Karasawa K et al. (2015) Enzymes "Overview of PAF-Degrading Enzymes."

Karasawa K, Inoue K

[Enzymes, 2015]

Because the acetyl group of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) is essential for its biological activity, the degradation of PAF is the most important mechanism that regulates the level of PAF. The enzyme that catalyzes the hydrolysis of acetyl group at the sn-2 position of PAF was termed PAF-acetylhydrolase (PAF-AH). Subsequent research revealed that the PAF-AH family includes intracellular forms called PAF-AH I and PAF-AH II as well as an extracellular isoform, plasma PAF-AH. PAF-AH I forms a complex consisting of catalytic subunits 1, 2, and regulatory subunits. PAF-AH I was identified from the brain, and previous studies focused on the role of PAF-AH I in brain development. However, subsequent studies found that PAF-AH I is involved in diverse functions such as spermatogenesis, amyloid- generation, cancer pathogenesis, and protein trafficking. Another intracellular enzyme, PAF-AH II, has no homology with PAF-AH I, although this enzyme shares sequence similarity to plasma PAF-AH. Because PAF-AH preferentially hydrolyzes oxidatively modulated or truncated phospholipids, it is considered to play a protective role against oxidative stress. Homologs of this enzyme are widely distributed among evolutionarily diverse organisms. For example, studies of Caenorhabditis elegans PAF-AH II demonstrate its contribution to epidermal morphogenesis. Extracellular plasma PAF-AH associates strongly with plasma lipoproteins. Because PAF-AH is mainly associated with LDL particles, it is considered to play an anti-inflammatory role by removing oxidized phospholipids generated in LDLs exposed to oxidative stress. In this overview, we describe the crucial roles of these three PAF-degrading enzymes in cell function and cell pathology.

Inoue T et al. (2004) Proc Natl Acad Sci U S A "Type II platelet-activating factor-acetylhydrolase is essential for epithelial morphogenesis in Caenorhabditis elegans."

Inoue T, Inoue K, Yamamoto M, Arai H, Sugimoto A, Suzuki Y, Tsujimoto M, Aoki J

[Proc Natl Acad Sci U S A, 2004]

Type II platelet-activating factor-acetylhydrolase [PAF-AH (II)] is an N-myristoylated enzyme that contains a lipase/esterase catalytic motif and selectively hydrolyzes the sn-2 acetyl ester of PAF and other short-chain acyl groups attached to phosphoglycerides. However, the physiological role of this enzyme remains to be elucidated. PAF-AH (II) is conserved in a variety of species ranging from a simple multicellular organism, Caenorhabditis elegans, to mammals. C. elegans possesses two homologous PAF-AH (II) genes, named paf-1 and paf-2. In this study, we generated these two loss-of-function mutants to elucidate the in vivo PAF-AH (II) function. Surprisingly, mutants of paf-2, a major isoform of C. elegans PAF-AH (II)s, exhibits gross defects in epithelial sheet formation, resulting in unsuccessful subsequent morphogenesis with complete penetrance. Moreover, paf-2 RNA interference worms show a variable abnormal morphology, including ectopic protrusions and a lumpy shape at the late embryonic and early larval stages due to epithelial organization defects. Consistent with these phenotypes, PAF-AH (II) is predominantly expressed in epithelial cells of C. elegans. This study demonstrates that PAF-AH (II) is essential for epithelial morphogenesis.

Radoff DT et al. (2014) Elife "The accessory helix of complexin functions by stabilizing central helix secondary structure."

Dittman JS, Bai J, Snead D, Eliezer D, Radoff DT, Dong Y

[Elife, 2014]

The presynaptic protein complexin (CPX) is a critical regulator of synaptic vesicle fusion, but the mechanisms underlying its regulatory effects are not well understood. Its highly conserved central helix (CH) directly binds the ternary SNARE complex and is required for all known CPX functions. The adjacent accessory helix (AH) is not conserved despite also playing an important role in CPX function, and numerous models for its mechanism have been proposed. We examined the impact of AH mutations and chimeras on CPX function in vivo and in vitro using C. elegans. The mouse AH fully restored function when substituted into worm CPX suggesting its mechanism is evolutionarily conserved. CPX inhibitory function was impaired when helix propagation into the CH was disrupted whereas replacing the AH with a non-native helical sequence restored CPX function. We propose that the AH operates by stabilizing CH secondary structure rather than through protein or lipid interactions.

Taneva SG et al. (2012) Biochim Biophys Acta "The amphipathic helix of an enzyme that regulates phosphatidylcholine synthesis remodels membranes into highly curved nanotubules."

