A Split-Marker System for CRISPR-Cas9 Genome Editing in Methylotrophic Yeasts.

Methylotrophic yeasts such as Ogataea polymorpha and Komagataella phaffii (sin. Hansenula polymorpha and Pichia pastoris, respectively) are commonly used in basic research and biotechnological applications, frequently those requiring genome modifications. However, the CRISPR-Cas9 genome editing approaches reported for these species so far are relatively complex and laborious. In this work we present an improved plasmid vector set for CRISPR-Cas9 genome editing in methylotrophic yeasts. This includes a plasmid encoding Cas9 with a nuclear localization signal and plasmids with a scaffold for the single guide RNA (sgRNA). Construction of a sgRNA gene for a particular target sequence requires only the insertion of a 24 bp oligonucleotide duplex into the scaffold. Prior to yeast transformation, each plasmid is cleaved at two sites, one of which is located within the selectable marker, so that the functional marker can be restored only via recombination of the Cas9-containing fragment with the sgRNA gene-containing fragment. This recombination leads to the formation of an autonomously replicating plasmid, which can be lost from yeast clones after acquisition of the required genome modification. The vector set allows the use of G418-resistance and LEU2 auxotrophic selectable markers. The functionality of this setup has been demonstrated in O. polymorpha, O. parapolymorpha, O. haglerorum and Komagataella phaffii.

Pr-SNTX, a short-chain three-finger toxin from Papuan pigmy mulga snake, is an antagonist of muscle-type nicotinic acetylcholine receptor (α2ßδε).

Three-finger toxins (3FTxs) are one of the major components in snake venoms. In this study, we isolated a cDNA encoding a short-chain 3FTx, Pr-SNTX, from Pseudechis rossignolii. The amino acid sequence of Pr-SNTX is nearly identical to that of its ortholog in Pseudechis australis. Pr-SNTX protein inhibited muscle-type (α2ßδε), but not neuronal α7 nicotinic acetylcholine receptor (nAChR) activity.

Inhibition of nicotinic acetylcholine receptors, a novel facet in the pleiotropic activities of snake venom phospholipases A2.

Phospholipases A2 represent the most abundant family of snake venom proteins. They manifest an array of biological activities, which is constantly expanding. We have recently shown that a protein bitanarin, isolated from the venom of the puff adder Bitis arietans and possessing high phospholipolytic activity, interacts with different types of nicotinic acetylcholine receptors and with the acetylcholine-binding protein. To check if this property is characteristic to all venom phospholipases A2, we have studied the capability of these enzymes from other snakes to block the responses of Lymnaea stagnalis neurons to acetylcholine or cytisine and to inhibit α-bungarotoxin binding to nicotinic acetylcholine receptors and acetylcholine-binding proteins. Here we present the evidence that phospholipases A2 from venoms of vipers Vipera ursinii and V. nikolskii, cobra Naja kaouthia, and krait Bungarus fasciatus from different snake families suppress the acetylcholine- or cytisine-elicited currents in L. stagnalis neurons and compete with α-bungarotoxin for binding to muscle- and neuronal α7-types of nicotinic acetylcholine receptor, as well as to acetylcholine-binding proteins. As the phospholipase A2 content in venoms is quite high, under some conditions the activity found may contribute to the deleterious venom effects. The results obtained suggest that the ability to interact with nicotinic acetylcholine receptors may be a general property of snake venom phospholipases A2, which add a new target to the numerous activities of these enzymes.

Water-soluble LYNX1 residues important for interaction with muscle-type and/or neuronal nicotinic receptors.

