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The YAP-TEAD protein-protein interaction mediates YAP oncogenic functions downstream of the Hippo pathway. To date, available YAP-TEAD pharmacologic agents bind into the lipid pocket of TEAD, targeting the interaction indirectly via allosteric changes. However, the consequences of a direct pharmacological disruption of the interface between YAP and TEADs remain largely unexplored. Here, we present IAG933 and its analogs as potent first-in-class and selective disruptors of the YAP-TEAD protein-protein interaction with suitable properties to enter clinical trials. Pharmacologic abrogation of the interaction with all four TEAD paralogs resulted in YAP eviction from chromatin and reduced Hippo-mediated transcription and induction of cell death. In vivo, deep tumor regression was observed in Hippo-driven mesothelioma xenografts at tolerated doses in animal models as well as in Hippo-altered cancer models outside mesothelioma. Importantly this also extended to larger tumor indications, such as lung, pancreatic and colorectal cancer, in combination with RTK, KRAS-mutant selective and MAPK inhibitors, leading to more efficacious and durable responses. Clinical evaluation of IAG933 is underway., (© 2024. The Author(s).)

During inflammatory response, blood leukocytes adhere to the endothelium. This process involves numerous adhesion molecules, including a transmembrane chemokine, CX3CL1, which behaves as a molecular cluster. How this cluster assembles and whether this association has a functional role remain unknown. The analysis of CX3CL1 clusters using native electrophoresis and single molecule fluorescence kinetics shows that CX3CL1 is a homo-oligomer of 3 to 7 monomers. Fluorescence recovery after photobleaching assays reveal that the CX3CL1-transmembrane domain peptide self-associates in both cellular and acellular lipid environments, while its random counterpart (i.e. peptide with the same residues in a different order) does not. This strongly indicates that CX3CL1 oligomerization is driven by its intrinsic properties. According to the molecular modeling, CX3CL1 does not associate in compact bundles but rather with monomers linearly assembled side by side. Finally, the CX3CL1 transmembrane peptide inhibits both the CX3CL1 oligomerization and the adhesive function, while its random counterpart does not. This demonstrates that CX3CL1 oligomerization is mandatory for its adhesive potency. Our results provide a new direction to control CX3CL1-dependent cellular adherence in key immune processes.

Aberrant activation of the JAK/STAT pathway is thought to be the critical event in the pathogenesis of the chronic myeloproliferative neoplasms, polycythemia vera, essential thrombocythemia and primary myelofibrosis. The most frequent genetic alteration in these pathologies is the activating JAK2V617F mutation, and expression of the mutant gene in mouse models was shown to cause a phenotype resembling the human diseases. Given the body of genetic evidence, it has come as a sobering finding that JAK inhibitor therapy only modestly suppresses the JAK2V617F allele burden, despite showing clear benefits in terms of reducing splenomegaly and constitutional symptoms in patients. To gain a better understanding if JAK2V617F is required for maintenance of myeloproliferative disease once it has evolved, we generated a conditional inducible transgenic JAK2V617F mouse model using the SCL-tTA-2S tet-off system. Our model corroborates that expression of JAK2V617F in hematopoietic stem and progenitor cells recapitulates key hallmarks of human myeloproliferative neoplasms, and exhibits gender differences in disease manifestation. The disease was found to be transplantable, and importantly, reversible when transgenic JAK2V617F expression was switched off. Our results indicate that mutant JAK2V617F-specific inhibitors should result in profound disease modification by disabling the myeloproliferative clone bearing mutant JAK2., Competing Interests: Some of the authors are full-time employees of Novartis Pharma AG (Emilie A. Chapeau, Emeline Mandon, Vincent Romanet, Nicolas Ebel, Rita Andraos-Rey, Zhiyan Qian, Miltos Kininis, Sabine Zumstein-Mecker, Ralph Tiedt, Francesco Hofmann, Matthias Mueller, Fabienne Baffert), or have been full-time employees of Novartis Pharma AG (Violetta Powajbo, Moriko Ito, Bernd Kinzel, Masato Murakami, Thomas Radimerski). Novartis Pharma AG had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Ruxolitinib is a marketed product of Incyte, for which Novartis Pharma AG received exclusive development and commercialization rights outside of the United States. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Accumulating evidence supports the remarkable presence at the membrane surface of cancer cells of proteins, which are normally expressed in the intracellular compartment. Although these proteins, referred to as externalized proteins, represent a highly promising source of accessible and druggable targets for cancer therapy, the mechanisms via which they impact cancer biology remain largely unexplored. The aim of this study was to expose an externalized form of cytokeratin 8 (eK8) as a key player of colorectal tumorigenesis and characterize its mode of action. To achieve this, we generated a unique antagonist monoclonal antibody (D-A10 MAb) targeting an eight-amino-acid-long domain of eK8, which enabled us to ascertain the pro-tumoral activity of eK8 in both KRAS -mutant and wild-type colorectal cancers (CRC). We showed that this pro-tumoral activity involves a bidirectional eK8-dependent control of caspase-mediated apoptosis in vivo and of the plasminogen-induced invasion process in cellulo. Furthermore, we demonstrated that eK8 is anchored at the plasma membrane supporting this dual function. We, therefore, identified eK8 as an innovative therapeutic target in CRC and provided a unique MAb targeting eK8 that displays anti-neoplastic activities that could be useful to treat CRC, including those harboring KRAS mutations.

