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Autotaxin (ATX) is a secreted enzyme responsible for producing lysophosphatidic acid (LPA). The ATX/LPA signaling axis is typically activated in wound healing and tissue repair processes. The ATX/LPA axis is highjacked and upregulated in the progression and persistence of several chronic inflammatory diseases, including cancer. As ATX inhibitors are now progressing to clinical testing, innovative diagnostic tools such as positron emission tomography (PET) are needed to measure ATX expression in vivo accurately. The radiotracer, [18F]PRIMATX, was recently developed and tested for PET imaging of ATX in vivo in a murine melanoma model. The goal of the present work was to further validate [18F]PRIMATX as a PET imaging agent by analyzing its in vivo metabolic stability and suitability for PET imaging of ATX in models of human 8305C thyroid tumor and murine 4T1 breast cancer. [18F]PRIMATX displayed favorable metabolic stability in vivo (65% of intact radiotracer after 60 min p.i.) and provided sufficient tumor uptake profiles in both tumor models. Radiotracer uptake could be blocked by 8-12% in 8305C thyroid tumors in the presence of ATX inhibitor AE-32-NZ70 as determined by PET and ex vivo biodistribution analyses. [18F]PRIMATX also showed high brain uptake, which was reduced by 50% through the administration of ATX inhibitor AE-32-NZ70. [18F]PRIMATX is a suitable radiotracer for PET imaging of ATX in the brain and peripheral tumor tissues.

Emerging studies are reporting associations between skeletal muscle abnormalities and survival in cancer patients. Cancer prognosis is associated with depletion of essential fatty acids in erythrocytes and plasma in humans. However the relationship between skeletal muscle membrane fatty acid composition and survival is unknown. This study investigates the relationship between fatty acid content of phospholipids in skeletal muscle and survival in cancer patients. Rectus abdominis biopsies were collected during cancer surgery from 35 patients diagnosed with cancer. Thin-layer and gas chromatography were used for quantification of phospholipid fatty acids. Cutpoints for survival were defined using optimal stratification. Median survival was between 450 and 500 days when patients had arachidonic acid (AA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in muscle phospholipid below the cut-point compared to 720–800 days for patients above. Cox regression analysis revealed that low amounts of AA, EPA and DHA are risk factors for death. The risk of death remained significant for AA [HR 3.5 (1.11–10.87), p = 0.03], EPA [HR 3.92 (1.1–14.0), p = 0.04] and DHA [HR 4.08 (1.1–14.6), p = 0.03] when adjusted for sex. Lower amounts of essential fatty acids in skeletal muscle membrane is a predictor of survival in cancer patients. These results warrant investigation to restore bioactive fatty acids in people with cancer.

Human cytomegalovirus (HCMV) infects 40–70% of adults in developed countries. HCMV proteins and DNA are detected in tumors and metastases, suggesting an association with increased invasion. We investigated HCMV infection in human breast cancer cell lines compared to fibroblasts, a component of tumors, and the role of platelet-derived growth factor receptor-α (PDGFRα). HCMV productively infected HEL299 fibroblasts and, to a lesser extent, Hs578T breast cancer cells. Infection of another triple-negative cell line, MDA-MB-231, and also MCF-7 cells, was extremely low. These disparate infection rates correlated with expression of PDGFRA, which facilitates HCMV uptake. Increasing PDGFRA expression in T-47D breast cancer and BCPAP thyroid cancer cells markedly increased HCMV infection. Conversely, HCMV infection decreased PDGFRA expression, potentially attenuating signaling through this receptor. HCMV infection of fibroblasts promoted the secretion of proinflammatory factors, whereas an overall decreased secretion of inflammatory factors was observed in infected Hs578T cells. We conclude that HCMV infection in tumors will preferentially target tumor-associated fibroblasts and breast cancer cells expressing PDGFRα. HCMV infection in the tumor microenvironment, rather than cancer cells, will increase the inflammatory milieu that could enhance metastasis involving lysophosphatidate.

Targeted therapy is increasingly used to manage metastatic papillary thyroid cancer. The focus of the present study was to examine glucose metabolism and tumor responses for thyroid cancer xenografts expressing the glycolytic pathway modulators platelet-derived growth factor receptor (PDGFR) and BRAFV600E. Radiolabelled glucose derivative [18F]FDG was used to analyze the effects of PDGFR blockade with imatinib, BRAF blockade with vemurafenib, as well as combined PDGFR and BRAF blockade in vitro and in vivo with PET. Dynamic PET data was correlated with immunohistochemistry staining and kinetic analysis for facilitative glucose transporter 1 (GLUT1) and hexokinase-II (HK2). Vemurafenib decreased [18F]FDG uptake in BCPAP cells in vitro; however, it was increased by ~70% with imatinib application to BCPAP cells. This metabolic response to tyrosine kinase inhibition required BRAFV600E as it was not seen in cell lines lacking mutated BRAF (TPC1). In xenografts, imatinib therapy in BCPAP thyroid tumour-bearing mice significantly increased [18F]FDG uptake and retention (>30%) in BCPAP tumours with PDGFRβ or both (α+β) isoforms. Kinetic analysis revealed that the increased glucose uptake is a consequence of increased phosphorylation and intracellular trapping of [18F]FDG confirmed by an increase in HK2 protein expression and activity, but not GLUT1 activity. BRAF inhibition alone, or combined PDGFR and BRAF inhibition, reduced (~60%) [18F]FDG uptake in both types of BCPAP (β or α+β) tumours. In terms of tumour growth, combination therapy with imatinib and vemurafenib led to a near abolition of the tumors (~90% reduction), but single therapy for BCPAP with PDGFRα expression was much less effective. In summary, imatinib led to a paradoxical increase of [18F]FDG uptake in xenografts that was reversed through BRAFV600E inhibition. The present data show that metabolic reprogramming in thyroid cancer occurs as a consequence of BRAF-mediated upregulation of HK2 expression that may permit tumour growth with isolated blockade of upstream tyrosine kinase receptors.

