Effect of C-Terminal S-Palmitoylation on D2 Dopamine Receptor Trafficking and Stability

We have used bioorthogonal click chemistry (BCC), a sensitive non-isotopic labeling method, to analyze the palmitoylation status of the D2 dopamine receptor (D2R), a G protein-coupled receptor (GPCR) crucial for regulation of processes such as mood, reward, and motor control. By analyzing a series of D2R constructs containing mutations in cysteine residues, we found that palmitoylation of the D2R most likely occurs on the C-terminal cysteine residue (C443) of the polypeptide. D2Rs in which C443 was deleted showed significantly reduced palmitoylation levels, plasma membrane expression, and protein stability compared to wild-type D2Rs. Rather, the C443 deletion mutant appeared to accumulate in the Golgi, indicating that palmitoylation of the D2R is important for cell surface expression of the receptor. Using the full-length D2R as bait in a membrane yeast two-hybrid (MYTH) screen, we identified the palmitoyl acyltransferase (PAT) zDHHC4 as a D2R interacting protein. Co-immunoprecipitation analysis revealed that several other PATs, including zDHHC3 and zDHHC8, also interacted with the D2R and that each of the three PATs was capable of affecting the palmitoylation status of the D2R. Finally, biochemical analyses using D2R mutants and the palmitoylation blocker, 2-bromopalmitate indicate that palmitoylation of the receptor plays a role in stability of the D2R.     

Bioorthogonal click chemistry to assay mu-opioid receptor palmitoylation using 15-hexadecynoic acid and immunoprecipitation

We have developed a modification of bioorthogonal click chemistry to assay the palmitoylation of cellular proteins. This assay uses 15-hexadecynoic acid (15-HDYA) as a chemical probe in combination with protein immunoprecipitation using magnetic beads in order to detect S-palmitoylation of proteins of interest. Here we demonstrate the utility of this approach for the mu-opioid receptor (MOR), a G-protein-coupled receptor (GPCR) responsible for mediating the analgesic and addictive properties of most clinically relevant opioid agonist drugs. This technique provides a rapid, non-isotopic, and efficient method to assay the palmitoylation status of a variety of cellular proteins, including most GPCRs.     

Multimer Formation Explains Allelic Suppression of PRDM9 Recombination Hotspots

Genetic recombination during meiosis functions to increase genetic diversity, promotes elimination of deleterious alleles, and helps assure proper segregation of chromatids. Mammalian recombination events are concentrated at specialized sites, termed hotspots, whose locations are determined by PRDM9, a zinc finger DNA-binding histone methyltransferase. Prdm9 is highly polymorphic with most alleles activating their own set of hotspots. In populations exhibiting high frequencies of heterozygosity, questions remain about the influences different alleles have in heterozygous individuals where the two variant forms of PRDM9 typically do not activate equivalent populations of hotspots. We now find that, in addition to activating its own hotspots, the presence of one Prdm9 allele can modify the activity of hotspots activated by the other allele. PRDM9 function is also dosage sensitive; Prdm9^+/- heterozygous null mice have reduced numbers and less active hotspots and increased numbers of aberrant germ cells. In mice carrying two Prdm9 alleles, there is allelic competition; the stronger Prdm9 allele can partially or entirely suppress chromatin modification and recombination at hotspots of the weaker allele. In cell cultures, PRDM9 protein variants form functional heteromeric complexes which can bind hotspots sequences. When a heteromeric complex binds at a hotspot of one PRDM9 variant, the other PRDM9 variant, which would otherwise not bind, can still methylate hotspot nucleosomes. We propose that in heterozygous individuals the underlying molecular mechanism of allelic suppression results from formation of PRDM9 heteromers, where the DNA binding activity of one protein variant dominantly directs recombination initiation towards its own hotspots, effectively titrating down recombination by the other protein variant. In natural populations with many heterozygous individuals, allelic competition will influence the recombination landscape.     