Membrane proteins (MP) are stable in their native lipid environment. To enable structural and functional investigations, MP need to be extracted from the membrane. This is a critical step that represents the main obstacle for MP biochemistry and structural biology. General guidelines and rules for membrane protein solubilization remain difficult to establish. This review aims to provide the reader with a comprehensive overview of the general concepts of MP solubilization and stabilization as well as recent advances in detergents innovation. Understanding how solubilization and stabilization are intimately linked is key to facilitate MP isolation toward fundamental structural and functional research as well as drug discovery applications. How to manage the tour de force of destabilizing the lipid bilayer and stabilizing MP at the same time is the holy grail of successful isolation and investigation of such a delicate and fascinating class of proteins., (Copyright © 2018. Published by Elsevier Inc.)

Inhibitors of double minute 2 protein (MDM2)-tumor protein 53 (TP53) interaction are predicted to be effective in tumors in which the TP53 gene is wild type, by preventing TP53 protein degradation. One such setting is represented by the frequent CDKN2A deletion in human cancer that, through inactivation of p14ARF , activates MDM2 protein, which in turn degrades TP53 tumor suppressor. Here we used piggyBac (PB) transposon insertional mutagenesis to anticipate resistance mechanisms occurring during treatment with the MDM2-TP53 inhibitor HDM201. Constitutive PB mutagenesis in Arf - /- mice provided a collection of spontaneous tumors with characterized insertional genetic landscapes. Tumors were allografted in large cohorts of mice to assess the pharmacologic effects of HDM201. Sixteen out of 21 allograft models were sensitive to HDM201 but ultimately relapsed under treatment. A comparison of tumors with acquired resistance to HDM201 and untreated tumors identified 87 genes that were differentially and significantly targeted by the PB transposon. Resistant tumors displayed a complex clonality pattern suggesting the emergence of several resistant subclones. Among the most frequent alterations conferring resistance, we observed somatic and insertional loss-of-function mutations in transformation-related protein 53 ( Trp53 ) in 54% of tumors and transposon-mediated gain-of-function alterations in B-cell lymphoma-extra large ( Bcl-xL ), Mdm4 , and two TP53 family members, resulting in expression of the TP53 dominant negative truncations ΔNTrp63 and ΔNTrp73. Enhanced BCL-xL and MDM4 protein expression was confirmed in resistant tumors, as well as in HDM201-resistant patient-derived tumor xenografts. Interestingly, concomitant inhibition of MDM2 and BCL-xL demonstrated significant synergy in p53 wild-type cell lines in vitro. Collectively, our findings identify several potential mechanisms by which TP53 wild-type tumors may escape MDM2-targeted therapy.

