surface-localized, phosphate headgroup recognition centre connected
to an interior hydrophobic pocket that adaptively expands to ensheath
differing-length lipid chains using a cleft-like gating mechanism.
The two-layer, a-helically-dominated ‘sandwich’ topology identifies
CPTP as the prototype for a new glycolipid transfer protein fold13
subfamily.CPTPresides in the cell cytosol but associates with the trans-
Golgi network, nucleus and plasma membrane. RNA interferenceinduced
CPTP depletion elevates C1P steady-state levels and alters
Golgi cisternae stack morphology. The resulting C1P decrease in
plasma membranes and increase in the Golgi complex stimulates
cPLA2a release of arachidonic acid, triggering pro-inflammatory
eicosanoid generation."

J. Phys. Chem B, Jun 24, 2013

N-Acyldopamines (NADAs), which are present in mammalian nervous tissues, exhibit interesting bio... more N-Acyldopamines (NADAs), which are present in mammalian
nervous tissues, exhibit interesting biological and pharmacological properties.
In the present study, a homologous series of NADAs with varying acyl chains
(n = 12−20) have been synthesized and characterized. Differential scanning
calorimetric studies show that in the dry state the transition temperatures,
enthalpies, and entropies of NADAs exhibit odd−even alternation with the
values corresponding to the even chain length series being slightly higher.
Both even and odd chain length NADAs display a linear dependence of the
transition enthalpies and entropies on the chain length. However, odd−even
alternation was not observed in the calorimetric properties upon hydration,
although the transition enthalpies and entropies exhibit linear dependence.
Linear least-squares analyses yielded incremental values contributed by each
methylene group to the transition enthalpy and entropy and the
corresponding end contributions. N-Lauroyldopamine (NLDA) crystallized in the monoclinic space group C2/c with eight
symmetry-related molecules in the unit cell. Single-crystal X-ray diffraction studies show that NLDA molecules are organized in
the bilayer form, with a head-to-head (and tail-to-tail) arrangement of the molecules. Water-mediated hydrogen bonds between
the hydroxyl groups of the dopamine moieties of opposing layers and N−H···O hydrogen bonds between the amide groups of
adjacent molecules in the same layer stabilize the crystal packing. These results provide a thermodynamic and structural basis for
investigating the interaction of NADAs with other membrane lipids, which are expected to provide clues to understand how they
function in vivo, e.g., as signaling molecules in the modulation of pain.

Phosphoinositol 4-phosphate adaptor protein-2 (FAPP2) plays a key role in glycosphingolipid (GSL)... more Phosphoinositol 4-phosphate adaptor protein-2 (FAPP2) plays a key role in glycosphingolipid (GSL) production using its C-terminal domain to transport newly synthesized glucosylceramide away from the cytosol-facing glucosylceramide synthase in the cis-Golgi for further anabolic processing. Structural homology modeling against human glycolipid transfer protein (GLTP) predicts a GLTP-fold for FAPP2 C-terminal domain, but no experimental support exists to warrant inclusion in the GLTP superfamily. Here, the biophysical properties and glycolipid transfer specificity of FAPP2-C-terminal domain have been characterized and compared with other established GLTP-folds. Experimental evidence for a GLTP-fold includes: i) far-UV circular dichroism (CD) showing secondary structure with high alpha-helix content and a low thermally-induced unfolding transition (~ 41 °C); ii) near-UV-CD indicating only subtle tertiary conformational change before/after interaction with membranes containing/lacking glycolipid; iii) Red-shifted tryptophan (Trp) emission wavelength maximum (λmax ~ 352 nm) for apo-FAPP2-C-terminal domain consistent with surface exposed intrinsic Trp residues; iv) ‘signature’ GLTP-fold Trp fluorescence response, i.e., intensity decrease (~ 30%) accompanied by strongly blue-shifted λmax (~ 14 nm) upon interaction with membranes containing glycolipid, supporting direct involvement of Trp in glycolipid binding and enabling estimation of partitioning affinities. A structurally-based preference for other simple uncharged GSLs, in addition to glucosylceramide, makes human FAPP2-GLTP more similar to fungal HET-C2 than to plant AtGLTP1 (glucosylceramide-specific) or to broadly GSL-selective human GLTP. These findings along with the distinct mRNA exon/intron organizations originating from single-copy genes on separate human chromosomes suggest adaptive evolutionary divergence by these two GLTP-folds.► FAPP2 enables glycosphingolipid synthesis in the Golgi by transfer of glucosylceramide. ► FAPP2 contains a modified GLTP-fold that can also transfer other neutral glycosphingolipids. ► Glycolipid selectivity of FAPP2-GLTP is more focused than human GLTP. ► Glycolipid compartmentation appears to mute evolutionary selection pressure and divergence.

The miscibility and phase behavior of hydrated binary mixtures of two N-acylethanolamines (NAEs),... more The miscibility and phase behavior of hydrated binary mixtures of two N-acylethanolamines (NAEs), N-myristoylethanolamine (NMEA), and N-palmitoylethanolamine (NPEA), with the corresponding diacyl phosphatidylethanolamines (PEs), dimyristoylphosphatidylethanolamine (DMPE), and dipalmitoylphosphatidylethanolamine (DPPE), respectively, have been investigated by differential scanning calorimetry (DSC), spin-label electron spin resonance (ESR), and 31P-NMR spectroscopy. Temperature-composition phase diagrams for both NMEA/DMPE and NPEA/DPPE binary systems were established from high sensitivity DSC. The structures of the phases involved were determined by 31P-NMR spectroscopy. For both systems, complete miscibility in the fluid and gel phases is indicated by DSC and ESR, up to 35 mol % of NMEA in DMPE and 40 mol % of NPEA in DPPE. At higher contents of the NAEs, extensive solid-fluid phase separation and solid-solid immiscibility occur depending on the temperature. Characterization of the structures of the mixtures formed with 31P-NMR spectroscopy shows that up to 75 mol % of NAE, both DMPE and DPPE form lamellar structures in the gel phase as well as up to at least 65°C in the fluid phase. ESR spectra of phosphatidylcholine spin labeled at the C-5 position in the sn-2 acyl chain present at a probe concentration of 1 mol % exhibit strong spin-spin broadening in the low-temperature region for both systems, suggesting that the acyl chains pack very tightly and exclude the spin label. However, spectra recorded in the fluid phase do not exhibit any spin-spin broadening and indicate complete miscibility of the two components. The miscibility of NAE and diacyl PE of matched chainlengths is significantly less than that found earlier for NPEA and dipalmitoylphosphatidylcholine, an observation that is consistent with the notion that the NAEs are most likely stored as their precursor lipids (N-acyl PEs) and are generated only when the system is subjected to membrane stress.