Lysophosphatidate (LPA) signaling through 6 receptors is regulated by the balance of LPA production by autotaxin (ATX) vs. LPA degradation by lipid phosphate phosphatases (LPPs). LPA promotes an inflammatory cycle by increasing the synthesis of cyclooxygenase-2 and multiple inflammatory cytokines that stimulate further ATX production. We aimed to determine whether the anti-inflammatory glucocorticoid (GC) dexamethasone (Dex) functions partly by decreasing the ATX-LPA inflammatory cycle in adipose tissue, a major site of ATX secretion. Treatment of human adipose tissue with 10-1000 nM Dex decreased ATX secretion, increased LPP1 expression, and decreased mRNA expressions of IL-6, TNF-α, peroxisome proliferator-activated receptor (PPAR)-γ, and adiponectin. Cotreatment with rosiglitazone (an insulin sensitizer), insulin, or both abolished Dex-induced decreases in ATX and adiponectin secretion, but did not reverse Dex-induced decreases in secretions of 20 inflammatory cytokines and chemokines. Dex-treated mice exhibited lower ATX activity in plasma, brain, and adipose tissue; decreased mRNA levels for LPA and sphingosine 1-phosphate (S1P) receptors in brain; and decreased plasma concentrations of LPA and S1P. Our results establish a novel mechanism for the anti-inflammatory effects of Dex through decreased signaling by the ATX-LPA-inflammatory axis. The GC action in adipose tissue has implications for the pathogenesis of insulin resistance and obesity in metabolic syndrome and breast cancer treatment.-Meng, G., Tang, X., Yang, Z., Zhao, Y., Curtis, J. M., McMullen, T. P. W., Brindley, D. N. Dexamethasone decreases the autotaxin-lysophosphatidate-inflammatory axis in adipose tissue: implications for the metabolic syndrome and breast cancer.

Introduction Receptor tyrosine kinase (RTK) platelet-derived growth factor receptor-alpha (PDGFRα) was recently identified as a molecular switch for dedifferentiation in thyroid cancer that predicts resistance to therapy as well as recurrence of disease in papillary thyroid cancer. Here we describe the radiolabeling and functional characterization of an imaging probe based on a PDGFRα-specific monoclonal antibody (mAb) for immuno-PET imaging of PDGFRα in papillary thyroid cancer. Methods Antibody D13C6 (Cell Signaling) was decorated with chelator NOTA using bioconjugation reaction with 2-(p-NCS-Bz)-NOTA. Radiolabeling was carried out using 40 μg of antibody-NOTA conjugate with 143–223 MBq of [64Cu]CuCl2 in 0.25 M NaOAc (pH 5.5) at 30 °C for 1 h. The reaction mixture was purified with size-exclusion chromatography (PD-10 column). PDGFRα and mock transfected B-CPAP thyroid cancer cells lines for validation of 64Cu-labeled immuno-conjugates were generated using LVX-Tet-On technology. PET imaging was performed in NSG mice bearing bilaterally-induced PDGFRα (+/−) B-CPAP tumors. Results Bioconjugation of NOTA chelator to monoclonal antibody D13C6 resulted in 2.8 ± 1.3 chelator molecules per antibody as determined by radiometric titration with 64Cu. [64Cu]Cu-NOTA-D13C6 was isolated in high radiochemical purity (>98%) and good radiochemical yields (19–61%). The specific activity was 0.9–5.1 MBq/μg. Cellular uptake studies revealed a specific radiotracer uptake in PDGFRα expressing cells compared to control cells. PET imaging resulted in SUVmean values of ~5.5 for PDGFRα (+) and ~2 for PDGFRα (−) tumors, after 48 h p.i.. After 1 h, radiotracer uptake was also observed in the bone marrow (SUVmean ~5) and spleen (SUVmean ~8.5). Conclusion Radiolabeled antibody [64Cu]Cu-NOTA-D13C6 represents a novel and promising radiotracer for immuno-PET imaging of PDGFRα in metastatic papillary thyroid cancer. Advances in knowledge and implications for patient care The presented work has the potential to allow physicians to identify papillary thyroid cancer patients at risk of metastases by using the novel immuno-PET imaging assay based on PDGFRα-targeting antibody [64Cu]Cu-NOTA-D13C6.

We have previously established that adipose tissue adjacent to breast tumors becomes inflamed by tumor-derived cytokines. This stimulates autotaxin (ATX) secretion from adipocytes, whereas breast cancer cells produce insignificant ATX. Lysophosphatidate produced by ATX promotes inflammatory cytokine secretion in a vicious inflammatory cycle, which increases tumor growth and metastasis and decreases response to chemotherapy. We hypothesized that damage to adipose tissue during radiotherapy for breast cancer should promote lysophosphatidic acid (LPA) signaling and further inflammatory signaling, which could potentially protect cancer cells from subsequent fractions of radiation therapy. To test this hypothesis, we exposed rat and human adipose tissue to radiation doses (0.25-5 Gy) that were expected during radiotherapy. This exposure increased mRNA levels for ATX, cyclooxygenase-2, IL-1β, IL-6, IL-10, TNF-α, and LPA1 and LPA2 receptors by 1.8- to 5.1-fold after 4 to 48 h. There were also 1.5- to 2.5-fold increases in the secretion of ATX and 14 inflammatory mediators after irradiating at 1 Gy. Inhibition of the radiation-induced activation of NF-κB, cyclooxygenase-2, poly (ADP-ribose) polymerase-1, or ataxia telangiectasia and Rad3-related protein blocked inflammatory responses to γ-radiation. Consequently, collateral damage to adipose tissue during radiotherapy could establish a comprehensive wound-healing response that involves increased signaling by LPA, cyclooxygenase-2, and other inflammatory mediators that could decrease the efficacy of further radiotherapy or chemotherapy.-Meng, G., Tang, X., Yang, Z., Benesch, M. G. K., Marshall, A., Murray, D., Hemmings, D. G., Wuest, F., McMullen, T. P. W., Brindley, D. N. Implications for breast cancer treatment from increased autotaxin production in adipose tissue after radiotherapy.

