Hyun Ah Sun et al. (2006) East Asia C. elegans Meeting "Molecular Cloning of Caenorhabditis elegans HMG-CoA Synthase Gene and Its Regulation in the Dauer Stage"

Young-Ki Paik, Hyun Ah Sun

[East Asia C. elegans Meeting, 2006]

HMG-CoA synthase (HMGS) has been known as one of the key enzymes involved in cholesterol biosynthesis (in cytosol) and ketone body formation (in mitochondrion) in animals, depending on the physiological condition. In C. elegans, it has not previously been well established as to the presence of this enzyme or its regulation under various physiological states of this nematode. In the course of our study for the daumone-related biology, we were interested in cloning and characterization of HMGS gene. Since one of the characteristic physiological features of dauer larvae of C. elegans is believed to be an yet to be characterized efficient fat mobilization and ketone body formation to meet the energy demands for survival, we wanted to investigate how this enzyme might be involved in energy metabolism in the daumone-induced dauer larvae. To understand a functional role of this enzyme in dauer state, we first isolated F25B4.6 clone, an ortholog of human HMGS gene, by searching through the C. elegans genome database. Our data from DNA sequencing, RNAi and RT-PCR experiments suggest that this clone indeed encodes an authentic C. elegans HMGS and its mRNA level appears to be regulated developmentally, which peaks the early L2 stage. With the availability of the over-expressed recombinant protein (Mr.. 51.4 kDa), functional characterization and molecular regulation of HMGS is now underway ([Supported by a grant from BioGreen 21 Project to YKP]).

Hyun-Ah Sun et al. (2007) International Worm Meeting "Molecular Cloning of the HMG-CoA Synthase Gene and Its Regulation during the Dauer Stage of Caenorhabditis elegans."

Young-Ki Paik, Nan-Young Yoon, Jeeyong Lee, Hyun-Ah Sun

[International Worm Meeting, 2007]

HMG-CoA synthase (HMGS) has been known to be one of the key enzymes involved in cholesterol biosynthesis (in the cytosol) and ketone body formation (in mitochondria) in animals, depending on the physiological conditions. In C. elegans, neither the presence of this enzyme nor its regulation under various physiological states has been well established. In the course of our study of daumone-related biology, we were interested in the cloning and characterization of HMGS gene. Since one of the characteristic physiological features of dauer larvae of C. elegans is believed to be efficient fat mobilization and ketone body formation to meet the energy demands for survival, we wanted to investigate how this enzyme might be involved in energy metabolism in the daumone-induced dauer larvae. To understand the functional role of this enzyme in the dauer state, we first isolated the F25B4.6 clone, an ortholog of the human HMGS gene, by searching the C. elegans genome database. Our data from DNA sequencing, RNAi and RT-PCR experiments suggest that this clone indeed encodes an authentic C. elegans HMGS and its mRNA level appears to be regulated developmentally, which peaks in the early L2 stage. With the availability of the over-expressed recombinant protein (Mr 51.4 kDa), functional characterization and molecular regulation of HMGS is now underway. (Supported by a grant from the BioGreen 21 Project to YKP.).

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.

Baudrimont A et al. (2010) PLoS Genet "Leptotene/zygotene chromosome movement via the SUN/KASH protein bridge in Caenorhabditis elegans."

Pasierbek P, Machacek T, Penkner A, Klein F, Fridkin A, Woglar A, Baudrimont A, Jantsch V, Gruenbaum Y, Gloggnitzer J, Wegrostek C

[PLoS Genet, 2010]

The Caenorhabditis elegans inner nuclear envelope protein matefin/SUN-1 plays a conserved, pivotal role in the process of genome haploidization. CHK-2-dependent phosphorylation of SUN-1 regulates homologous chromosome pairing and interhomolog recombination in Caenorhabditis elegans. Using time-lapse microscopy, we characterized the movement of matefin/SUN-1::GFP aggregates (the equivalent of chromosomal attachment plaques) and showed that the dynamics of matefin/SUN-1 aggregates remained unchanged throughout leptonene/zygotene, despite the progression of pairing. Movement of SUN-1 aggregates correlated with chromatin polarization. We also analyzed the requirements for the formation of movement-competent matefin/SUN-1 aggregates in the context of chromosome structure and found that chromosome axes were required to produce wild-type numbers of attachment plaques. Abrogation of synapsis led to a deceleration of SUN-1 aggregate movement. Analysis of matefin/SUN-1 in a double-strand break deficient mutant revealed that repair intermediates influenced matefin/SUN-1 aggregate dynamics. Investigation of movement in meiotic regulator mutants substantiated that proper orchestration of the meiotic program and effective repair of DNA double-strand breaks were necessary for the wild-type behavior of matefin/SUN-1 aggregates.

Minn I et al. (2009) Mol Biol Cell "SUN-1 and ZYG-12, mediators of centrosome-nucleus attachment, are a functional SUN/KASH pair in Caenorhabditis elegans."