Human LYNX1, belonging to the Ly6/neurotoxin family of three-finger proteins, is membrane-tethered with a glycosylphosphatidylinositol anchor and modulates the activity of nicotinic acetylcholine receptors (nAChR). Recent preparation of LYNX1 as an individual protein in the form of water-soluble domain lacking glycosylphosphatidylinositol anchor (ws-LYNX1; Lyukmanova, E. N., Shenkarev, Z. O., Shulepko, M. A., Mineev, K. S., D'Hoedt, D., Kasheverov, I. E., Filkin, S. Y., Krivolapova, A. P., Janickova, H., Dolezal, V., Dolgikh, D. A., Arseniev, A. S., Bertrand, D., Tsetlin, V. I., and Kirpichnikov, M. P. (2011) NMR structure and action on nicotinic acetylcholine receptors of water-soluble domain of human LYNX1. J. Biol. Chem. 286, 10618-10627) revealed the attachment at the agonist-binding site in the acetylcholine-binding protein (AChBP) and muscle nAChR but outside it, in the neuronal nAChRs. Here, we obtained a series of ws-LYNX1 mutants (T35A, P36A, T37A, R38A, K40A, Y54A, Y57A, K59A) and examined by radioligand analysis or patch clamp technique their interaction with the AChBP, Torpedo californica nAChR and chimeric receptor composed of the α7 nAChR extracellular ligand-binding domain and the transmembrane domain of α1 glycine receptor (α7-GlyR). Against AChBP, there was either no change in activity (T35A, T37A), slight decrease (K40A, K59A), and even enhancement for the rest mutants (most pronounced for P36A and R38A). With both receptors, many mutants lost inhibitory activity, but the increased inhibition was observed for P36A at α7-GlyR. Thus, there are subtype-specific and common ws-LYNX1 residues recognizing distinct targets. Because ws-LYNX1 was inactive against glycine receptor, its "non-classical" binding sites on α7 nAChR should be within the extracellular domain. Micromolar affinities and fast washout rates measured for ws-LYNX1 and its mutants are in contrast to nanomolar affinities and irreversibility of binding for α-bungarotoxin and similar snake α-neurotoxins also targeting α7 nAChR. This distinction may underlie their different actions, i.e. nAChRs modulation versus irreversible inhibition, for these two types of three-finger proteins.

Dimeric α-cobratoxin X-ray structure: localization of intermolecular disulfides and possible mode of binding to nicotinic acetylcholine receptors.

In Naja kaouthia cobra venom, we have earlier discovered a covalent dimeric form of α-cobratoxin (αCT-αCT) with two intermolecular disulfides, but we could not determine their positions. Here, we report the αCT-αCT crystal structure at 1.94 Å where intermolecular disulfides are identified between Cys(3) in one protomer and Cys(20) of the second, and vice versa. All remaining intramolecular disulfides, including the additional bridge between Cys(26) and Cys(30) in the central loops II, have the same positions as in monomeric α-cobratoxin. The three-finger fold is essentially preserved in each protomer, but the arrangement of the αCT-αCT dimer differs from those of noncovalent crystallographic dimers of three-finger toxins (TFT) or from the κ-bungarotoxin solution structure. Selective reduction of Cys(26)-Cys(30) in one protomer does not affect the activity against the α7 nicotinic acetylcholine receptor (nAChR), whereas its reduction in both protomers almost prevents α7 nAChR recognition. On the contrary, reduction of one or both Cys(26)-Cys(30) disulfides in αCT-αCT considerably potentiates inhibition of the α3ß2 nAChR by the toxin. The heteromeric dimer of α-cobratoxin and cytotoxin has an activity similar to that of αCT-αCT against the α7 nAChR and is more active against α3ß2 nAChRs. Our results demonstrate that at least one Cys(26)-Cys(30) disulfide in covalent TFT dimers, similar to the monomeric TFTs, is essential for their recognition by α7 nAChR, although it is less important for interaction of covalent TFT dimers with the α3ß2 nAChR.

Aversion to nicotine is regulated by the balanced activity of ß4 and α5 nicotinic receptor subunits in the medial habenula.

Nicotine dependence is linked to single nucleotide polymorphisms in the CHRNB4-CHRNA3-CHRNA5 gene cluster encoding the α3ß4α5 nicotinic acetylcholine receptor (nAChR). Here we show that the ß4 subunit is rate limiting for receptor activity, and that current increase by ß4 is maximally competed by one of the most frequent variants associated with tobacco usage (D398N in α5). We identify a ß4-specific residue (S435), mapping to the intracellular vestibule of the α3ß4α5 receptor in close proximity to α5 D398N, that is essential for its ability to increase currents. Transgenic mice with targeted overexpression of Chrnb4 to endogenous sites display a strong aversion to nicotine that can be reversed by viral-mediated expression of the α5 D398N variant in the medial habenula (MHb). Thus, this study both provides insights into α3ß4α5 receptor-mediated mechanisms contributing to nicotine consumption, and identifies the MHb as a critical element in the circuitry controlling nicotine-dependent phenotypes.