// Esther Ekpe-Adewuyi 1 , Ana Lopez-Campistrous 1 , Xiaoyun Tang 2 , David N. Brindley 2 , Todd P. W. McMullen 1, 3 1 Department of Surgery, University of Alberta, Edmonton, Canada 2 Signal Transduction Research Group, Department of Biochemistry, University of Alberta, Edmonton, Canada 3 Division of Surgical Oncology, Cross Cancer Institute, University of Alberta, Edmonton, Canada Correspondence to: Todd P. W. McMullen, email: todd.mcmullen@albertahealthservices.ca Keywords: papillary thyroid cancer (PTC), platelet derived growth factor receptor-alpha (PDGFRα), epithelial to mesenchymal transition (EMT), invadopodia, crenolanib Received: June 30, 2016 Accepted: October 22, 2016 Published: November 11, 2016 ABSTRACT Recently platelet derived growth factor receptor-alpha (PDGFRα) was recognized as a potential target to treat aggressive papillary thyroid cancer given its strong association with lymph node metastases. However, it is unclear how PDGFRα potentiates metastases and if it works through the canonical MAPK pathway traditionally linked to PTC oncogenesis. We explored the phenotypic changes driven by PDGFRα activation in human papillary thyroid cancer (PTC) cells and the downstream signalling cascades through which they are effected. We demonstrate that PDGFRα drives an impressive phenotypic change in PTC cell lines as documented by significant cytoskeletal rearrangement, increased migratory potential, and the formation of invadopodia. Cells lacking PDGFRα formed compact and dense spheroids, whereas cells expressing active PDGFRα exhibited invadopodia in three-dimensional culture. To achieve this, active PDGFRα provoked downstream activation of the MAPK/Erk, PI3K/Akt and STAT3 pathways. We further confirmed the role of PDGFRα as a transformative agent promoting the epithelial to mesenchymal transition of PTC cells, through the augmentation of Snail and Slug expression. Crenolanib, a small molecule inhibitor of PDGFRα, suppressed the levels of Snail and Slug and almost completely reversed all the phenotypic changes. We demonstrate that PDGFRα activation is an essential component that drives aggressiveness in PTC cells, and that the signaling pathways are complex, involving not only the MAPK/Erk but also the PI3K/Akt and STAT3 pathways. This argues for upstream targeting of the PDGFRα given the redundancy of oncogenic pathways in PTC, especially in patients whose tumors over-express this tyrosine kinase receptor.

A quarter-century after the discovery of autotaxin in cell culture, the autotaxin-lysophosphatidate (LPA)-lipid phosphate phosphatase axis is now a promising clinical target for treating chronic inflammatory conditions, mitigating fibrosis progression, and improving the efficacy of existing cancer chemotherapies and radiotherapy. Nearly half of the literature on this axis has been published during the last five years. In cancer biology, LPA signaling is increasingly being recognized as a central mediator of the progression of chronic inflammation in the establishment of a tumor microenvironment which promotes cancer growth, immune evasion, metastasis, and treatment resistance. In this review, we will summarize recent advances made in understanding LPA signaling with respect to chronic inflammation and cancer. We will also provide perspectives on the applications of inhibitors of LPA signaling in preventing cancer initiation, as adjuncts extending the efficacy of current cancer treatments by blocking inflammation caused by either the cancer or the cancer therapy itself, and by disruption of the tumor microenvironment. Overall, LPA, a simple molecule that mediates a plethora of biological effects, can be targeted at its levels of production by autotaxin, LPA receptors or through LPA degradation by lipid phosphate phosphatases. Drugs for these applications will soon be entering clinical practice.

Autotaxin (ATX) is a secreted enzyme, which produces extracellular lysophosphatidate (LPA) from lysophosphatidylcholine (LPC). LPA activates six G protein-coupled receptors and this is essential for vasculogenesis during embryonic development. ATX is also involved in wound healing and inflammation, and in tumor growth, metastasis, and chemo-resistance. It is, therefore, important to understand how ATX is regulated. It was proposed that ATX activity is inhibited by its product LPA, or a related lipid called sphingosine 1-phosphate (S1P). We now show that this apparent inhibition is ineffective at the high concentrations of LPC that occur in vivo. Instead, feedback regulation by LPA and S1P is mediated by inhibition of ATX expression resulting from phosphatidylinositol-3-kinase activation. Inhibiting ATX activity in mice with ONO-8430506 severely decreased plasma LPA concentrations and increased ATX mRNA in adipose tissue, which is a major site of ATX production. Consequently, the amount of inhibitor-bound ATX protein in the plasma increased. We, therefore, demonstrate the concept that accumulation of LPA in the circulation decreases ATX production. However, this feedback regulation can be overcome by the inflammatory cytokines, TNF-α or interleukin 1β. This enables high LPA and ATX levels to coexist in inflammatory conditions. The results are discussed in terms of ATX regulation in wound healing and cancer.