Influenza A virus displays one of the highest infection rates of all human viruses and therefore represents a severe human health threat associated with an important economical challenge. Influenza matrix protein 2 (M2) is a membrane protein of the viral envelope that forms a proton selective ion channel. Here we report the expression and native isolation of full length active M2 without mutations or fusions. The ability of the influenza virus to efficiently infect MDCK cells was used to express native M2 protein. Using a Calixarene detergents/surfactants based approach; we were able to solubilize most of M2 from the plasma membrane and purify it. The tetrameric form of native M2 was maintained during the protein preparation. Mass spectrometry shows that M2 was phosphorylated in its cytoplasmic tail (serine 64) and newly identifies an acetylation of the highly conserved Lysine 60. ELISA shows that solubilized and purified M2 was specifically recognized by M2 antibody MAB65 and was able to displace the antibody from M2 MDCK membranes. Using a bilayer voltage clamp measurement assay, we demonstrate a pH dependent proton selective ion channel activity. The addition of the M2 ion channel blocker amantadine allows a total inhibition of the channel activity, illustrating therefore the specificity of purified M2 activity. Taken together, this work shows the production and isolation of a tetrameric and functional native M2 ion channel that will pave the way to structural and functional characterization of native M2, conformational antibody development, small molecules compounds screening towards vaccine treatment., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Membrane proteins play crucial role in many cellular processes including cell adhesion, cell-cell communication, signal transduction and transport. To better understand the molecular basis of such central biological machines and in order to specifically study their biological and medical role, it is necessary to extract them from their membrane environment. To do so, it is challenging to find the best solubilization condition. Here we describe, a systematic screening method called BMSS (Biotinylated Membranes Solubilization & Separation) that allow screening 96 conditions at once. Streptavidine magnetic beads are used to separate solubilized proteins from remaining biotinylated membranes after solubilization. Relative quantification of dot blots help to select the best conditions to be confirmed by classical ultra-centrifugation and western blot. Classical detergents with different physical-chemical characteristics, novel calixarene based detergents and combination of both, were used for solubilization trials to obtain broad spectrum of conditions. Here, we show the application of BMSS to discover solubilization conditions of a GPCR target (MP-A) and a transporter (MP-B). The selected conditions allowed the solubilization and purification of non-aggregated and homogenous native membrane proteins A and B. Taken together, BMSS represent a rapid, reproducible and high throughput assessment of solubilization toward biochemical/functional characterization, biophysical screening and structural investigations of membrane proteins of high biological and medical relevance., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A variety of cancers depend on JAK2 signaling, including the high-risk subset of B cell acute lymphoblastic leukemias (B-ALLs) with CRLF2 rearrangements. Type I JAK2 inhibitors induce paradoxical JAK2 hyperphosphorylation in these leukemias and have limited activity. To improve the efficacy of JAK2 inhibition in B-ALL, we developed the type II inhibitor CHZ868, which stabilizes JAK2 in an inactive conformation. CHZ868 potently suppressed the growth of CRLF2-rearranged human B-ALL cells, abrogated JAK2 signaling, and improved survival in mice with human or murine B-ALL. CHZ868 and dexamethasone synergistically induced apoptosis in JAK2-dependent B-ALLs and further improved in vivo survival compared to CHZ868 alone. These data support the testing of type II JAK2 inhibition in patients with JAK2-dependent leukemias and other disorders., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Although clinically tested JAK inhibitors reduce splenomegaly and systemic symptoms, molecular responses are not observed in most myeloproliferative neoplasm (MPN) patients. We previously demonstrated that MPN cells become persistent to type I JAK inhibitors that bind the active conformation of JAK2. We investigated whether CHZ868, a type II JAK inhibitor, would demonstrate activity in JAK inhibitor persistent cells, murine MPN models, and MPN patient samples. JAK2 and MPL mutant cell lines were sensitive to CHZ868, including type I JAK inhibitor persistent cells. CHZ868 showed significant activity in murine MPN models and induced reductions in mutant allele burden not observed with type I JAK inhibitors. These data demonstrate that type II JAK inhibition is a viable therapeutic approach for MPN patients., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation., Methodology/principal Findings: Anionic calix[4]arene based detergents (C4Cn, n=1-12) were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5-24 nm, with the critical micellar concentration (CMC) being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein), a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM). They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux) much more efficiently than SDS (sodium dodecyl sulphate), FC12 (Foscholine 12) or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein., Conclusion/significance: These compounds seem promising to extract in a functional state membrane proteins obeying the positive inside rule. In that context, they may contribute to the membrane protein crystallization field.

