Enzymes and receptors of prostaglandin pathways with arachidonic acid-derived versus eicosapentaenoic acid-derived substrates and products

Dietary fish oil containing omega 3 highly unsaturated fatty acids has cardioprotective and anti-inflammatory effects. Prostaglandins (PGs) and thromboxanes are produced in vivo both from the omega 6 fatty acid arachidonic acid (AA) and the omega 3 fatty acid eicosapentaenoic acid (EPA). Certain beneficial effects of fish oil may result from altered PG metabolism resulting from increases in the EPA/AA ratios of precursor phospholipids. Here we report in vitro specificities of prostanoid enzymes and receptors toward EPA-derived, 3-series versus AA-derived, 2-series prostanoid substrates and products. The largest difference was seen with PG endoperoxide H synthase (PGHS)-1. Under optimal conditions purified PGHS-1 oxygenates EPA with only 10% of the efficiency of AA, and EPA significantly inhibits AA oxygenation by PGHS-1. Two- to 3-fold higher activities or potencies with 2-series versus 3-series compounds were observed with PGHS-2, PGD synthases, microsomal PGE synthase-1 and EP1, EP2, EP3, and FP receptors. Our most surprising observation was that AA oxygenation by PGHS-2 is only modestly inhibited by EPA (i.e. PGHS-2 exhibits a marked preference for AA when EPA and AA are tested together). Also unexpectedly, TxA(3) is about equipotent to TxA(2) at the TP alpha receptor. Our biochemical data predict that increasing phospholipid EPA/AA ratios in cells would dampen prostanoid signaling with the largest effects being on PGHS-1 pathways involving PGD, PGE, and PGF. Production of 2-series prostanoids from AA by PGHS-2 would be expected to decrease in proportion to the compensatory decrease in the AA content of phospholipids that would result from increased incorporation of omega 3 fatty acids such as EPA.     

Effect of temperature and low pH on structure and stability of matrix porin in micellar detergent solutions

Thermal and low pH stabilities of matrix porin (Omp F) solubilized in the micellar solutions of ionic (SDS) and nonionic detergents were investigated by the methods of circular dichroism, intrinsic fluorescence, light scattering and sedimentation velocity. The stability of porin structure in solution is much higher in the presence of beta-octyl glucoside than with SDS. In the presence of SDS, sharp transitions were detected by all parameters measured, above 55 degrees C at neutral pH and below pH 4.5 at 20 degrees C. These transitions involve at least three concomitant processes: unfolding of protein, dissociation of trimers to monomers and the disruption of the protein-detergent micellar complexes, all events being irreversible in the presence of SDS. The nonionic detergent, beta-octyl glucoside, increases the stability of porin in acidic conditions, since neither dissociation nor denaturation was observed in the pH region between 7.5 and 2.0. However, at pH less than 3.5, small, reversible changes in protein structure became evident. The thermal stability of porin is also increased by beta-octyl glucoside as evidenced by a transition temperature 15-20 degrees C higher as compared to SDS. A considerable degree of native porin structure was regained after heat treatment in the presence of beta-octyl glucoside, though the reconstituted trimers were not identical to the native ones. The addition of lipopolysaccharide and divalent cations (Ca2+, Mg2+) to the experimental system did not improve the thermal reversibility.     

Prognostic and Predictive Significance of MYC and KRAS Alterations in Breast Cancer from Women Treated with Neoadjuvant Chemotherapy

Breast cancer is a complex disease, with heterogeneous clinical evolution. Several analyses have been performed to identify the risk factors for breast cancer progression and the patients who respond best to a specific treatment. We aimed to evaluate whether the hormone receptor expression, HER2 and MYC genes and their protein status, and KRAS codon 12 mutations may be prognostic or predictive biomarkers of breast cancer. Protein, gene and mutation status were concomitantly evaluated in 116 breast tumors from women who underwent neoadjuvant chemotherapy with doxorubicin plus cyclophosphamide. We observed that MYC expression was associated with luminal B and HER2 overexpression phenotypes compared to luminal A (p<0.05). The presence of MYC duplication or polysomy 8, as well as KRAS mutation, were also associated with the HER2 overexpression subtype (p<0.05). MYC expression and MYC gain were more frequently observed in early-onset compared to late-onset tumors (p<0.05). KRAS mutation was a risk factor of grade 3 tumors (p<0.05). A multivariate logistic regression demonstrated that MYC amplification defined as MYC/nucleus ratio of ≥2.5 was a protective factor for chemotherapy resistance. On the other hand, age and grade 2 tumors were a risk factor. Additionally, luminal B, HER2 overexpression, and triple-negative tumors presented increased odds of being resistant to chemotherapy relative to luminal A tumors. Thus, breast tumors with KRAS codon 12 mutations seem to present a worse prognosis. Additionally, MYC amplification may help in the identification of tumors that are sensitive to doxorubicin plus cyclophosphamide treatment. If confirmed in a large set of samples, these markers may be useful for clinical stratification and prognosis.     