Delayed fluorescence in photosynthesis

Photosynthesis is a very efficient photochemical process. Nevertheless, plants emit some of the absorbed energy as light quanta. This luminescence is emitted, predominantly, by excited chlorophyll a molecules in the light-harvesting antenna, associated with Photosystem II (PS II) reaction centers. The emission that occurs before the utilization of the excitation energy in the primary photochemical reaction is called prompt fluorescence. Light emission can also be observed from repopulated excited chlorophylls as a result of recombination of the charge pairs. In this case, some time-dependent redox reactions occur before the excitation of the chlorophyll. This delays the light emission and provides the name for this phenomenon—delayed fluorescence (DF), or delayed light emission (DLE). The DF intensity is a decreasing polyphasic function of the time after illumination, which reflects the kinetics of electron transport reactions both on the (electron) donor and the (electron) acceptor sides of PS II. Two main experimental approaches are used for DF measurements: (a) recording of the DF decay in the dark after a single turnover flash or after continuous light excitation and (b) recording of the DF intensity during light adaptation of the photosynthesizing samples (induction curves), following a period of darkness. In this paper we review historical data on DF research and recent advances in the understanding of the relation between the delayed fluorescence and specific reactions in PS II. An experimental method for simultaneous recording of the induction transients of prompt and delayed chlorophyll fluorescence and decay curves of DF in the millisecond time domain is discussed.     

The recombinational anatomy of a mouse chromosome

Among mammals, genetic recombination occurs at highly delimited sites known as recombination hotspots. They are typically 1-2 kb long and vary as much as a 1,000-fold or more in recombination activity. Although much is known about the molecular details of the recombination process itself, the factors determining the location and relative activity of hotspots are poorly understood. To further our understanding, we have collected and mapped the locations of 5,472 crossover events along mouse Chromosome 1 arising in 6,028 meioses of male and female reciprocal F1 hybrids of C57BL/6J and CAST/EiJ mice. Crossovers were mapped to a minimum resolution of 225 kb, and those in the telomere-proximal 24.7 Mb were further mapped to resolve individual hotspots. Recombination rates were evolutionarily conserved on a regional scale, but not at the local level. There was a clear negative-exponential relationship between the relative activity and abundance of hotspot activity classes, such that a small number of the most active hotspots account for the majority of recombination. Females had 1.2x higher overall recombination than males did, although the sex ratio showed considerable regional variation. Locally, entirely sex-specific hotspots were rare. The initiation of recombination at the most active hotspot was regulated independently on the two parental chromatids, and analysis of reciprocal crosses indicated that parental imprinting has subtle effects on recombination rates. It appears that the regulation of mammalian recombination is a complex, dynamic process involving multiple factors reflecting species, sex, individual variation within species, and the properties of individual hotspots.     

Serotonergic activity of HP 184: Does spontaneous release have a role?

Examination of HP 184, [N-n-propyl)-N-(3-fluoro-4-pyridinyl)-1H-3-methylindodel-1-amine hydrochloride], in a variety of tests for serotonergic activity revealed some unique properties of this compound. We report here that 100 μM HP 184 enhanced spontaneous release of [³H]serotonin (5-HT) from rat hippocampal slices. This release was independent of the uptake carrier. In vivo assays confirmed that HP 184 (20 mg/kg, i.p.) lacked significant interactions at the norepinephrine (NE) or 5-HT uptake carrier itself. Notably, HP 184 (15 mg/kg, i.p.) reduced drinking behavior in schedule-induced polydipsic (SIP) rats. We previously reported that some selective 5-HT reuptake inhibitors decrease SIP 30–40% after a 14–21 day treatment. In the current study, HP 184 decreased SIP beginning with the first treatment, and this reduction (30%) was maintained for 28 days. We further investigated HP 184 and serotonin metabolite levels. One hour after i.p. administration of 30 mg/kg HP 184, the ratio of whole brain 5-hydroxyindolacetic acid (5-HIAA) to 5-HT was increased, suggesting serotonergic activation. Under these conditions, the brain: plasma ratio of HP 184 was approximately 2∶1, with brain concentrations of 1.6 μg/gram. We speculate that the spontaneous release effects of HP 184 may be responsible for the behavioral effects observed.     

The effects of biologically active substances in medicinal plants on the metabolic activity of neutrophils

Neutrophils are the typical effector cells of the innate immune response because they are the first leukocytes to be recruited to an inflammatory site where they engulf invading microorganisms and destroy them by multiple oxidative and non-oxidative mechanisms. The destructive potential of neutrophils requires the tight control of their recruitment into tissue compartments and the production of inflammatory mediators such as reactive oxygen species. These oxidants can be highly toxic not only for infectious agents but also for neighbouring host tissues resulting in various autoimmune and inflammatory diseases. Thus, a significant attention in medicine is paid to approaches designed to modulate the metabolic activity of neutrophils. Synthetic steroid and non-steroid compounds with adverse side effects are commonly used for this purpose. The effects of natural substances which can modulate the metabolic activity of neutrophils and which simultaneously would not exert any significant unfavourable side effects have recently been investigated. Suitable candidates for this purpose might be compounds contained in herbs. These include especially polysaccharides and polyphenols, but also terpenes. The aim of the present paper is to summarize contemporary knowledge on the effects of compounds from herbs on the metabolic activity of mammalian neutrophils.     