ABCG2 is an ATP-binding cassette (ABC) transporter preferentially expressed by immature human hematopoietic progenitors. Due to its role in drug resistance, its expression has been correlated with a protection role against protoporhyrin IX (PPIX) accumulation in stem cells under hypoxic conditions. We show here that zinc mesoporphyrin, a validated fluorescent heme analog, is transported by ABCG2. We also show that the ABCG2 large extracellular loop ECL3 constitutes a porphyrin-binding domain, which strongly interacts with heme, hemin, PPIX, ZnPPIX, CoPPIX, and much less efficiently with pheophorbide a, but not with vitamin B12. K(d) values are in the range 0.5-3.5 μm, with heme displaying the highest affinity. Nonporphyrin substrates of ABCG2, such as mitoxantrone, doxo/daunorubicin, and riboflavin, do not bind to ECL3. Single-point mutations H583A and C603A inside ECL3 prevent the binding of hemin but hardly affect that of iron-free PPIX. The extracellular location of ECL3 downstream from the transport sites suggests that, after membrane translocation, hemin is transferred to ECL3, which is strategically positioned to release the bound porphyrin to extracellular partners. We show here that human serum albumin could be one of these possible partners as it removes hemin bound to ECL3 and interacts with ABCG2, with a K(d) of about 3 μm.

Targeting of ribosome-nascent chain complexes to the translocon in the endoplasmic reticulum is mediated by the concerted action of the signal recognition particle (SRP) and the SRP receptor (SR). Ribosome-stripped microsomes were digested with proteases to sever cytoplasmic domains of SRalpha, SRbeta, TRAM, and the Sec61 complex. We characterized protein translocation intermediates that accumulate when Sec61alpha or SRbeta is inactivated by proteolysis. In the absence of a functional Sec61 complex, dissociation of SRP54 from the signal sequence is blocked. Experiments using SR proteoliposomes confirmed the assembly of a membrane-bound posttargeting intermediate. These results strongly suggest that the Sec61 complex regulates the GTP hydrolysis cycle of the SRP-SR complex at the stage of signal sequence dissociation from SRP54.

Proteins and glycolipids are glycosylated, sulfated and phosphorylated in the lumen of the Golgi apparatus. The nucleotide substrates of these reactions must first be translocated from the cytosol into the Golgi lumen by specific transporters (antiporters). These are hydrophobic, transmembrane spanning proteins that appear to regulate post-translational modifications in the Golgi lumen.

We have studied the duration of the ACTH inhibition effect on the incorporation and transformation of [1-14 C] eicosatrienoic acid, in isolated adrenocortical cells of normal rats. The effect of esculetin, indomethacin and nordihydroguaiaretic acid alone, or in the presence of ACTH or diBucAMP on arachidonate biosynthesis was also investigated. ACTH and diBucAMP produced a significant inhibition in arachidonic acid biosynthesis. The inhibition produced by ACTH on delta 5 desaturating activity was considered to be a short-term effect. Nordihydroguaiaretic acid and esculetin provoked a depression in the uptake of 20:3 (n-6) acid and an inhibition in the formation of 20:4 (n-6) acid by adrenocortical cells. This effect was potentiated when the cells were currently treated with either ACTH or diBucAMP. Indomethacin produced no changes in the uptake of 20:3 (n-6) acid, while induced an increment on delta 5 desaturation activity. This effect would indicate that, normally, the metabolites produced by the cyclooxygenase pathway would operate by depressing arachidonic acid biosynthesis. Considering the negative regulation of the delta 5 desaturase activity system produced by ACTH through cAMP, and the positive modulation inferred by the results obtained in this work, it is possible to assume that there are, at least, two mechanisms that take place on 20:4 (n-6) acid formation. These mechanisms seem to work independently from one another and they probably interact when effecting a bi-directional control.