Strategies for crystallizing membrane proteins

Crystallizing membrane proteins remains a challenging endeavor despite the increasing number of membrane protein structures solved by X-ray crystallography. The critical factors in determining the success of the crystallization experiments are the purification and preparation of membrane protein samples. Moreover, there is the added complication that the crystallization conditions must be optimized for use in the presence of detergents although the methods used to crystallize most membrane proteins are, in essence, straightforward applications of standard methodologies for soluble protein crystallization. The roles that detergents play in the stability and aggregation of membrane proteins as well as the colloidal properties of the protein-detergent complexes need to be appreciated and controlledbefore and during the crystallization trials. All X-ray quality crystals of membrane proteins were grown from preparations of detergent-solubilized protein, where the heterogeneous natural lipids from the membrane have been replaced by ahomogeneous detergent environment. It is the preparation of such monodisperse, isotropic solutions of membrane proteins that has allowed the successful application of the standard crystallization methods routinely used on soluble proteins. In this review, the issues of protein purification and sample preparation are addressed as well as the new refinements in crystallization methodologies for membrane proteins. How the physical behavior of the detergent, in the form of micelles or protein-detergent aggregates, affects crystallization and the adaptation of published protocols to new membrane protein systems are also addressed. The general conclusion is that many integral membrane proteins could be crystallized if pure and monodisperse preparations in a suitable detergent system can be prepared.In memory of Glenn D. Garavito.     

Exposure to 2500 Lux Increases Serum Melatonin in Venezuelan Equine Encephalomyelitis

When mice infected with the Venezuelan equine encephalomyelitis (VEE) virus were exposed to 2500 lux with a 12 h light: 12 h dark photoperiod, the serum levels of melatonin (MLT) remained constantly elevated. In mice exposed to 400 lux low levels of serum MLT were detected during the day and high levels during the night. An increase in the survival rate of the infected mice from 6 to 13 days after virus inoculation was also observed. The significant increment in the concentration of serum MLT produced by the high intensity light could be responsible for the longer survival rate of mice infected with the VEE virus.     

The membrane binding domains of prostaglandin endoperoxide H synthases 1 and 2. Peptide mapping and mutational analysis.

Prostaglandin endoperoxide H synthases 1 and 2 (PGHS-1 and -2) are the major targets of nonsteroidal anti-inflammatory drugs. Both isozymes are integral membrane proteins but lack transmembrane domains. X-ray crystallographic studies have led to the hypothesis that PGHS-1 and -2 associate with only one face of the membrane bilayer through a novel, monotopic membrane binding domain (MBD) that is comprised of four short, consecutive, amphipathic alpha-helices (helices A-D) that include residues 74-122 in ovine PGHS-1 (oPGHS-1) and residues 59-108 in human PGHS-2 (hPGHS-2). Previous biochemical studies from our laboratory showed that the MBD of oPGHS-1 lies somewhere between amino acids 25 and 166. In studies reported here, membrane-associated forms of oPGHS-1 and hPGHS-2 were labeled using the hydrophobic, photoactivable reagent 3-trifluoro-3-(m-[(125)I]iodophenyl)diazirine, isolated, and cleaved with AspN and/or GluC, and the photolabeled peptides were sequenced. The results establish that the MBDs of oPGHS-1 and hPGHS-2 reside within residues 74-140 and 59-111, respectively, and thus provide direct provide biochemical support for the hypothesis that PGHS-1 and -2 do associate with membranes through a monotopic MBD. We also prepared HelA, HelB, and HelC mutants of oPGHS-1, in which, for each helix, three or four hydrophobic residues expected to protrude into the membrane were replaced with small, neutral residues. When expressed in COS-1 cells, HelA and HelC mutants exhibited little or no catalytic activity and were present, at least in part, as misfolded aggregates. The HelB mutant retained about 20% of the cyclooxygenase activity of native oPGHS-1 and partitioned in subcellular fractions like native oPGHS-1; however, the HelB mutant exhibited an extra site of N-glycosylation at Asn(104). When this glycosylation site was eliminated (HelB/N104Q mutation), the mutant lacked cyclooxygenase activity. Thus, our mutational analyses indicate that the amphipathic character of each helix is important for the assembly and folding of oPGHS-1 to a cyclooxygenase active form.     

