Model-Driven Understanding of Palmitoylation Dynamics: Regulated Acylation of the Endoplasmic Reticulum Chaperone Calnexin

Cellular functions are largely regulated by reversible post-translational modifications of proteins which act as switches. Amongst these, S-palmitoylation is unique in that it confers hydrophobicity. Due to technical difficulties, the understanding of this modification has lagged behind. To investigate principles underlying dynamics and regulation of palmitoylation, we have here studied a key cellular protein, the ER chaperone calnexin, which requires dual palmitoylation for function. Apprehending the complex inter-conversion between single-, double- and non- palmitoylated species required combining experimental determination of kinetic parameters with extensive mathematical modelling. We found that calnexin, due to the presence of two cooperative sites, becomes stably acylated, which not only confers function but also a remarkable increase in stability. Unexpectedly, stochastic simulations revealed that palmitoylation does not occur soon after synthesis, but many hours later. This prediction guided us to find that phosphorylation actively delays calnexin palmitoylation in resting cells. Altogether this study reveals that cells synthesize 5 times more calnexin than needed under resting condition, most of which is degraded. This unused pool can be mobilized by preventing phosphorylation or increasing the activity of the palmitoyltransferase DHHC6.     

Involvement of the alternative oxidase in respiration of Yarrowia lipolytica mitochondria is controlled by the activity of the cytochrome pathway

The degree of involvement of cyanide-resistant alternative oxidase in the respiration of Yarrowia lipolytica mitochondria was evaluated by comparing the rate of oxygen consumption in the presence of cyanide, which shows the activity of the cyanide-resistant alternative oxidase, and the oxidation rate of cytochrome c by ferricyanide, which shows the activity of the main cytochrome pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was associated with oxygen consumption due to the functioning of the alternative oxidase. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation) completely inhibited oxygen consumption by the mitochondria. Under these conditions, the inhibition of the alternative oxidase by benzohydroxamic acid (BHA) failed to affect the reduction of ferricyanide at the level of cytochrome c. BHA did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These data indicate that the alternative system is unable to compete with the cytochrome respiratory chain for electrons. The alternative oxidase only transfers the electrons that are superfluous for the cytochrome respiratory chain.     

Information about a form (on the dynamic laws of morphogenesis)

How a developing embryo becomes "informed" about its form?" This problem remains obscure and controversial. We argue that the "information about a form" is distributed throughout three main components: the dynamic laws, the parameters and the initial/boundary conditions. In the absence of a dynamic law two other components are "blind", that is, do not contain any unambiguous information. We present a version of a dynamic law of morphogenesis, based upon the presumption of a feedback between passive and active mechanical stresses. We explore several models of shape formation based upon this law and show that, as depending upon the parameters values, they generate a large set of realistic shapes. Genetic and epigenetic basis of the models parameters is discussed.     

A computational framework for the design of optimal protein synthesis

Despite the establishment of design principles to optimize codon choice for heterologous expression vector design, the relationship between codon sequence and final protein yield remains poorly understood. In this work, we present a computational framework for the identification of a set of mutant codon sequences for optimized heterologous protein production, which uses a codon-sequence mechanistic model of protein synthesis. Through a sensitivity analysis on the optimal steady state configuration of protein synthesis we are able to identify the set of codons, that are the most rate limiting with respect to steady state protein synthesis rate, and we replace them with synonymous codons recognized by charged tRNAs more efficient for translation, so that the resulting codon-elongation rate is higher. Repeating this procedure, we iteratively optimize the codon sequence for higher protein synthesis rate taking into account multiple constraints of various types. We determine a small set of optimized synonymous codon sequences that are very close to each other in sequence space, but they have an impact on properties such as ribosomal utilization or secondary structure. This limited number of sequences can then be offered for further experimental study. Overall, the proposed method is very valuable in understanding the effects of the different properties of mRNA sequences on the final protein yield in heterologous protein production and it can find applications in synthetic biology and biotechnology.     

Impact of iron reduction on the metabolism of Clostridium acetobutylicum

Iron is essential for most living organisms. In addition, its biogeochemical cycling influences important processes in the geosphere (e.g., the mobilization or immobilization of trace elements and contaminants). The reduction of Fe(III) to Fe(II) can be catalysed microbially, particularly by metal-respiring bacteria utilizing Fe(III) as a terminal electron acceptor. Furthermore, Gram-positive fermentative iron reducers are known to reduce Fe(III) by using it as a sink for excess reducing equivalents, as a form of enhanced fermentation. Here, we use the Gram-positive fermentative bacterium Clostridium acetobutylicum as a model system due to its ability to reduce heavy metals. We investigated the reduction of soluble and solid iron during fermentation. We found that exogenous (resazurin, resorufin, anthraquinone-2,6-disulfonate) as well as endogenous (riboflavin) electron mediators enhance solid iron reduction. In addition, iron reduction buffers the pH, and elicits a shift in the carbon and electron flow to less reduced products relative to fermentation. This study underscores the role fermentative bacteria can play in iron cycling and provides insights into the metabolic profile of coupled fermentation and iron reduction with laboratory experiments and metabolic network modelling.     

Universal,easy access to geotemporal information: FetchClimate

Geotemporal information, information associated with geographical space and time, has always been critical to climate and environmental science. However, this information is certainly not universally or easily accessible. In fact, obtaining and using geotemporal information often comes with a considerable technical overheads, impeding research progress. To address this, we introduce FetchClimate: a cloud service designed to provide easy, universal access to geotemporal information. FetchClimate enables and accelerates the use of geotemporal information by enabling it to be accessed programmatically from a Web service (such as the statistical software R) or non‐programmatically using a Web browser. We intend the service to accelerate the pace of ecological and environmental research by eliminating the technical overheads currently needed to obtain geotemporal information. The software, online manual, and user support are freely available at < http://www.fetchclimate.com >.     

Exome‐based proteogenomics of HEK‐293 human cell line: Coding genomic variants identified at the level of shotgun proteome

Genomic and proteomic data were integrated into the proteogenomic workflow to identify coding genomic variants of Human Embryonic Kidney 293 (HEK‐293) cell line at the proteome level. Shotgun proteome data published by Geiger et al. (2012), Chick et al. (2015), and obtained in this work for HEK‐293 were searched against the customized genomic database generated using exome data published by Lin et al. (2014). Overall, 112 unique variants were identified at the proteome level out of ～1200 coding variants annotated in the exome. Seven identified variants were shared between all the three considered proteomic datasets, and 27 variants were found in any two datasets. Some of the found variants belonged to widely known genomic polymorphisms originated from the germline, while the others were more likely resulting from somatic mutations. At least, eight of the proteins bearing amino acid variants were annotated as cancer‐related ones, including p53 tumor suppressor. In all the considered shotgun datasets, the variant peptides were at the ratio of 1:2.5 less likely being identified than the wild‐type ones compared with the corresponding theoretical peptides. This can be explained by the presence of the so‐called “passenger” mutations in the genes, which were never expressed in HEK‐293 cells. All MS data have been deposited in the ProteomeXchange with the dataset identifier PXD002613 ( http://proteomecentral.proteomexchange.org/dataset/PXD002613 ).