NMR structure and action on nicotinic acetylcholine receptors of water-soluble domain of human LYNX1.

Discovery of proteins expressed in the central nervous system sharing the three-finger structure with snake α-neurotoxins provoked much interest to their role in brain functions. Prototoxin LYNX1, having homology both to Ly6 proteins and three-finger neurotoxins, is the first identified member of this family membrane-tethered by a GPI anchor, which considerably complicates in vitro studies. We report for the first time the NMR spatial structure for the water-soluble domain of human LYNX1 lacking a GPI anchor (ws-LYNX1) and its concentration-dependent activity on nicotinic acetylcholine receptors (nAChRs). At 5-30 µM, ws-LYNX1 competed with (125)I-α-bungarotoxin for binding to the acetylcholine-binding proteins (AChBPs) and to Torpedo nAChR. Exposure of Xenopus oocytes expressing α7 nAChRs to 1 µM ws-LYNX1 enhanced the response to acetylcholine, but no effect was detected on α4ß2 and α3ß2 nAChRs. Increasing ws-LYNX1 concentration to 10 µM caused a modest inhibition of these three nAChR subtypes. A common feature for ws-LYNX1 and LYNX1 is a decrease of nAChR sensitivity to high concentrations of acetylcholine. NMR and functional analysis both demonstrate that ws-LYNX1 is an appropriate model to shed light on the mechanism of LYNX1 action. Computer modeling, based on ws-LYNX1 NMR structure and AChBP x-ray structure, revealed a possible mode of ws-LYNX1 binding.

A new type of thrombin inhibitor, noncytotoxic phospholipase A2, from the Naja haje cobra venom.

Thrombin is a key enzyme in the blood coagulation cascade and is also involved in carcinogenesis; therefore, its inhibitors are of fundamental and clinical importance. Snake venoms are widely used as sources of proteins that affect blood coagulation. We have isolated a new protein, called TI-Nh, from the Naja haje cobra venom. TI-Nh is a mixed-type inhibitor of thrombin (K(i) of 72.8 nM for a synthetic peptide substrate) and effectively inhibits thrombin-induced platelet aggregation with an IC(50) value of 0.2 nM. At concentrations up to approximately 50 nM, at which the thrombin-clotting time is substantially prolonged, TI-Nh exerts no detectable effects on both the intrinsic and extrinsic pathways of the coagulation cascade. It does not hydrolyze either fibrinogen or thrombin. Although TI-Nh bears structural features typical of group IB phospholipases A(2) (PLA(2)s), it possesses relatively weak enzymatic activity and is nontoxic to PC12 cells at concentrations up to 15 microM. Nevertheless, TI-Nh evokes neurite outgrowth in these cells at a concentration of approximately 1 microM, similar to cytotoxic snake PLA(2)s with strong enzymatic activity. TI-Nh is the first thrombin inhibitor found in the venom of the Elapidae snake family, and it is the first phospholipase shown to inhibit thrombin.

Cysteine-rich venom proteins from the snakes of Viperinae subfamily - molecular cloning and phylogenetic relationship.

Cysteine-rich proteins found in animal venoms (CRISP-Vs) are members of a large family of cysteine-rich secretory proteins (CRISPs). CRISP-Vs acting on different ion channels were found in venoms or mRNA (cDNA) encoding CRISP-Vs were cloned from snakes of three main families (Elapidae, Colubridae and Viperidae). About thirty snake CRISP-Vs were sequenced so far, however no complete sequence for CRISP-V from Viperinae subfamily was reported. We have cloned and sequenced for the first time cDNAs encoding CRISP-Vs from Vipera nikolskii and Vipera berus vipers (Viperinae). The deduced mature CRISP-V amino acid sequences consist of 220 amino acid residues. Phylogenetic analysis showed that viper proteins are closely related to those of Crotalinae snakes. The presence of CRISP-V in the V. berus venom was revealed using a combination of gel-filtration chromatography, electrophoresis and MALDI mass spectrometry. The finding of the putative channel blocker in viper venom may indicate its action on prey nervous system.