International audience; Lipin-1 is a Mg2+-dependent phosphatidic acid phosphatase (PAP) that in mice is necessary for normal glycerolipid biosynthesis, controlling adipocytes metabolism and adipogenic differentiation. Mice carrying inactivating mutations in the Lpin1 gene display the characteristic features of human familial lipodystrophy. Very little is known on the roles of lipin-1 in human adipocyte physiology. Apparently fat distribution and weight is normal in humans carrying LPIN1 inactivating mutations, but a detailed analysis of adipose tissue appearance and functions in these patients has not been available so far. In this study, we performed a systematic histopathological, biochemical and gene expression analysis of adipose tissue biopsies from human patients harbouring LPIN1 biallelic inactivating mutations and affected by recurrent episodes of severe rhabdomyolysis. We also explored the adipogenic differentiation potential of human mesenchymal cell populations derived from lipin-1 defective patients. White adipose tissue from human LPIN1 mutant patients displayed a dramatic decrease in lipin-1 protein levels and PAP activity, with a concomitant moderate reduction of the adipocyte size. Nevertheless the adipose tissue develops without obvious histological signs of lipodystrophy and with a normal qualitative composition of the storage lipids. The increased expression of key adipogenic determinants such as SREBP1, PPARG and PGC1A shows that specific compensatory phenomena can be activated in vivo in human adipocytes under deficiency of a functional lipin-1.

This review deals with the effect of variations in phospholipid and sterol structure on the nature and magnitude of lipid-sterol interactions in lipid bilayer model membranes. The first portion of the review covers the effect of Chol itself on the thermotropic phase behavior and organization of a variety of different glycero- and sphingolipid membrane lipid classes, varying in the structure and charge of their polar headgroups and in the length and structure of their fatty acyl chains. The second part of this review deals with the effect of variations in sterol structure on the thermotropic phase behavior and organization primarily of the well studied DPPC model membrane system. In the third section, we focus on some of the contributions of sterol functional group chemistry, molecular conformation and dynamics, to sterol-lipid interactions. Using those studies, we re-examine the results of recently published experimental and computer-modeling studies to provide a new more dynamic molecular interpretation of sterol-lipid interactions. We suggest that the established view of the rigid sterol ring system and extended alkyl side-chain obtained from physical studies of cholesterol-phospholipid mixtures may not apply in lipid mixtures differing in their sterol chemical structure.

Aqueous dispersions of cholesterol-containing phosphatidylethanolamine (PE) bilayers were examined by a combination of high-sensitivity differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) and 31P-nuclear magnetic resonance spectroscopy. Regardless of hydrocarbon chain length, the incorporation of low levels of cholesterol into these bilayers causes progressive reductions in the temperature, enthalpy and overall cooperativity of the lipid hydrocarbon chain-melting phase transition. Moreover, at low cholesterol levels, the heating and cooling thermograms observed for the cholesterol/PE binary mixtures are similar, indicating comparable levels of lateral miscibility of cholesterol with PE bilayers in the gel and liquid-crystalline states. However, at higher levels of cholesterol incorporation, marked differences between the heating and cooling thermograms are noted. Upon heating, complex multicomponent thermograms are observed in PE bilayers containing large amounts of cholesterol, and the temperature and overall enthalpy values increase discontinuously from the pattern of monotonic decrease observed at lower cholesterol levels. Moreover, these discontinuities begin to emerge at progressively lower cholesterol concentrations as PE hydrocarbon chain length increases. Upon cooling, a simpler pattern of thermotropic behavior is observed, and the measured temperature and enthalpy values continue to decrease monotonically with increases in cholesterol content. These results suggest that at higher concentrations cholesterol exhibits a decreased degree of lateral miscibility in the gel or crystalline as compared to the liquid-crystalline states of PE bilayers, particularly in the case of the longer-chain PEs. Our FTIR and 31P-nuclear magnetic resonance spectroscopic studies also show that the thermotropic events observed with mixtures of low cholesterol content are analogous to the gel/liquid-crystalline phase transitions exhibited by the pure PEs. However, lamellar crystalline phases readily form when mixtures of high cholesterol content are cooled to low temperatures. Moreover, these crystalline phases are spectroscopically indistinguishable from those formed by the pure PEs, indicating that cholesterol is excluded from such phases. Upon subsequent heating, the melting of these crystalline phases gives rise to the complex thermograms detected by DSC and to the discontinuities in the phase transition temperature and enthalpy noted above. This pattern of behavior differs markedly from that observed with the corresponding phosphatidylcholines (PCs), where comparable degrees of cholesterol miscibility are observed in the gel and liquid-crystalline states even at high cholesterol concentrations, and where cholesterol inhibits rather than facilitates the formation of lamellar crystalline phases. We also find that the presence of cholesterol does not result in the hydrophobic mismatch-dependent shifts in the phase transition temperature in PE bilayers previously observed in PC bilayers of varying thickness. We attribute these differences in the effects of cholesterol on phospholipid thermotropic phase behavior to stronger electrostatic and hydrogen bonding interactions at the surfaces of PE and compared to PC bilayers.

Compared to normal tissues, many cancer cells overexpress autotaxin (ATX). This secreted enzyme produces extracellular lysophosphatidate, which signals through 6 GPCRs to drive cancer progression. Our previous work showed that ATX inhibition decreases 4T1 breast tumor growth in BALB/c mice by 60% for about 11 d. However, 4T1 cells do not produce significant ATX. Instead, the ATX is produced by adjacent mammary adipose tissue. We investigated the molecular basis of this interaction in human and mouse breast tumors. Inflammatory mediators secreted by breast cancer cells increased ATX production in adipose tissue. The increased lysophosphatidate signaling further increased inflammatory mediator production in adipose tissue and tumors. Blocking ATX activity in mice bearing 4T1 tumors with 10 mg/kg/d ONO-8430506 (a competitive ATX inhibitor, IC90 = 100 nM; Ono Pharma Co., Ltd., Osaka, Japan) broke this vicious inflammatory cycle by decreasing 20 inflammatory mediators by 1.5-8-fold in cancer-inflamed adipose tissue. There was no significant decrease in inflammatory mediator levels in fat pads that did not bear tumors. ONO-8430506 also decreased plasma TNF-α and G-CSF cytokine levels by >70% and leukocyte infiltration in breast tumors and adjacent adipose tissue by >50%. Hence, blocking tumor-driven inflammation by ATX inhibition is effective in decreasing tumor growth in breast cancers where the cancer cells express negligible ATX.