Lee JM, Cornell RB, Taneva SG

[Biochim Biophys Acta, 2012]

CTP:phosphocholine cytidylyltransferase (CCT) is an amphitropic protein regulating phosphatidylcholine synthesis. Lipid-induced folding of its amphipathic helical (AH) membrane-binding domain activates the enzyme. In this study we examined the membrane deforming property of CCT in vitro by monitoring conversion of vesicles to tubules, using transmission electron microscopy. Vesicle tubulation was proportional to the membrane density of CCT and proceeded either as growth from a pre-formed surface bud, or as a global transformation of roughly spherical vesicles into progressively thinner tubules. The tubulation pathway depended on the lipid compositional heterogeneity of the vesicles, with heterogeneous mixtures supporting the bud-extension pathway. Co-existence of vesicles alongside thick and thin tubules suggested that CCT can discriminate between flat membrane surfaces and those with emerging curvature, binding preferentially to the latter. Thin tubules had a limiting diameter of ~12nm, likely representing bilayer cylinders with a very high density of 1 CCT/50 lipids. The AH segment was necessary and sufficient for tubulation. AH regions from diverse CCT sources, including C. elegans, had tubulation activity that correlated with -helical length. The AH motifs in CCT and the Parkinson's-related protein, -synuclein, have similar features, however the CCT AH was more effective in its membrane remodeling function. That CCT can deform vesicles of physiologically relevant composition suggests that CCT binding to membranes may initiate deformations required for organelle morphogenesis and at the same time stimulate synthesis of the PC required for the development of these regions.

Hecht RM et al. (1982) Dev Biol "DNA replication may be uncoupled from nuclear and cellular division in temperature-sensitive embryonic lethal mutants of Caenorhabditis elegans."

Bartel AH, Oro JA, Schomer DF, Hecht RM, Wall SM

[Dev Biol, 1982]

The nuclear and DNA contents of 18 temperature-sensitive embryonic lethal mutants of Caenorhabditis elegans have been determined at the time of arrest. After each mutant was shifted to restrictive temperature, the embryonic arrest stage was recorded by the number of nuclei counted in embryonic squashes and as DNA nuclear equivalents recorded by flow cytometry. Together the two methods complemented each other and provided qualitative and quantitative information concerning the nuclear number, DNA content, morphological stage of arrest, and presence of anucleate embryonic cells. The arrest stage for most of the embryonic lethal mutants demonstrated their potential to continue nuclear division and DNA replication beyond their respective temperature-sensitive periods. These results suggested that the mutants' primary defect did not reside on a pathway closely coupled to DNA replication or nuclear division. For example, B65 contained up to 650 DNA nuclear equivalents beyond the end of its temperature-sensitive period at the 20- to 30-cell stage. B65 is of additional interest because it continued DNA synthesis beyond the normal 550-cell stage and at the same time failed to progress beyond the morphogenetic period. The presence of enlarged nuclei that contained extra DNA demonstrated that DNA replication was independent of nuclear and cellular division. For example, B1 and B244 arrested with 1 to 50 and 1 to 200 nuclei, respectively. However, they both contained 250 to 400 nuclear equivalents of DNA. Conversely, the presence of embryonic cells without nuclei suggested that cell division may also be independent of nuclear division or nuclear migration. The enlarged nuclear and anucleate embryonic cells were observed only in those mutants requiring a normal parental

Hecht RM et al. (1981) J Histochem Cytochem "Simple adaptations to extend the range of flow cytometry five orders of magnitude for the DNA analysis of uni-and multicellular systems."

Bartel AH, Hecht RM, Hungerford III EV, Oro JA, Schomer DF

[J Histochem Cytochem, 1981]

Procedures and instrumentation are described to extend the capability of a cytometry system to record samples that exhibit a wide range of fluorescence such as multicellular systems. The methods employs a log amplifier in combination with a set of neutral density filters that reduces the incident light reaching the photomultiplier tube. With any given filter, signals within an intensity range of 200-fold can be measured; different filters can be used to obtain an extended overall range. Polystyrene fluorescent microspheres and a variety of mithramycin stained biological samples ranging from yeast cells to Paramecium were processed by the system. The relative DNA content of individual multicellular embryos was determined for a heterogeneous population of embryonic stages isolated from the nematode, Caenorhabditis elegans. As part of the evaluation of the procedure, the practical upper limit of range extension was determined. The most intense fluorescent signal was produced when untreated pecan pollen stained with ethidium bromide fluoresced with a factor (8.4 +/- 1.3) x 10*4 more than ethidium bromide stained with E. coli cells.