We investigated long-chain fatty-acyl-CoA synthetase activity in rat testicular microsomes. The apparent Michaelis constants (Km's) for the substrate fatty acids increased while their corresponding maximal velocities decreased in the order 18:3(n-3), 20:3(n-6), and 18:0. The reaction with 20:3 as substrate was diminished in the presence of a constant amount of either 18:0, 18:2(n-6), or 18:3(n-3) in a manner consistent with their action as simple competitive inhibitors, with the Ki values for 18:0 and 18:3(n-3) being of the same order of magnitude as their respective Km's. Adrenocorticotrophin and/or dexamethasone administration to intact rats caused a significant decrease in the thioesterification of all three substrates without producing any alteration in the fatty-acid composition of the microsomal membranes. These results indicate the presence of a broad-specificity activating enzyme in testis whose function is subject to hormonal regulation.

The biosynthesis of arachidonic acid in Sertoli and Leydig cells isolated from the testes of mature rats has been investigated. Both types of cells incorporated [2-14C]eicosatrienoic acid from the incubation medium and transformed it into arachidonic acid. The administration of adrenocorticotropin (ACTH) to the rats decreased the delta 5 desaturating activity in the isolated testicular cells, while ACTH produced no changes in the uptake of the substrate. Similar results were obtained when ACTH was added to the incubation medium of cells isolated from non-hormone treated rats. The total fatty acid composition of the Sertoli cells isolated from ACTH-treated rats showed a significant increase in the relative percentage of 18:2n-6 and a decrease in the C20 and C22 polyenes. This may indicate that ACTH exerts an inhibitory effect on delta 6, delta 5 and delta 4 desaturase activities. Addition of corticosterone to the incubation medium also produced a significant decrease in arachidonic acid biosynthesis. Because ACTH is known to stimulate the release of corticosterone in vivo, both hormones may act cumulatively in the regulation of arachidonic acid metabolism in Sertoli and Leydig cells.

Serine palmitoyltransferase, 3-dehydrosphinganine reductase and sphinganine N-acyltransferase are responsible for the first steps in sphingolipid biosynthesis forming 3-oxosphinganine, sphinganine, and dihydroceramide, respectively. We confirmed the localization of these enzymes in the endoplasmic reticulum (ER) using highly purified mouse liver ER and Golgi preparations. Mild digestion of sealed "right-side out" mouse liver ER derived vesicles with different proteolytic enzymes under conditions where latency of mannose-6-phosphatase was 90% produced approximately 60-80% inactivation of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase activities. These sphingolipid biosynthetic activities (serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase) are not latent, indicating that they face the cytosolic side of the ER, so that substrates have free access to their active sites. Moreover, the membrane-impermeable compound, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, which binds to a large number of ER proteins, inhibits serine palmitoyltransferase and sphinganine N-acyltransferase activities by 30-70%.

Addition of exogenous sphingosine homologues (D-erythro configuration) with different alkyl chain lengths (12 and 18 carbon atoms) to the medium of primary cultured cerebellar cells resulted in a decrease of serine palmitoyltransferase activity in a time- and concentration-dependent manner. This enzyme catalyzes the first committed step in sphingolipid biosynthesis. Half-maximal reduction of enzyme activity occurred after a 4-h treatment with 25 microM sphingoid bases. Maximal decrease (approx. 80%) was obtained after treating the cells for 4-8 h with 50 microM long-chain bases. When a biosynthetically inert sphingoid, azidosphingosine (10-50 microM), was fed to the cells, decrease of 3-ketosphinganine formation was much slower, reaching its maximum (approx. 80%) after 24 h. In contrast to D-erythro-sphingosine, L-threo-C18-sphingosine did not yield any decrease of serine palmitoyltransferase activity when added to the cells under identical experimental conditions. Decrease of serine palmitoyltransferase activity was fully reversible after removal of the long-chain bases from the culture medium. Activities of other enzymes of lipid metabolism, ceramide synthase, long-chain acyl-CoA synthase and choline phosphotransferase, were not affected by the addition of sphingoid bases, indicating that the down regulation of serine palmitoyltransferase is quite specific.

