A key uncertainty in interpreting observations of bimodal merging galaxy clusters is the unknown angle between the subcluster separation vector and the plane of the sky. We present a new method for ...constraining this key parameter. We find analogs of observed systems in cosmological n-body simulations, and quantify their likelihood of matching the observed projected separation and relative radial velocities between subclusters, as a function of viewing angle. We derive constraints on the viewing angle of many observed bimodal mergers including the Bullet Cluster (1E 0657-558) and El Gordo (ACT-CL J0102-4915). We also present more generic constraints as a function of projected separation and relative radial velocity, which can be used to assess additional clusters as information about them becomes available. The constraints from these two observables alone are weak (typically 70°-75° at 68% confidence and 55°-60° at 95% confidence), but they incorporate much more cosmological context than the classical timing argument, marginalizing over many realizations of substructure, peculiar velocities, and so on. Compared with the MCMAC code, which implements the timing argument on NFW halos, our constraints generally predict subcluster separation vectors closer to the plane of the sky. This is because in realistic mergers, the subcluster velocity vectors are not entirely parallel to the separation vector (i.e., the mergers are not perfectly head-on). As a result, observation of a non-zero relative radial velocity does not exclude a separation vector in the plane of the sky, as it does in the head-on timing argument employed by MCMAC.
CTP:phosphocholine cytidylyltransferase (CCT) catalyzes a rate-limiting and regulated step in the CDP-choline pathway for the synthesis of phosphatidylcholine (PC) and PC-derived lipids. Control of ...CCT activity is multi-layered, and includes direct regulation by reversible membrane binding involving a built-in lipid compositional sensor. Thus CCT contributes to phospholipid compositional homeostasis. CCT also modifies the curvature of its target membrane. Knowledge of CCT structure and regulation of its catalytic function are relatively advanced compared to many lipid metabolic enzymes, and are reviewed in detail. Recently the genetic origins of two human developmental and lipogenesis disorders have been traced to mutations in the gene for CCTα.
The amphipathic helical (AH) membrane binding motif is recognized as a major device for lipid compositional sensing 1. We explore the function and mechanism of sensing by the lipid biosynthetic ...enzyme, CTP:phosphocholine cytidylyltransferase (CCT). As the regulatory enzyme in phosphatidylcholine (PC) synthesis, CCT contributes to membrane PC homeostasis. CCT directly binds and inserts into the surface of bilayers that are deficient in PC and therefore enriched in lipids that enhance surface charge and/or create lipid packing voids. These two membrane physical properties induce the folding of the CCT M domain into a ≥60 residue AH. Membrane binding activates catalysis by a mechanism that has been partially deciphered. We review the evidence for CCT compositional sensing, and the membrane and protein determinants for lipid selective membrane-interactions. We consider the factors that promote the binding of CCT isoforms to the membranes of the ER, nuclear envelope, or lipid droplets, but exclude CCT from other organelles and the plasma membrane. The CCT sensing mechanism is compared with several other proteins that use an AH motif for membrane compositional sensing. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.
Display omitted
•Homeostatic control of membrane PC content relies on a PC sensing device in CCT.•The PC sensor is an inducible membrane surface-binding amphipathic helix (domain M).•It recognizes features of PC-depleted membrane surfaces: negative charge and packing defects.•Membrane binding of domain M disrupts its auto-inhibitory contact with the active site.•CCT activation restores membrane PC content.
