Alexander A. Tokmakov

Evidence that phosphatidylinositol 3-kinase is involved in sperm-induced tyrosine kinase signaling in Xenopus egg fertilization

BackgroundStudies have examined the function of PI 3-kinase in the early developmental processes that operate in oocytes or early embryos of various species. However, the roles of egg-associated PI 3-kinase and Akt, especially in signal transduction at fertilization, are not well understood.ResultsHere we show that in Xenopus eggs, a potent inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), LY294002 inhibits sperm-induced activation of the tyrosine kinase Src and a transient increase in the intracellular concentration of Ca2+ at fertilization. LY294002 also inhibits sperm-induced dephosphorylation of mitogen-activated protein kinase, breakdown of cyclin B2 and Mos, and first embryonic cleavage, all of which are events of Ca2+-dependent egg activation. In fertilized eggs, an 85-kDa subunit of PI 3-kinase (p85) undergoes a transient translocation to the low-density, detergent-insoluble membranes (membrane microdomains) where Src tyrosine kinase signaling is operating. However, the tyrosine phosphorylation of p85 in fertilized eggs is not as evident as that in H2O2-activated eggs, arguing against the possibility that PI 3-kinase is activated by Src phosphorylation. Nevertheless, sperm-induced activation of PI 3-kinase has been demonstrated by the finding that Akt, a serine/threonine-specific protein kinase, is phosphorylated at threonine-308. The threonine-phosphorylated Akt also localizes to the membrane microdomains of fertilized eggs. Application of bp(V), an inhibitor of PTEN that dephosphorylates PIP3, the enzymatic product of PI 3-kinase, promotes parthenogenetic activation of Xenopus eggs. In vitro kinase assays demonstrate that PIP3 activates Src in a dose-dependent manner.ConclusionsThese results suggest that PI 3-kinase is involved in sperm-induced egg activation via production of PIP3 that would act as a positive regulator of the Src signaling pathway in Xenopus fertilization.

Analysis of signal transduction in cell-free extracts and rafts of Xenopus eggs

Intracellular signaling during egg activation/fertilization has been extensively studied using intact eggs, which can be manipulated by microinjection of different mRNAs, proteins, or chemical drugs. Furthermore, egg extracts, which retain high CSF activity (CSF-arrested extracts), were developed for studying fertilization/activation signal transduction, which have significant advantages as a model system. The addition of calcium to CSF-arrested extracts initiates a plethora of signaling events that take place during egg activation. Hence, the signaling downstream of calcium mobilization has been successfully studied in the egg extracts. Moreover, despite disruption of membrane-associated signaling compartments and ordered compartmentalization during extract preparation, CSF-arrested extracts can be successfully used to study early signaling events, which occur upstream of calcium release during egg activation/fertilization. In combination with the CSF-arrested extracts, activated egg rafts can reproduce some events of egg activation, including PLCgamma activation, IP3 production, transient calcium release, MAPK inactivation, and meiotic exit. This becomes possible due to complementation of the sperm-induced egg activation signaling machinery present in the rafts with the components of signal transduction system localized in the extracts. Herein, we describe protocols for studying molecular mechanisms of egg fertilization/activation using cell-free extracts and membrane rafts prepared from metaphase-arrested Xenopus eggs.

Calcium Signaling and Meiotic Exit at Fertilization in Xenopus Egg

Calcium is a universal messenger that mediates egg activation at fertilization in all sexually reproducing species studied. However, signaling pathways leading to calcium generation and the mechanisms of calcium-induced exit from meiotic arrest vary substantially among species. Here, we review the pathways of calcium signaling and the mechanisms of meiotic exit at fertilization in the eggs of the established developmental model, African clawed frog, Xenopus laevis. We also discuss calcium involvement in the early fertilization-induced events in Xenopus egg, such as membrane depolarization, the increase in intracellular pH, cortical granule exocytosis, cortical contraction, contraction wave, cortical rotation, reformation of the nuclear envelope, sperm chromatin decondensation and sister chromatid segregation.