Studies carried out on the adrenal glands of experimental diabetic rats have shown an important inhibition in polyenoic fatty acid biosynthesis. This effect was demonstrated by testing the activities of long-chain fatty acyl-CoA synthetase, the delta 5- and delta 6-desaturases of the (n-6) essential fatty-acid series and the delta 6-desaturase of the (n-3) series in liver and adrenal microsomes. The depression in desaturating activity in the insulin-deprived animals was independent of that produced on acyl-CoA-thioester biosynthesis. Experiments measuring the incorporation and transformation of [1-14C]eicosa-8,11,14-trienoic acid in adrenocortical cells isolated from streptozotocin-diabetic animals demonstrated a significant inhibition of arachidonic acid biosynthesis compared to controls. Insulin injections in diabetic rats partially restored the delta 5- and delta 6-desaturase activities. This effect could result from direct action by the hormone since the restoration was reproduced when arachidonic acid biosynthesis was measured after insulin was added to the incubation medium of adrenocortical cells isolated from diabetic animals. The results of the present study provide new information about the implication of this abnormal metabolism in the adrenal gland of diabetic rats.

The in vivo and in vitro effect of epinephrine on the incorporation and desaturation of [1-14C]eicosa-8,11,14-trienoic acid was studied in isolated adrenocortical cells of rats. Control cells incubated at different substrate concentrations and for different periods of time were able to incorporate eicosatrienoic acid and to desaturate it to arachidonic acid. The ultrastructural study demonstrated that most of the cells belonged to the zona fasciculata and presented a good preservation. When the cells were isolated from rats killed 7 and 12 h after the administration of a single dose of epinephrine, a decrease in the incorporation and desaturation of 20:3n6 was observed. The effect on the desaturation was independent from the incorporation of the acid, and it was also observed in the microsomes of the decapsulated adrenal gland from rats treated with epinephrine. The fine structure of adrenocortical cells did not show changes after the treatment with epinephrine. The addition of the hormone to the incubation medium containing cells isolated from untreated rats produced no effect on arachidonic acid biosynthesis, indicating that the effect of epinephrine would be indirect, through either a metabolic or a hormone mediator.

Delta-6 and delta 5 desaturation activity of rat adrenal gland microsomes was studied to determine the effect of microsomal protein and the substrate saturation curves. This tissue has a very active delta 6 desaturase for linoleic and alpha-linolenic acids. The administration of epinephrine (1 mg/kg body weight) 12 hr before killing, produced approximately a 50% decrease in desaturation of [1-14C]linoleic acid to gamma-linolenic acid, [1-14C]alpha-linolenic acid to octadeca-6,9,12,15-tetraenoic acid and [1-14C]eicosa-8,11,14-trienoic acid to arachidonic acid. A 30% decrease in delta 5 desaturation activity was also shown after 7 hr of epinephrine treatment. The changes on the oxidative desaturation of the same fatty acids in liver microsomes were similar. No changes were observed in the total fatty acid composition of adrenal microsomes 12 hr after epinephrine treatment. Mechanisms of action of the hormone on the biosynthesis of polyunsaturated fatty acids in the adrenal gland are discussed.

The effect of columbinic acid (5 trans, 9 cis, 12 cis, octadeca-trienoic acid) supplemented to a fat-free diet on the fatty acid composition and its correlation to the physical properties of several tissues of rats, was studied. The absence of lipids in the diet produced the typical changes in the fatty acid composition characteristic of essential fatty acid (EFA) deficiency, namely a significant increase in the relative percentage of monoenoic fatty acids with a concomitant decrease in linoleic and arachidonic acids and a rise in eicosa-5,8,11-trienoic acid in liver, kidney, lung and spleen homogenates. Columbinic acid supplemented to a fat-free diet for 24 or 48 hr was incorporated into the different tissues and was partially elongated to 7 trans, 11 cis, 14 cis eicosatrienoic acid, but it was not desaturated. It modified the fatty acid spectrum of the lipids in the different tissues returning it to a similar composition of non-EFA deficient animals, except for a decrease of linoleic acid. The absence of lipids in the diet produced an increase in the 1-6 diphenyl-1,3,5-hexatriene (DPH) steady-state fluorescence anisotropy (rs) in liver microsomes, that was corrected by the administration of columbinic acid for 24 hr. It is concluded that columbinic acid produced a change in the pattern of total fatty acid composition of the different tissues studied which induced a favorable effect on the physical properties of the liver microsomal membranes (rs), leading to an improvement on the fatty acid deficiency in those membranes. Besides, columbinic acid would also exert a favorable effect in the short term, but not in the long-term eicosanoids production.