CTP:phosphocholine cytidylyltransferase-alpha (CCTα) and CCTβ catalyze the rate-limiting step in phosphatidylcholine (PC) biosynthesis. CCTα is activated by association of its α-helical M-domain with ...nuclear membranes, which is negatively regulated by phosphorylation of the adjacent P-domain. To understand how phosphorylation regulates CCT activity, we developed phosphosite-specific antibodies for pS319 and pY359+pS362 at the N- and C-termini of the P-domain, respectively. Oleate treatment of cultured cells triggered CCTα translocation to the nuclear envelope (NE) and nuclear lipid droplets (nLDs) and rapid dephosphorylation of pS319. Removal of oleate led to dissociation of CCTα from the NE and increased phosphorylation of S319. Choline depletion of cells also caused CCTα translocation to the NE and S319 dephosphorylation. In contrast, Y359 and S362 were constitutively phosphorylated during oleate addition and removal, and CCTα-pY359+pS362 translocated to the NE and nLDs of oleate-treated cells. Mutagenesis revealed that phosphorylation of S319 is regulated independently of Y359+S362, and that CCTα-S315D+S319D was defective in localization to the NE. We conclude that the P-domain undergoes negative charge polarization due to dephosphorylation of S319 and possibly other proline-directed sites and retention of Y359 and S362 phosphorylation, and that dephosphorylation of S319 and S315 is involved in CCTα recruitment to nuclear membranes.
While most of the articles in this issue review the workings of integral membrane enzymes, in this review, we describe the catalytic mechanism of an enzyme that contains a soluble catalytic domain ...but appears to catalyze its reaction on the membrane surface, anchored and assisted by a separate regulatory amphipathic helical domain and inter-domain linker. Membrane partitioning of CTP: phosphocholine cytidylyltransferase (CCT), a key regulatory enzyme of phosphatidylcholine metabolism, is regulated chiefly by changes in membrane phospholipid composition, and boosts the enzyme's catalytic efficiency >200-fold. Catalytic enhancement by membrane binding involves the displacement of an auto-inhibitory helix from the active site entrance-way and promotion of a new conformational ensemble for the inter-domain, allosteric linker that has an active role in the catalytic cycle. We describe the evidence for close contact between membrane lipid, a compact allosteric linker, and the CCT active site, and discuss potential ways that this interaction enhances catalysis.
Display omitted
•CCT is activated by membrane binding.•CCT seeks negatively charged surfaces and lipid packing voids for insertion of an amphipathic helix.•The amphipathic helix is a silencing device in the CCT soluble form.•Membrane binding facilitates remodeling of a malleable inter-domain allosteric linker.•Linker folding pulls the active site close to the membrane and shields it from solvent.
CTP:phosphocholine cytidylyltransferase (CCT) interconverts between an inactive soluble and active membrane-bound form in response to changes in membrane lipid composition. Activation involves ...disruption of an inhibitory interaction between the αE helices at the base of the active site and an autoinhibitory (AI) segment in the regulatory M domain and membrane insertion of the M domain as an amphipathic helix. We show that in the CCT soluble form the AI segment functions to suppress kcat and elevate the Km for CTP. The crystal structure of a CCT dimer composed of the catalytic and AI segments reveals an AI-αE interaction as a cluster of four amphipathic helices (two αE and two AI helices) at the base of the active sites. This interaction corroborates mutagenesis implicating multiple hydrophobic residues within the AI segment that contribute to its silencing function. The AI-αE interaction directs the turn at the C-terminal end of the AI helix into backbone-to-backbone contact with a loop (L2) at the opening to the active site, which houses the key catalytic residue, lysine 122. Molecular dynamics simulations suggest that lysine 122 side-chain orientations are constrained by contacts with the AI helix-turn, which could obstruct its engagement with substrates. This work deciphers how the CCT regulatory amphipathic helix functions as a silencing device.
A biosensor that uses ion-channel switches Cornell, B. A; Braach-Maksvytis, V. L. B; King, L. G ...
Nature (London),
06/1997, Letnik:
387, Številka:
6633
Journal Article
Recenzirano
Biosensors are molecular sensors that combine a biological recognition mechanism with a physical transduction technique. They provide a new class of inexpensive, portable instrument that permit ...sophisticated analytical measurements to be undertaken rapidly at decentralized locations. However, the adoption of biosensors for practical applications other than the measurement of blood glucose is currently limited by the expense, insensitivity and inflexibility of the available transduction methods. Here we describe the development of a biosensing technique in which the conductance of a population of molecular ion channels is switched by the recognition event. The approach mimics biological sensory functions and can be used with most types of receptor, including antibodies and nucleotides. The technique is very flexible and even in its simplest form it is sensitive to picomolar concentrations of proteins. The sensor is essentially an impedance element whose dimensions can readily be reduced to become an integral component of a microelectronic circuit. It may be used in a wide range of applications and in complex media, including blood. These uses might include cell typing, the detection of large proteins, viruses, antibodies, DNA, electrolytes, drugs, pesticides and other low-molecular-weight compounds.