The existence of relatively large and long-lived detergent-insoluble, sphingolipid- and cholesterol-enriched, liquid-ordered lipid raft domains in the plasma membranes of eukaryotic cells has become widely accepted. However, we believe that the evidence for their existence is not compelling despite extensive work on both lipid bilayer model and biological membranes. We review here the results of recent studies, which in our view call into question the existence of lipid rafts in membranes, at least in the form commonly depicted.

The influence of the lipidic phase structures on their susceptibility to solubilization by the nonionic detergent C12E8 has been explored. To this aim, a phase diagram for dimyristoylphosphatidylch...

Our understanding of the role of cholesterol in biological membranes requires a detailed knowledge of the forces which mediate cholesterol-lipid interactions and of the lateral organization of cholesterol in natural and model membranes. The recent literature amply illustrates the strength of a multidisciplinary approach in determining the effects of cholesterol on the host bilayer lipids. To confidently apply our current knowledge of cholesterol-lipid interactions and cholesterol organization to natural membranes, however, a systematic analysis of the effects of cholesterol on a much broader range of host lipid bilayers will be required.

The extracellular lipase from Penicillium camembertiihas unique substrate specificity restricted to mono- anddiglycerides. The enzyme is a member of a homologousfamily of Upases from filamentous fungi. Four of theseproteins, from the fungi Rhizomucor miehei, Humicolalanuginosa, Rhizopus dele mar and P.camembertii, havehad their structures elucidated by X-ray crystallography.In spite of pronounced sequence similarities the enzymesexhibit significant differences. For example, the thenno-stability of the P.camembertii lipase is considerably lowerthan that of the H. lanuginosa enzyme. Since only theP.camembertii enzyme lacks the characteristic longdisulfide bridge, corresponding to Cys22-Cys268 in theH.lanuginosa lipase, we have engineered this disulfideinto the former enzyme in the hope of obtaining a signi-ficantly more stable fold. The properties of the doublemutant (Y22C and G269C) were assessed by a variety ofbiophysical techniques. The extra disulfide link was foundto increase the melting temperature of the protein from 51to 63°C. However, no difference is observed under reducingconditions, indicating an intrinsic instability of the newdisulfide. The optimal temperature for catalytic activitydecreased by 10°C and the optimum pH was shifted by 0.7units to more acidic.Keywords: enzyme engineering/protein stability/site-specificmutagenesis/thermostabi 1 ityIntroductionLipases are ubiquitous enzymes capable of hydrolyzinginsoluble acylglycerols. The enzymatic activity of lipasesincreases dramatically upon adsorption to an oil-water inter-face, a phenomenon known as interfacial activation (Desnuelleetal., 1960). Relatively recent investigations into the structure-function relationships in lipases revealed common features. Todate, all structurally characterized lipases belong to the a/phydrolase superfamily and have catalytic chymotrypsin-liketriads, Asp(Glu)-His-Ser, at their active sites. Interfacialactivation is believed to be triggered by conformational changesin the enzymes, although the molecular properties of thesubstrate may also play an important role (Brzozowski et at.,1991; Derewenda etal., 1992a, 1994b; Grochulski etal., 1993;van Tilbeurgh et al, 1993; Derewenda, 1995).Microbial lipases attract considerable attention owing totheir potential applications in industry, one of which is infood processing (Harwood, 1989). However, this particularexample requires enzymes with discrete substrate specificities.An extracellular lipase from Penicillium camembertii, PcL, isthe only known microbial lipase with a mono- and diacylgly-cerol specificity. The enzyme has been well characterized atboth protein and DNA levels (Yamaguchi and Mase, 1991;Yamaguchi et al., 1992). It is a member of a homologousfamily of extracellular lipases from filamentous fungi, whichalso comprises enzymes from Humicola lanuginosa (H1L),Rhizomucor miehei (Rml) and Rhizopus delemar (RdL), all ofwhich have been structurally characterized by X-ray crystallo-graphy at 2.0 A (PcL), 1.8 A (H1L), 1.9 A (Rml) and 2.6 A(RdL) resolution (Derewenda et al, 1992b, 1994a,b). Theyshare a similar tertiary fold, closely reminiscent of a typicala/p hydrolase paradigm (Ollis et al, 1992). Unfortunately, therelatively low thermostability of PcL precludes routine use ofthis enzyme in industry.Rml, H1L and RdL each have three disulfide bonds, one ofwhich is conserved and spans over 90% of the amino acidsequence connecting residues 22 and 268 (H1L numbering).This linkage is missing in PcL (Figure 1). We have thereforeengineered this disulfide into the PcL molecule using site-specific mutagenesis in the hope of elevating the enzyme'sdenaturation temperature {T^ without compromising its cata-lytic performance. In this paper we describe the results of thisexperiment.Materials and methodsMolecular modelingMolecular modeling was based on the refined X-ray structureof H1L (entry 1T1L in PDB) at 1.8 A resolution, the Rmlmodel (3TGL) at 1.9 A resolution and the 2.0 A model ofwild-type PcL (Derewenda et al, 1994a). All modeling wascarried out using FRODO (Jones, 1978) on an ESV20 work-station. Solvent accessibilities were calculated using VADAR(D.Wishard and B.D.Sykes, unpublished work).Plasmid construction and expression of PcL Y22C/G269Cdouble mutantOligonucleotide-directed mutations were incorporated into theintronless PcL gene (Yamaguchi et al, 1992) according toKunkel (1985) by ligating the appropriate DNA fragments.The nucleotide sequence of the construct was verified bysequence analysis using the dideoxynucleotide chain termina-tion method (Sanger et al, 1977). Mutagenic primers, 5'-TCCGCTTCACAGTATGACG-3' for the Y22C mutant and 5'-GGGGCCCTTGCAAGCATCAAC-3' for the G269C mutant,were synthesized using a Cyclone Plus (Milligene/Biosearch,Bedford, MA) DNA synthesizer. After separate introductionof each mutation, the complete double-mutant gene wasobtained by ligating the appropriate DNA fragments. Theconstruct was sequenced using the dideoxynucleotide chain-termination method (Sanger et al, 1977). Both wild-type andmutant genes were inserted into the HindTR site of the