Acyl-CoA synthetase activity with various long-chain fatty acid substrates and its kinetic properties were measured in rat adrenal microsomes. The apparent Michaelis constants (Km) for substrate fatty acids increased in the order eicosa-8,11,14-trienoic acid less than alpha-linolenic acid less than linoleic acid less than palmitic acid. The maximum velocities with these fatty acids decreased in the order linolenic greater than eicosa-8,11,14-trienoic acid greater than palmitic acid. The synthesis of radioactivity palmitoyl-CoA, linoleyl-CoA, alpha-linolenyl-CoA and eicosa-8,11,14-trienoyl-CoA from the respective radioactive substrates decreased in the presence of all the other fatty acids mentioned above. These effects were inversely correlated with their apparent Km values. These results support the idea of a single long-chain fatty acyl-CoA synthetase in the adrenal microsomal fraction for the acid tested. After testing the influence of different hormones, it was shown that the administration of epinephrine, ACTH and dexamethasone caused a significant decrease in the activity of the long-chain fatty acid-CoA synthetase. This inhibition is independent of the one produced by the same hormones on the desaturation of linoleic to gamma-linolenic acid.

Both the capacity of isolated adrenocortical cells to incorporate and transform [1-14C]linoleic and [1-14C]alpha-linolenic acids and the effect of ACTH on the biosynthesis of polyunsaturated fatty acids from [1-14C]alpha-linolenic acid were investigated. The cells were able to incorporate both labeled precursor acids and convert them into higher homologs. This transformation increases along the incubation time tested. When linoleic acid was the precursor, the biosynthesis of higher homologs was carried out following the desaturating-elongating route. Both pathways, the desaturating-elongating and the elongating, were detected when the substrate was alpha-linolenic acid. The results proved the existence of delta 6, delta 5 and delta 4-desaturases in this type of cells. Isolated adrenocortical cells obtained from rats treated with ACTH showed an increase in the amount of [1-14C]alpha-18:3 that remained in the cells without metabolization and, consequently, a decrease in the last product formed (20:5 n-3) was evident compared to the controls. Simultaneously, the desaturation-elongation products decreased significantly. Similar results were obtained when cells isolated from untreated rats were incubated for 3 h in the presence of ACTH. In this case, the values obtained returned to normal levels 6 h after incubation. These results were mimicked by dibutyryl-cyclic AMP. It can be concluded that the effect of ACTH on the biosynthesis of polyunsaturated fatty acids from alpha-linolenic acid was mediated through an enhancement of the intracellular levels of cAMP.

High carbohydrate and high protein diets administered to rats for only three days decreased and increased, respectively, the arachidonate/linoleate ratio (20:4 n6/18:2 n6) in total lipids of lung, kidney, and liver microsomes. In liver and kidney this correlated with a significant decrease in the first dietary condition, and with an increase in the second, of delta 6 desaturase activity, measured by the rate of conversion of 1-14C linoleate n6 to 1-14C linolenate n6. Such an enzymatic activity was not detectable in lung microsomes, probably because of the low capacity of this tissue to produce the coenzyme A ester of the substrate used, since in lung the effects of dietary manipulation on the 20:4/18:2 ratios were as large as in liver. Significant differences were observed in microsomes of the three tissues examined in the steady state fluorescence anisotropy (rs) of diphenylhexatriene, which correlated with their cholesterol/phospholipid ratios. Both were lower in microsomes from liver than from the other two tissues, and both remained unchanged in each tissue under the dietary regimens studied. The results indicate that the observed effects of high carbohydrate and high protein diets on fatty acid delta 6 desaturation activity do not lead to apparent alterations in the physical properties of microsomal membranes.