phosphocholine cytidylyltransferase (CCT) is the key regulatory enzyme in the synthesis of phosphatidylcholine, the most abundant phospholipid in eukaryotic cell membranes. The CCT-catalyzed transfer ...of a cytidylyl group from CTP to phosphocholine to form CDP-choline is regulated by a membrane lipid-dependent mechanism imparted by its C-terminal membrane binding domain. We present the first analysis of a crystal structure of a eukaryotic CCT. A deletion construct of rat CCTα spanning residues 1–236 (CCT236) lacks the regulatory domain and as a result displays constitutive activity. The 2.2-Å structure reveals a CCT236 homodimer in complex with the reaction product, CDP-choline. Each chain is composed of a complete catalytic domain with an intimately associated N-terminal extension, which together with the catalytic domain contributes to the dimer interface. Although the CCT236 structure reveals elements involved in binding cytidine that are conserved with other members of the cytidylyltransferase superfamily, it also features nonconserved active site residues, His-168 and Tyr-173, that make key interactions with the β-phosphate of CDP-choline. Mutagenesis and kinetic analyses confirmed their role in phosphocholine binding and catalysis. These results demonstrate structural and mechanistic differences in a broadly conserved protein fold across the cytidylyltransferase family. Comparison of the CCT236 structure with those of other nucleotidyltransferases provides evidence for substrate-induced active site loop movements and a disorder-to-order transition of a loop element in the catalytic mechanism.
phosphoethanolamine cytidylyltransferase (Pcyt2) is the main regulatory enzyme for de novo biosynthesis of phosphatidylethanolamine by the CDP-ethanolamine pathway. There are two isoforms of Pcyt2, ...-α and -β; however, very little is known about their specific roles in this important metabolic pathway. We previously demonstrated increased phosphatidylethanolamine biosynthesis subsequent to elevated activity and phosphorylation of Pcyt2α and -β in MCF-7 breast cancer cells grown under conditions of serum deficiency. Mass spectroscopy analyses of Pcyt2 provided evidence for isoform-specific as well as shared phosphorylations. Pcyt2β was specifically phosphorylated at the end of the first cytidylyltransferase domain. Pcyt2α was phosphorylated within the α-specific motif that is spliced out in Pcyt2β and on two PKC consensus serine residues, Ser-215 and Ser-223. Single and double mutations of PKC consensus sites reduced Pcyt2α phosphorylation, activity, and phosphatidylethanolamine synthesis by 50–90%. The phosphorylation and activity of endogenous Pcyt2 were dramatically increased with phorbol esters and reduced by specific PKC inhibitors. In vitro translated Pcyt2α was phosphorylated by PKCα, PKCβI, and PKCβII. Pcyt2α Ser-215 was also directly phosphorylated with PKCα. Mapping of the Pcyt2α- and -β-phosphorylated sites to the solved structure of a human Pcyt2β showed that they clustered within and flanking the central linker region that connects the two catalytic domains and is a novel regulatory segment not present in other cytidylyltransferases. This study is the first to demonstrate differences in phosphorylation between Pcyt2 isoforms and to uncover the role of the PKC-regulated phosphorylation.
Background: Post-translational regulation of CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) is largely unexplored.
Results: Pcyt2 isoforms are characteristically phosphorylated within a newly identified central linker segment not present in other cytidylyltransferases.
Conclusion: Pcyt2 phosphorylation influences cell growth and is regulated by conventional PKCs and serum deficiency.
Significance: The isoform-specific phosphorylation reveals a post-translational regulation of Pcyt2 that is distinct from other cytidylyltransferases.
PDF