We have studied the effects of cholesterol on the thermotropic phase behavior of aqueous dispersions of a homologous series of linear saturated phosphatidylcholines, using high-sensitivity differential scanning calorimetry and an experimental protocol which ensures that broad, low-enthalpy phase transitions are accurately monitored. We find that the incorporation of small amounts of cholesterol progressively decreases the temperature and the enthalpy, but not the cooperativity, of the pretransition of all phosphatidylcholines exhibiting such a pretransition and that the pretransition is completely abolished at cholesterol concentrations above 5 mol % in all cases. The incorporation of increasing quantities of cholesterol also alters the main or chain-melting phase transition of these phospholipid bilayers in both hydrocarbon chain length-dependent and hydrocarbon chain length-independent ways. At cholesterol concentrations of from 1 to 20-25 mol %, the DSC endotherms of all phosphatidylcholines studied consist of a superimposed sharp and broad component, the former ascribed to the melting of cholesterol-poor and the latter to the melting of the cholesterol-rich phosphatidylcholine domains. The temperature and cooperativity of the sharp component are reduced only slightly and in a chain length-independent manner with increasing cholesterol concentration, an effect we ascribe to the colligative effect of the presence of small quantities of cholesterol at the domain boundaries. Moreover, the enthalpy of the sharp component decreases and becomes zero at 20-25 mol % cholesterol for all of the phosphatidylcholines examined.(ABSTRACT TRUNCATED AT 250 WORDS)

We have examined the effects of cholesterol on the thermotropic phase behavior and organization of aqueous dispersions of a homologous series of linear disaturated phosphatidylserines by high-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy. We find that the incorporation of increasing quantities of cholesterol progressively reduces the temperature, enthalpy, and cooperativity of the gel-to-liquid-crystalline phase transition of the host phosphatidylserine bilayer, such that a cooperative chain-melting phase transition is completely or almost completely abolished at 50mol % cholesterol, in contrast to the results of previous studies. We are also unable to detect the presence of a separate anhydrous cholesterol or cholesterol monohydrate phase in our binary mixtures, again in contrast to previous reports. We further show that the magnitude of the reduction in the phase transition temperature induced by cholesterol addition is independent of the hydrocarbon chain length of the phosphatidylserine studied. This result contrasts with our previous results with phosphatidylcholine bilayers, where we found that cholesterol increases or decreases the phase transition temperature in a chain length-dependent manner (1993. Biochemistry , 32:516–522), but is in agreement with our previous results for phosphatidylethanolamine bilayers, where no hydrocarbon chain length-dependent effects were observed (1999. Biochim. Biophys. Acta, 1416:119–234). However, the reduction in the phase transition temperature by cholesterol is of greater magnitude in phosphatidylethanolamine as compared to phosphatidylserine bilayers. We also show that the addition of cholesterol facilitates the formation of the lamellar crystalline phase in phosphatidylserine bilayers, as it does in phosphatidylethanolamine bilayers, whereas the formation of such phases in phosphatidylcholine bilayers is inhibited by the presence of cholesterol. We ascribe the limited miscibility of cholesterol in phosphatidylserine bilayers reported previously to a fractional crystallization of the cholesterol and phospholipid phases during the removal of organic solvent from the binary mixture before the hydration of the sample. In general, the results of our studies to date indicate that the magnitude of the effect of cholesterol on the thermotropic phase behavior of the host phospholipid bilayer, and its miscibility in phospholipid dispersions generally, depend on the strength of the attractive interactions between the polar headgroups and the hydrocarbon chains of the phospholipid molecule, and not on the charge of the polar headgroups per se.

We have carried out a comparative study of the effect of cholesterol on the thermotropic phase behavior of the distearoyl and dielaidoyl molecular species of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine using high-sensitivity differential scanning calorimetry. For both molecular species of phosphatidylcholine, cholesterol incorporation produces bimodal endotherms at lower and unimodal endotherms at higher sterol concentrations. In both cases, heating and cooling endotherms are identical, and high concentrations of cholesterol (50 mol %) completely abolish the gel to liquid-crystalline phase transition. For the distearoyl molecular species of phosphatidylserine and phosphatidylethanolamine, heating and cooling endotherms are not identical, and cholesterol exhibits a considerably reduced miscibility in the gel as compared to the liquid-crystalline phase, particularly in the latter case. Thus, in neither case does the addition of 50 mol % cholesterol completely abolish the cooperative hydrocarbon chain-melting phase transition. However, the dielaidoyl molecular species of phosphatidylserine and phosphatidylethanolamine exhibit much closer correspondence in the heating and cooling modes than do the distearoyl species, and 50 mol % cholesterol is sufficient to almost or completely abolish the gel to liquid-crystalline phase transition of dielaidoylphosphatidylethanolamine and dielaidoylphosphatidylserine. In general, there is an inverse correlation between the strength of intermolecular phospholipid-phospholipid interactions, as manifested by the relative gel to liquid-crystalline phase transition temperatures of the pure phospholipids, and the miscibility of cholesterol in bilayers, particularly gel-state bilayers, formed from these phospholipids. These results indicate that the nature of cholesterol-phospholipid interactions, and thus the miscibility of cholesterol in the bilayer, depends on both the structure of the phospholipid polar headgroup and the hydrocarbon chains, as well as on the temperature and phase state of the phospholipid bilayer.