Unfertilized frog eggs die by apoptosis following meiotic exit.

BackgroundA characteristic feature of frog reproduction is external fertilization accomplished outside the females body. Mature fertilization-competent frog eggs are arrested at the meiotic metaphase II with high activity of the key meiotic regulators, maturation promoting factor (MPF) and cytostatic factor (CSF), awaiting fertilization. If the eggs are not fertilized within several hours of ovulation, they deteriorate and ultimately die by as yet unknown mechanism.ResultsHere, we report that the vast majority of naturally laid unfertilized eggs of the African clawed frog Xenopus laevis spontaneously exit metaphase arrest under various environmental conditions and degrade by a well-defined apoptotic process within 48 hours after ovulation. The main features of this process include cytochrome c release, caspase activation, ATP depletion, increase of ADP/ATP ratio, apoptotic nuclear morphology, progressive intracellular acidification, and egg swelling. Meiotic exit seems to be a prerequisite for execution of the apoptotic program, since (i) it precedes apoptosis, (ii) apoptotic events cannot be observed in the eggs maintaining high activity of MPF and CSF, and (iii) apoptosis in unfertilized frog eggs is accelerated upon early meiotic exit. The apoptotic features cannot be observed in the immature prophase-arrested oocytes, however, the maturation-inducing hormone progesterone renders oocytes susceptible to apoptosis.ConclusionsThe study reveals that naturally laid intact frog eggs die by apoptosis if they are not fertilized. A maternal apoptotic program is evoked in frog oocytes upon maturation and executed after meiotic exit in unfertilized eggs. The meiotic exit is required for execution of the apoptotic program in eggs. The emerging anti-apoptotic role of meiotic metaphase arrest needs further investigation.

Bioinformatics Analysis and Optimization of Cell-Free Protein Synthesis

Cell-free protein synthesis offers substantial advantages over cell-based expression, allowing direct access to the protein synthetic reaction and meticulous control over the reaction conditions. Recently, we identified a number of statistically significant correlations between calculated and predicted properties of amino acid sequences and their amenability to heterologous cell-free expression. These correlations can be of practical use for predicting expression success and optimizing cell-free protein synthesis. In this chapter, we describe our approach and demonstrate how computational and predictive bioinformatics can be used to analyze and optimize cell-free protein expression.

Comparative homology modeling of pyruvate dehydrogenase kinase isozymes from Xenopus tropicalis reveals structural basis for their subfunctionalization

Structural–functional divergence is responsible for the preservation of highly homologous genes. Protein functions affected by mutagenesis in divergent sequences require investigation on an individual basis. In the present study, comparative homology modeling and predictive bioinformatics analysis were used to reveal for the first time the subfunctionalization of two pyruvate dehydrogenase kinase (PDK) isozymes in the western clawed frog Xenopus tropicalis. Three-dimensional structures of the two proteins were built by homology modeling based on the crystal structures of mammalian PDKs. A detailed comparison of them revealed important structural differences that modify the accessibility of the nucleotide binding site in the two isozymes. Based on the generated models and bioinformatics data analysis, the differences between the two proteins in terms of kinetic parameters, metabolic regulation, and tissue distribution are predicted. The results obtained are consistent with the idea that one of the xtPDKs is the major isozyme responsible for metabolic control of PDC activity in X. tropicalis, whereas the other one has more specialized functions. Hence, this study provides a rationale for the existence of two closely related PDK isozymes in X. tropicalis, thereby enhancing our understanding of the functional evolution of PDK family genes.

Content of intrinsic disorder influences the outcome of cell-free protein synthesis.