The role of arginine 120 of human prostaglandin endoperoxide H synthase-2 in the interaction with fatty acid substrates and inhibitors.

Arg-120 is located near the mouth of the hydrophobic channel that forms the cyclooxygenase active site of prostaglandin endoperoxide H synthases (PGHSs)-1 and -2. Replacement of Arg-120 of ovine PGHS-1 with a glutamine increases the apparent Km of PGHS-1 for arachidonate by 1,000-fold (Bhattacharyya, D. K., Lecomte, M., Rieke, C. J., Garavito, R. M., and Smith, W. L. (1996) J. Biol. Chem. 271, 2179-2184). This and other evidence indicate that the guanido group of Arg-120 forms an ionic bond with the carboxylate group of arachidonate and that this interaction is an important contributor to the overall strength of arachidonate binding to PGHS-1. In contrast, we report here that R120Q human PGHS-2 (hPGHS-2) and native hPGHS-2 have very similar kinetic properties, but R120L hPGHS-2 catalyzes the oxygenation of arachidonate inefficiently. Our data indicate that the guanido group of Arg-120 of hPGHS-2 interacts with arachidonate through a hydrogen bond rather than an ionic bond and that this interaction is much less important for arachidonate binding to PGHS-2 than to PGHS-1. The Km values of PGHS-1 and -2 for arachidonate are the same, and all but one of the core residues of the active sites of the two isozymes are identical. Thus, the results of our studies of Arg-120 of PGHS-1 and -2 imply that interactions involved in the binding of arachidonate to PGHS-1 and -2 are quite different and that residues within the hydrophobic cyclooxygenase channel must contribute more significantly to arachidonate binding to PGHS-2 than to PGHS-1. As observed previously with R120Q PGHS-1, flurbiprofen was an ineffective inhibitor of R120Q hPGHS-2. PGHS-2-specific inhibitors including NS398, DuP-697, and SC58125 had IC50 values for the R120Q mutant that were up to 1,000-fold less than those observed for native hPGHS-2; thus, the positively charged guanido group of Arg-120 interferes with the binding of these compounds. NS398 did not cause time-dependent inhibition of R120Q hPGHS-2, whereas DuP-697 and SC58125 were time-dependent inhibitors. Thus, Arg-120 is important for the time-dependent inhibition of hPGHS-2 by NS398 but not by DuP-697 or SC58125.     

Dehydration and embedding temperatures affect the antigenic specificity of tubulin and immunolabeling by the protein A-colloidal gold technique

Qualitative and quantitative tests were performed to determine whether the temperature at which dehydration and embedding occur affects the antigenic specificity of tubulin and the protein A-gold (pAg) immunolabeling technique. The analysis indicates that low temperature (-35 degrees C) treatment increased the specificity and density of pAg labeled anti-tubulin antibodies to Leishmania tropica subpellicular microtubules as compared to samples prepared at 0 degrees C or 20 degrees C.     

A novel, multilayer structure of a helical peptide.

X-ray diffraction analysis at 1.5 A resolution has confirmed the helical conformation of a de novo designed 18-residue peptide. However, the crystal structure reveals the formation of continuous molecular layers of parallel-packed amphiphilic helices as a result of much more extensive helix-helix interactions than predicted. The crystal packing arrangement, by virtue of distinct antiparallel packing interactions, segregates the polar and apolar surfaces of the helices into discrete and well-defined interfacial regions. An extensive "ridges-into-grooves" interdigitation characterizes the hydrophobic interface, whereas an extensive network of salt bridges and hydrogen bonds dominates the corresponding hydrophilic interface.