It has been proposed that the lower levels of exogenous cholesterol incorporation into the membranes of the sterol-non-requiring as compared to the sterol-requiring mycoplasmas may be due to the much higher glycolipid content of the former and to the reduced ability of glycolipids, as opposed to phospholipids, to incorporate sterols [Efrati et al. (1986) Arch. Biochem. Biophys. 248, 282-288]. In order to test this hypothesis, we have investigated the interaction of cholesterol with the major membrane glyco- and phospholipids of the sterol-non-requiring mycoplasma Acholeplasma laidlawii B, utilizing elaidic acid-homogenous membranes in order to obviate any differences in the nature of cholesterol-lipid interactions due to variations in the fatty acid composition of the different membrane components. Specifically, we have studied the effect of increasing quantities of cholesterol on the thermotropic phase behavior of aqueous dispersions of phosphatidylglycerol, diglucosyl diacylglycerol, and monoglucosyl diacylglycerol, as well as the total membrane polar lipids of this organism, using high-sensitivity differential scanning calorimetry. We find that cholesterol is highly miscible in both the lamellar gel and liquid-crystalline states of phosphatidylglycerol but exhibits limited miscibility in the two neutral glycolipids, particularly in their lamellar gel and crystalline states. We also demonstrate that cholesterol has a limited miscibility in both the lamellar gel and liquid-crystalline states of bilayers composed of the total A. laidlawii B membrane polar lipids. These results demonstrate that the nature of cholesterol-lipid interactions depends markedly on the structure of the glycerolipid polar headgroup and suggests that the incorporation of lower levels of cholesterol into the membranes of the sterol-non-requiring mycoplasmas may indeed be due, at least in part, to their high glycolipid contents. We also show that cholesterol stabilizes the lamellar liquid-crystalline phase of the monoglucosyl diacylglycerol relative to the inverted hexagonal phase at all sterol concentrations, in contrast to the effects of cholesterol on dielaidoylphosphatidylethanolamine, which destabilizes the lamellar liquid-crystalline phase at low concentrations.

In this study we have examined the effects of analogues of cholesterol differing with respect to alkyl side-chain length and structure on the thermotropic phase behavior of bilayers formed from 1-stearoyl-2-oleoyl- sn -glycero-3-phosphocholine (SOPC), an important subclass of naturally occurring phosphatidylcholines (PCs). The synthetic sterols we studied contained either a terminally unbranched (n-series) or a single methyl-branched (iso-series) side chain of 3 to 10 carbon atoms. The phase transition behavior was examined by high-sensitivity differential scanning calorimetry (DSC). The main phase transition endotherm of SOPC/sterol bilayers consists of superimposed sharp and broad components, which represent the hydrocarbon chain melting of sterol-poor and sterol-rich phospholipid domains, respectively. The transition temperature and the cooperativity of the sharp component are moderately reduced upon sterol incorporation and the enthalpy decreases to zero when sterol levels of 20–30 mol% are reached. The enthalpy of the broad component transition initially increases to a maximum around 25 or 25–30 mol% sterol and thereafter decreases with further increases in sterol concentration. However, the broad transition of SOPC bilayers containing both short (C-22, i-C5 and n-C3) and long (i-C9 and i-C10) side-chain sterols still persists at levels of 50 mol% sterol. Thus the effective stoichiometry of SOPC-sterol interactions varies with changes in sterol alkyl side-chain length. The incorporation of short linear or branched side-chain sterols (C-22, n-C3, n-C4, i-C5) causes the broad component transition temperature and cooperativity to decrease dramatically, whereas the incorporation of medium- and long-chain sterols in both the n- and iso-series has less effect on the transition temperature and cooperativity of the broad component. Overall, no significant differences were found between the n- and iso-series sterols for a given side-chain length. A comparison of the phase behavior of dipalmitoylphosphatidylcholine (DPPC)/sterol (McMullen et al. (1995) Biophys. J. 69, 169–176) and SOPC/sterol mixtures indicates that the primary factor responsible for changes in the thermotropic phase behavior of these systems is the extent of the hydrophobic mismatch between the sterol and the host lipid bilayer. However, sterol miscibility in PC bilayers, and thus the stoichiometry of lipid-sterol interactions, also appears to depend on the degree of unsaturation of the host lipid bilayer.

We have investigated the thermotropic phase behavior of dipalmitoylphosphatidylcholine (DPPC) bilayers containing a series of cholesterol analogues varying in the length and structure of their alkyl side chains. We find that upon the incorporation of up to approximately 25 mol % of any of the side chain analogues, the DPPC main transition endotherm consists of superimposed sharp and broad components representing the hydrocarbon chain melting of sterol-poor and sterol-rich phospholipid domains, respectively. Moreover, the behavior of these components is dependent on sterol side chain length. Specifically, for all sterol/DPPC mixtures, the sharp component enthalpy decreases linearly to zero by 25 mol % sterol while the cooperativity is only moderately reduced from that observed in the pure phospholipid. In addition, the sharp component transition temperature decreases for all sterol/DPPC mixtures; however, the magnitude of the decrease is dependent on the sterol side chain length. With respect to the broad component, the enthalpy initially increases to a maximum around 25 mol % sterol, thereafter decreasing toward zero by 50 mol % sterol with the exception of the sterols with very short alkyl side chains. Both the transition temperature and cooperativity of the broad component clearly exhibit alkyl chain length-dependent effects, with both the transition temperature and cooperativity decreasing more dramatically for sterols with progressively shorter side chains. We ascribe the chain length-dependent effects on transition temperature and cooperativity to the hydrophobic mismatch between the sterol and the host DPPC bilayer (see McMullen, T. P. W., Lewis, R. N. A. H., and McElhaney, R. N. (1993) Biochemistry 32:516-522). Moreover, the effective stoichiometry of sterol/DPPC interactions is altered by a significantly large degree of hydrophobic mismatch between the sterol and the DPPC bilayer. Thus the short chain sterols appear to exhibit considerable immiscibility in gel state DPPC bilayers, effectively limiting their interaction with adjacent phospholipid molecules.