Cell-free protein synthesis is used to produce proteins with various structural traits. Recent bioinformatics analyses indicate that more than half of eukaryotic proteins possess long intrinsically disordered regions. However, no systematic study concerning the connection between intrinsic disorder and expression success of cell-free protein synthesis has been presented until now. To address this issue, we examined correlations of the experimentally observed cell-free protein expression yields with the contents of intrinsic disorder bioinformatically predicted in the expressed sequences. This analysis revealed strong relationships between intrinsic disorder and protein amenability to heterologous cell-free expression. On the one hand, elevated disorder content was associated with the increased ratio of soluble expression. On the other hand, overall propensity for detectable protein expression decreased with disorder content. We further demonstrated that these tendencies are rooted in some distinct features of intrinsically disordered regions, such as low hydrophobicity, elevated surface accessibility and high abundance of sequence motifs for proteolytic degradation, including sites of ubiquitination and PEST sequences. Our findings suggest that identification of intrinsically disordered regions in the expressed amino acid sequences can be of practical use for predicting expression success and optimizing cell-free protein synthesis.

Organization of the multiaminoacyl-tRNA synthetase complex and the cotranslational protein folding

Aminoacyl‐tRNA synthetases (ARSs) play an essential role in the protein synthesis by catalyzing an attachment of their cognate amino acids to tRNAs. Unlike their prokaryotic counterparts, ARSs in higher eukaryotes form a multiaminoacyl‐tRNA synthetase complex (MARS), consisting of the subset of ARS polypeptides and three auxiliary proteins. The intriguing feature of MARS complex is the presence of only nine out of twenty ARSs, specific for Arg, Asp, Gln, Glu, Ile, Leu, Lys, Met, and Pro, regardless of the organism, cell, or tissue types. Although existence of MARSs complex in higher eukaryotes has been already known for more than four decades, its functional significance remains elusive. We found that seven of the nine corresponding amino acids (Arg, Gln, Glu, Ile, Leu, Lys, and Met) together with Ala form a predictor of the protein α‐helicity. Remarkably, all amino acids (besides Ala) in the predictor have the highest possible number of side‐chain rotamers. Therefore, compositional bias of a typical α‐helix can contribute to the helixs stability by increasing the entropy of the folded state. It also appears that position‐specific α‐helical propensity, specifically periodic alternation of charged and hydrophobic residues in the helices, may well be provided by the structural organization of the complex. Considering characteristics of MARS complex from the perspective of the α‐helicity, we hypothesize that specific composition and structure of the complex represents a functional mechanism for coordination of translation with the fast and correct folding of amphiphilic α‐helices.

Identification of multiple physicochemical and structural properties associated with soluble expression of eukaryotic proteins in cell-free bacterial extracts.

Bacterial extracts are widely used to synthesize recombinant proteins. Vast data volumes have been accumulated in cell-free expression databases, covering a whole range of existing proteins. It makes possible comprehensive bioinformatics analysis and identification of multiple features associated with protein solubility and aggregation. In the present paper, an approach to identify the multiple physicochemical and structural properties of amino acid sequences associated with soluble expression of eukaryotic proteins in cell-free bacterial extracts is presented. The method includes: (1) categorical assessment of expression data; (2) calculation and prediction of multiple properties of expressed sequences; (3) correlation of the individual properties with the expression scores; and (4) evaluation of statistical significance of the observed correlations. Using this method, a number of significant correlations between calculated and predicted properties of amino acid sequences and their propensity for soluble cell-free expression have been revealed.

Monitoring gene expression in a single Xenopus oocyte using multiple cytoplasmic collections and quantitative RT-PCR.

Oocytes and eggs of the African clawed frog, Xenopus laevis, are commonly used in gene expression studies. However, monitoring transcript levels in the individual living oocytes remains challenging. To address this challenge, we used a technique based on multiple repeated collections of nanoliter volumes of cytoplasmic material from a single oocyte. Transcript quantification was performed by quantitative RT‐PCR. The technique allowed monitoring of heterologous gene expression in a single oocyte without affecting its viability. We also used this approach to profile the expression of endogenous genes in living Xenopus oocytes. Although frog oocytes are traditionally viewed as a homogenous cell population, a significant degree of gene expression variation was observed among the individual oocytes. A lognormal distribution of transcript levels was revealed in the oocyte population. Finally, using this technique, we observed a dramatic decrease in the content of various cytoplasmic mRNAs in aging unfertilized eggs but not in oocytes, suggesting a link between mRNA degradation and egg apoptosis.