Minn I, Hanna-Rose W, Rolls MM, Malone CJ

[Mol Biol Cell, 2009]

Klarsicht/ANC-1/Syne/homology (KASH)/Sad-1/UNC-84 (SUN) protein pairs can act as connectors between cytoplasmic organelles and the nucleoskeleton. Caenorhabditis elegans ZYG-12 and SUN-1 are essential for centrosome-nucleus attachment. Although SUN-1 has a canonical SUN domain, ZYG-12 has a divergent KASH domain. Here, we establish that the ZYG-12 mini KASH domain is functional and, in combination with a portion of coiled-coil domain, is sufficient for nuclear envelope localization. ZYG-12 and SUN-1 are hypothesized to be outer and inner nuclear membrane proteins, respectively, and to interact, but neither their topologies nor their physical interaction has been directly investigated. We show that ZYG-12 is a type II outer nuclear membrane (ONM) protein and that SUN-1 is a type II inner nuclear membrane protein. The proteins interact in the luminal space of the nuclear envelope via the ZYG-12 mini KASH domain and a region of SUN-1 that does not include the SUN domain. SUN-1 is hypothesized to restrict ZYG-12 to the ONM, preventing diffusion through the endoplasmic reticulum. We establish that ZYG-12 is indeed immobile at the ONM by using fluorescence recovery after photobleaching and show that SUN-1 is sufficient to localize ZYG-12 in cells. This work supports current models of KASH/SUN pairs and highlights the diversity in sequence elements defining KASH domains.

Hao H et al. (2019) Nucleus "SUN/KASH interactions facilitate force transmission across the nuclear envelope."

Starr DA, Hao H

[Nucleus, 2019]

LINC complexes (Linker of Nucleoskeleton and Cytoskeleton), consisting of inner nuclear membrane SUN proteins and outer nuclear membrane KASH proteins, are essential for nuclear positioning, cell migration and chromosome dynamics. Crystal structures of the central SUN-KASH interaction domains revealed close interfaces predicted to mediate LINC complex formation and function. However, the in vivo significance of these conserved interactions were unclear. Cain et al (2018) used a combination of Caenorhabditis elegans genetics, imaging in cultured NIH 3T3 fibroblasts, and Molecular Dynamic simulations, to study SUN-KASH interactions in cells. The study found that a conserved aromatic residue at the -7 position of the C-terminal KASH domain and conserved cysteines in both the KASH and SUN domains play important roles in force transmission across the nuclear envelope. Other properties of LINC complexes may also play significant roles in transmitting mechanical forces generated by the cytoskeleton across the nuclear envelope. For example, the helicies preceding the SUN domain activate SUN proteins and may be important for activating KASH-SUN interactions or for association of SUN and/or KASH proteins with the membrane. The longer coiled-coils spanning the perinuclear space may also play a role in mediating force transmission. Finally, LINC complexes may be arranged in higher-order structures to facilitate high force-bearing processes.

Sugimoto A et al. (2000) Japanese Worm Meeting "PAF-acetylhydrolase type II is essential for hypodermal cell epithelialization in C. elegans"

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

[Japanese Worm Meeting, 2000]

PAF-acetylhydrolase (PAF-AH), which hydrolyzes the acetyl group, is distributed widely in tissues and plasma. In mammals, tissue cytosol contains two types of PAF-AH, type I and type II. Type II PAF-AH (PAF-AH (II)) is a 40-kDa monomeric enzyme and N-myristoylated at the N-terminus. Since PAF-AH (II) effectively hydrolyzes oxidized phospholipids as well as PAF, it is suggested that PAF-AH (II) functions to protect cells against oxidative stress by removing oxidized phospholipids. In this study, we discovered two PAF-AH (II) genes from C. elegans and disrupted those genes to elucidate the biological function in this organism. Amino acid sequences of two PAF-AH (II) paf-1 and paf-2 exhibited 70% identity with each other and 35% identity with mammalian PAF-AH (II)s. Both paf-1 and paf-2 concerved a Gly-X-Ser-X-Gly catalytic center and the N-myristoylation sequence. As mammalian PAF-AH (II), C elegans PAF-AH (II)s expressed in E. coli hydrolyzed PAF and oxidized phospholipid but not phospholipids with long acyl chains. Then we made knockout C. elegans and analyzed their phenotypes. paf-2 (-/-) was lethal at the embryonic stage, whereas paf-1 (-/-) grew normally. paf-2 (-/-) embryo was examined using a scanning electron microscope and immuno-staining. paf-2 (-/-) embryo had a profound defect in the formation of epitherial cells, especially in hypodermal cells possibly because of very low expression level of a certain adhesion molecule at this stage. We also examined the expression of paf-2 by injecting paf-2-GFP fusion gene in wild type. paf-2 was expressed in the epithelial cells and in gut at high levels during embryogenesis. At adult stage paf-2 was expressed in the phrynx, vulva and intestine. These data demonstrate that PAF-AH (II) plays an important role in the formation of epithelial cells and suggest that oxidative stress against cell membranes is increased during epithelialization.

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.

Cain NE et al. (2014) J Cell Biol "The SUN protein UNC-84 is required only in force-bearing cells to maintain nuclear envelope architecture."

McDonald KL, Cain NE, Tapley EC, Starr DA, Cain BM

[J Cell Biol, 2014]

The nuclear envelope (NE) consists of two evenly spaced bilayers, the inner and outer nuclear membranes. The Sad1p and UNC-84 (SUN) proteins and Klarsicht, ANC-1, and Syne homology (KASH) proteins that interact to form LINC (linker of nucleoskeleton and cytoskeleton) complexes connecting the nucleoskeleton to the cytoskeleton have been implicated in maintaining NE spacing. Surprisingly, the NE morphology of most Caenorhabditis elegans nuclei was normal in the absence of functional SUN proteins. Distortions of the perinuclear space observed in unc-84 mutant muscle nuclei resembled those previously observed in HeLa cells, suggesting that SUN proteins are required to maintain NE architecture in cells under high mechanical strain. The UNC-84 protein with large deletions in its luminal domain was able to form functional NE bridges but had no observable effect on NE architecture. Therefore, SUN-KASH bridges are only required to maintain NE spacing in cells subjected to increased mechanical forces. Furthermore, SUN proteins do not dictate the width of the NE.