Ding Z et al. (2012) J Biol Chem "A 22-mer segment in the structurally pliable regulatory domain of metazoan CTP: phosphocholine cytidylyltransferase facilitates both silencing and activating functions."

Chen N, Taneva SG, Campbell SA, Semenec L, Huang HK, Ding Z, Cornell RB

[J Biol Chem, 2012]

CTP:phosphocholine cytidylyltransferase (CCT), an amphitropic enzyme that regulates phosphatidylcholine synthesis, is composed of a catalytic head domain and a regulatory tail. The tail region has dual functions as a regulator of membrane binding/enzyme activation and as an inhibitor of catalysis in the unbound form of the enzyme, suggesting conformational plasticity. These functions are well conserved in CCTs across diverse phyla, although the sequences of the tail regions are not. CCT regulatory tails of diverse origins are composed of a long membrane lipid-inducible amphipathic helix (m-AH) followed by a highly disordered segment, reminiscent of the Parkinson disease-linked protein, -synuclein, which we show shares a novel sequence motif with vertebrate CCTs. To unravel features required for silencing, we created chimeric enzymes by fusing the catalytic domain of rat CCT to the regulatory tail of CCTs from Drosophila, Caenorhabditis elegans, or Saccharomyces cerevisiae or to -synuclein. Only the tail domains of the two invertebrate CCTs were competent for both suppression of catalytic activity and for activation by lipid vesicles. Thus, both silencing and activating functions of the m-AH can tolerate significant changes in length and sequence. We identified a highly amphipathic 22-residue segment in the m-AH with features conserved among animal CCTs but not yeast CCT or -synuclein. Deletion of this segment from rat CCT increased the lipid-independent V(max) by 10-fold, equivalent to the effect of deleting the entire tail, and severely weakened membrane binding affinity. However, membrane binding was required for additional increases in catalytic efficiency. Thus, full activation of CCT may require not only loss of a silencing conformation in the m-AH but a gain of an activating conformation, promoted by membrane binding.

Pasquinelli AE (2002) Trends Genet "MicroRNAs: deviants no longer."

Pasquinelli AE

[Trends Genet, 2002]

Almost ten years ago, the Ambros laboratory made the extraordinary discovery that a gene essential for development in Caenorhabditis elegans encoded a 22-nucleotide, untranslated RNA. Further genetic studies in this nematode revealed the existence of a second tiny RNA gene that turned out to be conserved in animals as diverse as flies and humans. Now, the Ambros, Bartel and Tuschl laboratories have proven that those odd RNAs were just the first examples of a large family of RNAs, termed microRNAs (miRNAs). Although untranslated RNA genes, such as transfer RNAs and ribosomal RNAs, perform essential housekeeping roles in all living organisms, growing numbers of other RNAs, some widely conserved across phyla and others limited to certain species, are being uncovered and shown to fulfill specific duties. The discovery of miRNAs establishes a new class of regulatory RNAs and highlights the existence of unexpected RNA genes that, although ancient, are not extinct.

Robins H et al. (2005) Proc Natl Acad Sci U S A. "Incorporating structure to predict microRNA targets."

Li Y, Robins H, Padgett RW

[Proc Natl Acad Sci U S A., 2005]

MicroRNAs (miRNAs) are a recently discovered set of regulatory genes that constitute up to an estimated 1% of the total number of genes in animal genomes, including Caenorhabditis elegans, Drosophila, mouse, and humans [Lagos-Quintana, M., Rauhut, R., Lendeckel, W. M Tuschl, T. (2001) Science 294, 853-858; Lai, E. C., Tomancak, P., Williams, R. W. M Rubin, G.M. (2003) Genome Biol. 4, R42; Lau, N. C., Lim, L. P., Weinstein, E. G. M Bartel, D. P. (2001) Science 294, 858-862; Lee, R. C. M Ambros, V. (2001) Science 294, 862-8644; and Lee, R. C., Feinbaum, R. L. M Ambros, V. (1993) Cell 115, 787-798]. In animals, miRNAs regulate genes by attenuating protein translation through imperfect base pair binding to 3' UTR sequences of target genes. A major challenge in understanding the regulatory role of miRNAs is to accurately predict regulated targets. We have developed an algorithm for predicting targets that does not rely on evolutionary conservation. As one of the features of this algorithm, we incorporate the folded structure of mRNA. By using Drosophila miRNAs as a test case, we have validated our predictions in 10 of 15 genes tested. One of these validated genes is mad as a target for bantam. Furthermore, our computational and experimental data suggest that miRNAs have fewer targets than previously reported.