Shear stress and pressure modulate saphenous vein remodeling ex vivo

Vein graft failure remains an important clinical challenge, but factors contributing to vein graft failure have not clearly been defined. We investigated the role of the mechanical environment in vein remodeling in an ex vivo perfusion system. Porcine saphenous veins were subjected to five different ex vivo hemodynamic environments, including one mimicking an arterial bypass graft, for one week in order to independently assess the effects of shear stress and pressure on vein remodeling. The extent of intimal hyperplasia decreased with culture under increasing shear stress, with veins cultured under the lowest levels of shear stress exhibiting the greatest ratio of intimal/medial area, 0.15+/-0.03, which was greater than that of fresh veins (0.06+/-0.01, p<0.05). All perfused veins displayed characteristics of both medial hypertrophy and eutrophic remodeling, with those veins cultured under elevated pressures showing greater increases in mass and area than those cultured under venous pressures. Medial area correlated with the average pressure under which veins were cultured (R2=0.95, p<0.001), with veins cultured under bypass graft conditions, which were exposed to the greatest pressure during the one week culture, exhibiting the largest medial area (1.69+/-0.15 mm2), which was significantly greater than that of fresh veins (1.08+/-0.05 mm2, p<0.05). However, pulsatility was not a necessary stimulus for medial growth, as increases in medial area were observed in culture conditions in which steady flow and pressure were present. Our results suggest that pressure and shear stress act independently to regulate vein remodeling, influencing changes in vessel size as well as the nature of the remodeling.     

Characterization of unique modification of flagellar rod protein FlgG by Campylobacter jejuni lipid A phosphoethanolamine transferase, linking bacterial locomotion and antimicrobial peptide resistance

Gram-negative bacteria assemble complex surface structures that interface with the surrounding environment and are involved in pathogenesis. Recent work in Campylobacter jejuni identified a gene encoding a novel phosphoethanolamine (pEtN) transferase Cj0256, renamed EptC, that serves a dual role in modifying the flagellar rod protein, FlgG, and the lipid A domain of C. jejuni lipooligosaccharide with a pEtN residue. In this work, we characterize the unique post-translational pEtN modification of FlgG using collision-induced and electron transfer dissociation mass spectrometry, as well as a genetic approach using site-directed mutagenesis to determine the site of modification. Specifically, we show that FlgG is modified with pEtN at a single site (Thr(75)) by EptC and demonstrate enzyme specificity by showing that EptC is unable to modify other amino acids (e.g. serine and tyrosine). Using Campylobacter strains expressing site-directed FlgG mutants, we also show that defects in motility arise directly from the loss of pEtN modification of FlgG. Interestingly, alignments of FlgG from most epsilon proteobacteria reveal a conserved site of modification. Characterization of EptC and its enzymatic targets expands on the increasingly important field of prokaryotic post-translational modification of bacterial surface structures and the unidentified role they may play in pathogenesis.     

A Highlights from MBoC Selection: EWSR1 affects PRDM9-dependent histone 3 methylation and provides a link between recombination hotspots and the chromosome axis protein REC8

Meiotic recombination in most mammals requires recombination hotspot activation through the action of the histone 3 Lys-4 and Lys-36 methyltransferase PRDM9 to ensure successful double-strand-break initiation and repair. Here we show that EWSR1, a protein whose role in meiosis was not previously clarified in detail, binds to both PRDM9 and pREC8, a phosphorylated meiosis-specific cohesin, in male meiotic cells. We created a Ewsr1 conditional knockout mouse model to deplete EWSR1 before the onset of meiosis and found that absence of EWSR1 causes meiotic arrest with decreased histone trimethylation at meiotic hotspots, impaired DNA double-strand-break repair, and reduced crossover number. Our results demonstrate that EWSR1 is essential for promoting PRDM9-dependent histone methylation and normal meiotic progress, possibly by facilitating the linking between PRDM9-bound hotspots and the nascent chromosome axis through its component cohesin pREC8.