We have investigated the effects of cholesterol on the thermotropic phase behavior of annealed and unannealed aqueous dispersions of dipalmitoylphosphatidylcholine (DPPC) using high-sensitivity differential scanning calorimetry (DSC), concentrating particularly on the cholesterol concentration range from 0 to 20 mol%. We find that the incorporation of cholesterol into low-temperature annealed DPPC bilayers decreases the enthalpy of the subtransition without affecting the transition temperature, such that the subtransition is abolished by 20 mol% cholesterol. Similarly, the incorporation of cholesterol progressively decreases the temperature and enthalpy of the pretransition and abolishes it entirely at cholesterol concentrations above 5 mol%. The incorporation of increasing quantities of cholesterol also alters the main or chain-melting phase transition. At cholesterol concentrations of 2 to 20 mol% cholesterol, the DSC endotherm arising from the main transition consists of superimposed sharp and broad components, the former due to the melting of cholesterol-poor and the latter to the melting of the cholesterol-rich DPPC domains. The temperature and cooperativity of the sharp component decreases slightly with increasing cholesterol concentration whereas the enthalpy decreases markedly, becoming zero at 20–25 mol% cholesterol. In contrast, the temperature and enthalpy of the broad component increases, and the cooperativity decreases markedly over this same range of cholesterol concentrations. An apparent increase in cooperativity of the overall DPPC endotherm at 7 mol% cholesterol is shown to arise because of a convergence in the transition temperatures of the sharp and broad components of the DSC endotherms. Some of our experimental findings, particularly the absence of any evidence for the existence of a triple point near 7.5 mol% cholesterol, do not accord with a recently proposed DPPC/cholesterol phase diagram derived from DSC and 2H-NMR data (see Vist, M.R. and Davis, J.H. (1990) Biochemistry 29, 451–464). In addition, we examined the effect of cholesterol on phosphatidylcholines (PCs) of different chain lengths and confirm that a eutectic point does not exist for any of these PC/cholesterol mixtures. We then propose a new, more complete DPPC/cholesterol phase diagram based on our high-sensitivity DSC data as well as some recent spectroscopic data on PC/cholesterol mixtures and explore some of its biological implications.

We have investigated the comparative effects of the incorporation of increasing quantities of androstenol and cholesterol on the thermotropic phase behavior of aqueous dispersions of members of a homologous series of linear saturated diacyl PCs1 using high sensitivity DSC. We have also employed FTIR and 31P-NMR spectroscopy to study the comparative effects of androstenol and cholesterol incorporation on the organization of the host PC bilayer in both the gel and liquid-crystalline states. The effects of androstenol and cholesterol incorporation on the thermotropic phase behavior of shorter chain PCs like 14:0 PC are generally similar but not identical. The incorporation of either sterol progressively decreases the temperature and enthalpy, but not the cooperativity, of the pretransition and completely abolishes it at sterol concentrations above 5 mol%. Moreover, at sterol concentrations of 1 to 20-25 mol%, both androstenol and cholesterol incorporation produce DSC endotherms consisting of superimposed sharp and broad components, the former due to the hydrocarbon chain melting of sterol-poor and the latter to the melting of sterol-rich 14:0 PC domains. The temperature and cooperativity of the sharp component are reduced slightly with increasing concentration of androstenol or cholesterol, and the enthalpy of the sharp component decreases progressively and becomes zero at 20-25 mol% sterol. As well, at cholesterol or androstenol concentrations above 20-25 mol%, the enthalpy of the broad component also decreases linearly with increasing sterol incorporation and becomes zero at sterol levels of about 50 mol%. However, whereas cholesterol incorporation progressively increases the temperature of the broad component of the DSC endotherm, androstenol incorporation decreases the temperature of this component. In contrast, the effects of androstenol and cholesterol incorporation on the thermotropic phase behavior of the intermediate and longer chain PCs studied here are considerably different. Although the incorporation of cholesterol increases the main phase transition temperature of 16:0 PC slightly and decreases the phase transition of 18:0 PC and 21:0 PC, androstenol incorporation decreases the main phase transition temperatures of all three PCs rather markedly. Moreover, androstenol is less effective in reducing the enthalpy and cooperativity of the broad component of the DSC endotherm of 16:0 PC and especially 18:0 PC bilayers in comparison to cholesterol. Androstenol incorporation (> 5 mol%) also results in the appearance of a second, low temperature endotherm in the DSC traces of the intermediate and longer chain PC dispersions that is not observed in similar cholesterol/PC dispersions. FTIR and 31P-NMR results suggest that this endotherm arises from a temperature-induced dissolution of androstenol in the gel phase PC bilayers. This second endotherm occurs at lower androstenol concentrations and increases in area at a given androstenol level as the chain length of the host PC bilayer increases. We ascribe the increasing immiscibility of androstenol in both the gel and liquid-crystalline states of PC bilayers of increasing thickness to an increasing degree of hydrophobic mismatch between the androstenol molecule and the host phospholipid bilayer.